Studies on the biological functions of interaction between components in Wnt, TGF-β and HIF pathways for

cancer progression

KARTHIK ARIPAKA

Department of Medical Biosciences Umeå 2019

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

Copyright © Karthik Aripaka

Dissertation for PhD

ISBN: 978-91-7855-140-8

ISSN: 0346-6612

New series no: 2060

Electronic version available at: http://umu.diva-portal.org/

Printed by: Cityprint i Norr AB

Umeå, Sweden 2019

Cover picture front: Cancer cell signalling and targeting

Cover picture back: Immunofluorescence image showing protein expression of LRP5 (red)

and TRAF6 (green) in PC3U cells with nuclei stained by DAPI (blue). Co-localisation of

LRP5 and TRAF6 was observed (yellow) in the merged image (bottom right)

Cover picture design and composition: Designed by Karthik Aripaka using vector

resources from Freepik.com

You have to dream before your dreams can come true.

The dream is not that which you see while sleeping; it i s something

that does not let you s leep.

Dream, Dream, Dream; Dream transform into thoughts and

thoughts result in action.

- A P J Abdul Kalam

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To my father and mother

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Table of contents

TABLE OF CONTENTS .............................................................................................. V ABSTRACT ............................................................................................................... VII ORIGINAL PAPERS .................................................................................................. IX ABBREVIATIONS ........................................................................................................ X ENKEL SAMMANFATTNING PÅ SVENSKA .................................................... XIII INTRODUCTION / BACKGROUND ......................................................................... 1 1. Cancer: ................................................................................................................... 1

1.1. Prostate Cancer: ............................................................................................ 3 1.2. Renal Cell Carcinoma: .................................................................................. 6

2. Wnt signalling Pathway: ....................................................................................... 9 2.1. Canonical Wnt/β-catenin Pathway ............................................................. 11 2.2. Non-canonical Wnt pathways ..................................................................... 14 2.3. Wnt signalling in development ................................................................... 16 2.4. Wnt signalling in adult tissue homeostasis................................................. 18 2.5. Wnt signalling in cancer .............................................................................. 19

3. TGF-β signalling pathway: ................................................................................. 21 3.1. Smad signalling (Canonical): ...................................................................... 22 3.2. Non-Smad signalling (Non-Canonical): ..................................................... 24 3.3. TGF-β signalling in cancer: ........................................................................ 26

4. HIF (Hypoxia-inducible factor) system.............................................................. 28 4.1. Components of the HIF pathway ................................................................ 29 4.2. HIF signalling: ............................................................................................. 32

5. Epithelial to mesenchymal transition (EMT) .................................................... 34 6. Ubiquitination: ..................................................................................................... 36 7. TRAF6: ................................................................................................................. 39 AIMS ............................................................................................................................. 43 MATERIALS AND METHODS ................................................................................ 44 Materials: ..................................................................................................................... 44

Chemicals and reagents: ............................................................................................ 44 Instruments: ............................................................................................................... 45 Cell lines: ................................................................................................................... 46 Plasmids: .................................................................................................................... 47 siRNA: ....................................................................................................................... 47 Patients and tumour samples: .................................................................................... 48

Methods: ....................................................................................................................... 48 Invitro cell culture and transfections: ........................................................................ 48 Immunoblotting (IB) and Immunoprecipitation (IP): ................................................ 49 Immunofluorescence: ................................................................................................ 52 Dual-Luciferase assay: ............................................................................................... 52 Proximity Ligation assay (PLA): ............................................................................... 52 Ubiquitination Assay: ................................................................................................ 53 Quantitative real-time PCR: ....................................................................................... 53 Invasion Assay: .......................................................................................................... 54 Label-free monitoring of a TRAF6-LRP5 protein interaction: .................................. 54 In silico gene expression analysis: ............................................................................. 55

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Statistical analysis: ..................................................................................................... 55 Zebrafish Strains and Maintainance: ......................................................................... 55 CRISPR/Cas9 mediated generation Zebrafish TRAF6 mutants: ............................... 56 Zebrafish DNA - RNA extraction and quantitative RT-PCR: ................................... 56 Whole Mount staining for Zebrafish bone: ................................................................ 57

RESULTS AND DISCUSSION .................................................................................. 58 Paper I: ......................................................................................................................... 58 Paper II:........................................................................................................................ 61 Paper III: ...................................................................................................................... 64 CONCLUDING REMARKS ...................................................................................... 67 ACKNOWLEDGEMENTS ........................................................................................ 69 REFERENCES ............................................................................................................ 74

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Abstract

Cancer is a disease that involves aggressive changes in the genome and aberrant signals between the living cells. Signalling pathways such as TGF-β (Transforming growth factor-β), Wnt, EGF (epidermal growth factor) and HIF (Hypoxia-inducible factor) evolved to regulate growth and development in mammals are also implicated for tumorigenesis due to failure or aberrant expression of components in these pathways. Cancer progression is a multistep process, and these steps reflect genetic alterations driving the progressive transformation of healthy human cells into highly malignant derivatives. Many types of cancers are diagnosed in the human population, such as head & neck, cervical, brain, liver, colon, prostate, uterine, breast, and renal cell cancer.

Prostate cancer is the second most common cancer and one of the foremost leading cancer-related deaths in men in the world. Aberrant Wnt3a signals promote cancer progression through the accumulation of β-Catenin. In the first paper, we have elucidated intriguing functions for TRAF6 (Tumour necrosis factor receptor-associated factor-6) as a coregulatory factor for the expression of Wnt-target genes which was confirmed in vivo by using CRISPR/Cas9 genomic editing, in zebrafish. Our data suggest that Wnt3a promotes TRAF6 interaction with Wnt components, and TRAF6 is required for gene expression of β-Catenin as well as for the Wnt-ligand co-receptor LRP5. From the in vivo studies, we elucidated positive regulation of TRAF6, which is crucial for survival and development of zebrafish. This study identifies TRAF6 as an evolutionary conserved co-regulatory protein in the Wnt pathway that also promotes the progression of prostate and colorectal cancer due to its positive effects on Wnt3a signalling.

Hypoxia is a condition due to O2 deprivation, and Hypoxia-inducible factors (HIF) transcription factors are responsible for the maintenance of oxygen homeostasis in living cells. Irregularities in these HIF transcription factors trigger pathological cellular responses for initiation and progression of malignant cancers. Renal cell carcinoma, a malignant cancer arising in renal parenchyma and renal pelvis and, hypoxia plays a

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vital role in its progression. In the second paper, we have investigated the clinicopathological relevance of several hypoxic and TGF-β component proteins such as HIF-1α/2α/3α, TGF-β type 1 receptor (ALK5-FL) and the intracellular domain of ALK5 (ALK5-ICD), SNAI1 and PAI-1 with patient survival in clear cell renal cell carcinoma (ccRCC). We showed that HIF-2α associated with low cancer-specific survival. HIF-2α and SNAI1 positively correlated with ALK5-ICD, pSMAD2/3, PAI-1 and SNAI1 with HIF-2α; HIF-1α positively correlated with pSMAD2/3. Further, under normoxic conditions, our data suggest that ALK5 interacts with HIF-1α and HIF-2α, and promotes their expression and target genes such as GLUT1 and CA9, in a VHL dependent manner through its kinase activity. These findings shed light on the critical aspect of cross-talk between TGF-β signalling and hypoxia pathway, and also the novel finding of an interaction between ALK5 and HIF-α might provide a more in-depth understanding of mechanisms behind tumour progression

In the third paper, an ongoing study, we investigated the role of HIF-3α in the progression of Renal cell carcinoma and its association with the components of TGF-β and HIF pathways. We have observed increased levels of HIF-3α in ccRCC and pRCC (papillary renal cell carcinoma) which are associated with advanced tumour stage, metastasis and larger tumours. Also, we found HIF-3α show a significant positive association with pro-invasive gene SNAI1, which is a crucial regulator of epithelial to mesenchymal transition. TRAF6 an E3 ligase known to be a prognostic marker in RCC and we observed HIF-3α associates with TRAF6.

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Original Papers

I. TRAF6 function as a novel co-regulator of Wnt3a target genes in prostate cancer.

Karthik Aripaka, Shyam Kumar Gudey, Guangxiang Zang, Alexej Schmidt, Samaneh Shabani Åhrling, Lennart Österman, Anders Bergh, Jonas von Hofsten, Marene Landström. EBioMedicine. 2019 Jun 28. Volume 45, Pages 192–207

II. Interactions between TGF-β type I receptor and hypoxia-inducible factor-α mediates a synergistic crosstalk leading to poor prognosis for patients with clear cell renal cell carcinoma.

Pramod Mallikarjuna, Tumkur Sitaram Raviprakash, Karthik Aripaka, Börje Ljungberg & Marene Landström. Cell Cycle. 2019 Jul 24. Volume 18, Pages 2141-2156

III. Expression and association of HIF-3α with hypoxic and TGF-β signalling components in Renal cell carcinoma. (on-going manuscript )

Pramod Mallikarjuna, Karthik Aripaka, Tumkur Sitaram Raviprakash, Börje Ljungberg & Marene Landström.

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Abbreviations

ALK5 Activin A receptor type II-like kinase 5 AMH Anti-Mullerian Hormone Angptl4 Angiopoietin-like 4 APC Adenomatous polyposis coli ARNT Aryl hydrocarbon receptor nuclear translocator ATF2 Activating Transcription Factor 2 ATP Adenosine triphosphate BCL2/9 B-cell lymphoma 2/9 BHD Birt-Hogg-Dube syndrome bHLH Basic helix-loop-helix domain BMP Bone morphogenetic protein BRCA1&2 Breast Cancer Type 1 Susceptibility Protein 1&2 CA9 Carbonic anhydrase 9 CAFs Cancer-associated fibroblasts CAMKII Ca2+/calmodulin-dependent kinase II Cas9 CRISPR associated protein-9 nuclease CBP cAMP response element-binding protein (CREB)-

binding protein CCND1 Cyclin D1 ccRCC Clear cell renal cell carcinoma CDK Cyclin-dependent kinases CK1α Casein kinase 1α CRISPR Clustered regularly interspaced short Palindromic

repeats CRPC Castration-resistant prostate cancer DAAM1 Dishevelled associated activator of morphogenesis DAG 1,2 diacylglycerol DAPK Death-associated protein kinase DKK Dickkopf DUB Deubiquitinating enzymes DVL Dishevelled EGF Epidermal growth factor EMT Epithelial to mesenchymal transition EPHB3 Ephrin Type-B Receptor 3 ERG Erythroblast transformation-specific related gene FGF Fibroblast growth factors

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FH Fumarate hydratase FZD Frizzled GDFs Growth and differentiation factors GLUT1 Glucose transporter 1 GRHL2 Grainyhead Like Transcription Factor 2 GSK3β Glycogen synthase kinase-3β HCC Hepatocellular carcinoma HDAC Histone deacetylases HIF Hypoxia inducible factor HOXB13 Homeo Box B13 HPC1/2 Hereditary prostate cancer 1/2 HRE Hypoxia-responsive elements hTERT Human telomerase reverse transcriptase IGF-I Insulin growth factor-I IP3 Inositol 1,4,5-triphosphate JNK c-Jun N-terminal kinases LDLR Low-density lipoprotein receptor LGR5 Leucine-rich repeat-containing G-protein coupled

receptor 5 LRP5/6 Low-density lipoprotein receptor-related protein

5/6 LTBP Latent TGF-β binding protein MAPK Mitogen-activated protein kinase MIR Mortality to incidence ratio MMP7 Matrix Metalloproteinase 7 MMTV Mouse Mammary tumour virus MSX2 Msh Homeobox 2 mTOR Mammalian target of rapamycin NFAT Nuclear factor of activated T-cells NFκB Nuclear factor kappa-light-chain-enhancer of

activated B cells NICD Notch intra cellular domain OEA Ovarian endometrioid adenocarcinomas PAI-1 Plasminogen activator inhibitor-1 PAX6 Paired box protein Pax-6 PCP Planar cell polarity PI3K Phosphatidylinositol-3´kinase PKC Protein kinase C

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pRCC Papillary renal cell carcinoma PS1 Presenilin 1 PSA Prostate-specific antigen RCC Renal cell carcinoma RING Really interesting new gene RNF43 Ring finger 43 ROCK Rho-associated, coiled-coil containing protein

kinase ROR2 Receptor Tyrosine Kinase Like Orphan Receptor 2 RSPO2 R-Spondin 2 RYK Receptor Like Tyrosine Kinase sFRPs Secreted Frizzled-related proteins SGF Sarcoma growth factor SOST Sclerostin TACE TNF alpha converting enzyme TAK1 TGF-β activated kinase 1 TCFLEF T-cell factor/lymphoid enhancer-binding factor TGF-β Transforming growth factor- β TGIF Transforming Growth Factor-β Induced Factor TIRAP Toll/interleukin-1 receptor adaptor protein TMPRSS2 Transmembrane Serine Protease 2 TNF-α Tumour necrosis factor- α TRAF6 Tumour necrosis factor receptor-associated factor

6 TβRI TGFβ receptor I VEGF Vascular endothelial growth factor VHL Von Hippel-Lindau Wg Wingless WIF1 Wnt-interacting factor Wls/Evi Wnt Ligand Secretion Mediator ZEB1/2 Zinc finger E-box-binding homeobox 1/2 ZNFR3 Zinc and ring finger 3 β-Trcp β-transducin repeats-containing proteins

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Enkel sammanfattning på svenska

Cancer är en sjukdom som innefattar aggressiva förändringar i genomet och abnormala signaler mellan levande celler. Signalvägar såsom Transforming Growth Factor-β (TGF-β), Wnt, Epidermal Growth Factor (EGF) och Hypoxia-inducible factor (HIF) utvecklades i mammalieceller för att reglera tillväxt och utveckling och är också involverade i tumörutveckling på grund av bristande eller abnormala uttryck av komponenter i dessa signalvägar. Progression av cancer sker i flera steg och dessa steg reflekterar genetiska förändringar som driver progressiv transdifferentiering av friska humana celler till att bli mycket maligna cancerceller. Ett flertal olika cancerformer diagnostiseras hos människor som t.ex huvud- och halscancer, hjärntumörer, levercancer, tjocktarmscancer, prostatacancer, livmodercancer, bröstcancer och njurcancer.

Prostatacancer är den näst vanligaste cancerformen och en av de vanligaste cancer-relaterade dödsorsakerna hos män globalt sett. Avvikande Wnt3a-signalering befrämjar progression av cancer pga av att β-catenin accumuleras. I den första artikeln, har vi belyst nya och spännande funktioner för Tumour necrosis factor Receptor-Associated Factor 6 (TRAF6) som en co-regulerande faktor för att reglera gener som regleras av Wnt. Detta fynd konfirmerades in vivo genom användandet av den s.k. gensaxen det vill säga CRISPR/Cas9 i zebrafisk. Våra data antyder att Wnt3a befrämjar att TRAF6 fysiskt kan binda till komponenter i Wnt-signaleringskedjan och att TRAF6 krävs för att stimulera genuttrycket av β-catenin samt för en Wnt-ligand co-receptor som heter LRP5. Från dessa in vivo studier har vi belyst hur TRAF6 är ett evolutionärt bevarat protein i Wnt-signaleringskedjan som också befrämjar tumörprogression av både prostatacancer och tjocktarmscancer pga av sina positiva effekter på Wnt3a-signalering,

Hypoxi är ett tillstånd som beror på syrebrist och hypoxia-inducible factors (HIF) som är transkriptionsfaktorer som är ansvariga för upprätthållandet av syre homeostasis i levande celler. Avvikelser i dessa HIF

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transkriptionsfaktorer utlöser patologiska cellulära svar som leder till initiering och progression av maligna celler. Njurcancer uppstår i njurparenkymet och njurbäckenet och syrebrist spelar en viktig roll för att stimulera till progression av denna cancerform. I den andra artikeln har vi undersökt den kliniska och patologiska betydelsen av flera hypoxi och TGF-β signalkomponenter såsom HIF-1α/2α/3α, TGF-β Typ I receptor (ALK5-full längd) och den intracellulära domänen av ALK5 (ALK5-ICD), SNAI1 och PAI-1 med patientöverlevnad för patienter med klarcellig njurcancer (ccRCC). Vi visade att uttrycket av HIF-2α är associerat med låg cancer-specifik överlevnad. HIF-2α och SNAI1 är positivt korrelerade med ALK5-ICD, pSMAD2/3, PAI-1 och SNAI1 med HIF-2α; HIF-1α var positivt korrelerat med pSMAD2/3. Dessutom, under normala syreförhållanden visar våra data att ALK5 binder till HIF-1α och HIF-2α, och befrämjar deras uttryck samt uttrycket av deras s.k. målgener såsom GLUT1 och CA9, på ett VHL- och ALK5-kinase-beroende sätt. Dessa fynd belyser kritiska aspekter av ett samspel mellan TGF-β signalering och hypoxi, och erbjuder också en ökad förståelse genom vilka mekanismer tumörprogression styrs.

I det tredje manuskriptet, som är en pågående studie har vi undersökt rollen av HIF-3α för tumörprogressionen av njurcancer, och sambandet mellan HIF-3α och olika komponenter i TGFβ och HIF signaleringskedjan. Vi har funnit ökade nivåer av HIF-3α i klarcellig njurcancer (ccRCC) och papillär njurcancer, som var associerade med avancerat tumörstadium, metastaser och stora tumörer. Dessutom fann vi att HIF-3α uppvisade en signifikant, positiv association med den pro-invasiva genen SNAI1 som är en kritisk regulator av epitelial till mesenkymal transdifferentiering. TRAF6 som är ett E3 ubiquitin-ligase, är en prognostisk markör för njurcancer och vi har observerat att HIF-3α binder till TRAF6.

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Introduction / Background

1. Cancer:

Living organisms need a systematic and regulated development which is

one of the most complex and intricate phenomena in nature. Growth,

differentiation and maintenance have a synergistic approach in the process

of development. As the maximum degree of differentiation is reached, the

growth comes to a halt. Multicellular organisms can regenerate/repair their

tissues during the process of injury and healing by regulated cell division

which is governed by several complex signalling networks which maintain

the tight control. In certain instances, this growth, to a certain extent, skips

from the control of the organism and expresses properties such as

uncontrolled and unlimited proliferation along with disorganised

differentiation. This type of growth further results in progressive invasion

and metastasis, leading to the destruction of healthy neighbouring tissues

and ultimately lethal to the whole organism. This phenomenon/condition

of abnormal growth is known as cancer (1, 2).

Cancers are caused due to the contribution of several factors such as

somatic or germline mutations, environmental factors, chronic infections

and dietary factors (3). Recent studies have shown that common solid

tumours display subtle mutations in ~140 driver genes out of which 95%

are single-base substitutions which confer a selective growth advantage to

the mutated cells. Driver genes through several signalling pathways,

regulate core cellular functions such as cell fate determination, cell

survival and genome maintenance. These core functions are compromised

due to mutations in the driver genes directing the cells towards cancer

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progression (4). Cancer cells evolve progressively from healthy cells by

attaining biological capabilities gradually in the multistep process of

tumorigenesis. In 2011, Hanahan and Weinberg (5) proposed eight

hallmarks of cancer along with two enabling characteristics for

understanding the architecture behind the diversity of cancer development

(Fig.1).

Fig 1: The figure illustrates the Emerging Hallmarks of cancer and enabling characteristics proposed by Hanahan and Weinberg (5).

Various Growth factor signalling pathways are deregulated, resulting in

cancer progression, which is one of the hallmarks. Growth factors such as

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Transforming growth factor- β (TGF-β), Fibroblast growth factors (FGF),

Tumour necrosis factor- α (TNF-α), Vascular endothelial growth factor

(VEGF), interleukins and Wnt are responsible for the release of growth-

promoting signals and regulation of cell cycle to maintain homeostasis in

the normal cells. These cancer cells attain competence to survive without

these external growth signals and develop capabilities like producing

growth factor ligands or by destroying the normal control mechanisms

which prevent negative feedback mechanism or by constitutive activation

of downstream components in a signalling pathway. Evading growth

suppressors, resisting apoptosis, uncontrolled replication, sustained

angiogenesis, activating invasion and metastasis, abnormal metabolism

and escaping immune destruction were the other proposed hallmarks (5).

1.1. Prostate Cancer:

Prostate cancer is one of the most common forms of cancer in men.

Prostate cancer is the second most frequently diagnosed cancer and the

fifth leading cause of cancer-related death in men. According to

GLOBOCAN statistics in 2018, it is estimated that the incidence of 1.3

million new cases of prostate cancer with 359,000 related deaths

worldwide (3.8% of all cancer-related deaths in men) (6, 7). High

incidence rates of prostate cancer were observed in North America,

Northern Africa, Australia and Europe, especially in Northern Europe.

High cancer-related deaths and high mortality rates were reported in the

Caribbean and African countries. Due to improved treatment with better

diagnosis and early screening methods, the prostate cancer-related death

rates were low in countries like United States, Australia, Northern and

Western Europe. Mortality to incidence ratio (MIR) of prostate cancer is

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very low in developed countries with best health care systems such as the

United States, Norway, Sweden, Ireland and Australia (6, 8). In Sweden,

prostate cancer is the most commonly diagnosed cancer with a high

number of deaths in men compared to other cancer forms. In 2017, about

10,300 men were diagnosed, and about 2300 died with prostate cancer

(Swedish Cancer Registry, The National Board of Health and Welfare -

2017).

Fig 2: Illustration shows the anatomy of male reproductive organs with the

prostate gland and cancerous tumour. “Prostate anatomy image ©American

Cancer Society, 2017. Used with permission.”

Prostate cancer is a multifaceted disease, which can be asymptomatic as

well as a hostile, aggressive disease with poor prognosis. Prostate cancer

initially spreads to glandular acini, further extends to the outer tissue of

prostate gland, seminal vesicles and rectum (Fig 2). In the later high-grade

stages, metastasis occurs to the bone, lymph nodes and lungs (9). Most

common symptoms of this disease are painful and frequent urination,

nocturia, blood in urine, erectile dysfunction and prostatitis. In the

advanced stages, urinary retention and pain in the back might occur due to

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metastasis in bone (10, 11). Recent research has made advances in

understanding the underlying mechanism of prostate cancer. Prostate-

specific antigen (PSA) is the widely adopted screening method, but its

application remains controversial because of the uncertainty around its

benefits and risks (12, 13). The variation in the incidence rate of prostate

cancer between the populations is linked to differences in diagnostic usage

of PSA screening. Countries with high sociodemographic index have high

incidence rates due to better screening (12, 14).

Individual biological and lifestyle factors may contribute to the risk of

developing prostate cancer such as age, ethnicity, genetics, family history,

diet and other factors. In men, the risk of prostate cancer increases with

age. Men with age over 55 years have a 17-fold higher chance of

developing prostate cancer, especially with high incidence in African-

American men and low in Asian men (14, 15). African-American men

have common chromosome variants in 8q24, shown to associate with the

occurrence of prostate cancer (16). Also, mutations in genes regulating

apoptosis such as BCL2 has been reported to cause prostate cancer in

African American men (17). Established risk factors for the incidence of

prostate cancer also linked to the genetic mutations, which can be either

dominant, recessive or X-linked. Mutations in genes like BRCA1&2 (11),

HOXB13 (18) and HPC1 & HPC2 (ELAC2) (19) are linked to the

increased risk of hereditary prostate cancer. Several genome-wide studies

in prostate cancer have reported 77 SNPs associated with the disease. One

of the SNPs is located in a non-coding region close to the c-Myc oncogene,

affecting its expression (20). TMPRSS2: ERG gene fusion is the most

common genetic alteration observed in prostate cancer patients (21).

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ELAC2, known to be involved in prostate cancer incidence, binds to

activated SMAD2 and potentiates TGF-β regulated transcriptional

responses (22). Increased Insulin growth factor (IGF)-I levels correlate

with an elevated risk of prostate cancer (23). Disorders in androgen

receptor signalling like androgen receptor gene amplification associated

with castration-resistant prostate cancer (CRPC) (24). Dietary habits with

its range of effects also contribute to the risk of prostate cancer. High

consumption of saturated animal fats, red meat, dairy products shown to

be linked with the prostate cancer risk in several studies (15, 25).

Currently, most effective treatment methods involve histopathological

grading using a scoring system named Gleason grade, radical

prostatectomy, radiation therapy, chemotherapy, immunotherapy, bone-

targeting treatment and androgen-deprivation hormonal therapy (13, 24,

26). Relapse is a very common feature of this disease due to the

progression of cancer from hormone-sensitive to hormone-refractory state

(24). Molecular mechanisms underlying this relapse still need to be

addressed. Development of better prognostic markers and effective

treatment methods are essential future goals for the cancer researchers in

this field.

1.2. Renal Cell Carcinoma:

Renal cell carcinoma (RCC) is a common malignant cancer in adults

arising in renal parenchyma and renal pelvis. In 2012, the incidence of new

cases was estimated at 338,000 cases, with an estimated 143,000 deaths in

the world (27). According to GLOBOCAN 2018, the incidence of new

cases increased to 403,262 cases with 175,098 death cases (6). RCC is the

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11th most common type of cancer in Sweden, and in 2018 the incidence of

new cases and the number of deaths are estimated at 1299 and 629

respectively with high incidence rates in men (6). The highest incidence of

RCC was found in European countries mainly, Czech Republic, Lithuania,

Slovakia and Denmark, followed by North America and Australia (27-29).

Established risk factors include obesity, smoking, alcohol consumption,

hypertension and familial inheritance. Incidence rates of renal cell

carcinoma increase with the increase in age with the highest incidence at

age 75 years (29, 30). Symptoms include flank pain, haematuria, palpable

abdominal mass, weight loss and anaemia (31).

RCC comprises of several histological subtypes. Clear cell renal cell

carcinoma (ccRCC) accounts for 70% of RCC followed by 10-15% of

papillary renal cell carcinoma, 5% of chromophobe renal cell carcinoma

and 1% of other rare forms (32). ccRCC also called conventional RCC,

consists of cells with clear cytoplasm and arises from proximal renal

convoluted tubular epithelial cells (33). ccRCC occurrence is usually

sporadic, but also have rare familial inheritance patterns, i.e., germline

mutations in the von Hippel-Lindau gene (VHL) which is located on the

chromosome 3p25. This mutation results in a disease called von Hippel-

Lindau disease, and patients develop ccRCC due to inherited disease (34).

Loss of heterozygosity, implying loss of one allele on chromosome 3p,

which contains a VHL gene leads to sporadic ccRCC. Also, hyper-

methylation of the VHL gene is found in 11-19% of sporadic ccRCC (35-

37).

Papillary renal cell carcinoma is the second most common RCC, which

predominantly affects males. Papillary RCC originates from distal

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convoluted tubule with papillary or tubular architectural pattern (35).

Based on the cytoplasmic and nuclear staining, they were subdivided into

type I and type II. Missense mutations in the tyrosine kinase domain of c-

MET proto-oncogene are shown to cause type I hereditary papillary renal

cell carcinoma. Germline mutations in the FH gene encode for fumarate

hydratase; a Krebs cycle enzyme which causes type II hereditary

leiomyomatosis papillary renal-cell cancer syndrome (37, 38). Sporadic

Papillary RCC involves trisomies of chromosomes 7 and 17 and loss of Y

chromosome (33, 35).

Chromophobe renal cell carcinoma is characterised by large polygonal

cells with distinct cell border and reticulated cytoplasm and known to

originate from intercalated cells of collecting duct in the kidney. These

tumours have slow growth and infrequent metastasis with better prognosis

(33, 35, 37). 30% of chromophobe RCC reported to have mutations in the

p53 gene and also the loss of one allele in chromosome 17p was detected

in 78% of tumours (39). Hereditary chromophobe RCC characterised with

mutations in gene BHD encodes for protein folliculin, a tumour repressor

gene. These mutations lead to a rare autosomal dominant disorder called

Birt-Hogg-Dubé syndrome characterised by Hair-follicle hamartomas of

face and neck (37, 40, 41).

Treatment for RCC includes surgery (Radical and partial nephrectomy),

anti-angiogenic treatment to target VEGF and mTOR pathways with drugs

(sunitinib, sorafenib and bevacizumab), chemotherapy and

immunomodulatory therapies with interferon-α and Interleukin-2 (33, 38,

42, 43).

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2. Wnt signalling Pathway:

Wnt signalling pathway is evolutionarily highly conserved developmental

signalling network, which operates an array of cellular processes during

development and homeostasis in every tissue of an organism. These

cellular processes involve cell proliferation, cell migration, cell polarising

intracellular signalling, cell fate specification and stem cell renewal (44,

45). Genomic sequencing of ancient metazoans have given insights about

the specificity of Wnt genes, and they are confined only to metazoans. In

unicellular eukaryotes and fungi, the existence of Wnt genes was not

reported. This shows evidence that organisms which are multicellular and

with patterned body axis exhibit complete Wnt pathway but not unicellular

organisms (46-49).

Roel Nusse and Harold E Varmus first identified Mouse Wnt1 gene in

1982 while working on Mouse Mammary tumour virus (MMTV) induced

mammary hyperplasia. Wnt1 originally called as Int-1. This gene has a

proviral integration site for MMTV and is also essential for embryonic

development in mice (50). In Drosophila, a homologue of Wnt1 known as

wingless (wg) which controls the segment polarity during larval

development (51). Thus, the genes with similar function in embryogenesis

wg and Int1 became Wnt, means Wingless- related integration site.

Wnt signalling is a short-range extracellular communication system. Cells

respond functionally to Wnt signals upon Wnt ligand stimuli. Cell surface

receptors (LRP5/6 and Frizzled) receive these ligand signals and transmit

through downstream components for cellular functional responses (52).

Wnt signalling is broadly classified into two pathways. Canonical Wnt/β-

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catenin pathway and Non-canonical pathway. The non-canonical pathway

involves two signalling cascades Wnt/PCP and Wnt/Ca2+, which are β-

catenin independent (53).

Genome sequencing revealed 19 Wnt genes categorised into 12 conserved

subfamilies in mammals, 7 in Drosophila and 5 in C.elegans (54). Wnt

proteins are characteristic proteins with conserved cysteine-rich residues.

These proteins have an N-terminal signal peptide and are relatively

insoluble due to post-translational modification by an enzyme called

palmitoyltransferase Porcupine. This enzyme covalently attaches

palmitoleic acid to cysteine residues on Wnt ligands, which brings

hydrophobic nature to the ligands. Palmitoylation is essential for Wnt

ligand signalling and secretion (45, 55, 56). Further, transmembrane

proteins Evi/Wls transport these lipid-modified Wnt ligands to the plasma

membrane, making them available for release to perform diverse roles

(57). From earlier studies, it is reported that canonical Wnts are mainly

Wnt1, Wnt3a and Wnt8 which activates Wnt/β-catenin pathway and non-

canonical Wnts are Wnt5a and Wnt11 which transduces β-catenin

independent pathway (58)

Wnt ligands bind to the cell surface receptors Frizzled (FZD) and LRP5/6

(Low-density lipoprotein receptor-related protein) family proteins. FZD

proteins contain 120 amino acid and are seven-pass transmembrane

proteins with extracellular N-terminal cysteine-rich domain (CRD). There

are ten different FZD genes in humans; FZD 1-10 (59). FZD receptors are

involved in β-catenin dependent and independent signalling pathways

(58). However, LRP5 and LRP6 are involved in canonical Wnt pathway,

which acts as co-receptors along with FZD for signal transduction. The

11

two closely related proteins LRP5 and LRP6 belong to LDLR family. In

Drosophila, Arrow is homologous to LRP5 and LRP6 with 45%

similarities (60). These are single-pass transmembrane proteins with a long

extracellular domain containing four YWTD type β-propeller domains and

three LDLR type-A repeats. Intracellular domain harbours five PPPSPxS

motifs in both LRP5 and LRP6, which are phosphorylation sites. These

motifs phosphorylate upon Wnt stimulation for recruitment and activation

of downstream components (59).

2.1. Canonical Wnt/β-catenin Pathway

A hallmark of canonical Wnt signalling is stabilisation and nuclear

accumulation of transcriptional co-activator β-catenin. In the absence of

Wnt ligands, a destruction complex forms which comprises scaffolding

protein AXIN and adenomatous polyposis coli (APC) along with Ser/Thr

kinases Casein kinase 1α (CK1α) and glycogen synthase kinase-3β

(GSK3β). AXIN phosphorylation by GSK3β is an essential event in the

assembly of destruction complex (61). This destruction complex recruits

β-catenin and promotes phosphorylation of β-catenin by CK1α at Ser 45

residue and GSK3β phosphorylates at Thr 41, Ser 37 and 33. These

phosphorylation events create a binding site on β-catenin for E3 ubiquitin

ligase β-Trcp. β-catenin gets ubiquitinated by β-Trcp and directs it towards

degradation by 26S proteasome in the cytoplasm. Absence of nuclear β-

catenin recruits histone deacetylases (HDAC) to promoter regions of target

genes mediated by TCF/LEF and Groucho transcription factors leading to

the transcriptional repression (62, 63) (Fig 3).

12

The canonical pathway is activated when Wnt ligand binds to FZD and co-

receptors LRP5/6. This event recruits Dishevelled (DVL) to the FZD

intracellular domain in the cytoplasm. Binding of Wnt ligand to the

receptors induces phosphorylation of LRP5/6 cytoplasmic domains by

GSK3β and CK1α at PPSPxS motifs, creating a docking site for AXIN.

Further AXIN translocates to the binding sites on LRP5/6 and complexes

with DVL-FZD-LRP5/6 (59). Binding of AXIN to phospho-LRP5/6

promotes AXIN dephosphorylation that brings conformational changes in

AXIN structure, inhibiting it from interacting with destruction complex

(61). The complex consisting of LRP5/6-FZD-AXIN-GSK3β-CK1α-DVL

undergoes polymerisation through DVL-DIX domain, which results in the

formation of membrane-associated “Signalosomes” (64). The

signalosomes formation inactivates the destruction complex and leads to

β-catenin stabilisation and translocation into the nucleus (65). Inside the

nucleus stabilised β-catenin associates with TCF/LEF transcription factors

along with co-activators cAMP response element-binding protein

(CREB)-binding protein (CBP), B-cell lymphoma 9 (Bcl-9), and pygopus

(66). This transcriptional activator complex carries transcription of

downstream target genes such as c-MYC, VEGF, MMP7, Cyclin D, c-Jun,

Fibronectin and also genes of the Wnt pathway itself. Most of the target

genes are involved in the developmental signalling and adult homeostasis

(45, 67)

Canonical Wnt signalling is regulated at multiple levels by various

secreted antagonists and agonists. At ligand level, signalling is negatively

regulated by proteins Notum, sFRPs (secreted Frizzled-related proteins)

and WIF1 (Wnt-interacting factor). Notum inhibits signalling by removing

13

the acyl group attached to Wnt ligands and hinder ligand interaction to

FZD-LRP5/6 receptors (68). sFRPs and WIF1 proteins which exert its

inhibitory effect on Wnt signalling because of their homology with FZD

receptors compete with FZD-LRP5/6 receptors and binds to Wnt ligand

proteins to prevent signalling (69, 70). At the receptor level, Dickkopf

Fig 3: Wnt signalling pathway. Representative image showing the canonical pathway in the absence of active Wnt ligand and the presence of active Wnt ligand. Adapted from Zhan et al. 2017 (71).

(DKK), a secreted antagonist plays an essential role in negatively

regulating Wnt signalling by interacting with the LRP6 receptor and

regulates signalling (72). LGR5 (Leucine-rich repeat-containing G-protein

14

coupled receptor 5) is a seven-transmembrane receptor that acts as a

modulator of Wnt signalling. LGR5 is an interstitial stem cell marker.

LGR5 binds to ligand R-spondin and exerts its negative and positive

effects on Wnt signalling (73). In the absence of R-spondins, two

transmembrane E3 ubiquitin ligases ZNFR3/RNF43 (zinc and ring finger

3/ ring finger 43) targets FZD receptors by ubiquitination and degradation

(74) leads to inhibition of Wnt signalling. Binding of R-spondins to LGR5

receptor inhibits the activity of ZNFR3/RNF43, which leads to the

accumulation of FZD receptors on the cell surface, thereby causing

amplification of the Wnt signalling (71, 75). Norrin, an agonist of Wnt

signalling, enhances signalling by binding with FZD4 receptor and LRP-

dependent activation of the pathway (76, 77).

2.2. Non-canonical Wnt pathways

Non-canonical Wnt signalling is characterised by β-catenin independent

signal transduction mechanism. This pathway is mainly classified into

Wnt/PCP (planar cell polarity) pathway and the Wnt/Ca2+ pathway (Fig 4).

The Wnt/PCP pathway is activated upon binding of Wnt ligands to FZD

and the tyrosine kinase co-receptors ROR2 and RYK. This binding

activates and recruits DVL to the complex at the cell membrane where

activation occurs through DAAM1 (Dishevelled associated activator of

morphogenesis 1). These events lead to downstream signalling by small

GTPases RHOA (Ras-homology gene family member A) and RAC1 (Ras-

Related C3 Botulinum Toxin Substrate 1). These GTPases further trigger

activation of ROCK (Rho-associated, coiled-coil containing protein

kinase) and JNK (c-JUN-N terminal kinase) (78, 79). These signalling

15

events triggers activation of transcription factor ATF2 for driving the

transcription of target genes responsible for several developmental

processes like the neuronal arrangement, embryonic heart development

and anterior-posterior patterning along with the significant role in actin

polymerisation, cytoskeleton scaffolding and coordinated spatial

polarisation of cells (78, 80, 81).

Fig 4: Non-canonical Wnt signalling pathway. Representative image showing Planar cell polarity pathway and Wnt /calcium pathway. Interaction of Wnt ligand to ROR2/RYK and FZD receptors activate the pathway. Reprinted from the publisher (82) which permits unrestricted non-commercial use. Pez, Floriane, et al. Journal of hepatology, 59.5 (2013): 1107-1117.

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Wnt/Ca2+ pathway mainly regulates the release of calcium from

endoplasmic reticulum and maintenance of intracellular calcium levels.

Wnt/Ca2+ pathway promotes ventral cell fate, regulates tissue movements

during gastrulation and regulates dorsal axis formation during

embryogenesis (78, 80). Upon Wnt ligand binding to FZD receptor

triggers activation of phospholipase C, which in turn activates signalling

molecules, inositol 1,4,5-triphosphate (IP3) and 1,2 diacylglycerol (DAG).

IP3 and DAG stimulate the release of calcium which leads to an increase

in calcium levels in the cell. Further, amplified calcium levels trigger the

activation of CAMKII (Ca2+/calmodulin-dependent kinase II),

calcineurin and PKC (protein kinase C) (83). NFAT (nuclear factor of

activated T-cells) a transcription factor is activated by calcineurin and

boost the expression of genes responsible for polarisation of cells during

gastrulation and pro-inflammatory genes (84).

2.3. Wnt signalling in development

Wnt signalling is widely studied for its roles in several developmental

events across the animal kingdom. In most of the bilaterians (animals with

bilateral symmetry as an embryo), Wnt genes are expressed in the posterior

poles, while in pre-bilaterians the expression is polarised along the primary

axis. Wnt signalling is mainly responsible for developing posterior axis

features, and inhibition of Wnt genes in the anterior axis plays a vital role

in the body plan development in most vertebrates such as mouse, Xenopus

and zebrafish and cephalochordates (44, 85, 86).

In Xenopus embryos during early blastulae, after cortical rotation

accumulation of nuclear β-catenin in dorsal side cells occurs. This

17

phenomenon is essential for the transcriptional programme required for the

formation of Spearman organiser (85, 87). β-catenin is involved in the

Anterior-posterior (AP) axis formation. In mouse embryos, the first β-

catenin activity observed in extraembryonic visceral endoderm. During the

initiation of gastrulation in the epiblast β-catenin is distributed in the cells

adjacent to embryonic-extra embryonic junctions, and further β-catenin

signalling was detected in primitive streak (88). In Xenopus and zebrafish,

deletion of β-catenin or overexpression of DKK1 results in anteriorization

of AP axis with loss of posterior body and overexpression of Wnt8 causes

posteriorization of AP axis (89, 90). Wnt pathway synergistically cross-

talk with other signalling pathways such as the BMP pathway for the

ventral-posterior region establishment and FGF pathway for signalling

events in dorsal and posterior axis (91, 92).

Wnt signalling is responsible for the integration of components (lens and

retina) during the formation of an eye from neural crest cells in chick

embryos. TGF-β and canonical Wnt pathways transcriptionally repress

PAX6 activity for the formation of a functional eye (93). Wnt plays a vital

role in the formation of Cochlea (94), and both canonical and non-

canonical pathways are involved in muscle development (95). In mouse

embryos, Wnt1 and Wnt10b are responsible for the formation of the

midbrain and anterior hindbrain (96). In mammals and zebrafish Wnt

signalling play a crucial role in the early development of lungs and swim

bladder, respectively (97). Also, Wnt signalling is responsible for the

development of tail in zebrafish along with BMP and nodal signalling

pathways (98).

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2.4. Wnt signalling in adult tissue homeostasis

Wnt signalling is widely studied for its role in the maintenance and

activation of stem cell reservoirs. Stem cells are a niche of cells that have

a high potential to maintain tissue homeostasis in adults by renewing

themselves by cell division. Lineage tracing experiments provided

evidence that Wnt signalling is crucial for stem cell niches in several

tissues such as intestine, stomach, bone, liver, skin and hair follicles (99).

In the intestinal epithelium, Wnt co-receptor and target gene LGR5

identified as a novel stem cell marker. In intestinal crypt base columnar

cells, LGR5 is exclusively expressed (100). These basal crypt stem cells

are highly proliferative and regenerate throughout adult life. Wnt and R-

spondin promote the self-renewal of the basal crypt LGR5+ stem cells

which migrate to intestinal villi and further differentiate into intestinal cell

types for maintaining intestinal homeostasis (101, 102).

Bone remodelling is a crucial process that occurs throughout adult life for

maintaining calcium homeostasis and meeting the functional demands due

to the mechanical stress of bone. Bone resorption by osteoclasts and new

bone formation and maintenance by osteoblasts and osteocytes occurs in

the process of remodelling. Wnt signalling increases the bone mass by its

activity through Wnt co-receptor LRP5 on mesenchymal progenitor cells.

SOST (sclerostin), a Wnt repressor secreted by osteoclasts reduces

osteoblast formation by inhibiting LRP5 activity, thus maintaining balance

in the process of bone remodelling (103-105).

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2.5. Wnt signalling in cancer

Wnt signalling is associated with cancer, and its aberrant activation plays

a crucial role in multiple pathogeneses along with cancer. Targeting Wnt

signalling have a great significance in the treatment of cancer. Dysfunction

of components in Wnt canonical signalling was known to develop several

types of cancers such as colorectal, ovarian, hepatocellular carcinoma

(HCC), breast, myelomas, respiratory and prostate. Loss or gain of

function mutations in Wnt signalling components have a high chance of

cancer-promoting or cancer-driving capabilities. Majority of tumour

causing mutations of Wnt components occurs in APC, AXIN1-2 and β-

catenin, which leads to the inappropriate stabilisation of β-catenin to

transactivate the downstream target genes (c-Myc, CCND1, etc.)

persistently (45, 54, 63).

Different mutations in APC and β-catenin are found to be the leading cause

of colorectal carcinogenesis (45, 106). In multiple myelomas, epigenetic

alterations affect the Wnt signalling such as promoter methylation of wnt

antagonists DKK1-2 and sFRP1,2&4 and also overexpression of

transcriptional factor BCL9 and Wnt co-receptor LGR4 (107). 17%

inactivating mutations in AXIN1-2 and 15-33% of activating mutations in

β-catenin were reported in sporadic HCC (108, 109). 16-54% of Ovarian

endometrioid adenocarcinomas (OEA) characterised by common

mutations in β-catenin resulted in a two-fold increase of β-catenin/TCF

target genes such as BMP4, CCND1, MSX2, MMP7, FGF9, CD44 and

EPHB3 (110). In prostate cancer, 18% of alterations in the Wnt signalling

pathway have been identified (111). 5% of tumours harbours mutations in

the β-catenin gene and recurrent mutations have been identified in APC

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and βTRC genes. Mutations in E3 ligases ZNRF3 and RNF43 were

identified and were exclusive with APC mutations (111, 112). A fusion of

genes GRHL2- RSPO2 and RSPO2 overexpression due to gene

rearrangement, increases R-spondin levels, an agonist of Wnt signalling.

These fusion events discovered in metastatic castration-resistant prostate

cancer (mCRPC) (111, 113). Frequent hypermethylation of the APC gene

was identified during prostate cancer progression (114, 115). Canonical

Wnt pathway is known to regulate cellular metabolism in OEA and breast

cancer by its effects on downstream target genes, which control oxidative

phosphorylation, glutamine and fatty acid metabolism (110, 116, 117). C-

Myc an important Wnt target gene known to control mitochondrial

bioenergetics and lipid synthesis and also synergistic cross-talk between

Canonical Wnt pathway and c-Myc in cancer cells regulate tumour cell

metabolism and promotes aerobic glycolysis in cancer cells (117, 118).

These examples represent the importance of Wnt signalling in the

regulation of cancer development and progression.

21

3. TGF-β signalling pathway:

TGF-β (Transforming growth factor-β) is an intriguing cytokine, belongs

to a large family of secreted proteins, known as TGF-β superfamily that

have a wide range of properties including cell proliferation, migration and

apoptosis during embryonic development, tissue homeostasis and immune

system modulation. TGF-β is also known to drive cancer progression by

its effects through immunosuppression, proangiogenesis and inducing

epithelial plasticity leading to Epithelial-Mesenchymal transition (EMT).

Also, TGF-β can induce antiproliferative effects by regulating the genes

responsible for cell cycle progressions such as cyclin-dependent kinases

(Cdks) and downregulation of c-Myc. Thus TGF-β showing its properties

of a potent growth inhibitor in normal epithelial tissues and display both

tumour suppressor and promoter properties (119-121).

TGF-β was discovered in 1978 while working on murine sarcoma virus-

transformed cells, initially known as Sarcoma growth factor (SGF) (122).

In humans, the TGF-β superfamily consists of nearly 33 members which

can be further subdivided into two subfamilies depending on the type of

signalling they activate; TGF-β/nodal/activin subfamily and BMP (bone

morphogenetic protein) subfamily include BMPs and GDFs (growth and

differentiation factors) (119, 120, 123).

TGF-β signalling is activated through its TGF-β ligands which exist in

three isoforms TGF-β1, TGF-β2 and TGF-β3; these are secreted into the

extracellular matrix in its latent form with its propeptide and LTBP (latent

TGF-β binding protein) (124). Propeptide and LTBP further cleaved by

proteases from TGF-β ligand for final activation (125, 126). Other ligands

22

involved in the TGF-β superfamily signalling are BMP-2/4/6, GDF-5,

activin, AMH (Anti-Mullerian Hormone) and OP-1 (123). TGF-β ligand

signals through two pairs of heterodimeric serine/threonine receptor

complexes type I (ALK 1-7) and type II (TβRII, ActR-IIA, ActR-IIB,

BMPRII and AMHRII).

Mature TGF-β ligand binds to the constitutive kinase active TβRII, which

recruits and phosphorylates active kinase domain of TβRI (ALK5) and

results in the formation of receptor hetero-tetrameric complex with two

TβRII and two TβRI receptors. This complex further activates receptor

(R)-Smads (Smad signalling) and also several pathways which do not

involve conventional Smads (Non-Smad signalling) (120, 127, 128)

3.1. Smad signalling (Canonical):

The signals from the activated receptor-ligand complex are transmitted

into the cytoplasm through R-Smads. Further, R-Smads form complexes

with Common-mediator Smads (Co-Smad), which enters the nucleus for

driving the expression target genes. Smads are vertebrate ortholog to

MAD protein in Drosophila. There are three different kinds of Smads

based on their functionality; R-Smads (Smad-1/2/3/5/8), Co-Smad (Smad-

4) and inhibitory (I)-Smads (Smad-6/7). R-Smads 2/3 involved in TGF-

β/Activin signalling, and R-Smads 1/5/8 transduces BMP signalling. I-

Smads inhibit TGF-β signalling by binding to R-Smads or receptors (129,

130)

R-Smad and Co-Smads have conserved structural domains N-terminal

MH1 and C-terminal MH2 connected through a proline-rich linker region.

The linker domain contains phosphorylation sites for kinases like MAPKs

23

and CDKs, and R-Smads have a PY motif an interaction site for E3

ubiquitin ligase Smurf1/2. These phosphorylation and ubiquitination

events inhibit Smads from nuclear transport and result in proteasomal

degradation, along regulating the TGF-β signalling. I-Smads lack MH1

domain. MH1 domain interacts with Smad binding elements (SBE) on the

promoter sites of target genes. MH1 domain is also responsible for nuclear

localisation signal for both R-Smad and Co-Smads. MH2 domain is highly

conserved in all the Smads, which is responsible for receptor interaction.

R-Smads-MH2 domain contains specific motifs SSXS, which are

phosphorylated by TβRI receptors. These phosphorylation motifs are

absent in Co-Smads and I-Smads (131).

When the TGF-β ligand interaction activates the TβRI and TβRII, this

allows binding of MH1 domain of R-Smads to the c-terminal intracellular

domain of TβRI. This results in the phosphorylation of Smad-2 and Smad-

3 by activated TβRI at SSXS motifs. Phosphorylation of R-Smads recruits

Smad-4, which they form a heteromeric complex and further dissociates

from the receptor complex (Fig 5). These complexes translocate into the

nucleus and carry a wide range of target gene transcriptional regulation

(120, 123, 129, 130). R-Smad and Smad4 complexes bind to the SBE

elements on the promoter sites of several target genes like p15INK4B,

p21CIP1, c-Myc, PAI-1, ZEB1/2, SNAI1, Twist1, Smad7 and many more

that are involved in apoptosis, immune responses, embryonic

development, cell differentiation and migration (132-135). Smad

complexes bind to several transcription factors (Runx, bHLH, NFκB and

forkhead, etc.) and co-activators (CBP/p300, TGIF, c-Ski) to mediate cell-

24

specific or pathway-specific responses to TGF-β signalling (123, 127, 136,

137).

3.2. Non-Smad signalling (Non-Canonical):

Apart from conventional Smad activation, TGF-β signals also transduce

several non-canonical activations through proteins Ras-ERKs

(extracellular signal-regulated kinases)-MAPK (mitogen-activated protein

kinase), Par6-Rho GTPases, PI3K (phosphatidylinositol-3´kinase)-AKT-

mTOR (mammalian target of rapamycin), and TAK1 (TGF-β activated

kinase 1)/TRAF6- p38/JNK (c-Jun N-terminal kinases) (128) (Fig 5).

Fig 5: TGF-β signalling pathway: Canonical pathway transmits its signals through Smad 2/3 and 4 while in the non-canonical pathway several factors are involved in signal transmissions such as PI3K, TRAF6, MAPKinases, Rho GTPases and ERK proteins. Reprinted with permission from the publisher (128)

25

In epithelial cells and fibroblasts, phosphorylated TβRI can activate the

MAPK pathway to activate ERK 1/2 via Ras to revive stress signals (138).

TβRII phosphorylates Par6, which inhibits small GTPase Rho, through

recruitment of Smurf1 to regulate actin reorganisation and cell polarization

(139, 140). TGF-β signals activate the PI3K pathway through subunit p85

and induce rapid phosphorylation of AKT and activate mTOR. These

signals are essential during protein synthesis and cell cycle progression.

Besides, the PI3K/AKT pathway through TGF-β signalling involved in

EMT, invasion and migration in cancer progression by its effects through

SNAI1 (141-143).

TRAF6, an E3 ubiquitin ligase interacts with TβRI at a consensus motif.

This TRAF6-TβRI complex undergoes oligomerisation when the TGF-β

ligand binds to the TβRII-TβRI complex (144). The oligomerisation of the

TRAF6-TβRI leads to autoubiquitination and activation of TRAF6 and

recruitment of TAK1. TAK1 gets activated by Lys-63 ubiquitination

causing activation of TAK1-p38/JNK pathway, which promotes apoptosis

and EMT (144-146). TGF-β mediated TRAF6 ubiquitination of TACE

mediates cleavage of TβRI at the ectodomain region. Also, TRAF6

activates and recruit PS1 (Presenilin 1), a component of the γ-secretase

complex through TGF-β mediated Lys-63 polyubiquitination to cleave

TβRI at the transmembrane region generating an ICD (Intracellular

domain). This TβRI-ICD translocates into the nucleus and drives the

expression of SNAI1, Jagged1, MMP2 by interacting with NICD (Notch-

ICD) and transcription co-activator p300 (147, 148).

26

3.3. TGF-β signalling in cancer:

TGF-β is known to be both tumour-suppressor and tumour promoter with

its effects through growth regulation and cancer progression. It is an

essential mediator of cell proliferation, migration and apoptosis, in

contrast, it also mediates cancer cell invasion, metastasis and angiogenesis

(121, 123). Understanding the context-based regulation through TGF-β

provides crucial insights for the therapeutic strategies.

In epithelial cells and during the tumour initiation, TGF-β is mainly

responsible for growth inhibition via Smad dependent signalling. TGF-β

exerts its pro-apoptotic effects through both Smad-dependent and

independent pathways by induction of pro-apoptotic proteins like Death-

associated protein kinase (DAPK), BIM and caspase 9 (149). Mutations in

Endoglins, an agonist of TGF-β signalling, is reported to cause

hemorrhagic telangiectasia syndrome, mirroring TGF-β tumour

suppressor properties (150). Many reports with TβRI and TβRII mutations

were known to cause various cancer such as prostate, breast, colon,

ovarian, lung and pancreas, and these mutations are common in tumours

with microsatellite instabilities (151-153). hTERT (human telomerase

reverse transcriptase), a protein which is known to stabilise and extend

chromosomal telomers is inhibited in normal cells by TGF-β via

interaction of Smad3 to hTERT promoter and direct cells for apoptosis,

reflecting its role for inhibiting cell immortality. In most of the cancers,

the inhibitory effect of TGF-β on hTERT is lost, making them immortal

and with dysregulated growth (154-156). Further, the antiproliferative

roles of TGF-β are explained by transcriptional downregulation of c-Myc.

TGF-β induced downregulation of c-Myc results in the induction of CDK

27

inhibitors p15 and p21 for cell cycle arrest. Induction of p15 and p21 can

also happen independently of c-Myc but through TGF-β direct induction

(120, 157, 158).

Tumour promoting roles of TGF-β is widely studied. TGF-β can switch

from tumour suppressor to tumour promoter role in the presence of

oncogenic and epigenetic events. In the late stages of cancer progression

due to the mutational burden in its components, TGF-β drive cancer

towards aggressive mode accompanied by EMT, angiogenesis, invasion

and metastasis. TGF-β promotes tumorogenesis by modulating tumour

stroma. Several clinical investigations demonstrated that TGF-β levels are

elevated in tumour stroma and linked to poor prognosis (159, 160). TGF-

β promotes transcription of SNAI1, which results in loss of E-Cadherin

and epithelial cells acquires EMT phenotype to cancer cells (161). Cancer-

associated fibroblasts (CAFs) in tumour stroma mature from resident

fibroblast by TGF-β stimulation and inturn these CAFs promotes

metastasis through secretion of IL-6/11, which promote of JAK-STAT

oncogenic signalling (162). TGF-β promotes cancer progression by

upregulating several target genes which promote angiogenesis such as

FGF-2 (fibroblast growth factor-2), Angptl4 (Angiopoietin-like 4) and

TGF-β mediated stromal-derived angiogenic factors like VEGF (Vascular

endothelial growth factor), CTGF (connective tissue growth factor) and

PDGF (Platelet-derived growth factor) (163-166).

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4. HIF (Hypoxia-inducible factor) system

Oxygen is an essential component of metabolism in aerobic organisms. It

is required for survival, and it is essential in generating the cellular

adenosine triphosphate (ATP) during oxidative phosphorylation in

mitochondria, the energy resource of the cell. Oxygen (O2) is transported

by red blood cells throughout the body to enable normal cellular functions

(167). Normoxia, a normal physiological O2 level, is different between

different tissues. In adult human tissues, the physiological oxygen levels

vary between 1%-14%, which provides a microenvironment for cells. Low

oxygen tension is found in venous blood, retina, bone marrow and brain.

Arterial blood, lung tissue, heart, liver and kidney shows high oxygen

tension (168, 169). When the O2 levels fall below the physiological level,

the condition is termed as hypoxia. Localised transient O2 deficiency leads

to a metabolic crisis and can produce irreversible cellular damage.

Hypoxia is a driver of several cellular functions and occurs when the

demand for O2 exceeds supply. Hypoxia can contribute to a wide range of

pathophysiological conditions and also normal physiological processes

such as embryonic development. Various pathological conditions involve

chronic lung disease, stroke, obstructive sleep apnea and cancer (170-172).

Normal mammalian embryonic development occurs in a hypoxic

environment where tissue growth is higher than available oxygen, and here

HIFs (Hypoxia-inducible factors) play a vital role in embryos to enable

survival in low oxygen conditions (173). Other important physiological

processes activated by HIFs during hypoxia are hyperventilation,

erythropoiesis, activation of glucose metabolism and angiogenesis (174).

Angiogenesis is a process of formation of new blood vessels and involves

29

activation and migration of endothelial cells, which branch from existing

blood vessels to form vascular branches. VEGF, a significant contributor

to angiogenesis, is a direct target of HIFs and is activated upon hypoxia.

TGF-β, FGFs, MMPs and Angiopoietin-1&2 also contribute to

angiogenesis (175, 176). Angiogenesis is essential in embryogenesis and

also in cancer progression. Hypoxia, along with inappropriate activation

of HIFs cause tumorigenesis (177).

4.1. Components of the HIF pathway

The HIF family of transcription factors are responsible for the maintenance

of oxygen homeostasis in living cells. HIFs are composed of oxygen labile

α and stable β heterodimer subunits. There are three different α subunits

HIF-1α, HIF-2α and HIF-3α; and one β subunit HIF- β.

HIF proteins:

HIF-α proteins are oxygen labile transcription factors, and in low oxygen

tension conditions, HIF-α protein levels increase rapidly. Conversely, the

HIF-β subunit is constitutively expressed independent of oxygen

concentration. Three subunits HIF-1α, HIF-2α and HIF-3α, are encoded

by genes HIF1A, EPAS1 and HIF3A. HIF-β is also known as aryl

hydrocarbon receptor nuclear translocator (ARNT) encoded by gene

ARNT (178). HIF-α proteins binds to HIF- β and translocate to the nucleus

where they bind to hypoxia-responsive elements (HREs) of the target

genes (175). HIF-1α, HIF-2α and HIF-β have been characterised

thoroughly. They possess N-terminal basic helix-loop-helix domain

(bHLH) responsible for binding to DNA, followed by PAS (Per-Arnt-

30

Sim)-A, PAS-B, PAC (PAS-associated COOH) domain which is

responsible for heterodimerisation of HIF-α and β subunit. HIF-1α and

HIF-2α possess oxygen-dependent degradation domain (ODD) followed

by two transactivation domains NTAD and CTAD at the C-terminal, while

HIF-β has only CTAD but lacks ODD and NTAD (178, 179) (Fig 6). HIF-

1α is ubiquitously expressed, while HIF-2α is detected only in specific

tissues, including endothelium, lung, kidney, brain and heart (180).

Fig 6: Diagrammatic overview of the domain structures of HIF-α and HIF-β isoforms. Adapted from Yang SL et al. 2015 (181).

The HIF-3α subunits are the most recently identified members of the HIF

family and are a relatively weak transcriptional activator of hypoxia-

responsive genes. Human HIF-3α has ten splice variants, namely, HIF3α1-

10. Several splice variants of HIF-3α are due to unique transcription

initiation sites in three different exons, namely exon 1a, 1b and 1c of

HIF3A gene (182). Structurally, HIF-3α1, 2 and 4 variants posses bHLH,

31

PAS domains and HIF-3α1-3 have an oxygen-sensing ODD domain but

HIF-3α4-6 lack ODD domain. The CTAD domain is absent in HIF-3α, but

HIF-3α1 has a unique DNA and protein binding domain LZIP. HIF-3α4 is

similar to mouse inhibitory PAS (IPAS), which lacks the ODD and LZIP

domain (Fig 6). HIF-3α4 dimerise with HIF-β and also with HIF-1α, which

results in a transcriptionally inactive dimer, thus acting as a dominant-

negative regulator of HIF-1α and HIF-2α (182-185).

VHL:

VHL is known to be a tumour suppressor gene, and germline mutations in

this gene cause a hereditary cancer syndrome called von Hippel-Lindau

disease, which leads to the development of clear cell renal carcinoma and

hemangioblastomas of the central nervous system and retina (186, 187).

VHL inactivation in both the alleles is frequent in sporadic ccRCC (32).

VHL (von Hippel-Lindau) protein is an E3 RING-type ubiquitin ligase

know to target several proteins for proteasomal degradation. The important

proteins which were polyubiquitinated by VHL are HIF-1α, HIF-2α and

HIF-3α during normoxia, which makes VHL an important negative

regulator of HIF pathway (170, 179, 188). The VHL protein is ubiquitously

expressed and evolutionarily conserved across species. The human VHL

gene is located in chromosome 3p25 contains 4.5 kb mRNA with three

exons (187). There are two isoforms of VHL, VHL30 and VHL19. VHL30 is

a larger protein with 30kD with 213 amino acids and other VHL19 derived

from a 54th methionine codon residue of the coding exon 1 with 160 amino

acids (189). VHL30 is mainly localised to the cytoplasm, endoplasmic

reticulum (ER) and minimal concentrations in the nucleus and VHL19 is

32

localised to both cytosol and nucleus, but not in ER (190). VHL protein

has two functional domains α subunit and β subunit. VHL α subunit

associates with elongin C, elongin B, Cul2, and Rbx1 for its ubiquitin

ligase activity with which it targets proteins. β subunit is responsible for

binding to prolyl hydroxylated HIFα (187).

4.2. HIF signalling:

Protein stability of HIF-α is dependent on oxygen tension in the tissues,

while HIF-β is insensitive to oxygen tension and stable. During normoxia,

when oxygen is available, a family of dioxygenase enzymes called PHDs

(prolyl-hydroxylases) hydroxylate two prolyl residues in the amino- and

the carboxy-terminal oxygen-dependent degradation (ODD) domains in

HIF-α (191). The hydroxylated prolines create a recognition site for VHL

protein, an E3 ubiquitin ligase, which binds to and polyubiquitinates HIF-

α by K-48 linked ubiquitins and directs it for proteasomal degradation (Fig

7) (192, 193).

In hypoxia, when oxygen concentration drops, PHDs become inactive,

resulting in HIF-α accumulation and HIF-α dimerises with HIF-β and

translocates into the nucleus and bind to the consensus motif HREs

(Hypoxia responsive elements) on the promoter regions of HIF-target

genes to activate the transcription of genes in order to adapt to the hypoxic

stress (170, 179, 188) (Fig 7). In the nucleus, HIF-α and β complex bind

to coactivators like CBP/p300 to form an active transcriptional complex

for driving the target genes (194).

33

Fig 7: Hypoxia-inducible factors (HIFs) activity under normoxia and hypoxic conditions. Adapted from Simon et al. 2016 (195).

HIF activates a large number of genes in response to hypoxia, which is

required for many cellular processes including energy metabolism,

apoptosis, angiogenesis, cytoskeleton formation, autophagy, cell

differentiation and maintaining oxygen homeostasis (196). HIF factors

contribute to epithelial to mesenchymal transition through its effects on

VEGF, which enhances angiogenesis in cancer (175, 176). Hypoxia also

activates important transcription factors, such as p53, NF-κB, AP-1 and

c-Myc (197, 198).

34

5. Epithelial to mesenchymal transition (EMT)

The epithelial to mesenchymal transition (EMT) is characterised by loss

of cell-cell adhesion, increased motility and changes in polarization of the

cells. EMT is an important phenomenon that occurs during embryogenesis

and cancer progression (199, 200). Epithelial cells acquire mesenchymal

phenotype, leading to enhanced motility and invasion. During EMT,

epithelial cells lose E-cadherin expression, which holds the cell-cell

junctions and attains mesenchymal markers, such as vimentin and

fibronectin. EMT is a crucial process before metastasis can occur (121,

201).

Epithelial cells are closely connected by tight junctions, with an

apicobasal polarity, actin cytoskeleton polarisation and are bound by

a basal lamina at their basal surface. Mesenchymal cells lack polarisation

and are characterised by spindle-shaped morphology (202). Cancer cell

dissemination is vital in cancer progression leading to invasion and

metastasis. Dissemination of cancer cells from the primary tumour is

initiated by loss of cell-cell adhesion, increased motility of cells and

breakthrough of basement membrane causing invasion and spreading into

surrounding tissues or distant organs using bloodstream or lymph (200).

The mesenchymal transition of epithelial cells is characterised by

expression of vimentin, and the increased deposition of extracellular

matrix (ECM) proteins, including collagens and fibronectin and loss of E-

cadherin, promotes high expression of N-Cadherin, which attains high

motility for cells. Loss of polarity to cells allows them to migrate easily.

Increased expression of matrix metalloproteinases (MMP7, MMP9) allow

35

the cells to degrade basement membrane and escape into surrounding

tissue (Fig 8) (121, 203, 204).

Fig 8: Schematic representation of epithelial-mesenchymal transition. EMT characterised by gain of a mesenchymal phenotype, loss of cell-to-cell adhesion, loss of polarity, and acquire migratory and invasive properties.

TGF-β is a potent driver of EMT. Canonical TGF-β signalling regulates

the expression of EMT-inducing transcription factors Snail/Slug, ZEB1/2,

and Twist (121, 204). Non-canonical TGF-β signalling contributes to the

increased motility, invasion, and actin reorganisation (202). Hypoxia also

contributes to EMT (172). Hypoxia upregulates HIF-1α/HIF-2α and in

turn modulates EMT, angiogenesis and metastasis. HIFs under hypoxia

activates several EMT transcriptional regulators such as Twist1, Snail,

Slug, ZEB1/2, and E12/E47 which further bind to the promoter sequences

of EMT marker genes to facilitate EMT (205, 206). Sequestration of β-

catenin in the cytoplasm is an essential function of E-cadherin at the cell

36

junctions. Loss of E-Cadherin from cell junctions due to EMT results in

the release of β-catenin into the cytoplasm and translocate into nucleus to

drive the transcription of mesenchymal markers such as fibroblast-

specific protein 1 (FSP1), Snail, vimentin, slug; which further enhances

migration and invasion (115, 207-209).

6. Ubiquitination:

Ubiquitination is a process of post-translational modification of proteins

through 76 aminoacid polypeptide known as ‘Ubiquitin’. Ubiquitin family

proteins display a remarkable similarity in their structure. Other members

of the family include Nedd8, Sumo1, ThiS and Fat10 etc. (210, 211). These

ubiquitins are small regulatory proteins and are ubiquitously expressed in

most tissues in all eukaryotes. These ubiquitins are covalently attached to

its target protein at the amino group of lysine residue through an isopeptide

bond (212). Substrates marked with ubiquitins are targeted by multisubunit

ATP dependent protease called 26S proteasome and form ubiquitin-

proteosome system (UPS), which regulates the turnover of misfolded and

damaged proteins and also controls the stability of short-lived proteins

such as transcription factors (213). The ubiquitination process also affects

proteins in several ways like controlling protein activity, signal

transduction, cellular localisation of proteins and prevent or promote

protein interactions. Many biological processes are regulated by

ubiquitination such as inflammatory responses, cell cycle regulation, DNA

repair, endocytosis and antigen presentation (214-216).

The human ubiquitin is encoded by four genes UbB, UbC, UBA52 and

RPS27A/UBA80 and they are rapidly processed by DUB

37

(deubiquitinating enzymes) family of enzymes to produce free ubiquitin

proteins in the cytoplasm (217). DUBs are mainly responsible for

reversing the process of ubiquitination. This process of deubiquitination is

equally crucial as ubiquitination for maintaining the cellular homeostasis

and regulation of cellular signals. Ubiquitination involves three essential

steps catalysed by three different enzyme groups namely ubiquitin-

activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and

ubiquitin ligases (E3) which mark activation, conjugation and ligation

respectively (Fig 9). In humans, there are two ubiquitin E1s, ~40 E2s

(UbcH5A, UbcH7/E2-F1, UbcH8, Ubc13, BRUCE, E2-EPF) and ~600

E3s (E3α/Ubr1, Mdm2, TRAF6, IAPs, Smurf1, E6-AP). E3 interacts with

its target protein through one of the two structural domains either HECT

domain or RING domain and are thus categorised into HECT Domain E3s

and RING domain E3s (218-220).

Ubiquitins must be activated to bind to the substrate proteins at the C-

terminus. At first, ubiquitin is adenylated by E1 enzyme using ATP source.

The activated ubiquitin in a complex with E1 called E1-ubiquitin thiol

ester is covalently linked to the large group of E2 conjugating enzymes.

Further, E2 proteins transiently carry the activated complex and catalyse

substrate ubiquitination either alone or in conjunction with a ubiquitin-

protein ligase, E3. E3 ligases, transfer activated ubiquitin from E2 to

substrate lysine residue (monoubiquitination) or ubiquitin lysine residue

(polyubiquitination) (212, 216, 219, 220).

38

Fig 9: Schematic illustration of the ubiquitin-proteasome system. K48- linked polyubiquitination drives proteins for proteasome-mediated degradation. K63- linked polyubiquitination signals for DNA repair, translation, signal transduction and endocytosis. Reprinted with permission from the publisher (213).

Protein modifications can occur either by a single ubiquitin-protein called

monoubiquitination or chain of ubiquitin proteins known as

polyubiquitination. Secondary ubiquitin always links to lysine residue (K)

or the N-terminal methionine of preceding ubiquitin protein. Ubiquitin has

seven lysine residues K6, K11, K27, K29, K33, K48 and K63, all of which

conjugate with other ubiquitin in the process of polyubiquitination (221).

Each lysine binding has a different cellular response. K48-linked

polyubiquitination directs proteins to proteasomal degradation (212, 219)

whereas K63-linked polyubiquitination performs processes like DNA

repair, endocytosis and signal transduction (218).

39

7. TRAF6:

TRAF6 belongs to the family of TRAFs (TNF (Tumor necrosis factor)-

receptor-associated factors) which have essential roles in intracellular

signal transduction of IL-1 (Interleukin-1) receptor/TLR (toll-like

receptor), BCR /TCR (B-cell/T-cell receptors) and TNFR (Tumor necrosis

factor receptor) superfamily (222, 223). TRAFs are a family of adapter

proteins and ubiquitin ligases and are known to associate with cytoplasmic

domains of TNF receptors. TRAFs activate several signalling pathways,

including NF-κB (nuclear factor-Kappa B), MAPK and JNK p38 (224).

TRAFs regulate apoptosis, haematopoiesis, adaptive and innate immune

responses and bone metabolism (222). The TRAF family includes seven

different TRAFs (TRAF1-7) in mammals (224, 225). TRAF2, 3 and 6 are

expressed ubiquitously in most cell and tissue types (226), while TRAF1

and TRAF5 are confined to spleen, lungs and thymus (171, 172) and

TRAF4, is mainly expressed in neuroepithelium during embryogenesis

(227). TRAFs are characterised by an N-terminal RING (Really interesting

new gene) finger domain, one to seven Zinc finger (ZF) domains followed

by conserved and distinct carboxy-terminal containing a coiled-coil (CC)

domain (TRAF-N) and a TRAF-C domain. TRAF1 does not have a RING-

finger domain (223). The Carboxyl domain of TRAF6 is a trimeric

structure. It is mushroom-shaped with the TRAF-C domain as the head and

coiled-coil as the stalk for trimeric structure. The TRAF-C is mainly

responsible for interaction with receptors, other TRAFs and various

adapter proteins (228, 229). The RING domain and adjacent ZFs are

essential for the activation of downstream signalling (230). All TRAFs are

localised to cytoplasm except for TRAF4, which is also found in the

40

nucleus (231). In breast cancer, TRAF6 has also been found in the nucleus

in hypoxic cells (232).

TRAF6 is an adapter protein and E3 ubiquitin ligase known to promote

various inflammatory responses through different signalling pathways

such as NF-κB and Mitogen-activated protein kinases (MAPKs) (233).

TRAF6 was first identified by a yeast two-hybrid screen and found to be a

signal transducer of CD40 and also by EST library screening by using

TRAF2 sequence homology (223, 234). TRAF6 contains a coiled-coil

domain (TRAF-N), four zinc fingers, and a RING domain (Fig 10) (235).

Fig 10: Diagrammatic representation of the structure of TRAF6 with its domains. N-terminal RING domain and Zinc fingers 1-4; C-terminal coiled-coil (CC) and TRAF-C domains. Adapted from Yin et al. (235)

TRAF6 is pleiotropic in participating in various signal transduction

pathways unlike other TRAFs, thus showing its distinct features and

physiological functions. TRAF6 has a unique TRAF-C domain among

TRAFs with least homology and with an overall sequence identity of

~30% when compared to other TRAFs (223). TRAF6 is ubiquitously

expressed (223, 234). TRAF6 interacts with other protein through

consensus motif Pro-X-Glu-X-X-(Ar/Ac), where Ar is an aromatic and

Ac an acidic residue in its TRAF-C domain (228). TRAF1, 2, 3 and 5 do

not share the same consensus binding motif, and they bind instead to

Pro/Ser/Thr/Ala)-X-(Gln/Glu)-Glu motif (236, 237). TRAF6 bind to

41

consensus motifs of CD40, IRAKs (Interleukin-1 receptor-associated

kinase) and RANK (Receptor activator of nuclear factor κ B). The TRAF6

binding region is required for CD40-mediated NF-kB activation, which is

known to play an essential role in adaptive immunity and, maturation and

survival of B-cells and dendritic cells (238, 239). Mutations in the three

residues of the consensus motif in CD40 significantly reduce NF-kB

reporter activity (228, 234). TRAF6 can transduce signals from both IL-

1R/TLR family and TNFR family proteins. In IL-1R/TLR signalling

TRAF6 binds to kinases-IRAK through recruitment of adapter proteins

MyD88, TRIF-related adaptor molecule (TRAM) and toll/interleukin-1

receptor adaptor protein (TIRAP) which elicit NF-kB activation and IL-1-

mediated JNK activation (223, 240). RANK and TRAF6 interaction have

a functional role in bone metabolism, where TRAF6 is a critical adapter in

RANK-mediated differentiation of osteoclasts maintaining the bone

homeostasis (239-242).

TRAF6 N-terminal RING and zinc fingers are rigid and elongated

structures and exist in dimeric structure in both crystal and solution (235)

(Fig 11). The C-terminal CC domain and TRAF-C have a trimeric

mushroom-shaped structure. This trimeric structure is transient and

attained upon signal reception to modulate downstream signalling. The

trimer is the functional form of TRAFs, and trimeric structure of the

TRAF6 C-terminal is an important post-translation event for its role in

interaction with different proteins (243).

In its function as an E3 ubiquitin ligase, the RING domain of TRAF6 is

known to facilitate downstream signalling by K63-linked

polyubiquitination driving the proteins for DNA repair signal transduction

42

and endocytosis. Upon activation with relevant signalling ligand, TRAF6

promotes its activation by K63-linked autoubiquitination (244, 245).

Further, in conjunction with E2 ubiquitin-conjugating enzymes Ubc13-

UeV1A, the RING domain attain E3 ligase activity and catalyses the

formation of K63-linked polyubiquitination of target proteins, resulting in

activation of the pathways such as NFκB and MAPK (235, 245).

Fig 11: Dimeric structure of TRAF6 protein with RING domain and Three zinc fingers 1-3 (Z1-3). PDB ID: 3HCS (235)

Previous studies reported that TRAF6 knockout mice develop

osteopetrosis, exencephaly, defects in mammary and lymph nodes (233,

239, 246), and hypohidrotic ectodermal dysplasia (247). TRAF6

deficiency in knock out mice shows reduced responses to IL-1R, TNFR

signalling family members, causing an imbalance between immune

activation and suppression (248). TRAF6 is involved in pathogenesis and

progression of several cancers, and overexpression of TRAF6 is reported

in Multiple myelomas (249), lung cancer (250), colon cancer (251), gastric

cancer (252), hepatocellular carcinoma (253), breast cancer (232) and

prostate cancer (254, 255). TRAF6 promotes prostate cancer growth and

invasion by activation of the AKT pathway (256, 257).

43

Aims

The thesis aims to study the biological functions of interactions between several components in different pathways such as Wnt, TGF-β and HIF for cancer progression

Paper I:

To study functional role for TRAF6 in Wnt3a signalling and prostate cancer progression.

Paper II:

To investigate the potential crosstalk between TGF-β and HIF pathways and understanding the biological significance of interactions between components of TGF-β and HIF pathways in clear cell renal cell carcinoma.

Paper III:

To study the expression and association of HIF-3α with the components of HIF and TGF-β pathways and its biological relevance in the progression of renal cell carcinoma.

44

Materials and Methods

Materials: Chemicals and reagents:

4, 6-Diamidino-2-phenylindole Vector Laboratories dihydrochloride (DAPI) 3-8%, 7% Tris-acetate Invitrogen polyacrylamide precast gels 4-12%, 10%, 12% Bis-Tris Invitrogen polyacrylamide precast gels 4% formaldehyde APL Pharma specials Agarose Sigma-Aldrich Alizarin red Sigma-Aldrich Antigen Retrieval Reagents Biocare Medical BCA protein assay kit Thermo Scientific Bovine serum albumin (BSA) Roche Cas9 nuclease, S. pyogenes New England BioLabs Chloroform Sigma-Aldrich COCl2 Tocris DMEM Sigma-Aldrich Dual-luciferase® reporter Promega assay system Duo-link detection kit Sigma-Aldrich ECL kit GE Healthcare EMEM ATCC Ethanol Sigma-Aldrich Ethylenediaminetetraacetic acid Sigma-Aldrich (EDTA) Foetal bovine serum (FBS) Sigma-Aldrich Fugene 6, Fugene HD Promega Glutamine Sigma-Aldrich Glycerol Sigma-Aldrich Hydrogen peroxide (H2O2) solution Sigma-Aldrich Invasion assay kit cell Biolabs LDS sample buffer Invitrogen Methanol Sigma-Aldrich NP-40 Calbiochem

45

Nucleospin® RNA XS columns MACHEREY-NAGEL Odyssey blocking buffer Licor Biosciences Oligofectamine Invitrogen PBS tablets Sigma-Aldrich Pefabloc Roche Penicillin-streptomycin (PEST) Sigma-Aldrich Power SYBR Green PCR master mix Applied Biosystems Prestained PageRuler protein ladder Thermo Fisher Scientific Protein-G Sepharose beads GE Healthcare Recombinant human Wnt3a R&D systems Recombinant TRAF6-Flag Origene RepSox Tocris RIPA buffer Thermo Fisher Scientific RNAimax Thermo Fisher Scientific RNeasy Mini Kit Qiagen RPMI 1640 Sigma-Aldrich Sodium chloride (NaCl) Sigma-Aldrich Sodium citrate Sigma-Aldrich Sodium orthovanadate Sigma-Aldrich Spectra Multicolor High Range Thermo Fisher Scientific Protein ladder TGF-β1 R&D system ThermoScript RT-PCR system Invitrogen Tricaine mesylate Sigma-Aldrich Tris Sigma-Aldrich TritonX-100 Sigma-Aldrich TRIzol Thermo Fisher Scientific Trypsin-EDTA solution Sigma-Aldrich Tween Sigma-Aldrich T7 mMESSAGE mMACHINE Ambion transcription kit

Instruments:

Agilent 2100 Bioanalyzer Agilent Applied Biosystems 7900HT Applied Biosystems fast Real-time PCR system Amersham Imager 600 GE Healthcare

46

BLItz system FORTÉBIO Bright-field microscope 3D Histech Compact desktop centrifuge Eppendorf ELISA scanner Thermo Scientific GloMax®96 Luminometer Promega Interferometry Bio-Layer detection BLItz System Laminar flow hood Labogene NanoDrop 1000 Spectrophotometer Thermo Fisher Scientific Nikon D5200 digital camera Nikon Nikon SMZ1500 stereomicroscope Nikon Odyssey CLx Imaging System Licor Biosciences PCR thermocycler Applied Biosystems See-saw rocker Stuart equipment Tissuelyser II Qiagen Transblot Turbo transfer system Bio-Rad Laboratories ZEISS Axio Imager 2 Carl Zeiss MicroImaging Zeiss LSM 710 Meta microscope Carl Zeiss MicroImaging

Cell lines:

A498 Human Renal cancer cell line which has a loss of expression of VHL due to functional mutation in the VHL gene.

ACHN Human Renal cancer cell line which has intact expression VHL. ACHN is of the clear cell type of RCC.

DU145 Human prostate carcinoma cell line derived from metastatic brain site. DU145 are not hormone-sensitive and lack prostate-specific antigen (PSA) expression.

HEK293 Human embryonic kidney (HEK) cells derived from human embryonic kidney used for expression of LRP5-ICD plasmids and further protein purification.

LNCaP Human prostate cancer androgen-dependent cell line derived from lymph node metastatic site. Epithelial in morphology and adherent.

PC3U Human prostate cancer androgen-independent cell-line. A clonal derivative of a PC3 cell line (258). Derived from the metastatic bone site. Epithelial in morphology and adherent.

47

SW480 Human colon adenocarcinoma cell line. Epithelial in morphology and adherent.

Plasmids:

3xHA-tagged wild-type ubiquitin a gift from V. M. Dixit, Genetech, USA

TCF-reporter TOPflash firefly plasmid Merck-Millipore

pGL4.73 [hRluc/SV40] renilla plasmid Promega

LRP51407–1615HA-(His)6 WT A synthetic gene encoding for LRP51407–1615 HA-(His)6 E1425A the intracellular domain of

human LRP5 from amino acid 1407 until 1615 as WT or with a mutant TRAF6 consensus binding motif ordered from Eurofinsgenomics, Germany and further cloned into pCMV tag5.

pDR274 Zebrafish single guide RNA (sgRNA) expression vector, Addgene

ALK5-HA (TβRI) Gift from P. ten Dijke, University of Leiden, the Netherlands

pcDNA 3.1-VHL Origene technologies

siRNA:

TRAF6 siGENOME human Dharmacon Research

SMARTpool siRNA

Non-specific control siRNA Dharmacon Research

siVHL Ambion, Thermo Fisher Scientific

48

Patients and tumour samples:

The study involves 154 patients with ccRCC (Fig 12). All clinical samples used were collected after obtaining informed and signed consent from patients. The study is approved by the institutional review board and the ethical committee of northern Sweden. Tumour and adjacent kidney cortex samples were obtained after nephrectomy from the patients. The samples were used for the protein studies, and the number of samples for each protein depended on the sample availability. Patient follow-up performed in a scheduled program and used for survival analysis.

Fig 12: Categorisation of ccRCC Patients involved in the study based on gender, age, tumour size, stage, grade and patient follow up.

Methods:

Invitro cell culture and transfections:

Cell lines were grown in the corresponding media as represented in Table 1. Cells were transfected with corresponding plasmids using Fugene 6 or Fugene HD according to the manufacturer's protocol. siRNA was transfected using oligofectamine or RNAimax according to Manufactures protocol. Cells were starved for 12 hrs (ACHN & A498) or 16-18 hrs

49

(PC3U, DU145, LNCaP, SW480) and further stimulated with Wnt3a (50 ng/ml) or TGF-β (10 ng/ml) for different time points.

Cell line Culture Conditions Growth Media Starving media Paper

A498 37 ⁰C + 5% CO2

RPMI1640 + 10% FBS + 1% L-Glutamine RPMI1640 + 2% FBS II&III

ACHN 37 ⁰C + 5% CO2

EMEM + 10% FBS + 1% L-Glutamine EMEM + 2% FBS II&III

DU145 37 ⁰C + 5% CO2

RPMI1640 + 10% FBS + 1% PEST + 1% L-

Glutamine

RPMI1640 + 1% FBS + 1% PEST + 1% L-

Glutamine I

HEK293 37 ⁰C + 5% CO2

EMEM + 10% FBS + 1% PEST + 1% L-Glutamine I

LNCaP 37 ⁰C + 5% CO2

RPMI1640 + 10% FBS + 1% PEST + 1% L-

Glutamine

RPMI1640 + 1% FBS + 1% PEST + 1% L-

Glutamine I

PC3U 37 ⁰C + 5% CO2

RPMI1640 + 10% FBS + 1% PEST + 1% L-

Glutamine

RPMI1640 + 1% FBS + 1% PEST + 1% L-

Glutamine I

SW480 37 ⁰C + 5% CO2

DMEM + 10% FBS + 1% PEST

DMEM + 1% FBS + 1% PEST I

Table 1: Culture conditions and growth media for different cell lines used in the study.

Immunoblotting (IB) and Immunoprecipitation (IP):

Cells were harvested after stimulation and lysis of cells was carried out using RIPA buffer (50 mM Tris (pH 8), 150 mM NaCl, 1% TritonX-100, 10% (v/v) glycerol, 1 mM aprotinin, 1 mM Pefabloc and 1 mM Sodium orthovanadate) and total cell proteins were extracted. Protein concentrations were determined by BCA kit. Total cell lysates were subjected to immunoprecipitation using corresponding antibodies overnight and incubated with protein-G beads for 1hr at 4⁰C. Beads were washed four times, and LDS sample buffer was added to the beads and boiled for 5 min to elute the immunocomplexes. Further samples were loaded on to precast SDS PAGE gels and blotted on to nitrocellulose filters using iBlot or Transblot Turbo transfer system. Membranes were blocked in 5% BSA or Odyssey blocking buffer for 1h at room temperature and

50

incubated overnight at 4°C with gentle agitation with the corresponding primary antibodies (Table 2). Primary antibodies were detected using HRP-conjugated antibodies or light chain specific HRP-antibodies or IRDye® secondary antibodies (Licor) (Table 2) and developed using the enhanced chemiluminescence (ECL) kit from GE Healthcare or Odyssey CLx Imaging System. Image Studio SystemTM software version 3.1 (Licor Biosciences) was used to measure the density of proteins and β-actin was used as an internal control for measuring relative density values for all the proteins.

Antibody Species Catalogue number

Company Application

TRAF6 Rabbit ab40675 Abcam IB, IP

TRAF6; D10 Mouse sc-8409 Santa Cruz IF

TRAF6 Rabbit ab33915 Abcam PLA

K63 Mouse BML-PW0600 Enzo Life Sciences Inc PLA

LRP5 (D80F2) Rabbit 5731 Cell signalling technology IB, IP, IF

LRP5 Goat ab36121 Abcam PLA, IF

β-Catenin Mouse 610154 BD Biosciences IB,IP,IF

Active β-Catenin Rabbit 8814 Cell signalling

technology IB

β-Actin Mouse A5441 Sigma-Aldrich IB

c-Myc Rabbit 5605S Cell signalling technology IB

Anti-HA Mouse 11666606001 Roche IB

HIF-1α Rabbit NB100-134 Novus Biologicals IB, IP, PLA

HIF-2α Rabbit, Mouse

NB100-122, NB100-132 Novus Biologicals IB, IP, PLA

HIF-3α Rabbit ab10134 Abcam IB, IP, PLA

SNAI1 Rabbit 3879 Cell Signaling Technology IB

HA Rabbit 2367 Cell Signaling Technology IB, IP, PLA

51

TβRI or ALK5 (V-22) Rabbit sc-398 Santa Cruz

Biotechnology IB, IP

Phospho-SMAD2 Rabbit 3108 Cell Signaling

Technology IB

SMAD2 Mouse 3103 Cell Signaling Technology IB

PAI-1/Serpine1 Rabbit NBP1-19773 Novus Biologicals IB

VHL (G-7) Mouse SC-17780 Santa Cruz Biotechnology IB

VHL Rabbit NB100-485 Novus Biologicals IB

CA9 Rabbit ab15086 Abcam IB

GLUT-1 Rabbit PA1-46152 Thermo Fischer Scientific IB

E-Cadherin Mouse ab76055 Abcam IB

N-Cadherin Rabbit ab18203 Abcam IB

Danio rerio TRAF6 Rabbit GenScript HK Ltd. IB

Secondary Antibodies for IB and IP

Goat anti-rabbit HRP Rabbit P0448 Dako

Goat anti-mouse HRP Mouse P0447 Dako

Light chain anti-rabbit Rabbit 211-032-171 Jackson Immuno-

research laboratories

Light chain anti-mouse Mouse 115-035-174 Jackson Immuno-

research laboratories

IRDye® 800CW Goat anti-Rabbit

Rabbit 925-32211 Licor Biosciences

IRDye® 680RD goat anti-

Mouse Mouse 925-68070 Licor Biosciences

Secondary Antibodies for Immunofluorescence (IF)

Alexa Flour 555 donkey anti-rabbit

Rabbit A-31572 Invitrogen

Alexa Flour 488 goat anti-

mouse Mouse A-10680 Invitrogen

Alexa Flour 555 Rabbit anti-goat

Goat A-21431 Invitrogen

52

Table 2: List of antibodies used in the studies. Primary and secondary antibodies used for Immunoblotting, Immunoprecipitation and Immunofluorescence.

Immunofluorescence:

PC3U cells were grown on sterile coverslips, and after stimulation, cells were collected and washed with PBS. Then the cells were fixed and permeabilised for 10 min each with 4% formaldehyde and 2% Triton-X100 and blocked with 5% BSA for 1 hr. Cells were incubated with primary and secondary antibodies for 1 hr each and mounted onto a glass slide with mounting media containing DAPI. Co-localisation of proteins were visualised using confocal microscopy.

Dual-Luciferase assay:

TCF reporter plasmid TOPflash is transfected along with either siTRAF6 or siCtrl to measure the Wnt signalling activity in the presence and absence of TRAF6 in PC3U and SW480 cells. Renilla Luciferase is also transfected into cells to serve as internal transfection control. After cells were treated with Wnt3a, lysates were made, and Dual-Luciferase assay is performed using Luminometer according to the manufacturers' protocol.

Proximity Ligation assay (PLA):

In situ Proximity ligation assay in cells:

Cells were grown in eight-well or six-well dishes and stimulated with Wnt3a or TGF-β accordingly. After stimulation cells were fixed, permeabilised and blocked using 4% formaldehyde, 2% Triton-X100 and Duo-link blocking buffer respectively. The further assay was performed according to the manufacturer's protocol using Duo-link detection kit (Sigma). Cells were mounted on to slide using mounting media containing DAPI and images were taken at 40× objective lens using confocal microscopy. The analysis was performed using Duo-link image tool software to quantify the signals.

53

In situ Proximity ligation assay in prostate adenocarcinoma tissue sections:

Malignant tissue sections from prostate adenocarcinoma were obtained from male patients who had undergone through prostatectomy. Tissue sections were rehydrated in an ethanol gradient, and heat-induced antigen retrieval performed followed by blocking with Duolink blocking solution or 5% normal goat serum for 1 hr. Tissue sections were further incubated with primary antibodies overnight at 4⁰C and treated with PLA Duolink probes. For fluorescent imaging, amplification was performed with 1x amplification buffer red and counterstained with phalloidin –Alexa 488, and for brightfield detection, amplification was performed followed by bright-field detection, substrate development and nuclear staining. Sections were mounted, and images were obtained either by using Zeiss Imager Z2, with objectives plan-Apochromat dry NA 0.8 and Plan-Apochromat oil immersion NA 1.4 or bright field microscope. From the whole scanning pictures, three different images with at least 300 cells were taken at random, and the mean PLA dots versus total cells (mean ± S.E.M) were counted and analysed by Mann-Whitney U test.

Ubiquitination Assay:

PC3U cells were transfected with HA-tagged ubiquitin and harvested after stimulation with Wnt3a. Cells were heated at 95⁰C in the presence of 1% SDS and diluted with lysis buffer containing 0.5% NP-40, followed by centrifugation. The slimy layer was collected and incubated with TRAF6 primary antibody overnight and then incubated with protein-G beads for 1hr at 4⁰C. Beads were washed four times, and sample buffer along with a reducing agent, was added to the beads and boiled for 5 min (144).

Quantitative real-time PCR:

RNA was extracted from cancer cell lines using the RNeasy Mini Kit according to the manufacturer's protocol and the RNA concentration, was measured using a NanoDrop 1000 Spectrophotometer. cDNA synthesised

54

from total RNA using the ThermoScript RT-PCR system. qRT PCR was performed using Power SYBR Green PCR master mix, and forward and reverse primers (primers used presented in Table 3) in Applied Biosystems 7900HT Fast Real-time PCR system.

Gene Forward Primer Reverse primer

TRAF6 GTTGCTGAAATCGAAGCACA CGGGTTTGCCAGTGTAGAAT

β-Catenin CCAATAGGGGCACAAAACGT CCTCTTCCCCATCTCTGTCA

LRP5 ACGTCAGCCTGAAGACCATC GCGCCACTTCGATTCTGTTG

c-Myc CTTCTCTGAAAGGCTCTCCTTG GTCGAGGTCATAGTTCCTGTTG

GAPDH CTCTGGTAAAGTGGATATTGT GGTGGAATCATATTGGAACA

Table 3: Primers for human prostate cancer cell line (PC3U) mRNA qRT-PCR analysis.

Invasion Assay:

Cells were transfected with respective siRNA or plasmids and starved for 12 hrs. The invasion kit chambers were coated with Bovine Type I collagen and the assay was performed according to the manufacturers' protocol. Trypsinised cells were seeded into the upper chamber containing media with 2% FBS. The chambers are immersed into a 24-well plate containing 10% FBS with media. Cells were stimulated with either Wnt3a (50 ng/ml) or TGF-β (10 ng/ml) for 24 hrs. Upon stimulation, invasive cells migrate from the upper chamber to the 24-well plate through collagen filter and captured in the filter. The process was stopped by removing the upper chambers from 24-well plate, and collagen filter with cells was stained with a staining solution and photographed with a Zeiss light microscope. Next, the invaded cells were lysed, and the optical density (OD) was measured at 560 nm.

Label-free monitoring of a TRAF6-LRP5 protein interaction:

To measure the protein interaction between two proteins in real-time, we have used interferometry Bio-layer detection with BLItz system.

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Recombinant LRP51407–1615HA-(His)6WT was immobilised on Ni-NTA sensor, washed once with PBS and Kon determined by adding TRAF6-Flag at a concentration of 167 nM or 822 nM. Next, Koff determined by washing with PBS until a stable binding curve was achieved.

In silico gene expression analysis:

cBioPortal, an online repository to study gene expression using Provisional TCGA databases in prostate cancer and renal cell carcinoma, was used. Gene expression correlations between the genes were quantified using these databases. We also obtained the survival analysis data from the Human Protein Atlas using TCGA databases where Kaplan-Meier plots showing the correlation between patient survival and mRNA expression levels. GENEVESTIGATOR®, a public database, was used to find the TRAF6 mRNA expression levels among various cancers. KM-plotter and OncoLnc databases were also used to obtain cancer-specific and overall survival curves for RCC patients.

Statistical analysis:

All the experiments were performed at least three times independent of each other unless otherwise stated. IBM SPSS Statistics 24.0 was the software used to perform statistical analysis. Student's t-test and Mann-Whitney U test was used to show the difference in expression of two independent variables and bivariate correlations between two proteins were analysed using Spearman's correlation. Kaplan-Meier curves were used to show the survival data in cancer patients and zebrafish. Error bars indicate standard error of the mean (S.E.M). P values <0.05 were considered statistically significant.

Zebrafish Experiments:

Zebrafish Strains and Maintainance:

Zebrafish (Danio rerio) was used in the experiments and the mutants generated were under permits from the ethical board (permit: A-13-15;

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Umeå Djurförsöksetiska nämnd). Either London wild Type or tupfel long fin strains were used to obtain embryos and Larvae. Zebrafish (ZF) were maintained by standard procedures at the zebrafish facility at Umea University.

CRISPR/Cas9 mediated generation Zebrafish TRAF6 mutants:

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein-9 nuclease (Cas9)-CRISPR/Cas9 technique was employed to generate ZF TRAF6 mutants. TRAF6 exon-2 was targeted for the generating mutations using customised 20 nucleotide sequence pair; 5′-CTGTCTGATGGGTCTCCGCT-3′ (right) and 5′-AGCGGAGACCCATCAGACAG-3′ (left). CAS9 nuclease along with transcribed sgRNA containing nucleotide sequence pair injected into single-cell staged zebrafish embryos. Mutated DNA loci in Fo fish were identified by a restriction enzyme MbiI recognises incorporated restriction site in each customised target nucleotide sequence. Sequencing is later performed to confirm the mutations in the DNA sequence.

Zebrafish DNA - RNA extraction and quantitative RT-PCR:

Heterozygous TRAF6 male and female were incrossed, and seven days post fertilisation (dpf) embryos were collected for RNA extractions. Single embryos were transferred into Eppendorf tubes with TRIzol and homogenised in a tissuelyser. After chloroform added, the samples were centrifuged and the transparent top aqueous layer used for RNA extraction and bottom organic layer for DNA extraction. RNA containing aqueous layer precipitated with 70% RNA and applied to Nucleospin® RNA XS columns. Further procedures were performed according to the manufacturer's protocol. RNA quality is assessed using Agilent bioanalyser. cDNA and qRT PCR performed as described in the above sections. Primers used in qRT PCR presented in Table 4.

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Zebrafish gene Forward Primers Reverse Primer

β-Catenin1 CCATTGGCAGCAACAGTCTT GAAGGACTGGTTGAAGCCCT

β-Catenin2 CCATGGAGCAGGACCGTAA CCTGCTCCCACTCGTACAG

LRP5 AGTAGGACGAGCAAACGGAC GATCTCTCGCTGTAGACTTTGC

Cyclin D1 TTGTTTTGCTGCGAAGTGGA TCGCGACGATTTTCCTCATT

CBX8a GAATATTCTGGATGAGCGTC GCTTTTGCCTTCATTAGAAAC

CBX8b CCACAGGACTAATCCACAAT TGAATAGTGCTTCAGGTGTT

MSXd AGTTTGGTCAGAGGAGAAAT CTTGTGGAGTCTTGAGAGAA

β-Actin CGAGCTGTCTTCCCATCCA TCACCAACGTAGCTGTCTTTCTG

Table 4: Primers for zebrafish mRNA qRT-PCR analysis

Whole Mount staining for Zebrafish bone:

Adult WT and TRAF6-/- zebrafish (50 dpf) were treated with a lethal dose of tricaine mesylate and fixed in 10% formalin. Visceral organs were surgically removed, and the body cavity was rinsed with distilled water. Further clearing procedures were performed by placing fish in 1% KOH + 3% H2O2 solution for 2 hr followed by 2 hr with half this concentration, rinsed with distilled water twice and transferred into 30% saturated sodium tetraborate solution and incubated overnight. Soft tissues were digested using a solution with 1% trypsin and 2% borax for 4 hrs or more depending on the amount of tissue left. Then fishes were stained in 1% KOH + Alizarin red solution for 12 hrs or more. Specimens were rinsed with distilled water several times to remove excess stain and transferred to 100% glycerol before a series of 1% KOH: glycerol solutions (80:20, 60:40, 30:70) (259). Specimens were photographed using Nikon SMZ1500 stereomicroscope and a Nikon D5200 digital camera.

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Results and Discussion

Paper I: TRAF6 function as a novel co-regulator of Wnt3a target genes in

prostate cancer

Prostate cancer is the second most frequently diagnosed cancer and the

fifth leading cause of cancer-related death in men (6, 7). Wnt signalling is

a vital growth factor signalling pathway involved in embryo development

and is also implicated in cancer progression (45, 53, 54). Aberrant Wnt

signalling due to either genetic or epigenetic changes in Wnt components

leads to the development of castration-resistant prostate cancer, but the

underlying molecular mechanisms remained elusive. Targeting Wnt

signalling by downregulation could prevent the progression of prostate

cancer (115, 260). In this paper, we have elucidated an important co-

regulatory role of TRAF6 in the Wnt pathway in human prostate and

colorectal cancer cells, which was confirmed in vivo by using

CRISPR/Cas9 genomic editing, in zebrafish.

TRAF6, an E3 ubiquitin ligase and an adapter protein promote

inflammatory responses to various cytokines (223). We found that TRAF6

has a functional role as a positive co-regulator of Wnt components and

their target genes. We found that TRAF6 interacts and co-localises with

Wnt hallmark protein β-Catenin and the Wnt co-receptors LRP5 and LRP6

upon Wnt3a stimulation in human prostate cancer cell lines and also in

human prostate cancer tissues. TRAF6-LRP5 interaction was also

observed in SW480 cells, a colon adenocarcinoma cell line, demonstrating

that interaction also occurs in other cancer forms. TRAF6

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autoubiquitination is an essential event for its activation (244), and we

found that upon Wnt3a stimulation TRAF6 undergoes K63-linked

ubiquitination for its enzymatic activity. By in silico analysis, we looked

for a possible consensus binding motif (Pro-X-Glu-X-X-(aromatic/acidic

residue)) (228, 235) for TRAF6 in the LRP5 protein structure and found

the motif PHEYVS. We speculate that phospho-serine in the last position

of the consensus sequence in LRP5 may substitute the properties of acidic

amino acid, making it a binding site for TRAF6. By mutagenesis studies

in PC3U cell lines we observed reduced association between TRAF6-

LRP5 observed in mutant LRP5. No direct interaction between the

recombinant proteins was observed, suggesting that it is likely due to that

both TRAF6 and LRP5 undergo post-translational modifications in cancer

cells, which contribute to the regulation of their binding activity. From

these findings, we speculate that Wnt3a promotes TRAF6 interaction with

Wnt components (LRP5, LRP6 and β-Catenin), both in vivo and in vitro.

By using siRNA in prostate cancer and colon cancer cell lines, we found

that TRAF6 is required for gene expression of β-Catenin as well as for the

Wnt-ligand co-receptor LRP5 and effects c-MYC mRNA and protein

expression. Earlier studies reported that the high expression of both

TRAF6 and c-Myc is correlated with poor survival in patients with prostate

cancer (261-263). We further investigated a functional role for TRAF6 in

Wnt3a-induced cancer responses, by silencing TRAF6 expression in

prostate cancer (PC3U) and colon adenocarcinoma (SW480) cell lines and

observed that expression of TRAF6 was required in both cancer cell lines

to promote Wnt3a-induced invasion of cancer. Our observations from the

invasion assay explain the potential role of TRAF6 in cancer cells for their

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invasion capabilities, which is identical with earlier studies in the lung,

gastric and colon cancer (250-252). This positive regulation of TRAF6 is

essential for Wnt3a induced responses in prostate cancer and colon cancer.

We generated CRISPR/Cas9 mediated TRAF6 deficient zebrafish, where

the majority of TRAF6 mutant zebrafish did not survive into

adulthood, which is consistent with survival studies on TRAF6 deficient

mice. Previous studies show that TRAF6 and LRP5 were involved in bone

metabolism, and defects in both the genes were reported to have a severe

bone malformation in humans and mice (54, 239, 246, 264, 265). Similar

to the effects in mice, TRAF6 deficient zebrafish show signs of defective

bone formation with a fusion of tail vertebrae which resulted in the

abnormal tail formation and a malformed body axis. TRAF6 deficient

zebrafish also show decreased mRNA expression levels of the Wnt

components LRP5, β-Catenin1/2 and other target genes such as Cyclin

D1, Msxd, CBX8a/b. TRAF6 induced positive regulation of these genes is

crucial for the survival and development of zebrafish.

This study identifies TRAF6 as an evolutionary conserved co-regulatory

protein for transduction of the canonical Wnt3a pathway that also

promotes progression and invasion of prostate cancer through its positive

effects on Wnt signalling. In future, TRAF6 may contribute as a

therapeutic target for inhibiting prostate cancer progression and improving

the prognosis for patients with prostate cancer

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Paper II: Interactions between TGF-β type I receptor and hypoxia-inducible

factor-α mediates a synergistic crosstalk leading to poor prognosis for

patients with clear cell renal cell carcinoma.

Effective delivery of oxygen is essential in multicellular organisms to

perform normal metabolic activities. Hypoxia is a condition with deprived

oxygen content in the tissues. Hypoxia can lead to several physiological

and pathophysiological conditions like cancer. HIFs (Hypoxia-inducible

factors) regulate the hypoxia-mediated cellular responses. HIF pathway

deregulation such as epigenetic silencing of the VHL gene is one of the

common cause in Renal Cell Carcinoma (RCC) (266). RCC is a common

malignancy in adults which originates from renal tubular epithelium in

kidneys. In this study we have investigated the possible cross-talk between

HIF and TGF-β pathways in clear cell Renal cell carcinoma (ccRCC)

We demonstrated that HIF-1α and HIF-2α were significantly higher in

ccRCC tumour samples compared to adjacent kidney cortex. Earlier

studies also elaborated the increased roles of HIF-1α and HIF-2α in cancer

progression (267, 268). Survival analysis showed increased expression of

HIF-2α, which was associated with poor cancer-specific survival but not

with HIF-1α. Expression of HIF-1α negatively correlated with VHL but

not with HIF-2α. HIF-1α is reported to be degraded by hypoxia-associated

factors (HAFs) in normoxia, and it is dependent on VHL. However, HIF-

2α was regulated differently by HAFs, driving them towards

transactivation. HIF-2α was reported to be associated with chronic

prolonged hypoxia, and HAFs regulate the switch from HIF-1α to HIF-2α,

i.e., during early hypoxia to sustained hypoxia (269, 270). Also, we found

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HIF-1α associated with smaller tumours and HIF-2α associated with

advanced tumour stage supporting the earlier observations.

Further, we investigated the association between the proteins in HIF and

TGF-β signalling pathways in ccRCC. HIF-2α significantly correlated

with ALK5-ICD, pSMAD2/3, PAI-1 and SNAI1 proteins and a close to

positive association trend with ALK5-FL was observed while HIF-1α was

found to be associated with only pSMAD2/3. Also, SNAI1 was found to

be significantly correlated with all the TGF-β components. From earlier

reports, it is known that HIF-1α mediated hypoxia induces upregulation of

Smad dependent TGF-β pathway (271), PAI-1 is a known target gene of

both HIF-1α and HIF-2α (272) and SNAI1 is known to be induced by both

TGF-β and hypoxia (273) which is consistent with our data.

It is reported that ectopic ALK5 expression in Renal cell carcinoma cell

lines leads to activation of TGF-β signalling and ALK5-ICD is correlated

with poor prognosis and tumour progression (274). Also, VHL is known

to ubiquitinate ALK5 for its proteasomal degradation (275). From these

observations, we speculate that ALK5 is a crucial player in the regulation

of TGF-β and hypoxia signalling, and we, therefore, investigated its role

in the functional crosstalk. We found that in normoxia, ectopic expression

of ALK5 induced increased expression of HIF-1α and HIF-2α and their

classic target genes CA9 and GLUT1 (276, 277). Also, VHL expression is

negatively correlated with levels of HIF-1α, HIF-2α and PAI-1 levels.

Further we demonstrated that ALK5 kinase activity is needed for its

expression and induction of TGF-β and Hypoxic factors along with their

target genes. We also reported that ALK5 induced invasiveness of RCC

cell lines and leads to reduced E-cadherin expression. Further, we

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confirmed an increased expression of ALK5 under hypoxic conditions

using CoCl2, a chemical inducer of hypoxia (278, 279). Importantly, we

showed that ALK5 interacts with both HIF-1α and HIF-2α, and these

complexes were localised both in cytoplasm and nucleus.

From these results, we conclude that cancer progression is mediated by

crosstalk between various signalling pathways and here our findings shed

light on the critical aspects of Hypoxia and TGF-β induced ccRCC tumour

progression by their synergistic crosstalk in which ALK5 potentially

drives the expression of HIF-1α and HIF-2α even under normoxic

conditions. Also, the novel finding of an interaction between ALK5 and

HIF-α might provide a more in-depth understanding of mechanisms

behind tumour progression.

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Paper III: Expression and association of HIF-3α with hypoxic and TGF-β

signalling components in Renal cell carcinoma.

HIF-3α exists in ten different functional splice variants, which makes it

challenging to characterise their biological importance during hypoxia.

HIF-3α is known to be relatively weak transcriptional activators of

hypoxia-responsive genes. HIF-3α has dual roles that are regulation of its

target genes and suppressing the levels of HIF-1α and HIF-2α by

competitive binding with HIF-β subunit and thereby regulating the target

genes of HIF-1α and HIF-2α (184). Also, HIF-3α4 has a dominant-

negative function of inactivating HIF-1-mediated transcription. This HIF-

3α4 is down-regulated in ccRCC patients leading to increased HIF-1α

levels, suggesting potential significance in renal tumorigenesis (280, 281).

HIF-3α has unique c-terminal LZIP domain which enables it to bind to

DNA and proteins, thereby promoting HIF-3α-specific gene transcription

(282).

In this paper, which is an ongoing study, we investigated the role of HIF-

3α in the progression of Renal cell carcinoma and its association with the

components of TGF-β. We investigated ccRCC and pRCC patient tumour

tissue samples and observed that HIF-3α protein levels were increased in

patient tumour samples compared to adjacent kidney cortex. We further

analysed the clinicopathological parameters and found that HIF-3α levels

were associated with advanced tumour stage and metastasis in ccRCC and

associated with larger tumours in pRCC. It has previously been reported

that HIF-3α is known to be associated with prolonged hypoxia (269). Data

from public databases show that HIF-3α is a significant prognostic factor

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associated with improved survival in pRCC patients and total RCC

patients. However, HIF-3α was not associated with cancer-specific

survival in both patient sample data and also from public databases.

In our previous study (Paper-II) (283), we have shown a significant

association between HIF-1α and HIF-2α and TGF-β components. In this

study, we observed that HIF-3α show a significant positive correlation

with HIF-1α and HIF-2α in pRCC and, in ccRCC, it has a significant

positive correlation with VHL. Besides, in ccRCC HIF-3α show a

significant positive association with pro-invasive gene SNAI1, which is a

crucial regulator of epithelial to mesenchymal transition.

TRAF6 an E3 ligase known to be a prognostic marker in RCC and we

observed that HIF-3α associates with TRAF6. From earlier studies,

TRAF6 is known to be a positive regulator of HIF-1α levels independent

of oxygen availability. TRAF6 is also known to polyubiquitinate HIF-1α

and enhances its expression (284, 285). In this study, using cBioportal

TCGA provisional database, we found that TRAF6 has a significant

positive correlation with HIF-3α and TRAF6 was associated with better

overall survival in both pRCC and ccRCC. From the in vitro studies by

using immunoprecipitation and PLA, we have found that HIF-3α interacts

with TRAF6 in ACHN cell line and TGF-β enhances the expression of

HIF-3α and TRAF6.

From these data, we conclude that HIF-3α could play a vital role in the

RCC progression via its effects through increased levels and significant

association with clinicopathological parameters. The interactions with

TGF-β components might have positive effects on migration and invasion

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primarily through SNAI1, and also TRAF6 could be a key player. As a low

expression of TRAF6 related to poor prognosis, its normal levels might be

essential to maintain homeostasis and regulation of HIF-3α through its

direct interaction. We are still working on the other experiments to

investigate if TRAF6 can ubiquitinate HIF-3α and regulate its role in

hypoxia and normoxia.

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Concluding remarks

PAPER I:

• TRAF6 is an E3 ligase which interacts with Wnt components

LRP5, β-Catenin and positively regulates their expression.

• TRAF6 is an important co-regulator of Wnt3a signalling and

thereby promote activation of Wnt target genes and drives prostate

cancer progression.

• TRAF6 regulates Wnt3a-induced invasion of cancer cells and

promote cancer progression.

• TRAF6 is crucial in the embryonic development and crucial for

survival and bone development zebrafish through its positive

regulation of Wnt target genes.

PAPER II:

• HIF-1α and HIF-2α protein levels were increased in ccRCC

tumours, and they are positively associated with TGF-β

components such as ALK5, ALK5-ICD, pSMAD2/3, PAI-1 and

SNAI1.

• HIF-1α and HIF-2α associated with smaller tumours and advanced

tumour stage, respectively. In addition, increased expression of

HIF-2α significantly associated with poor cancer-specific survival

but not HIF-1α.

• ALK5, through its kinase activity, potentially drives the expression

and increases HIF-1α and HIF-2α levels under normoxic

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conditions and further effects expression of hypoxic target genes

CA9 and GLUT1. Further VHL dictates the expression of hypoxic

factors under normoxia.

• Hypoxia induces the expression of ALK5 and also phosphorylates

SMADs in RCC cell lines. ALK5 also induces invasiveness in

renal cell carcinoma.

• ALK5 interacts with both HIF-1α and HIF-2α in normoxic

conditions.

PAPER III:

• HIF-3α protein levels were increased in both ccRCC and pRCC

patient tumour samples. HIF-3α associated with advanced tumour

stage and metastasis in ccRCC and larger tumours in pRCC.

• HIF-3α significantly associates with SNAI1 and VHL in ccRCC,

and in pRCC, it is associated with HIF-1α and HIF-2α.

• HIF-3α interacts with TRAF6 in vitro, and TGF-β enhances the

expression of HIF-3α and TRAF6

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Acknowledgements

It was a great journey. Full of beautiful memories and happy moments. I have experienced both the roller-coaster rides and smooth rides. Ups and downs. Failures and success. Disappointments and achievements. ‘Your best teacher is your last mistake’ – I made many mistakes, and I earned many teachers. I learnt from my mistakes and always tried to make new mistakes. The journey of my PhD happened with years of groundwork not only from me but also from my family and my teachers. In the journey of my PhD, I got a chance to meet different people with different backgrounds. It was a great experience getting to know people which helped me in paving my path towards PhD studies. I want to thank each and everyone who helped me to reach my goals.

I want to thank Umea University and the funding agencies Knut and Alice Wallenberg Foundation (KAW 2012.0090), Swedish Cancer Society (CAN 2017/544), Swedish Medical Research Council (2016-02513), Prostatacancerförbundet, Konung Gustaf den V:s och Drottning Victorias Frimurarestiftelse and Cancerforskningsfonden Norrland for financially supporting my PhD studies.

Firstly, I want to thank and express my immense gratitude to my supervisor, Marene Landström, who believed in me and provided the opportunity to do a PhD. You are an inspiration to me. During the experiments, the immense support and encouragement you gave enabled me to work with a positive attitude. No matter the result, your support always drove me towards the final destination. You are an excellent teacher, providing scientific insights and guided me throughout the journey. Your caring, patience and motivation helped to complete my PhD with flying colours. Thank you for everything.

I want to express sincere thanks to Jonas von Hofsten, my co-supervisor. I started my research journey at your lab as a master student during my early days in Umea. You taught me right from basics in research and handling fishes. I haven’t met such a calm and a composed person like you in the field of research. You always supported when Marene and I wanted to generate TRAF6 mutants and encouraged us to go further with CRISPR technology. We had excellent talks in the fish room during genotyping and discussions about TV series. I hope you finished watching all the seasons of ‘walking dead’. I always annoyed you with my time sense and my lethargic work in cleaning fish tanks, but you tolerated me so much (I am getting better with cleaning nowadays). Thank you so much for guidance and help during my PhD studies.

I want to thank my co-supervisor Carl-Henrik Heldin for your feedback on my TRAF6 project and always enjoyed your views and inputs on our projects during the KAW meetings and conferences. You are such an inspiration. I also want to thank my co-supervisor Ruth Palmer for your inputs on TRAF6 project and discussions about possible work with Drosophila during the initial days of my PhD.

I would like to acknowledge Börje Ljungberg for his advice and scientific inputs for the RCC projects.

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I would like to acknowledge Anders Bergh and Pernilla Anderson for helping in providing patient tissue samples and pleasant interactions during prostate cancer meetings along with Pernilla Wikström, Emma Persson, Maria Brattsand and all other.

I especially want to thank my present and past colleagues from Marene’s group with whom I grew up in research.

Shyam and Reshma, who initially guided me in the lab when I joined as a student and taught me everything related to cell culture and many.Thank you, Shyam for introducing me to TRAF6-Wnt project. I have much enjoyed your company in the lab, and the excellent time we spent together. My wishes to Ameya. I always enjoyed Reshma’s oven biryani. Hope we will have more food in future together. Thanks for your support.

I am grateful to Yabing Mu and Guangxiang for their suggestions and help with experiments in TRAF6 project. Thank you

Stina Rudolfsson, you are always welcoming. Thanks for teaching me the basics of prostate cancer during the initial days of my PhD. I really enjoyed our pleasant times during the conferences in Leiden and Uppsala.

Jie Song, we shared office space right throughout our PhD studies. We shared sweet talks, jokes, gossips and many. We had delightful times during the conferences. Thank you for your help with my experiments, especially PLA experiments (I troubled you with a lot of questions). All the best for your research carrier.

I also want to thank Alexej Schmidt for your help in the TRAF6 project. Your contribution to the project has increased the standard of the paper. Your feedback and inputs in the manuscript helped a lot. We had a pleasant conversation about food, culture, beer.. many. I like your multilingual email about the lab duties (PS: Our Indian lab members do not speak Hindi). Thank you.

Lennart, you are such a happy man. Our never-ending discussions on the whiteboard. They were fun. Your constant new ideas about Wnt and TRAF6 really helped in completing the project. You made work easy with so many plasmid constructs. I hope we will have many more ideas for designing new projects. Thank you for your contributions.

Olena Rakhimova, you are such a nice person with all positive vibes. I always enjoyed our conversations, and I cannot forget your instant help when I asked for a phone conversation with airport people. I still remember your piano skills (at a meeting in Olofsfors bruk). Thank you.

Samaneh, I am always jealous of your hard work and your skills with managing things. Your contribution to TRAF6 project really helped in speeding up the project. I enjoyed your food and a delightful dinner with your family. Thank you for your contributions.

I would also like to thank past members of Marene’s group Linda Köhn – for great times and discussions in the lab and during TGF-β conferences. Anders Wallenius – we used to sit next to each other in the conferences. I used to like your abstract drawings in your

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notes during conference talks and the ideas about your tattoos and of course, surfing. Thanks for sharing your thoughts about CRISPR. Also, I would like to thank Maria, Gizem, Li Wang, Chunyan Li, Yang Zhou, Tommy Etekal and Dincer for beautiful times during my PhD studies in the lab.

I would like to thank Ravi, Vidya and little Kanasu. We had delightful times with our families on many occasions with yummy food. I always love your instincts Ravi, and your humour and jokes. Our lively conversations at Kahls for years together and your help during gowthami pregnancy really boosted our energies. Thanks for always being with us. Thank you for introducing me to ccRCC project which helped me for the completion of my PhD studies. Thank you for everything.

Thanks to Pramod and Neetha for our wonderful times together in Umea. Pramod, I don’t know where to start because you are with me all the time. We started our journey together and happy that it is still going and hope it continues further. We experienced everything together. You are a great friend and always supportive. I always feel so glad that our relationship is happening in both the personal and professional lives of ours. Our journey together is a roller-coaster ride. We shared our happy and sad moments together. I am glad that we have such kind of relationship between us. I would like to congratulate you and wish you all the best for your PhD thesis defence. It's always lovely to see Praneetha, Aaryan and Raaga grow up together. Thanks for your encouragement and support all through these years.

I want to specially acknowledge the administrative staff of Medical Biosciences, Terry Persson, Clas Wikström, Carina Ahlgren, Ida Laestander, Karin Boden and Åsa Lundsten for their continuous and constant help all throughout my PhD studies. Thank you for your unconditional support.

Terry, you are such a sweet person. I still remember your constant emails to both Migrationsverket and US embassy for my visa issues. Oh... I cannot ask you for more. Also during my thesis writing, you took good care of me with chocolates and fruits late in the evenings. Thanks for everything.

I would like to acknowledge Maria Brattsand, for introducing me to cross-country skiing (which I failed miserably by not following basics) during winter 2014. It was a lifetime experience.

I also want to thank Karin Nylander, Jonathan Gilthorpe and Gunilla Olivercrona for their advice and encouragements.

I would like to thank colleagues from Jonas von Hofsten group. Hanna Nord - you introduced me to the research work environment during my master thesis. Thanks for your patience and help in teaching me the basics about zebrafish. Thanks for your advice in generating CRISPR knockout fishes. Nils – we had wonderful conversations together. I always enjoyed your company. Your inputs with TALENS designing really helped me.

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Thanks for your help with genotyping. I would also like to acknowledge the past and current members of the zebrafish group.

Building 6M, 2nd floor, I spent more time here than home. The people around made it a wonderful place. I had great times with my fellow PhD students and also other people in the department. I am privileged to get to know about Xingru, Zahra, Lee-Ann, Manuela, Maria N, Ela, Erik B, Erik D, Isil, Elin, Denise, Björn, Kestin, Johan B, Vicky, Sandhya, Mahmood, Anika, Mona, Helena, Max, Saba, Greg, Rob, Parviz and many more. The trips to Horse-back riding and high rope challenge and beer-corners were unforgettable.

I would like to thank Celia, Maria, Ela and Anika for making their presence during my wedding and the lovely time we spent in India. I always cherish those moments.

Chaitu, you are always a good friend of mine. We cherished our best moments during our masters in India. We dreamt together to pursue higher studies abroad. We worked really hard for it. I wouldn’t be here in Umea if you are not here. I lost hope in the journey, but you always encouraged me. Your support and affection towards me are unconditional. I always admire you for your determination. Thank you for everything. All the very best for your future.

My Indian friends in Umea, it is a big list. They made my stint in Umea a memorable one. Cricket, cooking food, movies, photoshoots, conversations, volleyball, badminton.. so many. I never felt bored. The trips to Narvik and Stockholm cruise brought lifetime memories. These moments I can cherish throughout my life. I made many lifetime friends on this journey. I would like to thank Harsha, Jani, Raghu, Edward, Sampath, Gaurav, Mahesh, Kamraj, Vishnu, Saravana, Ganesh, Vivek, Sridhar, Mahesh N, Srinivas Oruganti, Mohan, Edvin, Yugnadhar, Sharat, Krishna, Anil, Mridula, Hareesha, Lalitha, Jonitta, Lokesh, Praneeth, Faizal, Bharat and my recent Indian friends Raviteja, Maruthi, Bala, Vijay, Prasad and Raghav. Faizal and Bharat (raja’s) we still have to go for long Euro road trip on open-top mini cooper as we planned. You both are such sweet friends and companions to cherish the moments for life.

Venky, Bindu and Eeshu, you people are really close to us. We had wonderful times together along with our families. Venky (Guruvu garu), you are always like a mentor giving expert advice in crucial situations. Hoping for beautiful times together in future in India. I also like to thank Vijay & Kamala, Munender & Sharvani and Chandra Kiran & Kalyani for their support and parenting tips to make life easy. Also I would like to thank Raghav and Yashasvini for lively conservations and dinners together. I also want to acknowledge Anubhav forhis well wishes and constant support and of course tasty dinners are unforgettable.

I was always inspired by people and nature around me. I was greatly influenced by my teachers, who helped in guiding me towards biological sciences. I would like to acknowledge and thank my natural science teacher Suchala Devi madam and Zoology teacher Vishnu sir who greatly influenced me to pursue my carrier in science.

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My friends are my significant assets who always inspired me and always prompted me to concentrate on my goals. I would like to thank my school friends DV, Rama Krishna, Rajesh, Soma Sekhar and many. I always cherished great moments with my friends Anil, Vikram, Praveen, Manikanth, Santhosh, Sai, Reddy, Naveen Raj Kumar and Gopi baaa. A special mention about a wonderful person Umapathi Anna, who always took good care of me and supported in every part of my dreams for pursuing PhD. Never got bored when Harsha RV,Venu Gopal, Srinivas and Kiran around me. Full of fun and adventure. Harsha, we shall finish what we are dreaming now, the target is getting close. Rohita, Santhi, Deepthi, Maitu, Sahithi and Suma I always remember the good times we spent. I also want to remember my juniors during my masters Anil, Kuttu, Deepika, Yugandhar, Amulya, Mitra, Deepthi and many more for our constant talk about research and fun

I am deeply and emotionally connected to my best buddies Deviram, Sushma, Vishnu Priya and Chiranjeevi. You guys always there with me every part of my life. Your encouragements and love towards me were still inspiring. Thank you for being in my life.

I would also like to thank my in-laws for their constant support and encouragement. Shivani, you will reach great heights, I always like the way you think. Thanks for your endless love and affection.

My family has more confidence in me than I have on myself. They always believed in me. Daddy and Mommy, I dedicate this thesis to you. It wouldn’t have been possible without your constant support. You made my dreams come true. Whatever I achieved or will achieve is because of you. You sacrificed all through your life to fulfil our dreams. I will always work hard to make you proud. Thank you for everything. My brother Sanjay is a significant asset for me. We went through life events together. You have always supported me and also believed in me. I will always be there for you. All the best for your PhD studies.

My two strands of DNA, the amazing twins, Raaganjali and Aaryan. My life took an unimaginable turn by their birth. They are my stress busters. Such cute and beautiful twins. I love you guys. I will always try to make you proud.

Finally, everything comes to life partner. It is always sweet when someone is always next to you to give a little push for the next level. Gowthami, you are such a lovely and admirable person. You added that tinge of spice to my life. I am bad in expressing my feeling directly to you, but all these years, your presence made my life more lively and happy. During my PhD studies, you tolerated me so much. We had good and bad days, but we survived together with more love and affection. Your support and belief in me inspired to go further. The way you handle the situations with kids during my busy times is incredible. You are a wonderful mother and best wife. Thanks for coming into my life and making it colourful.

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