Wnt/²-catenin Signaling and Epigenetic Deregulation - DiVA Portal

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Wnt/β-catenin Signaling and Epigenetic Deregulation in Breast Cancer and Parathyroid TumoursList of Papers
This thesis is based on the following papers, which are referred to in the text by their roman numerals.
I Björklund, P., Svedlund, J., Olsson, A-K., Åkerström, G., Westin, G. The internally truncated LRP5 receptor presents a therapeutic target in breast cancer. PLoS One 2009, 4:e4243.
II Svedlund, J., Aurén, M., Sundström, M., Dralle, H., Åkerström, G., Björklund, P., Westin, G. Aberrant WNT/β- catenin signaling in parathyroid carcinoma. Molecular Cancer 2010, 9:294.
III Svedlund, J., Koskinen Edblom, S., Marquez, V.E., Åkerström, G., Björklund, P., Westin, G. Hypermethylated in cancer 1 (HIC1), a tumour suppressor gene epigenetically deregulated in hyperparathyroid tumours by histone H3 lysine modification. Journal of Clinical Endocrinology & Metabolism 20121, jc.2011-3136; doi:10.1210/jc.2011-3136.
IV Svedlund, J., Åkerström, G., Björklund, P., Westin, G. EZH2 is highly expressed in hyperparathyroid tumours and sustains parathyroid tumour cell proliferation. Manuscript.
1 Copyright 2012, The Endocrine Society Reprints were made with permission from the respective publishers.
Diagnosis and treatment .................................................................... 20 Molecular background ....................................................................... 20
Parathyroid tumours ............................................................................... 20 Diagnosis and treatment .................................................................... 21 Molecular background ....................................................................... 22
Aims of the study ........................................................................................ 24
Materials and methods ................................................................................ 25 Summary of materials and methods ....................................................... 25
Tissues ............................................................................................... 25 DNA/RNA isolation and cDNA synthesis......................................... 25 RT-PCR and semi-quantitative RT-PCR........................................... 25 qRT-PCR ........................................................................................... 26 Bisulfite pyrosequencing ................................................................... 26 DNA sequencing................................................................................ 26 Western blot....................................................................................... 26 Immunohistochemistry ...................................................................... 27 Immunoprecipitation.......................................................................... 27 Cell culturing ..................................................................................... 27 Transfection experiments................................................................... 28 Conditioned medium ......................................................................... 28 Cell viability and apoptosis ............................................................... 28
Summary of included papers ...................................................................... 31 Paper I. The internally truncated LRP5 receptor presents a therapeutic target in breast cancer............................................................................. 31 Paper II. Aberrant WNT/β-catenin signaling in parathyroid carcinoma 33 Paper III. Hypermethylated in cancer 1 (HIC1), a tumour suppressor gene epigenetically deregulated in hyperparathyroid tumours by histone H3 lysine modification............................................................... 33 Paper IV. EZH2 is highly expressed in hyperparathyroid tumours and sustains parathyroid tumour cell proliferation........................................ 35
Concluding remarks .................................................................................... 37
Acknowledgements..................................................................................... 42
References................................................................................................... 44
ADRB2 Beta-2-adrenergic receptor ATF7IP ATF7-interacting protein Atoh1 Atonal homolog 1 APC Adenomatous polyposis coli Axin Growth-suppressive protein Aza Demethylating agent 5-Aza-2’-deoxycytidine BRCA1/2 Breast cancer 1/2 Cadherin-1 Calcium-dependent adhesion protein type 1 CCND1 Cyclin D1 CDKN2A/B Cyclin-dependent kinase inhibitor 2A/B ChIP Chromatin immunoprecipitation CK1 Casein kinase 1 c-Myc Oncogene CpG Cytosine-phosphate-guanine CXCR7 C-X-C chemokine receptor type 7 DKK Dickkopf DNA Deoxyribonucleic acid DNMT1 DNA methyltransferase 1 DNMT3A DNA methyltransferase 3A DNMT3B DNA methyltransferase 3B DNMT3L DNA methyltransferase 3L Dvl/Dsh Dishevelled DZNep Global histone methylation inhibitor 3-
deazaneplanocin A EED Embryonic ectoderm development E2F1 E2F transcription factor 1 EFNA1 Ephrin-A1 ER Estrogen receptor EZH2 Enhancer of zeste homolog 2 FGF23 Fibroblast growth factor 23 FGFBP1 Fibroblast growth factor binding protein 1 FHH Familial hypocalciuric hypercalcemia G9a Histone methyltransferase of H3K9 GLP G9a-like-protein GSK3 Glycogen synthase kinase 3 H3 Histone 3
H4 Histone 4 H2A Histone 2A H2B Histone 2B H3K4me Histone 3 lysine 4 methylation H3K27me Histone 3 lysine 27 methylation H3K9me Histone 3 lysine 9 methylation H3K9Ac Histone 3 lysine 9 acetylation HEK-293T Human embryonic kidney 293 cell line HeLa Human epithelial cervical cancer cell line HER2 Human Epidermal growth factor Receptor 2 HIC1 Hypermethylated in cancer 1 HOTAIR HOX antisense intergenic RNA HPT Hyperparathyroidism HPT-JT Hyperparathyroidism-jaw tumour syndrome HRPT2 Parafibromin, CDC73 KCNQ1OT1 KCNQ1 overlapping transcript 1 Klotho Anti-aging gene LRP5 Low-density lipoprotein receptor-related protein 5 LRP5Δ Truncated low-density lipoprotein receptor-related
protein 5 MCF7 Human breast adenocarcinoma cell line MEN1 Multiple endocrine neoplasia type 1 MEN2A/B Multiple endocrine neoplasia type 2A/B MLL1-4 Mixed-lineage leukemia 1-4 mRNA Messenger ribonucleic acid NSHPT Neonatal severe primary hyperparathyroidism p18 Tumour suppressor p27 Tumour suppressor p53 Protein 53, tumour suppressor pHPT Primary hyperparathyroidism PR Progesterone receptor PRC2 Polycomb repressive complex 2 Ptch1 Protein patched homolog 1 PTEN Phosphatase and tensin homolog PTH Parathyroid hormone RASSF1 Ras association domain-containing protein 1 RB1 Retinoblastoma gene RBBP7/4 Retinoblastoma-binding protein 7/4 RET Proto-oncogene RIZ1 Retinoblastoma protein-interacting zinc-finger 1 RNA Ribonucleic acid SCID mice Severe combined immune deficiency mice Set1a/b SET domain-containing protein 1a/b SETDB1 Histone methyltransferase of H3K9
SFRP Secreted frizzled-related proteins sHPT Secondary hyperparathyroidism sHPT-1 Parathyroid tumour cell line SIRT1 Sirtuin 1 SOX SRY-box containing genes Suv39h Suppressor of variegation 3-9 homolog Suz12 Suppressor of zeste 12 homolog T47D Human ductal breast epithelial tumour cell line TCF Transcription factor WIF1 Wnt inhibitory factor 1 Wnt Wingless-type WNT1, WNT3, WNT3A Wnt ligands WT1 Wilms tumour protein 1 XIST X-inactive specific transcript
Wnt/β-catenin signaling Wnt/β-catenin signaling directs cell proliferation and cell fate during embryonic development and adult homeostasis. In the absence of Wnt ligand or presence of Wnt antagonists, the axin/adenomatous polyposis coli (APC)/casein kinase 1 (CK1)/glycogen synthase kinase 3 (GSK3) protein complex binds and phosphorylates β-catenin resulting in ubiquitination and proteosomal degradation of β-catenin. The pathway is activated when a Wnt ligand binds to the extracellular frizzled receptor and its co-receptor low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6). After Wnt ligand binding, the protein dishevelled (Dvl) is recruited resulting in phosphorylation of LRP5/6 and recruitment of axin to the receptor, thus disrupting the axin/APC/CK1/GSK3 protein complex. Nonphosphorylated active β-catenin is then accumulated and transported to the nucleus. β-catenin forms complexes with the TCF/LEF transcription factors and activates Wnt target genes (Fig. 1) [1]. Two examples of target genes are c-Myc and cyclin D1 that are associated with proliferation [2, 3].
Figure 1. A simplified view of the Wnt/β-catenin signaling pathway.
Wnt ligands and inhibitors In humans there are 19 secreted Wnt ligands acting both on the secreting cell and neighbouring cells and 10 frizzled receptors that can activate the canonical (Wnt/β-catenin), or non-canonical (Wnt/PCP or Wnt/Ca+) Wnt pathways [1, 4, 5]. Moreover, parafibromin encoded by the gene associated with the Hyperparathyroidism-jaw tumour syndrome (HRPT2, see below), is found to transactivate Wnt signaling by direct binding to β-catenin [6]. Several Wnt inhibitors have been identified such as the extracellular Dickkopfs (DKK), secreted Frizzled-related proteins (SFRP1-5) and Wnt inhibitory factor 1 (WIF1) that prevent ligand-receptor interactions [1]. Intracellular inhibitors are DACT that interacts with Dvl [7], Wilms tumour protein 1 (WT1) that promote β- catenin ubiquitination and degradation [8], the nuclear proteins SRY-box containing genes (SOX) and the transcriptional repressor Hypermethylated in cancer 1 (HIC1) that interacts with the β- catenin/TCF/LEF complex, inhibiting transcription of Wnt target genes [9, 10]. Furthermore, menin encoded by the MEN1 (Multiple endocrine neoplasia type 1) gene (see below), inhibits the transcriptional activity of β-catenin by transporting β-catenin out of the nucleus [11].
Wnt/β-catenin signaling and cancer Aberrant activation of Wnt/β-catenin signaling has been linked to several cancers. This can be caused by genetic and epigenetic alterations. Examples include stabilizing mutations in β-catenin [12, 13], deletions or other inactivating mutations in APC and WTX [12, 14-16], inactivating mutations in Axin1 and Axin2 [17, 18], and SFRP1 [19] as well as expression of a truncated alternatively spliced LRP5 receptor (LRP5Δ) with resistance to DKK1 inhibition [20]. Examples of epigenetic alterations are promoter DNA hypermethylation of APC, Axin2, SFRPs, WIF1, DKK, SOX and HIC1 [21-23]. DACT3 is reported to be epigenetically repressed due to bivalent chromatin marks (H3K4me3 and H3K27me3) [24] and EZH2-mediated repression (H3K27me3) of AXIN2 and SFRP5 have been revealed [25].
Epigenetics Epigenetics signifies heritable changes in gene function that do not alter the primary DNA sequence. The most studied epigenetic modifications include DNA methylation and different histone modifications.
DNA methylation occurs at cytosines followed by guanines (CpGs) in the DNA sequence. Unmethylated CpGs are enriched in 60 % of promoters often including the first exon and these CpG-rich regions are called CpG islands. Most repetitive DNA is methylated probably to prevent chromosomal instability. DNA methylation controls genomic imprinting and in certain cases play roles in tissue-specific gene expression [26]. Different methylation patterns in CpG island shores (located 2 kb distant from CpG islands) are shown to distinguish tissue types [27]. Methylated CpG islands are associated with repressed gene activity, also including microRNA genes [28, 29]. Another level of epigenetic modification occurs at the nucleosomes. DNA, 146 bp, is generally wrapped around the nucleosome consisting of two copies of the four core histone proteins H3, H4, H2A and H2B [30]. The N-terminal histone tails show many different posttranslational modifications affecting chromatin structure and function, such as acetylation, methylation, and phosphorylation. Acetylation of histone lysines and methylation of histone 3 lysine 4 (H3K4me) are associated with an open chromatin structure and active gene transcription, whereas methylation of histone 3 lysine 9 (H3K9me) and lysine 27 (H3K27me) are associated with a closed and transcriptionally repressed chromatin [26].
DNA methyltransferases Mammals have three different DNA methyltransferases DNMT1, DNMT3A and DNMT3B. DNMT1 is called a maintainance methyltransferase since it has a preference for hemimethylated DNA over unmethylated DNA. It is responsible for maintaining the methylation patterns during cell divisions and is highly expressed in proliferating somatic cells [31]. Homozygous germline mutation of DNMT1 in mice leads to a reduction in DNA methylation and embryonic lethality [32]. The DNMT3 family includes DNMT3A, DNMT3B and DNMT3L. DNMT3A and DNMT3B are active methyltransferases that show no preference between hemimethylated and unmethylated DNA and are therefore called de novo methyltransferases. They are responsible for the establishment of DNA methylation patterns in early mammalian development and in germ cells. DNMT3L lacks catalytic activity and function as a regulatory factor in germ cells. DNMT3A and DNMT3B are highly expressed in embryonic stem cells and tissues, and knockout mice embryos show developmental defects and die in the uterus or shortly after birth [31].
Histone methyltransferases The histone methyltransferases G9a and GLP are mainly responsible for mono- and dimethylation of the repressive mark H3K9 (associated with heterochromatin), whereas Suv39h is responsible for the trimethylation. SETDB1 can alone dimethylate H3K9, and when associated with its cofactor ATF7IP it trimethylates the same residue. It has been shown that all these enzymes can participate in a complex, cooperating to establish the different degrees of H3K9 methylation [33]. The polycomb repressive complex 2 (PRC2) is responsible for mono- di- and trimethylation of the repressive mark H3K27. It consists of the four components Suz12, EED, RBBP7/4 and the enzymatic subunit EZH2. The H3K27me mark is associated with heterochromatin during development and occupies many genes in embryonic stem cells. It has been revealed that PRC2 is involved in differentiation, maintaining cell identity, proliferation and stem-cell plasticity [34]. Lately it has been shown that long noncoding RNAs are important participants in PRC2 function and recruitment such as XIST (involved in X chromosome inactivation), KCNQ1OT1 (involved in imprinting of KCNQ1) and HOTAIR (promotes PRC2 binding) [35-41]. Mammalian cells have six characterized histone methyltransferases, SET1a, SET1b and MLL1-4, which are responsible for the methylation of the active mark H3K4. MLL1 and MLL2 have been shown to cooperate with histone acetyltransferases and to interact with the transcription machinery [42]. Moreover menin (MEN1) has been shown to interact with MLL, PRC2 and histone deacetylases [43-47].
Epigenetics and cancer Tumour DNA is generally found to be hypomethylated and CpG islands of putative tumour suppressor genes hypermethylated, in comparison to DNA from corresponding normal tissue. The loss of methylation in repetitive elements contributes to chromosomal instability and promotes chromosomal rearrangements. Hypermethylated genes with concomitantly reduced or absent expression are often involved in cell cycle regulation, apoptosis, angiogenesis or DNA repair, thereby promoting cellular transformation and tumour growth [26]. Methylation changes in CpG island shores, involved in tissue-specific differentiation, have been implicated in colon cancer [27]. Moreover, aberrant methyltransferase activity due to mutation of the DNA methyltransferase DNMT3A have been revealed in acute myeloid leukemia [48, 49]. DNA hypermethylation of genes is further associated with loss of the active histone marks acetylation and H3K4me, and gain of the repressive marks
H3K9me and H3K27me [26]. Genes that become hypermethylated in tumours are often found to have “bivalent” chromatin marks (H3K4me and H3K27me) in embryonic stem cells. It is suggested that if the additional repressive mark H3K9me is added to these genes, H3K27me and H3K9me predispose to promoter hypermethylation [50]. Aberrant methyltransferase activity of EZH2, the H3K27 methyltransferase, due to either activating or inactivating mutations has been observed in lymphoma and myeloid neoplasms, respectively [34, 51, 52]. Both chromosomal loss of histone acetyltransferase genes and gene copy number gain of histone methyltransferases have been linked to several cancers [26].
Low-density lipoprotein receptor-related protein 5 (LRP5) The LRP5 receptor was first cloned in 1998 and was identified as an apolipoprotein E binding receptor expressed in hepatocytes and adrenal cortex [53, 54]. It was later discovered to function as a co-receptor to the Frizzled receptor in Wnt signaling [55, 56]. LRP5 activity is inhibited through binding of the Wnt antagonist DKK1 [57-59]. Mutations in LRP5 have been related to several bone diseases such as Osteoporosis pseudoglioma (associated with reduction of skeletal mass and blindness) and high-bone-mass diseases [60-67]. Furthermore LRP5 mutations have been shown to reduce DKK1 inhibition [68].
Adenomatous polyposis coli (APC) The tumour suppressor APC is part of the degradation complex that regulates the amount of active β-catenin in Wnt signaling. APC is found to be involved in many processes such as cell migration, cell adhesion, transcriptional activation, neuronal differentiation and apoptosis [69]. Germline mutations in APC cause Familial adenomatous polyposis, resulting in multiple adenomas in the rectum and colon. Sporadic mutations leading to inactivation of APC have been strongly implicated in the cause of approximately 80 % of colorectal cancers [1, 70-72]. APC expression can also be reduced through DNA hypermethylation of the APC 1A promoter in several malignancies [73-77].
Hypermethylated in cancer 1 (HIC1) HIC1 was first identified as a potential tumour suppressor gene in 1995 and frequently shows loss of heterozygosity or epigenetic silencing by DNA hypermethylation in several types of solid tumours and leukemias [22, 23, 78]. Heterozygous HIC1+/- mice show development of several spontaneous gender- and age-specific tumours [79]. It was recently found that targeted methylation of HIC1 and RASSF1A alone could transform human mesenchymal stem cells into cancer initiating cells [80]. HIC1 is a transcriptional repressor and the number of known targets is increasing. It participates in complex regulatory loops modulating p53 and E2F1 DNA-damage responses through repression of the deacetylase SIRT1 [78, 81-84]. Furthermore HIC1 has been shown to cooperate with Ptch1 and the H3K27 methyltransferase EZH2 in repressing transcription of Atoh1 involved in malignant growth of medulloblastoma [85, 86]. Other targets of HIC1 are FGFBP1 involved in blood vessel growth [87], CXCR7 involved in cell adhesion and tumour development [88, 89], ADRB2 involved in migration and invasion [90] as well as EFNA1 involved in tumour growth, invasion, metastasis and angiogenesis [91, 92]. Moreover HIC1 attenuates Wnt signaling by binding to TCF4 and β- catenin with recruitment to discrete nuclear structures [10].
Enhancer of zeste homolog 2 (EZH2) EZH2 is the enzymatic subunit of the PRC2 complex responsible for mono- di- and trimethylation of H3K27 [93]. It has also been shown to interact with DNA methyltransferases [94, 95]. The PRC2 complex occupies many genes involved in key pathways regulating embryonic development, differentiation, stem-cell biology and cell fate decisions [96]. Furthermore EZH2 has been shown to enhance Wnt/β-catenin signaling and estrogen signaling by directly interacting with β-catenin and the ER receptor in breast cancer [97, 98]. Recently, EZH2 was shown to also influence Wnt/β-catenin signaling in hepatocellular carcinoma by epigenetically repressing several Wnt antagonists including Axin2 and SPRP5 [25]. EZH2 is overexpressed in several cancers such as prostate, breast, bladder, gastric, lung and hepatocellular carcinoma, and is associated with invasive and metastatic disease [51]. Amplification of EZH2 has been observed in several cancers [99, 100]. EZH2 is considered an oncogene and heterozygous missense mutations at amino acid Y641 was shown to cause higher catalytic efficiency in lymphoma [93, 101]. Though, EZH2 has also been suggested to have a tumour suppressor function with inactivating mutations in myeloid neoplasms [102, 103].
Breast cancer Breast cancer is the most common malignancy among women in the Western world. The incidence is low for younger women but increases with age. In 2009, 158/100000 Swedish women were diagnosed with breast cancer [104]. Breast cancer is divided into two main groups, in situ carcinomas (non-invasive) and invasive carcinomas. In situ carcinomas are associated with good prognosis and are further classified into lobular and ductal carcinomas. Invasive breast cancer is classified as ductal (40-75 %), lobular (5-15 %), tubular (2-7 %), medullary (1-7 %) and mucinous carcinomas (2 %) [105, 106]. An international staging system is used (Table 1, American Joint Committee on Cancer). Predictive factors in breast cancer include the presence of estrogen receptors (ER), progesterone receptors (PR) and increased expression of the growth factor receptor HER2 in the tumour [105, 106]. Later research has proposed classification of breast tumours based on their gene expression profiles. With this approach the tumours are divided into basal-like (Breast cancer 1 (BRCA1) negative and ER negative), HER2+ (HER2 amplification and ER negative), Luminal B (other amplification, BRCA1/2 negative and ER negative) and Luminal A (BRCA1/2 negative and ER positive), with the worst prognosis for basal-like and HER2+ tumours [107, 108].
Table 1
Stage Criteria
I Invasive breast cancer
Invasive breast cancer • no tumour but spread to the axillary lymphnode or • ≥2cm tumour and spread to the axillary lymphnode or • >2 and ≤5cm tumour and no spread to the axillary lymphnode
Invasive breast cancer • >2cm and ≤5cm tumour and spread to the axillary lymphnode or • >5cm tumour and no spread to the axillary lymphnode
Invasive breast cancer • no tumour but spread to the axillary lymphnode or spread to lymph nodes near the
breastbone or • ≤5cm tumour and spread to the axillary lymphnode or • >5cm tumour and spread to the axillary lymphnode
Invasive breast cancer Tumour of any size and spread to chest wall and/or skin of breast and
• Spread to axillary lymphnode or spread to lymph nodes near the breastbone • Inflammatory breast cancer
Invasive breast cancer No tumour or tumour of any size and spread to chest wall and/or skin of breast and
• Spread to lymph nodes above or below the collarbone and • Spread to axillary lymphnode or spread to lymph nodes near the breastbone
IV Invasive breast cancer
• Spread to other organs of the body (usually lungs, liver, bone or brain)
Diagnosis and treatment Breast cancer is diagnosed by clinical examination, mammography and cytology (and/or core biopsy). In situ carcinomas are treated by either breast-conserving surgery or mastectomy with reconstruction of the breast. If the patient had breast- conserving surgery for ductal in situ carcinoma there could be a risk of relapse and radiotherapy is used postoperatively. Invasive carcinomas are treated by mastectomy or breast-conserving surgery. A sentinel node biopsy is analyzed during the surgery and in presence of metastatic cancer cells, axillary lymph node ectomy is performed. Postoperative therapies depend on the stage of cancer, presence of metastases and predictive factors. Radiotherapy, chemotherapy, immunotherapy and hormonal therapy are used solely or in combinations. Hormonal therapy with tamoxifen can be used in patients with tumours positive for ER and/or PR and with herceptin in Her2 positive tumours [106]. For most breast cancer patients the prognosis is good with a survival of 10-15 years, but the risk of relapse is present for several years [105].
Molecular background Ten percent of breast cancer cases are hereditary with an early onset of disease. Hereditary disease is mostly caused by mutations in two genes, BRCA1 and BRCA2. Both genes are involved in DNA repair and mutations result in an inactive protein. Women with mutations in one of these genes have a risk of 60-80 % of developing breast cancer [109]. Other hereditary mutations are in the tumour suppressor genes p53 and PTEN linked to the Li-Fraumeni Syndrome and Cowden Disease, respectively [110]. Sporadic breast cancers demonstrate amplifications of c-Myc, CCND1 (cyclin D1) and HER2, as well as mutation in the p53 and CDH1 (E- cadherin) genes [111]. Aberrant activation of the Wnt/β-catenin pathway has been observed in 60 % of breast cancers [112-115]. Mutations in APC, AXIN or β-catenin are rare [116-120], but epigenetic inactivation of the Wnt antagonists WIF1, SFRP1 and DKK1 have been revealed [121-124].
Parathyroid tumours Patients with hyperparathyroidism (HPT) display hypersecretion of parathyroid hormone (PTH) from enlarged parathyroid glands. PTH plays a role in calcium homeostasis by tightly regulating serum calcium
concentrations and bone mineralization. PTH increases serum calcium directly by stimulating the kidney to reabsorb calcium and by bone resorption. PTH also has an indirect effect by enhancing expression of the enzyme 25-hydroxyvitamin D3 1α-hydroxylase, thus increasing the production of active vitamin D in the kidney. Active vitamin D [1,25(OH)2D3] stimulates the absorption of calcium from the intestine and bone mineralization [125-127]. A negative feedback loop exists where active vitamin D represses PTH mRNA transcription and cell proliferation in the parathyroid glands [128] (Fig. 2). Primary HPT (pHPT) is characterized by one or several enlarged parathyroid glands due to adenoma (80-85 %), hyperplasia or multiglandular disease (~15 %) or cancer (<1 %) causing hypersecretion of PTH and generally hypercalcemia. Secondary HPT (sHPT) commonly develops due to uremia leading to phosphate retention, hypocalcemia and reduced active vitamin D levels. Hypocalcemia consequently stimulates production of PTH causing hyperplastic parathyroid glands and eventually hypercalcemia [125-127]. Long-term treatment with lithium in manodepressive disease can also cause sHPT. The prevalence of pHPT is ~1 % and increases with age [129]. A higher prevalence of 2 % have been reported in postmenopausal women [130].
Figure 2. Regulation of calcium homeostasis by the parathyroid hormone (PTH).
Diagnosis and treatment pHPT is diagnosed by elevated PTH and ionized calcium levels in serum and is treated by removing the enlarged gland/glands surgically. In the
rare case of carcinoma, during surgery the enlarged gland may display a hard, white and fibrotic texture with common local invasion into the thyroid. Consequently part of the thyroid together with possibly affected lymph nodes is routinely removed together with the parathyroid tumour. sHPT most commonly results from parathyroid gland stimulation in patients with chronic kidney disease. The sHPT patients are initially hypocalcemic, but in later stages tend to display hypercalcemia due to autonomous parathyroid gland function with progressive disease. sHPT can be treated with vitamin D, phosphate-restricted diet, calcium supplementation (if hypocalcemic), calcimimetics (cinacalcet, MimparaR) or kidney transplantation. In case of persistently elevated PTH and ionized calcium levels in serum the enlarged glands often have to be surgically removed, also because of considerable risk for adverse effects on cardiovascular morbidity and mortality associated with the calcium-phosphate regulatory disturbance [131]. Surgery for HPT cures the patients in 95-99 %. In the case of carcinoma, the 10 year survival rate is 49 % and several operations are often needed to reverse sometimes severe hypercalcemia by removal of recurrent metastase during an often extended disease course [129, 132].
Molecular background The familiar forms with risk of developing pHPT are apart from MEN1 [133, 134], familial hypocalciuric hypercalcemia and neonatal severe primary hyperparathyroidism (FHH and NSHPT, due to heterozygote or homozygote mutations in the calcium sensing receptor gene) [126], Multiple endocrine neoplasia type 2A (MEN2A due to mutation in the RET proto-oncogene) [135], rarely or ever Multiple endocrine neoplasia type 2B (MEN2B) and Hyperparathyroidism-jaw tumour syndrome (HPT-JT, due to mutation in HRPT2 encoding for parafibromin, a regulator of transcription and histone modifications) [136] . The first identified oncogene in sporadic pHPT was the CCND1 (cyclin D1) gene, where a pericentromeric inversion resulted in a fusion protein between the PTH gene promoter and CCND1 [137]. In 30 % of sporadic pHPT loss of one MEN1 allele has been observed, and 15 % had a somatic mutation in the remaining allele [138]. Two target genes of menin, p18 and p27 [139], showed reduced expression in parathyroid tumours of primary and secondary HPT [140]. Somatic mutations in p27 have been found in pHPT (4.7 %) and not in sHPT [141, 142]. Aberrant activation of the Wnt/β-catenin pathway due to an aberrantly spliced internally truncated LRP5 receptor was observed in tumours of both pHPT (86 %) and sHPT (100 %) [20]. Stabilizing mutations of β-catenin in parathyroid adenomas was reported in one study (7.3 %) and not in three other studies [143-146]. Recently, a study of 57 adenomas from
Italy found one (1.8 %) β-catenin stabilizing mutation [147]. Furthermore, the anti-aging gene Klotho was downregulated in pHPT [148] and may represent a tumour suppressor gene. Klotho works together with the phosphaturic factor FGF23 in regulating 25- hydroxyvitamin D3 1α-hydroxylase activity and PTH secretion [149- 151]. Parathyroid carcinoma revealed reduced expression of the RB1 (retinoblastoma) gene and this was suggested as a marker for malignant parathyroid tumours [152, 153], however there are contradictory results whether RB1 gene alterations are specific for parathyroid carcinoma [154-157]. Moreover, increased expression of CCND1 (cyclin D1) has been shown [158]. Mutations in MEN1 was found in 13 % of sporadic parathyroid carcinomas and HRPT2 mutations have been observed in frequencies varying between 15-100 %, together with absent staining for parfibromin [159-163]. Additionally, absent staining for APC was recently suggested as an even more efficient marker for parathyroid carcinoma [164]. Hypermethylation of the tumour suppressor genes RIZ1, RASSF1, HPRT2, APC, CDKN2A/B, WT1 and SFRPs have been shown in parathyroid tumours [165-168].
Aims of the study
The overall aim was to study genetic and epigenetic defects in breast cancer and parathyroid tumours to identify disease mechanisms and find possible targets for therapy. The specific aims of the study were: Paper I. To investigate the expression of an aberrantly spliced internally truncated LRP5 receptor (LRP5Δ) and its role in aberrant activation of Wnt/β-catenin signaling in breast cancer. Paper II. To examine the expression of the tumour suppressor gene APC (Adenomatous polyposis coli) and its role in aberrant activation of Wnt/β-catenin signaling in malignant parathyroid tumours. Paper III. To study the expression and epigenetic regulation of the tumour suppressor gene HIC1 (Hypermethylated in cancer 1) in benign and malignant parathyroid tumours. Paper IV. To investigate the expression of the histone methyltransferase EZH2 (Enhancer of zeste homolog 2) and its role in aberrant activation of Wnt/β-catenin signaling in benign and malignant parathyroid tumours.
Materials and methods
Summary of materials and methods The following is a brief summary of the materials and methods used in this thesis, a detailed description can be found in the individual papers.
Tissues All tissues, except for two carcinomas given by prof. Henning Dralle, Germany, were collected from the clinical routine at Uppsala University Hospital (paper I, II, III, IV). Approval of ethical committee and informed consent were achieved. The breast cancer cDNA panel (paper I) was ordered from OriGene Technologies.
DNA/RNA isolation and cDNA synthesis DNA was extracted using QIAamp DNA Mini kit (Qiagen) (paper II) or AllPrep DNA/RNA Mini Kit (Qiagen) (paper III and IV). RNA was extracted using Trizol (Life Technologies) and Nucleospin RNA II kit (Machery-Nagel) (paper I, II). Alternatively RNA was extracted using AllPrep DNA/RNA Mini Kit and RNase-Free DNase Set (Qiagen) (paper III, IV). Successful DNase tretments were established by PCR analysis of all RNA preparations. DNA free RNA was reverse transcribed using random hexamer primers with the first strand cDNA synthesis kit (GE Healthcare) (paper I, II) or using the RevertAid First strand cDNA Synthesis kit (Fermentas) (paper III, IV).
RT-PCR and semi-quantitative RT-PCR RT-PCR was performed using primary and nested primers targeting both LRP5wt and LRP5Δ, alternatively nested primers only targeting LRP5Δ (paper I). Semi-quantitative RT-PCR was performed to examine the relative mRNA levels with primers for APC and GAPDH endogenous control. The number of PCR cycles to avoid saturation was determined (paper II). PCR was performed in Gene Amp 9700 thermal cycle (Applied Biosystems).
qRT-PCR Quantitative RT-PCR was performed using Taqman PCR core Reagent kit and assays for LRP5tot and 28S rRNA endogenous control (Applied biosystems). DKK1 primers were used with iQ SYBR Green supermix (Bio-Rad Laboratories). qRT-PCR reactions were performed on MyiQ Single-color Real-Time PCR Detection System (Bio-Rad Laboratories) (paper I). Taqman assays for APC, HIC1, DNMT1, Klotho, EZH2, Axin2, Cyclin D1, LRP5, EED, Suz12 and GAPDH endogenous control were used together with Taqman gene expression Master Mix and analysed on StepOnePlusTM Real-Time PCR systems (Applied Biosystems) (paper II, III, IV). Each cDNA sample was analyzed in triplicate.
Bisulfite pyrosequencing Bisulfide treatment was performed using the EpiTect Bisulfite Kit (Qiagen). Prior to pyrosequencing, a PCR was performed using the HotStarTaq® Plus Master Mix (Qiagen) and primers for APC, HIC1 P0, HIC1 P1, Klotho and EZH2. The PCRs were performed in Gene Amp 9700 thermal cycle (Applied Biosystems). Pyrosequencing was done with the PyroMarkTM Q24 system (Qiagen) (paper II, III, IV). The assay for HIC1 P0, HIC1 P1, Klotho and EZH2 were validated to distinguish equal amplification of unmethylated and methylated DNA. This was done by analysing a dilution series of bisulfide treated CpGenomeTM
Universal Methylated DNA (Millipore) and placenta DNA (paper III, IV).
DNA sequencing Sequencing was performed on the ABI 373A using the ABI Prism dye terminator cycle seguencing ready reaction kit (Applied Biosystems) and primers for LRP5Δ (paper I) or on the 3130xl Genetic Analyser using the Big Dye terminator v1.1 cycle sequencing kit (Applied Biosystems) and primers for APC (paper II).
Western blot Protein was extracted using CytobusterTM protein extraction reagent (Novagen) supplemented with Complete mini protease inhibitor cocktail tablets (Roche Diagnostics). Primary antibodies used were anti-active-β- catenin mouse monoclonal antibody (05-665 Upstate), anti-LRP5 goat polyclonal antibody (sc-21390 Santa Cruz), anti-β-catenin goat polyclonal antibody (sc-1496 Santa Cruz), anti-APC rabbit polyclonal
antibody (sc-896 Santa Cruz), anti-DNMT1 rabbit polyclonal antibody (ab16632 abcam), anti-flag rabbit polyclonal antibody (sc-807 Santa Cruz), anti-EZH2 mouse monoclonal antibody (17-662 Millipore), anti- PARP rabbit polyclonal antibody (AB16661 Millipore), anti-actin goat polyclonal antibody (sc-1616 Santa Cruz) and anti-β-tubulin rabbit polyclonal antibody (sc-9104 Santa Cruz). After incubation with the appropriate secondary antibody, bands were visualized using the enhanced chemiluminescence system (GE Healthcare) (paper I, II, III, IV).
Immunohistochemistry Cryosections (6 µM) were fixed with acetone and blocked with 0.3 % H2O2 in PBS to inhibit peroxidase activity. The sections were then blocked using an avidin-biotin blocking kit (Vector Laboratories Inc.) and normal horse serum. Immunostaining was done using an anti-APC mouse monoclonal antibody diluted 1/5 (sc-9998 Santa Cruz). After incubation with biotinylated secondary horse-anti-mouse antibody an avidin-biotin complex (Vector Laboratories Inc.) were applied. The sections were then incubated with 3-amino-9-ethylcarbazole followed by counterstaining with Mayer’s hematoxylin. As positive control adenomas were also immunostained (paper II).
Immunoprecipitation Cells were resuspended in 300 μl buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.5 % NP-40, 50 mM NaF and 1 mM EDTA supplemented with Complete mini protease inhibitor cocktail tablets (Roche Diagnostics)) and kept on ice for 20 min. Then 20 μl anti-LRP5 goat polyclonal antibody (sc-21390 Santa Cruz) was added and the lysate was incubated over night in 4°C with gentle agitation. The lysate was further incubated 6 h with 50 μl of Protein G Plus-agarose (sc-2002 Santa Cruz) in 4°C with gentle agitation. After centrifugation the agarose beads were boiled in 40 μl Laemmli sample buffer for 10 min and 10-20 μl was analyzed by Western Blot (paper I).
Cell culturing MCF7 cells, HEK 293T cells, HeLa cells, sHPT-1 cells, parathyroid pHPT primary cells, parathyroid sHPT primary cells, and parathyroid carcinoma primary cells were cultured in DMEM/10 % fetal bovine serum (Sigma). T47D cells were cultured in RPMI-1640/10 % fetal bovine serum (Sigma) (paper I, II, III, IV).
Transfection experiments For transfection experiments 2x105 cells were distributed onto 35-mm dishes and transfected in triplicates. The expression vectors LRP5wt, LRP5Δ, HIC1, shEZH2-1 and shEZH2-2, the luciferase reporters TOPFLASH/FOPFLASH (Upstate) and pTOPGlow/pFOPGlow, the CMV-Lac reference plasmid, the DKK1 promotor luciferase constructs WT short and TBE3,4mut short were transfected using transfection reagent jetPEI (Poly-Plus Transfection SAS) (paper I) or by using FuGENE® 6 transfection reagent (Roche Diagnostics) (paper III, IV). Luciferase reporter activity was determined luminometrically after 24 h and normalized to β-galactosidase activity (paper I). siRNA against LRP5wt, LRP5Δ, LRP5tot, β-catenin and control non-silencing siRNA (Qiagen) were transfected using jetSI-ENDO and harvested after 96h (paper I). DNMT1 siRNA, LRP5tot siRNA, β-catenin siRNA and control non-silencing siRNA (Qiagen) were transfected for 96 h or 120 h with InterferinTM siRNA transfection reagent (Polyplus Transfection) (paper III, IV). For EZH2 siRNA, cells were transfected with InterferinTM siRNA transfection reagent (Polyplus Transfection) for 48 h and then transfected again and further incubated for 72 h (paper III, IV). For some experiments the cells were first transfected with the luciferase reporter and after 24 h transfected with siRNA or incubated with anti- LRP5 antibody, luciferase activity was then determined after 96 h or 24 h, respectively (paper I).
Conditioned medium Conditioned medium of WNT1, WNT3, WNT3A and DKK1 was produced in HEK293T cells transfected for 24 h with pCIN4/Wnt1, pLNC Wnt-3HA, PON-Wnt-3a or pCS2/Dkk1 using the transfection reagent Fugene 6 (Roche Diagnostics) (paper I). The conditioned medium might contain uncharacterized signaling molecules induced by the WNTs or DKK1. The medium was added to the cells 6 h after transfection of luciferase reporter.
Cell viability and apoptosis Cell death and cell viability was measured using the Nucleocounter (ChemoMetec) (paper I) or cell viability was measured with the cell proliferation reagent WST-1 (Roche Molecular Biochemicals) (paper I, II, III). Furthermore, DNA synthesis was measured by incubating the cells with 2 μl 3H-thymidine/well. After 12 h incubation unspecifically incorporated 3H-thymidine was precipitated and discarded before lysing the cells. Radioactivity was monitored in a β-counter (paper III, IV).
Apoptosis was detected using the Cell Death Detection ELISA kit (Roche Molecular Biochemicals, Mannheim, Germany) (paper I, II, III). As positive control for apoptosis, cells were incubated with 0.1 μg/ml Camptothesin. Apoptosis was also detected by western blot using an anti-PARP antibody (paper III).
Mouse xenograft model Two to three week old female Fox Chase severe combined immunodefiency (SCID) mice were used. The mice were anesthetized with isoflurane (Forene;Abbot) during the procedures. Both flanks were injected subcutaneously with 200 μl MCF7 cells together with BD Matrigel Matrix (BD Biosciences Clontech) (1:1) after transfection of 106 cells for 24 h. The animals were monitored every day and sacrificed after 5 weeks. The animal experiment was approved by the Uppsala University board of animal experimentation and was performed according to the United Kingdom Coordinating Committee on Cancer Research guidelines for the welfare of animals in experimental neoplasia (paper I).
Treatment of cell cultures with antibody or drugs MCF7 cells, HeLa cells and T47 D cells were incubated for 96 h with 10 μg/ml and 20 μg/ml anti-LRP5 goat polyclonal antibody (Santa Cruz) (paper I). Primary parathyroid cells were incubated in triplicates with 5 μM and 10 μM 5-Aza-2'-Deoxycytidine (Aza) (Sigma-Aldrich) for 24 h and 48 h (paper II) or 2.5 μM and 5 μM 3-deazaneplanocin A (DZNep) for 48 h (paper III). sHPT-1 cells were incubated in triplicates with 5 μM and 10 μM Aza or 2,5 μM and 5 μM DZNep for 72 h (paper III). Media was changed every 24 h.
Primary cell preparation Primary cells were prepared directly after operation. The tissue was minced and put on a shaker at 37 °C for 1 h in 1 mg/ml collagenase (Sigma), 0.05 mg/ml DNase I, 1.5 % bovine serum albumin and 0.95 mM CaCl in Nutrient Mixture F-10 Ham (Sigma) pH 7.4. Cells were then centrifuged and resuspended in 0.14 M NaCl, 6.7 mM KCl, 8.5 mM HEPES and 1 mM EGTA pH 7.4 and purified by centrifugation through 25 % and 75 % standard isotonic percoll (Amersham). Live cells were counted with a Bürker chamber using Trypan Blue (paper II, III).
Chromatin immunoprecipitation sHPT-1 cells (2x107) and primary parathyroid tumour cells (8x106) were distributed onto 97-mm dishes. ChIP was performed using the EZ-ChIP Kit (Millipore) and crosslinking was done with 1 % formaldehyde (Merck, Germany). DNA was sheared with four 10 second pulses using a sonicator 2 mm tip set to 30 % amplitude. Antibodies used for immunoprecipitation were H3 monomethyl K9 polyclonal antibodies (ab8896 and ab9045 abcam), H3 dimethyl K9 polyclonal antibody (ab1220 abcam), H3 trimethyl K9 polyclonal antibody (ab8898 abcam), H3 methyl K9 polyclonal antibody (4069 Cell Signaling Technology), H3 dimethyl K27 polyclonal antibody (ab24684 abcam), H3 trimethyl K27 polyclonal antibody (07-449 Millipore), H4 trimethyl K20 polyclonal antibody (ab9053 abcam) and EZH2 monoclonal antibody (17-662 Millipore). Primers used were HIC1 P1 and GAPDH (control) promoter primers (paper III).
Colony formation assay sHPT-1 cells (2x105) were distributed onto 35-mm dishes and transfected in triplicates with 2 μg of pcDNA3-Flag-HIC1 or pcDNA3 empty vector using FuGENE® 6 transfection reagent (Roche Diagnostics). Alternatively, cells were transfected with 1 μg of two different shRNAs to EZH2 (pCEP-shEZH2-1, pCEP-shEZH2-2) or two control shRNAs (pCEP-shluc, pCEP-shcontrol-1). After 24 h cells were seeded at 2.0x103 in 6-well plates and the following day 0.1 mg/ml G418 or 0.1 mg/ml Hygromycin B were added to culture medium. Media was changed every 4th day. After 10 days in selection for HIC1 vector and 8 days for EZH2 shRNA vectors, cells were fixed with 10 % acetic acid/10 % methanol and stained with 0.4 % crystal violet and visible colonies were photographed and counted (paper III, IV).
Statistical analysis Statistical analyses were performed with Statistica 6 (paper I) or PASW Statistic 20 (paper II, III, IV). Data was presented as arithmetical mean ± SEM and unpaired t test was used. A p value of < 0.05 was considered significant.
Summary of included papers
Paper I. The internally truncated LRP5 receptor presents a therapeutic target in breast cancer Aberrant activation of Wnt/β-catenin signaling, leading to accumulation of β-catenin, has been observed in 60 % of breast cancers [112-115]. Mutations in components of the Wnt/β-catenin pathway are rare in breast cancer, but epigenetic inactivation of Wnt antagonists have been revealed [116-124]. This paper investigates the expression and function of the aberrantly spliced internally truncated LRP5 receptor (LRP5Δ) in breast cancer. The LRP5Δ receptor has a 142 amino acid in frame deletion (amino acids 666-809), making it insensitive to the Wnt antagonist DKK1 and is strongly implicated in deregulated Wnt/β- catenin signaling in parathyroid tumours [20]. Nineteen breast cancer specimens and four normal breast tissues were studied regarding expression of LRP5Δ as well as the level of nonphosphorylated active β-catenin. In addition, a commercially available cDNA panel covering eight breast cancer stages (0, I, IIA, IIB, IIIA, IIIB, IIIC, IV) was screened for expression of LRP5Δ. Sixteen of the nineteen analyzed breast carcinomas expressed LRP5Δ compared to none of the normal tissues. The tumours expressing LRP5Δ also demonstrated accumulation of nonphosphorylated active β-catenin. In the cDNA panel covering different breast cancer stages, 79 out of 95 carcinomas from all disease stages expressed LRP5Δ. The role for LRP5Δ in the accumulation of nonphosphorylated active β- catenin was studied by transfecting MCF7 cells with siRNAs against LRP5Δ, LRP5wt and LRP5tot (targets both Wt and Δ). Reduced expression of active β-catenin was observed with siRNA against LRP5Δ and LRP5tot, but not with siLRP5wt. This indicated that continued expression of LRP5Δ was required for maintained level of nonphosphorylated active β-catenin. To investigate if LRP5Δ also was required for the endogenous β-catenin transcriptional activity, MCF7 cells were transfected with TCF/β-catenin luciferase reporters or the natural β-catenin responsive DKK1 promotor luciferase construct together with siRNAs against LRP5Δ, LRP5wt and LRP5tot. The endogenous β-catenin activity was decreased with siRNA against LRP5Δ and LRP5tot for all constructs investigated, this was not
the case with siLRP5wt. Also the mRNA level of DKK1 in MCF7 was reduced after transfection with LRP5Δ siRNAs, further supporting the dependence of continuous expression of LRP5Δ on endogenous β- catenin transcriptional activity. The Wnt/β-catenin signaling ligands WNT1, WNT3 and WNT3A are expressed in MCF7 cells [123]. To reveal their effects on the endogenous β-catenin transcriptional activity in the absence and presence of LRP5Δ, MCF7 cells were cotransfected with a TCF/β-catenin luciferase reporter and expression vectors for either LRP5wt or LRP5Δ compared to empty control vector. The cotransfected cells were then stimulated with conditioned medium containing WNT1, WNT3 or WNT3A. Only WNT3 activated the endogenous β-catenin activity in MCF7 cells. All three WNTs were able to activate β-catenin transcriptional activity in the presence of cotransfected LRP5wt and LRP5Δ, although the highest activation was seen with WNT3 and LRP5Δ. The effect of the Wnt antagonist DKK1 on endogenous β-catenin transcriptional activity was then investigated in MCF7 cells and HEK293T control cells. A TCF/β-catenin luciferase reporter was transfected and the cells were stimulated with WNT3, WNT3+DKK1 and DKK1 conditioned medium. DKK1 was unable to inhibit WNT3- induced endogenous β-catenin activity in MCF7 cells, in contrast to the result for the HEK293T control cells. Thus, indicating that DKK1 inhibition failure contributed to the aberrant activation of Wnt/β-catenin signaling in LRP5Δ positive tumours. The dependence of LRP5Δ on cell viability and apoptosis was analysed by transfecting MCF7 cells with siRNAs against LRP5Δ, LRP5wt and LRP5tot. siRNA against LRP5Δ and LRP5tot both reduced cell viability and induced apoptosis whereas siLRP5wt had no effect. Tumour growth in vivo was analyzed in a xenograft SCID mouse model by transfecting MCF7 cells with siRNAs against LRP5Δ and LRP5tot. Both siRNAs reduced tumour growth when compared to control non- silencing siRNA. MCF7 cells, HeLa cells (only expressing LRP5wt) and T-47D breast cancer cells (expressing both LRP5Δ and LRP5wt) were incubated with an anti-LRP5 goat polyclonal antibody, and the level of nonphosphorylated active β-catenin was analyzed as well as endogenous β-catenin activity, cell viability and apoptosis. The LRP5 antibody reduced the level of nonphosphorylated active β-catenin and endogenous β-catenin transcriptional activity in MCF7 cells and T-47D cells. The antibody also reduced cell viability and induced apoptosis of MCF7 cells and T-47D cells, but not of HeLa cells. The results indicate that accumulation of nonphosphorylated active β-catenin and breast tumour cell growth is dependent on LRP5Δ. The possibility of antibody therapy directed against LRP5Δ should be evaluated in the future.
Paper II. Aberrant WNT/β-catenin signaling in parathyroid carcinoma In this study we wanted to investigate the tumour suppressor gene adenomatous polyposis coli (APC) and its possible role in aberrant Wnt/β-catenin signaling in parathyroid carcinoma. Five parathyroid carcinoma specimens were compared to eight normal parathyroid tissues regarding APC mRNA and protein expression, promoter methylation, as well as protein levels of total and nonphosphorylated active β-catenin. Primary parathyroid carcinoma cells were treated with the demethylating agent 5-aza-2’-deoxycytidine (Aza) and cell viability, apoptosis, APC expression and nonphosphorylated active β-catenin expression were examined. The expression of APC was reduced or undetectable in all investigated parathyroid carcinomas. This was accompanied by a higher expression of nonphosphorylated active β-catenin, demonstrating aberrant Wnt/β- catenin signaling in parathyroid carcinoma. No mutations were detected in the APC gene, but the APC 1A promoter was found to be hypermethylated in the carcinomas compared to the normal tissues. Treatment of primary carcinoma cells, prepared directly after operation, with Aza increased APC expression and reduced the expression of nonphosphorylated active β-catenin. Furthermore, the Aza treated parathyroid carcinoma cells showed reduced cell viability and apoptosis. Based on the results presented we suggest that adjuvant epigenetic therapy could be considered in patients with metastatic or recurrent parathyroid carcinoma.
Paper III. Hypermethylated in cancer 1 (HIC1), a tumour suppressor gene epigenetically deregulated in hyperparathyroid tumours by histone H3 lysine modification The HIC1 tumour suppressor gene first caught our attention because HIC1+/- mice developed pancreatic islet cell carcinoma in addition to other tumours. Loss of function of the other allele in the tumours occurred only by DNA methylation [79]. Furthermore, HIC1 negatively regulates SIRT1, which in turn regulates Wnt antagonists SFRPs and Dishevelled proteins to promote Wnt signaling [81, 169, 170]. HIC1 can also antagonize Wnt signaling by binding to TCF4 and β-catenin with recruitment to discrete nuclear structures [10]. Thus, inactivation or reduced expression of HIC1 may contribute to the overall level of β- catenin in a cell.
In order to investigate whether HIC1 is involved in parathyroid tumourigenesis we first determined the mRNA expression levels, by quantitative RT-PCR, in pHPT tumours (both single adenomas and multiple tumours from the same patient) and sHPT tumours as well as in parathyroid carcinomas and normal parathyroid tissues. Generally, HIC1 mRNA (P1 transcripts with exon 1a-2, Fig. 3) was significantly underexpressed in the tumour groups. Also all patients with multiple parathyroid tumours showed underexpression of HIC1, suggesting that disturbed expression of HIC1 represent an early event during parathyroid tumour development. Furthermore, overexpressing HIC1 in the parathyroid cell line sHPT-1 [171] resulted in reduced viability without induction of apoptosis and also in suppressed colony formation, supporting a tumour suppressor role for HIC1 in the parathyroid gland.
Figure 3. The structure of the HIC1 locus, the transcripts and location of the methylation measurements (figure adopted from Svedlund et al., 2012 Journal of Clinical Endocrinology & Metabolism, jc.2011-3136; doi:10.1210/jc.2011- 3136.
Next, we determined the CpG methylation level of the HIC1 P0 promoter (exon 1b) that has been found to be hypermethylated in many tumour types [22, 23]. Methylation here has been suggested to repress expression also from the upstream P1 (exon 1a) promoter [79]. Quantitative bisulfite pyrosequencing analysis revealed relatively increased methylation level in the parathyroid carcinomas, only in 4 out of 39 pHPT tumours, and in none of the sHPT tumours. Thus, generally no relation of HIC1 hypermethylation to gene expression was observed for the parathyroid adenomas and secondary hyperplastic parathyroid glands. In contrast, the sHPT-1 parathyroid cell line showed 94 % methylation level at the HIC1 P0 promoter (exon 1b) and treatment with the demethylating agent 5-aza-2’-deoxycytidine (Aza) induced expression strongly (15-fold) from P1 (not methylated, data not shown). As expected, Aza inhibited cell growth and induced apoptosis. To determine whether demethylation at the P0 promoter was responsible also for the induced expression from the P1 promoter, sHPT-1 cells were transfected with siRNA directed to the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. The methylation level at the P0 promoter was clearly reduced only in DNMT1-silenced cells, but without induction of transcription from the P1 promoter. The hypermethylated
Klotho gene, which was used as a positive control, showed induced transcription in DNMT1-silenced cells. These results suggested that the apparent induction of the P1 promoter with Aza was not due to demethylation at P0, but rather to an indirect effect. In order to investigate whether repressive histone modifications might be responsible for the observed deregulated HIC1 expression from the P1 promoter, sHPT-1 cells and primary parathyroid tumour cell cultures were treated with the global histone methylation inhibitor 3- deazaneplanocin A (DZNep) [172]. This inhibitor clearly induced HIC1 P1 expression in the cell cultures and in addition, cell growth inhibition and apoptosis were observed. Experiments using chromatin immunoprecipitation (ChIP) of sHPT-1 cells, a primary sHPT cell culture and a primary parathyroid carcinoma cell culture suggested involvement of the repressive histone modification H3K27me, and EZH2-silenced sHPT-1 cells showed an increase in HIC1 expression. Thus, the observed underexpression of HIC1 could be explained by epigenetic deregulation involving aberrant histone methylation, possibly by EZH2, rather than CpG methylation.
Paper IV. EZH2 is highly expressed in hyperparathyroid tumours and sustains parathyroid tumour cell proliferation. Paper IV investigates EZH2 (Enhancer of zeste homolog 2) in parathyroid tumours and its role in aberrant activation of Wnt/β-catenin signaling. EZH2 is the methyltransferase of the repressive histone mark H3K27me and has been found to interact with β-catenin and to epigenetically repress Wnt antagonists, including Axin2 [25, 96, 98]. The expression of EZH2 was generally increased in pHPT and sHPT tumours and most apparent in parathyroid carcinomas. By reducing the expression of EZH2 in the parathyroid tumour cell line sHPT-1, the viability and colony formation capacity of the cells were markedly reduced, suggesting that EZH2 may function as an oncogene in parathyroid tumours. Methylation analysis upstream of the EZH2 promoter showed no difference in the methylation levels between parathyroid tumours and normal parathyroid tissue, indicating that the increased expression of EZH2 was not due to loss of promoter hypermethylation in the tumours. Furthermore, depletion of EZH2 and LRP5tot in sHPT-1 cells reduced the amount of nonphosphorylated active β-catenin, indicating that they both contributed to maintain a level of β-catenin necessary for tumour cell growth. Furthermore, decreased expression of EZH2 reduced the expression of the β-catenin target gene
cyclin D1 as well as increased the expression of Axin2. Thus, the overexpression of EZH2 could contribute to the increased levels of nonphosphorylated active β-catenin in parathyroid tumours and EZH2 may represent a novel therapeutic target [173].
Concluding remarks
We have found deregulation of Wnt/β-catenin signaling in breast cancer through the expression of an aberrantly spliced internally truncated LRP5 receptor (LRP5Δ). LRP5Δ was frequently expressed in breast cancer of different cancer stage and maintained expression of LRP5Δ was required for the aberrant activation of Wnt/β-catenin signaling and breast tumour cell growth. Sixty percent of breast cancers have shown aberrant activation of the Wnt/β-catenin signaling pathway [112-115], possibly explained by epigenetic inactivation of Wnt antagonists and rarely by mutations in different components of the pathway [116-124]. Thus, our finding demonstrates a new possible aberration causing activation of Wnt/β-catenin signaling in breast cancer. We revealed aberrant activation of Wnt/β-catenin signaling in the five investigated parathyroid carcinomas, probably due to epigenetic inactivation of the tumour suppressor gene APC by CpG island hypermethylation. Mutations in the genes HPRT2 (encoding parafibromin) and MEN1 (encoding menin) have been demonstrated in various frequencies in parathyroid carcinomas [159-163]. Both parafibromin and menin have been revealed to influence Wnt/β-catenin signaling [6, 11], further supporting the importance of deregulated Wnt/β-catenin signaling in parathyroid carcinogenesis. Parathyroid carcinomas have demonstrated overexpression of the β-catenin target gene cyclin D1 [158] and our finding that Wnt/β-catenin signaling may be aberrantly activated could explain this findings. Moreover we revealed that expression of the tumour suppressor gene HIC1 was reduced in parathyroid tumours and retained HIC1 expression reduced viability and suppressed colony formation in the parathyroid tumour cell line sHPT-1, supporting a tumour suppressor role in the parathyroid gland. The repressed expression of HIC1 could be explained by epigenetic deregulation involving aberrant histone methylation rather than DNA methylation as observed in other tumours [22, 23], and may involve the histone methyltransferase EZH2. HIC1 attenuates Wnt/β- catenin signaling at several levels [10, 81, 169, 170] and the reduced expression of HIC1 may then also contribute to deregulated Wnt/β- catenin signaling in parathyroid tumours [174]. Lastly we demonstrated overexpression of the histone methyltransferase EZH2 in parathyroid tumours. Depletion of EZH2 in sHPT-1 reduced
viability and colony formation capacity of the cells, suggesting that EZH2 may function as an oncogene in parathyroid tumours. Furthermore, reduced expression of EZH2 decreased the amount of nonphosphorylated active β-catenin, probably due to the increased expression of the Wnt antagonist Axin 2. The expression of cyclin D1, known to be overexpressed in a fraction of parathyroid tumours [143, 158, 175, 176] and regulated by β-catenin in parathyroid cells [171], was also reduced. This indicates that the overexpression of EZH2 could contribute to the aberrant activation of Wnt/β-catenin signaling and that EZH2 may represent a novel therapeutic target in parathyroid tumours. Methylation analysis upstream of the EZH2 promoter revealed no difference in the methylation levels between parathyroid tumours and normal parathyroid tissue that could explain the increased expression. However, c-Myc has been demonstrated to be overexpressed in parathyroid tumours of primary and secondary origin [174] and EZH2 expression was found to be stimulated by c-Myc [177], suggesting one possibility for the observed increased expression. Moreover, parafibromin have been found to down-regulate c-Myc [178], though it is not known if c-Myc is overexpressed in parathyroid carcinomas. Other possibilities for the increased expression of EZH2 could be gene amplification or aberrant microRNA regulation of EZH2, as has been shown in other tumour types [99, 100, 179-181]. Another speculation is that the protein stability of EZH2 is increased in the tumours, since some had EZH2 mRNA levels within the normal range but showed increased levels of EZH2 protein. The fact that menin (MEN1) interacts with PRC2 [43] and that EZH2 interacts with the ER receptor [98] (pHPT is more frequent among postmenopausal women) makes the role of EZH2 in parathyroid tumourigenesis even more interesting. The findings that HIC1 was underexpressed and EZH2 overexpressed in parathyroid tumours regardless of the hyperparathyroid disease state may represent a possibility for a common pathway in parathyroid tumour development.
Summary of the thesis in Swedish
Wnt/β-catenin signalering och felaktig epigenetisk reglering i bröstcancer och bisköldkörteltumörer Bröstcancer är den vanligaste maligniteten hos kvinnor i västvärlden. Risken för att få bröstcancer är låg hos den yngre befolkningen men ökar med åldern. 10 % av bröstcancerfallen är ärftliga och beror på mutationer i generna BRCA1 eller BRCA2. Av de kvinnor som har en sådan mutation utvecklar 60-80 % bröstcancer, oftast vid en yngre ålder. Bröstcancer behandlas genom att ta bort tumören kirurgiskt samt att ge olika kombinationer av strålning, cytostatika, hormonterapi och immunoterapi.
Bisköldkörteltumörer orsakar sjukdomen hyperparathyroidism vilket leder till förhöjda nivåer av PTH (bisköldkörtelhormon). PTH reglerar kalciumbalansen i kroppen och vid hyperparathyroidism orsakar den ökade nivån av PTH att kalcium frisätts från bland annat ben, detta leder till urkalkat skelett och höga halter av kalcium i blodet. Risken att utveckla tumörer är relativt hög hos den äldre befolkningen (~1 %) och än högre för kvinnor efter klimakteriet (2 %). Tumörerna är oftast benigna men i mindre än 1 % av fallen är de maligna. Hyperparathyroidism behandlas genom att ta bort tumören kirurgiskt, i de fall då tumörerna uppstått på grund av njurfel kan njurtransplantation och behandling med aktivt vitamin D tillsammans med calcimimetica vara tillräckligt.
Wnt/β-catenin signalering styr cellers öde och tillväxt under den embryonala utvecklingen samt stabiliteten hos mogna celler. Vid cancer växer cellerna okontrollerat och det kan delvis bero på felaktig aktivering av Wnt/β-catenin signaleringsvägen. När signaleringsvägen är aktiverad transporteras en aktiv form av proteinet β-catenin till cellkärnan där det sätter igång uttrycket av flera olika målgener som reglerar bland annat cellens tillväxt. Wnt/β-catenin signaleringen kan aktiveras av att komponenter i signaleringsvägen är muterade eller epigenetiskt nedreglerade. Epigenetisk reglering definieras som ärftliga förändringar i genuttryck som inte beror på förändringar i DNA sekvensen. Exempel på epigentisk reglering är metylering av DNA samt metylering av svansen på histoner (histoner är de proteiner som packar ihop DNA-strängen i cellen till kromosomer). Metylering av DNA kopplas ihop med minskat
genuttryck och metylering av histonsvansar kan kopplas både med ökat och minskat genuttryck.
Syftet med det här arbetet har varit att studera genetiska och epigenetiska defekter i bröstcancer och bisköldkörteltumörer, både för att förstå uppkomsten av tumörerna samt att hitta möjlig terapi.
Det första arbetet undersöker förekomsten av LRP5Δ i bröstcancer. LRP5 är en cellmembranreceptor som är involverad i Wnt/β-catenin signaleringsvägen. LRP5Δ är en alternativ form av LRP5 där en del av receptorn saknas. Den del som saknas är ett bindningställe för DKK1, ett protein som normalt binder till LRP5 och reglerar Wnt/β-catenin signaleringen negativt. Vi har tidigare visat att LRP5Δ uttrycks och är betydelsefull för aktivering av Wnt/β-catenin signalering samt tumörtillväxt i bisköldkörteltumörer. LRP5Δ visade sig vara uttryckt i bröstcancer och tumörerna hade även förhöjda nivåer av aktivt β-catenin. Genom att minska uttrycket av LRP5Δ i bröstcancerceller kunde vi reducera tumörtillväxten i möss samt minska nivåerna av aktivt β-catenin, reducera celltillväxt och inducera celldöd. Bröstcancerceller behöver följaktligen uttrycka LRP5Δ för att aktivera Wnt/β-catenin signaleringsvägen och för att kunna växa. Genom att behandla bröstcancerceller med en antikropp riktat mot LRP5 kunde vi se reducerad celltillväxt och inducerad celldöd, därför skulle terapi med en LRP5 antikropp kunna vara användbart i behandlingen av bröstcancer.
Arbete två studerar uttrycket av proteinet APC i maligna bisköldkörteltumörer. APC är en komponent i Wnt/β-catenin signaleringen där det reglerar nivåerna av aktivt β-catenin. Mutationer i APC genen är starkt kopplat till utvecklingen av koloncancer i ungefär 80 % av fallen. APC uttrycket visade sig vara väldigt lågt i maligna bisköldkörteltumörer, detta berodde troligen på att APC promotorn var DNA-metylerad. Tumörerna hade även förhöjda nivåer av aktivt β- catenin vilket tyder på att Wnt/β-catenin signaleringsvägen är felaktig aktiverad. Vi preparerade celler från en malign bisköldkörteltumör direkt efter operation och behandlade de primära cellerna med en drog som tar bort DNA-metylering (Aza). Behandlingen med Aza ökade uttrycket av APC och minskade uttrycket av aktivt β-catenin, behandlingen reducerade även celltillväxten samt inducerade celldöd. Vi föreslår därför att adjuvant epigenetisk terapi skulle kunna användas hos patienter som inte går att operera eller patienter med återkommande maligna bisköldkörteltumörer.
Det tredje arbetet utreder uttrycket av proteinet HIC1 i benigna och maligna bisköldkörteltumörer. Genen som kodar för HIC1 är ofta DNA- metylerad i flera cancrar. HIC1 förhindrar uttrycket av olika målgener och reglerar Wnt/β-catenin signaleringsvägen negativt på flera sätt. Vi
har tidigare visat att Wnt/β-catenin signaleringsvägen är aktiv i bisköldkörteltumörer, därför skulle ett minskat uttryck av HIC1 kunna bidra till den totala ökade nivån av aktivt β-catenin i en cell. Uttrycket av HIC1 var lägre i benigna och maligna bisköldkörteltumörer jämfört med normala bisköldkörtlar. HIC1 promotorn var endast metylerad i ett fåtal benigna tumörer samt hos alla maligna tumörer, därför kan det minskade uttrycket av HIC1 inte förklaras av DNA- metylering hos de benigna tumörerna. Vi har tidigare utvecklat en cellinje kallad sHPT-1 som kommer från en benign sekundär bisköldkörteltumör, denna cellinje hade ett reducerat HIC1 uttryck samt uppvisade DNA-metylering i HIC1 promotorn. Genom att manipulera sHPT-1 kunde vi se att ökat uttryck av HIC1 minskade celltillväxten och kolonibildningen, vi kunde även se att om vi reducerade DNA- metyleringen av HIC1 promotorn så påverkade det inte HIC1 uttrycket. Vi behandlade då sHPT-1 samt primära cellkulturer preparerade från bisköldkörteltumörer direkt efter operation med en drog som tar bort viss histonmetylering (DZNep). Behandlingen med DZNep ökade HIC1 uttrycket samt minskade celltillväxten. Vi kunde även hitta histonmetyleringen H3K27me i HIC1 promotorn och genom att minska uttrycket av EZH2 (enzymet som orsakar H3K27me) i sHPT-1 så ökade HIC1 uttrycket. Studien visar att ökat uttryck av HIC1 minskar tumörcellernas tillväxt samt att HIC1 uttrycket verkar vara reducerat på grund av histonmetylering och inte DNA-metylering i bisköldkörteltumörer.
Arbete fyra studerar uttrycket av EZH2 i benigna och maligna bisköldkörteltumörer. EZH2 är det enzym som orsakar metylering av H3K27 och det har visat sig vara överuttryckt i flera cancrar. Nyligen upptäckte man att EZH2 epigenetiskt nedreglerar Axin2, en komponent i Wnt/β-catenin signaleringen som reglerar nivåerna av aktivt β-catenin. Uttrycket av EZH2 var högre i benigna och framförallt i maligna bisköldkörteltumörer jämfört med normala bisköldkörtlar. Vi hittade inga skillnader i DNA-metylering som skulle kunna förklara det ökade uttrycket. När vi minskade uttrycket av EZH2 i sHPT-1 kunde vi se minskad celltillväxt och kolonibildning samt minskat uttryck av aktivt β- catenin och cyclin D1 (en målgen för β-catenin) samt ökat uttryck av Axin2. Därmed kan det ökade uttrycket av EZH2 bidra till den ökade mängden av aktivt β-catenin i bisköldkörteltumörer. Vidare skulle EZH2 kunna vara ett möjligt terapimål i behandlingen av dessa tumörer.
Sammanfattningsvis har vi påvisat olika genetiska och epigenetiska defekter i bröstcancer och bisköldkörteltumörer som bidrar till den okontrollerade celltillväxten. Dessa defekter är viktiga för att förstå varför tumörerna uppstår, de är även potentiella terapimål för att underlätta behandling.
This thesis work was carried out at the Department of Surgical Sciences, Uppsala University. I want to express my gratitude to all of you that have helped and supported me along the way. My main supervisor professor Gunnar Westin. I couldn’t have been more fortunate to have you as my supervisor. With your great knowledge, enthusiasm, encouragement and dedication you are the best supervisor one could wish for, I could never thank you enough! My supervisor Peyman Björklund. Thank you for introducing me to the group and teaching me (almost) everything I know in the lab. I admire your expertise and dedication and I will never forget all you have done for me. My supervisor professor Göran Åkerström. Thank you for all the encouragement and great knowledge. To me you will always be the parathyroid-expert. Birgitta Bondeson and Peter Lillhager. Thanks for everything you both have done for me, from all the help in the lab to the interesting discussions and laughters. Present and former members in the group of endocrine surgery. Katarina Edfeldt, Alberto Delgado Verdugo, Tobias Åkerström, Joakim Crona, Cihan Cetinkaya, Johanna Sandgren, Ola Hessman, Per Hellman, Peter Stålberg, Ulrika Segersten and Daniel Lindberg. Thank you for being good friends, for the great discussions, the company on coffee-breaks, lunches and conferences, the laughters and the inputs on everything from research to life in general. My room mates Anna-Stina Sahlqvist and Carl Mårten Lindqvist. What can I say, without you I would have laid on the floor tearing my hair.
Tijana Krajisnik, Margareta Halin-Lejonklou and Konstantin Galichanin. Thank you for all the interesting discussions and the great company in the lab (and outside!), at conferences and courses. Everyone at the Clinical Research Centre at Uppsala University/Uppsala University Hospital, no one mentioned and no one forgotten! Thanks to my students for all your hard work and your contributions to this thesis. Thanks to my family and friends: My parents Virve and Sven-Åke, for your endless love and support. My sister Tessan, without you my life would be miserable and boring. My brothers Mathias and Björn, for being the best brothers in the world. All my old friends and horse-friends that remind me that there is a life on the outside. My horse Ciara and my sisters dog Tyson for never asking me any questions. My love (nöje) Johan, you are the best. You have always supported me although you don’t really know what I am doing. Without all of you this would never have been possible! I love you all! Thank You!
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