Loss of NF2 induces TGF-β receptor 1 mediated Non ......of the Hippo/YAP pathway (5), Ras signaling...

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Loss of NF2 induces TGF-β receptor 1 mediated non-canonical and oncogenic TGF-β signaling:

Implication of the therapeutic effect of TGF-β receptor 1 inhibitor on NF2 syndrome

Jung-Hyun Cho1,*, Ah-Young Oh1,*, Soyoung Park1, So-mi Kang1, Min-Ho Yoon1, Tae-Gyun Woo1,

Shin-Deok Hong1, Jihwan Hwang2, Nam-Chul Ha3, Ho-Young Lee4, Bum-Joon Park1,#

Running title: Therapeutic effect of TGF-βR 1 inhibition on NF2 syndrome

1Department of Molecular Biology, College of Natural Science, Pusan National University, BUSAN,

Korea (Republic of)

2Department of Microbiology, College of Natural Science, Pusan National University, BUSAN, Korea

(Republic of)

3Department of Food engineering, College of Agricultural Science, Seoul National University,

SEOUL, Korea (Republic of)

4Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam, Korea

(Republic of)

*These authors equally contribute at this work

#Corresponding author: Bum-Joon Park, Pusan National University, 2, Busandaehak-ro 63beon-gil,

Geumjeong-gu, Busan, Korea (Republic of) 46241, Phone: 82-51-510-2220; Fax: 82-51-513-9258; E-

mail: [email protected]

Conflicts of Interesting: Authors declared that there are no conflicts of interesting

on April 3, 2020. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 22, 2018; DOI: 10.1158/1535-7163.MCT-17-1210

http://mct.aacrjournals.org/

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Abstract

Neurofibromatosis type 2 (NF2) syndrome is a very rare human genetic disease and there is no proper

treatment for it until now. In our recent study, it has been reported that the loss of NF2 activates

MAPK signaling through reduction of RKIP in a mesothelioma model. Here, we show that loss of

NF2 induces reduction of the TGF-β receptor 2 (TβR2) expression and an overwhelming expression

of TGF-β receptor 1 is activated by physical stimuli such as pressure or heavy materials. Activated

TβR1 induces the phosphorylation and degradation of RKIP. RKIP reduction consequently results in

MAPK activation as well as Snail-mediated p53 suppression and occurrence of EMT in NF2-deficient

cells by physical stimuli. Thus, TβR1 kinase inhibitors restore cell differentiation and induce growth

suppression in NF2 deficient Schwannoma cell line and MEF. Moreover, TEW7197, a specific TβR1

kinase inhibitor, reduces tumor formation in the NF2-model mouse (Postn-Cre;NF2f/f). Gene

expression profiling reveals that TEW7197-treatment induces the expression of lipid metabolism-

related gene set such as NF2-restored cells in HEI-193 (NF2-deficient Schwannoma). Our results

indicate that reduction or deletion of TβR2 or NF2 induces the TβR1-mediated oncogenic pathway,

and therefore inhibition of the unbalanced TGF-β signaling is a putative strategy for NF2-related

cancers (NF2 syndrome and mesothelioma) and TβR2 mutated advanced cancers.




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Introduction

NF2 (Neurofibromatosis type 2) syndrome is a very rare genetic disorder, where patients are afflicted

by Schwannoma (benign tumor of Schwann cell) in the peripheral nervous system (1). Loss of hearing

is common feature of NF2 syndrome, due to presence of tumor in the vestibular nervous system.

Furthermore, tumors are also detected in the spinal ganglion in NF2 patients (2). Genetic alteration of

NF2 is revealed a loss of the NF2/Merlin (2). At the cellular level, NF2/Merlin functions as a linker

between the cytoskeleton and membrane (3, 4). Additionally, it is suggested to be a negative regulator

of the Hippo/YAP pathway (5), Ras signaling cascade, and mTORC2 (6). Thus, according to previous

literatures, it is clear that NF2/Merlin is an important tumor suppressor protein. However, these

functions are unable to explain why loss of NF2/Merlin induces Schwannoma without the

development of a general carcinoma.

In our previous study, we revealed that treatment of silica activates the Erk-Snail signaling and

suppresses the p53 pathway via RKIP reduction in mesothelioma (7). In addition, loss of NF2

promotes RKIP reduction and suppresses p53 activation (7). Indeed, NF2 is an important regulator of

physical stresses and is frequently found to be deleted in asbestos-induced mesothelioma (5, 6).

During the development of the neuro-system, Schwann cells (peripheral nerve system) and

oligodendrocytes (central nerve system) need to physically bind to the axon of neurons. Indeed,

contact of Schwann precursor cells and neural axons is an important trigger for differentiation of

Schwann cells (8). For Schwann cell differentiation and cell death, TGF-β signaling is critical (9). In

particular, there is an association between TGF-β signaling and axonal neurogulin-1 during the

attachment of Schwann cell to neural axon. Considering this fact, it is plausible that TGF-β signaling

components, including TGF-β receptors, would be associated with the physical-contact induced

signaling cascade.

Mesothelioma and Schwannoma commonly occur due to the deregulation of the physical contact-

induced signaling cascade. Malignant mesothelioma occurs in the thin layer surrounding the organs,

namely mesothelium (10), and some weighted material (maybe asbestos) are implicated as the




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causative agents of this cancer. Thus, we hypothesize that NF2 would work as an important regulator

of physical contact-induced cellular signaling, with an additional involvement of TGF-β signaling.

To examine the physical stimulation induced signaling cascade and relevance with NF2 syndrome, we

investigate the pressure-induced cellular response and role of NF2 in this signaling network. Although

asbestos is the best trigger for mesothelioma-related physical stimulation, its use is completely

forbidden for any purpose. Thus, we used silica as a replacement for asbestos (11-13). In addition,

pressure was generated on the cell by overlaying with a coverslip. Our experiments indicate that TβR1,

but not TβR2, works as a receptor for silica and pressure; loss of NF2 induces the down regulation of

TβR2; in the absence of TβR2, physical stimulation activates TβR1 which in turn induces RKIP

reduction and p53 inactivation. Taken together, we believe that inhibition of TβR1 can induce cell

death in NF2 deficient Schwannoma as well as mesothelioma.

Material and Methods

Cell culture and Reagents

ACHN (NF2 mutant) and MDA-MB-231 (NF2 mutant) cells were purchased from the Korean Cell

Line Bank. A549, HEK293, NCI-H28, NCI-H2452 and MDA-MB-468 cell lines were obtained from

American Type culture collection (ATCC, Manassas, VA). LNcaP (TβR1 null) was provided by Dr.

Chi SG (Korea University, Seoul, KOREA). HCT116 cell lines were obtained from Dr. Vogelstein B

(Johns Hopkins University, Baltimore, USA) and human Schwannoma cell line HEI-193 (NF2

deficient) was provided by Dr. Zadeh G (University Health Network, Toroto, CANADA). Mouse

schwann cells derived from NF2flox mice (14) were subjected to in vitro Cre-mediated deletion and

then cells were transduced with pMSCV-hygro retroviral rescue constructs encoding either full length

Merlin isoform 1(MSchw-WT), the truncated version that was encoded by the SKY mutant allele

(MSchw-SKY), or the empty vector (MSchw-KO). The cell lines were kindly provided from Dr.

Greer P (Queen's University, Ontario, CANADA). All cells were maintained in a 5% CO2 humidified




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incubator at 37℃. ACHN, HEI-193, HEK293, L132, LNCaP and MDA-MB-468 were cultured in

liquid DEME medium supplemented with 10% FBS and 1% antibiotics. A549, HCT116, NCI-H28,

NCI-H2452, MDA-MB-231were cultured in RPMI-1640 supplemented with 10% FBS and 1%

antibiotics. Primary tumor cells isolated from the NF2 syndrome model mice were cultured in DMEM

medium supplemented with 15% FBS and 1% antibiotics. Mouse embryonic fibroblast (MEF) cells

were isolated from 14.5 day embryos using a standard protocol and cultured in DMEM medium

supplemented with 15% FBS and 1% antibiotics. The appearance and growth characteristics of all

cells used in this study were compared with published information to ensure their authenticity.

Reagents

All culture reagents used in the experiments were procured as follows, unless indicated otherwise:

Silica (S5631; silicon dioxide), SB431542 and 4-OHT from Sigma Aldrich (St, Louis, Mo, USA);

Adriamycin from Calbiochem (Darmstadt, Hessen, Germany); Porcine TGF-β1 from R&D systems

(Minneapolis, MN, USA); TEW7197 was provided by Dr. Kim SJ (CHA University, School of

Medicine, Seongnam, KOREA); other TβR1 kinase inhibitors (Alectinib, PF-06463922, Certinib and

Crizotinib) from Selleck Chemicals (Houston, TX, USA).

Physical stimulation

To know how cells responded to physical stress, we used silica or cover glass. Cells were seeded at a

density of 0.5x104 cells on a cover glass (12 mm circle; thickness, 0.13-0.17 mm). The cover glass

was flipped up-side-down and incubated for indicated time in serum free medium. The intensity of the

pressure was changed by placing a second cover glass over the seeded cover glass. Silica was directly

administered to 2 x 105 cells under SF (serum free) conditions. The schematic diagram is described in

Supplementary Fig. S1A.

Silica-Resistant cell line




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To obtain the silica-resistant A549 (A549T) cell line, A549 cells were seeded in a cell culture dish and

exposed to high concentration silica (100 μg/ml). After 3-4 days incubation, the viable cells were

washed with media and cultured. This method was repeated for one month to obtain surviving cell

population resistant to silica. Experiments involving A549T were performed using the resistant cell

line thus developed.

Vectors and Transfection

pCMV RKIP-HA was provided by Keum G (David Geffen School of Medicine at University of

California, Los Angeles, CA, USA). pCMV RKIP-T101A-HA and pCMV RKIP-T101D-HA were

generated by Dr. Hwang JH (Pusan National University, Pusan, KOREA). Snail-FLAG vector were

provided by Dr. Hung MC (MD Anderson cancer center, TX, USA). The pcDNA3 NF2-FLAG, pRK5

TGF beta type 1 receptor-FLAG, pRK5 TGF beta type 1 receptor-CA (T202D)-FLAG, pCMV5b TGF

beta type 2 receptor-HA and pWZL E-cadherin-GFP were obtained from Addgene (Cambridge, MA,

USA). HA-p53 vectors were a kind gift from Dr. Kim S (Seoul National University, Seoul, KOREA).

Transfection was performed using the Jetpei transfection agent (Polyplus New York, USA) for

mammalian expression of these vectors. As described previously (15), cells were seeded at a density

of 2 x 105 cells per well in 12 well plates, and cultured overnight before transfection. The vector (1.5

μg) was mixed with 1.5 μl of Jetpei reagent in 150 mM NaCl solution. The mixture was incubated for

15 min at room temperature, after which it was added to the cells. After 3 hours, the serum-free

medium was replaced by medium supplemented with 10% FBS.

SiRNA and Transfection

For in vitro gene knock down, si-RNA against target proteins were generated (Cosmo Genetech, Seoul,

Korea).

Si-NF2-1: 5’-ATCATGATCCAGTACCTTCTTGTCC-3’

Si-NF2-2: 5’-CAGCCTGTCTTTCGACTTCAA-3’




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Si-Snail: 5'- GCGAGCTGCAGGAC

The INTERFERin® transfection reagent (Polyplus New York, USA) for siRNA was used for

transfection. Briefly, cells were seeded at a density of 2 x 105 per well in 12 well plates and incubated

overnight before transfection. A total of 1.5 pmoles (21 ng) of siRNA duplexes were mixed with 4 µl

of INTERFERin® in 100 µl medium without serum. The mixture was incubated for 15 min at room

temperature to allow formation of the INTERFERin®/siRNA complexe. This mixture was then added

to the cells And incubated for a further 4 hours, after which the serum-free medium was replaced by

medium supplemented with 10% FBS.

Western Blot Analysis

As described previously (16), proteins were extracted from cells with RIPA buffer (50 mM Tris-Cl,

pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS and 10% sodium deoxycholate). Samples were applied

to SDS-PAGE and transferred onto a PVDF membrane. Blotted membranes were incubated with

primary antibodies for 1 hr to overnight at 4°C, followed by incubation with HRP-conjugate species-

matched secondary antibodies for 1 hr at room temperature. Peroxidase activity was detected by

chemi-luminescence using the ECL kit (Intron, Seoul, Korea). For immunoprecipitation (IP) analysis,

whole-cell lysates were incubated first with the appropriate antibodies for 4 hr at 4°C, and then with

protein A/G agarose beads (Invitrogen, California, USA) for 2 hr at 4°C. After centrifugation and

washing with RIPA, the precipitated immune-complexes were subjected to SDS-PAGE and western

blot analysis. The following antibodies were used in this study: HA (sc-7392), GFP (sc-9996), GST

(sc-138), RKIP (sc-5426), Snail (sc-28199), p53 (sc-126), NF2 (sc-331), TGFβ R1(sc-398), TGFβ

R2(sc-400), Vimentin (sc-7557), Actin (sc-1616) and E-cadherin (sc-8426), all purchased from Santa

Cruz Biotechnology (Santa Cruz, Texas, USA). Anti-phospho-Erk (9101L), anti-total-Erk (9102) and

anti-p-smad2/3 (8828S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Anti-FLAG (F3165) was obtained from Sigma-Aldrich (St. Louis, Mo, USA).




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Immunofluorescence Staining

Cells were seeded on a cover glass and transfected with the indicated vectors or treated with the

appropriate chemicals. After fixing with Me-OH for 30 min, the cells were incubated with blocking

buffer [PBS + anti-human-antibody (1:500)] for 1 hr. Cells were then washed with PBS, incubated

with anti-γ-tubulin antibody in blocking buffer (1: 100 to 200) for 4 hr, and subsequently with FITC-

conjugated or Rhodamine-conjugated secondary antibodies in blocking buffer (1: 500) for 2 hr.

Nuclear staining was achieved with DAPI. After washing with PBS, cover glasses were mounted with

mounting solution (Vector Laboratories, Cambridgeshire, UK), and the immunofluorescence signal

was detected through fluorescence microscopy (Zeiss, Jena, Germany) (17).

Recombinant proteins, Immunoprecipitation and GST pull-down assays

Glutathione S-transferase (GST)-pull down assay and immunoprecipitation (IP) assay were performed

to evaluate the protein-protein interaction. For GST-pull down, agarose-bead conjugated GST

(negative control) or GST-target protein was incubated with cell lysate or recombinant protein in RIPA

buffer for 1 h at 4°C. IP assay was performed with cell lysate or recombinant protein with RIPA buffer.

The whole lysates were incubated with appropriate primary antibodies for 2 hr at 4°C, and reacted

with agarose bead conjugated protein A/G (Invitrogen, Carlsbad, CA, USA) for 2 h. After

centrifugation, the precipitates were washed with RIPA buffer twice and subjected to SDS-PAGE and

WB analysis (17).

Co-precipitation of heavy material (Silica and FeO2)

For analyzing the association of silica with cell surface proteins, precipitation assay was performed

using heavy materials (FeO2 and silica). The silica was treated to react with cell surface protein 4 hr

before lysis using RIPA buffer. To avoid artifacts, the same amount of heavy material (40 μg/ml) was

added to the control sample. Furthermore, protein A/G-agarose (Invitrogen, Carlsbad, CA, USA) was




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also added to the control sample and the whole cell lysate. Immediately, centrifugation was performed.

The precipitated material was washed with RIPA buffer and analyzed by SDS-PAGE and WB. (related

Fig. 1E; Supplementary Fig. A2A and S2B)

In vitro kinase assay

To check the phosphorylation of RKIP by TβR1, whole cell lysates transfected with Flag-TβR1 were

incubated with 15 mg of GST-fused RKIP protein for 4 h at 37℃ in 25 ml of 50 mM Tris (pH 8.0)

buffer containing 1 mM ATP, 10 mM MgCl2 and 10 mM 2-mercaptoethanol. WB analysis was

performed to detect phosphorylated RKIP using anti-phospho-Smad2 (Ser465/467) / Smad3

(Ser423/425) antibody (Cell Signaling, Danver, MA, USA) (18, 19).

MTT Assay

To measure the cell viability, cells were treated with the indicated chemicals for 4 days. For the MTT

assay, cells were incubated with 0.5 mg/ml of MTT solution (Calbiochem, Darmstadt, Germany) for 4

hr at 37°C. After removing the excess solution, the precipitated material was dissolved in 200 μl

DMSO and quantified by measuring the absorbance at 540 nm.

Luciferase Assay

To estimate the transcriptional activity of TGF-β signaling, 3TP-Luc vectors were transfected into

cells for 24 hr, and cells were exposed to the indicated chemicals for appropriate duration. After

washing with wash buffer (Promega, Wisconsin, USA), the cells were lysed using lysis buffer

(Promega, Wisconsin, USA), and luciferase activity was determined by a luminometer (MicroDigital,

Gyeonggi-do, South Korea).

In vitro migration assay

For the analysis of in vitro cell migration, transwell assay was performed using Polycarbonate




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Membrane Transwell Inserts (3422; Corning, NY, USA). Briefly, 0.6 ml media containing 10% FBS

was added to each well of the well plate. Cells were re-suspended in serum-free medium, and 0.1 ml

of the cell suspension was added to the inside compartment. The plate was incubated with or without

silica (10 μg/ml, 60 hr) in 5% CO2 incubator. Cells in the upper chamber were then discarded, and the

attached cells in the bottom section were fixed using 4% PFA for 30 min. After fixation, migratory

cells are stained by 0.05% Trypan blue solution (Gibco, BRL, Paisley, UK) and quantified. The

migration rate was quantified by counting the migration cells in six random fields using a light

microscope.

RT-PCR

For RT-PCR, total cellular RNA was extracted using the Qiagen (Hilden, Germany) RNA extraction

kit. After measuring the RNA concentration, 1 μg of total RNA was reverse transcribed into cDNA

using the MMLV RT (Invitrogen, Carlsbad, CA, USA) and a random hexamer. RT-PCR was

performed with the specific primers. The primer sequences were as follows: for

HMGCS1 Forward 5′-TCCCACTCCAAATGATGACA-3′

HMGCS1 Reverse 5′-CTTCAGGTTCTGCTGCTGTG-3′

LDLR Forward 5′-TCTGTCGTGTGTGTTGGGAT-3′

LDLR Reverse 5′-ACGACAAGATTGGGGAAGTG-3′

INSIG1 Forward 5′-CAACACCTGGCATCATCG-3′

INSIG1 Reverse 5′-CTCGGGGAAGAGAGTGACAT-3′

DDIT4 Forward 5′-CGAGTCCCTGGACAGCAG-3′

DDIT4 Reverse GGTCACTGAGCAGCTCGAAG-3′

MMP2 Forward 5′-CCCTGATGTCCAGCGAGTG-3′

MMP2 Reverse 5′-ACGACGGCATCCAGGTTATC-3′

GAPDH Forward 5’-ATCTTCCAGGAGCGAGATCCC-3’

GAPDH Revere 5’-AGTGAGCTTCCCGTTCAGCTC-3’




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Mice

All experimental procedures using laboratory animals were approved by the animal care committee of

Pusan National University. NF2 (FVB/NJ) mice were obtained from Dr. Clapp DW (Indiana

University, Indianapolis, USA). Before commencing the experiments, all mice were maintained under

temperature- and light-controlled conditions (20–23oC, 12 h/12 h light/dark cycle) and provided

autoclaved food and water ad libitum. The Postn-cre transgene was detected by PCR analysis using

the following primers:

Postn-Cre Forward 5’-ATG-TTT-AGC-TGG-CCC-AAA-TG-3’

Postn-Cre Reverse 5’-CGA-CCA-CTA-CCA-GCA-GAA-CA-3’

Nf2flox.flox and Nf2Δ bands were detected by PCR analysis with the following primer.

Full length NF2 FORWARD 5’-CTTCCCAGACAAGCAGGGTTC-3’

Full length NF2 Reverse5’-GAAGGCAGCTTCCTTAAGTC-3’

ΔNF2 Forward 5’-CTCTATTTGAGTGCCTGCCATG-3’

ΔNF2 Reverse 5’- GAAGGCAGCTTCCTTAAGTC-3’

Band sizes and primers were described in Giovannini et al (14, 20).

Drug treatment in vivo

NF2 (3 month-old, N=10) mice were intraperitoneally (i.p.) administered the carrier, TEW7197 (5

mg/kg), 3 times a week. After terminating the experiment of each group, mice were dissected and

tumors were isolated. Throughout the experimental period, the body weight were measured 3 times a

week.

Image Acquisition of [18F] FDG PET/CT

To acquire [18F] -FDG PET/CT image, each mouse was fasted at least 6 h. [18F] FDG (500±23 uCi)

was intravenously administered through tail vein. After administration of [18F] FDG, mouse was




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placed in the dimmed lighted cage for 60 min. Each mouse was maintained under anesthesia with

isoflurane (2.5% flow rate) for the duration of the scan. Animals were positioned prone in the

standard mouse bed. Limbs were positioned lateral to the body to acquire uniform CT images. Whole

brain CT images were acquired with a Micro-PET/CT scanner (nanoPET/CT, Bioscan Inc.,

Washington DC, USA). For CT image acquisition, the X-ray source was set to 200 µA and 45 kVp

with 0.5 mm. The CT images were reconstructed using cone-beam reconstruction with a Shepp filter

with the cutoff at the Nyquist frequency and a binning factor of 4, resulting in an image matrix of 480

× 480 × 632 and a voxel size of 125 µm (21).

Histological Analysis

At the end of the experimental period, the mice were sacrificed, and the tumors were dissected; tissues

were fixed using 10 % formalin in PBS for 24 h and embedded in paraffin blocks according to the

basic tissue processing procedure. For histological analysis, the embedded tissues were cut in 5 μm

slices using a Leica microtome (Wetzlar, Germany), and transferred onto adhesive-coated slides

(Marienfeld laboratory glassware, Germany). After deparaffinization and rehydration, sections were

stained with H&E for routine examination. For IHC staining, the rehydrated tissue sections were

incubated with antibodies to TGFβ R1 (sc-398) and TGFβ R2 (sc-400). Antigen retrieval was

performed using 10 mM sodium citrate (pH 6.0), twice at 95oC for 10 min each, and endogenous

peroxidase activity was blocked with 3% hydrogen peroxidase for 10 min. The treated slides were

then dehydrated following a standard procedure, and sealed with cover glass using mounting solution

(22, 23).

Microarray

For differential gene expression analysis by NF2 transfection and TEW7197 treatment, microarray

was performed using the Affymetrix GeneChip (Human Gene 2.0 ST Array; DNA Link, Inc, Korea).

Total RNA obtained from HEI-193 transfected with NF2 (about 60% transfection yield) or treated




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with TEW7197 (10 μM for 12 hr or 24 hr) was quantified and used for analysis. Array data extract

were performed using Affymetrix GeneChip® Command Console® Software (AGCC). For

differentially expressed gene (DEG) analysis, probes were selected if the difference in expressions

were 1.5 fold compared to the HEI-193 control, and were statistically filtered by t-test (p<0.05). Fold

value was calculated in this way (Absolute Fold Change = [case/control] if case > control,

[control/case] if control > case). For the functional analysis, only DEG probes were used. Web tool

DAVID (24, 25), data base of which are Gene Ontology, KEGG, BIOCARTA and OMIM_DISEAE,

was used for functional grouping of probes. All statistical and functional analysis were performed by

DNA Link, Inc. The full data set is available in the NCBI’s Gene Expression Omnibus (GEO)

database (GEO GSE115359; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE115359).

Statistical Analysis

All results are expressed as the mean + s.e.m. and performed at least n=3 per group. Student’s t-test

was performed for obtaining the statistical significance.




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Results Loss of NF2 provides the resistance to pressure-induced growth suppression

To generate physical stress, cells were seeded on cover glass, which was subsequently flipped upside-

down (Supplementary Fig. S1A). The effect of NF2 on pressure-induced cell viability was assessed

first by using mouse embryonic fibroblast (MEF), obtained from Ubc-Cre-ER; NF2f/f mouse. In the

absence of 4-OHT, NF2 was normally expressed (Supplementary Fig. S1B) and viability of MEF

gradually decreased with increasing incubation time (Fig. 1A). However, elimination of NF2

expression by 4-OHT treatment (Supplementary Fig. S1B) increased the cell viability (Fig. 1A).

Pressure-induced p53 and RKIP induction were not observed in NF2-deficient MEF (Fig. 1B). To

confirm this, the same experiment was performed using HEI-193 cells (NF2-deficient Schwannoma

cell line) (26, 27). Resistance to pressure-induced growth suppression was restored by re-expression

of NF2 (Fig. 1C). In addition, pressure-induced RKIP and p53 reduction was abolished by NF2

transfection in HEI-193 cells (Fig. 1D). We also observed that the reduction of p53 and RKIP in

response to silica treatment (Supplementary Fig. S1A) that induced RKIP reduction in mesothelioma,

was completely blocked by NF2 restoration (Fig. 1E). Numerous cell lines were evaluated in this

experiment. In non-transformed L132, pressure suppressed the cell viability. In contrast, ACHN cells

(NF2 mutated kidney cancer cell line; 28) were resistant to physical stimulus (Supplementary Fig.

S1C), and HCT116 was partially resistant to physical stimulation (29). Our results indicate that

elimination of NF2 or RKIP provides the resistance to pressure-induced growth suppression in L132

(Supplementary Fig. S1D), indicating that RKIP and NF2 are critical for physical stress-induced




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signaling.

TβR1 associates with silica and induces NF2 and RKIP reduction

Although physical stresses reduce NF2 and RKIP, it is as yet unknown how cells recognize these

stimuli. To address this, we checked the association of silica with cell surface proteins. Among tested

proteins (E-cadherin, TβR1, TβR2, and Her2/Neu), only TβR1 (Fig. 1F and Supplementary Fig. S1F)

showed dose-dependent expression to silica (Supplementary Fig. S2A). To avoid artifacts, we the

precipitation assay was repeated using FeO2 and protein A/G-agarose. Similar to silica, FeO2 co-

precipitated with TβR1 (but not Her2/Neu; Supplementary Fig. S2B). However, protein A/G-agarose-

beads showed no interaction with TβR1 (Supplementary Fig. S2B). Treatment of silica reduced the

RKIP and NF2 expression in TβR2-deficient HCT116 but not in TβR1 deleted LNCaP (30-32) (Fig.

1G), suggesting that TβR1 is responsible for physical stimulation. This result is consistent with our

previous result that HCT116 is resistance to press-induced growth suppression (Supplementary Fig.

S1C). Indeed, TβR1 transfection, but not TβR2, suppressed the RKIP and NF2 expression and

induced Snail expression (Fig. 1H). Moreover, constitutively active TβR1 was more effective on RKIP,

NF2 and Snail expression than wild type TβR1 (Supplementary Fig. S2C). Furthermore, we observed

a dose-dependent reduction of RKIP and NF2 in response to TβR1 (Supplementary Fig. S2D). Silica-

induced RKIP and NF2 reduction were also observed in elevated TβR1 expressed cell lines, as

compared to TβR2 (Supplementary Fig. S2E). In addition, TβR1 expression was induced by silica

treatment (H28, HEI-193 in Supplementary Fig. S2E). However, silica-treatment did not activate 3TP-

luciferase (Supplementary Fig. S3A) or induce the R-Smad phosphorylation (Supplementary Fig. S3B)

or translocation (Supplementary Fig. S3C). Next, we monitored the involvement of Hippo/Yap

pathway via analysis of the promotor activity of CTGF, target of YAP-TEAD in NF2 deficient human

Schwannoma (HEI-193) and mouse Schwann cell (Mschw). But, treatment of silica did not alter the

promotor activity (Supplementary Fig. S3D). This result indicated that silica did not activate

Hippo/Yap pathway. Although our results indicate that physical stress uses the unusual TβR1-




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mediated signaling, it is as yet unknown how TβR1 recognizes silica or heavy material.

NF2 stabilizes TβR2 receptor and suppresses EMT

To evaluate the effect of NF2 on TβR1-mediated signaling, we assessed the effect of siNF2 on TβR1

and TβR2 expression. The endogenous and exogenous TβR2 expressions were reduced by eliminating

NF2 using siRNA in HEK293 and HCT116 cells (Fig. 2A and B; Supplementary Fig. S4A), and a

similar reduction was also observed in NF2 knock out MEF (Fig. 2C). NF2-deficient MEF showed the

reduction of RKIP and TβR2 and induction of p-Erk in response to silica (Fig. 2D). Conversely, the

restoration of NF2 into HEI-193 induced the expression of TβR2 expression as well as RKIP and p53

(Supplementary Fig. S4B). We obtained the similar results from NF2 transfected ACHN

(Supplementary Fig. S4C) and si-NF2 transfected L132 (Supplementary Fig. S4D), where NF2

enhanced the TβR2 expression. Pulse-chase analysis revealed that NF2 extended the half-life of TβR2

(Fig. 2E) but not TβR1 (Supplementary Fig. S4E and S4F). Indeed, the proteasome inhibitor, MG132

could block the reduction of NF2, RKIP and TbR2 without altering the mRNA expression (Fig. S4G).

To confirm the role of NF2 in TβR2 expression and RKIP, we examined the expression of these

proteins in MSchw cell (mouse NF2 deficient Schwann cell line) and its stable transfected cell lines

with empty (MSchw-EV), wild type NF2 (MSchw-WT) and mutant NF2 (terminated by a splice

mutation that causes a R477C; MSchw-SKY). We observed that reductions of RKIP, p53 and TbR2 by

silica-treatment were suppressed in the MSchw-WT cell line (Supplementary Fig. S4H), strongly

supporting the hypothesis that NF2 is a critical factor for stability of RKIP and TβR2. In addition,

from this experiment, we observed that silica suppressed p-Smad2/3 in regardless of NF2 status. We

also checked the p-YAP S127 expression at the same samples. Although induction of p-YAP S127

(inactivation form) was obviously detected by NF2 transfection, treatment of silica did not alter the p-

YAP S127 expression (Supplementary Fig. S4H). Taken together with our previous results that silica-

treatment suppressed p-Smad2/3 in A549 (Supplementary Fig. S3B), silica itself was not related with

canonical TGF-β signaling as well as Hippo/Yap pathway.




17

Since reduction of RKIP induces Snail and promote EMT (7, 33, 34) and numerous malignancies are

known to harbor TβR2 mutation (28, 29, 35, 36), we evaluated the effect of silica on cancer cell

invasion using the transwell assay. As expected, silica promoted cell invasion (Fig. 2F; Supplementary

Fig. S5A). Interestingly, TβR2 expression blocked the cancer cell invasion by promoting the NF2 and

RKIP overexpression. Indeed, silica and pressure suppressed E-cadherin (Supplementary Fig. S5B

and S5C) and induced the mesenchymal marker vimentin (Fig. 2G; Supplementary Fig. S5B). We

obtained similar results from the E-cadherin luciferase system, wherein the reduction of E-cadherin by

silica was eliminated by TβR2, RKIP and NF2 expression (Supplementary Fig. S5D). However,

elimination of Snail did not suppress the cell viability of HEI-193 (Supplementary Fig. S5E),

suggested that induction of Snail would be related only with EMT but not cell proliferation at NF2

deficient condition. Since TβR1-mediated physical stress-induced signaling reduced RKIP and NF2

expression, we assumed that balance between TβR1 and TβR2 could block the RKIP and NF2

reduction. In fact, TβR2 transfection could block the silica-induced RKIP, NF2 and p53 reduction (Fig.

2H). These results suggest that TβR1-mediated signaling cascade is oncogenic, and TβR2 has the

capability to block it.

Activated TβR1 reduces NF2 and RKIP expression via kinase activity

Since reduction of RKIP is a critical event in the TβR1-mediated oncogenic pathway, we speculated

how TβR1 alone suppresses RKIP. To address this, we assessed the interaction of RKIP and TGF-β

signaling components. Among the tested proteins, TβR1 showed a strong binding affinity with RKIP

(Fig. 3A), which was inhibited in the presence of NF2 (Supplementary Fig. S6A and S6B). Also,

inhibitors of TβR1 kinase (37-41) induced the RKIP and NF2 as well as p53 expression (Fig. 3B) and

block the silica-induced RKIP and NF2 reduction (Fig. 3C). Furthermore, TβR1 inhibitor blocked the

interaction of TβR1 and RKIP (Fig. 3D). We also detected the affinity of RKIP for p-Smad antibody

when recombinant RKIP was reacted with cell lysate obtained from silica-treated TβR1-transfectant

(Fig. 3E). This implies that RKIP is a target of TβR1 kinase. Analysis of the RKIP amino acid




18

sequence reveals well conserved SSXS/T motif, which is the TβR1 kinase consensus phosphorylation

site (38). To confirm this, we two different RKIP mutants were generated (T101A and T101D), and

we found that phospho-mimetic RKIP mutant had a very low protein stability, and the RKIP T101A

showed resistance to the silica-mediated reduction (Fig. 3F; Supplementary Fig. S7B). In addition,

this mutant blocked the NF2 reduction (Fig. 3F; Supplementary Fig.S7B). Essentially, RKIP T101A

was resistant to TβR1-mediated reduction (Supplementary Fig. S7C and S7D) and increased NF2

expression (Supplementary Fig. S7E and S7F). We further observed an increase in the TβR2

expression by RKIP transfection, particularly by RKIP T101A (Supplementary Fig. S7C and S7D).

TβR1 kinase inhibitors can induce NF2 and RKIP expression in NF2 deficient cell lines

This experiment reveals that inhibition of TβR1 kinase blocks the RKIP and NF2 reduction, implying

that TβR1 inhibitor is useful for NF2-deficient tumors, such as NF2 syndrome Schwannoma and

mesothelioma as well as TβR2 mutated cancers. Indeed, treatment of TEW7197 dose-dependently

induced RKIP and p53 in Schwannoma (HEI-193), mesothelioma (H28) and HCT116 (Fig. 4A). In

addition, TEW7197 blocked the TβR1-induced RKIP and NF2 reduction (Fig. 4B; Supplementary Fig.

S8A); and TEW7197 and SB431542 induced the NF2, RKIP, and p53 expression in HCT116

(Supplementary Fig. S8B). In mesothelioma, co-treatment of TEW7197 and adriamycin exerted a

synergic effect on p53 expression (Fig. 4C) and growth suppression (Fig. 4D). However, TEW7197

did not show growth suppression on restoring the NF2 in ACHN (Supplementary Fig. S8C) or normal

MEF (Supplementary Fig. S8D). Comparing to Rad001 that is recently employed in clinical trials for

NF2 syndrome (42, 43), the TβR1 inhibitor exerted an anti-proliferative effect on Schwannoma cells

(Fig. 4E; Supplementary Fig. S8E), with obvious induction of p53 and RKIP in HEI-193 (Fig. 4F).

TEW7197 is most suitable candidate drug for NF2 syndrome

Due to the recent developments of numerous selective TβR1 inhibitors, we evaluated the effect to

choose the ideal chemical candidate for NF2 syndrome and mesothelioma. First, we assessed the




19

efficacy of 6 chemicals, including TEW7197 and SB431532 (Supplementary Fig. S9A), on the cell

viability of HEI-193 (Fig. 4G) and H28 (Supplementary Fig. S9B). In both cell lines, Certinib and

Crizotinib induced obvious effects, whereas Alectinib and PF-06463922 were less effective than

TEW7197 (Fig. 4G; Supplementary Fig. S9B). Although all tested chemicals, except Crizotinib, could

block the RKIP reduction by silica (Supplementary Fig. S9C), two chemicals (Certinib and Crizotinib)

showed toxicity towards normal MEF, together with their obvious cytotoxic activity on NF2 null cells

(Supplementary Fig. S9D). In addition, TEW7197 was most effective on RKIP induction in HEI-193

(Fig. 4H). Thus, we our results determined TEW7197 as an ideal drug candidate for further studies.

TEW7197 promotes Schwannoma cell differentiation

To know the global effect of TEW7197, we performed the microarray with HEI-193 (Supplementary

Fig. S10A). Compared to the control, TEW7197-treatment altered the expression of about 5000 genes,

which differed based on the treatment time. NF2-transfection also altered 4229 gene expressions (Fig.

5A). Of these, 1183 genes were commonly altered by TEW7197 and NF2 transfection. Many of these

were grouped as cholesterol biosynthesis or adipose tissue formation (Fig. 5A; Supplementary Fig.

S10B-S10E). We also obtained similar results by Pathway & Biocarta analysis (Supplementary Fig.

S10F-S10H), and confirmed the induction of several genes by RT-PCR (Fig. 5B). Since Schwann

cells accumulated cholesterol and other lipids for myelin sheet formation, the gene expression profile

indicates that TEW7197 and NF2 transfection induce Schwann cell differentiation (44, 45). Indeed,

HEI-193 showed a differentiated phenotype after treatment with TEW7197 (Fig. 5C). To confirm this,

we checked other Schwann cell differentiation markers such as sox2 and GFAP (46). As expected,

TEW7197 suppressed the sox2 expression and GFAP induction (Fig. S11A). In addition, MPZ, a

marker of myelinated Schwann cells (46), was increased after exposure to TEW7197 (Fig. S11B). We

further confirmed the reduction of sox2 and induction of GFAP by treatment of TEW7197 in HEI-193

through IF staining (Fig. S11C and D). These results strongly suggest that TEW-7197 promotes the

differentiation of Schwannoma into Schwann cells. Next, we evaluated the expression of TβR1 and




20

TβR2 in the NF2 syndrome mouse model for a more concrete and logical analysis. We observed a

reduction of TβR2 in the dorsal root ganglion of Postn-Cre;NF2f/f mice (Fig. 5D; Supplementary Fig.

S12).

Therapeutic effect of TEW7197 on NF2 syndrome mouse model

Since TEW7197 is a specific TβR1 kinase inhibitor that suppresses the viability of NF2-deficient cells,

we tested the in vivo efficacy of TEW7197 in the NF2 syndrome mouse model (14, 20). TEW7197 is

now in the phase 2 clinical trial, implying that there is severe toxicity. To do this, we injected 10

mg/kg of TEW7197 intraperitoneally (3 times/week) for 6-8 weeks (Supplementary Fig. S13A).

Through the PET/CT analysis, we observed that TEW7197 suppresses the tumor formation (Fig. 5E;

Supplementary Fig. S13B). Histological analysis confirmed that TEW7197 diminishes the tumor

formation (Fig. 5F). Indeed, large tumors presented in the dorsal root ganglion of Postn-Cre; NF2f/f

were not detected in TEW7197 treated mice (Fig. 5F; Supplementary Fig. S13C). Moreover, this

chemical increased the body weight (Supplementary Fig. S13D). The vehicle-treated mouse presented

with very large tumors (Supplementary Fig. S14A), from which we isolated and cultured the primary

tumor cells. These cells also revealed the differentiated phenotype in response to TEW7197

(Supplementary Fig. S14B). Resistance to pressure-induced growth suppression was also overcome

by TEW7197 (Supplementary Fig. S14C). These results strongly suggest that this specific inhibitor of

TβR1 is a strong anti-cancer candidate for NF2 or RKIP deregulated cancers, including mesothelioma

and NF2 syndrome-related tumors.

Discussion

This study revealed that physical stresses such as pressure or attachment of macro-materials activate

the TβR1-mediated unusual oncogenic signaling (Fig. 5G). Activated TβR1 by physical stimuli

induced phosphorylation-induced RKIP degradation. In contrast, NF2 induced TβR2 expression and




21

maintains a balance between TβR1 and TβR2 (Fig. 5G). Thus, loss of NF2 induces a disturbance in

the balance of TβR, and facilitates TβR1-mediated oncogenic signaling. Since we previously found

that RKIP reduction by NF2 loss promotes Snail stabilization and p53 suppression (7), TβR1-

mediated RKIP-NF2 reduction would suppress p53 activity. Indeed, we observed the induction of p53

by specific TβR1 inhibitor, accompanied with RKIP/NF induction (Fig. 4A). In addition, we found

that TβR1-mediated oncogenic signaling promotes EMT and cell migration (Fig. 2F) (47-49). We also

observed the inhibitory effect on cell migration by transfection of TβR2 as well as NF2. These results

indicate that re-balancing between TβR1 and TβR2 can block the TβR1-mediated oncogenic signaling,

and that NF2-mediated stabilization of RKIP also blocks the TβR1-mediated oncogenic property.

Since the final step of TβR1-mediated oncogenic signaling is achieved by reduction of RKIP, wild

type NF2 exerts an anti-oncogenic feature in TβR2 deficient conditions, such as in HCT116. Hence,

HCT116 is moderately resistant to pressure-induced apoptosis (Fig. S1C). These results also imply

why cancer cells show frequent mutations in TβR2 (35, 50, 51).

We also demonstrate the association of FeO2 with TβR1 (Supplementary Fig. S2B). This result

indicates that any other heavy material is capable of activating the TβR1-mediated oncogenic

signaling. Thus, this new oncogenic pathway may provide the answer for wood dust-induced nasal

cavity carcinoma (52, 53) or asbestos-induced mesothelioma (54). Considering that micro-dust is

significantly increased globally, an increase of related cancers induced by air-pollution is therefore

predicted. Thus, our mechanism would be useful for providing a potential therapy to cancers such as

mesothelioma and lung cancer. Moreover, we have shown the TEW7197 suppresses tumor

progression in the NF2 syndrome mouse model. In contact with neuro-fibers, Schwann cell precursor

is differentiated into Schwann cell. Based on our hypothesis, this contact would be the physical

stimuli. Under conditions of intact NF2, the Schwann cell precursor will differentiate into Schwann

cell. However, under NF2-deficient conditions, this physical contact would trigger the TβR1-mediated

oncogenic signaling and reduction of RKIP. Hence, inhibition of TβR1 kinase is one of the plausible

targets for NF2 syndrome. This hypothesis is strongly supported by the outcome of our in vivo results.




22

Indeed, similar effects could be obtained after treatment with other TβR1 inhibitors (Supplementary

Fig S9). The inhibitors selected for our experiments provided representative chemicals for similar

backbone and clinical usage (all have approval for clinical trials from FDA). All the tested chemicals,

except Crizotinib, block the RKIP reduction and suppress the cell viability (except PF-06463922).

However, Alectinib and Certinib also showed strong cytotoxicity in normal cells. Considering these

results, TEW7197 would be the most plausible candidate as a therapeutic drug for NF2 syndrome.

Alectinib and Certinib would be more suitable for aggressive cancer treatments, such as mesothelioma.

Since clinical trial of TEW7197 has been approved, it would be applied to NF2 syndrome after

adjustment of proper dosage with in near future. In addition, this chemical can also be applied to

mesothelioma since this malignancy shows very low RKIP expression and/or NF2 deletion (7).




23

Acknowledgments

This research was supported by Basic Science Research Program through the National Research

Foundation of Korea (NRF) funded by the Korea government(MSIP) (NRF-2017R1A2B2007355; B.-

J. Park) and by the Bio & Medical Technology Development Program of the NRF funded by the

Korean government, MSIP (NRF-2016M3A9D9945477)




24

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Figure legends

Figure 1.




31

Loss of NF2 protects physical stress-induced cell death. A, Loss of NF2 provided the resistance to

physical stress-induced cell death in MEF cells. Cell viability was measured by the MTT assay under

serum free (SF) conditions. Asterisk indicates statistical significance (P<0.05, Student’s t-test). B,

Increased p53 and RKIP by pressure in normal MEF were not observed in NF2-deleted cells. Ubc-

Cre-ER; NF2f/f MEF were incubated with 4-OHT for 4 days to induce NF2 deletion, and pressure was

stimulated using 12 mm cover glass for indicated times. C, Restoration of NF2 sensitized pressure-

induced cell death in the NF2-deficient HEI-193 Schwannoma cells. Cells were transfected with NF2

for 24 hr and incubated under cover glass for indicated time. Asterisk indicates statistical significance

(P<0.05, Student’s t-test). D, Transfection of NF2 blocked the reduction of RKIP and p53 in HEI-193

cells. Cells, transfected with NF2 for 24hr, were incubated under cover glass for indicated time under

SF conditions. Actin was used as the loading control. E, Reduction of p53 and RKIP by silica were

completely blocked by NF2 transfection in NF2-deficient HEI-193 cell line. HEI-193 cells were

transfected with NF2 expression vectors for 24 hr. Cells were exposed to silica under SF conditions at

the indicated concentrations and time. EV indicates empty vector control. F, In co-precipitated

material with silica, only TβR1 associated with silica. HEK293 cells were transfected with indicated

vector, exposed to silica for 4 hr (40 μg/ml) under SF conditions, and then precipitated using the silica.

For the negative control, GFP (EV), Her2/Neu, E-cadherin (E-cad) were examined. However, these

proteins were not co-precipitated with silica. PPT and SUP indicate precipitated materials and

supernatants, respectively. G, TβR1-null LNCaP does not respond to silica treatment, while TβR2-

deficient HCT116 was responsive. Cells were incubated with indicated concentrations of silica for 24

hr under SF conditions. In LNCaP, there was no reduction of RKIP and NF2. The number under bands

indicates the ratio of each protein expression to actin, determined by image j software. H,

Overexpression of TβR1, but not TβR2, reduced the RKIP and NF2, whereas Snail expression was

increased by TβR1 and TβR2. HEK293 cells were co-transfected with indicated vectors for 24 hr. The

number under bands indicates the ratio of each protein expression to actin, determined by image j

software.




32

Figure 2.

Loss of NF2 induces an imbalance in the TβR1 and TβR2 expression. A and B, Elimination of NF2

using siRNA suppressed the TβR2 expression in HEK293 and HCT116 cells. Both cells, transfected

with TβR1 or TβR2, were co-transfected with si-NF2 for 36 hr. C, NF2 knock-out MEF showed

reduction of TβR2 and RKIP. Ubc-Cre-ER; NF2f/f MEF exposed to 4-OHT induces NF2 deletion.

Ubc-Cre-ER; NF2f/f MEF were incubated with 4-OHT (1 μg/ml) for 4 days and subjected to WB

analysis. D, Silica induced rapid activation of Erk in Ubc-Cre-ER; NF2f/f MEF. After elimination of

NF2 through 4 day-incubation with or without 4-OHT, MEF cells were treated with 30 μg/ml of silica

for indicated time. Loss of NF2 induced the RKIP and TβR2 reduction. E, NF2 extended the TβR2

half-life, and promoted TβR1 turn-over. HEK293 cells were transfected with indicated vectors for 24

hr. Protein half-life was measured after treatment of CHX (cycloheximide; 100 μg/ml) for indicated

time. F, Transfection of RKIP or NF2, which induce TβR2 expression, blocked the cell migration in

response to silica, such as TβR2 overexpression. In the transwell migration assay, exposure to silica

(10 μg/ml) was for 60 hr. More detailed pictures are provided in Fig. S5A. G, Silica or pressure

induced the mesenchymal marker Vimentin. HCT116 cells, incubated under cover-slip or with silica

for 72 hr, were fixed and incubated with anti-Vimentin antibody and FITC-conjugated secondary

antibody (Green). DAPI was used as the DNA stain. H, Re-expression of TβR2 into HCT116 blocked

the silica-induced NF2 and RKIP reduction. Cells were transfected with indicated vectors for 24 hr.

Cells were subjected to silica exposure and pressure under SF conditions for the indicated

concentrations and time. In WB assay (A-E and H), Actin was used as the loading control for protein

expression. The number under bands indicates the ratio of each protein expression to actin,

determined by image j software.

Figure 3.




33

TβR1 kinase activity is required for RKIP and NF2 suppression. A, RKIP was interacted with TβR1.

Bead conjugated GST-RKIP was incubated with cell lysates and transfected with the indicated vector.

After 0.5 hr incubation under rotation, co-precipitated materials with GST-RKIP were detected by WB

analysis. PPT and SUP indicate precipitates and supernatants, respectively. B, TβR1 kinase inhibitors

induced RKIP and NF2 in HCT116 (TβR2-deficient cells). Cells were transfected with the indicated

vectors for 24 hr and then incubated with TEW7197 (10 μΜ) and SB431542 (10 μΜ) for additional

24 hr. The number under bands indicates the ratio of each protein expression to actin, determined by

image j software. C, TEW7197 blocked the silica-induced NF2 and RKIP reduction. In addition,

TEW7197 induces p53 expression. TEW7197 (10 μΜ) was treated for 12 hr in SF condition, followed

by exposure to silica for the indicated times and concentrations. The number under bands indicates the

ratio of each protein expression to actin, determined by image j software. D, TβR1 kinase inhibitors

blocked the interaction between RKIP and TβR1 and increase the NF2 and RKIP interaction. GST

pull-down assay was performed using bead-GST-RKIP and transfected cell lysates with the indicated

vectors. PPT and SUP indicate the precipitates and supernatants, respectively. The number under

bands indicates the ratio of each protein expression to RKIP (GST), determined by image j software.

E, RKIP was phosphorylated by TβR1. In vitro kinase assay was performed using the GST-RKIP

protein. '+ S' indicates a cell lysate to which silica was added after lysis. 'S4h' indicates a cell lysate

treated with silica (4 hr) before lysis. Right panel presents a brief scheme of this experiment. F, RKIP

T101A (RKIP T/A), the resistant form of TβR1-mediated phosphorylation, was not reduced by silica

and blocked the NF2 reduction. This mutant also blocked the p53 reduction. Actin was used as

loading controls for protein expression. HCT116 cells were transfected with indicated vectors for 24

hr. Silica was treated for the indicated times and concentrations. T/A and T/D indicates RKIP mutant

forms in which the threonine 101 residue is substituted with an alanine (A) or aspartic acid (D).

Figure 4.

The effect of TβR1 kinase inhibitors on NF2-deficient cells. A, Treatment of TEW7197 induced RKIP




34

and p53 expression in Schwannoma (HEI-193), mesothelioma (H28) and HCT116. TEW7197 was

treated with the indicated concentration for 48 hr in SF condition. B, TEW7197 blocked the TβR1-

induecd RKIP reduction and induced the NF2 and p53 expression in HCT116. HCT116 cells

transfected with FLAG- TβR1 were incubated with silica for 24 hr, with or without 12 hr pre-

treatment with TEW7197 (10 μM). Actin was used as the loading control. L.E and S.E indicate long

exposure and short exposure, respectively. C, TEW7197 sensitized to DNA damaging agent

(Adriamycin; Adr) induced p53 expression in mesothelioma cell lines (H28). TEW7197 (10 μM) was

treated for 12 hr, followed by Adriamycin (0.1, 0.2 μg/ml) treatment for 6 hr in SF condition. D,

Synergisticc cytotoxic effect with Adriamycin was seen only after exposure to TβR1 kinase inhibitor

in mesothelioma cell line. All chemicals were treated for 48 hr in SF condition. Cell viability was

measured by MTT assay. Asterisk indicates statistical significance (P<0.05, Student’s t-test) and N.S

indicates not significant. E, TEW7197 induced cell death in NF2-deficient Schwannoma cell line

(HEI-193). TβR1 kinase inhibitors were more effective than RAD001 (Anti-Neurofibromatosis Type

2 drug) in HEI-193 cell. All chemicals were treated for 72 hr in SF condition. Cell viability was

measured by MTT assay. Asterisk indicates statistical significance (P<0.05, Student’s t-test). F,

Induction of p53 and RKIP by TEW7197 in HEI-193. Cells were incubated with the indicated

concentration of TEW7197 for 24 hr. G, The effect on cell viability of various TβR1 inhibitors.

Although Certinib and Crizotinib induced obvious cell death, they also showed similar cytotoxicity in

normal cells (Fig. S9C and D). H, Among the tested chemicals, the TEW7197-induced RKIP was

most apparent. HEI-193 cells were incubated with the indicated chemicals for 24 hr. Actin was used

as loading control for protein expression.

Figure 5.

Therapeutic effect of TEW7197 in the NF2 model mouse. A, Restoration of NF2 and TEW7197

treatment showed similar gene expression profile in lipid metabolism. To evaluate the effect of

TEW7197 on global gene expression, we performed the microarray using HEI-193 and NF2-




35

transfected or TEW9197 treated HEI-193. Among the differentially expressed genes (1.5 fold cut-off;

EV-transfected HEI-193 (HEI-193) vs NF2 transfected HEI-193 (NF2), HEI-193 vs TEW7197 treated

HEI-193 (TEW7197 12 hr or TEW7197 24 hr), 1183 genes were commonly detected. Many of them

were involved in lipid metabolism such as cholesterol biosynthesis and adipose tissue related gene set

(See Fig EV11; 24, 25). B, Gene expression was confirmed by RT-PCR. Representative 5 genes were

assessed to confirm the microarray data. GAPDH was used as the loading control. C, TEW7197

induced cell differentiation of HEI-193. Cells treated with TEW7197 spread and obviously reduced

cell proliferation. D, Reduction of TβR2 in dorsal root ganglion (DRG) of NF2 syndrome model

mouse (Postn-Cre;NF2f/f). To confirm our hypothesis, we dissected the DRG and performed the IHC

with TβR1 and TβR2 antibody. Compared to the wild type mouse (WT), we detected reduction of

TβR2 in Postn-Cre;NF2f/f mice(in particular Schwan cells). DRG was isolated from 6 month old mice.

E, Through PET/CT analysis, we found that TEW7197 suppresses the tumor formation. Postn-

Cre;NF2f/f (FVB/NJ) mice (3 month-old, N=9) mice were intraperitoneal (i.p.) injected with vehicle

(N=4) and TEW7197 (5 mg/kg; N=5), 3 times a week for 6-8 weeks. Compared to the vehicle-treated

mice, the number of tumors (red arrows) were reduced after TEW7197 treatment. F, Histological

analysis: consistent with the PET-CT analysis, tumors were found in 23 week-old Postn-Cre;NF2f/f

mouse (vehicle, Male). In contrast, no apparent tumors were seen in TEW7197 treated mice (30

week-old male). G, Diagram. Under healthy conditions, physical stress or contact did not activate

TβR1 due to the inhibitory effect of TβR2. However, under NF2-deficient or TβR2 suppressed

conditions, extracellular physical stresses may activate TβR1 kinase and reduce RKIP expression.

Consequentially, activated MAPK and Snail promote tumor formation via p53 suppression, E-

cadherin reduction and cell cycle promotion. Thus, inhibition of TβR1 is a potential therapeutic

strategy for NF2 syndrome as well as TβR2 defected cancers.




Published OnlineFirst August 22, 2018.Mol Cancer Ther Jung-Hyun Cho, Ah-Young Oh, Soyoung Park, et al.

R1 inhibitor on NF2 syndromeβeffect of T signaling; Implication of Therapeuticβand oncogenic TGF-

receptor 1 mediated Non-canonicalβLoss of NF2 induces TGF-

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