Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β

E XP ER I ME NTAL C E LL RE S E ARCH

] (]]]]) ]]]–]]]

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Research Article

Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β Sunita Chopra, Neeraj Kumar, Annapoorni Rangarajan, Paturu Kondaiahn Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India

article information

abstract

Article Chronology:

Actions of transforming growth factor-β are largely context dependent. For instance, TGF-β is

Received 22 December 2014

growth inhibitory to epithelial cells and many tumor cell-lines while it stimulates the growth of

Received in revised form

mesenchymal cells. TGF-β also activates fibroblast cells to a myofibroblastic phenotype. In order

2 March 2015

to understand how the responsiveness of fibroblasts to TGF-β would change in the context of

Accepted 4 March 2015

transformation, we have compared the differential gene regulation by TGF-β in immortal

Keywords:

cell-line (HT1080). The analysis revealed regulation of 6735, 4163, and 3478 probe-sets by TGF-β

β-catenin

in hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively. Intriguingly, 5291 probe-sets were

fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and a human fibrosarcoma

Calcium

found to be either regulated in hFhTERT or hFhTERT-LTgRAS cells while 2274 probe-sets were

ERK1/2

regulated either in hFhTERT or HT1080 cells suggesting that the response of immortal hFhTERT

P38

cells to TGF-β is vastly different compared to the response of both the transformed cells hFhTERT-

Transformation

LTgRAS and HT1080 to TGF-β. Strikingly, WNT pathway showed enrichment in the hFhTERT cells in Gene Set Enrichment Analysis. Functional studies showed induction of WNT4 by TGF-β in hFhTERT cells and TGF-β conferred action of these cells was mediated by WNT4. While TGF-β activated both canonical and non-canonical WNT pathways in hFhTERT cells, Erk1/2 and p38 Mitogen Activated Protein Kinase pathways were activated in hFhTERT-LTgRAS and HT1080 cells. This suggests that transformation of immortal hFhTERT cells by SV40 large T antigen and activated RAS caused a switch in their response to TGF-β which matched with the response of HT1080 cells to TGF-β. These data suggest context dependent activation of non-canonical signaling by TGF-β. & 2015 Published by Elsevier Inc.

Introduction

components of TGF-β signaling axis are found to be mutated in different types of cancers like colon, gastric, pancreas, breast and

TGF-β plays dual roles in cancer. TGF-β is a known inhibitor of epithelial cell growth and helps maintain homeostasis in a normal tissue context [1]. Consistent with its tumor suppressive role, several

other solid tumors [2–5]. In contrast, cancers harboring an intact TGFβ pathway are refractory to the growth inhibitory effects of TGF-β and become more aggressive resulting in poor prognosis of the patients. Increased TGF-β expression by tumor cells correlated with tumor

n

Corresponding author. Fax: þ91 80 23600999. E-mail address: [email protected] (P. Kondaiah).

progression in non-small cell lung carcinoma (NSCLC), colorectal cancer, prostate cancer, and gastric carcinoma. Additionally, intense

http://dx.doi.org/10.1016/j.yexcr.2015.03.001 0014-4827/& 2015 Published by Elsevier Inc.

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

2

E XP E RI ME N TAL CE L L R ES E ARC H

TGF-β staining has also been positively correlated with metastasis in breast carcinoma, prostate cancer, and colorectal cancer [6–9]. Of late an increasing amount of data detailing the role of tumor microenvironment in invasion and metastasis has been reported. The tumor microenvironment principally comprises of activated fibroblasts producing several growth factors and cytokines which help the tumor cells to become more aggressive [10,11]. An important cytokine present in the tumor micro-environment is TGF-β. CAFs (cancer associated fibroblasts) or activated fibroblasts isolated from xenograft breast tumors are found to have an active TGF-β signaling [12]. Fibroblasts on treatment with TGF-β undergo trans-differentiation into myofibroblasts (activated fibroblasts) [13–15]. Thus activated, fibroblasts have a higher proliferation rate; secrete various cytokines, collagens, matrix metallo-proteases and other components of the ECM which aid the cancer cells in invasion [10]. Contrary to its growth inhibitory effects on epithelial cell growth, TGF-β stimulates the growth of mesenchymal cells in cultures [16–18]. All these data point to a differential role of TGF-β carried out by signaling pathways activated differentially depending on the context. In the canonical TGF-β signaling pathway, ligand binds to the constitutively active serine–threonine kinase receptor, TβRII, comma causing it to heterodimerize and to activate another serine–threonine kinase receptor TβRI (ALK5). After activation, the TβRI phosphorylates the Smad2 and Smad3 proteins which bind Smad4 and translocate to the nucleus. The Smad dependent pathway has been implicated in both pro- and anti-tumor actions of TGF-β [19,20]. Many Smad independent signaling pathways influenced by TGF-β have also been identified [21]. TGF-β has been shown to activate MAPK, WNT, and PI3K-AKT etc signaling pathways in different contexts [21–25]. But what decides the context and how the context defines the distinct TGF-β responses is still open for investigation. A previous study has shown that TGF-β treatment led to an activation of c-Abl receptor tyrosine kinase in fibroblast cultures but not in the epithelial cultures [26,27]. But not much is known about such “differential” activations and how they might contribute to the distinct outcomes. Could transformed or the normal status of a cell act as a context for TGF-β to activate different signaling pathways? Would TGF-β activate different pathways in normal epithelial, fibroblast and tumor cells? One way to address these questions is to delineate the TGF-β regulated genes in different fibroblast, epithelial and transformed cells and reconstruct the signaling pathways which could lead to such gene expression changes. In the, past several studies have identified genes regulated by TGF-β using microarray approach [28–30]. However, differential regulation of genes by TGF-β in normal and tumor cells is not clearly understood. A study by our group in which a comparison of gene expression profiles in immortalized lung epithelial cells with the A549 lung carcinoma cells has been reported in which a major conclusion was on the proposed role of MAPK signaling in the differential regulation of genes by TGF-β in normal versus tumor cells [31]. Another group has identified matrix remodeling genes as differentially regulated by TGF-β in primary mouse keratinocytes versus HaRas transformed keratinocytes [32]. However there has been no attempt to study the differential gene expression by TGF-β in fibroblasts as compared to their transformed counterparts. Hence, in order to identify the differential actions of TGF-β in normal and transformed fibroblasts, in the present work, we have studied the differential gene expression changes and signaling pathways activated by TGF-β in immortal (hFhTERT) and transformed (HT1080 and hFhTERT-LTgRAS) fibroblast cells. We report that TGF-β treatment has contrasting responses on these cells. Most importantly, our

] (]]]]) ]]]–]]]

findings revealed activation of MAPK signaling pathways in transformed cells as opposed to the activation of canonical WNT and noncanonical WNT signaling pathways in the immortal fibroblasts by TGF-β.

Materials and methods Cell-lines, maintenance and treatments HT1080 (dermal fibrosarcoma, ATCC) [33], hFhTERT (human foreskin fibroblasts immortalized with human terminal telomerase) [34], hFhTERTLTgRAS (hFhTERT transformed with SV40 large T antigen and activated RAS) [34], primary hF cells [34], hFF (primary human foreskin fibroblasts) [35] and NIH3T3 (mouse embryonic fibroblasts, ATCC), cells were cultured in DMEM (Sigma Aldrich, USA) supplemented with 10% Fetal Bovine Serum (FBS), 100 units/ml penicillin and 100 mg/ml streptomycin (Invitrogen Life Sciences, USA). All the cell-lines were maintained at 37 1C in a humid atmosphere with 5% CO2. For all treatments cells were grown to 80% confluence in the growth media followed by serum free washes (3 times) for 24 h. rhTGF-β1 (R&D systems, USA) and EGF (Sigma Aldrich, USA) treatments were given at 5 ng/ml and 20 ng/ml respectively. The MAPK pathway inhibitors used were SB203580 (p38 inhibitor) and PD98059 (ERK1/2 inhibitor), both from Sigma Aldrich, USA. SB 431542 (SigmaAldrich, USA) was used to block TβRI while SIS3 (Sigma-Aldrich, USA)

Fig. 1 – TGF-β treatment led to induction of TGF-β responsive luciferase activities in hFhTERT, hFhTERT-LTgRAS and HT1080 cells. (A) pSBE-lux and (B) p3TP-lux reporter assay: Y-axis shows the relative luciferase units (firefly/renilla) before and after TGF-β treatment. Each bar represents mean7SE of triplicate samples. For statistical significance student's unpaired t-test was used (*, **, and *** represent po0.05, o0.01 and o0.0001 respectively).

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

was used to block the Smad3 signaling. All the inhibitor treatments were given at 10 mM concentration for one hour prior to giving TGF-β treatments.

RNA isolation and purification For isolating RNA for both microarray and RT-PCR analysis, TGF-β1 treatment was given for 1, 4 and 12 h. Untreated cells served as controls for each time point. Total RNA was isolated from cells using TRI reagent (Sigma-Aldrich, USA) according to manufacturer's protocol and further purified using RNAeasy columns (Qiagen, GmbH, Germany). The RNA quantity and quality were assessed by OD260 and OD280 measurements on a spectrophotometer (Nanodrop ND1000 from Thermo-scientific, USA) and the integrity was determined on a MOPS-formaldehyde denaturing agarose gel.

3

] (]]]]) ]]]–]]]

E X PE R IM EN TA L C ELL R E S EA RC H

Microarray labeling, hybridization and data analysis Agilent Low RNA Input Linear Amplification Two-Color Kit PLUS (Agilent Technologies, USA) was used to label the samples with cyanine 3- or cyanine 5-labeled CTP as per the manufacturer's protocol. Briefly, total RNA is first converted to cDNA using primers containing T7 polymerase binding sequence and oligo dT. The resulting cDNA is further used for transcription by T7 RNA polymerase which amplifies the amount of input RNA incorporating Cy-3 and Cy-5 tagged CTP. cRNA from the control samples was labeled with Cy3-CTP and cRNA from the TGF-β1 treated samples was labeled with Cy5-CTP. The labeled cRNA was purified using RNAeasy columns (Qiagen, GmbH, Germany) and quantified using Spectrophotometer (Nanodrop ND1000 from Thermo-scientific, USA). Equal amounts of Cy3-CTP and Cy5-CTP labeled cRNA from each time point was mixed in hybridization buffer and then hybridized onto 4  44K Agilent whole genome human oligonucleotide microarray slides according to the manufacturer's protocol.

Fig. 2 – Expression profiling of TGF-β target genes in hFhTERT (A), hFhTERT-LTgRAS (B) and HT1080 (C) cells. Microarrays were hybridized with labeled cRNAs isolated from cells treated with TGF-β for 1, 4 and 12 h with respective untreated samples serving as controls. All the arrays were in duplicates. Hierarchical clustering was done using Euclidean distance metric.

Table 1 – Probe-sets differentially regulated in hFhTERT, hFhTERT-LTgRAS and HT1080 cells after 1, 4 and 12 h of TGF-β treatment. (U – Upregulated; D – downregulated, by TGF-β compared to untreated controls). Probe-sets differentially regulated by TGF-β ( Z1.5 fold/ r -1.5 fold and po0.05)

1h 4h 12 h

hFhTERT

hFhTERT-LTgRAS

HT1080

356Uþ1282D 948Uþ1750D 2029Uþ2023D

398Uþ617D 1122Uþ1507D 605Uþ441D

170Uþ254D 477Uþ1099D 639Uþ1318D

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

4

E XP E RI ME N TAL CE L L R ES E ARC H

The slides were incubated at 60 1C at 10 rpm on rotisserie in the Agilent hybridization oven for 17 h. The slides were then washed with Gene Expression Wash Buffer 1 and 2 for 1 min each, dried and scanned. Scanning was done in the Agilent Scanner. Intensity values were extracted from the scanned images using Agilent Feature Extraction Software. The spots of compromised quality and low intensity were omitted from the analysis. Data was normalized using LOWESS algorithm using limma package in R-conductor [36]. Cy5:Cy3 intensity ratios were calculated. TMeV software was used to identify statistically significant genes [37].

Real time PCR analysis For cDNA synthesis, two micrograms of total RNA was reverse transcribed using the ABI cDNA Archive kit (Applied Biosystems, USA). The sequences of the primers used are shown in Supplementary Table 1. All the PCR reactions have been done using Dynamo SYBR green mix (Finnzymes, Finland) in ABI Prism 7900HT sequence

] (]]]]) ]]]–]]]

detection system (Applied Biosystems, USA). The analysis has been done using SDS 2.1 software (Applied Biosystems, USA). For normalization of RT-PCR data, RPL-35A expression [31] was used.

Western Blot analysis To monitor endogenous protein levels, cells were harvested in lysis buffer containing 50 mM Tris–Cl pH-7.5 (Sigma-Aldrich, USA), 150 mM NaCl (Sigma-Aldrich, USA), 0.1% SDS (Calbiochem, Germany), 0.5% NP40 (Amresco, USA) and protease inhibitor cocktail, PIC (Calbiochem, Germany). Total protein content in the cell lysates was estimated by Bradford's assay (Biorad, USA). 50–100 mg of total protein was loaded onto 10–12.5% SDS-PAGE gels and transferred onto PVDF membranes (ImmobilonP, Millipore Corporation, Germany) by using wet electrophoresis Biorad transfer apparatus. After the transfer, membranes were blocked for an hour at RT with 5% nonfat dry milk (Fluka, Switzerland) in TBS (containing 0.1% Tween-20, Sigma-Aldrich USA) and incubated overnight at 4 1C with primary

Fig. 3 – Hierarchical clustering of genes differentially regulated by TGF-β between (A) hFhTERT and hFhTERT-LTgRAS cells, and (B) hFhTERT and HT1080 cells. 2-Way ANOVA was performed on the normalized log2transformed Cy5/Cy3 ratios on all samples of HT1080 and hFhTERT/hFhTERT-LTgRAS and hFhTERT separately to find out the differentially regulated genes. Data was clustered using Complete Linkage Rule. Hierarchical clustering was done using Euclidean distance metric. (C) Hierarchical clusters of 786 probe-sets commonly differentially regulated by TGF-β in hFhTERT-LTgRAS vs. hFhTERT and HT1080 vs. hFhTERT cells. Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

E X PE R IM EN TA L C ELL R E S EA RC H

5

] (]]]]) ]]]–]]]

Table 2 – Probe-sets induced by TGF-β in hFhTERT cells and marginally induced, not regulated or repressed by TGF-β in HT1080 and hFhTERT-LTgRAS cells. Gene name

TSPAN2 ANKRD38 FZD8 NOX4 PLN TSPAN2 CTGF BQ130701 THC2385873 WNT4 ESM1 CTPS TMEM49 IGF1 AF086187 CR600305 KLRC1 SOX9 NGEF AI379175 CA423842 FOXA2 RINT1 RASGRF1 KCTD4 OLFML2B

hFhTERT

HT1080

hFhTERT-LTgRAS

1h

4h

12 h

1h

4h

12 h

1h

4h

12 h

0.12 0.50 0.62 0.28 0.06 0.13 1.28 0.31 1.68 0.07 0.16 0.13 0.70 0.24 0.05 0.10 0.18 0.45 0.44 0.05 0.58 0.91 0.14 0.49 1.49 0.01

4.41 5.17 2.81 4.24 0.21 2.85 4.27 3.81 3.40 4.01 2.81 2.18 3.65 2.13 1.13 0.75 0.53 0.51 0.41 0.91 0.94 0.36 0.07 0.31 1.10 1.38

6.47 6.00 5.24 5.23 5.01 4.97 4.72 4.15 3.99 3.84 3.82 3.75 3.72 3.64 3.61 3.58 3.40 0.86 1.32 1.70 0.00 1.32 0.61 1.19 0.45 1.86

0.16 0.72 0.08 0.20 0.07 0.08 0.39 0.07 0.50 0.09 0.24 0.18 0.13 0.35 0.32 0.31 0.11 0.43 0.24 0.09 1.70 0.64 0.18 0.12 0.54 0.04

0.27 0.20 0.21 1.31 0.05 0.50 1.76 1.33 0.55 0.27 0.11 0.33 0.48 0.29 0.09 0.17 0.03 0.96 0.07 0.38 0.45 1.27 0.11 0.02 0.08 0.23

1.58 0.00 0.24 2.79  0.81 2.16 1.27 1.33  1.01  1.31 1.16 0.14 0.63 0.41 0.42  0.02  0.49  1.20  0.18 0.07  2.69  0.93 0.09  0.20  1.23  0.11

 0.24 0.09 0.24  0.14  0.27 0.44  0.05 0.56 0.19 0.04  0.23  0.14  0.41 0.05 0.25 0.05  0.23  0.21  0.13  0.21  0.66  0.90  0.78  0.36  0.38 0.53

0.34 0.05 0.07 0.02 0.35 0.10 0.22 1.69 0.01 0.28 0.03 0.16 0.57 0.21 0.19 0.02 0.05 0.79 0.36 0.55 0.52 1.53 0.28 0.02 0.24 0.43

 0.09 0.41  0.23 0.13  0.12  0.45 0.26 1.75 0.21 0.17 0.10 0.17  0.32  0.11 0.06 0.59 0.06  0.73  0.72  0.71  0.66  0.63  0.62  0.61  0.56  0.55

Note: The log2 fold change 40.58/o 0.58 is shown in bold.

Table 3 – Probe-sets repressed by TGF-β in hFhTERT cells and not regulated or induced by TGF-β in HT1080 and hFhTERTLTgRAS cells. Genes

KIT KIAA1199 BDKRB2 LOC285016 TMTC1 PLAU ADRB2 NPTX1 LMO1 MAF NOTCH1 ID2 TIAM2 KRTAP3 3 SMAD6 ID2 AF229166 VASN C8orf47

hFhTERT

HT1080

hFhTERT-LTgRAS

1h

4h

12 h

1h

4h

12 h

1h

4h

12 h

0.09 0.04 0.03 0.49 0.02 0.25 0.10 0.08 1.06 0.18 0.45 0.52 0.12 0.34 0.45 0.83 0.62 0.28 0.03

3.14 1.72 1.90 0.92 1.18 1.71 1.20 0.21 0.46 0.67 2.21 0.96 1.65 0.95 0.54 0.92 1.29 0.84 0.80

4.02 3.88 2.49 2.47 2.25 1.73 1.65 1.60 1.51 1.50 1.38 1.17 1.16 1.04 1.01 0.94 0.86 0.63 0.54

0.83 0.04 0.44 0.45 0.97 0.44 0.84 0.36 0.40 0.16 0.37 1.81 0.08 1.08 1.15 2.25 0.50 0.67 2.38

0.11 0.68 1.10 1.31  0.40 0.55  0.93 0.67 1.64 1.77  0.33 1.77 1.54  0.11 2.33 1.70  0.30 1.68 0.12

1.67 0.86 2.26 1.12  0.54 0.80  0.79 2.35 2.36 2.40  0.36 1.07 1.40 0.76 0.98 1.14  0.43 1.89 0.97

0.84 0.25  0.10 0.33  0.35 0.15 0.45 0.44 0.16 0.08 0.19 2.56 0.00 0.43  0.01 2.49 0.64 0.93 0.34

0.78 0.71 0.18  0.13  0.10  0.13 0.67 0.41 0.29 0.18 0.18 0.03 0.53 1.00 0.08  0.39 1.01 0.38 0.80

0.34 0.41 0.15 0.12 0.05 0.26 0.24 0.30 0.07 0.32 0.27 0.46 0.51 0.57 0.16 0.35 0.50 0.48 0.40

Note: The log2 fold change 40.58/o 0.58 is shown in bold.

antibodies. Secondary antibodies were incubated for 45–60 min at 25 1C. Membranes were probed with Super Signal West Femto Enhanced Luminiscence Kit (Thermo-scientific, USA). ERK1/2, Phospho-

ERK1/2, p38, Phospho-p38, Smad2/3, and Phospho-Smad2 antibodies were procured from Cell Signaling Technologies, USA. β-actin and β-catenin antibodies were purchased from Sigma Aldrich, USA. Anti-

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

6

E XP E RI ME N TAL CE L L R ES E ARC H

α-SMA antibody was procured from Abcam. HRP-conjugated goat anti-mouse (Sigma-Aldrich, USA) or HRP-conjugated goat anti-rabbit (Sigma Aldrich USA) secondary antibodies were used.

Immunocytochemistry For immunocytochemical studies, cells were grown on sterile glass coverslips in 12 well culture plates till they reached 50% confluence. After the TGF-β treatment, cells were washed with chilled phosphate buffered saline (DPBS, Invitrogen, USA) and fixed in methanol at 20 1C for 10 min. Blocking was done in 10% FBS in PBS for 60 min followed by overnight incubation with β-catenin or α-SMA antibody in a humidified chamber at 4 1C. Cells were washed thrice with PBS after the overnight incubation and kept in anti-rabbit secondary antibody, Alexa Flor 488 (Invitrogen, USA) in PBS for 1 h. After that, cells were again washed thrice and counterstained with 1 mg/ml of propidium iodide (Sigma Aldrich, USA) for 15 min and mounted in the VectaShield (Invitorgen, USA) solution on clean slides. The fluorescence was visualized under the confocal microscope (Zeiss LSM, GmbH, Germany) and photographed.

Table 4 – Pathways enriched in hFhTERT cells compared to hFhTERT-LTgRAS cells: the differentially regulated gene-list was fed into the KEGG pathway analysis and pathways enriched in hFhTERT cells compared to hFhTERT-LTgRAS cells are listed below. Pathways enriched in hFhTERT cells compared to hFhTERTLTgRAS cells

Size

MAPK signaling pathway Cytokine cytokine receptor interaction Regulation of actin cytoskeleton WNT signaling pathway Neuroactive ligand receptor interaction JAK STAT signaling pathway T cell receptor signaling pathway PPAR signaling pathway Adipocytokine signaling pathway Hedgehog signaling pathway

103 97 78 72 65 58 44 32 32 26

] (]]]]) ]]]–]]]

INDO-1-AM assay for detection of intracellular Ca2þ The UV-excitable calcium indicator, Indo-1-AM {1-[2-amino-5-(6carboxy-2-indolyl)phenoxy]-2-(2-amino-5-methylphenoxy ethane-N, N,N0 ,N0 -tetraacetic acid, pentaacetoxymethyl ester} is a cell permeable and Ca2þ dependent ratiometric dye. Upon excitation at 355 nm, a shift in the emission spectra of Indo-1-AM is observed from 480 nm to 408 nm when bound to Ca2þ. Cells to be analyzed for Ca2þ levels after TGF-β treatment were plated in 6 well plates and serum starved when they reached 80% confluence. TGF-β treatment was given for 12 h after which cells were trypsinized and treated with 2 mM Indo-1-AM (Molecular Probes, Invitrogen, USA) in the presence of 0.2% Pluronic acid F-127 (Molecular Probes, Invitrogen, USA) detergent for 30 min at 37 1C and 5% CO2 in dark with frequent tapping. Cells were washed thrice with ice-cold 17 mM potassium phosphate buffer (pH 6.4) at 4000 rpm at 4 1C. Cells were then re-suspended in minimum volume of potassium phosphate buffer and mounted on a glass slide with a drop of water on the cover slip. Cells were excited with UV light of 355 nm and the fluorescence of Ca2þ bound Indo-1-AM was observed at 405 nm using an inverted fluorescence microscope (Olympus DSU, USA).

Table 6 – Pathways enriched in hFhTERT cells compared to HT1080 cells: the differentially regulated gene-list was fed into the KEGG pathway analysis and pathways enriched in hFhTERT cells compared to HT1080 cells are listed below. Pathways enriched in hFhTERT cells compared to HT1080 cells

Size

MAPK signaling pathway WNT signaling pathway Natural killer cell mediated cytotoxicity JAK STAT signaling pathway p53 signaling pathway MTOR signaling pathway PPAR signaling pathway Adipocytokine signaling pathway

87 48 42 41 27 20 20 16

Table 5 – Pathways enriched in hFhTERT-LTgRAS cells compared to hFhTERT cells: the differentially regulated gene-list was fed into the KEGG pathway analysis and pathways enriched in hFhTERT-LTgRAS cells compared to hFhTERT cells are listed below.

Table 7 – Pathways enriched in HT1080 cells compared to hFhTERT cells: the differentially regulated gene-list was fed into the KEGG pathway analysis and pathways enriched in HT1080 cells compared to hFhTERT cells are listed below.

Pathways enriched in hFhTERT-LTgRAS cells compared to hFhTERT cells

Size

Pathways enriched in HT1080 cells compared to hFhTERT cells

Size

Pathways in cancer Calcium signaling pathway Chemokine signaling pathway Cell cycle Insulin signaling pathway Neurotrophin signaling pathway TGF beta signaling pathway TOLL like receptor signaling pathway p53 signaling pathway ERBB signaling pathway VEGF signaling pathway MTOR signaling pathway

170 68 67 62 62 48 46 37 37 35 32 23

Pathways in cancer Cytokine cytokine receptor interaction Focal adhesion Endocytosis Regulation of actin cytoskeleton Chemokine signaling pathway Calcium signaling pathway Cell cycle Neurotrophin signaling pathway Insulin signaling pathway TGF beta signaling pathway ERBB signaling pathway

113 67 61 58 57 51 48 46 43 41 36 36

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

E X PE R IM EN TA L C ELL R E S EA RC H

Dual luciferase assay DLR assay system (Promega) was used to perform dual luciferase assays. Briefly, cells are grown in 12 well plates and transfected with 0.5 mg either of the pSBE-lux [38], p3TP-lux [39] or the pTOP-FLASHlux (Upstate, Cell Signaling Solutions, USA) along with 50 ng of the pRL-TK (renilla luciferase) construct for 6 h in plain DMEM using lipofectamine (Invitrogen, USA) reagent. Cells are lysed after the respective treatments are over in 200 ml of 1  passive lysis buffer. Both firefly and renilla readings were taken in a luminometer (TD-20/ 20, Turner Designs). The ratio of firefly and renilla (relative luciferase units, RLU) was calculated and plotted.

Retrovirus mediated transduction in hFhTERT cells HEK293T cells were grown in 60 mm dishes to 80% confluence and tranfected with either the pBABE-puro-empty vector or the pBABEpuro-WNT4 (1 μg) along with helper plasmids, pUMVC3 (900 ng) and pVSVG (100 ng) using lipofectamine (Invitrogen Life Sciences, USA) for 6 h in plain DMEM. After 6 h plain DMEM was removed and 3 ml of reconstituted media was added. 30 h later the spent media was discarded and 1.5 ml of fresh reconstituted media was added and spent media containing viral particles was collected every 12 h for three times. The spent media was passed through a 0.4 mm filter before adding to hFhTERT cells growing in 60 mm dishes. 6 h later media was removed and fresh reconstituted media was added. 48 h later puromycin (1 mg/ml) selection was started. After all the mock transduced cells had died, the EV and WNTO.E. cells were plated for

7

] (]]]]) ]]]–]]]

RNA and immunocytochemical studies. For knockdown of WNT 4 also, a similar lentiviral mediated transduction approach was used. shRNAs against WNT4 in a pGFP-V-RS backbone (Origene Technologies Inc.) were used for the same.

Results Comparison of TGF-β induced gene expression profiles between hFhTERT, hFhTERT-LTgRAS and HT1080 cells TGF-β is known to have a growth inhibitory effect on epithelial cells and several tumor cell-lines while it stimulates the growth of fibroblasts. In our previous study we had shown that TGF-β induced gene expression profiles are different in immortal epithelial cells when compared to carcinoma cells [31]. Presently we have looked at the differential response of immortal fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and fibrosarcoma (HT1080) tumor cells to TGF-β. All the three cell lines responded to the TGF-β treatment as indicated by the induction of TGF-β responsive luciferase constructs, pSBE-lux and p3TP-lux (Fig. 1). For undertaking microarray analyses, each of these cell types was treated with TGF-β for a period of 1, 4, and 12 h. Untreated cells served as control at each of the time-points. The probe-sets which were either significantly up or down regulated (1.5 fold; Pr0.05) in the TGF-β treated cells compared to the respective untreated cells at any of the three time points were considered for further analysis. The data has been submitted to the GEO database (Accession no. GSE 40315). A total of 6735, 4163, and 3478 probe-sets showed regulated expression in

Table 8 – Components of the MAPK pathway regulated by TGF-β in hFhTERT cells compared to the hFhTERT-LTgRAS and the HT1080 cells. Gene name

hFhTERT

hFhTERT-LTgRAS

HT1080

Gene name

hFhTERT

hFhTERT-LTgRAS

HT1080

RPS6KA5 MAP3K5 TGFB3 RASA2 MAP2K6 FGF13 DUSP1 IL1B PLA2G4A MAP4K4 CACNG5 PAK1 RPS6KA5 RAC2 RASGRP2 MAPK12 PRKACA PPP3CC AKT1 FGF7 ELK1 ARRB2 MYC MKNK2 MAPK3 MAPK11 MAPK14

2.87 2.09 2.05 1.94 1.93 1.88 1.55 1.53 1.44 1.07 1.03 1.02 1.01 1.00 0.97 0.96 0.93 0.89 0.87 0.83 0.76 0.72 0.64 0.63 0.62 0.61 0.59

– – – – – –  0.81 – – – – – – – – – – – – – – – – – – – –

– – – – – – – –  0.71      0.72 – – – – – – – – – – – – –

FLNB PPP3CA FAS FGFR1 CASP3 RELB HSPB1 MAPK7 HSPA8 NR4A1 TGFB2 ZAK FOS HSPB1 MAP3K2 TGFB1 MRAS FASLG CACNA1C SRF FGF2 FGF18 GADD45B GADD45A NFKB2 PRKACB RASGRP1

0.62 0.63 0.65 0.66 0.70 0.72 0.74 0.77 0.82 0.87 0.90 0.95 0.98 1.01 1.03 1.13 1.33 1.34 1.39 1.77 1.98 3.21 4.49 – – – –

– – 0.60 – – – – –

– – – – – – – – – 0.83 – – – – – – 1.6 – – – 0.97 0.95 1.91 – – 0.85 1.02

1.14 – – – – – – – – – 1.02 – – – 1.01 0.84 – –

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

8

E XP E RI ME N TAL CE L L R ES E ARC H

] (]]]]) ]]]–]]]

Fig. 4 – TGF-β activates ERK1/2 and p38 MAPK pathways in HT1080 and hFhTERT-LTgRAS cells but not in the fibroblast cell-lines, hFhTERT and NIH3T3 or in the primary hF cells. (A) Lysates of hFhTERT and HT1080 cells treated with TGF-β1 (5 ng/ml) for 1, 6, 12, 24 h and respective untreated controls were subjected to immunoblotting with anti-phospho ERK1/2 and anti-phospho p38 antibodies. (B) Cell lysates from hFhTERT-LTgRAS, hFhTERT, NIH3T3, and hF cells treated with TGF-β1 (5 ng/ml), EGF (20 ng/ml) for 12 h and UV light for 30 min along with untreated controls were subjected to immunoblotting and probed for phospho-ERK1/2 and phospho-p38. Antibodies against total ERK1/2 and total p38 proteins were used as normalizing controls in both A and B. (C) p3TP-luciferase assay performed after TGF-β treatment in the presence of inhibitors SIS3, PD98059, and SB203580 in HT1080, hFhTERT-LTgRAS and hFhTERT cells. The bars represent mean7SE of triplicate experiments. Student's unpaired t-test was used for statistical analysis (*, **, and *** represent po0.05, o0.01 and o0.0001 respectively; ns – not significant).

Table 9 – Components of the WNT pathway regulated by TGF-β in hFHTERT cells compared to the hFhTERT-LTgRAS and the HT1080 cells. Gene name

hFhTERT

hFhTERT-LTgRAS

HT1080

Gene name

hFhTERT

hFhTERT-LTgRAS

HT1080

WNT4 FZD8 WNT11 NFATC1 FZD4 DKK1 PPP2CB FRAT2 CTNNB1 TCF7 FZD9 PPP3CA PPP2CA DKK4 CCND1 MYC

5.51 4.76 2.34 2.31 1.94 1.85 0.83 0.79 0.73 0.70 0.64 0.63 0.62 0.59  0.58  0.64

– – – – – – – – – – – – – – 0.84 –

– – – 1.57 – – – – – – – – – – – –

APC DVL1 LEF1 TBL1X PPP3CC CAMK2G PPP2R5A PRKACA RAC2 SFRP1 SMAD3 CAMK2D AXIN2 SFRP2 BTRC CTNNBIP1

0.64 –0.69 0.71 0.85 0.89 0.90 0.92 0.93 1.00 1.11 1.14 1.47 1.84 2.18 – –

– – 0.95 – – – – – – – – – – – 0.65 –

– – – – – – – 0.82 0.70 – 0.90 – – – – 1.21

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

E X PE R IM EN TA L C ELL R E S EA RC H

hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively after TGF-β treatment compared to their respective untreated controls at least at one of the time-points (Fig. 2 and Table 1). Supplementary Tables 2– 10 give lists of top 40 highly induced or repressed genes in hFhTERT, hFhTERT-LTgRAS, and HT1080 cells at various time points. To understand how responsiveness to TGF-β changes after RAS transformation, we performed a two-way ANOVA on the Cy5–Cy3 ratios across all the time points to find out genes differentially regulated between the hFhTERT and hFhTERT-LTgRAS by TGF-β. Two-way ANOVA was done on the two cell-lines as variables using data from different time points. Median Cy5/Cy3 values across the time points was compared between the cell-lines and probe-sets which passed the test (po0.05) were considered differentially regulated by TGF-β between the celllines. A total of 5291 probe-sets were found to be significantly differentially regulated between hFhTERT and hFhTERT-LTgRAS cells (Fig. 3a). We also compared the TGF-β regulated gene expression profiles of hFhTERT and HT1080 cells by performing a two-way ANOVA. We found that a total of 2274 probe-sets were differentially regulated by TGF-β in hFhTERT Vs. HT1080 cells (Fig. 3b). Also 787 probe-sets were commonly differentially regulated between hFhTERT vs. hFhTERT-LTgRAS and hFhTERT vs. HT1080 cells (Fig. 3c and Tables 2 and 3). A KEGG Pathway Analysis performed on the differentially regulated probe-sets revealed that some of the pathways differentially enriched between hFhTERT and HT1080 and differentially enriched between hFhTERT and hFhTERT-LTgRAS cells are similar (Tables 4–7). The microarray results were validated by testing the expression of a small set of genes by real time RT-PCR. A few of the known TGF-β target genes were validated such as TGFBI, TMEPAI, CTGF, EDN1, ID2, IL6, SMAD7,PAI1, etc. A number of genes were found to be novel targets of TGF-β in the hFhTERT cells like APCDD1, AXIN2 and DACT1 (all regulators of the WNT signaling pathway). IGF1, WNT4 and WNT11 were also found to be regulated by TGF-β in the hFhTERT cells only (Supplementary Tables 10–12).

Differential activation of MAPK pathways in immortal and transformed or tumor fibroblasts after TGF-β treatment MAPK pathway is known to be affected by TGF-β [22,23] and this pathway is a known regulator of cellular growth [40,41]. In the KEGG Pathway Analysis the MAPK signaling pathway came up as an enriched pathway in hFhTERT cells treated with TGF-β compared to both hFhTERT-LTgRAS and HT1080 cells. Indeed many components of the MAPK pathway were regulated by TGF-β in hFhTERT cells compared to both hFhTERT-LTgRAS and HT1080 cells (Table 8). Hence, in order to understand whether TGF-β affected MAPK signaling pathways in these cell-lines, we determined the phosphorylation status of ERK1/2 and p38 proteins after treating the cells with TGF-β. ERK1/2 was phosphorylated in response to TGF-β in a time dependent manner in the HT1080 cells (Fig. 4a). In hFhTERT cells while the basal phosphorylated levels of ERK1/2 were very high, there was no induction by TGF-β (Fig. 4a). p38 MAPK was also phosphorylated post TGF-β treatment in HT1080 cells while we could not detect any phosphorylated p38 before and after TGF-β treatment in hFhTERT cells (Fig. 4a). In the hFhTERT-LTgRAS cells also TGF-β treatment led to the activation of both ERK1/2 and p38 MAPK pathways (Fig. 4b). But we could not detect activation of the ERK1/2 and p38 MAPK pathways by TGF-β in other fibroblast cell-line NIH3T3 and primary hF cells. (Fig. 4b). EGF (20 ng/ml) treatment and UV exposure (30 min) were given to these cells which served as positive controls for Erk1/2 and p38 MAPK activation respectively. In order to

9

] (]]]]) ]]]–]]]

determine if the activation of the MAPK pathways by TGF-β is required for the gene expression changes conferred by TGF-β in HT1080 and hFhTERT-LTgRAS cells, we made use of the p3TP-lux plasmid which can be activated by activating either the smads or the AP1 transcription factors. We observed that in both the hFhTERTLTgRAS and the HT1080 cells, a pre-treatment with either of the p38, ERK1/2 or the Smad3 inhibitors diminished the ability of TGF-β to induce the p3TP-luc activity (Fig. 4c). On the contrary in the hFhTERT cells, it was only in the presence of the Smad3 inhibitor that TGF-β was unable to induce the activity of the same (Fig. 4c).

Differential activation of the WNT signaling pathway by TGF-β in hFhTERT, hFhTERT-LTgRAS and HT1080 cells WNT signaling pathway components were also enriched in the TGF-β regulated gene expression profiles of the hFhTERT cells compared to both hFhTERT-LTgRAS and the HT1080 cells (Table 9). Several WNT pathway targets [42] were found to be regulated by TGF-β in the hFhTERT cells but not in the hFhTERT-LTgRAS and the HT1080 cells (Table 10 and Fig. 5a). Indeed TGF-β treatment led to an induction in the activity of the WNT pathway reporter plasmid, pTOP-FLASH-luc in hFhTERT cells as against HT1080 (a very marginal induction) and hFhTERT-LTgRAS cells (no induction) (Fig. 5b). However there was not much change in the total β-catenin levels or its nuclear localization (Fig. 5c) in all the cell-lines.

Table 10 – Targets of the WNT signaling regulated by TGF-β in the hFhTERT, hFhTERT-LTgRAS and HT1080 cells in the microarray data. Gene name

hFhTERT

HT1080

hFhTERT-LTgRAS

IGF1 IL6 FGF18 GREM2 SALL4 WISP1 KRT18 KRT7 VEGF RUNX2 MET EDN1 GREM1 MMP9 SOX1 CDX1 SOX17 CCND1 EGFR LEF1 WISP2 ID2 KRT33A VEGFC FGF9 KRT19 JUN JAG1 AXIN2

13.88 9.35 5.69 4.73 4.26 2.71 3.01 2.56 2.44 2.38 1.89 1.87 1.69  1.64  3.47  1.74  2.74  1.56  1.58  1.63  1.72  2.01  2.04  2.37  2.85  2.94  3.41  2.95  2.54

– 2.68 1.50 – – 1.58 – – 1.54 – – 5.59 – – – – – – – 1.50 – 2.14 – – – – – 1.73 –

– – – – – 1.90 – – – – – 3.80 – – – – – 1.80 – 1.70 – – – – – – – – –

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

10

E XP E RI ME N TAL CE L L R ES E ARC H

] (]]]]) ]]]–]]]

Fig. 5 – TGF-β regulates WNT signaling in hFhTERT cells (A) TGF-β regulates the expression of WNT target genes in hFhTERT cells. Yaxis represent mean7SE of six replicates. Student's paired t-test was used for statistical significance (*, **, and *** represent po0.05, o0.01 and o0.0001 respectively; ns – not significant). (B) pTOP-FLASH reporter activity in HT1080, hFhTERT-LTgRAS and hFhTERT cells in the presence or absence of TGF-β. The Y-axis shows the relative luciferase units (pTOP-FLASH/pRL-TK) before and after TGFβ treatment. The bars represent mean7SE of triplicate experiments. Student's unpaired t-test was used for statistical analysis (*, **, and *** represent po0.05, o0.01 and o0.0001 respectively; ns – not significant). (C) Immunocytochemistry was done on HT1080, hFhTERT-LTgRAS, hFhTERT and hFF cells grown on glass coverslips using anti β-catenin antibody (Sigma-Aldrich). Anti-rabbit fluorescent tagged secondary antibody, Alexa-488 (Invitrogen Life Sciences, USA) was used for visualization. Propidium iodide (Sigma Aldrich, USA) was used at 1 mg/ml for nuclear staining. Green represents β-catenin and red represents PI stain in the nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

WNT4 mediates activation of fibroblasts by TGF-β WNT4 and WNT11 were identified as novel targets of TGF-β in hFhTERT cells from the microarray data. RT-PCR analysis of WNT4 and WNT11 after TGF-β treatment in hFhTERT cells confirmed the microarray findings (Fig. 6a). On the other hand, in HT1080 and the hFhTERT-LTgRAS cells, there was no induction of WNT4 or WNT11 after TGF-β treatment (Fig. 6b). In carcinoma and epithelial cell-lines we could not observe any induction of WNT4 and WNT11 by TGF-β while in fibroblasts of different origin, WNT4 was found to be induced by TGF-β while WNT11 was induced only in the primary hF cells (Fig. 6c). WNT4 and WNT11 mediate activation of the non-canonical WNT pathway which is predominantly calcium (Ca2þ) dependent and feeds into three different signaling pathways, CamKII-PKC, Phospholipase-PDE and the planar cell polarity pathways [43,44]. Hence, we studied the levels of Ca2þ in the cytoplasm of hFhTERT cells after TGF-β treatment using INDO-1-AM, a cell permeable calcium binding dye. In hFhTERT cells Ca2þ levels increased after TGF-β treatment (Fig. 6d). Also, downstream activation of CamKII was observed after TGF-β treatment in both hFhTERT and hFF cells (Fig. 6e). Ca2þ signaling plays a crucial role in the activation of fibroblasts. The TGF-β conferred activation of both hFhTERT and hFF cells was also found to be dependent on the presence of Ca2þ ions (Fig. 7a). Since WNT4 causes activation of Ca2þ dependent signaling pathways and WNT4 is necessary for the maintenance of the

phenotype of the smooth muscle cells [45], we speculated that WNT4 should also lead to activation of the fibroblast cells. Hence, we overexpressed WNT4 in the hFhTERT cells and found that in the WNT4 overexpressing cells, α-SMA levels were induced suggesting an activation of the hFhTERT cells by WNT4 (Fig. 7b and c). Not only that, the TGF-β conferred activation of hFhTERT cells was also found to be dependent on the presence of WNT4 in the cells. Down-regulating WNT4 expression using shRNA constructs in the hFhTERT cells abolished TGF-β mediated activation of these cells (Fig. 7d and e).

Discussion The question of dual role of TGF-β in cancer has intrigued scientists for a long time [1,23]. Although copious amount of literature is available on TGF-β signaling, we have a poor understanding of the TGF-β signals driving the diverse responses seen in various epithelial, mesenchymal and tumor cells. As an initial step towards apprehending the complex TGF-β biology we have succeeded in identifying two signaling pathways differentially activated by TGF-β in immortal and transformed fibroblast cells. We have shown that TGF-β treatment on immortal fibroblast cells led to activation of WNT signaling pathway. An induction of WNT4 was observed only in the cells of mesenchymal origin. Mobilization of Ca2þ and activation of the non-canonical WNT

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

E X PE R IM EN TA L C ELL R E S EA RC H

] (]]]]) ]]]–]]]

11

Fig. 6 – WNT4 induction by TGF-β and activation of the Ca2þ dependent CaMKII pathway in the hFhTERT cells (A) TGF-β treatment induces the expression of WNT4 and WNT11 mRNAs by several fold in hFhTERT cells. Y-axis represent mean7SE of six replicates. Student's paired t-test was used for statistical significance (*, **, and *** represent po0.05, o0.01 and o0.0001 respectively). (B) No induction of WNT4 and WNT11 mRNAs was observed in both HT1080 and hFhTERT-LTgRAS cells after TGF-β treatment. (C) TGF-β induction of WNT4 exclusively observed only in the fibroblast cells. (D) Cytoplasmic calcium levels visualized with INDO-1-AM dye before and after TGF-β treatment in hFhTERT cells. (E) Lysates of hFhTERT and hFF cells treated with TGF-β1 (5 ng/ml) for 12 h and respective untreated controls were subjected to immunoblotting with anti-phospho CamKII (Abcam) antibody. Total CamKII anibody (Abcam) was used as normalizing control.

signaling was also observed in the hFhTERT and hFF cells by TGFβ. On the other hand treatment of HT1080 and hFhTERT-LTgRAS cells with TGF-β led to the activation of Erk1/2 and p38 MAPK pathways while no induction of WNT4 and WNT11 and WNT signaling was seen in these cells after treatment with TGF-β. It has previously been reported that TGF-β can induce the formation of calcium waves in pulmonary fibroblasts which have an effect on the transcription of several matrix proteins [46]. It has also been shown that TGF-β regulates actin cytoskeleton by modulating calcium levels in murine mesangial cells [47]. We have also shown that Ca2þ modulation by TGF-β is crucial for the activation of the human fibroblast cells and TGF-β conferred activation is dependent on WNT4. A previous study by Nik et al. reported that TGF-β treatment on both tumoral and non-tumoral fibroblasts led to an increase in activated β-catenin levels [48]. In the present work we failed to see increase or nuclear localization of β-catenin upon TGF-β treatment in either the transformed HT1080 and hFhTERTLTgRAS cells or the immortal hFhTERT and primary hFF cells whilst several β-catenin targets were actually regulated after TGF-

β treatment in the immortal hFhTERT cells but not in either the HT080 or the hFhTERT-LTgRAS cells. This discrepancy could be explained in the light of observations where smads and β-catenin cooperatively regulate the expression of several target genes [49,50]. It has also been shown that smad3 can regulate the expression of Xtwn gene in Xenopus independent of β-catenin [50]. Indeed the pTOP-FLASH reporter activity after TGF-β treatment was induced in the case of hFhTERT cells but not in both HT1080 and hFhTERT-LTgRAS cells. These findings suggest an activation of both canonical and non-canonical WNT signaling in the non-transformed fibroblasts but not in the transformed fibroblasts. Activation of ERK and p38 MAPK kinases by TGF-β has been well characterized in the context of tumor cells [21–23,51]. Classically it has been shown that activation of the ERK1/2 MAPK pathway can cause an increase in the growth of cells [18,19]. But several studies in the realm of TGF-β biology have shown that TGF-β induced activation of ERK1/2 MAPK pathway in epithelial and several tumor cell-lines actually led to or accompanied TGF-β

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

12

E XP E RI ME N TAL CE L L R ES E ARC H

] (]]]]) ]]]–]]]

Fig. 7 – Activation of hFhTERT cells by WNT4. (A) Immunocytochemistry using anti-α-SMA antibody (Abcam) was performed upon hFhTERT and hFF cells growing on glass coverslips. Cells were grown to confluence, serum starved and pre-treated with EGTA (1.5 mM) for 2 h before treating with TGF-β (5 ng/ml) for 48 h. (B) PCR analysis of WNT 4 and α-SMA in pBABE-puro empty vector (EV) and pBABE-puro-WNT4 (WNT O.E.) transduced hFhTERT cells. (C) ICC with anti-α-SMA antibody in WNT4 O.E. and EV transduced hFhTERT cells. (D) Real-time RT-PCR analysis of WNT4 after TGF-β treatment in hFhTERT cells after these cells were transduced with four different shRNA constructs (Origene) against WNT4. Mock transduced and shScrm served as controls. The bars represent mean7SE of triplicates. Student's unpaired t-test was used for statistical analysis (*, **, and *** represent po0.05, o0.01 and o0.0001 respectively). (E) ICC with anti-α-SMA antibody in hFhTERT cells after knockdown of WNT4 expression. Antirabbit fluorescent tagged secondary antibody, Alexa-488 (Invitrogen Life Sciences, USA) was used for visualization in all the ICC experiments. Propidium iodide (Sigma Aldrich, USA) was used at 1 mg/ml for nuclear staining. Green represents α-SMA and red represents PI stain in the nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

induced growth inhibition of these cells [18,52]. We also observed an activation of the ERK1/2 MAPK in HT1080 and hFhTERTLTgRAS cells while we failed to detect any induction of phosphorylated ERK1/2 levels in the hFhTERT cells and some other fibroblast cell-lines. The p38 MAPK pathway was also found to be activated by TGF-β only in the HT1080 and hFhTERT-LTgRAS cells but not in any of the fibroblast cell-lines. A previous study has shown that TGF-β treatment results in the activation of c-Abl receptor tyrosine kinase in fibroblast cultures but not in the epithelial cultures [26]. They also reported that this activation was dependent on phosphatidylinositol 3-kinaseactivated PAK2 which is activated by TGF-β in mesenchymal cells [27]. These findings also throw light on the differential activation of downstream signaling pathways by TGF-β in different cellular contexts. Our data advocates differential activation of WNT and MAPK pathways as central to the differential effects of TGF-β in different cellular contexts. An important finding of the present study is the switch observed in the responsiveness of hFhTERT cells to TGF-β on being transformed with SV40 Large T antigen and activated RAS. TGF-β treatment of the transformed hFhTERTLTgRAS cells did not induce the expression of either WNT4 or

WNT11 while it did activate the ERK1/2 and p38 MAPK pathways. We have also established that WNT4 plays a crucial role in TGF-β mediated activation of the immortal hFhTERT cells. This assumes significance in the light of recent findings where spread of cancers is linked to the activation status of fibroblasts [10,11]. It would be interesting to find out status of WNT4 in the CAFs of various types of cancers and how that correlates with tumor aggression. The delineation of signaling pathways specifically activated in different contexts by TGF-β could provide reference points which should be studied in greater depth to understand how various TGF-β effects are dependent on such distinct signals. As an example, previous reports from our lab have identified that while S100A2 regulation by TGF-β itself was dependent on the activation of the non-canonical MAPK signaling, we have shown that presence of S100A2 is critical for the activation of smad3 signaling and induction of several TGF-β target genes [53,54]. This goes on to establish the importance of non-canonical signaling by TGF-β for its canonical signal transduction. Hence it is critical to identify and delineate non-canonical context dependent signals. Such information would facilitate in identifying and targeting specific TGF-β signals which drive the tumor growth and aggression while

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

E X PE R IM EN TA L C ELL R E S EA RC H

retaining those which subjugate it. This would greatly enhance our understanding of TGF-β actions in the tumor context which eventually could be exploited in the management of aggressive tumors. In conclusion, in this study we provided evidence for the differential activation of WNT and MAPK pathways by TGF-β in transformed vs. normal mesenchymal cellular contexts.

[13] [14] [15]

Acknowledgments Financial support for this work was by the Department of Biotechnology, Government of India and Indian Institute of Science partnership program. We are grateful to Dr. K. Satyamoorthy for the hFF cells. We thank Ms. Meenakshi and Ms. Deepti at the IISc Confocal facility for images and Dr. Praveen Kumar for help with the immunocytochemical experiments. The infrastructure support to MRDG by the departments of DST, DBT and UGC is gratefully acknowledged.

Appendix A.

[16]

[17] [18]

Supporting information [19]

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2015.03.001. [20]

references [21] [1] J. Massague, TGF-beta in cancer, Cell 134 (2008) 215–230. [2] L. Levy, C.S. Hill, Alterations in components of the TGF-beta superfamily signaling pathways in human cancer, Cytokine Growth Factor Rev. 17 (2006) 41–58. [3] S. Markowitz, J. Wang, L. Myeroff, R. Parsons, L. Sun, et al., Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability, Science 268 (1995) 1336–1338. [4] S.A. Hahn, A.B. Seymour, A.T. Hoque, M. Schutte, L.T. da Costa, et al., Allelotype of pancreatic adenocarcinoma using xenograft enrichment, Cancer Res. 55 (1995) 4670–4675. [5] S. Thiagalingam, C. Lengauer, F.S. Leach, M. Schutte, S.A. Hahn, et al., Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers, Nat. Genet. 13 (1996) 343–346. [6] Y. Hasegawa, S. Takanashi, Y. Kanehira, T. Tsushima, T. Imai, et al., Transforming Growth-beta1 level correlates with angiogenesis, tumor progression, and prognosis in patients with nonsmall cell lung carcinoma, Cancer 91 (2001) 964–971. [7] H. Saito, S. Tsujitani, S. Oka, A. Kondo, M. Ikeguchi, et al., The expression of transforming growth factor-beta1 is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric carcinoma, Cancer 86 (1999) 1455–1462. [8] H. Tsushima, S. Kawata, S. Tamura, N. Ito, Y. Shirai, et al., High levels of transforming growth factor beta 1 in patients with colorectal cancer: association with disease progression, Gastroenterology 110 (1996) 375–382. [9] P. Wikstrom, P. Stattin, I. Franck-Lissbrant, J.E. Damber, A. Bergh, transforming growth factor beta 1 is associated with angiogenesis, metastasis, and poor clinical outcome in prostate cancer, Prostate 37 (1998) 19–29. [10] K. Pietras, A. Ostman, Hallmarks of cancer: interactions with the tumor stroma, Exp. Cell Res. 316 (2010) 1324–1331. [11] T.D. Tlsty, P.W. Hein, Know thy neighbor: stromal cells can contribute oncogenic signals, Curr. Opin. Genet. Dev. 11 (2001) 54–59. [12] Y. Kojima, A. Acar, E.N. Eaton, K.T. Mellody, C. Scheel, et al., Autocrine TGF-beta and stromal cell-derived factor-1 (SDF-1)

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

] (]]]]) ]]]–]]]

13

signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts, Proc. Natl. Acad. Sci. USA 107 (2010) 20009–20014. D.M. Bissell, Chronic liver injury, TGF-beta, and cancer, Exp. Mol. Med. 33 (2001) 179–190. G. Serini, G. Gabbiani, Mechanisms of myofibroblast activity and phenotypic modulation, Exp. Cell Res. 250 (1999) 273–283. S. Hashimoto, Y. Gon, I. Takeshita, K. Matsumoto, S. Maruoka, et al., transforming growth factor-beta 1 induces phenotypic modulation of human lung fibroblasts to myofibroblast through a cJun-NH2-terminal kinase-dependent pathway, Am. J. Respir. Crit. Care Med. 163 (2001) 152–157. A.B. Roberts, M.A. Anzano, L.M. Wakefield, N.S. Roche, D.F. Stern, et al., Type beta transforming growth factor: a bifunctional regulator of cellular growth, Proc. Natl. Acad. Sci. USA 82 (1985) 119–123. J. Massague, The transforming growth factor-beta family, Annu. Rev. Cell Biol. 6 (1990) 597–641. Y. Chatani, S. Tanimura, N. Miyoshi, A. Hattori, M. Sato, et al., Cell type-specific modulation of cell growth by transforming growth factor beta 1 does not correlate with mitogen-activated protein kinase activation, J. Biol. Chem. 270 (1995) 30686–30692. P.M. Siegel, W. Shu, R.D. Cardiff, W.J. Muller, J. Massague, transforming growth factor beta signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis, Proc. Natl. Acad. Sci. USA 100 (2003) 8430–8435. A.B. Roberts, F. Tian, S.D. Byfield, C. Stuelten, A. Ooshima, et al., Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis, Cytokine Growth Factor Rev. 17 (2006) 19–27. Y.E. Zhang, Non-Smad pathways in TGF-beta signaling, Cell Res. 19 (2009) 128–139. M.K. Lee, C. Pardoux, M.C. Hall, P.S. Lee, D. Warburton, et al., TGFbeta activates ERK MAP kinase signalling through direct phosphorylation of ShcA, EMBO J. 26 (2007) 3957–3967. L. Yu, M.C. Hebert, Y.E. Zhang, TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses, EMBO J. 21 (2002) 3749–3759. M.C. Wilkes, S.J. Murphy, N. Garamszegi, E.B. Leof, Cell-typespecific activation of PAK2 by transforming growth factor beta independent of Smad2 and Smad3, Mol. Cell Biol. 23 (2003) 8878–8889. F. Caraci, E. Gili, M. Calafiore, M. Failla, C. La Rosa, et al., TGFbeta1 targets the GSK-3beta/beta-catenin pathway via ERK activation in the transition of human lung fibroblasts into myofibroblasts, Pharmacol. Res. 57 (2008) 274–282. M.C. Wilkes, E.B. Leof, Transforming growth factor beta activation of c-Abl is independent of receptor internalization and regulated by phosphatidylinositol 3-kinase and PAK2 in mesenchymal cultures, J. Biol. Chem. 281 (2006) 27846–27854. M.C. Wilkes, H. Mitchell, S.G. Penheiter, J.J. Dore, K. Suzuki, et al., Transforming growth factor-beta activation of phosphatidylinositol 3-kinase is independent of Smad2 and Smad3 and regulates fibroblast responses via p21-activated kinase-2, Cancer Res. 65 (2005) 10431–10440. F. Verrecchia, M.L. Chu, A. Mauviel, Identification of novel TGFbeta /Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach, J. Biol. Chem. 276 (2001) 17058–17062. R.C. Chambers, P. Leoni, N. Kaminski, G.J. Laurent, R.A. Heller, Global expression profiling of fibroblast responses to transforming growth factor-beta 1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching, Am. J. Pathol. 162 (2003) 533–546. Y.C. Yang, E. Piek, J. Zavadil, D. Liang, D. Xie, et al., Hierarchical model of gene regulation by transforming growth factor beta, Proc. Natl. Acad. Sci. USA 100 (2003) 10269–10274.

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001

14

E XP E RI ME N TAL CE L L R ES E ARC H

[31] P. Ranganathan, A. Agrawal, R. Bhushan, A.K. Chavalmane, R.K. Kalathur, et al., Expression profiling of genes regulated by TGFbeta: differential regulation in normal and tumour cells, BMC Genomics 8 (2007) 98. [32] D.S. Bae, N. Blazanin, M. Licata, J. Lee, A.B. Glick, Tumor suppressor and oncogene actions of TGFβ1 occur early in skin carcinogenesis and are mediated by Smad3, Mol. Carcinog. 48 (5) (2009) 441–453. [33] S. Sheed, W.A. Nelson-Rees, E.M. Toth, P. Arnstein, M.B. Gardner, Characterisation of a newly derived human sarcoma cell-line (HT-1080), Cancer 33 (1974) 1027–1033. [34] A. Rangarajan, S.J. Hong, A. Gifford, R.A. Weinberg, Species- and cell type-specific requirements for cellular transformation, Cancer Cell 6 (2004) 171–183. [35] I. Khan, N. Kumar, I. Pant, S. Narra, P. Kondaiah, Activation of TGFβ pathway by areca nut constituents: a possible cause of oral submucous fibrosis, Plos One 7 (12) (2012) e51806. [36] G.K. Smyth, T. Speed, Normalization of cDNA microarray data, Methods 31 (2003) 265–273. [37] A.I. Saeed, N.K. Bhagabati, J.C. Braisted, W. Liang, V. Sharov, E.A. Howe, et al., TM4 microarray software suite, Methods Enzymol. 411 (2003) 134–193. [38] J.C. Jonk, S. Itoh, C.H. Heldin, P. Dijke, W. Kruijer, Identification and Functional characterization of a smad binding element (SBE) in the JunB promoter that acts as the transforming growth factorβ/activin and bone morphogenetic protein inducible enhancer, J. Biol. Chem. 273 (2000) 21145–21152. [39] J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J. Doody, et al., TGF-β signals througha heteromeric protein complex, Cell 71 (6) (1992) 1003–1014. [40] G. Pearson, F. Robinson, T. Beers Gibson, B.E. Xu, M. Karandikar, et al., Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions, Endocr. Rev. 22 (2001) 153– 183. [41] T.S. Lewis, P.S. Shapiro, N.G. Ahn, Signal transduction through MAP kinase cascades, Adv. Cancer Res. 74 (1998) 49–139. [42] The WNT homepage. 〈http://web.stanford.edu/group/nusselab/ cgi-bin/wnt/main〉. [43] M.T. Veeman, J.D. Axelrod, R.T. Moon, A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling, Dev. Cell 5 (2003) 367–377. [44] A.D. Kohn, R.T. Moon, Wnt and calcium signaling: beta-cateninindependent pathways, Cell Calcium 38 (2005) 439–446.

] (]]]]) ]]]–]]]

[45] P. Itaranta, L. Chi, T. Seppanen, M. Niku, J. Tuukkanen, et al., Wnt4 signaling is involved in the control of smooth muscle cell fate via Bmp-4 in the medullary stroma of the developing kidney, Dev. Biol. 273 (2006) 473–483. [46] S. Mukherjee, M. Kolb, F. Duan, L.J. Janssen, Transforming growth factor-β evokes Ca2þ waves and enhances gene expression in human pulmonary fibroblasts, Am. J. Respir. Cell Mol. Biol. 46 (2012) 757–764. [47] T.A. Mcgowan, M. Madesh, Y. Zhu, L. Wang, M. Russo, L. Deelman, R. Henning, S. Joseph, G. Hajnoczky, K. Sharma, TGF-β-induced Ca2þ influx involves the type III IP3 receptor and regulates actin cytoskeleton, Am J. Physiol. Ren. Physiol. 282 (2002) F910–F920. [48] S.A. Nik, R.P. Ebrahim, K.V. Dam, J.J. Cassiman, S. Tejpar, TGF-β modulates β-Catenin stability and signaling in mesenchymal proliferations, Exp. Cell Res. 313 (2007) 2887–2895. [49] S.S. Cheon, Q. Wei, A. Gurung, A. Youn, T. Bright, et al., Beta catenin regulates wound size and mediates the effect of TGF-beta in cutaneous healing, FASEB J. 20 (2006) 692–701. [50] E. Labbe, A. Letamendia, L. Attisano, Association of smads with LEF1/TCF mediates cooperative signaling by the TGF-beta and the WNT pathways, Proc. Natl. Acad. Sci. 97 (15) (2000) 8358–8363. [51] A.J. Galliher, W.P. Schiemann, Src Phosphorylates Tyr284 in TGFB Type II receptor and regulates TGF-B stimulation of p38 MAPK during breast cancer cell proliferation and invasion, Cancer Res. 67 (2007) 8. [52] M.T. Hartsough, K.M. Mulder, Transforming growth factor-β activation of p44 MAPK in proliferating cultures of epithelial cells, J. Biol. Chem. 120 (1995) 7117–7124. [53] S. Naz, M. Bashir, P. Ranganathan, P. Bodapati, V. Santosh, et al., Pro-tumorigenic actions of S100A2 involve regulation of PI3/AKT signaling and functional interaction with smad3, Carcinogenesis 35 (1) (2014) 14–23. [54] S. Naz, P. Ranganathan, P. Bodapati, A.H. Shastry, L.N. Mishra, et al., Regulation of S100A2 expression by TGF-beta induced MEK/ ERK signaling and its role in cell migration/invasion, Biochem. J. 447 (1) (2012) 81–91.

Please cite this article as: S. Chopra, et al., Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.001