Twist1 Is a Key Regulator of Cancer-associated Fibroblasts

There are no conflicts of interest. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Cancer-associated fibroblasts (CAF) are key contributors to malignant progression, but their critical regulators remain largely unknown. In this study, we examined the role of Twist1, a central regulator of epithelial-mesenchyme transition in carcinoma cells, in the transdifferentiation of normal quiescent fibroblasts to CAF and we defined its upstream controls and downstream effectors. Primary human gastric fibroblast and CAF cultures were established from gastrectomy specimens and validated as non-tumor cells by somatic mutation analyses. In these cultures, exposure to the pro-inflammatory cytokine IL-6 commonly expressed in tumors was sufficient to induce Twist1 expression in normal fibroblasts and transdifferentiate them into CAF via STAT3 phosphorylation. In xenograft models, tumor infiltration of Twist1-expressing CAF was enhanced strongly by ectopic IL-6 expression in gastric or breast cancer cells. We found that Twist1 expression was necessary and sufficient for CAF transdifferentiation. Enforced expression of Twist1 in normal fibroblasts was also sufficient to drive CAF marker expression and malignant character in gastric cancer cells both in vitro and in vivo. Conversely, silencing the expression of Twist1 in CAF abrogated their tumor-promoting properties. Downstream of Twist1 we defined the chemokine CXCL12 as a transcriptional target. Clinically, CXCL12 and Twist1 expression were correlated in CAF present in gastric tumor specimens. Lastly, ectopic expression of Twist1 in normal fibroblasts suppressed premature senescence, whereas Twist1 attenuation accelerated senescence in CAF. Our findings define Twist1 as a compelling target to deprogram the tumor supporting features of the cancer microenvironment. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.


Introduction
Tumor growth and progression are markedly influenced by the tumor microenvironment (TME), which comprises the extracellular matrix (ECM) and a variety of stromal cells, such as fibroblast, monocytes, and endothelial cells (1,2).Fibroblasts are the most common cells in connective tissue, and their main function is to maintain the ECM and regulate epithelial differentiation by stromal-epithelial crosstalk (3,4).Fibroblasts display a wide spectrum of activation states: normal fibroblasts show thin, wavy, small spindle cell morphology, whereas activated fibroblasts are large, plump, spindle-shaped mesenchymal cells with stress fibers (1,3).Among activated fibroblasts, those found in association with cancer cells are known as cancer-associated fibroblasts (CAFs) (3).
CAFs promote cancer progression through remodeling of the ECM, induction of angiogenesis, and recruitment of inflammatory cells and directly stimulate cancer cell proliferation via the secretion of growth factors and mesenchymal-epithelial cell interactions (5).Although CAF's biological and clinical significance in tumor microenvironment has been widely recognized, the mechanism by which normal fibroblasts are transformed into CAFs remain vastly unknown.
In our previous study, we showed that Twist1 is expressed in stromal fibroblasts in gastric cancer tissues and that Twist1 expressing fibroblasts possess CAF-like properties (6).Twist1 is a basic Helix-Loop-Helix transcription factor that is essential for the development of mesodermally-derived tissues (7,8).During mouse embryogenesis, Twist1 is expressed in various mesodermal tissues including neural crest-derived mesenchyme, branchial arch, muscle, bone, and fibroblasts (9,10).
After birth, Twist1 expression is barely detectable in normal mesenchymal cells of on April 20, 2017.© 2014 American Association for Cancer Research.cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on November 3, 2014; DOI: 10.1158/0008-5472.CAN-14-0350 adult tissues and limited to mesenchymal stem cell (11,12) and human white adipocyte (13).On the other hand, Twist1 is known to be an important inducer of epithelial-mesenchymal transition (EMT) and Twist1 overexpression has been reported in a variety of epithelial cancer cells with clinical correlation with poor prognosis (14)(15)(16).However, despite the abundance of Twist 1 expressing stromal fibroblasts within cancer tissue, its clinical significance and regulating mechanism remain almost completely unknown.
In the present study, we investigated molecular mechanism by which Twist1 is induced in cancer associated fibroblasts and the role of Twist1 in driving the transdifferentiation of quiescent fibroblasts into CAFs.In addition, we showed that Twist1 expression is necessary and sufficient for CAF transdifferentiation.

Cell lines
The gastric normal fibroblast cell line Hs738 was obtained from the American Type Culture Collection (ATCC).Hs738 and the stomach cancer cell lines MKN1, MKN28, MKN74, NCI-N87, SNU638, and SNU668, and the breast cancer cell line MCF-7 were used.Cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea) that performs cell line characterizations using DNA fingerprinting analysis and passaged in our laboratory for fewer than 6 months.Maintenance procedures are described in Supplementary Methods.

Isolation and culture of fibroblasts
Human stomach tumor specimens were obtained from patients undergoing

Incubation of fibroblast with conditioned Media from gastric cancer cells
Detailed procedures are described in Supplementary Methods.

Tissue specimens and construction of tissue microarrays (TMA)
A total of 332 FFPE gastric tissue samples were included in this study.All patients provided written informed consent according to institutional guidelines.Detailed procedures are described in Supplementary Methods.

Immunohistochemical analysis
Detailed methods and list of antibodies used were described in Supplementary Methods.

Cell viability/proliferation assay
Stomach cancer cells were cultured with CM from normal and CAFs for 2 days.
Cells were detached, seeded in 96 well plates, and cultured for 24 hrs.Detailed procedures are described in Supplementary Methods.and MCF-7+GFP cells were infected with IL-6 expressing lentivirus.Tumor cells (3×10 6 ; SNU638 and 2×10 6 ; MCF7) were implanted subcutaneously in the right flank in 0.1 ml of serum-free medium and growth factor reduced matrigel (1:1).Tumor volume was determined every 2 weeks.

Lentivirus transduction.
Detailed procedures are described in Supplementary Methods.Detailed procedures and primer sequences are described in Supplementary Methods.

SDS-PAGE
Detailed procedures are described in Supplementary Methods.

Invasion/Migration assay
Detailed procedures are described in Supplementary Methods.

ChIP assay
CAF#14 cells were grown to 70í80% confluency and fixed with 1% formaldehyde.
Nuclear extracts were sonicated to shear the DNA into fragments smaller than 500 bp.Twist1-DNA complexes were immunoprecipitated using Protein G Dynabeads (Cat.10003D, Invitrogen, USA) conjugated with anti-Tiwst1 IgG or control mouse IgG.
After the precipitated DNA was eluted, crosslinks were reversed by 0.3 M NaCl and purified with the QIAquick PCR Purification Kit (28104, Qiagen, CA).PCR was performed on the purified DNA using primer sets as described in Supplementary Methods.

Luciferase assay
The pGL3.CXCL12 promoter (í1442 to +28) luciferase vector was generated by inserting cxcl12 promoter sequence.The 293T cells (5×10 4 cells per well) were plated into 24 well plates and incubated for 24 h.Promoter vectors were cotransfected with either pSG5.htwist1.HA or pSG5.HA vector into cells using Lipofectamine 2000 (Invitrogen).Luciferase reporter gene assays were performed using the Luciferase assay system (Promega).Sequences were available in Supplementary Methods.

Site-directed mutagenesis
The Twist1 binding E-BOX-1 region (CAGGTG) and non-binding E-BOX-2 region (CATCTG) on the CXCL12 promoter were mutated using the pGL3.CXCL12 (í1442 to +28) luciferase vector as the template DNA.The PCR amplification for sitedirected mutagenesis was performed using the corresponding mutant primers.The sequence changes for the E-BOX-1(í514 bp to í509 bp) htwist1 binding site were as follows: WT, GAGGTG and mutant E-BOX-1, TTGGTG.The sequence changes for the E-BOX-2(í1042 to í1037) were as follows: WT, CATCTG; and mutant E-BOX-2, TTTCTG.Sequences were available in Supplementary Methods.

Immunofluorescence assay
Paraffin sections from MKN74, SNU638 and MCF7 xenograft models were used and detailed procedures are described in Supplementary Methods.

TCGA Data
mRNA expression data were obtained for 9 tumors of TCGA carcinoma project through the cBio cancer genomics portal (http://www.cbioportal.org/public-portal).

Gene expression microarray
Data have been deposited in Gene Expression Omnibus (accession number GSE62740).Expression profiling analysis was performed using the Agilent Oligo

High throughput sequencing
Genomic DNA was isolated from two patients for each cancer cell, matched CAFs, and matched normal fibroblasts.Library construction and targeted exome capture (505 genes) using the Agilent SureSelect Target Enrichment System were performed.
Sequencing of exome-enriched libraries was performed using Illumina's MiSeq System.Average target coverage was 138.3 for the six tested samples and more than 90% of target bases achieving 30X coverage were obtained in all six samples.Detail methods and sequencing metrics are available in Supplementary Methods.

Statistical Analysis
Detailed methods are described in Supplementary Methods.

Twist1 upregulation by the IL-6/STAT3 axis in quiescent fibroblasts with the expression of CAF markers
In a previous study, we showed that Twist1 is expressed in CAFs derived from gastric cancer tissues with significant associations with CAF markers expression and poor clinical outcome (6).Despite these results suggesting the importance of Twist1 in CAFs, the mechanism by which Twist1 is activated in fibroblasts remains unknown.
To address this issue, two primary normal fibroblast cultures (NF# 19  were established from gastric non-tumor tissues.When we cultured these normal fibroblasts with conditioned medium (CM) from four gastric cancer cell lines (MKN28, 74, NCI-N87, and SNU668), only CM from SNU668 significantly up-regulated mRNA levels of CAF markers, including FAPĮ, FSP1, PDGFRĮ, and Twist1 (Figure 1A, B).
Next, we searched for cytokines selectively secreted by SNU668 because cytokines are important soluble factors in CM for CAF activation (17).The mRNA levels of cytokine genes obtained from the BioCarta data set for gastric cancer cell lines were validated using gene expression data from TCGA project.The results showed that IL-6 mRNA levels were distinctly high in SNU668 cells compared to those of other cell lines (Figure 1C).IL-6 protein levels were also markedly increased in the CM of SNU668 (Figure 1D), suggesting that IL-6 is a major inducer of CAF transdifferentiation.To test this hypothesis, the effect of IL-6 blocking on CAF transdifferentiation was evaluated by adding IL6-blocking antibody to CM from SNU668 cells, which abolished the enhanced expression of Twist1 and CAF markers induced by CM of SNU668 (Figure 1E).Furthermore, acquired CAF phenotype in fibroblast by IL6 was maintained even after removal of IL6 (Supplementary Figure 1).Next, gastric normal fibroblasts were treated with IL-6 at 50ng/ml for 2 days and its impact on the expression of CAF markers was assessed by western blot analyses.
Twist1 and CAF markers such as FAPĮ, PDGFRĮ, PDGFRȕ, Į-SMA, and FSP1 were significantly upregulated by IL-6 in normal fibroblasts (Figure 1F and Supplementary Figure 2A).This IL-6 induced upregulation of CAF markers was repressed by silencing of Twist1 expression using shRNA (Figure 1G and Supplementary Figure 2B).Because IL-6 signaling is mediated by STAT3 phosphorylation in other types of cells (18), we examined whether STAT3 is on April 20, 2017.© 2014 American Association for Cancer Research.cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on November 3, 2014; DOI: 10.1158/0008-5472.CAN-14-0350 phosphorylated in gastric fibroblasts in response to IL-6 signaling.The results was that IL-6 increased pSTAT3 levels as well as Twist1 in two gastric fibroblast lines in a dose-dependent manner, and IL-6 induced upregulation of Twist1 was abrogated by a STAT3 inhibitor (JSI-124) (Figure 1H and Supplementary Figure 2C).These results indicated that Twist1 expression is activated in fibroblast by the IL-6/STAT3 axis.
The effect of IL-6 on generation of Twist1-expressing CAFs was further investigated in in vivo models.Ectopic IL-6 expressing SNU638-GFP cancer cells (SNU638 GFP/IL6 ) and MCF7-GFP cells (MCF7 GFP/IL6 ), which were originally negative for IL-6 (Figure 2A and 2B) were generated and injected subcutaneously into immune compromised nude mice (Figure 2C).Tumorigenicity of ectopic IL6 expressing SNU638 and MCF7 was remarkably superior to respective controls (Figure 2C).Furthermore, Twist1 expressing stromal cells were much more frequent in xenograft tumors formed by these IL6 expressing SNU638 GFP/IL6 cells and MCF7 GFP/IL6 cells compared with controls (P<0.001)(Figure 2D,E).These Twist1 expressing stromal cells were negative for GFP but positive for Tenascin-C (Tn-C), indicating that these cells were CAFs not originated from cancer cells (Figure 2D).This experiment was repeated using another gastric cancer cell, MKN74 and identical result was obtained (Supplementary Figure 3).The expressions of IL-6, pSTAT3, and Twist1 in gastric cancer tissues were studied using immunohistochemistry and IL-6 expression was prevalent in the cancer stroma in all cases (n=46).pSTAT3 expression was detected in both cancer cells and stromal fibroblasts, whereas Twist1 expression was mainly limited to CAFs.
There was a significant correlation between pSTAT3 and Twist1 expression in CAFs (P<0.001) (Figure 2F).

Twist1 expression is sufficient for transdifferentiation of quiescent fibroblasts to CAFs
To determine whether Twist1 alone confers normal fibroblast with CAF like properties, gastric normal fibroblasts (Hs738, NF#19, and NF#32) were induced to express Twist1 using lentivirus, and we found that the protein levels of CAF markers, such as FAPĮ, PDGFRĮ, PDGFRȕ, Į-SMA, and FSP1, were enhanced by Twist1 in these normal fibroblasts (Figure 3A and Supplementary Figure 2D).Next, we collected CM from control fibroblasts (NF#14 GFP , NF#32 GFP ), ectopic Twist1expressing fibroblasts (NF#14 Twist1 , NF#32 Twist1 ), and patient matched CAFs (CAF#14, #32), then cultured gastric cancer cells with these CM (Figure 3B).CM from Twist1expressing NF#14 Twist1 & NF#32 Twist1 significantly enhanced the proliferation of MKN28 and MKN74 cells to the same extent as CM from CAF#14, 32 did (Figure 3B and supplementary figure 4), and remarkably increased the migratory and invasive abilities of gastric cancer cells (Figure 3C).Finally, the effect of Twist1-exprssing fibroblasts on tumorigenicity of gastric cancer cells was examined in vivo using a xenograft model.Specifically, human SNU638 and SNU668 gastric cancer cells (5×10 5 cells each) were co-injected subcutaneously into nude mice in a suspension with either Twist1-expressing fibroblast or control fibroblasts (1.5×10 6 cells each).
Xenografts containing Twist1-expressing CAFs grew significantly larger and faster than both of xenografts infused with Twist1-negative fibroblasts and xenografts by cancer cells alone (no fibroblasts) (P<0.001)(Figure 3D).Microvessel density and cancer cells' proliferation rate within xenograft tumor were also significantly increased when Twist1-expressing fibroblasts were added (Figure 3E, 3F).These data indicated that ectopic Twist1-expressing normal fibroblasts acquired both the functional and expressional attributes of CAFs, strongly suggesting that Twist1 is a sufficient condition for CAF transdifferentiation.

Twist1 expression in CAFs is essential for the maintenance of the CAF phenotype
To investigate Twist1's role in CAF, primary cultures of CAFs (CAF#14 and 32) were established from stomach cancer patients' tissues and both CAFs expressed Twist1.Then, both CAFs' somatic mutational profiles were compared to those of matched cancer cells using high throughput sequencing technology to exclude contamination by cancer cells.The results was that cancer cells and CAFs had distinctive mutational patterns in both patients, with cancer cells showing frequent mutations, whereas no mutations, except for one synonymous mutation, were identified in CAFs (Figure 4A).The detailed somatic mutation profiles of the isolated cancer cells and CAFs in the two patients were exported to an Excel table.The different somatic mutation patterns in CAFs and cancer cells suggested that these CAFs were neither contaminated cancer cells nor CAFs originated from cancer cells.Silencing Twist1 expression in both gastric CAFs, CAF#14 and CAF#32, using shRNA markedly reduced the expression of CAF markers, including FAPĮ, PDGFRĮ, PDGFRȕ, Į-SMA, and FSP1 (Figure 4B and Supplementary Figure 2E).Furthermore, the enhanced proliferation of MKN28 and MKN74 gastric cancer cells induced by CM derived from endogenously Twist1 expressing CAF#14 and CAF#32 was not observed when these cancer cells were treated with CM from Twist1-depleted CaF#14 and CaF#32 cells (Figure 4C).The ability of CAFs to facilitate the migration and invasion of gastric cancer cells was also remarkably reduced by knock-down of Twist1 in gastric CAFs (Figure 4D).These results indicate that Twist1 is necessary for CAF transdifferentiation and essential for the maintenance of the CAF phenotype.

Genome-wide analyses of the effect of Twist1 on mRNA expressional changes in fibroblasts identified CAF phenotype-related Twist1 target genes
To elucidate the functional mechanism of Twist1 in CAFs, changes in mRNA expression profiles induced by Twist1 were analyzed using mRNA expression microarrays.To improve specificity of this analysis, we designed two experimentations; (1) gain-of-function effect of twist1 in normal fibroblasts (NF#14 and NF#32) and ( 2) loss-of-function effect of twist1 in patient matched CAFs (CAF#14 and CAF#32).These experimentations were repeated 2 times.Genes upregulated in the two Twist1 induced fibroblast lines (NF#14 and #32) and those downregulated in the two Twist1 deleted CAFs (CaF#14 and #32) are shown using a volcano plot (Figure 4E).Among approximately 60,000 probes annotating 20,000 genes, 221 genes were significantly upregulated in Twist1 induced fibroblasts and 889 genes were significantly downregulated in Twist1 deleted CAF (cutoff point: fold change>1.5 and P <0.05).Among them, 23 genes whose expression was increased in Twist1 induced fibroblasts and simultaneously reduced in Twist1 deleted CAFs were considered candidate target genes upregulated by Twist1.As shown in Figure 4F, these genes include many CAF phenotype-related genes involved in remodeling of cancer microenvironment, such as SULF2, lysyl oxidase (LOX), CXCL12, Tn-C (TNC), delta-sarcoglycan (SGCD), and HAPLN1.

Twist1 in fibroblasts with clinical relevance
Based on the results of our gene-chip study and functional significance of CXCL12 as a strong tumor-promoting chemokine, we suspect that CXCL12 is a key target of Twist1 in CAFs (19,20).First, we validated our mRNA microarray results in both mRNA and protein levels.We confirmed that the secretion of CXCL12 was markedly enhanced by Twist1 alone in normal fibroblast (NF#32) and Twist1 is also essential for CXCL12 secretion in CAF (CaF#32) using ELISA assay (Figure 5A).Besides, Twist1 overexpression increased the mRNA level of CXCL12 in normal fibroblasts (NF#14 and NF#32), whereas knock-down of Twist1 decreased the mRNA level of CXCL12 in CAFs (CAF#14 and CAF#32) (Figure 5B).The association between Twist1 and CXCL12 was analyzed in other types of cancer using public mRNA expression data from TCGA, which showed a significant correlation between Twist1 and CXCL12 expression in various cancers, such as breast, colorectal, ovarian, endometrial, lung, thyroid, and renal cancers, and glioblastoma (Supplementary Figure 5).These results suggest that Twist1 regulates CXCL12 expression at the transcriptional level.
To further examine the regulation of CXCL12 by Twist1, we performed luciferase reporter assays using the pGL3.CXCL12 vector carrying the CXCL12 promoter region (Figure 5C).The pGL3.CXCL12 and pSG5.hTwist1.HA vectors were transiently co-transfected into NIH3T3 cells.As shown in Figure 5D (17).Twist1 bound specifically to the positive control (PDGFRĮ; í1839 to í1834 bp) and the proximal E-box CAGGTG (Figure 5E), whereas no amplification was detected in the distal E-box (E-box2) or in the NC site containing no E-box sequence (Figure 5E).These results indicate that Twist1 binds directly to the endogenous CXCL12 promoter.
In gastric cancer tissues, CXCL12 is expressed in both cancer cells and stromal cells and there is a significant correlation in stromal expressions between CXCL12 and Twist1 (P<0.001) (Figure 5F).In addition, stromal expression of CXCL12 was associated with an unfavorable clinical outcome (P=0.0232)(Figure 5G).Patients' group with both Twist1 and CXCL12 expressing CAFs had the worst prognosis, whereas the other group in which CAFs were negative for both Twist1 and CXCL12 had the best prognosis, and the difference was statistically significant (P<0.001)(Figure 5G).Additional results for clinical relevance of CXCL12 in gastric cancer were described in Supplementary Results & Tables 1-4 and Supplementary Figure

Twist1 contributes to perpetual CAF activation by repressing the senescence of fibroblasts and CAFs
One of the CAF-specific properties that distinguishes them from normal activated fibroblasts such as myofibroblast is their constitutive activation (3).In contrast to CAFs, myofibroblasts activated during conventional inflammation regress to a normal state through senescence or apoptosis after the source of inflammation is removed (3).Because Twist1 suppresses cellular senescence in various types of epithelial cancer cells (21), we hypothesized that Twist1 induced the constitutive activation of CAFs through repression of senescence.To validate this hypothesis, the effect of Twist1 on cellular senescence was examined in both normal fibroblasts and CAFs.
Twist1-overexpression significantly reduced SA staining and downregulated the expressions of p15 and p16, representative inducers of senescence, in gastric normal fibroblasts (NF#5 and NF#15) (Figure 6A).On the other hand, silencing Twist1 expression by shRNA accelerated senescence in CAFs, as detected by increased SA staining in CAF#13 and CAF#36 (Figure 6B), and upregulated p15 and p16 expressions in all three gastric CAFs (CAF#13, CAF#14, and CAF#32).Our results strongly suggest that Twist1 contributes to the generation of CAFs by repressing cellular senescence.Together, our findings support a model in which Twist1 is a key regulator of cancer associated fibroblasts (Figure 6C).

Discussion
In our previous study, we showed that Twist1 is frequently overexpressed in stromal fibroblasts surrounding gastric cancer cells (6).And these Twist1-expressing stromal fibroblasts also expressed CAF markers such as FSP1 and PDGFRĮ with association with poor prognosis (6).Despite these results, there was no direct evidence for functional role of Twist1 as a key regulator of CAFs.Therefore, in the present study, we established fibroblasts and CAFs models from gastrectomy specimens and confirmed that Twist1 expression is necessary and sufficient for CAF transdifferentiation.In addition, IL6/STAT3 axis was discovered to be a key upstream control of Twist1.Microarray analysis of Twist1's effect on mRNA expression in fibroblasts identified CXCL12 as a key Twist1's target in CAFs.Moreover, Twist1 was revealed to suppress cellular senescence of normal fibroblasts and CAFs.
Since Yang et al. first reported that Twist1 is a critical player in cancer metastasis (16), most studies focused on Twist1's role in cancer cells (22)(23)(24), with little attention paid to its possible role in the tumor microenvironment, including CAFs.In contrast to rare expression of Twist1 in adult normal fibroblast, Twist1 is frequently and strongly expressed in embryonic fibroblast (9,11,25) and pathologic fibroblasts in idiopathic pulmonary fibrosis (26,27) and desmoid tumor (28).Bridges et al. (26) reported that Twist1 enhanced survival and accumulation of fibroblasts in fibrotic lung disease, which is consistent with results of our present study.And Bacac et al. (28) reported that Twist1 is highly expressed in desmoid tumors (neoplastic myofibroblastic lesions) compared with nodular fasciitis (non-tumorous proliferation of fibroblasts).Furthermore, Spaeth et  fibroblasts and CAFs.Indeed, Twist1 was revealed to be induced by the IL6/STAT3 axis in gastric normal fibroblasts and was indispensable for IL-6 induced CAF transdifferentiation (Figure 1).Furthermore, IL-6 was abundant in the cancer stroma and a significant positive correlation between pSTAT3 and Twist1 was observed in cancer tissues (Figure 2F).
In the present study, we searched for Twist1 target genes in CAFs using mRNA microarray and most of identified candidate genes, such as CXCL12 and Tn-C were previously reported to be CAF phenotype-related (19,20,40).Orimo et al. reported that CXCL12 secreted by CAFs promotes angiogenesis and recruitment of additional inflammatory cells (20).Kojima et al. reported that CXCL12 plays a role in the maintenance of the CAF phenotype through formation of an auto-stimulatory loop (19).Furthermore, CXCL12 has recently emerged as a therapeutic target in various cancers (41)(42)(43).However, the transcription factor that regulates CXCL12 remains unknown.In the present study, we showed that CXCL12 was transcriptionally regulated by Twist1 and the expressions of CXCL12 and Twist1 were significantly correlated to each other in cancer tissues with significant clinical relevance.In our previous study, we examined the genome-wide transcriptional regulation by Twist1 in synovial sarcoma combining both ChIP-SEQ (chromatin immunoprecipitationsequencing) and mRNA array (23).In that study, we found that Twist1 upregulated CXCL12 expression in synovial sarcoma and bound to a similar sequence in the proximal E-box site (í509 bp), which strongly supports the results of the present study (23).represses premature senescence by abrogating regulators of p53-and Rbdependent pathways in breast cancer cells (21).Our results are highly consistent with those of previous studies and suggest that Twist1 is likely to promote CAF transdifferentiation through the suppression of senescence.
Fibroblast is a highly heterogeneous entity.Chang et al. showed that fibroblasts from different anatomical regions display characteristic different phenotypes (44).In addition to the differences between fibroblasts from different anatomical sites, fibroblasts separated from a single tissue are not composed of a homogeneous population (45).Likewise, the expressional profiles of CAFs may show considerable heterogeneity depending on the anatomical region.Therefore, fibroblasts and CAFs used in study should be derived from the same organ to minimize unexpected bias (3).In the present study, we established primary fibroblast and CAF cultures from gastric cancer tissues to study the interaction between gastric cancer cells and CAFs.
In conclusion, we identified Twist1 as a novel and key transcription factor that regulates CAFs.Based on our results, Twist1 is suggested as a potent therapeutic target to reverse unfavorable reprogramming of cancer microenvironment.
cancer cells subcutaneously implanted in balb/c-nude mice as the tumor model.To examine the effect of Twist1-positive fibroblasts on xenograft tumor growth, 5×10 5 stomach cancer cells were co-injected with 1.5×10 6 Twist1-positive fibroblasts [Hs738(Twist1)].As controls, the same number of cancer cells were injected alone or with Twist1-negative fibroblast [Hs738(GFP)].To investigate the effect of IL-6 secreted by cancer cells on surrounding CAFs in vivo, SNU638+GFP , the CXCL12 promoter-reporter activity was significantly affected when Twist1 was introduced into the cells.Two E-box sequences located at 509 bp and at 1037 bp upstream of the transcription start site were identified in the promoter region of CXCL12.To confirm on April 20, 2017.© 2014 American Association for Cancer Research.cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on November 3, 2014; DOI: 10.1158/0008-5472.CAN-14-0350 the role of these E-boxes, mutations were introduced [CAGGTG (í509) to TTGGTG and CATCTG (í1037) to TTTCTG] and the mutant vectors were transfected into NIH3T3 cells.Mutation of the proximal E-box (í509) abrogated the effect of Twist1 on CXCL12 promoter activity, whereas mutation of the distal E-box (í1037) made no difference, indicating that the proximal E-box is crucial for Twist1 function.Next, we performed chromatin immunoprecipitation (ChIP) assays in endogenous Twist1expressing CAFs (CaF#14).Specific primer sets for the known Twist1-binding E-box CACATG (PDGFRĮ; í1839 to í1834 bp) in the PDGFRĮ promoter were used as a positive control
Although the identity of CAFs remains obscure, one of the characteristics that distinguish CAFs from conventional activated fibroblasts is their constitutive on April 20, 2017.© 2014 American Association for Cancer Research.cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on November 3, 2014; DOI: 10.1158/0008-5472.CAN-14-0350 activation.During normal inflammation, once a wound is repaired, the number of activated fibroblasts decreases and resting phenotype is restored (3).Unlike wound healing, CAFs at tumor site remain perpetually activated (3).In the present study, we showed that Twist1 suppressed cellular senescence in fibroblasts.In fact, the suppression of senescence by Twist1 was previously reported in various types of cells.Maestro et al. reported that Twist1 inhibits apoptosis and bypasses p53induced growth arrest by direct and indirect modulation of the ARF/MDM2/p53 pathway in mouse embryonic fibroblasts (25).Ansieau et al. reported that Twist1

Figure 1 .
Figure 1.Twist1 is required for IL6 induced differentiation of quiescent

Figure 3 .
Figure 3. Twist1 is sufficient for transition of normal fibroblast to CAF. (A) The

Figure 5 .Figure 6 .
Figure 5. Twist1 directly upregulated the expression of CXCL12 with clinical Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on November 3, 2014; DOI: 10.1158/0008-5472.CAN-14-0350 surgery at Samsung Medical Center of SungKyunKwan University of Medicine, Seoul, Korea.An experienced pathologist grossly examined and obtained representative samples of the tumor tissues (CAF, Human Gastric Cancerassociated Fibroblast) and distal normal tissues (NF, Human Gastric Normal Fibroblast).Detailed procedures are described in Supplementary Methods.
on April 20, 2017.© 2014 American Association for Cancer Research.cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on November 3, 2014; DOI: 10.1158/0008-5472.CAN-14-0350 on April 20, 2017.© 2014 American Association for Cancer Research.cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.