Connective Tissue Growth Factor Activates Pluripotency Genes and Mesenchymal–Epithelial Transition in Head and Neck Cancer Cells

Published OnlineFirst May 16, 2013; DOI: 10.1158/0008-5472.CAN-12-4085

Cancer Tumor and Stem Cell Biology Research

Cheng-Chi Chang1, Wen-Hao Hsu1, Chen-Chien Wang1, Chun-Hung Chou9, Mark Yen-Ping Kuo2, Been-Ren Lin3, Szu-Ta Chen4,12, Shyh-Kuan Tai5, Min-Liang Kuo7,8, and Muh-Hwa Yang6,9,10,11

Abstract The epithelial–mesenchymal transition (EMT) is a key mechanism in both embryonic development and cancer metastasis. The EMT introduces stem-like properties to cancer cells. However, during somatic cell reprogramming, mesenchymal–epithelial transition (MET), the reverse process of EMT, is a crucial step toward pluripotency. Connective tissue growth factor (CTGF) is a multifunctional secreted protein that acts as either an oncoprotein or a tumor suppressor among different cancers. Here, we show that in head and neck squamous cell carcinoma (HNSCC), CTGF promotes the MET and reduces invasiveness. Moreover, we found that CTGF enhances the stem-like properties of HNSCC cells and increases the expression of multiple pluripotency genes. Mechanistic studies showed that CTGF induces c-Jun expression through avb3 integrin and that c-Jun directly activates the transcription of the pluripotency genes NANOG, SOX2, and POU5F1. Knockdown of CTGF in TW2.6 cells was shown to reduce tumor formation and attenuate E-cadherin expression in xenotransplanted tumors. In HNSCC patient samples, CTGF expression was positively correlated with the levels of CDH1, NANOG, SOX2, and POU5F1. Coexpression of CTGF and the pluripotency genes was found to be associated with a worse prognosis. These ﬁndings are valuable in elucidating the interplay between epithelial plasticity and stem-like properties during cancer progression and provide useful information for developing a novel classiﬁcation system and therapeutic strategies for HNSCC. Cancer Res; 73(13); 1–11. 2013 AACR.

Introduction fibrosis, and cancer metastasis (1, 2). A recent breakthrough Epithelial–mesenchymal transition (EMT), which is hall- in EMT research has shown that EMT is capable of introducing marked by suppression of the adherent protein E-cadherin, is stem-like properties to epithelial cells (3, 4). In human cancers, a fundamental process in embryonic development, organ the acquisition of stem-like properties in cancer cells is crit- ically involved in disease progression and treatment resistance (5). Different mechanisms have been shown to be responsible Authors' Affiliations: 1Graduate Institute of Oral Biology; 2Graduate for the EMT-induced stem-like properties of cancer cells. For Institute of Clinical Dentistry, School of Dentistry; Departments of 3Surgery and 4Pediatrics, National Taiwan University Hospital; 5Department of instance, the EMT inducer Zeb1 inhibits the expression of the Otolaryngology; 6Division of Hematology–Oncology, Department of Med- microRNA 200 family, resulting in the upregulation of the icine, Taipei Veterans General Hospital; 7Graduate Institute of Toxicology, polycomb protein Bmi1 and the induction of stemness in College of Medicine; 8Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University; 9Institute of Clinical Medicine, pancreatic cancer (6). In head and neck cancers, we previously 10Cancer Research Center and Genomic Research Center, National Yang- showed that the other EMT inducer Twist1 directly activates 11 Ming University; Genomic Research Center, Academia Sinica, Taipei; BMI1 transcription and that Twist and Bmi1 act cooperatively and 12Department of Pediatrics, National Taiwan University Hospital Yun- Lin Branch, Yun-Lin, Taiwan to promote the EMT and stemness (7). However, the role of pluripotency genes in EMT-induced stemness remains unclear. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Recently, the most fascinating progress in stem cell research is the direct reprogramming of somatic cells into an embryonic W.H. Hsu and C.C. Wang contributed equally to this work. stem cell (ESC)-like state by defined factors, as in the gener- Corresponding Authors: Muh-Hwa Yang, Institute of Clinical Medicine, ation of induced pluripotent stem cells (iPSC; refs. 8–10). A Cancer Research Center and Genomic Research Center, National Yang-Ming University, No. 155, Sec. 2, Li-Nong Street, Taipei 112, further conceptual advance in iPSC research is that mesen- Taiwan. Phone: 886-2-28267000, ext. 7911; Fax: 886-2-28235870; E-mail: chymal–epithelial transition (MET), the reverse process of [email protected]; or Min-Liang Kuo, Graduate Institute of Toxi- EMT, is essential for the generation of iPSCs (11, 12). Moreover, cology, College of Medicine and Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, No. 1, Sec. E-cadherin, an important adhesion molecule that is down- 1, Jen-Ai Road, Taipei 100, Taiwan. Phone: 886-2-23123456 ext. 88607; regulated during the EMT, has been shown to be highly Fax: 886-2-23410217; E-mail: [email protected] expressed in mouse ESCs and reprogrammed cells, and E- doi: 10.1158/0008-5472.CAN-12-4085 cadherin itself induces pluripotency (13). All of these results 2013 American Association for Cancer Research. from independent study groups show the critical role of the

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MET and E-cadherin in the induction of pluripotency in Spheroid formation assay somatic cells. However, these findings seem to conflict with Cells (1 104) were suspended in serum-free DMEM/F-12 the concept of EMT-induced stemness in cancer cells (14). (Gibco-BRL) containing N2 supplement, 10 ng/mL human Clarification of the relationship between the EMT/MET and recombinant bFGF, and 10 ng/mL EGF (all from R&D Systems stem cell properties in cancer cells is necessary. Inc.). After cultivation for 14 days, primary spheroids were Connective tissue growth factor (CTGF, also known as harvested by centrifugation and then dissociated and resus- CCN2) is a secreted protein that acts as a multifunctional pended in this medium. The number of secondary spheroids signaling modulator in various biologic or pathologic process- larger than 100 mm was counted after 14 days. es (15). In human cancers, the pleiotropic functions of CTGF have been investigated among different types of cancers (16). cDNA microarray CTGF acts as an oncoprotein in glioma and breast cancer (17, The Affymetrix HG-U133 plus 2.0 whole-genome array was 18). However, we previously showed the tumor-suppressive used for cDNA microarray. The microarray data were depos- effect of CTGF in lung cancer and colon cancer (19, 20). Head ited in the NCBI GEO database with the accession number and neck squamous cell carcinoma (HNSCC), including GSE30423. cancers originating in the oral cavity, oropharynx, hypophar- ynx, and larynx, is one of the leading causes of cancer-related Prediction of putative transcription factors death worldwide. Local progression and lymph node involve- TESS (25) and PROMO 3.0 (26) were used to predict the ment are the major causes of HNSCC-related mortality and the putative transcriptional factors that regulate the expression of incidence of distal organ metastasis is relatively rare compared pluripotency genes. with other cancers (21). However, the mechanism responsible for the local progression of HNSCC is unclear. We recently Cloning of the proximal promoter regions of POU5F1, showed that CTGF attenuates the invasiveness of oral squa- SOX2, NANOG, and JUN, generation of the promoter mous cell carcinoma cells through miR504 and FOXP1 (22). In reporter constructs, and luciferase reporter assay this report, we further show the unique function of CTGF in The genomic regions flanking the promoter region of human HNSCC in the induction of the MET and stem-like properties, POU5F1 (1464 þ53 bp to ATG), NANOG (1544 þ160 bp which results in hindering the dissemination but promoting to ATG), SOX2 (1580 þ300 bp to ATG), and JUN (1140 the local progression of this cancer. þ80 bp to ATG) were amplified by PCR and inserted into the SacI/BglII sites of the pGL4.2 vector to generate the corresponding reporter constructs (Fig. 3E and 5A). Promoter Materials and Methods constructs containing mutated c-Jun–binding sites were gen- Cells and plasmids erated by site-directed mutagenesis (Fig. 5A). The luciferase HEK-293T and the human hypopharyngeal cancer cell line reporter assay was conducted by transfecting the reporter FaDu were obtained from the Bioresource Collection and construct with or without the siRNA vector into the indicated Research Center of Taiwan. The human oral cancer cell line cell lines. A plasmid expressing the bacterial b-galactosidase OECM-1 was originally provided by Dr. Ching-Liang Meng of gene (pCMV-bgal) or the renilla luciferase gene (pRL-TK) was the National Defense Medical College in Taiwan (23). The cotransfected in each experiment as an internal control for human oral cancer cell lines SAS and HSC3 were provided by transfection efficiency. Cells were harvested after 24 hours of Dr. Cheng-Chi Chang of National Taiwan University. The transfection, and the luciferase activities were assayed as TW2.6 cells were provided by Dr. Mark Y.B. Kuo of National previously described (7). All values are expressed as the fold Taiwan University (24). The characteristics of the other HNSCC change in luciferase activity after normalization to the b-galac- cell lines are described in our previous study (22). All of the cell tosidase activity. lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS, except for FaDu and OECM-1, which Statistical analysis were cultured in Roswell Park Memorial Institute (RPMI)-1640 An independent Student t test was used to compare the 2 medium with 10% FBS. continuous variables between 2 groups, and a c test was applied The pcDNA3-CTGF was generated by inserting the open for the comparison of dichotomous variables. A Kaplan–Meier reading frame of CTGF into the pcDNA3.1 vector, and pCDH- estimation and a log-rank test were used to compare the JUN was generated by inserting the open reading frame of JUN difference in the survival period between patient groups. The into the pCDH-MCV-MCS-EF1-puro vector. The short-inter- level of statistical significance was set at 0.05 for all tests. fering RNA (siRNA) vectors pSUPER-si-CTGF, pSUPER-si-JUN, Please see Supplementary Methods for the other methods and pSUPER-si-ESR1 were generated by inserting a short- used in this study. hairpin sequence against the target genes into the pSUPER. puro vector, and a control vector for the siRNA experiments Results was constructed by inserting a scrambled sequence. Supple- CTGF induces mesenchymal–epithelial transition in mentary Table S1 lists the sequences used for the production of head and neck cancer cells the siRNA constructs. Stable clones were generated by trans- Because CTGF has been shown to play different roles, either fection of the expression vectors and/or siRNA plasmids and to promote or inhibit metastasis in different types of human selected using the appropriate antibiotics. cancers (16), we herein investigated the effect of CTGF on the

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migration of HNSCC cells. First, we screened the expression inhibit migration in HNSCC. To this end, we examined the level of CTGF in 4 HNSCC cell lines, including SAS, HSC3, FaDu, EMT phenotype in our established stable cell lines. A switch and TW2.6. CTGF was found to be signiﬁcantly higher in TW2.6 from N-cadherin to E-cadherin was shown in the SAS and FaDu than in the other 3 cell lines (Supplementary Fig. S1A). We CTGF transfectants (Fig. 1D and E), which indicates the therefore selected SAS and FaDu as parental cell lines to occurrence of the MET. Consistently, the suppression of generate stable CTGF overexpression lines and TW2.6 to CTGF in TW2.6 caused a shift from E-cadherin to N-cadherin establish stable CTGF knockdown lines. The ectopic expres- (Fig. 1F). In the SAS and FaDu cells, overexpression of CTGF sion of CTGF inhibited the migration of the SAS and FaDu cells induced an epithelial morphology and expression of membra- (Fig. 1A and B), and knockdown of endogenous CTGF in the nous E-cadherin (Fig. 1G and H). In contrast, knockdown of TW2.6 cells augmented their migratory ability (Fig. 1C). How- CTGF dissociated membranous E-cadherin in the TW2.6 cells ever, CTGF did not have signiﬁcant impact on proliferation of (Fig. 1I). Collectively, these results suggest that CTGF inhibits the SAS and FaDu cells (Supplementary Fig. S1B), suggesting migration and promotes the MET in HNSCC cells. that CTGF reduces cellular migration without affecting pro- liferative ability in HNSCC cells. CTGF promotes stem-like properties and upregulates Because the EMT is a crucial process in promoting cancer pluripotency genes cell migration and metastasis (2), we investigated whether Because the EMT generates cells with stem-like properties CTGF could induce the MET, the reverse process of EMT, to (3, 4), we reasoned that CTGF may reduce cellular stemness in

A SAS-Neo SAS-C1 D G SAS SAS-Neo SAS-C1 0 h SAS-Neo 1.5 Neo C1 C2 SAS-C1 SAS-C2 CTGF 12 h 1 E-cadherin

N-cadherin 24 h 0.5 β-Acn 0 Fold change of migrated cells 36 h 12 24 36 h

FaDu-Neo FaDu-C1 BEHFaDu-Neo FaDu-C1 FaDu 0 h FADU-Neo 1.5 FaDu-Neo FADU-C1FaDu-C1 Neo C1 C2 FADU-C2FaDu-C2 CTGF 24 h 1 E-cadherin

0.5 48 h N-cadherin β-Acn 0 72 h Fold change of migrated cells 24 48 72 h

CITW2.6-scr TW2.6-siC1 F TW2.6-scr TW2.6-siC1 TW2.6 4 TW2.6-Scr 0 h TW2.6-siC1 scr siC1 siC2 TW2.6-siC2 3 CTGF 6 h E-cadherin 2 N-cadherin 1 12 h β-Acn

Fold change of migrated cells 0 24 h 6 12 24 h

Figure 1. CTGF induces MET in HNSCC. A–C, wound-healing migration assay of SAS cells stably transfected with CTGF (SAS-C1 and SAS-C2) versus a control vector (SAS-Neo; A), FaDu cells stably transfected with CTGF (FaDu-C1 and FaDu-C2) versus a control vector (FaDu-Neo; B) and TW2.6 cells receiving siRNA against CTGF (TW2.6-siC1 and TW2.6-siC2) versus a scrambled sequence (TW2.6-scr; C). Left, photos of wound-healing assay. Right, relative migratory ability at different time points. D–F, Western blot analysis of CTGF, E-cadherin, and N-cadherin in SAS (D), FaDu (E), and TW2.6 stable cells (F). G–I, phase contrast image (top) and immunoﬂuorescence staining of E-cadherin (bottom) in SAS (G), FaDu (H), and TW2.6 stable cells (I). Scale bar, 200 mm for phase contrast image; 20 mm for immunoﬂuorescence image.

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HNSCC. To examine this notion, we conducted experiments to cells, which produce a low level of CTGF, and then observed the evaluate the stem-like properties of HNSCC cells using our impact of exogenous CTGF on the stem-like properties of these þ stable cell lines, including the analysis of the putative HNSCC cells. rCTGF increased the proportion of CD44 cells and þ stem cell marker CD44 (27), aldehyde dehydrogenase 1 enhanced the colony-forming and spheroid-forming abilities (ALDH1) activity, the proportion of side-population cells, in both the SAS and FaDu cells (Supplementary Fig. S4). anchorage-independent growth, and spheroid-forming ability. Next, we investigated the correlation between the expres- To our surprise, in the SAS cells, the ectopic expression sion of CTGF and the pluripotency genes in HNSCC cells. We þ of CTGF increased the proportion of CD44 (Fig. 2A), ﬁrst tested the expression of 14 human ESC-enriched genes (9) þ side-population (Supplementary Fig. S2A), and ALDH1 cells in the stable CTGF-manipulated SAS and TW2.6 cell lines. Most (Supplementary Fig. S2B). Ectopic CTGF also enhanced the of the ESC-enriched genes were upregulated in the CTGF- anchorage-independent growth and spheroid-forming ability overexpression cells and downregulated in the CTGF-knock- (Fig. 2B and C). This result was also shown in another HNSCC down cells (Fig. 2D). Among these genes, we investigated the þ cell line FaDu: an increased proportion of CD44 cells and impact of CTGF on the expression of POU5F1 (POU class 5 enhanced colony-forming and spheroid-forming abilities were homeobox 1, which encodes the Oct4 protein), NANOG, and shown in FaDu–CTGF transfectants (Supplementary Fig. S3A– SOX2 due to their importance in both stem cells and cancers C). Knockdown of CTGF in TW2.6 cells, which express a high (28, 29). First, we compared the expression level of the plur- þ level of endogenous CTGF, reduced the CD44 population, ipotency genes among different HNSCC cell lines and found spheroid-forming ability, and anchorage-independent growth that the cells with low endogenous CTGF (e.g., SAS and HSC3) (Fig. 2A–C). Because CTGF is a secreted protein, we used the tended to have a lower level of POU5F1/NANOG/SOX2.In recombinant CTGF protein (rCTGF) to treat SAS and FaDu contrast, in cells with high endogenous CTGF (e.g., TW2.6),

A B Isotype CD44 SAS-Neo SAS-C1 SAS-C2 TW2.6-scr TW2.6-siC1

Isotype CD44

SAS-Neo 0.94% 2.05% TW2.6-scr 0.03% 27.83% 80 300 SSC SSC

SAS-C1 * 0.5% 31.99% 60 * 200 40 * 0.04% TW2.6-siC1 14.78% 100 10,000 cells 20 10,000 cells SAS-C2 Sphere counts/ 0.96% 32.81% CD44 Sphere counts/ 0 0 SAS-Neo SAS-C1 SAS-C2 TW2.6-scr TW2.6-siC1 CD44

120 SAS-C1/ TW2.6-siC1/ C D fold E * SAS-Neo TW2.6-scr * SOX15 80 >4.5 SAS TW2.6 NANOG 4 Neo C1 C2 scr siC1 siC2 40 DPPA2 CTGF CTGF LIN28 3.5 POU5F1

Colony counts (>0.1mm) 0 SAS-Neo SAS-C1 SAS-C2 SOX2 3 Oct4 Oct4 OTX1 600 2.5 ESG1 Nanog Nanog 400 MYBL2 2 FOXD3 Sox2 Sox2 * 1.5 200 STELLA β β-Acn ZNF206 1 -Acn 0 TW2.6-scr TW2.6-siC1 REX1 Colony counts (>0.1mm) UTF1 0.5 <0.25

þ Figure 2. CTGF promotes stem-like properties of HNSCC cells. A, ﬂow cytometry for analyzing CD44 expression in SAS cells transfected with the CTGF-expressing vector (SAS-C1 and SAS-C2) or with an empty vector as a control (SAS-Neo; left) and TW2.6 cells receiving siRNA against CTGF (TW2.6- siC1) versus a scrambled sequence (TW2.6-scr; right). The percentage of CD44þ cells is shown in the right bottom quadrant. B, spheroid formation assay. Top, representative pictures. Bottom, quantiﬁcation (n ¼ 3). C, soft agar colony formation assay (n ¼ 3). Colonies larger than 0.1 mm were counted. D, a heat-map summarizing the results of relative expression of 14 stemness genes in SAS-C1 versus SAS-Neo or TW2.6-siC1 versus TW2.6-scr. E, Western blot analysis of CTGF, Oct4, Nanog, and Sox2 in SAS and TW2.6 stable cell lines. In B and C, data represent means SEM. , P < 0.05 by Student t test.

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the level of POU5F1/NANOG/SOX2 was relatively higher (Sup- and the induction of the MET. Because the pluripotency genes plementary Fig. S5A). Stable expression of CTGF in the SAS and were upregulated by CTGF at the mRNA level (Fig. 2D), we first FaDu cells increased the level of Oct4, Sox2, and Nanog (Fig. 2E aimed to identify the putative transcription factor(s) mediating left; Fig. S5B and S5C). Consistently, knockdown of CTGF in CTGF-induced pluripotency gene expression. To this end, TW2.6 cells reduced Oct4, Sox2, and Nanog (Fig. 2E right). cDNA microarray analysis was conducted in FaDu cells treated Exogenous CTGF upregulated the expression of the pluripo- with rCTGF versus vehicle control, and the transcripts that tency genes in a dose- and time-dependent manner, and the increased more than 2-fold were considered to be upregulated effect of rCTGF on inducing POU5F1/NANOG/SOX2 was more upon rCTGF treatment. In addition, we used 2 software sys- prominent than its effect on the other 2 pluripotency genes tems, TESS and PROMO 3.0, to predict the transcription factors KLF4 and MYC (Supplementary Fig. S6). Taken together, these that may simultaneously regulate the transcription of NANG, results suggest that CTGF promotes stem-like properties and SOX2,andPOU5F1. Putative transcription factors that are induces the expression of pluripotency genes, especially responsible for CTGF-mediated pluripotency gene induction POU5F1, NANOG, and SOX2, in HNSCC. were identified by overlapping these 3 datasets (Fig. 3A). Using this strategy, we identified 6 factors (TCFL2, ESR1, YY1, c-Jun is a major player in the CTGF-induced MET and MYB, LEF1, and JUN) as candidates that coordinate the expres- stem-like properties sion of NANOG, SOX2 and POU5F1 simultaneously. To validate Next, we investigated how CTGF induces the MET and stem- this result, quantitative RT-PCR analysis was conducted in the like properties in HNSCC. We hypothesized that CTGF regu- CTGF overexpression and knockdown systems. In the SAS lates the pluripotency genes POU5F1, NANOG,andSOX2 simul- cells, 3 of the 6 transcription factors (ESR1, LEF1, and JUN) were taneously, resulting in the enhancement of stem-like properties upregulated in the CTGF transfectants compared with the

A B SAS-Neo C 5 SAS-C1 Binding Binding 3 TW2.6-scr sites sites SAS-C2 * TW2.6-siC1 4 * predicon predicon TW2.6-siC2 by TESS by PROMO * 3 * 2 (n = 61) 6 (n = 46) * *

mRNA > 2x in (fold change) 1 FaDu+rCTGF (fold change) n 1 *

( = 53214) Relave gene expression *

Relave gene expression * * 0 0 TCFL2 ESR1 YY1 MYB LEF1 JUN LEF1 ESR1 JUN

DESAS 1 TSS F * Neo C1 C2 JUN 3 * * c-Jun JUN-Luc Luc 2.5 p-c-Jun (-1,140~+80bp) 14 * 15 luc * 2 β-Acn 12 luc luc 1.5 10 10 * TW2.6 8 1 renilla scr siC1 siC2 6 Fold change of 5 firefly luc/ renilla c-Jun 4 0.5 Fold change of Fold change of 2 p-c-Jun firefly luc/ renilla

firefly luc/ 0 0 0 (hr) β-Acn Neo C1 C2 Citric rCTGF JUN-Luc(0.2 μg/mL) + + ++ buffer rCTGF(0.2 μg/mL) - + ++ FaDu IgG(2 μg/mL) - - + - An-αvβ3(2 μg/mL) -- - +

Figure 3. c-Jun is a major factor responsible for CTGF-induced pluripotency genes expression, and CTGF activates JUN transcription through avb3 integrin. A, schematic representation of the strategy for mining the candidate transcriptional factors mediating CTGF-induced pluripotency genes expression. B, quantitative RT-PCR analysis of the mRNA levels of 6 candidate transcriptional factors in CTGF-expressing SAS stable clones (SAS-C1 and SAS-C2) and a control clone (SAS-Neo; n ¼ 3). C, relative mRNA levels of LEF1, ESR1, and JUN in TW2.6 cells receiving siRNA against CTGF (TW2.6-siC1 and TW2.6-siC2) or a scrambled sequence (TW2.6-scr). D, Western blot analysis of total c-Jun and phosphorylated c-Jun (p-c-Jun) in SAS (top) and TW2.6 (bottom) stable cells. E, top, schematic representation of the reporter construct of JUN promoter (JUN-Luc). TSS, transcription start site. Bottom left, relative luciferase activity of FaDu cells transfected with JUN-Luc and treated with rCTGF (0.2 mg/mL) or a vehicle control (n ¼ 3). Bottom right, relative luciferase activity of SAS stable cells transfected with JUN-Luc (n ¼ 3). F, relative luciferase activity in FaDu cells transfected with JUN-Luc and treated with rCTGF for 12 hours, with or without an avb3 neutralizing antibody. IgG was a control of the antibody neutralization experiment (n ¼ 3). In B, C, E, and F, data represent means SEM. , P < 0.05 by Student t test.

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control cells (Fig. 3B). In the TW2.6 cells, however, only ESR1 Next, we focused on the regulation of c-Jun by CTGF and JUN were signiﬁcantly decreased when CTGF was knocked because c-Jun is an important factor in cancer progression down (Fig. 3C). We therefore considered that c-Jun (the protein (30), and head and neck cancer is a male-predominant encoded by JUN) and ESR1 (estrogen receptor 1) are major disease (31, 32). In SAS cells, overexpression of CTGF factors contributing to the CTGF-induced expression of enhanced the expression of total and phosphorylated c-Jun; POU5F1/NANOG/SOX2. To conﬁrm this, we repressed the consistently, knockdown of CTGF in TW2.6 reduced the level expression of JUN and ESR1, respectively, using siRNA in the of total and phosphorylated c-Jun (Fig. 3D). Because JUN was SAS–CTGF transfectants. Repression of either JUN or ESR1 found to be upregulated by CTGF at the mRNA level, we reduced the CTGF-induced expression of POU5F1/NANOG/ assumedthatCTGFactivatesthetranscriptionofJUN. SOX2 in the SAS cells (Supplementary Fig. S7), suggesting that To address this notion, we generated a reporter construct both c-Jun and ESR1 contribute to CTGF-mediated pluripo- containing the proximal promoter of JUN and tested whether tency genes expression. CTGF could activate it. Both exogenous CTGF treatment and

A SAS-C1-scr SAS-C1-siJUN SAS-C1 scr siJUN c-Jun

E-Cadherin SAS-C1-scr SAS-C1-siJUN

N-Cadherin

β-Acn

SAS - SAS - BCCDH c-Jun Isotype CD44 D CTGF 0.17% 26.8% 30 SAS-C1 c-Jun -scr 25 20 E-Cadherin 15

γ-Catenin SSC 0.00% 3.04% 10 5 *

Vimen SAS-C1 % of CD44 expression 0 -siJUN SAS-C1 SAS-C1 N-Cadherin -scr -siJUN β-Acn CD44 E SAS-C1 F SAS-C1 G SAS-C1 scr siOct4 scr siSox2 scr siNanog Oct4 Sox2 Nanog

E-Cadherin E-Cadherin E-Cadherin

N-Cadherin N-Cadherin N-Cadherin

Vimenn Vimenn Vimenn

β-Acn β-Acn β-Acn

Figure 4. c-Jun is critical in CTGF-induced MET and stemness, and suppression of single pluripotency factor only partially affects the EMT markers. A, left, Western blot analysis of c-Jun, E-cadherin, and N-cadherin in SAS-C1 cells receiving siRNA against c-Jun (siJUN) or a scrambled control (scr). Right, phase contrast image (top) and immunofluorescence staining of E-cadherin (bottom). Scale bar, 200 mm for phase contrast image and 20 mmfor immunofluorescence. B, Western blot of CTGF, c-Jun, epithelial markers (E-cadherin and g-catenin), and mesenchymal markers (N-cadherin and vimentin) in SAS cells transfected with a c-Jun expression vector (SAS-c-Jun) or a control vector (SAS-CDH). C, representative results of flow cytometry þ þ for analyzing CD44 expression in SAS-C1-siJUN versus SAS-C1-scr. The percentage of CD44 cells was shown in the right top quadrant. D, quantification þ of the CD44 flow cytometry results (n ¼ 3). Data represent means SEM. , P < 0.05 by Student t test. E–G, Western blot analysis of the epithelial marker E-cadherin, mesenchymal marker N-cadherin and vimentin, and pluripotency factors Oct4/Nanog/Sox in SAS-C1 cells receiving siRNA against a scrambled control (scr) or Oct4 (E), Sox2 (F), or Nanog (G).

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ectopic CTGF expression activated the JUN promoter (Fig. regulation of E-cadherin, appearance of a mesenchymal-like 3E). Because CTGF has been shown to induce target gene morphology, and dissociation of E-cadherin at intercellular expression through interaction with avb3integrin(33),we junctions (Fig. 4A). Interestingly, the ectopic expression of c- investigated whether CTGF induces JUN transactivation Jun promoted the MET in SAS cells (Fig. 4B). Repression of c- þ through avb3 integrin. The data showed that in FaDu cells, Jun reduced the CD44 population in the SAS–CTGF trans- the activation of the JUN promoter by exogenous CTGF was fectants (Fig. 4C and D). However, knockdown of individual abrogated in the presence of an anti-avb3 antibody (Fig. 3F), pluripotency genes, including POU5F1, NANOG, and SOX2, only suggesting that avb3 is an essential receptor in CTGF- partially affected the expression of EMT markers in the SAS– induced JUN transactivation. CTGF cells. Repression of POU5F1 enhanced N-cadherin We next investigated the role of c-Jun and individual plur- expression (Fig. 4E), and silencing NANOG or SOX2 attenuated ipotency factors in the CTGF-induced MET and stem-like the levels of both E-cadherin and N-cadherin (Fig. 4F and 4G). characteristics. In stable SAS–CTGF cells, knockdown of JUN Taken together, these results indicate that CTGF induces reversed the MET, i.e., upregulation of N-cadherin and down- the transcriptional activation of JUN through avb3 integrin

SAS-Neo SAS + citric buffer SAS/Neo A B SAS-C1 C 5 SAS + rCTGF NANOG promoter (-1544~+160bp) 3.5 SAS/C1SAS-C2 * * 3 SAS/C2 * luc 4 * BS1 BS2 BS3 Luc 2.5 * 2 * 3 * * renilla Nanog-Luc TGAGTCA TGACTTC TGGGTCA 1.5 2 * 1 Nanog-Luc(mut) GTCTGAC GTCAGGA GTTTGAC (fold change) 1 0.5 Fold change of POU5F1 promoter 0 firefly luc/ 0 (-1464~+53bp) Relave luciferase acvity Nanog-Luc Oct4-Luc Sox2-Luc Oct4- Sox2- Nanog- BS1 BS2 BS3 Luc SAS-Neo Luc Luc Luc 2 2 SAS-C1 SAS + citric buffer Oct4-Luc GAAGTCA TGACTGG GAGGTCA luc 1.5 SAS-C2 1.5 SAS + rCTGF Oct4-Luc(mut) TCCTGAC GTCAGTT TCTTGAC

1 renilla 1 SOX2 promoter (-1580~+300bp)

BS2 Luc (fold change) 0.5 0.5

BS1 BS3 Fold change of ﬁreﬂy luc/

Relave luciferase acvity 0 0 TGACTCC TGACTGC TGACACA Sox2-Luc Nanog- mutaonOct4Oct4- mutaonSox2Sox2- NanogNanog- Oct4- Sox2Sox2- Sox2-Luc(mut) GTCAGAA GTCAGTA GTCACAC Luc(mut) Luc(mut) Luc(mut) mutaonLuc(mut) mutaonLuc(mut) mutaonLuc(mut)

1.5 1.5 D 1.5 E F

1 1 1 * * (fold change) (fold change) (fold change) 0.5 0.5 0.5 * * * Relave luciferase acvity Relave luciferase acvity Relave luciferase acvity 0 0 0 scr + - + - scr + - + - scr + - + - siJUN - + - + siJUN - + - + siJUN - + - + Nanog-Luc Nanog-Luc(mut) Oct4-Luc Oct4-Luc(mut) Sox2- Luc Sox2 –Luc(mut)

NANOG POU5F1 SOX2 promoter +1 TSS promoter +1 TSS promoter +1 TSS G P1 P2 P3 H P1 P2 P3 I P1 P2 P3 BS1 BS2 BS3 BS1 BS2 BS3 BS1 BS2 BS3 c-Jun IgG 10 c-Jun IgG c-Jun IgG * 1.4 1.2 * 8 1.2 1 * * 1 6 P1 0.8 * 0.8 P1 P1

P2 0.6 4 0.6 P2 % Input P2 % Input P3 * % Input * * 0.4 0.4 * P3 2 * * * P3 * 0.2 0.2 0 0 0

Figure 5. CTGF activates POU5F1/NANOG/SOX2 transcription through c-Jun. A, schematic representation of the wild-type and c-Jun–binding sites–mutated promoter constructs of NANOG (Nanog-Luc and Nanog-Luc-mut), POU5F1 (Oct4-Luc and Oct-Luc-mut), and SOX2 (Sox2-Luc and Sox2-Luc-mut). BS1, BS2, and BS3 indicate c-Jun–binding sites. B, luciferase reporter assay. The relative luciferase activity in SAS–CTGF stable cells transfected with the wild-type (top) or mutant (bottom) promoter reporter constructs (n ¼ 3). C, luciferase reporter assay. The relative luciferase activity in SAS cells transfected with the wild-type (top) or mutant (bottom) promoter reporter constructs and treated with an rCTGF 0.2 mg/mL or a vehicle control (n ¼ 3). D–F, relative luciferase activity in SAS-C1 cells cotransfected with the wild-type or mutant promoter reporter construct of NANOG (D), POU5F1 (E), or SOX2 (F), and the vector containing siRNA against c-Jun (si-JUN) or a scrambled sequence (scr; n ¼ 3). G–I, qChIP assay. Top, organization of the NANOG (F), POU5F1 (G), and SOX2 (H) promoter and schematic representation of the primer design. TSS, transcription start site. P1, P2, and P3 indicate the ampliﬁed sequences. BS1, BS2, and BS3 indicate the predicted c-Jun–binding sites. IgG was a control of immunoprecipitation experiments. n ¼ 3 for each experiment. In B–I, data represent means SEM. , P < 0.05 by Student t test.

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and c-Jun is a major factor involved in the CTGF-mediated the pluripotency genes POU5F1, NANOG, and SOX2. Upregula- MET and stemness in HNSCC. tion of multiple pluripotency genes results in enhanced stem- like properties and the MET in HNSCC cells. The induction of c-Jun directly regulates the pluripotency genes POU5F1/ the MET and stemness by CTGF promotes the local progres- NANOG/SOX2 sion but reduces the invasiveness of HNSCC. Next, we sought to determine whether CTGF transactivates POU5F1/NANOG/SOX2 through c-Jun or ESR1. Reporter constructs containing the proximal promoter of POU5F1, NANOG, Discussion or SOX2 were generated (Fig. 5A). In SAS cells, the ectopic The phenotypic association between the EMT and cancer CTGF or treatment with rCTGF activated the promoters of stem cells (CSC) has been firmly established. However, recent POU5F1, NANOG, and SOX2, and mutation of the c-Jun–binding studies implicate that self-renewal is not bound to the mes- sites abrogated this activation (Fig. 5B and C). Knockdown of enchymal phenotype and that epithelial-type cells are favor- JUN in SAS–CTGF cells reduced the CTGF-induced wild-type able for tumor colonization and proliferation. In prostate pluripotency gene promoter activation but had no effect on the cancers, the epithelial gene expression program is enriched c-Jun–binding site–mutated promoters (Fig. 5D–F). Silencing in tumor-initiating cells (35). In breast cancers, miR-200, a of ESR1 also abrogated the CTGF-induced promoters activa- major miRNA that is downregulated during the EMT and is tion (Supplementary Fig. S8). Here, we also focused on inves- associated with the epithelial phenotype, facilitates metastatic tigating the role of c-Jun in the regulation of POU5F1/NANOG/ colonization through the repression of Sec23a (36). The iPSC SOX2 by CTGF. A quantitative chromatin immunoprecipita- studies revealed that the MET, the reverse process of EMT, is tion (qChIP) assay confirmed the direct binding of c-Jun to the prerequisite for somatic cell reprogramming (11, 12). In this promoters of POU5F1/NANOG/SOX2 in 2 independent SAS– study, we showed that CTGF induces stem-like properties and CTGF transfectants (Fig. 5G–I). These results indicate that in local progression in HNSCC through the MET, and this is the CTGF-overexpressing HNSCC cells, c-Jun activates the tran- first study to show the impact of the secreted protein-induced scription of POU5F1, NANOG, and SOX2 through direct binding MET on tumor progression. This finding provides insight for to their promoters. understanding the interplay between epithelial–mesenchymal plasticity and self-renewal during cancer progression and also CTGF promotes tumor growth but inhibits invasiveness uncouples the "inevitable" association between the EMT and in vivo CSCs. We suggest that CSCs are derived from the EMT and Next, we evaluated the effect of CTGF on tumor growth and harbor migratory abilities or are derived from the MET with invasiveness in vivo using a xenotransplantion assay. Different lower invasiveness but higher colonizing abilities. doses of TW2.6-siCTGF or TW2.6-scr cells were subcutane- The critical role of the pluripotency transcription factors ously injected into nude mice. Knockdown of CTGF not only (e.g., Oct4, Nanog, and Sox2) in somatic cell reprogramming reduced the incidence of tumors but also decreased the volume has been extensively investigated. However, the association of the formed tumors (Fig. 6A and B). However, repression of between these factors and the EMT in cancer cells is poorly endogenous CTGF in TW2.6 promoted invasion of the tumor understood. Sporadic reports link a pluripotency gene signa- cells into the adjacent tissues and reduced the E-cadherin ture to the EMT. Among these reports, most of them suggest expression in the implanted tumors (Fig. 6C). Finally, we that pluripotency gene expression is associated with the confirmed the experimental results in HNSCC patient samples. mesenchymal phenotype and cancer invasiveness (37–39). Quantitative RT-PCR analysis was conducted on 78 pairs of However, 1 report showed the opposite result, i.e., the silencing HNSCC samples to detect the expression of CTGF, NANOG, of Oct4 promotes the EMT of cancer cells (40). In addition to POU5F1, SOX2, and CDH1. The mRNA level of CTGF positively the undefined role of pluripotency factors in the EMT, how correlated with the levels of POU5F1, NANOG, SOX2, and CDH1 these genes are regulated during cancer progression has also (Fig. 6D), suggesting the induction of epithelial and pluripo- been elusive. Here, we identify a novel pathway in which CTGF tency genes expression by CTGF in the HNSCC samples. CTGF coordinates the expression of the pluripotency genes through expression was significantly associated with the primary tumor c-Jun to promote the MET. Knockdown of the individual size (Fig. 6E), i.e., T-stage of the American Joint Committee on pluripotency factors is not able to completely reverse the MET Cancer staging system (34). However, CTGF expression did not process, suggesting that the pluripotency factors act collabo- significantly differ between patients with or without metasta- ratively rather than individually to regulate the epithelial sis (Fig. 6F). We then analyzed the prognostic impact of CTGF plasticity of cancer cells. and pluripotency gene expression in the HNSCC cases (Fig. 6G). CTGF is known to play distinct roles among different Patients with both high CTGF and high pluripotency genes cancers, acting either as an oncoprotein or tumor suppressor expression (group 3) or with low CTGF expression (group 2) (16–20). Here, we show a unique role for CTGF in HNSCC in tended to have a worse prognosis than those with both high that CTGF promotes local progression but reduces invasive- CTGF and low pluripotency genes expression (group 1). ness. Because both factors are important for HNSCC prognosis, We propose a model to summarize our findings in Fig. 7. In the impact of CTGF on HNSCC cells is relatively complicated, HNSCC cells, CTGF induces the MET and stemness via the and the overall impact of CTGF may rely on whether the following mechanism. CTGF induces the expression of c-Jun CTGF–stemness axis is activated. In this study, we found that through avb3, and c-Jun directly activates the transcription of patients with high CTGF and low pluripotency factor

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TW2.6-scr TW2.6-siC1 A B C TW2.6- TW2.6- TW2.6-scr TW2.6-siC1 H & E scr siC1 103 1/4 0/4 104 3/4 1/4 105 4/4 4/4 ) 3 2,000 CTGF 1,500 1,000 500 * 0 E-Cadherin

Tumor volume (mm TW2.6- TW2.6- scr siC1 D NANOG SOX2 POU5F1 CDH1 15 15 15 15 p<0.000001P<0.001 p<0.0002P<0.001 p<0.0003 P<0.00006P<0.001 P<0.001 10 10 10 10 CDH1 SOX2 NANOG of POU5F1 5 of 5 5 5 of of -∆∆CT -∆∆CT 2 -∆∆CT 0 2 0 0 0 -∆∆CT 2 2

-5 -5 -5 -5 CTGF low CTGF high CTGF low CTGF high CTGF low CTGF high CTGF low CTGF high E G F 1.0 P value 10.0 4 * 0.8 1 vs. 2=0.08 8.0 1 vs. 3=0.04 3 0.6 2 vs. 3=0.38 expression 6.0 * expression 2 0.4 CTGF

4.0 CTGF Group 1: CTGF high & 3 factors low 0.2 CTGF 2.0 1 Group 2: low

Proporon of survival Group 3: CTGF high & 3 factors high Relave

Relave 0.0 0 T1 T2 T3 T4 Metastasis (-) Metastasis (+) 5 10 2015 3025 Month(s)

Figure 6. CTGF induces MET, promotes tumor growth, and attenuates invasiveness in vivo. A, representative pictures of nude mice 6 weeks after injection of TW2.6-siC1 versus TW2.6-scr cells. The black arrows indicate the xenotransplanted tumor. Cell dose ¼ 1 105 cells. B, top, a table showing the result of xenotransplantation study (n ¼ 4 for each group). Bottom, comparison of the tumor volume of TW2.6-siC1 or TW2.6-scr formed xenotransplanted tumors (cell dose ¼ 1 105; n ¼ 4). C, top, H&E stain showing the implanted tumor. The black arrows indicate the border of implanted tumor, and the gray arrows indicate the muscle infiltration of tumor cells. Scale bar, 500 mm. Middle and bottom, immunohistochemistry of CTGF (middle) and E-cadherin (bottom) in implanted tumor. Scale bar, 200 mm. D, relative mRNA expression levels of POU5F1, NANOG, SOX2, and CDH1 in CTGF low (2 DDCT <1) versus high (2DDCT 1) HNSCC samples (n ¼ 78). The boxplots represent sample maximum (top end of whisker), top quartile (top of the box), median (band in the box), bottom quartile (bottom of the box), and sample minimum (bottom end of whisker). E, relative mRNA level of CTGF in HNSCC samples with different T stages. , P < 0.05 by Student t test. F, relative mRNA level of CTGF in HNSCC samples with or without lymph node/distant metastasis. G, comparison of the disease-free survival of HNSCC patients with different expression patterns of CTGF and pluripotency genes. P values were estimated by a log-rank test. expression had a better prognosis, whereas patients with both tasize to distal organs (21). According to our results, we suggest high CTGF and pluripotency factor expression and low CTGF that combining the CTGF and pluripotency gene expression expression had a worse prognosis. This result indicates that in profiles in HNSCC will be a better prognostic indicator than an patients with high expression of both CTGF and pluripotency individual marker. genes, the CTGF–stemness axis is activated and the stemness In summary, our study is the first one to show that the of the tumor cells is increased. In low CTGF cases, the tumors induction of pluripotency gene expression by a secreted pro- are more mesenchymal-like and invasive. Both of them will tein promotes the MET in cancer cells, which leads to local have a worse prognosis than those with high CTGF and low progression but reduces distal dissemination. This conceptual pluripotency factor expression. This finding provides a rea- breakthrough provides insight into understanding the inter- sonable explanation for the clinical observation that head and play between epithelial plasticity and stemness during cancer neck cancers severely destruct local tissues but rarely metas- progression. Furthermore, this report also provides useful

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Chang et al.

CTGF CTGF

CTGF CTGF CTGF CTGF

αvβ3

oﬀ POU5F1/SOX2/ NANOG on Figure 7. A model depicting the mechanism of CTGF-induced MET c-Jun JUN and stemness in HNSCC. on c-Jun POU5F1/SOX2 /NANOG

MET and stemness Mesenchymal type Epithelial type with with low stem-like properes stem-like properes

Tumor Tumor formaon invasion

information for developing a novel classification system and Writing, review, and/or revision of the manuscript: S.T. Chen, M.H. Yang Study supervision: C.C. Chang, M.L. Kuo, M.H. Yang therapeutic strategy for HNSCC according the expression patterns of CTGF and pluripotency genes. Grant Support fl This work was supported by the Taipei Veterans General Hospital, National Disclosure of Potential Con ict of Interest Taiwan University Hospital joint grant (VN101-02 to M.H. Yang and B.R. Lin); fl No potential con icts of interest were disclosed. National Science Council (101-2321-B-010-015 to M.H. Yang); Excellent Trans- lational Medicine Research Projects of National Taiwan University, College of Authors' Contributions Medicine, and National Taiwan University Hospital (100-C101-014 to C.C. Conception and design: C.C. Chang, W.H. Hsu, C.C. Wang, M.Y.P. Kuo, M.H. Chang); a grant from Ministry of Education, Aim for the Top University Plan Yang (M.H. Yang), and a grant from Department of Health, Center of Excellence for Development of methodology: C.C. Chang, W.H. Hsu, C.C. Wang, C.H. Chou Cancer Research (DOH101-TD-C-111-007 to M.H. Yang and DOH101-TD-PB-111- Acquisition of data (provided animals, acquired and managed patients, 007 to C.C. Chang and M.L. Kuo). provided facilities, etc.): C.C. Chang, W.H. Hsu, C.C. Wang, C.H. Chou, M.Y.P. The costs of publication of this article were defrayed in part by the Kuo, S.K. Tai payment of page charges. This article must therefore be hereby marked Analysis and interpretation of data (e.g., statistical analysis, biostatistics, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this computational analysis): C.C. Chang, W.H. Hsu, C.C. Wang, C.H. Chou, M.Y.P. fact. Kuo, B.R. Lin, M.L. Kuo, S.T. Chen, M.H. Yang Administrative, technical, or material support (i.e., reporting or orga- Received October 30, 2012; revised March 22, 2013; accepted March 25, 2013; nizing data, constructing databases): B.R. Lin, S.T. Chen, S.K. Tai published OnlineFirst May 16, 2013.

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Connective Tissue Growth Factor Activates Pluripotency Genes and Mesenchymal−Epithelial Transition in Head and Neck Cancer Cells

Cheng-Chi Chang, Wen-Hao Hsu, Chen-Chien Wang, et al.

Cancer Res Published OnlineFirst May 16, 2013.

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