Keratin 19 Expression in Hepatocellular Carcinoma Is Regulated by Fibroblast-Derived HGF Via a MET-ERK1/2-AP1 and SP1 Axis

Published OnlineFirst January 23, 2018; DOI: 10.1158/0008-5472.CAN-17-0988

Cancer Molecular Cell Biology Research

Keratin 19 Expression in Hepatocellular Carcinoma Is Regulated by Fibroblast-Derived HGF via a MET-ERK1/2-AP1 and SP1 Axis Hyungjin Rhee1, Hye-Young Kim1, Ji-Hye Choi2,3, Hyun Goo Woo2,3, Jeong Eun Yoo1, Ji Hae Nahm1, Jin-Sub Choi4, and Young Nyun Park1,5

Abstract

Keratin (KRT) 19 is a poor prognostic marker for hepatocellular activated KRT19 expression in HCC cells. In clinical specimens carcinoma (HCC); however, regulatory mechanisms underlying of human HCC (n ¼ 339), HGF and KRT19 protein expression its expression remain unclear. We have previously reported the correlated with CAF levels. In addition, HGF or MET presence of fibrous tumor stroma in KRT19-positive HCC, protein expression was associated with FOSL1 and KRT19 expres- suggesting that cross-talk between cancer-associated fibroblasts sion and was found to be a poor prognostic factor. Analysis of (CAF) and tumor epithelial cells could regulate KRT19 expression. data from The Cancer Genome Atlas also revealed KRT19 This was investigated in this study using an in vitro model of expression was closely associated with CAF and MET-mediated paracrine interaction between HCC cell lines (HepG2, SNU423) signaling activities. These results provide insights into the molec- and hepatic stellate cells (HSC), a major source of hepatic ular background of KRT19-positive HCC that display an aggres- myofibroblasts. HSCs upregulated transcription and translation sive phenotype. of KRT19 in HCC cells via paracrine interactions. Mechanistically, Significance: These findings reveal KRT19 expression in hepatocyte growth factor (HGF) from HSCs activated hepatocellular carcinoma is regulated by cross-talk between c-MET and the MEK–ERK1/2 pathway, which upregulated KRT19 cancer-associated fibroblasts and HCC cells, illuminating new expression in HCC cells. Furthermore, AP1 (JUN/FOSL1) therapeutic targets for this aggressive disease. Cancer Res; 78(7); and SP1, downstream transcriptional activators of ERK1/2, 1619–31. 2018 AACR.

Introduction resection or radiofrequency ablation (2, 3, 5–7). KRT19 protein expression or KRT19 expression–related signatures have also Keratin 19 (KRT19) is a small (approximately 40 kDa) type I emerged as predictors of HCC recurrence and survival after liver cytokeratin, which lacks the tail domain common among transplantation (8, 9). cytokeratins (1). KRT19 is a marker for hepatic stem/progenitor Despite the clinical significance of KRT19 expression in cells (2, 3). In the normal liver, mature hepatocytes express HCC, the regulatory mechanism underlying KRT19 expression KRT8 and KRT18, while cholangiocytes additionally express remains unclear. Previously, at least a subset of KRT19-positive KRT7 and KRT19 (4). Although most hepatocellular carcino- HCCs was considered to have originated from hepatic pro- mas (HCC) do not express KRT19 like normal hepatocytes, genitor cells expressing KRT19 (10). Conversely, a recent study 10%–28% of HCCs were reported to express KRT19, and were using lineage-tracing rodent HCC models demonstrated that found to display a more invasive phenotype and poorer out- KRT19-positive HCC cells originated from mature hepatocytes comes than those that do not, when treated using hepatic and were not observed in the early clonal expansion of hepatocarcinogenesis (11, 12). Therefore, KRT19 expression was 1Department of Pathology, Brain Korea 21 PLUS Project for Medical Science, proposed to be acquired during hepatocarcinogenesis. Integrated Genomic Research Center for Metabolic Regulation, Yonsei Univer- Increasing evidence supports the notion that tumor microen- sity College of Medicine, Seoul, Korea. 2Department of Physiology, Ajou Uni- vironmental components including cancer-associated fibroblasts versity School of Medicine, Suwon, Korea. 3Department of Biomedical Science, (CAF), the extracellular matrix, angiogenic cells, and immune cells 4 Graduate School, Ajou University, Suwon, Korea. Department of Surgery, are important factors for growth, migration/invasion, and angio- Yonsei University College of Medicine, Seoul, Korea. 5Severance Biomedical genesis in various tumors including HCC (13). CAFs, which Science Institute, Yonsei University College of Medicine, Seoul, Korea. produce the fibrous tumor stroma, are known to contribute Note: Supplementary data for this article are available at Cancer Research to an aggressive cancer phenotype, facilitating invasiveness, Online (http://cancerres.aacrjournals.org/). proliferation, and immune suppression (14). Moreover, they Current address for H. Rhee: Department of Radiology, Yonsei University were also reported to stimulate cancer cell stemness by activating College of Medicine, Seoul, Korea. the WNT or NOTCH signaling pathway in colon, breast, and Corresponding Author: Young Nyun Park, Department of Pathology, Yonsei prostate cancers (15). We have previously reported that HCCs University College of Medicine, 50-1, Yonsei-ro, Seodaemun-gu, Seoul 03722, expressing KRT19 exhibited fibrous tumor stroma. In contrast, Korea. Phone: 822-2228-1768; Fax: 822-362-0860; E-mail: [email protected] that there was little or no fibrous stroma in HCCs in the absence of doi: 10.1158/0008-5472.CAN-17-0988 KRT19 expression, while HCCs with abundant fibrous stroma 2018 American Association for Cancer Research. (namely, "scirrhous HCC") commonly expressed KRT19 (3, 16).

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The fibrous stroma of HCCs was reported to be generated by CAFs, IHC staining for KRT19, HGF, MET, and FOSL1 was per- recruited and transdifferentiated from peritumoral myofibro- formed using an automated staining system (Ventana Medical blasts through the cross-talk with HCC cells (17). Hepatic myofi- Systems, Inc.). a-Smooth muscle actin (aSMA) and CD90 broblasts are a heterogenous group of cells, and activated hepatic staining was performed using an Envision kit (Dako) according stellate cells represent one of the major sources of hepatic myofi- to the manufacturer's instructions. All slides were counter- broblasts (18, 19). In light of these results, we hypothesized that stained with hematoxylin. Detailed information on antibodies KRT19 expression in HCCs might be regulated by fibrous stromal and criteria for IHC analysis are described in Supplementary components of the tumor microenvironment. Tables S2–S4. Here, we established an in vitro interaction model of HCC The clinical outcomes of the patients were retrospectively cells and CAFs using HCC and hepatic stellate cell lines. We obtained by reviewing the electronic medical records at Severance discovered that the hepatic stellate cell-derived hepatocyte Hospital. The last update for the HCC patient cohort was Sep- growth factor (HGF) and the subsequent activation of the tember 2016. The endpoints were defined as follows: disease- MET-MEK-ERK1/2 pathway activates KRT19 transcription via specific survival was defined as the time from surgery to death in the activator protein 1 (AP1) and specificity protein 1 (SP1) patients with HCC involving >50% of the liver, HCC with exten- transcription factors. This regulatory axis was validated in a sive portal vein tumor thrombosis, or HCC with extrahepatic large cohort (n ¼ 339) of patients with HCC, with IHC staining metastasis (27); disease-free survival was defined as the time from and gene expression profiles from HCC cohorts from The surgery to an initial diagnosis of recurrence regardless of location. Cancer Genome Atlas (TCGA; n ¼ 371). Our results provide This study was in accordance with the ethical guidelines of the a better understanding of the molecular background of KRT19- Declaration of Helsinki, and was approved by the Institutional positive HCCs with an aggressive biological behavior. Review Board of the Severance Hospital (4-2016-0878), and the requirement for informed consent was waived.

Materials and Methods Gene expression profile analysis Cell lines and reagents Gene expression profiles of HCC cohorts were obtained from A human hepatoblastoma cell line, HepG2, and two hepa- TCGA (https://cancergenome.nih.gov, LIHC, n ¼ 371), excluding tocellular carcinoma cell lines, Hep3B and PLC/PRF/5, were the profiles of nontumor tissues. The expression enrichment of a purchased from ATCC (20–22). The Huh7, SNU182, SNU423, gene signature in an individual specimen was calculated by using and SNU475 cell lines were purchased from the Korean Cell the preranked gene-set enrichment analysis (GSEA, http://soft Line Bank (23, 24). Two hepatic stellate cell lines, hTERT-HSC ware.broadinstitute.org/gsea/index.jsp). and LX-2, were obtained as gifts from David Brenner (Univer- sity of California, San Diego, San Diego, CA; ref. 25) and Scott Statistical analysis Friedman (Icahn School of Medicine at Mount Sinai, New York, Statistical analyses were performed using the SPSS software NY 26), respectively. Cell lines were maintained in DMEM (version 23.0, SPSS Inc.). The Mann–Whitney U test, c2 test, Fisher supplemented with 10% FBS and penicillin/streptomycin exact test, or Spearman correlations were used as deemed appro- (Invitrogen). Cell lines were authenticated using short tandem priate. Survival analyses were performed using the Kaplan–Meier repeat analysis, and the databases of ATCC or Korean Cell Line method and the log rank test. Bank were used as reference. The primary human hepatic For detailed information on experimental procedures, please stellate cells were purchased from ScienCell, maintained in refer to the Supplementary Materials and Methods and Supple- dedicated media, and used in the experiments up to 8 passages. mentary Tables S5–S7. The cell lines in use were regularly screened to monitor myco- plasma infections per several months, using PCR-based detec- tion kit (iNtRON Biotechnology). U0126 was purchased from Results Cell Signaling Technology; mithramycin A was purchased from KRT19 expression in HCC is upregulated by paracrine factors Tocris Bioscience; SCH772984, SU11274, and PHA-665752 from hepatic stellate cells were purchased from Selleckchem; doxycycline and S3I-201 The correlation between KRT19 expression and fibrous stro- were purchased from Sigma-Aldrich. mal components was confirmed in a cohort of patients with HCC (n ¼ 339) using TMA. The expression of the KRT19 protein Human HCC tissue samples and clinicopathologic and IHC was higher in HCCs with a higher proportion of aSMA-positive analyses CAFs (Fig. 1A and B). We analyzed the degree of KRT19 expres- We enrolled a total of 339 patients with HCC who did not sion and the amount of aSMA-positive CAFs in a semiquanti- undergo any kind of preoperative treatment before hepatic resec- tative manner, and observed a significant correlation (Spearman tion. Consecutive HCC tissue samples were obtained by hepatic correlation coefficient r ¼ 0.155, P ¼ 0.004). There was also a resection between March 2006 and February 2011 at the Sever- positive correlation between the degree of KRT19 expression ance Hospital, Yonsei University Medical Center. Their clinico- and the amount of CD90-positive CAFs (r ¼ 0.124, P ¼ 0.023; pathologic characteristics are described in Supplementary Table Supplementary Fig. S1A). In addition, the association between S1. The representative paraffin-embedded sections of HCC were the expression of KRT19 and fibrous stromal markers including used for tissue microarray (TMA) construction and IHC analysis. ASMA, FAP, and VIM was evaluated using gene expression data For TMA construction, two core biopsies of 2 mm in diameter, from TCGA (LIHC, n ¼ 371). KRT19 expression was significantly were taken from individual HCC donor paraffin blocks and correlated with ASMA (r ¼ 0.25, P ¼ 7.56 10 7), FAP (r ¼ arranged into recipient TMA blocks using a trephine apparatus 0.30, P ¼ 4.06 10 9), and VIM expression (r ¼ 0.43, P ¼ 5.63 (Superbiochips Laboratories). 10 18), respectively (Fig. 1C).

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KRT19 in HCC Is Regulated by CAF via HGF-MET-AP1 and SP1

Figure 1. KRT19 expression in HCC is upregulated by paracrine factors from hepatic stellate cells. A, Representative cases of HCC with or without the aSMA-positive CAFs. Scale bars, 100 mm. B, Correlation between the aSMA-positive CAFs and KRT19 protein expression. C, Correlation between the gene expression level of KRT19 and fibrous stromal markers including ASMA, FAP, and VIM in HCC data from TCGA (n ¼ 371). D, mRNA and protein expression levels of KRT19 after treatment with hTERT-HSC CM. HCC cells (HepG2 and SNU423) were harvested at the indicated times, and mRNA and protein expression levels were analyzed by qRT-PCR or Western blot analysis. E, mRNA and protein expression levels of KRT19 after coculture with hTERT-HSC. F, Morphologic changes in HepG2 and SNU423 induced by coculture with hTERT-HSC or CM from hTERT-HSC. Scale bars, 100 mm. Each qRT-PCR experiment was independently conducted two times in triplicate, and a representative result is represented in the figure. The bar graph represents the mean SD, and statistical significance is indicated (, P < 0.05; , P < 0.01; , P < 0.001, t test).

To establish an in vitro model for evaluating the interactions increase in the expression of KRT19 in HepG2 cells, whereas between epithelial tumor cells and CAFs in HCC, we tested two CM from LX-2 did not (Supplementary Fig. S1D). hepatic stellate cell lines: hTERT-HSC and LX-2. hTERT-HSCs Next, we treated the HepG2 and SNU423 cell lines with CM showed a higher expression for ﬁbroblast activation-related from hTERT-HSCs for 12–72 hours. The CM from hTERT-HSCs markers, including FAP and aSMA (28), when compared with increased the KRT19 mRNA and protein expression levels, and that in LX-2 cells (Supplementary Fig. S1B and S1C). Interest- the difference was apparent at 24–72 hours (Fig. 1D). We also ingly, conditioned media (CM) from hTERT-HSCs induced an conducted indirect cocultures using permeable cell culture

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inserts with 1-mm pore sizes. The sharing of the culture media mechanism underlying the upregulation of KRT19 upon treat- between the HCC cell lines and hTERT-HSCs increased KRT19 ment with CM from hTERT-HSCs. The CM treatment induced mRNA and protein expression in HepG2 and SNU423 cells the phosphorylation of ERK1/2 in both HepG2 and SNU423, (Fig. 1E). Similarly, CM from primary hepatic stellate cells and the phosphorylation of STAT3 in HepG2. Other signaling increased KRT19 mRNA and protein expression (Supplementary pathways, including p38 MAPK, JNK, and AKT, were unaffected Fig. S1B, S1E and S1F). CM from hTERT-HSC or primary hepatic (Fig. 2A). Treatment with the STAT3 inhibitor (S3I-201) stellate cells, and indirect cocultures with hTERT-HSCs resulted decreased KRT19 expression in HepG2, but not SNU423, sug- in similar features, leading to cell scattering and elongation gesting that the STAT3 pathway is not a common regulator of (Fig. 1F; Supplementary Fig. S1G). These results suggest that KRT19 in both cell lines (Supplementary Fig. S2A). paracrine factors from hTERT-HSCs regulate KRT19 expression When the MEK–ERK1/2 pathway was inhibited with the in HCC cell lines. MEK inhibitor (U0126) or ERK1/2 inhibitor (SCH772984; ref. 29), the upregulation of KRT19 induced following treat- hTERT-HSC CM regulates KRT19 expression through the ment with CM from hTERT-HSCs was abolished in HepG2 MEK–ERK1/2 pathway and SNU423 cells (Fig. 2B). KRT19 expression also increas- Considering that KRT19-positive HCCs display an aggressive ed following 12-O-Tetradecanoylphorbol-13-acetate (TPA) biological behavior (3), oncogenic signaling pathways includ- treatment, and the combination of the MEK or ERK1/2 inhib- ing MAPK, AKT, and STAT3 were investigated to determine the itor with TPA decreased KRT19 expression (Supplementary

Figure 2. Conditioned media regulates KRT19 expression through the MEK–ERK1/2 pathway. A, The activation status of selected oncogenic pathways in HepG2 and SNU423 by CM from hTERT-HSC. B, KRT19 mRNA and protein levels after CM treatment combined with/without a MEK or ERK inhibitor. mRNA and protein expression levels were analyzed by qRT-PCR or Western blot analysis. C, Upregulation of KRT19 mRNA and protein levels in HepG2 and SNU475 cells by the transient expression of a constitutively active MEK1 (S218D/S222D) or ERK1. The amounts of plasmid used in transfections (per 35-mm dish) are indicated. Each qRT-PCR experiment was independently conducted two times in triplicate, and a representative result is represented in the ﬁgure. The bar graph represents the mean SD, and statistical signiﬁcance is indicated (, P < 0.05; , P < 0.01; , P < 0.001, t test).

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KRT19 in HCC Is Regulated by CAF via HGF-MET-AP1 and SP1

Fig. S2B). To further confirm that KRT19 expression is regu- and S4C). When MET selective inhibitors (SU11274 and lated by the MEK–ERK1/2 pathway, the constitutively active PHA-665752) were combined with CM, the upregulation of form of MEK1 (S218D/S222D) and wild-type ERK1 were KRT19 also decreased (Fig. 3G). Taken together, our data overexpressed in HepG2 and SNU475 cells (30). These cell suggest that HGF paracrine signaling from hepatic stellate cells lines were chosen for the overexpression experiment, instead upregulates KRT19 expression via MET and the MEK-ERK1/2 of SNU423, because the transfection efficiency of SNU423 was pathway. quite low, compared with that in other cells (Supplementary Fig. S2C). The overexpression of MEK1 (S218D/S222D) and The AP1 and SP1 transcription factor binding sites in the ERK1 increased KRT19 expression by 2- to 6-fold (Fig. 2C). KRT19 promoter are important for KRT19 expression Taken together, CM from hTERT-HSCs increased KRT19 To determine important cis-regulatory elements in the expression in HCC cell lines via the MEK–ERK1/2 pathway. KRT19 promoter, we constructed luciferase reporter plasmids of approximately 3 kb in length (2,885 bp from þ41 bp HGF in hTERT-HSC CM upregulates KRT19 via MET and the relative to the transcription start site) and serial deletion MEK–ERK1/2 pathway mutants (Fig. 4A). When measuring the promoter activity of To specify which paracrine factors and receptors activate the constructs in HepG2 and SNU423 cells, we found it to be the MEK–ERK1/2 pathway in HCC cells, a phospho-receptor commonly and significantly decreased at two segments: tyrosine kinase array was performed in HepG2 and SNU423 between 2,167 and 1,967 and between 152 and 69. cells and three MET, ALK, and FGFR3 receptors were found To confirm that these two promoter segments are truly impor- to be commonly phosphorylated(Fig.3A).Considering tant for KRT19 regulation, we also measured the promoter that MET is known to drive the invasive growth of HCC via activity of deletion constructs lacking these segments: D2167- various signaling pathways including MEK-ERK1/2 (31), MET 1967 and D152-69. The constructs elicited a >70% decrease and its well-known ligand, HGF, were further investigated. in promoter activity in the case of D2167–1967 and >25% for We confirmed the phosphorylation of MET upon treatment D152-69 (Fig. 4B). with CM from hTERT-HSCs in HepG2 and SNU423 cells by We next searched for putative transcription factor binding sites Western blot analysis (Fig. 3B). The concentration of HGF, at positions 21671967 and 15269 using online pro- determined by ELISA, was much higher in CM from hTERT- grams (PROMO; http://alggen.lsi.upc.es, and Tfsitescan; http:// HSCs (10.8 ng/mL), compared with that in CM from HepG2 www.ifti.org/cgi-bin/ifti/Tfsitescan.pl). And mutant constructs and SNU423 (<0.2 ng/mL for both; Fig. 3C). Treatment with for the AP1, MAZ, ELK, ETS, and SP1 binding sites were generated recombinant HGF upregulated KRT19 expression in both using site-specific mutagenesis (Fig. 4C). Of these mutant con- HCC cell lines, and the combined treatment with MAPK structs, mutants of the 2021 AP1 site and 72 SP1 site revealed a inhibitors decreased the HGF-induced upregulation of KRT19 significant reduction in activity. Therefore, AP1 and SP1, as expression (Fig. 3D). downstream transcriptional activators of ERK1/2, were identified In addition, CM from LX-2, where the HGF concentration to be important for KRT19 regulation in HCC. was very low (<0.1 ng/mL), did not increase KRT19 expression (Supplementary Fig. S3A). The HGF concentration in CM from The binding of JUN, FOSL1, and SP1 to the KRT19 promoter primary hepatic stellate cells (30.4 ng/mL) was even higher is important for the regulation of KRT19 expression than that in CM from hTERT-HSCs (Supplementary Fig. S3B). The AP1 binding site sequence at the 2021 position in the HCC cell lines with low MET expression, including SNU182 KRT19 promoter (TGACTCA) is a well-known TPA-responsive andPLC/PRF/5,alsoshowedlowKRT19expression(Supple- element, to which various heterodimers of the JUN and FOS mentary Fig. S3C). The transient overexpression of MET in family may bind (32). To identify specific AP1 proteins that SNU475 cells increased phospho-MET and phospho-ERK1/2, regulate KRT19 expression in HCC cell lines, we performed as well as KRT19 expression at both the mRNA and protein siRNA screening for AP1 family members. At first, we screened level. In PLC/PRF/5, the transient overexpression of MET also the transcriptional levels of seven JUN and FOS genes, including increased phospho-MET and phospho-ERK1/2, and KRT19 JUN, JUNB, JUND, FOS, FOSB, FOSL1,andFOSL2. Among mRNA levels. (Supplementary Fig. S3D). these seven genes, we selected JUN, JUND, FOSL1,andFOSL2, To determine whether HGF is indeed responsible for the which showed a relatively higher mRNA expression than other KRT19 upregulation induced by CM from hTERT-HSCs, we genes in the JUN and FOS family (Fig. 5A). To target these genes, established doxycycline-inducible knockdown versions of a transient knockdown with two pooled siRNAs was carried out; hTERT-HSCs (hT-shHGF-1, hT-shHGF-2). Two days of doxy- the knockdown efficiency was greater than 80% (Supplementary cycline treatment (1 mg/mL) prior to CM generation efficiently Fig. S5A). JUN or FOSL1 knockdown generally decreased KRT19 depleted HGF (7.2 ng/mL vs. 0.17 ng/mL in hT-shHGF-1, 8.4 expression in HepG2 and SNU423 cells (Fig. 5B). To confirm ng/mL vs. 0.13 ng/mL in hT-shHGF-2, Fig. 3E). Treatment of that these results did not represent off-target effects, each siRNA HepG2 and SNU423 cells with CM from nondoxycycline-trea- was transfected separately. By transfecting siJUN-1, siJUN-2, tedhT-shHGF-1orhT-shHGF-2upregulatedKRT19expression siFOSL1-1, and siFOSL1-2, both JUN and FOSL1 were success- at both the transcriptional and translational levels, whereas fully knocked down, at the transcriptional and translational treatment with CM from doxycycline-treated hT-shHGF-1 or level. Moreover, KRT19 mRNA levels also declined in both hT-shHGF-2 did not (Fig. 3F). In addition, doxycycline reversed HepG2 and SNU423 cells (Fig. 5C, Supplementary Fig. S5B). the cell scattering induced by the CM from hT-shHGF-1 and 2 Following treatment with an SP1 inhibitor (mithramycin A), (Supplementary Fig. S4A). Further, HGF neutralization with KRT19 expression decreased at both the transcriptional and anti-HGF immunoglobulins reversed the CM-induced KRT19 translational levels (Fig. 5D). Conversely, the overexpression upregulation and cell scattering (Supplementary Fig. S4B of JUN, FOSL1, a heterodimeric fusion protein of JUN and

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Figure 3. Hepatocyte growth factor upregulates KRT19 via MET and the MEK–ERK1/2 pathway. A, The phosphorylation status of receptor tyrosine kinases in HepG2 and SNU423 treated with CM from hTERT-HSC. The increased phosphorylation of three receptors (MET, ALK, and FGFR3) was commonly observed in HepG2 and SNU423 cells. B, Increased phosphorylation of MET following CM treatment was conﬁrmed in HepG2 and SNU423 cells. C, HGF concentrations in CM from hTERT-HSC, HepG2, and SNU423 cells determined by enzyme-linked immunosorbent assay. D, KRT19 mRNA and protein levels after HGF treatment combined with/without a MEK or ERK inhibitor. mRNA and protein expression levels were analyzed by qRT-PCR or Western blot analysis. E, HGF concentrations in complete media (DMEM þ 10% FBS) and CM from the hT-shHGF-1 and hT-shHGF-2 stable cell lines with/without doxycycline treatment. F, KRT19 mRNA and protein levels after treatment with CM from the hT-shHGF-1 and hT-shHGF-2stablecelllineswith/without doxycycline treatment. G, KRT19 mRNA and protein levels after CM treatment combined with/without MET inhibitors. For qRT-PCR experiments, the bar graph represents the mean SD, and statistical signiﬁcance is indicated (, P < 0.05; , P < 0.01; , P < 0.001, t test).

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KRT19 in HCC Is Regulated by CAF via HGF-MET-AP1 and SP1

Figure 4. AP1 and SP1 transcription factor binding sites in the KRT19 promoter are important for KRT19 expression. A, Luciferase reporter assay results for KRT19 promoter constructs. An approximately 3-kb-long KRT19 promoter and its serial deletion constructs were tested in HepG2 and SNU423 cells; the ﬁreﬂy luciferase results were normalized to the thymidine kinase-promoter-driven nano-Luc luciferase results. The bar graph represents the mean SD. B, Luciferase reporter assay results for KRT19 promoter constructs, in which the 21671967 or 15269 segments were deleted from the full-length KRT19 promoter using site-directed mutagenesis. C, Luciferase activity of mutant reporter plasmids, in which the putative transcription factor binding sites were altered by site-directed mutagenesis from the full-length KRT19 promoter.

FOSL1 (JUNFOSL1) (33), and SP1 increased KRT19 transcript downstream molecules, including FOSL1 and KRT19 were levels (Fig. 5E). In accordance with a previous report stating that evaluated by immunocytochemical analysis in our in vitro FOSL1 phosphorylation by ERK1/2 resulted in decreased pro- model (Supplementary Figs. S6 and S7), IHC in TMA samples teasomal degradation and increased stability (34), HepG2 and from the human HCC cohort (n ¼ 339; Figs. 1A and 6A), and SNU423 cells treated with CM from hTERT-HSCs displayed gene expression data from TCGA. increased phosphorylation and FOSL1 levels (Fig. 5F). The immunocytochemical staining demonstrated that HGF To conﬁrm the binding of JUN, FOSL1, and SP1 to the was weakly expressed in hTERT-HSCs, whereas HGF expression KRT19 promoter, we performed DNA pull-downs and ChIP was stronger in HepG2 or SNU423 cells after CM treatment, assays. We synthesized a 26-bp-long biotinylated double- suggesting an accumulation of HGF in tumor epithelial cells stranded DNA, mimicking the AP1 and SP1 sites in the KRT19 (Supplementary Fig. S6A). A time-course experiment after CM promoter (e.g., 2021 for the AP1 site, and 72 for the SP1 treatment showed the serial accumulation of HGF in HepG2 cells, site). Mutant biotinylated DNAs were also synthesized, in and its colocalization with MET, suggesting that HGF in HepG2 which AP1 or SP1 binding motifs were altered. Western blot cells is derived from CM (Supplementary Fig. S6B). We also analyses of the DNA binding assay revealed the binding of JUN carried out immunocytochemical staining for ERK1/2, phos- and FOSL1 to the 2021 AP1 site and that of SP1 binding to pho-ERK1/2, FOSL1, and phospho-FOSL1; the nuclear translo- the 72 SP1 site in HepG2 and SNU423 cells (Fig. 5G). ChIP cation of these proteins was observed after CM treatment (Sup- assays conﬁrmed the increased binding of JUN and FOSL1 at plementary Fig. S7A–S7D). the 2021 AP1 site and that of SP1 at the 72 SP1 site In the IHC analysis of TMAs in the human HCC cohort, the following CM treatment (Fig. 5H). membrane expression of MET and HGF, cytoplasmic expression of KRT19, and nuclear expression of FOSL1 were scored HGF and/or MET expression correlates with a higher KRT19 as described previously (3, 35, 36). When evaluating HGF and FOSL1 expression in human HCC tissue samples expression, its membrane distribution in epithelial tumor cells To validate our in vitro results regarding the regulatory axis of was focused where MET was expressed, although it was also KRT19, the expression levels of HGF, MET, and that of their observed in CAFs from the tumor stroma and the cytoplasm of

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Figure 5. JUN, FOSL1, and SP1 are important regulators of KRT19 expression. A, Relative mRNA expression levels of AP1 family genes that could bind TPA-responsive elements. mRNA expression was analyzed by qRT-PCR. B, KRT19 mRNA expression levels after transfection of two pooled siRNAs targeting JUN, JUND, FOSL1, or FOSL2. C, KRT19 mRNA expression levels after transfection of siRNAs targeting JUN or FOSL1. The knockdown efficiency was confirmed by Western blot analysis. D, KRT19 mRNA and protein levels after 24 hours of mithramycin A treatment combined with CM. mRNA and protein expression were analyzed by qRT-PCR and Western blot analysis, respectively. E, KRT19 mRNA levels after transient expression of JUN, FOSL1, JUNFOSL1 (heterodimeric fusion protein consisting of JUN, glycine-rich tether, and FOSL1), and SP1. F, Changes in phosphorylation and JUN and FOSL1 levels following a 24-hour CM treatment of HepG2 and SNU423 cells. G, Western blot analysis of eluted DNA-binding proteins from DNA pull-down assay. The nuclear extracts of HepG2 and SNU423 were incubated with DNA fragments mimicking the 2021 AP1 or 72SP1bindingsitesofKRT19.H, Chromatin immunoprecipitation analysis of HepG2 and SNU423 demonstrating increased JUN and FOSL1 binding at the 2021 AP1 site and increased SP1 binding at the 72 SP1 site following CM treatment. For qRT-PCR experiments, the bar graph represents the mean SD, and statistical significance is indicated (, P < 0.05; , P < 0.01; , P < 0.001, t test).

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KRT19 in HCC Is Regulated by CAF via HGF-MET-AP1 and SP1

Figure 6. FOSL1 and KRT19 expression and the prognosis for patients with HCC according to the HGF/MET expression status. A, Representative HCC tissue samples with or without HGF, MET, and FOSL1 expression. Scale bars, 100 mm. B, Association between HGF and MET expression and the percentage of aSMA- positive CAFs per tumor area. C, Association between KRT19 expression andHGF,MET,andFOSL1expression.D, Expression of FOSL1 and KRT19 compared with the combined expression status of HGF and MET. E, Differences in disease-speciﬁc survival and disease-free survival between HGF- or MET-positive patients with HCC and HGF- and MET-double negative patients with HCC.

epithelial tumor cells (Supplementary Fig. S7E). The membrane expression of MET was not signiﬁcantly different by the CAF distribution of HGF and MET was observed in 17% (56/339), levels in HCC (Fig. 6B). FOSL1 and KRT19 expression was and 31% (104/339) of HCCs, respectively. The expression of observed in 46% (157/339), and 10% (35/339) of HCCs, HGF was frequently observed in HCCs with 5% of CAF per respectively. The expression of KRT19 was frequently observed tumor area when compared with those without, whereas the in HCCs that expressed HGF or FOSL1, when compared with

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those that did not (P < 0.001, and P ¼ 0.001, respectively; Discussion Fig. 6C). We also analyzed the correlation between the pro- HCCs that express KRT19 are known for their aggressive portion of aSMA-positive CAFs and the degree of KRT19, HGF, biological behavior (3, 6); however, the underlying regulatory MET, and FOSL1 expression in a semiquantitative manner. A mechanism remains unclear. Interestingly, KRT19 expression significant positive correlation between the degree of HGF was closely correlated with CAFs in the fibrous tumor stroma in expression and the proportion of aSMA-positive CAFs was HCCs. Therefore, to explore the regulatory mechanism under- observed (r ¼ 0.160, P ¼ 0.003). Furthermore, the degree of lying KRT19 expression, we investigated the cross-talk between KRT19expressionalsoshowedasignificant positive correlation tumor epithelial cells and CAFs using an in vitro model of with that of HGF (r ¼ 0.288, P < 0.001), MET (r ¼ 0.158, P ¼ paracrine interaction between HCC cell lines and hepatic stel- 0.004), and FOSL1 (r ¼ 0.190, P < 0.001). Conversely, the late cell lines, and a large cohort of patients with HCC. Our in proportion of CD90-positive CAFs was not correlated with the vitro and human study demonstrated that KRT19 expression in HGF (r ¼ 0.073, P ¼ 0.182), MET (r ¼0.098, P ¼ 0.071), or HCCs was regulated by the CAF-derived HGF via the MET- FOSL1 expression (r ¼0.014, P ¼ 0.794). ERK1/2-AP1 and SP1 axis, thus highlighting the molecular When HCC cases were categorized according to HGF and background of clinically aggressive KRT19-positive HCCs (Sup- MET protein expression, FOSL1 and KRT19 expression were plementary Fig. S8). most frequently observed in the HGF and MET double positive Previously, a subset of HCCs exhibiting a stem/progenitor-like group and least frequently observed in the HGF and MET gene expression pattern, namely the hepatoblast signature, was double negative group (Fig. 6D, P < 0.001 at both). Patients reported to indicate a poor prognosis (39). In the aforemention- with HCC with HGF or MET expression showed higher serum ed study, HCCs with hepatoblast signatures highly expressed alpha-fetoprotein levels and more frequent microvascular markers of hepatic stem cells, including KRT19, and the AP1 invasion, compared with patients with HCC with HGF and transcription factor was proposed as a major driver of hepatoblast MET double negative expression (P < 0.05 for all; Supplemen- signature expression. Our results provide a functional connection tary Table S8). The expression of HGF or MET was a poor between HCCs with KRT19 expression and those with hepatoblast prognostic factor for disease-specific survival and disease-free signatures through the MET-ERK1/2-AP1 axis. Taken together, survival in our univariate analysis; however, it was not an these results suggest that the MET/HGF and AP1 signaling axis is independent prognostic factor (Fig. 6E; Supplementary Tables important for the regulation of stemness in HCCs, and KRT19 S9 and S10). represents a valuable marker of stemness and tumor-initiating The protein expression of phospho-MET was also evaluated in capacity. Accordingly, CAFs are considered a major source of HCC tissues by IHC staining; however, it was not detected in all HGF in addition to endothelial cells (40), which was recently cases (Supplementary Fig. S7F). The stability of the phospho-MET reported to regulate the tumor-initiating capacity of HCCs via the protein was assessed in our in vitro model. HepG2 cells treated HGF/MET pathway (36). with CM from hTERT-HSCs were harvested at various time points In fact, CAFs represent a heterogeneous group with respect to (0 to 30 minutes) after media exchange (CM was removed and their origin, biological properties, and differentiation markers exchanged with control media). We observed that phospho-MET (41). CAFs are usually considered to support the extracellular levels rapidly declined, whereas FOSL1 and KRT19 expression matrix and promote cancer progression via paracrine signaling levels did not change (Supplementary Fig. S7G). Therefore, the (14). However, in certain circumstances, CAFs could play a tumor- negative expression of phospho-MET in HCC tissues is linked to restraining role, including growth arrest, and promoting antitu- its instability. mor immune responses (41, 42). In HCCs, CAFs have been To further validate the association of KRT19 expression with reported to promote HCC progression, via the secretion of various aSMA-positive CAF and MET signaling, we evaluated the paracrine factors including the HGF, SDF-1, IL6, TGFb, EGF, and expression of CAF and MET-related gene signatures obtained FGF families (43). In this study, the proportion of aSMA-positive from previous studies (37, 38). After filtering out the genes CAFs was associated with HGF and KRT19 immunoreactivity and with frequent missing values greater than 50%, the enrichment a poor outcome, suggesting the tumor-promoting role of aSMA- scores of the upregulated CAF signature (CAF_UP, n ¼ 21) and positive CAFs. Conversely, the proportion of CD90-positive CAFs the up- and down-regulated MET signatures (i.e.,MET_UP,n ¼ was positively correlated with the degree of KRT19 expression, but 19, and MET_DOWN, n ¼ 4) were calculated from the HCC not with HGF, MET, or FOSL1 expression. The heterogeneity of data of TCGA (LIHC, n ¼ 371). We found that these gene CAFs in HCCs remains unclear, and the specific markers for signatures were closely correlated with KRT19 expression tumor-promoting and tumor-restraining CAFs remain to be across HCC samples (Fig. 7A, top). The analysis of gene sets uncovered. also revealed a significant correlation between KRT19 expres- MET, the HGF receptor, is a receptor tyrosine kinase that can sion and the enrichment scores for the CAF_UP (r ¼ 0.32, P ¼ activate downstream pathways including MAPK, PI3K, and 2.5 10 10), MET_UP (r ¼ 0.54, P ¼ 4.46 10 30), and RAC1-CDC42, and promote the invasive growth of cells MET_DOWN (r ¼0.44, P ¼ 1.05 10 19)signatures, (31). HCCs with high MET expression are also known to be respectively (Fig. 7A, bottom). Moreover, the stratification of associated with a poorer prognosis than those without (35, the patients based on the expression levels of the CAF or MET 44). MET is an emerging therapeutic target of HCC, and signatures also revealed significant differences in KRT19 currently, several agents targeting the HGF/MET signaling axis, expression levels between the groups (CAF_UP, P ¼ 4.61 including tivantinib, cabozantinib, and INC280 are undergo- 10 8; MET_UP, P ¼ 6.88 10 18;MET_DN,P ¼ 3.13 10 14; ing clinical trials (45). For an optimal therapeutic application permuted t test; Fig. 7B). These results strongly suggest that of MET inhibitors, a selective enrollment of patients with MET- KRT19 expression is closely associated with the expression of addicted HCC is crucial. Unfortunately, no practically effective CAF and MET-mediated signaling activities.

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KRT19 in HCC Is Regulated by CAF via HGF-MET-AP1 and SP1

Figure 7. External validation of the association of KRT19 expression with CAF and MET signatures. A, Expression heatmap of CAF_UP (n ¼ 21), MET_UP (n ¼ 19), and MET_DOWN gene signatures (n ¼ 4) according to the KRT19 gene expression in the HCC data of TCGA (n ¼ 371). B, Comparison of KRT19 expression according to the expression levels of the CAF or MET signatures. The high- and low-groups of HCC are stratiﬁed by the mean value of the enrichment scores for each signature, respectively.

marker is known for assessing the activation status of the HGF/ correlated with those of their downstream targets including MET axis (31). Considering the short half-life (less than 10 KRT19 and FOSL1. Further analysis of gene expression data minutes) of phospho-MET, as noted in our in vitro experiment, from TCGA also revealed that KRT19 expression was closely we doubt that a reliable assessment of the phosphorylation associated with CAF and MET-mediated signaling activities. status of MET could be performed in human HCC tissue Taken together, KRT19 may serve as an indirect marker for the samples, especially in formalin-fixed paraffin-embedded tis- activation of HGF/MET signaling and may represent a poten- sues by IHC staining. Although IHC staining may not reliably tial marker for therapeutic applications involving the inhibi- reveal its cellular source, because paracrine factors can bind to tion of HGF/MET signaling. the extracellular matrix (46), the positive immunoexpression In our study, AP1 (JUN/FOSL1 complex) and SP1 transcrip- of HGF was associated with a proportion of aSMA-positive tion factors were identified as crucial positive regulators of CAFs, and the degree of MET or HGF expression was also KRT19 expression in HCC. Accordingly, SP1 was reported as

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a transcriptional activator of KRT19 in pancreatic cancer cell Authors' Contributions lines (47). To our knowledge, the AP1-binding site located 2- Conception and design: H. Rhee, H.-Y. Kim, J.S. Choi, Y.N. Park kbp upstream of the transcription start site has not been Development of methodology: H. Rhee, H.-Y. Kim, Y.N. Park described previously, because previous studies have used rel- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Rhee, H.-Y. Kim, J.E. Yoo, J.H. Nahm, J.S. Choi, atively short (less than 2 kbp) mouse KRT19 promoter con- Y.N. Park structs to search for transcriptional activators. A previously Analysis and interpretation of data (e.g., statistical analysis, biostatistics, reported SP1-binding site in the mouse KRT19 promoter was computational analysis): H. Rhee, H.-Y. Kim, J.-H. Choi, H.G. Woo, Y.N. Park homologous with the SP1-binding site that we found in the Writing, review, and/or revision of the manuscript: H. Rhee, H.-Y. Kim, human KRT19 promoter (Supplementary Fig. S9). Interesting- H.G. Woo, Y.N. Park ly, the AP1-binding site is very well conserved in most mam- Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.H. Nahm, Y.N. Park mals (Supplementary Fig. S9), suggesting the functional impor- Study supervision: Y.N. Park tance of the AP1-driven regulation of KRT19. In conclusion, the expression of KRT19 in HCC is regulated Acknowledgments by CAF via the HGF-MET-ERK1/2-AP1 and SP1 axis. The HGF This research was supported by grants from the National Research and/or MET protein expression was well correlated with FOSL1 Foundation of Korea (NRF) funded by the Korean government (MSIP; and KRT19 expression in human HCC tissues. Our results grant number: NRF-2011-0030086, NRF-2016M3A9D5A01952416, NRF- provide insights into the molecular background of KRT19- 2017R1A2B4005871, and NRF-2017M3A9B6061512). positive HCC with an aggressive biological behavior. The The costs of publication of this article were defrayed in part by the payment assessment of these markers could indicate the activation of of page charges. This article must therefore be hereby marked advertisement MET signaling and predict poor outcomes for HCCs. in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Disclosure of Potential Conﬂicts of Interest Received April 8, 2017; revised August 12, 2017; accepted January 18, 2018; No potential conﬂicts of interest were disclosed. published OnlineFirst January 23, 2018.

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Keratin 19 Expression in Hepatocellular Carcinoma Is Regulated by Fibroblast-Derived HGF via a MET-ERK1/2-AP1 and SP1 Axis

Hyungjin Rhee, Hye-Young Kim, Ji-Hye Choi, et al.

Cancer Res 2018;78:1619-1631. Published OnlineFirst January 23, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-0988

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