Functional Genomics Identifies Hepatitis-Induced STAT3–TYRO3

Published OnlineFirst December 12, 2019; DOI: 10.1158/1078-0432.CCR-18-3531

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

Functional Genomics Identiﬁes Hepatitis-Induced STAT3–TYRO3–STAT3 Signaling as a Potential Therapeutic Target of Hepatoma Chia-Liang Tsai1,2, Jeng-Shou Chang1, Ming-Chin Yu3, Chern-Horng Lee4, Tse-Ching Chen5, Wen-Yu Chuang5, Wei-Liang Kuo1, Chen-Chun Lin1, Shi-Ming Lin1, and Sen-Yung Hsieh1,2

ABSTRACT ◥ Purpose: Hepatitis promotes the development and recurrence of ligand, to further activate TYRO3-mediated signaling. Further- hepatocellular carcinoma (HCC). Receptor tyrosine kinases (RTK) more, TYRO3 activation elicited intracellular SRC- and STAT3 play critical roles in the development of many cancers. We explored signaling. In mice, hepatitis and Tyro3 synergistically promoted the potential roles of RTKs in hepatitis-related liver cancers. HCC development. Silencing TYRO3 expression or inhibiting Experimental Design: We conducted loss-of-function screening its kinase activity suppressed xenograft HCC growth in nude to elucidate the roles of RTKs in the development of HCC in vitro mice. and in vivo. Conclusions: Many RTKs are simultaneously involved in Results: Many RTKs were coexpressed in HCC and were HCC development. Hepatitis exerts dual effects on the activation involved in tumor development and growth. Of these, TYRO3 of TYRO3-mediated signaling in HCC cells, which further elicits promoted tumor growth and was clinically associated with the “TYRO3–STAT3–TYRO3” signaling loop to facilitate tumor hepatitis activity and poor prognosis. In mice, chemical- growth. Our ﬁndings unveil a previously unrecognized link induced hepatitis transcriptionally activated Tyro3 expression between RTKs and hepatitis-associated HCC and suggest TYRO3 via IL-6/IL6R–STAT3 signaling. Moreover, hepatitis-associated as a marker and therapeutic target for the HCCs with higher apoptotic cells facilitated the presentation of GAS6, a TYRO3 hepatitis activity.

Introduction Chronic hepatitis caused by hepatitis B or C virus, alcoholism, chronic cholangitis, autoimmune diseases, or metabolic disorders Inflammation is a complex biological response to tissue injury (such as diabetes and hyperlipidemia) is the primary risk factor for triggered by wounded cells, chemical irritants, or invading pathogens hepatocellular carcinoma (HCC) development and recurrence (3–7). with the goal of eliminating the foreign pathogens and repairing tissue Indeed, over 90% of liver cancers, including HCC, are associated with damage. An association between chronic inflammation and cancer has chronic liver inflammation such as chronic hepatitis and cholangitis, been documented since the mid-19th century via the observation of and approximately 80% of these are associated with underlying infiltration of inflammatory cells in tumors by Rudolf Virchow, a cirrhosis (8). The association of hepatitis activity with HCC has been German pathologist. Indeed, tumor-associated inflammation is now thoroughly documented and the eradication of hepatitis B and C recognized as a hallmark of cancer (1), and chronic inflammation is viruses by interferons and antiviral agents has been found to signif- known to foster tumor formation and progression in many human icantly decrease the incidence of HCC in patients with chronic cancers, including buccal, esophageal, gastric, colorectal, and liver hepatitis B and C (9–12). However, the mechanistic relationship cancers (2). between chronic hepatitis and liver cancers is not yet fully understood. Although much has been shown for the roles of tumor-associated inflammatory cells in promoting HCC development (13), little is known about the mechanism by which the persistent inflammation 1Department of Gastroenterology and Hepatology, Chang Gung Memorial of the liver can drive the transformation of hepatocytes into tumor 2 Hospital, Taoyuan, Taiwan. Graduate Institute of Biomedical Science, College cells. of Medicine, Chang Gung University, Taoyuan, Taiwan. 3Department of General Receptor tyrosine kinases (RTK) are a subclass of cell-surface Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 4Department of General Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 5Depart- receptors that exhibit intrinsic, ligand-dependent protein tyrosine ment of Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan. kinase activity to transduce extracellular signals that regulate funda- Note: Supplementary data for this article are available at Clinical Cancer mental cellular processes, including proliferation, differentiation, sur- Research Online (http://clincancerres.aacrjournals.org/). vival, metabolism, migration, and cell cycle (14). The aberrant activation of RTKs has been implicated in the development of cancers, and C.-L. Tsai, J.-S. Chang, M.-C. Yu, and C.-H. Lee contributed equally to this article. the targeting of this aberrant RTK-associated signaling has been used Corresponding Author: Sen-Yung Hsieh, Department of Gastroenterology and clinically for anticancer therapies (15). Despite this, the role of RTKs in Hepatology, Chang Gung Memorial Hospital, No. 5, Fu-Hsin Rd, Kweishan, supporting the development and growth of liver cancers remains Taoyuan 333, Taiwan. Phone: þ886-975368031; Fax: 886-3328-2824; E-mail: [email protected] unclear. As such, we conducted a loss-of-function screening of the human RTKs and identified TYRO3 promoting HCC development Clin Cancer Res 2020;XX:XX–XX and progression. doi: 10.1158/1078-0432.CCR-18-3531 TYRO3 belongs to the TAM subfamily of RTKs, which comprises 2019 American Association for Cancer Research. TYRO3, AXL, and MER (TAM RTKs). The TAM RTKs are unique for

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Gene expression profiling Translational Relevance The details about gene expression profiling in human HCC and liver Targeting receptor tyrosine kinases (RTK) has successfully tissues, including the expression of the RTK family, are provided in treated many human cancers but not hepatocellular carcinoma Supplementary Information (24). (HCC). Our comprehensive survey revealed that many RTKs are coexpressed and involved in the development and growth of HCC. Cell culture, plasmids, siRNAs, and growth factors Among these, TYRO3 expression was upregulated in a subgroup of Human Huh7 and Hep3B HCC cell lines were used for the initial HCC strongly associated with hepatitis activity and poor clinical tumorigenicity screening because of their low to moderate tumor- outcomes. Mechanistically, hepatitis exerts dual effects on the igenicity (21). SK-Hep1 cell line was used for its high TYRO3 activation of TYRO3-mediated signaling: transcriptionally activat- expression but undetectable AXL expression, although it has been ing TYRO3 expression via IL6–STAT3 signaling and facilitating reported to be of endothelial origin and with epithelial morpho- GAS6 presentation by apoptotic cells. Moreover, TYRO3 is both a logy (25). Huh7 cells were used as the primary model throughout target and an inducer of STAT3-mediated signaling, thereby this study. forming the STAT3–TYRO3–STAT3 signaling loop in HCC cells promoting tumor development and growth. Silencing of TYRO3 shRNA library and RNAi screening for RTKs regulating expression or inhibition of its kinase activity suppressed HCC tumorigenicity of HCC growth in vitro and in vivo. TYRO3 (þ) HCCs highly overlap with shRNA clones targeting 54 members of the human RTK family the immune-specific class of HCC. TYRO3 is a potential marker (AATYK, AAATYK2, AATYK3, and DKFZa761P101 not included; 4–6 and therapeutic target for the HCCs with high hepatitis activity. clones targeting each RTK) were obtained from the National RNAi Core, Taiwan. Transduction, selection, and assays for tumor development and growth in nude mice and in soft agars were conduced as described previously (26). Xenograft tumors were scored as follows: their roles in the regulation of immune, reproductive, hematopoietic, þ1toþ5 represented a tumor volume of 120%, 140%, 160%, 180%, vascular, and neurologic systems. They are essential for the efficient and 200% of the controls, respectively; scores 1to5 represented a phagocytosis of apoptotic cells via the interaction between their tumor volume of 80%, 60%, 40%, 20%, and no growth compared to the ligands, GAS6, PROS, and phosphatidylserine on the apoptotic cell controls. All experiments were conducted in duplicate. Only genes membrane (16). They also act as pleiotropic inhibitors of the innate with consistent scores less than 3 in both the duplicated xenograft inflammatory response to pathogens (17). Deficiencies in TAM RTK- assay and the in vitro tumor-sphere formation assays when their mediated signaling may lead to chronic inflammatory and autoim- expression had been silenced were regarded as “high score for sup- mune diseases (16). On the other hand, aberrantly elevated TAM RTK pressing tumor growth” (Supplementary Table S3). expression is strongly associated with cancer progression, metastasis, and resistance to targeted therapies (18, 19). However, the underlying Quantitative RT-PCR and immunoblotting assays mechanisms remain to be elucidated. Immunoblotting assays and qRT-PCR were performed as previ- It is known that TAM RTKs are related to immune tolerance and ously described (21). Please refer to the Supplementary Information play essential roles in tissue homeostasis during acute and chronic liver for more details on the primer sequences and antibodies used in this injury (20). In this study, lentivirus-based shRNA screening (21) for study. RTKs involved in hepatoma growth identifed TYRO3. We then investigated the mechanism by which TYRO3 interplays with hepatitis Xenograft tumor formation for anti-TYRO3 treatment to promote HCC growth and found a hepatitis-mediated positive- Four- to 6-week-old male athymic nude mice (BALB/c-nu) were regulatory loop that constitutively activates oncogenic signaling in used for the xenograft tumor assays according to the Guide for the Care hepatoma cells. Our findings provide a mechanistic link between and Use of Laboratory Animals (National Academy of Sciences, 1985). hepatitis and liver cancers and a potential therapeutic target for Transduced Huh7 or Mahlavu cells (2 106/site) were either subcu- anti-HCC therapies. taneously injected into the dorsal flanks of athymic nude mice as previously described (21) or orthotopically injected into the largest lobe of the liver (n ¼ 6 for each set). For anti-TYRO3 treatment, Materials and Methods LDC1267 (20 mg/kg in PBS) was intraperitoneally injected daily for a Tissues and patients total of 15 days (27). See also Supplementary Information. We collected tumors and adjacent nontumor liver tissues from 120 and 8 patients who underwent hepatectomy for HCC and nonmalig- Induction of liver inflammation in mice and liver tumor nant etiologies, respectively, between 1999 and 2000 at our hospital. development Written informed consent from the patients was collected at the same Six to 8-week-old male C57BL/6 mice were administered a dose of time as tissue sample collection and was recorded in the Tissue Bank of 200 mg/kg/day thioacetamide (TAA, Sigma) in drinking water for Chang Gung Memorial Hospital (Taipei, Taiwan). The studies were 5 weeks. For hepatoma development, mice were treated with TAA for conducted in accordance with the ethical quidelines of Declaration of 24 weeks and then injected with TYRO3 transduced Hepa1–6 cells Helsinki and were approved by the Internal Review Board of Medical through the spleen. Mice without TAA treatment were used as a Ethics of Chang Gung Memorial Hospital. Hepatitis activity was control. See also Supplementary Information. graded by using Ishak's hepatitis activity index (22, 23). We defined tumor grading and staging according to the Edmondson grading Supplementary information system and the 7th edition of the American Joint Committee on The accession numbers of the microarray data include profiling Cancer (AJCC) TNM staging system, respectively. We conducted IHC, gene expression in paired HCC and their paratumor liver tissues from as described previously (21). 120 patients with HCC, and the microarray data of Huh7 cells without

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versus with silenced TYRO3 by using Affymetrix HG-U133 Plus2 randomly selected shRNAs targeting 6 RTK genes in addition to those arrays have been submitted to Gene Expression Omnibus under the targeting TYRO3, AXL, and MER (Supplementary Fig. S3E). The access numbers of GES101685 and GES119388, respectively. results are shown in Supplementary Fig. S4 and summarized in Supplementary Table S3. Xenograft tumor growth in nude mice and Statistical analysis tumor-sphere formation in soft-agar assays were suppressed by the The correlation between TYRO3 expression and the clinical man- individual silencing of 13 RTKs (MER, TYRO3, DDR1, EGFR, ERBB3, ifestations was measured by the c2 test. Five-year overall survival (OS) EPHA1, EPHA2, EPHA6, EPHB2, EPHB3, FGFR3, INSRR, and ROR2) and 3-year recurrence-free survival (RFS) were analyzed using and promoted by the individual silencing of 7 RTKs (ERBB4, PGFR2, Kaplan–Meier survival analysis and the log-rank test. The relationship IGF1R, RON, CSF1R, VEGFR1, and VEGFR3; Supplementary Fig. S4; between Tyro3 expression and the Ishak's scores was analyzed using Supplementary Table S3), suggesting that multiple RTKs are involved the Student t test. A P value less than 0.05 was considered statistically in both the positive or negative regulation of HCC formation and significant. growth. We further validated the results by using two individual shRNA clones for each RTK gene along with shV as a negative control in both xenograft-tumor growths in nude mice and tumor-sphere Results formation in soft-agar assays. The results are summarized in Supple- Multiple RTKs are coexpressed in human liver and HCCs mentary Table S3. Collectively, multiple RTKs were simultaneously To comprehensively survey the potential roles of the RTK family in expressed in each HCC and function as either promoters or suppres- the regulation of HCC growth, we examined the expression of all the sors of tumor growth. members of the human RTK family in 8 normal liver tissues, 8 early Of the identified oncogenic RTKs, we selected TYRO3 for further (T1) HCCs, and 16 advanced (T3) HCCs using gene expression study because silencing its expression consistently suppressed tumor microarray assays. Although the RTK expression profiles were variable growth both in vivo and in vitro and previous studies show that TYRO3 among the samples, there was an average of 30.4 (mean SD ¼ 30.4 has potential implications in immune regulation (16, 17, 20). 4.3) and 27.2 (mean SD ¼ 27.2 5.4) RTKs expressed in each normal liver and HCC sample, respectively (P ¼ 0.022; Fig. 1A; TYRO3 promotes human HCC development and growth Supplementary Fig. S1; Supplementary Table S1). In general, the To determine the roles of TYRO3 in cell proliferation and tumor- members of the TAM, DDR, EGFR, FGFR, and VEGFR subfamilies igenicity, we used two shRNA clones to suppress TYRO3 expression were frequently expressed in both the liver and HCC samples. (shTYRO3-1 and -2, Supplementary Fig. S5A) and found that the Hierarchical clustering analysis revealed that the RTK expression silencing of TYRO3 expression suppressed cell proliferation in both profile of HCCs is distinguishable from those of normal livers Huh7 and SK-Hep1 cells (Fig. 2A). In contrast, the ectopic expression (Fig. 1A; Supplementary Fig. S1). Moreover, there were fewer RTKs of the wild-type TYRO3 but not two kinase-dead mutants (D655A and expressed in advanced HCCs than in early HCCs (RTKs/tumor: T1 ¼ K550A; Supplementary Fig. S5B and S5C; ref. 28) or an empty vector 30.4 5.1; T3 ¼ 24.0 7.5; P < 0.01; Supplementary Table S1). facilitated the proliferation of Huh7 cells (Fig. 2B). Moreover, the To identify the RTKs that are deregulated in HCC, we compared the silencing of TYRO3 expression significantly suppressed the tumor- RTK expression profiles between the HCCs and their corresponding sphere formation of Huh7 and SK-Hep1 cells in the soft-agar assays paratumor liver tissues in a cohort of 264 HCC cases from a public (Fig. 2C), whereas the ectopic expression of the wild-type but not the database (GEO access number 25097). We found that the RTK kinase-dead mutants of TYRO3 promoted tumor-sphere formation of expression profile of HCCs were distinguishable from that of non- Huh7 and Hep3B cells in the soft-agar assays (Fig. 2D; Supplementary tumor liver tissues and there were more RTKs downregulated than Fig. S5D). Consistent with the findings of the RTK family screening, upregulated in HCCs (Fig. 1B). There were 11 (TYRO3, ERBB3, silenced TYRO3 expression further suppressed xenograft tumor EPHA1, EPHB2, EPHB4, FGFR4, INSR, PDGFRB, PTK7, ROS1, and growth in nude mice (Fig. 2E). To determine the role of TYRO3 in RYK) and 26 RTKs that were significantly upregulated and down- HCC development, we used Hep3B cells, a low tumorigenic HCC cell regulated in HCCs, respectively (Supplementary Fig. S2). line, and found that the ectopic expression of TYRO3 but not an empty The coexpression of multiple RTKs in HCCs was also evidenced at vector (V) promoted the development of xenograft tumors in nude the protein level using IHC with data obtained from a public database mice (Fig. 2F–H; Supplementary Fig. S5E). Collectively, these results (Human Protein Atlas, http://www.proteinatlas.org/; Supplementary suggest that TYRO3 promotes both the development and growth of Table S2). HCC. Together, these fi ndings indicate that multiple RTKs are coexpressed in each HCC or liver sample. Relatively fewer RTKs are TYRO3 upregulation associates with hepatitis activity and expressed in advanced HCC than in early HCCs. tumor inflammation First, we examined the relative expression levels of TYRO3 between Multiple RTKs are simultaneously involved in the regulation of the tumor and nontumor liver tissues in three independent HCC HCC growth cohorts retrieved from GEO databases (GSE25097; GSE14520; Next, we utilized RNA interference for loss-of-function screen- GSE10143). We found that TYRO3 was significantly upregulated in ing (21) of 54 members of the human RTK family in Huh7 cells to the HCCs compared to the paratumor liver tissues (Supplementary identify the RTKs involved in HCC development and growth. The Fig. S6A). Moreover, TYRO3 upregulation was observed at stages T1, transduced Huh7 cells were then subjected to xenograft tumor for- T2, and T3A of HCC (GSE36376; Supplementary Fig. S6B), suggesting mation in nude mice and tumor-sphere formation in soft-agar assays that TYRO3 upregulation is an early event during HCC development. (Supplementary Fig. S3A–S3C). We also used cells transduced with an We then used IHC to examine TYRO3 expression on tissue empty vector (shV) as a control. The silencing efficiency for the shRNA microarrays containing liver sections from 120 patients with HCC. library is shown in Supplementary Fig. S3D. We also validated the Relatively high TYRO3 level in HCC cells was observed in 34 cases silencing efficiency of these shRNA clones by quantitative RT-PCR in (28.3%). Overall, TYRO3 was upregulated in the HCCs compared with

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Figure 1. Expression pattern of receptor tyrosine kinases in livers and HCCs. A, The expression status for a given RTK is shown as expressed (red) or not expressed (pink). The presence and absence calls were analyzed using the Affymetrix MAS 5 method base on "Presence-Absence calls with Negative Probesets" (PANP), which uses sets of Affymetrix-reported probes with no known hybridization partners to deﬁne whether or not the gene is expressed. The expression pattern is based on the subfamilies of RTKs. See also Supplementary Fig. S1. B, Heatmap of the relative expression levels by two-dimensional hierarchical analysis. The gene expression data are based on an HCC cohort containing 264 HCCs and their paratumor liver tissues retrieved from a public domain database (GSE25097).

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Figure 2. TYRO3 promotes HCC development and progression. A and B, Cell proliferation assays. We used two shRNA clones of targeting TYRO3 (shTYRO3-1, and shTYRO3- 2), or an empty vector (shV) as the control. We carried out the ectopic expression of TYRO3 using wild-type (wt) or two kinase-dead mutants (D655A and K550A) in Huh7 cells (see also Supplementary Fig. S5A–S5C). C and D, Tumorsphere formation in soft-agar assays. Colony numbers were counted in six fields for each well. The experiment was performed in duplicate. , P < 0.001; , P < 0.001. E, Xenograft tumor assays in nude mice. Huh7 cells transduced with shV or shTYRO3-1 and shTYRO3-2 were injected into the left and right flanks, respectively, of the same mice (n ¼ 4, for each set, in duplicate). The results are summarized in the middle. Right, representative xenograft tumors. F–H, TYRO3 promotes Hep3B xenograft tumor development in nude mice. We transfected Hep3B cells (a low tumorigenic HCC cell line) with TYRO3 expression or an empty vector (V) as the control, then orthotopically inoculated the cells into mouse livers, and sacrificed the mice eight weeks later. F, Detection of tumors (T) by sonography and liver dissection. G, Summary of tumor numbers in the livers. H, Representative histology images. Scale bar, 100 mm.

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Figure 3. High TYRO3 expression is associated with hepatitis activity and poor prognosis. A, The immunohistochemistry analysis of TYRO3 in the HCC tissue sections of 120 HCC patients. Two representative cases are shown. The scatter plot shows the relative TYRO3 expression levels between the paired tumor (T) and paratumor liver tissues (N). Among these, 34 cases with high TYRO3 expression in their HCC tissues. B, The results of the Kaplan–Meier survival curves reveal an association between high TYRO3 expression and low survival (P ¼ 0.03 by log-rank test). C, High TYRO3 expression is associated with lower survival in an HCC cohort (n ¼ 361) from The Cancer Genome Atlas (TCGA) database. D and E, High levels of TYRO3 in HCC samples are associated with high serum AST and total bilirubin levels, and high Ishak's hepatitis indices in the paratumor liver tissues (see also Supplementary Fig. S8). H: high TYRO3; L: low TYRO3 level. F, The representative IHC results show high and low TYRO3 levels in HCC. Tissue sections were counterstained with hematoxylin for cellular nuclei. Scale bar, 100 mm.

their nontumor liver tissues (P < 0.001, via paired t test; Fig. 3A; database (Fig. 3C). As shown in Supplementary Table S4, a high Supplementary Table S4). The results of the Kaplan-Meier curves and TYRO3 level was not associated with patients' age, gender, tumor size, log-rank tests demonstrated an association between high TYRO3 level serum alpha-fetoprotein (AFP) level, or histologic grade. However, it and low overall survival (P ¼ 0.03; Fig. 3B), which was further was strongly associated with underlying cirrhosis, advanced tumor validated by an independent HCC cohort obtained from TCGA stages, virus hepatitis C, higher serum alamine transaminase (ALT),

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aspartate transaminase (AST), and total bilirubin levels, prolonged middle) and activation of STAT3 (nuclear localization of phosphor- prothrombin time (INR), a lower platelet count, and a higher Child– ylated STAT3, Fig. 4A, bottom). Because IL6 is a proinflammatory Pugh score (Fig. 3D; Supplementary Fig. S7). Histologically, TYRO3 cytokine, and the IL6/IL6R–STAT3 signaling pathway is known to be upregulation was significantly associated with higher Ishak's hepatitis related to hepatitis-induced hepatocellular carcinogenesis (29, 30), we indices including higher periportal hepatitis (P < 0.001), higher portal examined the effects of IL6-mediated inflammatory (STAT3) signaling inflammation (P ¼ 0.039), higher lobular inflammation (P < 0.01), and on the expression of TYRO3. IL6 induced TYRO3 expression by a higher confluent necrosis (P < 0.001; Fig. 3E). Figure 3F shows that the quantitative PCR assay (Fig. 4C). An increase in phosphorylated HCCs with high TYRO3 level exhibited a greater degree of necrosis STAT3 indicated the activation of STAT3-mediated signaling by IL6 withobviously infiltrating inflammatory cells compared with HCCs (Fig. 4D). Notably, the silencing of STAT3 expression with an shRNA with low TYRO3 levels. Collectively, these findings suggest that the targeting STAT3 prevented TYRO3 upregulation by IL6 treatment expression of TYRO3 in HCC cells is associated with hepatitis activity, (Fig. 4E). Collectively, these fi ndings indicate that hepatic inflamma- tumor inflammation, advanced cirrhosis, and poor clinical outcomes. tion induces TYRO3 expression at least in part via IL6–IL6R–STAT3– mediated signaling. Hepatitis induces TYRO3 expression via the IL6–IL6R–STAT3 inflammatory signaling pathway TYRO3 is a target of IL6–IL6R–STAT3 signaling Given that high TYRO3 levels are associated with hepatitis activity To determine how IL6–IL6R–TAT3 signaling upregulates TYRO3, and tumor inflammation, we hypothesized that inflammation induces we examined the TYRO3 promoter sequences and found a candidate TYRO3 expression in tumor cells. We treated mice with thioacetamide STAT3-binding site (Fig. 4F, top). Then, we used chromatin immu- (TAA), a hepatoxic agent, to induce liver inflammation (Fig. 4A, H noprecipitation assays and confirmed the binding of STAT3 to the and E, top) and found an increased expression of TYRO3 (Fig. 4B). TYRO3 promoter (Fig. 4G). We further cloned the TYRO3 promoter Moreover, TAA treatment led to the upregulation of IL6 (Fig. 4A, and used it to drive the expression of luciferase in cells, which was

Figure 4. Hepatitis activates TYRO3 expression. A, Mice were treated with or without thioacetamide (TAA) in drinking water for 4 weeks to induce liver inflammation. The representative liver sections with H&E staining as well as the immunohistochemistry for IL6 and phosphorylated STAT3 in the liver sections are shown. Notably, TAA induced inflammatory cell infiltration, hepatocyte necrosis, upregulated IL6, and the upregulation and nuclear translocation of phosphorylated STAT3 (P-STAT3). Scale bar, 100 mm. B, Immunoblotting for Tyro3 levels in the livers of mice treated or not treated with TAA. C and D, Quantitative RT-PCR (C) and immunoblotting assays (D) for the relative TYRO3 levels in Huh7 and SK-Hep1 cells treated with 10 ng/mL of IL6 versus the untreated cells. The phosphorylation of STAT3 was used to confirm the successful activation of the IL6–IL6R–STAT3 signaling pathway. E, Silencing STAT3 prevented the induction of TYRO3 expression by IL6. F, Top, the scheme illustrating the TYRO3 gene structure and the clones of the wild-type and mutant promoters of the TYRO3 gene. Stars indicate the nucleotides that were mutated at the STAT3 binding sites on the promoter to create a negative control. Bot- tom, the promoter activity assays show the luciferase activity driven by the wild-type (wt-Pt) versus STAT3-binding site mutated (mt-Pt) TYRO3 promoters in Huh7 cells with or without IL6 treatment. The results are representative of three independent experiments. VC: transfection with an empty vector. G, PCR results of the chromatin immunoprecipitation assays in Huh7 cells treated with IL6 using anti-STAT3 antibodies and non-specific immunoglobulin G (IgG), respectively. All experiments were conducted in duplicate.

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Figure 5. Apoptotic bodies increase TYRO3-mediated SRC/STAT3 oncogenic signaling in HCC cells. A, Intracellular signals were affected by the silencing of TYRO3 with siRNAs (siTYRO3) or by the ectopic expression of the wild-type (WT) versus kinase-dead mutant (K550A) in HCC cells. siRNAs containing scrambled sequences (siNS) and an empty vector (V) were used as the controls. B, The induction of TYRO3 phosphorylation by GAS6 at the indicated doses under serum-free culturing for 24 hours. C, TYRO3-dependent activation of STAT3 signaling by GAS6. D, Apoptotic cells enhance the activation of TYRO3–STAT3 signaling. Cells were cultured in serum-free media for 24 hours, followed by treatment with 160 mJ/cm2 of UV-B to induce cell apoptosis. Apoptotic bodies were then collected to treat Huh7 and SK- Hep1 cells with and without silencing TYRO3 expression. E, GAS6 further enhanced the activation of TYRO3 in the presence of apoptotic bodies. The experiments of (A–E) were performed at least in duplicate. F, Association of hepatocyte apoptosis with high TYRO3 expression in HCC tissues. We randomly selected six HCC sections with high or low TYRO3 levels, respectively, from the same cohort as shown in Fig. 3F to assay the ongoing apoptosis of hepatoma cells using IHC for BAX (top two panels) and cleaved caspase-3 (CAPS3; bottom two panels). Right, summary of the correlation between TYRO3 and BAX or cleaved CAPS3 levels. IHC score ¼ IHC intensity % of cells with positivity. Scale bar, 100 mm.

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further enhanced by treatment with IL6 (Fig. 4F; bottom). Ablation of Hepatitis supports TYRO3-mediated tumor development in the STAT3-binding sequences within the TYRO3 promoter abolished mice the induced luciferase activity by IL6 treatment (Fig. 4F; bottom). To test the interplay between TYRO3-mediated signaling and These findings suggest that TYRO3 is a target of STAT3-mediated hepatitis in the development of HCC, we inoculated Hepa1-6 cells signaling and that hepatitis activity may induce TYRO3 expression via (a low tumorigenic mouse hepatoma cells) transduced with Tyro3 (T3) the IL6–IL6R–STAT3 signaling pathway. or an empty vector (VC; Supplementary Fig. S9) via spleen injection into mice (age >32 weeks) with or without TAA-induced chronic TYRO3-mediated STAT3 signaling in human HCC cells hepatitis (24-week treatment; Fig. 6A and B). We found that small To identify TYRO3-mediated intracellular signals, we compared the liver nodules were detected by sonography in TAA (þ) mice inocu- gene expression profiles between the Huh7 cells with and without lated with Tyro3 (þ) Hepa1-6 cells [TAA(þ)/T3, Fig. 6A, right; silenced TYRO3 expression in the presence or absence of growth indicated by arrowheads), whereas none the control [TAA(-)/VC] or arrest-specific 6 (GAS6), a ligand of TYRO3, by using the gene TAA(-)/T3 mice had overt HCC (Fig. 6A, left and middle, respec- expression microarray assays. Then, we subjected the differentially tively). Histologically, dispersed hyperchromatic cells were identified expressed genes to reconstruct the intracellular signaling pathways in the liver sections from TAA(þ)/T3 mice but not the control using a software analyzer (MetaCore). We found that the immune [TAA(-)/VC] or TAA(-)/T3 mice (Fig. 6C). We then used IHC response signaling, including IL6 and JAK–STAT3, and the signaling staining for alpha-fetoprotein (AFP) and glypican 3 (GPC3) and pathways that regulate cell-cycle progression were the most affected confirmed that disperse HCC cells were detected in the livers of (Supplementary Fig. S8). We further examined the intracellular sig- TAA (þ)/T3 mice but not in TAA(-)/VC or TAA(-)/T3 mice naling pathways that are regulated by TYRO3 and found that silencing (n ¼ 6; Fig. 6D–F). These finding indicate that hepatitis facilitates TYRO3 expression suppressed the phosphorylation of SRC and TYRO3-mediated HCC development. STAT3 (Fig. 5A, left and middle). In contrast, the ectopic expression of TYRO3 significantly increased phosphorylated SRC and phosphor- TYRO3 is a potential target for anti-HCC therapy ylated STAT3, whereas the activation of SRC and STAT3 was partially We then tested whether inhibition of TYRO3 would prevent or prevented by a kinase-dead mutant of TYRO3 (K550A; Fig. 5A, right). suppress liver tumor development. We used LDC1267, a TYRO3/ These results suggest that TYRO3 mediates the activation of intracel- AXL/MER RTK inhibitor, to treat Huh7-derived xenograft tumors in lular SRC and STAT3 signaling pathways. nude mice. LDC1267 significantly suppressed the development of xenograft tumors when it had been delivered for 15 days starting at the Apoptotic cells enhance TYRO3-mediated intracellular time of tumor inoculation (Fig. 6G). IHC for proliferating cell nuclear signaling antigen (PCNA) demonstrated that cell proliferation of tumors was Inflammation is usually associated with cell apoptosis, during which suppressed after treatment with LDC1267 (Fig. 6H). Given that membranous phosphatidylserine (PS), a phospholipid on the inner cell anticancer therapy usually starts once the cancer is detected, we started membrane (cytosol-facing) switches and becomes an extracellular the treatment when the xenograft tumors were 500 mm3 and found surface phospholipid. GAS6 is a ligand of TYRO3 as well as AXL that LDC1267 significantly suppressed tumor growth (Fig. 6I and J). and MERTK (TAM). It is known that the PS of apoptotic cells Consistent antitumor effects of LDC1267 were observed when using facilitates the presentation of GAS6 to TAM receptors on macro- Mahlavu cells in tumorsphere formation in vitro and the xenograft- phages, which triggers the engulfment of apoptotic cell debris and tumor growth in nude mice (Supplementary Fig. S10). Notably, subsequently leads to anti-inflammatory macrophage polariza- LDC1267 also inhibits the kinase activity of AXL, MER, and other tion (31). As such, we investigated whether apoptotic cells induced targets in cell-free assays, such that part of the effect seen in vivo could by hepatitis activate TYRO3-mediated signaling in hepatoma cells. be due to the inhibition of other targets in addition to TYRO3. First, we demonstrated that a recombinant GAS6 was able to activate In summary, our findings suggest that hepatitis displays dual effects TYRO3–STAT3 signaling (Fig. 5B). The activation of STAT3 by GAS6 on the activation of TYRO3-mediated signaling by the upregulation of was found to be TYRO3-dependent because the silencing of TYRO3 TYRO3 expression via IL6–STAT3–mediated signaling and the acti- expression prevented the phosphorylation of STAT3 by GAS6 vation of TYRO3 via facilitating ligand presentation by inflammation- (Fig. 5C). Furthermore, we found that apoptotic cells further enhanced resulted apoptotic bodies (Fig. 6K). Moreover, TYRO3 serves as both a the activation of STAT3 by GAS6, and this was TYRO3-dependent target and an activator of the STAT3-mediated signaling to facilitate because the silencing of TYRO3 prevented further activation (Fig. 5D). HCC development and growth (Fig. 6K). On the other hand, the addition of GAS6 also enhanced the activation of TYRO3 by apoptotic bodies (Fig. 5E), indicating the synergistic effect of apoptotic bodies and GAS6 on the activation of TYRO3- Discussion mediated signaling. The IHC of BAX (induced by cell stress including The treatment of advanced HCC with anti-RTK targeted therapies apoptosis induction) and cleaved CASP3 (cleaved caspase-3, a marker has not yet been successful (32). In this study, we carried out a indicative of ongoing cell apoptosis) further confirmed the association comprehensive survey of the expression of RTK family members and between hepatitis-mediated apoptosis (higher BAX level and positivity their potential oncogenic roles in human HCCs. We found that for cleaved CASP3) and a high level of TYRO3 in clinical HCC samples multiple RTKs are coexpressed in both the HCC and nontumor (Fig. 5F). Collectively, hepatitis exerts dual effects: (i) transcriptional hepatocytes of each patient. Given the complexity of the liver micro- activation via the IL6–STAT3 signaling and (ii) activation of TYRO3 environment, especially in the case of chronic hepatitis and cirrhosis, via facilitated ligand presentation by apoptotic cells. On the other the coexpression of multi-RTKs in hepatocytes and hepatoma cells not hand, TYRO3 functions both as a target and an inducer of the STAT3- only endows them with survival benefits under harsh conditions but mediated signaling, “STAT3–TYRO3–STAT3” forms a positive feed- also promotes malignant transformation (33). In addition, the coex- back loop that might sustain the signaling to promote hepatocyte pression of multi-RTKs provides these cells with an alternative sig- transformation. naling pathway via the cross-talk between different RTKs to evade

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Figure 6. Hepatitis-induced STAT3–TYRO3–STAT3 oncogenic signaling may serve as therapeutic targets for HCC. Mice were treated with TAA (þ) or without treatment ()for 24 weeks. Hepa1-6 cells ectopically expressing Tyro3 (T3) or not (VC) were injected into the spleen of mice. A–E, Representative liver samples and images are shown. A, Tumor growth in the liver was monitored by sonography. Arrowheads indicate multiple small nodules in the liver of TAA (þ)/T(3) mice. B, TAA(þ) mice have a nodular liver surface, suggestive of cirrhosis. C, H&E staining reveals inflammatory cell infiltration and fibrosis in the liver sections of TAA (þ)/T(3) mice (right) but not in either control [TAA()/VC, left] or TAA()/T3 mice, middle]. IHC for AFP (D) and glypican 3 (GPC3; E) are shown. Scale bar, 100 mm. See also Supplementary Fig. S9 for the expression of Tyro3 in T3 and VC Hepa1-6 cells. F, Dot plots for the expression of AFP and GPC3. IHC score ¼ IHC intensity % of cells with positivity. n ¼ 6. , P < 0.01. G, Tumor growth curves. Nude mice inoculated with Huh7 cells were intraperitoneally injected with 20 mg/g LDC1267 weekly. n ¼ 6. Green bar: the duration of LDC1267 treatment. H, Left, H&E staining of tumor sections. Middle, IHC of PCNA. Scale bar, 100 mm. Right, quantification of cells positive for PCNA in tumor sections. I, The tumor growth curves of mice with versus without LDC1267 treatment when xenograft tumors were over 500 mm3. Green bar: the duration of LDC1267 treatment. The arrowhead: the time starting the treatment. J, Representative tumor sections by H&E staining. Scale bar, 100 mm. See also Supplementary Fig. S10. K, Schematic representation of the dual roles of hepatitis in TYRO3-mediated oncogenic signaling.

anticancer therapies. Targeting a single RTK or a subfamily of RTKs acquired resistance to treatment, so-called “evasion resistance.” may shift survival signaling from the original RTKs, such that tumor Indeed, such detour mechanisms have been found in both the primary cells can evade anticancer therapies, resulting in a primary and and acquired resistance of cancer cells to anti-EGFR therapies in many

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human cancers (34). Evasion resistance may also account for the points in tumor cells (44). This led us to hypothesize that TYRO3 clinical trial failuresof many RTK inhibitors being used to treat expression in the tumor-associated macrophages (Kupffer cells) in advanced HCC (35). As such, we hypothesize that targeting a single HCC tissues may suppress anti-tumor immunity. If so, targeting RTK will not be successful for the treatment of advanced HCC unless TYRO3 could reverse the suppression of anti-tumor immunity of the there are genetic, epigenetic, or microenvironmental cues whereby a M2 macrophages of HCCs. Cook and colleagues recently reported specific RTK will dominate tumor growth, so-called “oncogene addic- that the ablation of Mer (a member of the TAM family) in the tumor tion” (36, 37). Oncogene addiction has been previously found in leukocytes of tumor-bearing mice effectively suppressed tumor several types of cancers, including lung cancer, as an activation growth and metastasis by enhancing anti-tumor immune mutation of EGRF in many nonsmoking Asian females (38), or breast response (45). Cumulative evidence shows that TAMs function as cancer, as a gene amplification with constitutive activation of HER2. immune checkpoints and the inhibition of the GAS6- or PROS1- However, such a dominant but druggable oncogene required for the TAM interaction may block tumor-derived immune suppression, growth and survival of HCC has not yet been identified. providing a promising prospect for improving current anti-cancer By way of RNA interference screening, we identified TYRO3 as a efficacy (42). Indeed, many inhibitors targeting TAMs or blocking tumor promoter gene of HCC. Recently, by using qRT-PCR to the GAS6- or PROS1–TAM interaction are currently being studied in quantify TYRO3 expression in 55 HCC cases, Duan and colleagues clinical trials for both hematopoietic and solid cancers (43, 44, 46). reported that TYRO3 was upregulated in 42% of HCCs and was Further studies should be conducted using a combination of TAM associated with large tumor size and higher levels of serum ALT (39). inhibitors with immunotherapies, including immune checkpoint In this study, we measured the levels of bth TYRO3 transcripts and inhibitors, for advanced cancers, such as HCC. proteins. We found that TYRO3 was upregulated in 28% of the HCCs Our finding of the association between TYRO3 expression and examined and was associated with hepatitis activity and poor clinical hepatitis activity of HCC is reminiscent of a newly identified subgroup outcomes. Our mechanistic studies revealed that TYRO3 was a direct of HCC, namely, the immune-specific class (47). The HCCs of the target of STAT3 transcription factors and was upregulated by inflam- immune-specific class are characterized by inflammatory response matory cytokines, such as IL6, via STAT3 signaling. markers, including immune cell infiltration and upregulation of It has been shown that hepatitis leads to hepatocyte apoptosis. PS on immune regulatory molecules, such as PD-L1 and programmed cell the surface of apoptotic bodies binds to GAS6 and Protein S (ligands death 1 (PD-1). It is speculated that these HCC cells might be for TAM receptors) and facilitates GAS6/Protein S presentation to susceptible to therapeutic agents that regulate tumor immune TAM receptors on phagocytes to promote phagocytosis (16). Here, we response, such as immune checkpoint inhibitors. Interestingly, we show that apoptotic bodies facilitate TYRO3 activation in hepatoma found that HCCs with high TYRO3 expression had a significantlt cells, especially in the presence of GAS6. The activation of TYRO3 higher PD-L1 level (P ¼ 0.004; Supplementary Fig S11), indicating that further elicits downstream SRC- and STAT3-mediated signaling. the TYRO3-high subgroup of HCC overlaps with the immune-specific Notably, TYRO3 functions both as a target and an eliciting factor of class of HCC. TYRO3 may serve as both a marker and a potential the STAT3-mediated signaling pathway, thereby forming a positive therapeutic target for the immune subgroup of HCCs. regulatory loop to sustain this oncogenic signaling, which may further In summary, the coexpression of multi-RTKs in HCC may enable contribute to tumor transformation and progression. Our findings tumor cells to counteract various microenvironmental stresses and produced a novel model of hepatitis-mediated oncogenic signaling endows tumor cells with resistance to RTK-targeted therapies. Chronic that facilitates the malignant transformation of hepatocytes. inflammation (hepatitis) exerts dual roles in the activation of TYRO3- Our hypothesis that TYRO3-mediated oncogenic signaling may be mediated signaling: the upregulation of TYRO3 and the presentation used as a therapeutic target was confirmed by the fact that the silencing of GAS6 for the activation of TYRO3 by inflammation-induced of TYRO3 via RNA interference or a TAM inhibitor suppressed tumor apoptotic bodies. TYRO3 upregulation and activation result in the growth both in vitro and in vivo. Indeed, Jung and colleagues, recently activation of the “STAT3–TYRO3–STAT3” oncogenic signaling loop showed that small molecule inhibition of STAT3 effectively suppresses to sustain the transformation and progression of HCC (Fig. 6E). Our HCC growth both in vivo and in vitro (40). Kabir and colleagues findings provide not only a mechanistic link between hepatitis and reported that TYRO3 was a target of miR-7-5p (miR-7) and that the HCC development but also a therapeutic target for the suppression of ectopic expression of miR-7 suppressed HCC growth by inhibiting oncogenesis in HCC cells and the enhancement of anti-tumor immu- TYRO3-mediated signaling (41). We further hypothesize that the nity in the tumor microenvironment (18). inflammation-mediated “STAT3–TYRO3–STAT3” oncogenic signaling loop is also involved in the oncogenesis of other human cancers Disclosure of Potential Conflicts of Interest fl associated with chronic in ammation, including cervical, buccal, No potential conflicts of interest were disclosed. esophageal, gastric, colorectal, bile ductal, mesothelial, pancreatic, skin, and urinary bladder cancers and lymphomas. ’ TYRO3 is known to promote the M2-subtype differentiation of Authors Contributions macrophages, which suppresses inflammation and facilitates tissue Conception and design: S.-Y. Hsieh Development of methodology: J.-S. Chang, T.-C. Chen repair. Moreover, tumor-associated M2 macrophages promote Acquisition of data (provided animals, acquired and managed patients, provided tumor growth via the suppression of antitumor immunity (42). For facilities, etc.): C.-L. Tsai, J.-S. Chang, M.-C. Yu, C.-H. Lee, T.-C. Chen, instance, the activation of AXL suppressed the antitumor immunity W.-Y. Chuang, W.-L. Kuo, C.-C. Lin, S.-M. Lin of human glioblastomas and prolonged the survival time of mice Analysis and interpretation of data (e.g., statistical analysis, biostatistics, bearing glioblastomas when combined with anti-AXL and anti–PD-1 computational analysis): C.-L. Tsai, J.-S. Chang, T.-C. Chen, W.-L. Kuo, C.-H. Lee therapies (43). Recently, Birge and colleagues reported that the Writing, review, and/or revision of the manuscript: J.-S. Chang, M.-C. Yu, S.-Y. Hsieh ectopic expression of TAM RTKs on epithelial cells upregulated Administrative, technical, or material support (i.e., reporting or organizing data, programmed death-ligand 1 (PD-L1) expression, suggesting that constructing databases): C.-L. Tsai, J.-S. Chang, T.-C. Chen TAM RTKs may be involved in the regulation of immune check- Study supervision: S.-Y. Hsieh

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Acknowledgments The costs of publication of this article were defrayed in part by the We would like to thank Dr. Yun-Shien Lee, Jang-Hau Lian at the Genomic payment of page charges. This article must therefore be hereby marked advertisement Medicine Core Lab, and the Laboratory Animal Center of the Chang Gung Memorial in accordance with 18 U.S.C. Section 1734 solely to indicate Hospital for their technical assistance and the National RNAi Core of Taiwan for the this fact. lentivirus-based shRNA clones. This study was supported by grants from the Chang Gung Memorial Hospital (CMRPG3F1971-3; CMRPG3F1981-3; CMRPG3F1991-3), the Ministry of Science and Technology (MOST 105-2314-B-182A-144-MY3), and Received October 27, 2018; revised March 27, 2019; accepted December 9, 2019; the National Health Research Institute, Taiwan (NHRI-EX108-10806BI). published ﬁrst December 12, 2019.

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Functional Genomics Identifies Hepatitis-Induced STAT3− TYRO3−STAT3 Signaling as a Potential Therapeutic Target of Hepatoma

Chia-Liang Tsai, Jeng-Shou Chang, Ming-Chin Yu, et al.

Clin Cancer Res Published OnlineFirst December 12, 2019.

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