Functional Genomics Identifies Hepatitis-Induced STAT3-TYRO3-STAT3 Signaling As A

Author Manuscript Published OnlineFirst on December 12, 2019; DOI: 10.1158/1078-0432.CCR-18-3531 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Functional genomics identifies hepatitis-induced STAT3-TYRO3-STAT3 signaling as a

2 potential therapeutic target of hepatoma

3 Chia-Liang Tsai,1,5† Jeng-Shou Chang,1† Ming-Chin Yu,4† Chern-Horng Lee,2† Tse-Ching

4 Chen,3 Wen-Yu Chuang,3 Wei-Liang Kuo,1 Chen-Chun Lin,1 Shi-Ming Lin,1 and Sen-

5 Yung Hsieh1,5**

6 1Department of Gastroenterology and Hepatology, 2Department of General

7 Medicine, 3Department of Pathology, 4Department of General Surgery, Chang Gung

8 Memorial Hospital, Taoyuan 333, Taiwan

9 5Graduate Institute of Biomedical Science, College of Medicine, Chang Gung

10 University, Taoyuan 333, Taiwan

11 Running title: hepatitis induced TYRO3 oncogenic signaling

12 Keywords: hepatocellular carcinoma; hepatitis; receptor tyrosine kinases; targeted

13 therapy; TYRO3

14 Financial support: This study was supported by grants from the Chang Gung Memorial

15 Hospital (CMRPG3F1971-3; CMRPG3F1981-3; CMRPG3F1991-3), the Ministry of

16 Science and Technology (MOST 105-2314-B-182A-144-MY3), and the National Health

17 Research Institute, Taiwan (NHRI-EX108-10806BI).

18 Corresponding author: Sen-Yung Hsieh, Department of Gastroenterology and

19 Hepatology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan. Phone: 886-3-

Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 12, 2019; DOI: 10.1158/1078-0432.CCR-18-3531 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 3281200 ext 8128; Fax: 886-3-3272236; E-mail: [email protected];

2 [email protected]

3 † These authors contributed equally to this study.

4 Conflicts of interest: The authors declare that there are no conflicts of interest in

5 this study.

6 Electronic word count: 4,729

7 Number of figures and tables: 6

1 Translational Relevance

2 Targeting receptor-tyrosine-kinases (RTKs) has successfully treated many human

3 cancers but not hepatocellular carcinoma (HCC). Our comprehensive survey revealed

4 that many RTKs are co-expressed and involved in the development and growth of

5 HCC. Among these, TYRO3 expression was upregulated in a subgroup of HCC strongly

6 associated with hepatitis activity and poor clinical outcomes. Mechanistically,

7 hepatitis exerts dual effects on the activation of TYRO3-mediated signaling:

8 transcriptionally activating TYRO3 expression via IL-6—STAT3 signaling and

9 facilitating GAS6 presentation by apoptotic cells. Moreover, TYRO3 is both a target

10 and an inducer of STAT3-mediated signaling, thereby forming the STAT3—TYRO3—

11 STAT3 signaling loop in HCC cells promoting tumor development and growth.

12 Silencing of TYRO3 expression or inhibition of its kinase activity suppressed HCC

13 growth in vitro and in vivo. TYRO3 (+) HCCs highly overlap with the immune-specific

14 class of HCC. TYRO3 is a potential marker and therapeutic target for the HCCs with

15 high hepatitis activity.

1 Abstract

2 Purpose: Hepatitis promotes the development and recurrence of hepatocellular

3 carcinoma (HCC). Receptor tyrosine kinases (RTKs) play critical roles in the

4 development of many cancers. We explored the potential roles of RTKs in hepatitis-

5 related liver cancers.

6 Experimental Design: We conducted loss-of-function screening to elucidate the roles

7 of RTKs in the development of HCC in vitro and in vivo.

8 Results: Many RTKs were co-expressed in HCC and were involved in tumor

9 development and growth. Of these, TYRO3 promoted tumor growth and was

10 clinically associated with hepatitis activity and poor prognosis. In mice, chemical-

11 induced hepatitis transcriptionally activated Tyro3 expression via IL-6/IL6R—STAT3

12 signaling. Moreover, hepatitis-associated apoptotic cells facilitated the presentation

13 of GAS6, a TYRO3 ligand, to further activate TYRO3-mediated signaling. Furthermore,

14 TYRO3 activation elicited intracellular SRC- and STAT3 signaling. In mice, hepatitis

15 and Tyro3 synergistically promoted HCC development. Silencing TYRO3 expression or

16 inhibiting its kinase activity suppressed xenograft HCC growth in nude mice.

17 Conclusions: Many RTKs are simultaneously involved in HCC development. Hepatitis

18 exerts dual effects on the activation of TYRO3-mediated signaling in HCC cells, which

19 further elicits the “TYRO3—STAT3—TYRO3” signaling loop to facilitate tumor

1 growth. Our findings unveil a previously unrecognized link between RTKs and

2 hepatitis-associated HCC and suggest TYRO3 as a marker and therapeutic target for

3 the HCCs with higher hepatitis activity.

4 Significance: TYRO3 plays a crucial role in hepatitis-associated HCC. TYRO3 (+) HCCs

5 highly overlap with the immune-specific class of HCC reported by Sia et al.

6 (Gastroenterology 2017; 153:812-826). TYRO3 is a potential marker and therapeutic

7 co-target with immune-checkpoint inhibitors for the subgroup of HCCs with high

8 hepatitis activity.

1 Introduction

2 Inflammation is a complex biological response to tissue injury triggered by wounded

3 cells, chemical irritants, or invading pathogens with the goal of eliminating the

4 foreign pathogens and repairing tissue damage. An association between chronic

5 inflammation and cancer has been documented since the mid-19th century via the

6 observation of infiltration of inflammatory cells in tumors by Rudolf Virchow, a

7 German pathologist. Indeed, tumor-associated inflammation is now recognized as a

8 hallmark of cancer (1), and chronic inflammation is known to foster tumor formation

9 and progression in many human cancers, including buccal, esophageal, gastric,

10 colorectal, and liver cancers (2).

11 Chronic hepatitis caused by hepatitis B or C virus, alcoholism, chronic

12 cholangitis, autoimmune diseases, or metabolic disorders (such as diabetes and

13 hyperlipidemia) is the primary risk factor for hepatocellular carcinoma (HCC)

14 development and recurrence (3-7). Indeed, over 90% of liver cancers, including HCC,

15 are associated with chronic liver inflammation such as chronic hepatitis and

16 cholangitis, and approximately 80% of these are associated with underlying cirrhosis

17 (8). The association of hepatitis activity with HCC has been thoroughly documented

18 and the eradication of hepatitis B and C viruses by interferons and anti-viral agents

19 has been found to significantly decrease the incidence of HCC in patients with

1 chronic hepatitis B and C (9-12). However, the mechanistic relationship between

2 chronic hepatitis and liver cancers is not yet fully understood. While much has been

3 shown for the roles of tumor-associated inflammatory cells in promoting HCC

4 development (13), little is known about the mechanism by which the persistent

5 inflammation of the liver can drive the transformation of hepatocytes into tumor

6 cells.

7 Receptor tyrosine kinases (RTKs) are a subclass of cell-surface receptors that

8 exhibit intrinsic, ligand-dependent protein tyrosine kinase activity to transduce

9 extracellular signals that regulate fundamental cellular processes, including

10 proliferation, differentiation, survival, metabolism, migration and cell cycle (14). The

11 aberrant activation of RTKs has been implicated in the development of cancers, and

12 the targeting of this aberrant RTK-associated signaling has been used clinically for

13 anti-cancer therapies (15). Despite this, the role of RTKs in supporting the

14 development and growth of liver cancers remains unclear. As such, we conducted a

15 loss-of-function screening of the human RTKs and identified TYRO3 promoting HCC

16 development and progression.

17 TYRO3 belongs to the TAM subfamily of RTKs, which comprises TYRO3, AXL, and

18 MER (TAM RTKs). The TAM RTKs are unique for their roles in the regulation of

19 immune, reproductive, hematopoietic, vascular, and neurological systems. They are

1 essential for the efficient phagocytosis of apoptotic cells via the interaction between

2 their ligands, GAS6, PROS, and phosphatidylserine on the apoptotic cell membrane

3 (16). They also act as pleiotropic inhibitors of the innate inflammatory response to

4 pathogens (17). Deficiencies in TAM RTK-mediated signaling may lead to chronic

5 inflammatory and autoimmune diseases (16). On the other hand, aberrantly

6 elevated TAM RTK expression is strongly associated with cancer progression,

7 metastasis, and resistance to targeted therapies (18,19). However, the underlying

8 mechanisms remain to be elucidated.

9 It is known that TAM RTKs are related to immune tolerance and play essential

10 roles in tissue homeostasis during acute and chronic liver injury (20). In this study,

11 lentivirus-based shRNA screening (21) for RTKs involved in hepatoma growth

12 identifed TYRO3. We then investigated the mechanism by which TYRO3 interplays

13 with hepatitis to promote HCC growth and found a hepatitis-mediated positive-

14 regulatory loop that constitutively activates oncogenic signaling in hepatoma cells.

15 Our findings provide a mechanistic link between hepatitis and liver cancers and a

16 potential therapeutic target for anti-HCC therapies.

19 Experimental Procedures

1 Tissues and patients

2 We collected tumors and adjacent non-tumor liver tissues from 120 and eight patients

3 who underwent hepatectomy for HCC and non-malignant etiologies, respectively,

4 between 1999 and 2000 at our hospital. Written informed consent from the patients

5 was collected at the same time as tissue sample collection and was recorded in the

6 Tissue Bank of Chang Gung Memorial Hospital. The studies were conducted in

7 accordance with the ethical quidelines of Declaration of Helsinki and were approved

8 by the Internal Review Board of Medical Ethics of Chang Gung Memorial Hospital.

9 Hepatitis activity was graded by using Ishak’s hepatitis activity index (22,23). We

10 defined tumor grading and staging according to the Edmondson grading system and

11 the 7th edition of the American Joint Committee on Cancer (AJCC) TNM staging system,

12 respectively. We conducted immunohistochemistry, as previously described (21).

14 Gene expression profiling

15 The details about gene expression profiling in human HCC and liver tissues, including

16 the expression of the RTK family, are provided in Supplemental Information (24).

18 Cell culture, plasmids, siRNAs, and growth factors.

1 Human Huh7 and Hep3B HCC cell lines were used for the initial tumorigenicity

2 screening because of their low to moderate tumorigenicity (21). SK-Hep1 cell line

3 was uded for its high TYRO3 expression but undetectable AXL expression, although it

4 has been reported to be of endothelial origin and with epithelial morphology (25).

5 Huh7 cells were used as the primary model throughout this study.

7 shRNA library and RNAi screening for RTKs regulating tumorigenicity of HCC

8 shRNA clones targeting 54 members of the human RTK family (AATYK, AATYK2,

9 AATYK3, and DKFZp761P1010 not included; 4-6 clones targeting each RTK) were

10 obtained from the National RNAi Core, Taiwan. Transduction, selection, and assays

11 for tumor development and growth in nude mice and in soft agars were conduced as

12 described previously (26). Xenograft tumors were scored as follows: +1 to +5

13 represented a tumor volume of 120%, 140%, 160%, 180%, and 200% of the controls,

14 respectively; scores -1 to -5 represented a tumor volume of 80%, 60%, 40%, 20%,

15 and no growth compared to the controls. All experiments were conducted in

16 duplicate. Only genes with consistent scores less than -3 in both the duplicated

17 xenograft assay and the in vitro tumor-sphere formation assays when their

18 expression had been silenced were regarded as “high score for suppressing tumor

19 growth” (Table S3).

2 Quantitative RT–PCR and immunoblotting assays

3 Immunoblotting assays and qRT-PCR were performed as previously described

4 (21). Please refer to the Supplemental Information for more details on the primer

5 sequences and antibodies used in this study.

7 Xenograft tumor formation for anti-TYRO3 treatment.

8 Four- to six-week-old male athymic nude mice (BALB/c-nu) were used for the

9 xenograft tumor assays according to the Guide for the Care and Use of Laboratory

10 Animals (National Academy of Sciences, 1985). Transduced Huh7 or Mahlavu cells (2

11 × 106/site) were either subcutaneously injected into the dorsal flanks of athymic

12 nude mice as previously described (21) or orthotopically injected into the largest

13 lobe of the liver (n = 6 for each set). For anti-TYRO3 treatment, LDC1267 (20 mg/kg

14 in PBS) was intraperitoneally injected daily for a total of 15 days (27). See also

15 Supplemental Information.

17 Induction of liver inflammation in mice and liver tumor development

1 Six to eight-week-old male C57BL/6 mice were administered a dose of 200

2 mg/kg/day thioacetamide (TAA, Sigma) in drinking water for five weeks. For

3 hepatoma development, mice were treated with TAA for 24 weeks and then injected

4 with TYRO3 transduced Hepa1-6 cells through the spleen. Mice without TAA

5 treatment were used as a control. See also Supplemental Information.

7 Statistical analysis

8 The correlation between Tyro3 expression and the clinical manifestations was

9 measured by the chi-squared test. Five-year overall survival (OS) and 3-year

10 recurrence-free survival (RFS) were analyzed using Kaplan–Meier survival analysis and

11 the log-rank test. The relationship between Tyro3 expression and the Ishak's scores

12 was analyzed using the Student’s t-test. A P-value less than 0.05 was considered

13 significant.

16 Results

17 Multiple RTKs are co-expressed in human liver and HCCs

18 To comprehensively survey the potential roles of the RTK family in the regulation of

19 HCC growth, we examined the expression of all the members of the human RTK

1 family in 8 normal liver tissues, 8 early (T1) HCCs, and 16 advanced (T3) HCCs using

2 gene expression microarray assays. Although the RTK expression profiles were

3 variable among the samples, there was an average of 30.4 (mean ± SD = 30.4 ± 4.3)

4 and 27.2 (mean ± SD = 27.2 ± 5.4) RTKs expressed in each normal liver and HCC

5 sample, respectively (P = 0.022; Fig. 1A, Supplemental Fig. S1, and Supplemental

6 Table S1). In general, the members of the TAM, DDR, EGFR, FGFR, and VEGFR

7 subfamilies were frequently expressed in both the liver and HCC samples.

8 Hierarchical clustering analysis (HCA) revealed that the RTK expression profile of

9 HCCs is distinguishable from those of normal livers (Fig. 1A and S1). Moreover, there

10 were fewer RTKs expressed in advanced HCCs than in early HCCs (RTKs/tumor: T1 =

11 30.4 ± 5.1; T3 = 24.0 ± 7.5; P < 0.01; Table S1).

12 To identify the RTKs that are deregulated in HCC, we compared the RTK

13 expression profiles between the HCCs and their corresponding para-tumor liver

14 tissues in a cohort of 264 HCC cases from a public database (GEO access number

15 25097). We found that the RTK expression profile of HCCs were distinguishable from

16 that of non-tumor liver tissues and there were more RTKs downregulated than

17 upregulated in HCCs (Fig. 1B). There were 11 (TYRO3, ERBB3, EPHA1, EPHB2, EPHB4,

18 FGFR4, INSR, PDGFRB, PTK7, ROS1, and RYK) and 26 RTKs that were significantly

19 upregulated and downregulated in HCCs, respectively (Fig. S2).

1 The co-expression of multiple RTKs in HCCs was also evidenced at the protein

2 level using immunohistochemistry with data obtained from a public database

3 (Human Protein Atlas, http://www.proteinatlas.org/; Supplemental Table S2).

4 Together, these findings indicate that multiple RTKs are co-expressed in each

5 HCC or liver sample. Relatively fewer RTKs are expressed in advanced HCC than in

6 early HCCs.

8 Multiple RTKs are simultaneously involved in the regulation of HCC growth

9 Next, we utilized RNA interference for loss-of-function screening (21) of 54 members

10 of the human RTK family in Huh7 cells to identify the RTKs involved in HCC

11 development and growth. The transduced Huh7 cells were then subjected to

12 xenograft tumor formation in nude mice and tumor-sphere formation in soft-agar

13 assays (Supplemental Fig. S3A-C). We also used cells transduced with an empty

14 vector (shV) as a control. The silencing efficiency for the shRNA library is shown in

15 Fig. S3D. We also validated the silencing efficiency of these shRNA clones by

16 quantitative RT-PCR in randomly selected shRNAs targeting 6 RTK genes in addition

17 to those targeting TYRO3, AXL, and MER (Fig. S3E). The results are shown in Fig. S4

18 and summarized in Supplemental Table S3. Xenograft tumor growth in nude mice

19 and tumor-sphere formation in soft-agar assays were suppressed by the individual

1 silencing of 13 RTKs (MER, TYRO3, DDR1, EGFR, ERBB3, EPHA1, EPHA2, EPHA6,

2 EPHB2, EPHB3, FGFR3, INSRR, and ROR2) and promoted by the individual silencing of

3 7 RTKs (ERBB4, PGFR2, IGF1R, RON, CSF1R, VEGFR1, and VEGFR3; Supplemental Fig.

4 S4 and Table S3), suggesting that multiple RTKs are involved in both the positive or

5 negative regulation of HCC formation and growth. We further validated the results

6 by using two individual shRNA clones for each RTK gene along with shV as a negative

7 control in both xenograft-tumor growths in nude mice and tumor-sphere formation

8 in soft-agar assays. The results are summarized in Table S3. Collectively, multiple

9 RTKs were simultaneously expressed in each HCC and function as either promoters

10 or suppressors of tumor growth.

11 Of the identified oncogenic RTKs, we selected TYRO3 for further study because

12 silencing its expression consistently suppressed tumor growth both in vivo and in

13 vitro and previous studies show that TYRO3 has potential implications in immune

14 regulation (16,17,20).

16 TYRO3 promotes human HCC development and growth

17 To determine the roles of TYRO3 in cell proliferation and tumorigenicity, we used

18 two shRNA clones to suppress TYRO3 expression (shTYRO3-1 and -2, Fig. S5A) and

19 found that the silencing of TYRO3 expression suppressed cell proliferation in both

1 Huh7 and SK-Hep1 cells (Fig. 2A). By contrast, the ectopic expression of the wild-type

2 TYRO3 but not two kinase-dead mutants (D655A and K550A; Supplemental Fig. S5B,

3 S5C)(28) or an empty vector facilitated the proliferation of Huh7 cells (Fig. 2B).

4 Moreover, the silencing of TYRO3 expression significantly suppressed the tumor-

5 sphere formation of Huh7 and SK-Hep1 cells in the soft-agar assays (Fig. 2C),

6 whereas the ectopic expression of the wild-type but not the kinase-dead mutants of

7 TYRO3 promoted tumor-sphere formation of Huh7 and Hep3B cells in the soft-agar

8 assays (Fig. 2D; Fig. S5D). Consistent with the findings of the RTK family screening,

9 silenced TYRO3 expression further suppressed xenograft tumor growth in nude mice

10 (Fig. 2E). To determine the role of TYRO3 in HCC development, we used Hep3B cells,

11 a low tumorigenic HCC cell line, and found that the ectopic expression of TYRO3 but

12 not an empty vector (V) promoted the development of xenograft tumors in nude

13 mice (Fig. 2F-H; Fig. S5E). Collectively, these results suggest that TYRO3 promotes

14 both the development and growth of HCC.

16 TYRO3 upregulation associates with hepatitis activity and tumor inflammation

17 First, we examined the relative expression levels of TYRO3 between the tumor and

18 non-tumor liver tissues in three independent HCC cohorts retrieved from GEO

19 databases (GSE25097; GSE14520; GSE10143). We found that TYRO3 was significantly

1 upregulated in the HCCs compared to the para-tumor liver tissues (Supplemental Fig.

2 S6A). Moreover, TYRO3 upregulation was observed at stages T1, T2, and T3A of HCC

3 (GSE36376; Fig. S6B), suggesting that TYRO3 upregulation is an early event during

4 HCC development.

5 We then used immunohistochemistry to examine TYRO3 expression on tissue

6 microarrays containing liver sections from 120 HCC patients. Relatively high TYRO3

7 level in HCC cells was observed in 34 cases (28.3%). Overall, TYRO3 was upregulated

8 in the HCCs compared to their non-tumor liver tissues (P < 0.001, via paired t-test;

9 Fig. 3A; Supplemental Table S4). The results of the Kaplan-Meier curves and log-rank

10 tests demonstrated an association between high TYRO3 level and low overall survival

11 (P = 0.03; Fig. 3B), which was further validated by an independent HCC cohort

12 obtained from TCGA database (Fig. 3C). As shown in Supplemental Table S4, a high

13 TYRO3 level was not associated with patients’ age, gender, tumor size, serum alpha-

14 fetoprotein (AFP) level, or histological grade. However, it was strongly associated

15 with underlying cirrhosis, advanced tumor stages, virus hepatitis C, higher serum

16 alamine transaminase (ALT), aspartate transaminase (AST), and total bilirubin levels,

17 prolonged prothrombin time (INR), a lower platelet count, and a higher Child-Pugh

18 score (Fig. 3D; Fig. S7). Histologically, TYRO3 upregulation was significantly

19 associated with higher Ishak’s hepatitis indices including higher periportal hepatitis

1 (P < 0.001), higher portal inflammation (P = 0.039), higher lobular inflammation (P <

2 0.01), and higher confluent necrosis (P < 0.001; Fig. 3E). Figure 3F shows that the

3 HCCs with high TYRO3 level exhibited a greater degree of necrosis withobviously

4 infiltrating inflammatory cells compared to HCCs with low TYRO3 levels. Collectively,

5 these findings suggest that the expression of TYRO3 in HCC cells is associated with

6 hepatitis activity, tumor inflammation, advanced cirrhosis, and poor clinical

7 outcomes.

9 Hepatitis induces TYRO3 expression via the IL-6–IL6R–STAT3 inflammatory

10 signaling pathway

11 Given that high TYRO3 levels are associated with hepatitis activity and tumor

12 inflammation, we hypothesized that inflammation induces TYRO3 expression in

13 tumor cells. We treated mice with thioacetamide (TAA), a hepatoxic agent, to induce

14 liver inflammation (Fig. 4A; H&E, upper panel) and found an increased expression of

15 TYRO3 (Fig. 4B). Moreover, TAA treatment led to the upregulation of interleukin-6

16 (IL-6; Fig. 4A, middle panel) and activation of STAT3 (nuclear localization of

17 phosphorylated STAT3, Fig. 4A, lower panel). Since IL-6 is a pro-inflammatory

18 cytokine, and the IL-6/IL-6R-STAT3 signaling pathway is known to be related to

19 hepatitis-induced hepatocellular carcinogenesis (29,30), we examined the effects of

1 IL-6-mediated inflammatory (STAT3) signaling on the expression of TYRO3. IL-6

2 induced TYRO3 expression by a quantitative PCR assay (Fig. 4C). An increase in

3 phosphorylated STAT3 indicated the activation of STAT3-mediated signaling by IL-6

4 (Fig. 4D). Notably, the silencing of STAT3 expression with an shRNA targeting STAT3

5 prevented TYRO3 upregulation by IL-6 treatment (Fig. 4E). Collectively, these findings

6 indicate that hepatic inflammation induces TYRO3 expression at least in part via

7 IL6—IL-6R—STAT3-mediated signaling.

9 TYRO3 is a target of IL-6—IL6R–STAT3 signaling

10 To determine how IL6–IL-6R—TAT3 signaling upregulates TYRO3, we examined the

11 TYRO3 promoter sequences and found a candidate STAT3-binding site (Fig. 4F, upper

12 panel). Then, we used chromatin-immunoprecipitation assays and confirmed the

13 binding of STAT3 to the TYRO3 promoter (Fig. 4G). We further cloned the TYRO3

14 promoter and used it to drive the expression of luciferase in cells, which was further

15 enhanced by treatment with IL-6 (Fig. 4F; lower panel). Ablation of the STAT3-

16 binding sequences within the TYRO3 promoter abolished the induced luciferase

17 activity by IL-6 treatment (Fig. 4F; lower panel). These findings suggest that TYRO3 is

18 a target of STAT3-mediated signaling and that hepatitis activity may induce TYRO3

19 expression via the IL-6—IL6R – STAT3 signaling pathway.

2 TYRO3-mediated STAT3 signaling in human HCC cells

3 To identify TYRO3-mediated intracellular signals, we compared the gene expression

4 profiles between the Huh7 cells with and without silenced TYRO3 expression in the

5 presence or absence of growth arrest-specific 6 (GAS6), a ligand of TYRO3, by using

6 the gene-expression microarray assays. Then, we subjected the differentially

7 expressed genes to reconstruct the intracellular signaling pathways using a software

8 analyzer (MetaCore). We found that the immune response signaling, including IL-6

9 and JAK-STAT3, and the signaling pathways that regulate cell-cycle progression were

10 the most affected (Supplemental Fig. S8). We further examined the intracellular

11 signaling pathways that are regulated by TYRO3 and found that silencing TYRO3

12 expression suppressed the phosphorylation of SRC and STAT3 (Fig. 5A, left and

13 middle panels). By contrast, the ectopic expression of TYRO3 significantly increased

14 phosphorylated SRC and phosphorylated STAT3, whereas the activation of SRC and

15 STAT3 was partially prevented by a kinase-dead mutant of TYRO3 (K550A; Fig. 5A,

16 right panels). These results suggest that TYRO3 mediates the activation of

17 intracellular SRC and STAT3 signaling pathways.

19 Apoptotic cells enhance TYRO3-mediated intracellular signaling

1 Inflammation is usually associated with cell apoptosis, during which membranous

2 phosphatidylserine (PS), a phospholipid on the inner cell membrane (cytosol-facing)

3 switches and becomes an extracellular surface phospholipid. GAS6 is a ligand of

4 TYRO3 as well as AXL and MERTK (TAM). It is known that the PS of apoptotic cells

5 facilitates the presentation of GAS6 to TAM receptors on macrophages, which

6 triggers the engulfment of apoptotic cell debris and subsequently leads to anti-

7 inflammatory macrophage polarization (31). As such, we investigated whether

8 apoptotic cells induced by hepatitis activate TYRO3-mediated signaling in hepatoma

9 cells. First, we demonstrated that a recombinant GAS6 was able to activate TYRO3—

10 STAT3 signaling (Fig. 5B). The activation of STAT3 by GAS6 was found to be TYRO3-

11 dependent since the silencing of TYRO3 expression prevented the phosphorylation

12 of STAT3 by GAS6 (Fig. 5C). Furthermore, we found that apoptotic cells further

13 enhanced the activation of STAT3 by GAS6, and this was TYRO3-dependent since the

14 silencing of TYRO3 prevented further activation (Fig. 5D). On the other hand, the

15 addition of GAS6 also enhanced the activation of TYRO3 by apoptotic bodies (Fig.

16 5E), indicating the synergistic effect of apoptotic bodies and GAS6 on the activation

17 of TYRO3-mediated signaling. The immunohistochemistry of BAX (induced by cell

18 stress including apoptosis induction) and cleaved CASP3 (cleaved caspase 3, a marker

19 indicative of ongoing cell apoptosis) further confirmed the association between

1 hepatitis-mediated apoptosis (higher BAX level and positivity for cleaved CASP3) and

2 a high level of TYRO3 in clinical HCC samples (Fig. 5F). Collectively, hepatitis exerts

3 dual effects: 1) transcriptional activation via the IL-6—STAT3-signaling and 2)

4 activation of TYRO3 via facilitated ligand presentation by apoptotic cells. On the

5 other hand, TYRO3 functions both as a target and an inducer of the STAT3-mediated

6 signaling, “STAT3—TYRO3—STAT3” forms a positive feedback loop that might

7 sustain the signaling to promote hepatocyte transformation.

9 Hepatitis supports TYRO3-mediated tumor development in mice

10 To test the interplay between TYRO3-mediated signaling and hepatitis in the

11 development of HCC, we inoculated Hepa1-6 cells (a low tumorigenic mouse

12 hepatoma cells) transduced with Tyro3 (T3) or an empty vector (VC; Supplemental

13 Fig. S9) via spleen injection into mice (age >32 weeks) with or without TAA-induced

14 chronic hepatitis (24-week treatment; Fig. 6A and B). We found that small liver

15 nodules were detected by sonography in TAA (+) mice inoculated with Tyro3 (+)

16 Hepa1-6 cells [TAA(+)/T3, Fig. 6A, right panel; indicated by arrowheads), whereas

17 none the control [TAA(-)/VC] or TAA(-)/T3 mice had overt HCC (Fig. 6A, left and

18 middle panel, respectively). Histologically, dispersed hyperchromatic cells were

19 identified in the liver sections from TAA(+)/T3 mice but not the control [TAA(-)/VC]

1 or TAA(-)/T3 mice (Fig. 6C). We then used immunohistochemical staining for alpha-

2 fetoprotein (AFP) and glypican 3 (GPC3) and confirmed that disperse HCC cells were

3 detected in the livers of TAA (+)/T3 mice but not in TAA(-)/VC or TAA(-)/T3 mice (N =

4 6; Fig. 6D-F). These finding indicate that hepatitis facilitates TYRO3-mediated HCC

5 development.

7 TYRO3 is a potential target for anti-HCC therapy

8 We then tested whether inhibition of TYRO3 would prevent or suppress liver tumor

9 development. We used LDC1267, a TYRO3/AXL/MER RTK inhibitor, to treat Huh7-

10 derived xenograft tumors in nude mice. LDC1267 significantly suppressed the

11 development of xenograft tumors when it had been delivered for 15 days starting at

12 the time of tumor inoculation (Fig. 6G). Immunohistochemistry for proliferating cell

13 nuclear antigen (PCNA) demonstrated that cell proliferation of tumors was

14 suppressed after treatment with LDC1267 (Fig. 6H). Given that anti-cancer therapy

15 usually starts once the cancer is detected, we started the treatment when the

16 xenograft tumors were 500 mm3 and found that LDC1267 significantly suppressed

17 tumor growth (Fig. 6I and J). Consistent anti-tumor effects of LDC1267 were

18 observed when using Mahlavu cells in tumor-sphere formation in vitro and the

19 xenograft-tumor growth in nude mice (Supplemental Fig. S10). Notably, LDC1267

1 also inhibits the kinase activity of AXL, MER, and other targets in cell-free assays,

2 such that part of the effect seen in vivo could be due to the inhibition of other

3 targets in addition to TYRO3.

4 In summary, our findings suggest that hepatitis displays dual effects on the

5 activation of TYRO3-mediated signaling by the upregulation of TYRO3 expression via

6 IL-6—STAT3-mediated signaling and the activation of TYRO3 via facilitating ligand

7 presentation by inflammation-resulted apoptotic bodies (Fig. 6K). Moreover, TYRO3

8 serves as both a target and an activator of the STAT3-mediated signaling to facilitate

9 HCC development and growth (Fig. 6K).

12 Discussion

13 The treatment of advanced HCC with anti-RTK targeted therapies has not yet been

14 successful (32). In this study, we carried out a comprehensive survey of the

15 expression of RTK family members and their potential oncogenic roles in human

16 HCCs. We found that multiple RTKs are co-expressed in both the HCC and non-tumor

17 hepatocytes of each patient. Given the complexity of the liver microenvironment,

18 especially in the case of chronic hepatitis and cirrhosis, the co-expression of multi-

19 RTKs in hepatocytes and hepatoma cells not only endows them with survival benefits

1 under harsh conditions but also promotes malignant transformation (33). In addition,

2 the co-expression of multi-RTKs provides these cells with an alternative signaling

3 pathway via the crosstalk between different RTKs to evade anti-cancer therapies.

4 Targeting a single RTK or a subfamily of RTKs may shift survival signaling from the

5 original RTKs, such that tumor cells can evade anti-cancer therapies, resulting in a

6 primary and acquired resistance to treatment, so-called “evasion resistance.”

7 Indeed, such detour mechanisms have been found in both the primary and acquired

8 resistance of cancer cells to anti-EGFR therapies in many human cancers (34). Evasion

9 resistance may also account for the clinical trial failuresof many RTK inhibitors being

10 used to treat advanced HCC (35). As such, we hypothesize that targeting a single RTK

11 will not be successful for the treatment of advanced HCC unless there are genetic,

12 epigenetic, or microenvironmental cues whereby a specific RTK will dominate tumor

13 growth, so-called “oncogene addiction” (36,37). Oncogene addiction has been

14 previously found in several types of cancers, including lung cancer, as an activation

15 mutation of EGRF in many non-smoking Asian females (38), or breast cancer, as a

16 gene amplification with constitutive activation of HER2 . However, such a dominant

17 but druggable oncogene required for the growth and survival of HCC has not yet

18 been identified.

1 By way of RNA interference screening, we identified TYRO3 as a tumor promoter

2 gene of HCC. Recently, by using q-RT-PCR to quantify TYRO3 expression in 55 HCC

3 cases, Duan et al. reported that TYRO3 was upregulated in 42% of HCCs and was

4 associated with large tumor size and higher levels of serum ALT (39). In this study, we

5 measured the levels of bth TYRO3 transcripts and proteins. We found that TYRO3

6 was upregulated in 28% of the HCCs examined and was associated with hepatitis

7 activity and poor clinical outcomes. Our mechanistic studies revealed that TYRO3 was

8 a direct target of STAT3 transcription factors and was upregulated by inflammatory

9 cytokines, such as IL6, via STAT3-signaling.

10 It has been shown that hepatitis leads to hepatocyte apoptosis. PS on the

11 surface of apoptotic bodies binds to GAS6 and Protein S (ligands for TAM receptors)

12 and facilitates GAS6/Protein S presentation to TAM receptors on phagocytes to

13 promote phagocytosis (16). Here, we show that apoptotic bodies facilitate TYRO3

14 activation in hepatoma cells, especially in the presence of GAS6. The activation of

15 TYRO3 further elicits downstream SRC- and STAT3-mediated signaling. Notably,

16 TYRO3 functions both as a target and an eliciting factor of the STAT3-mediated

17 signaling pathway, thereby forming a positive regulatory loop to sustain this

18 oncogenic signaling, which may further contribute to tumor transformation and

1 progression. Our findings produced a novel model of hepatitis-mediated oncogenic

2 signaling that facilitates the malignant transformation of hepatocytes.

3 Our hypothesis that TYRO3-mediated oncogenic signaling may be used as a

4 therapeutic target was confirmed by the fact that the silencing of TYRO3 via RNA

5 interference or a TAM inhibitor suppressed tumor growth both in vitro and in vivo.

6 Indeed, Jung et al., recently showed that small molecule inhibition of STAT3

7 effectively suppresses HCC growth both in vivo and in vitro (40). Kabir et al. reported

8 that TYRO3 was a target of microRNA-7-5p (miR-7) and that the ectopic expression of

9 miR-7 suppressed HCC growth by inhibiting TYRO3-mediated signaling (41). We

10 further hypothesize that the inflammation-mediated “STAT3—TYRO3—STAT3”

11 oncogenic signaling loop is also involved in the oncogenesis of other human cancers

12 associated with chronic inflammation, including cervical, buccal, esophageal, gastric,

13 colorectal, bile ductal, mesothelial, pancreatic, skin, and urinary bladder cancers and

14 lymphomas.

15 TYRO3 is known to promote the M2-subtype differentiation of macrophages,

16 which suppresses inflammation and facilitates tissue repair. Moreover, tumor-

17 associated M2 macrophages promote tumor growth via the suppression of anti-

18 tumor immunity (42). For instance, the activation of AXL suppressed the anti-tumor

19 immunity of human glioblastomas and prolonged the survival time of mice bearing

1 glioblastomas when combined with anti-AXL and anti-PD-1 therapies (43). Recently,

2 Birge and colleagues reported that the ectopic expression of TAM RTKs on epithelial

3 cells upregulated programmed death-ligand 1 (PD-L1) expression, suggesting that

4 TAM RTKs may be involved in the regulation of immune checkpoints in tumor cells

5 (44). This led us to hypothesize that TYRO3 expression in the tumor-associated

6 macrophages (Kupffer cells) in HCC tissues may suppress anti-tumor immunity. If so,

7 targeting TYRO3 could reverse the suppression of anti-tumor immunity of the M2

8 macrophages of HCCs. Cook et al. recently reported that the ablation of Mer (a

9 member of the TAM family) in the tumor leukocytes of tumor-bearing mice

10 effectively suppressed tumor growth and metastasis by enhancing anti-tumor

11 immune response (45). Cumulative evidence shows that TAMs function as immune

12 checkpoints and the inhibition of the GAS6- or PROS1-TAM interaction may block

13 tumor-derived immune suppression, providing a promising prospect for improving

14 current anti-cancer efficacy (42). Indeed, many inhibitors targeting TAMs or blocking

15 the GAS6- or PROS1-TAM interaction are currently being studied in clinical trials for

16 both hematopoietic and solid cancers (43,44,46). Further studies should be

17 conducted using a combination of TAM inhibitors with immunotherapies, including

18 immune checkpoint inhibitors, for advanced cancers, such as HCC.

1 Our finding of the association between TYRO3 expression and hepatitis activity

2 of HCC is reminiscent of a newly identified subgroup of HCC, namely, the immune-

3 specific class (47). The HCCs of the immune-specific class are characterized by

4 inflammatory response markers, including immune cell infiltration and upregulation

5 of immune regulatory molecules, such as PD-L1 and programmed cell death 1 (PD1).

6 It is speculated that these HCC cells might be susceptible to therapeutic agents that

7 regulate tumor immune response, such as immune checkpoint inhibitors.

8 Interestingly, we found that HCCs with high TYRO3 expression had a significantlt

9 higher PD-L1 level (P = 0.004; Supplemental Figure S11), indicating that the TYRO3-

10 high subgroup of HCC overlaps with the immune-specific class of HCC. TYRO3 may

11 serve as both a marker and a potential therapeutic target for the immune subgroup

12 of HCCs.

13 In summary, the co-expression of multi-RTKs in HCC may enable tumor cells to

14 counteract various microenvironmental stresses and endows tumor cells with

15 resistance to RTK-targeted therapies. Chronic inflammation (hepatitis) exerts dual

16 roles in the activation of TYRO3-mediated signaling: the upregulation of TYRO3 and

17 the presentation of GAS6 for the activation of TYRO3 by inflammation-induced

18 apoptotic bodies. TYRO3 upregulation and activation result in the activation of the

19 “STAT3—TYRO3—STAT3” oncogenic signaling loop to sustain the transformation and

1 progression of HCC (Fig. 6E). Our findings provide not only a mechanistic link

2 between hepatitis and HCC development but also a therapeutic target for the

3 suppression of oncogenesis in HCC cells and the enhancement of anti-tumor

4 immunity in the tumor microenvironment (18)

7 Acknowledgments: We would like to thank Dr. Yun-Shien Lee, Mr. Jang-Hau Lian at

8 the Genomic Medicine Core Lab and the Laboratory Animal Center of the Chang

9 Gung Memorial Hospital for their technical assistance and the National RNAi Core of

10 Taiwan for the lentivirus-based shRNA clones.

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1 Support Information

2 The Supporting Information can be found at

3 The accession Numbers of the microarray data include profiling gene expression in

4 paired HCC and their paratumor liver tissues from 120 patients with HCC, and the

5 microarray data of Huh7 cells without versus with silenced TYRO3 by using

6 Affymetrix HG-U133 Plus2 arrays have been submitted to Gene Expression Omnibus

7 under the access numbers of GES101685 and GES119388, respectively.

8 Author Contributions: S.Y.H. conceived and designed the studies and secured

9 funding. CLT and JSC performed the experiments. S.Y.H., CLT, and JSC wrote the

10 manuscript. MCY, CHL, WLK, CCL, and SML collected patients and clinical information

11 and conducted clinical association analyses. TCC and WYC provided tissue

12 microarrays and were in charge of pathological interpretations.

1 Figure Legends

2 Figure 1. Expression pattern of receptor tyrosine kinases in livers and HCCs. (A) The

3 expression status for a given RTK is shown as expressing (red) or not expressing

4 (pink). The presence and absence calls were analyzed using the Affymetrix MAS 5

5 method base on "Presence-Absence calls with Negative Probesets" (PANP), which

6 uses sets of Affymetrix-reported probes with no known hybridization partners to

7 define whether or not the gene that is expressed. The expression pattern is based on

8 the subfamilies of RTKs. See also Supplemental Figure S1. (B) Heatmap of the relative

9 expression levels by two-dimensional hierarchical analysis. The gene expression data

10 are based on an HCC cohort containing 264 HCCs and their para-tumor liver tissues

11 retrieved from a public domain database (GSE25097).

13 Figure 2. TYRO3 promotes HCC development and progression. (A and B) Cell

14 proliferation assays. We used two shRNA clones of targeting TYRO3 (shTYRO3-1, and

15 shTYRO3-2), or an empty vector (shV) as the control. We carried out the ectopic

16 expression of TYRO3 using wild-type (wt) or two kinase-dead mutants (D655A and

17 K550A) in Huh7 cells (see also Supplemental Fig. S5A-C). (C and D) Tumorsphere

18 formation in soft-agar assays. Colony numbers were counted in six fields for each

19 well. The experiment was performed in duplicate. **, P < 0.001; ***, P < 0.001. (E)

1 Xenograft tumor assays in nude mice. Huh7 cells transduced with shV or shTYRO3-1

2 and shTYRO3-2 were injected into the left and right flanks, respectively, of the same

3 mice (n = 4, for each set, in duplicate). The results are summarized in the middle

4 panel. Right panel: representative xenograft tumors. (F-H) TYRO3 promotes Hep3B

5 xenograft tumor development in nude mice. We transfected Hep3B cells (a low

6 tumorigenic HCC cell line) with TYRO3 expression or an empty vector (V) as the

7 control, then orthotopically inoculated the cells into mouse livers, and sacrificed the

8 mice eight weeks later. (F) Detection of tumors (T) by sonography and liver

9 dissection. (G) Summary of tumor numbers in the livers. (H) Representative histology

10 images. Scale bar = 100 M.

12 Figure 3. High TYRO3 expression is associated with hepatitis activity and poor

13 prognosis. (A) The immunohistochemistry analysis of TYRO3 in the HCC tissue

14 sections of 120 HCC patients. Two representative cases are shown. The scatter plot

15 shows the relative TYRO3 expression levels between the paired tumor (T) and

16 paratumor liver tissues (N). Among these, 34 cases with high TYRO3 expression in

17 their HCC tissues. (B) The results of the Kaplan-Meier survival curves reveal an

18 association between high TYRO3 expression and low survival (P = 0.03 by log-rank

19 test). (C) High TYRO3 expression is associated with lower survival in an HCC cohort (n

1 = 361) from The Cancer Genome Atlas (TCGA) database. (D and E) High levels of

2 TYRO3 in HCC samples are associated with high serum AST and total bilirubin levels,

3 and high Ishak’s hepatitis indices in the paratumor liver tissues (see also

4 Supplemental Figure S8). H: high TYRO3; L: low TYRO3 level. (F) The representative

5 IHC results show high and low TYRO3 levels in HCC. Tissue sections were

6 counterstained with hematoxylin for cellular nuclei. Scale bar = 100 M.

8 Figure 4. Hepatitis activates TYRO3 expression. (A) Mice were treated with or

9 without thioacetamide (TAA) in drinking water for 4 weeks to induce liver

10 inflammation. The representative liver sections with H&E staining as well as the

11 immunohistochemistry for IL-6 and phosphorylated STAT3 in the liver sections are

12 shown. Notably, TAA induced inflammatory cell infiltration, hepatocyte necrosis,

13 upregulated IL-6, and the upregulation and nuclear translocation of phosphorylated

14 STAT3 (P-STAT3). Scale bar = 100 M. (B) Immunoblotting for Tyro3 levels in the

15 livers of mice treated or not treated with TAA. (C) Quantitative RT-PCR and (D)

16 immunoblotting assays for the relative TYRO3 levels in Huh7 and SK-Hep1 cells

17 treated with 10 ng/mL of IL-6 versus the untreated cells. The phosphorylation of

18 STAT3 was used to confirm the successful activation of the IL-6—IL-6R—STAT3

19 signaling pathway. (E) Silencing STAT3 prevented the induction of TYRO3 expression

1 by IL-6. (F) Upper panel: The scheme illustrating the TYRO3 gene structure and the

2 clones of the wild-type and mutant promoters of the TYRO3 gene. Stars indicate the

3 nucleotides that were mutated at the STAT3 binding sites on the promoter to create a

4 negative control. Lower panel: The promoter activity assays show the luciferase

5 activity driven by the wild-type (wt-Pt) versus STAT3 binding-site mutated (mt-Pt)

6 TYRO3 promoters in Huh7 cells with or without IL-6 treatment. The results are

7 representative of three independent experiments. VC: transfection with an empty

8 vector. (G) PCR results of the chromatin immunoprecipitation assays in Huh7 cells

9 treated with IL-6 using anti-STAT3 antibodies and non-specific immunoglobulin G

10 (IgG), respectively. All experiments were conducted in duplicate.

12 Figure 5. Apoptotic bodies increase TYRO3-mediated SRC/STAT3 oncogenic

13 signaling in HCC cells. (A) Intracellular signals were affected by the silencing of

14 TYRO3 with siRNAs (siTYRO3) or by the ectopic expression of the wild-type (WT)

15 versus kinase-dead mutant (K550A) in HCC cells. siRNAs containing scrambled

16 sequences (siNS) and an empty vector (V) were used as the controls. (B) The

17 induction of TYRO3 phosphorylation by GAS6 at the indicated doses under serum-

18 free culturing for 24 hours. (C) TYRO3-dependent activation of STAT3 signaling by

19 GAS6. (D) Apoptotic cells enhance the activation of TYRO3—STAT3 signaling. Cells

1 were cultured in serum-free media for 24 hours, followed by treatment with 160

2 mJ/cm2 of UV-B to induce cell apoptosis. Apoptotic bodies were then collected to

3 treat Huh7 and SK-Hep1 cells with and without silencing TYRO3 expression. (E) GAS6

4 further enhanced the activation of TYRO3 in the presence of apoptotic bodies. The

5 experiments of (A-E) were performed at least in duplicate. (F) Association of

6 hepatocyte apoptosis with high TYRO3 expression in HCC tissues. We randomly

7 selected six HCC sections with high or low TYRO3 levels, respectively, from the same

8 cohort as shown in Figure 3F to assay the ongoing apoptosis of hepatoma cells using

9 IHC for BAX (upper two panels) and cleaved Caspase 3 (CAPS3; lower two panels).

10 Right: summary of the correlation between TYRO3 and BAX or cleaved CAPS3 levels.

11 IHC score = IHC intensity × % of cells with positivity. Scale bar = 100 M.

13 Figure 6. Hepatitis-induced STAT3—TYRO3—STAT3 oncogenic signaling may serve

14 as therapeutic targets for HCC. Mice were treated with TAA (+) or without treatment

15 (-) for 24 weeks. Hepa1-6 cells ectopically expressing Tyro3 (T3) or not (VC) were

16 injected into the spleen of mice. (A-E) Representative liver samples and images are

17 shown. (A) Tumor growth in the liver was monitored by sonography. Arrowheads

18 indicate multiple small nodules in the liver of TAA (+)/T(3) mice. (B) TAA(+) mice have

19 a nodular liver surface, suggestive of cirrhosis. (C) H&E staining reveals inflammatory

1 cell infiltration and fibrosis in the liver sections of TAA (+)/T(3) mice (right panel) but

2 not in either control [TAA(-)/VC, left panel] or TAA(-)/T3 mice, middle panel].

3 Immunohistochemistry for AFP (D) and glypican 3 (GPC3; E) are shown. Scale bar =

4 100 M.See also Figure S9 for the expression of Tyro3 in T3 and VC Hepa1-6 cells. (F)

5 Dot plots for the expression of AFP and GPC3. IHC score = IHC intensity × % of cells

6 with positivity. N = 6. ** indicates P < 0.01 (G) Tumor growth curves. Nude mice

7 inoculated with Huh7 cells were intraperitoneally injected with 20 g/g LDC1267

8 weekly. N = 6. Green bar: the duration of LDC1267 treatment. (H) Left panel: H&E

9 staining of tumor sections. Middle: Immunohistochemistry of PCNA. Scale bar = 100

10 M. Right: quantification of cells positive for PCNA in tumor sections. (I) The tumor

11 growth curves of mice with versus without LDC1267 treatment when xenograft

12 tumors were over 500 mm3. Green bar: the duration of LDC1267 treatment. The

13 arrowhead: the time starting the treatment. (J) Representative tumor sections by

14 H&E staining. Scale bar = 100 M. See also Supplemental Figure S10. (K) Schematic

15 representation of the dual roles of hepatitis in TYRO3-mediated oncogenic signaling.

Expressed Not expressed Normal liver Early Advanced HCC

B N T

Figure 1

Figure 2 A B shV Huh7 shVcontrol SK-hep-1 WT Huh7 shTYRO3-1 shTYRO3-1shRNA1 D655A shTYRO3-2 shTYRO3-2 shRNA2 K550A

440 *** vector *** *** O.D

Day(s) Day(s) Day(s)

C Huh7 SK-Hep1 D Hep3B shV shTYRO3-1 shTYRO3-2 shV shTYRO3-1 shTYRO3-2 WT D655A K550A

colony diameter ≧ 50μm colony diameter ≧ 50μm colony diameter ≧ 50μm shV shV WT shTYRO3-1 shTYRO3-1 K550A shTYRO3-2 shTYRO3-2 D655A

＊＊＊＊＊＊＊＊＊＊＊ colonies / field / colonies ＊＊＊＊＊ E ***

) shV1 3 shV2 *** shTYRO3-1 shTYRO3-2 shV shTYRO3 *** tumor size(mmtumor tumor weight(g) tumor

wk(s)

F G H #1 Hep3B TYRO3 20 15 T T 10 Hep3B V 05 # nodules/mice 00 #2 Hep3B TYRO3 T V Ty T T T #1 1 17 T T #2 0 14 Hep3B TYRO3 #3 0 8

A B T

Case 1 N

Case 2 T

C D

ALT (IU/L) AST (IU/L) Bil (mg/dL) P = 0.057 P = 0.024 P = 0.0095 20 400400 400 15 300 300 10 200200 200 100 100 05 0000 000 00 HL HL HL

E Ishak’s score Periportal Confluent necrosis Portal Lobular Fibrosis P = 0.002 P < 0.001 P < 0.001 P = 0.039 P = 0.005 P = 0.07

20 4 6 4 3 6 15 3 3 4 2 4 10 2 2 2 1 2 5 1 1 0 0 0 0 0 0 H L H L H LH L H L H L

F #1 High Tyro3 #2 High Tyro3 #3 High Tyro3

#4 Low Tyro3 #5 Low Tyro3 #6 Low Tyro3

Figure 3

Figure 4

A C D TAA(-) TAA(+) Huh7 SK-Hep1 5 Huh7 SK-hep-1 14 *** *** IL-6 ─ + ─ + 12 4 Tyro3

H&E 10 3 8 p-Stat3 6 2 4 Stat3 foldchange 1 2 IL-6 0 0 β-actin IL-6 ― + ― +

F P-STAT3

B TAA - + - + Tyro3

β-actin

E Huh7 SK-Hep1 IL-6 ─ + ─ + ─ + ─ + shSTAT3 ─ ─ + + ─ ─ + + TYRO3 *** ** STAT3 *** ** pSTAT3

β-actin

G IL-6 + + IP Ab IgG STAT3 Input IP

A Huh7 SK-hep-1 Huh7 B Gas6 Huh7 SK-Hep-1 siNS siTYRO3 siNS siTYRO3 V WT K550A (ng/ml) — 50 100 200 — 50 100 200 TYRO3 pTYRO3 pSRC TYRO3 SRC pSTAT3 pSTAT3 5.2 1.0 2.9 1.0 STAT3 STAT3 1.3 1.0 2.1 1.0 β-actin β-actin

C D GAS6 GAS6 E Apoptotic bodies shTYRO3 ─ ─ + + shTYRO3 ─ ─ + + ─ ─ + + Gas6 ─ + ─ + apoptotic ─ + ─ + ─ + ─ + GAS6 ─ + ─ +

TYRO3 TYRO3 pTYRO3

pSTAT3 pSTAT3 TYRO3

STAT3 STAT3 β-actin

β-actin β-actin Huh7 Huh7 SK-Hep-1

F #1 High TYRO3 #2 High TYRO3 300 P < 0.001

200

#3 Low TYRO3 #4 Low TYRO3 100 Bax score IHC IHC for BAX for IHC 0 TYRO3 TYRO3 High Low

#1 High TYRO3 #2 High TYRO3 300 P < 0.001

200

#3 Low TYRO3 #4 Low TYRO3 100 clv clv Cas 3 score 0

IHC for cleaved CASP3 cleaved for IHC TYRO3 TYRO3 High Low Figure 5

Figure 6

A TAA(-) / VC TAA(-) / T3 TAA(+) / T3 F Sonograph B

G C Liver surface H&E

D AFP

E H GPC3

I K

J #1 #2 LDC1267 control

Functional genomics identifies hepatitis-induced STAT3-TYRO3-STAT3 signaling as a potential therapeutic target of hepatoma

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

Clin Cancer Res Published OnlineFirst December 12, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-3531

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