Oncogenic Hepatocytes Research Articles

Abstract 2869: The role of regulatory T cells in NASH-mediated HCC development

Abstract Objective Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease, ranging from simple steatosis to an advanced form of non-alcoholic steatohepatitis (NASH) without effective treatment. NASH can progress to liver cirrhosis and hepatocellular carcinoma (HCC) in association with increased production of liver-resident regulatory T cells (Treg). This study aims to identify the role of Tregs in NASH-driven HCC development. Methods Wild-type C57BL/6 mice were fed with a choline-low high-fat high-sugar diet (CL-HFS), which recapitulates the Western-pattern diet, to induce NASH, then received transplantation of oncogenic hepatocytes by intrasplenic injection to induce an orthotopic HCC. Wild-type mice fed with a normal diet were used for control. Hepatic immune cell profiling was characterized by flow cytometry to identify the cellular basis mediating CL-HFS-induced NASH. The metagenomic assay was applied to identify NASH-relevant gut microbiota in the liver tissue. Results CL-HFS caused successful development of NASH and liver fibrosis in wild-type mice. Fast orthotopic tumor growth was detected in NASH-bearing mice compared to wild-type mice. Strongly positive staining of glypican 3 (GPC3), increased frequency of Tregs, and increased expression of Col1α1, Col4α1, and α-SMA were detected in livers and tumors from NASH-bearing mice compared to wild-type mice by IHC and flow cytometry. Metagenomic assay detected the lipid-producing bacterial genus Bacillus in NASH liver, which was demonstrated to increase the frequency of Tregs in healthy spleen cells. Conclusion CL-HFS-caused NASH promotes HCC growth likely by increasing liver-resident Tregs and liver-infiltrating Bacillus. Keywords: Non-alcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), choline-low high-fat high-sugar diet (CL-HFS), regulatory T cells (Tregs), Bacillus, hepatocellular carcinoma (HCC). Citation Format: Ming Yang, Eric T. Kimchi, Kevin F. Staveley-O’Carroll, Guangfu Li. The role of regulatory T cells in NASH-mediated HCC development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2869.

Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance.

Senescence is a stress-responsive tumor suppressor mechanism associated with expression of the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells trigger their own immune-mediated elimination, which if evaded leads to tumorigenesis. Senescent parenchymal cells are separated from circulating immunocytes by the endothelium, which is targeted by microenvironmental signaling. Here we show that SASP induces endothelial cell NF-κB activity and that SASP-induced endothelial expression of the canonical NF-κB component Rela underpins senescence surveillance. Using human liver sinusoidal endothelial cells (LSECs), we show that SASP-induced endothelial NF-κB activity regulates a conserved transcriptional program supporting immunocyte recruitment. Furthermore, oncogenic hepatocyte senescence drives murine LSEC NF-κB activity in vivo. Critically, we show two distinct endothelial pathways in senescence surveillance. First, endothelial-specific loss of Rela prevents development of Stat1-expressing CD4+ T lymphocytes. Second, the SASP up-regulates ICOSLG on LSECs, with the ICOS-ICOSLG axis contributing to senescence cell clearance. Our results show that the endothelium is a nonautonomous SASP target and an organizing center for immune-mediated senescence surveillance.

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis.

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Abstract 4960: Studying the impact of gut microbiome to hepatocellular cancer in an orthotopic murine model

Abstract Background and Objective: As one of the most important components of human body, gut microbiota play a vital role in human health by participating in metabolomics and maintaining immunological homeostasis. Microbiota dysbiosis is significantly associated with initiation and progression of different diseases. Due to close anatomical and functional connection between gut and liver (gut-liver axis), gut microbiota are found to impact the development of liver diseases including hepatocellular cancer (HCC) without fully understanding mechanisms. The overall objective of present study is to investigate how microbiota modulate HCC growth by regulating intrahepatic immune response. Methods: Administration of carbon tetrachloride in combination with transplant of oncogenic hepatocytes by intrasplenic injection was used to make an orthotopic mouse model of HCC. A specific antibiotics cocktail (ABX) was selected to inhibit some kinds of gut microbiota. 16s rRNA sequencing was used to characterize gut microbiota profile in mice with the different treatments. Magnetic resonance imaging (MRI) was used to monitor tumor growth. Adoptive transfer of tumor antigen-specific CD8+ T cells following the treatment was used to investigate how ABX administration impacted intrahepatic immune response. Flow cytometry and multiplex cytokine assay were used to detect the alteration of effector CD8+ T cell in phenotype, proliferation, and function. Immunohistochemistry (IHC) was used to investigate immune cell tumor-infiltration. Results: The established murine model reflects the typical features of human HCC. ABX oral administration significantly changed gut microbiota composition, particularly in abundance of P. aeruginosa and Muribaculaceae. ABX-mediated alteration of gut microbiota significantly retarded HCC progression. Interestingly, ABX treatment was found to prevent adoptively transferred tumor antigen-specific CD8+ T cell from tumor-induced immune tolerance, as higher frequency of effector CD8+ T cell was detected in ABX-treated tumor-bearing mice compared to that in control tumor-bearing mice. Correspondingly, ABX treatment resulted in the increase in expression of CD69 and enhanced production of IFN-γ in tumor antigen-specific CD8+ T cells relevant to that in control mice. Conclusion: Modulating microbiota with antibiotics is able to shape intrahepatic immune responses and therapeutically suppress tumor growth, providing a new option in HCC control. Citation Format: Xiaoqiang Qi, Ming Yang, Joseph Stenberg, Guangfu Li, Kevin Staveley-O’Carroll. Studying the impact of gut microbiome to hepatocellular cancer in an orthotopic murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4960.

Transcriptomic analyses of oncogenic hepatocytes reveal common and different molecular pathways of hepatocarcinogenesis in different developmental stages and genders in krasG12V transgenic zebrafish

Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is mainly due to genetic changes in hepatocytes. However, molecular expression in hepatocytes during hepatocarcinogenesis has not been characterized. In this study, using an inducible kras transgenic zebrafish models for HCC, transcriptomic profiles of oncogenic hepatocytes from larvae, male and female adult fish following a brief induction of oncogenic kras were investigated. We found that oncogenic hepatocytes from all the three sources possess most of the cancer hallmarks at molecular level, including Sustaining proliferative signaling, Evading growth suppressors, Resisting cell death, Avoiding immune destruction, Inflammation, Reprogramming of energy metabolism, Angiogenesis, and Activating invasion and metastasis, suggesting the malignant transformation at molecular level could occur at the early stage of hepatocarcinogensis and can be captured in hepatocytes. However, each group of oncogenic hepatocytes also had their own characteristics. Larval oncogenic hepatocytes have cancer stem cell features. Female oncogenic hepatocytes showed resemblance to a mild human HCC subtype while male oncogenic hepatocytes resembled a severe HCC subtype, consistent with the observed sex disparity of HCC in both zebrafish and human. Finally, the two adult groups were more similar to each other than to the larval group, indicating an overwhelming effect of development over the gender.

Leptin induces muscle wasting in kras-driven hepatocellular carcinoma (HCC) model in zebrafish

ABSTRACTCancer cachexia affects up to 80% of patients with advanced solid cancer and leads to excessive muscle wasting. Here, using an inducible zebrafish hepatocellular carcinoma (HCC) model driven by oncogenic krasG12V, we observed a progressive muscle-wasting phenotype in adult zebrafish, characterized by significant loss of body weight and muscle fibers. By differential feeding, we observed that overfeeding caused fatty liver, accelerated carcinogenesis and muscle wasting. Interestingly, leptin, an obesity hormone, was upregulated in oncogenic hepatocytes and overfeeding groups. We also found that leptin expression progressively increased during human liver disease progression. By using leptin receptor (lepr)-knockout fish, we found that tumor fish in the lepr mutant background had a higher survival rate and significantly lower muscle-wasting level after tumor induction than the tumor fish in the wild-type background. Chemical inhibitors targeting leptin signaling also alleviated the muscle-wasting phenotype, indicating that leptin signaling may be a new therapeutic target for cancer patients with muscle wasting.

RETRACTED: Activation of Hepatic Stellate Cells During Liver Carcinogenesis Requires Fibrinogen/Integrin αvβ5 in Zebrafish

Hepatocellular carcinoma (HCC) is one of the most common cancers and it usually develops from a background of liver fibrosis or inflammation. The crosstalk between tumor cells and stromal cells plays an important and stimulating role during tumor progression. Previously we found in a krasV12–induced zebrafish HCC model that oncogenic hepatocytes activate hepatic stellate cells (HSCs) by up-regulation of serotonin and activate neutrophils and macrophages by up-regulation of cortisol. In the present study, we found a novel signaling transduction mechanism between oncogenic hepatocytes and HSCs. After krasV12 induction, fibrinogen was up-regulated in oncogenic hepatocytes. We reasoned that fibrinogen may bind to integrin αvβ5 on HSCs to activate HSCs. Consistent with this notion, pharmaceutical treatment using an antagonist of integrin αvβ5, cilengitide, significantly blocked HSC activation and function, accompanied by attenuated proliferation of oncogenic hepatocytes and progression of liver fibrosis. On the contrary, adenosine 5’-diphosphate, an agonist of αvβ5, activated HSCs significantly that further stimulated the tumor progression and liver fibrosis. Interestingly, in human liver disease samples, we detected an increased level of fibrinogen during tumor progression which indicated the potential role of fibrinogen signaling in HCC progression. Thus, we concluded a novel interaction between oncogenic hepatocytes and HSCs through the fibrinogen related pathway in both the zebrafish HCC model and human liver disease samples.

Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice

BACKGROUND & AIMSCeramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors.METHODSImmune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachlo-ride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8+ T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8+ T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay.RESULTSInjection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8+ T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8+ T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF.CONCLUSIONSIn mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8+ T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.

Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells.

The long noncoding RNA (lncRNA) MEG3 is significantly downregulated in pancreatic neuroendocrine tumors (PNETs). MEG3 loss corresponds with aberrant upregulation of the oncogenic hepatocyte growth factor (HGF) receptor c-MET in PNETs. Meg3 overexpression in a mouse insulin-secreting PNET cell line, MIN6, downregulates c-Met expression. However, the molecular mechanism by which MEG3 regulates c-MET is not known. Using chromatin isolation by RNA purification and sequencing (ChIRP-Seq), we identified Meg3 binding to unique genomic regions in and around the c-Met gene. In the absence of Meg3, these c-Met regions displayed distinctive enhancer-signature histone modifications. Furthermore, Meg3 relied on functional enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), to inhibit c-Met expression. Another mechanism of lncRNA-mediated regulation of gene expression utilized triplex-forming GA-GT rich sequences. Transfection of such motifs from Meg3 RNA, termed triplex-forming oligonucleotides (TFOs), in MIN6 cells suppressed c-Met expression and enhanced cell proliferation, perhaps by modulating other targets. This study comprehensively establishes epigenetic mechanisms underlying Meg3 control of c-Met and the oncogenic consequences of Meg3 loss or c-Met gain. These findings have clinical relevance for targeting c-MET in PNETs. There is also the potential for pancreatic islet β-cell expansion through c-MET regulation to ameliorate β-cell loss in diabetes.

Ribonucleotide reductase represents a novel therapeutic target in primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a highly aggressive B-cell malignancy that is closely associated with one of oncogenic viruses infection, Kaposi's sarcoma-associated herpesvirus (KSHV). PEL prognosis is poor and patients barely survive more than 6 months even following active chemotherapy interventions. There is therefore an urgent need to discover more effective targets for PEL management. We recently found that the ribonucleotide reductase (RR) subunit M2 is potentially regulated by the key oncogenic HGF/c-MET pathway in PEL (Dai et al., Blood. 2015;126(26):2821-31). In the current study, we set to investigate the role of RR in PEL pathogenesis and to evaluate its potential as a therapeutic target. We report that the RR inhibitor 3-AP actively induces PEL cell cycle arrest through inhibiting the activity of the NF-κB pathway. Using a xenograft model, we found that 3-AP effectively suppresses PEL progression in immunodeficient mice. Transcriptome analysis of 3-AP treated PEL cell lines reveals altered cellular genes, most of whose roles in PEL have not yet been reported. Taken together, we propose that RR and its signaling pathway may serve as novel actionable targets for PEL management.

Successful chemoimmunotherapy against hepatocellular cancer in a novel murine model

We have established a clinically relevant animal model of hepatocellular cancer (HCC) in immune competent mice to elucidate the complex dialog between host immunity and tumors during HCC initiation and progression. Mechanistic findings have been leveraged to develop a clinically feasible anti-tumor chemoimmunotherapeutic strategy. Intraperitoneal injection of carbon tetrachloride and intrasplenic inoculation of oncogenic hepatocytes were combined to induce progressive HCCs in fibrotic livers of immunocompetent mice. Immunization and adoptive cell transfer (ACT) were used to dissect the tumor antigen-specific immune response. The ability of the tyrosine kinase inhibitor sunitinib to enhance immunotherapy in the setting of HCC was evaluated. This new mouse model mimics human HCC and reflects its typical features. Tumor-antigen-specific CD8+ T cells maintained a naïve phenotype and remained responsive during early-stage tumor progression. Late tumor progression produced circulating tumor cells, tumor migration into draining lymph nodes, and profound exhaustion of tumor-antigen-specific CD8+ T cells associated with accumulation of programmed cell death protein 1 (PD-1)hi CD8+ T cells and regulatory T cells (Tregs). Sunitinib-mediated tumoricidal effect and Treg suppression synergized with antibody-mediated blockade of PD-1 to powerfully suppress tumor growth and activate anti-tumor immunity. Treg accumulation and upregulation of PD-1 provide two independent mechanisms to induce profound immune tolerance in HCC. Chemoimmunotherapy using Food and Drug Administration-approved sunitinib with anti-PD-1 antibodies achieved significant tumor control, supporting translation of this approach for the treatment of HCC patients. In the current study, we have established a clinically relevant mouse model which mimics human liver cancer. Using this unique model, we studied the response of the immune system to this aggressive cancer. Findings from this trial have led to the development of an innovative and clinically feasible chemoimmunotherapeutic strategy.

Abstract 4004: Development of a successful combination therapy for hepatocellular cancer by targeting Treg and PD-1

Abstract We have established a clinically relevant murine model to reveal tumor-induced immunotoerance in hepatocellular carcinoma (HCC). Critical factors are targeted to develop immune-based therapies for HCC control. Intrasplenic (ISPL) inoculation of oncogenic hepatocytes and intraperitoneal (IP) injection of carbon tetrachloride (CCl4) are combined to induce progressive HCCs in fibrotic livers of immunocompetent mice. We characterize the features of this model, examine tumor-antigen-specific (TAS) immunity during tumor initiation and progression, and identify the critical factors in tumor-induced immune tolerance. The established murine model recapitulates human HCC and reflects its typical features. TAS CD8+ T cells initially maintain a naive phenotype and function in early-stage tumor-bearing mice, then become profound exhaustion with the tumor progression to the advanced stage. The deep immunosuppression is associated with the significant upregulation of Programmed cell death protein 1(PD-1), Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and the increase of Tregs. While the changes in Tregs and PD-1 are systemic, they are particularly pronounced in the tumor microenvironment. Sunitinib-mediated reduction of Treg together with antibody-mediated blockade of PD-1 synergistically suppress tumor growth and activate anti-tumor immunity. Our data provide evidence that oncogenic hepatocytes escape immune surveillance during tumor initiation via immune ignorance, while later-stage established HCCs evade immune destruction via tumor-induced immunotolerance. Synergy of sunitinib and anti-PD-1 Ab generates favorable effect on suppressing tumor growth and activating anti-tumor immunity. This combinational strategy has high translational potential in HCC treatment. Citation Format: Dai Liu, Guangfu Li, Timothy Cooper, Eric Kimchi, Xiaoqiang Qi, Ningfei Li, Don Rockey, Todd Schell, Kevin Staveley-O’Carroll. Development of a successful combination therapy for hepatocellular cancer by targeting Treg and PD-1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4004.

The matricellular protein CCN1 suppresses hepatocarcinogenesis by inhibiting compensatory proliferation.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, and is on the rise in the United States. Previous studies showed that the matricellular protein CCN1 (CYR61) is induced during hepatic injuries and functions to restrict and resolve liver fibrosis. Here we show that CCN1 suppresses hepatocarcinogenesis by inhibiting carcinogen-induced compensatory hepatocyte proliferation, thus limiting the expansion of damaged and potentially oncogenic hepatocytes. Consistent with tumor suppression, CCN1 expression is down-regulated in human HCC. Ccn1ΔHep mice with hepatocyte-specific deletion of Ccn1 suffer increased HCC tumor multiplicity induced by the hepatocarcinogen diethylnitrosoamine (DEN). Knockin mice (Ccn1dm/dm) that express an integrin α6β1-binding defective CCN1 phenocopied Ccn1ΔHep mice, indicating that CCN1 acts through its α6β1 binding sites in this context. CCN1 effectively inhibits EGFR-dependent hepatocyte proliferation through integrin α6-mediated accumulation of reaction oxygen species (ROS), thereby triggering p53 activation and cell cycle block. Consequently, Ccn1dm/dm mice exhibit diminished p53 activation and elevated compensatory hepatocyte proliferation, resulting in increased HCC. Furthermore, we show that a single dose of the EGFR inhibitor erlotinib delivered prior to DEN-induced injury was sufficient to block compensatory proliferation and annihilate development of HCC nodules observed 8 months later, suggesting potential chemoprevention by targeting CCN1-inhibitable EGFR-dependent hepatocyte proliferation. Together, these results show that CCN1 is an injury response protein that functions not only to restrict fibrosis in the liver, but also to suppress hepatocarcinogenesis by inhibiting EGFR-dependent hepatocyte compensatory proliferation.

Stimulation of hepatocarcinogenesis by neutrophils upon induction of oncogenic kras expression in transgenic zebrafish

Background & AimsChronic inflammation is a major etiological factor for hepatocellular carcinoma (HCC), but how immune cells respond in the initiation of hepatocarcinogenesis remains uncharacterized. This study aims to investigate the response and roles of neutrophils in early hepatocarcinogenesis.MethodsBy inducible expression of oncogenic krasV12 in hepatocytes in transgenic zebrafish combined with live imaging of neutrophils in transparent larvae, the response of neutrophils to oncogenic liver was characterized and their roles investigated by pharmaceutical and genetic manipulations.ResultsWe found a rapid recruitment of neutrophils to the liver upon induction of krasV12 expression. Pharmaceutical stimulation of neutrophils resulted in further increases of neutrophils in oncogenic livers, liver size and tumor severity, while inhibition of neutrophils caused decreases of liver-associated neutrophils and liver size. Time-lapse video indicated that neutrophils had a stagnant migratory pattern meandering along the tumor edge but became relatively stationary upon entering the krasV12-expressing liver. Both oncogenic hepatocytes and tumor-associated neutrophils (TANs) were isolated via fluorescence-activated cell sorting. Molecular analyses indicated a pro-inflammatory microenvironment, as marked by increased tgfβ1a expression in krasV12-expressing hepatocytes and a loss of anti-tumor activities in TANs. Depletion of Tgf-β significantly reduced the number of TANs and the size of oncogenic liver.ConclusionsAn inflammatory cue from oncogenic hepatocytes upon induction of krasV12 expression causes a rapid recruitment of neutrophils to oncogenic liver and the neutrophils play a promoting role in early hepatocarcinogenesis.

Characterization of HGF/Met Signaling in Cell Lines Derived From Urothelial Carcinoma of the Bladder.

There is mounting evidence of oncogenic hepatocyte growth factor (HGF)/Met signaling in urothelial carcinoma (UC) of the bladder. The effects of three kinase inhibitors, cabozantinib, crizotinib and EMD1214063, on HGF-driven signaling and cell growth, invasion and tumorigenicity were analyzed in cultured UC cell lines. SW780 xenograft growth in SCID and human HGF knock-in SCID (hHGF/SCID) mice treated with cabozantinib or vehicle, as well as tumor levels of Met and pMet, were also determined. Met content was robust in most UC-derived cell lines. Basal pMet content and effector activation state in quiescent cells were low, but significantly enhanced by added HGF, as were cell invasion, proliferation and anchorage independent growth. These HGF-driven effects were reversed by Met inhibitor treatment. Tumor xenograft growth was significantly higher in hHGF/SCID mice vs. SCID mice and significantly inhibited by cabozantinib, as was tumor phospho-Met content. These studies indicate the prevalence and functionality of the HGF/Met signaling pathway in UC cells, suggest that paracrine HGF may contribute to UC tumor growth and progression, and that support further preclinical investigation of Met inhibitors for the treatment of UC is warranted.