Exploring the JAK/STAT Signaling Pathway in Hepatocellular Carcinoma: Unraveling Signaling Complexity and Therapeutic Implications.

Hepatocellular Carcinoma (HCC) continues to pose a substantial global health challenge due to its high incidence and limited therapeutic options. In recent years, the Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT) pathway has emerged as a critical signaling cascade in HCC pathogenesis. The review commences with an overview of the JAK/STAT pathway, delving into the dynamic interplay between the JAK/STAT pathway and its numerous upstream activators, such as cytokines and growth factors enriched in pathogenic livers afflicted with chronic inflammation and cirrhosis. This paper also elucidates how the persistent activation of JAK/STAT signaling leads to diverse oncogenic processes during hepatocarcinogenesis, including uncontrolled cell proliferation, evasion of apoptosis, and immune escape. In the context of therapeutic implications, this review summarizes recent advancements in targeting the JAK/STAT pathway for HCC treatment. Preclinical and clinical studies investigating inhibitors and modulators of JAK/STAT signaling are discussed, highlighting their potential in suppressing the deadly disease. The insights presented herein underscore the necessity for continued research into targeting the JAK/STAT signaling pathway as a promising avenue for HCC therapy.

Targeting EGFR/PI3K/AKT/mTOR Signaling in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) poses a significant global health concern, with its incidence steadily increasing. The development of HCC is a multifaceted, multi-step process involving alterations in various signaling cascades. In recent years, significant progress has been made in understanding the molecular signaling pathways that play central roles in hepatocarcinogenesis. In particular, the EGFR/PI3K/AKT/mTOR signaling pathway in HCC has garnered renewed attention from both basic and clinical researchers. Preclinical studies in vitro and in vivo have shown the effectiveness of targeting the key components of this signaling pathway in human HCC cells. Thus, targeting these signaling pathways with small molecule inhibitors holds promise as a potential therapeutic option for patients with HCC. In this review, we explore recent advancements in understanding the role of the EGFR/PI3K/AKT/mTOR signaling pathway in HCC and assess the effectiveness of targeting this signaling cascade as a potential strategy for HCC therapy based on preclinical studies.

Antitumor Effect of Low-Dose of Rapamycin in a Transgenic Mouse Model of Liver Cancer.

PURPOSE: We investigate whether low-dose rapamycin is effective in preventing hepatocellular carcinoma (HCC) growth and treating HCC after tumor development in transgenic mice. MATERIALS AND METHODS: We established transgenic mice with HCC induced by activated HrasG12V and p53 suppression. Transgenic mice were randomly assigned to five experimental groups: negative control, positive control, tacrolimus only, rapamycin only, and tacrolimus plus rapamycin. The mice were further divided into two groups according to time to commencement of immunosuppressant treatment: de novo treatment and post-tumor development. RESULTS: In the de novo treatment group, marked suppression of tumor growth was observed in the rapamycin only group. In the post-tumor development group, the rapamycin only group displayed no significant suppression of tumor growth, compared to the positive control group. In T lymphocyte subset analysis, the numbers of CD4+ effector T cells and CD4+ regulatory T cells were significantly lower in the positive control, tacrolimus only, and tacrolimus plus rapamycin groups than the negative control group. Immunohistochemical analysis revealed significantly higher expression of phosphorylated-mTOR, 4E-BP1, and S6K1 in the positive control group than in the rapamycin only group. CONCLUSION: Low-dose rapamycin might be effective to prevent HCC growth, but may be ineffective as a treatment option after HCC development.

Anti-Cancer Effects of YAP Inhibitor (CA3) in Combination with Sorafenib against Hepatocellular Carcinoma (HCC) in Patient-Derived Multicellular Tumor Spheroid Models (MCTS).

PURPOSE: To assess the expression levels of YAP and TAZ in patient-derived HCC tissue and identify the effects of YAP/TAZ inhibition depending on the baseline YAP/TAZ expression when combined with sorafenib using a patient-derived multicellular tumor spheroid (MCTS) model. METHODS: Primary HCC cell lines were established from patient-derived tissue. Six patient-derived HCC cell lines were selected according to YAP/TAZ expression on Western blot: high, medium, low. Then, MCTS was generated by mixing patient-derived HCC cells and stroma cells (LX2, WI38, and HUVECs) and YAP/TAZ expression was assessed using Western blot. Cell viability of MCTS upon 48 h of drug treatment (sorafenib, sorafenib with CA3 0.1 µM, and CA3 (novel YAP1 inhibitor)) was analyzed. RESULTS: Out of six patient-derived HCC cell lines, cell lines with high YAP/TAZ expression at the MCTS level responded more sensitively to the combination therapy (Sorafenib + CA3 0.1 µM) despite the potent cytotoxic effect of CA3 exhibited in all of the patient-derived HCCs. CONCLUSION: Targeting YAP/TAZ inhibition using the novel YAP1 inhibitor CA3 could be a promising therapeutic strategy to enhance sensitivity to sorafenib especially in HCCs with high YAP/TAZ expression in MCTS.

Target Therapy for Hepatocellular Carcinoma: Beyond Receptor Tyrosine Kinase Inhibitors and Immune Checkpoint Inhibitors.

Hepatocellular carcinoma (HCC) is a major health concern worldwide, and its incidence is increasing steadily. To date, receptor tyrosine kinases (RTKs) are the most favored molecular targets for the treatment of HCC, followed by immune checkpoint regulators such as PD-1, PD-L1, and CTLA-4. With less than desirable clinical outcomes from RTK inhibitors as well as immune checkpoint inhibitors (ICI) so far, novel molecular target therapies have been proposed for HCC. In this review, we will introduce diverse molecular signaling pathways that are aberrantly activated in HCC, focusing on YAP/TAZ, Hedgehog, and Wnt/ß-catenin signaling pathways, and discuss potential therapeutic strategies targeting the signaling pathways in HCC.

Activated TAZ induces liver cancer in collaboration with EGFR/HER2 signaling pathways.

BACKGROUND: Liver cancer is a major global health concern due to the steady increases in its incidence and mortality. Transcription factors, yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ) have emerged as critical regulators in human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), the two major types of primary liver cancer. However, our study as well as other previous reports have shown that activation of YAP and TAZ (YAP/TAZ) in adult murine livers is insufficient for the development of liver cancer, suggesting a requirement for an additional oncogenic collaborator for liver carcinogenesis in adulthood. Therefore, we sought to identify the oncogenic partners of YAP/TAZ that promote hepatocarcinogenesis in adults. METHODS: Data analysis of the transcriptome of patients with liver cancer was performed using the national center for biotechnology information (NCBI) gene expression omnibus (GEO) database and the cancer genome atlas (TCGA). The cancer therapeutics response portal (CTRP) was used to investigate the correlation between sensitivity to chemicals and the copy number of TAZ in human cancer cell lines. Transposons encoding constitutively activated forms of TAZ (TAZS89A), BRAF (BRAFV600E), and PIK3CA (PI3KE545K) were used for hydrodynamic tail vein injection. Mice were monitored at least twice per week and sacrificed when moribund. Tumor-bearing livers were formalin fixed for hematoxylin-eosin staining and immunohistochemistry. RESULTS: Through database analyses, we identified EGFR/HER2 signaling to be essential in human cancers with high TAZ activity. Furthermore, immunohistochemical analyses showed that human HCC and CC tissues with high YAP/TAZ activities exhibited concomitant activation of EGFR/HER2 signaling pathways. To demonstrate that EGFR/HER2 signaling promotes YAP/TAZ-mediated hepatocarcinogenesis, TAZS89A was simultaneously expressed in murine adult livers with BRAFV600E or PI3KE545K, activated forms of effector molecules downstream of EGFR/HER2 signaling pathways. Expression of TAZS89A plus BRAFV600E induced HCC, whereas TAZS89A and PI3KE545K led to the development of CC-like cancer. CONCLUSIONS: Our study demonstrates that TAZ collaborates with EGFR/HER2 signaling pathways to induce both HCC and CC.

Pharmacological Inhibition of Sonic Hedgehog Signaling Suppresses Tumor Development in a Murine Model of Intrahepatic Cholangiocarcinoma.

Cholangiocarcinoma (CCC) is the second most primary liver cancer with an aggressive biological behavior, and its incidence increases steadily. An aberrant up-regulation of the sonic hedgehog signaling pathway has been reported in a variety of hepatic diseases including hepatic inflammation, fibrosis, as well as cancer. In this study, we determined the effect of a sonic hedgehog inhibitor, vismodegib, on the development of CCC. Through database analyses, we found sonic hedgehog signaling was up-regulated in human CCC, based on overexpression of its target genes, GLI1 and GLI2. Further, human CCC cells were highly sensitive to the treatment with vismodegib in vitro. Based on the data, we investigated the in vivo anti-cancer efficacy of vismodegib in CCC employing a murine model of CCC developed by hydrodynamic tail vein injection method. In the murine model, CCC induced by constitutively active forms of TAZ and PI3K exhibited up-regulated sonic hedgehog signaling. Treatment of vismodegib significantly suppressed tumor development in the murine CCC model, based on comparison of gross morphologies and liver weight/body weight. It is expected that pharmacological inhibition of sonic hedgehog signaling would be an effective molecular target therapy for CCC.

c-Myc-driven Hepatocarcinogenesis.

BACKGROUND/AIM: Dysregulation of the c-Myc gene is frequently found in human hepatocellular carcinoma (HCC), often accompanied by genetic and epigenetic alterations in other cancer-related genes. Here, we investigated the tumorigenic potential of c-Myc in diverse genetic environments in which the Ras, Wnt/ß-catenin, Sonic hedgehog, or P53 pathways were either activated or inactivated. MATERIALS AND METHODS: Hydrodynamic tail vein injection was employed to administer expression transposons and generate transgenic livers expressing c-Myc together with a constitutively active form of RAS (HRASG12V), ß-catenin (ß-cateninS33Y), Smo (SmoM2), or short hairpin RNA targeting P53 (shp53). RESULTS: c-Myc was most tumorigenic when the RAS signaling pathway was activated, whereas no tumors were found in mice when either ß-cateninS33Y or SmoM2 was co-expressed with c-Myc. Approximately 40% of mice had HCC when c-Myc was over-expressed under P53 inactivation. Furthermore, we investigated the effect of mutation in c-Myc on hepatocarcinogenesis. CONCLUSION: No significant differences in tumorigenic potential were found between wild type c-Myc and c-MycT58A, minimizing the role of the mutation in hepatocarcinogenesis.

MAPK/ERK Signaling Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a major health concern worldwide, and its incidence is increasing steadily. Recently, the MAPK/ERK signaling pathway in HCC has gained renewed attention from basic and clinical researchers. The MAPK/ERK signaling pathway is activated in more than 50% of human HCC cases; however, activating mutations in RAS and RAF genes are rarely found in HCC, which are major genetic events leading to the activation of the MAPK/ERK signaling pathway in other cancers. This suggests that there is an alternative mechanism behind the activation of the signaling pathway in HCC. Here, we will review recent advances in understanding the cellular and molecular mechanisms involved in the activation of the MAPK/ERK signaling pathway and discuss potential therapeutic strategies targeting the signaling pathway in the context of HCC.

Knockdown of Atg7 suppresses Tumorigenesis in a murine model of liver cancer.

Hepatocellular Carcinoma (HCC) is the most common type of primary liver cancer in adults and a leading cause of cancer-related deaths worldwide. Studies have shown that autophagy is significantly involved in carcinogenesis, in particular, driven by activated RAS signaling. Autophagy related 7 (Atg7) is a critical component for the formation of autophagosome and required for autophagy processes. We investigated the role of autophagy in RAS-driven tumorigenesis in the liver, via the knockdown of Atg7 in the model. Transposon vectors encoding short hairpin RNAs targeting Atg7 (Atg7 shRNA) were constructed. Inhibition of autophagy via Atg7 knockdown was tested in Hep3B cells cultured in nutrient-starved medium. Formation of autophagosome was suppressed in nutrient-starved Hep3B cells expressing Atg7 shRNA, demonstrating that it efficiently inhibited autophagy in HCC cells. Transposons encoding Atg7 shRNA were mixed with those expressing HRASG12V and p53 shRNA, and subsequently used for hydrodynamic injection to 5-week-old C57BL/6 mice. Tumorigenesis in livers induced by HRASG12V and p53 shRNA was significantly suppressed by Atg7 knockdown. The inhibition of autophagy led to a decreased proliferation of cancer cells, as determined by Ki-67 staining. Our data indicate that knockdown of Atg7 led to a significant decrease in tumorigenesis in a murine HCC model induced by activated RAS. Inhibition of autophagosome formation is expected to be a therapeutic option for liver cancer.

YAP/TAZ Suppress Drug Penetration Into Hepatocellular Carcinoma Through Stromal Activation.

BACKGROUND AND AIMS: HCC is the most predominant type of liver cancer affecting 800,000 people globally each year. Various small-molecule compounds targeting diverse oncogenic signaling pathways have been tested for patients with HCC, and clinical outcomes were not satisfactory. In this study, we investigated molecular signaling that determines the efficiency of drug delivery into HCC. APPROACH AND RESULTS: Hydrodynamics-based transfection (HT) was performed to develop mouse models for HCC induced by various oncogenes. Mice bearing liver cancer were treated with verteporfin at 5 weeks after HT. Multicellular HCC organoid (MCHO) models were established that contained various types of stromal cells, such as hepatic stellate cells, fibroblasts, and endothelial cells together with HCC cells. Tumor organoids were treated with verteporfin, and distributions of the drug in the organoids were assessed using fluorescence microscopy. Murine HCC models developed by HT methods showed that a high Yes-associated protein/Transcriptional co-activator with PDZ-binding motif (YAP/TAZ) activity in HCC cells impaired verteporfin penetration into the cancer. Activation of tumor stroma was observed in HCC with a high YAP/TAZ activity. Consistent with the findings in the in vivo models of HCC, MCHOs with activated YAP/TAZ signaling showed stromal activation and impaired penetration of verteporfin into the tumor organoids. Inhibition of YAP/TAZ transcriptional activity in HCC cells significantly increased drug penetration into the MCHO. CONCLUSIONS: Drug delivery into liver cancer is impaired by YAP/TAZ signaling in tumor cells and subsequent activation of stroma by the signaling. Disrupting or targeting activated tumor stroma might improve drug delivery into HCC with an elevated YAP/TAZ activity.

Ras Mitogen-activated Protein Kinase Signaling and Kinase Suppressor of Ras as Therapeutic Targets for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a high incidence cancer and a major health concern worldwide. Among the many molecular signaling pathways that are dysregulated in HCC, the Ras mitogen-activated protein kinase (Ras/Raf/MAPK) signaling pathway has gained renewed attention from basic and clinical researchers. Mutations in Ras and Raf genes which are known to activate the Ras/Raf/MAPK signaling pathway have been infrequently detected in human HCC; however, the Ras/Raf/MAPK signaling pathway is activated in more than 50% of HCC cases, suggesting an alternative mechanism for the activation of the signaling pathway. Kinase suppressor of Ras acts as a molecular scaffold for facilitating the assembly of Ras/Raf/MAPK signaling pathway components and has been implicated in the regulation of this signaling pathway. In this review, we provide important insights into the cellular and molecular mechanisms involved in the activation of the Ras/Raf/MAPK signaling pathway and discuss potential therapeutic strategies for HCC.

Effects of transarterial chemoembolization on regulatory T cell and its subpopulations in patients with hepatocellular carcinoma.

BACKGROUND: Regulatory T cell (Treg) plays an essential role in regulating anti-tumor immunity. The aim of this study was to investigate the effect of transarterial chemoembolization (TACE) on Treg in hepatocellular carcinoma (HCC) patients. METHOD: The frequency of peripheral blood Tregs in 27 HCC patients who underwent TACE were measured at baseline and 1 month after TACE. The frequency of peripheral blood Tregs at baseline were compared with those in 23 healthy controls. Tregs were further classified into three subpopulations [Treg (I), Treg (II), Treg (III)] based on expression levels or markers and their function. The patients were divided into two groups according to tumor response after TACE; complete response group and incomplete response group. The correlations between the frequency of Treg and clinical factors were analyzed. RESULTS: The frequency of Treg in HCC patients (7.52%) was significantly higher than in healthy controls (4.99%) at baseline. Regarding Treg subpopulations, the frequency of Treg (II) was significantly higher in HCC patients (2.51%) than in healthy controls (0.60%). In comparison of Treg numbers at baseline and post-TACE by tumor response, the change of Treg (III) in complete response group from baseline to post-TACE was significantly decreased (63.8 → 53.2/mm3). Patients with a high post-TACE Treg (III) (3.8 months) exhibited a significantly shorter median time to progression than those with a low post-TACE Treg (III) (11.6 months). In multivariate analyses, hypoalbuminemia (hazard ratio 3.324; 95% CI 1.098-10.063, p = 0.034) and high post-TACE Treg (III) (hazard ratio 3.080; 95% CI 1.091-8.696, p = 0.034) were significant factors for associating with progression. CONCLUSIONS: The frequency of Tregs in HCC patients was significantly higher than in healthy controls. In addition, patients with a high post-TACE Treg (III) exhibited a significantly lower progression-free survival rate than those with a low post-TACE Treg (III).

Genetically Engineered Mouse Models for Liver Cancer.

Liver cancer is the fourth leading cause of cancer-related death globally, accounting for approximately 800,000 deaths annually. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, comprising approximately 80% of cases. Murine models of HCC, such as chemically-induced models, xenograft models, and genetically engineered mouse (GEM) models, are valuable tools to reproduce human HCC biopathology and biochemistry. These models can be used to identify potential biomarkers, evaluate potential novel therapeutic drugs in pre-clinical trials, and develop molecular target therapies. Considering molecular target therapies, a novel approach has been developed to create genetically engineered murine models for HCC, employing hydrodynamics-based transfection (HT). The HT method, coupled with the Sleeping Beauty transposon system or the CRISPR/Cas9 genome editing tool, has been used to rapidly and cost-effectively produce a variety of HCC models containing diverse oncogenes or inactivated tumor suppressor genes. The versatility of these models is expected to broaden our knowledge of the genetic mechanisms underlying human hepatocarcinogenesis, allowing the study of premalignant and malignant liver lesions and the evaluation of new therapeutic strategies. Here, we review recent advances in GEM models of HCC with an emphasis on new technologies.

High Risk of Hepatocellular Carcinoma Development in Fibrotic Liver: Role of the Hippo-YAP/TAZ Signaling Pathway.

Liver cancer is the fourth leading cause of cancer-related death globally, accounting for approximately 800,000 deaths annually. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, making up about 80% of cases. Liver fibrosis and its end-stage disease, cirrhosis, are major risk factors for HCC. A fibrotic liver typically shows persistent hepatocyte death and compensatory regeneration, chronic inflammation, and an increase in reactive oxygen species, which collaboratively create a tumor-promoting microenvironment via inducing genetic alterations and chromosomal instability, and activating various oncogenic molecular signaling pathways. In this article, we review recent advances in fields of liver fibrosis and carcinogenesis, and consider several molecular signaling pathways that promote hepato-carcinogenesis under the microenvironment of liver fibrosis. In particular, we pay attention to emerging roles of the Hippo-YAP/TAZ signaling pathway in stromal activation, hepatic fibrosis, and liver cancer.

Barrier to autointegration factor 1, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3, and splicing factor 3b subunit 4 as early-stage cancer decision markers and drivers of hepatocellular carcinoma.

An accurate tool enabling early diagnosis of hepatocellular carcinoma (HCC) is clinically important, given that early detection of HCC markedly improves survival. We aimed to investigate the molecular markers underlying early progression of HCC that can be detected in precancerous lesions. We designed a gene selection strategy to identify potential driver genes by integrative analysis of transcriptome and clinicopathological data of human multistage HCC tissues, including precancerous lesions, low- and high-grade dysplastic nodules. The gene selection process was guided by detecting the selected molecules in both HCC and precancerous lesion. Using various computational approaches, we selected 10 gene elements as a candidate and, through immunohistochemical staining, showed that barrier to autointegration factor 1 (BANF1), procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3), and splicing factor 3b subunit 4 (SF3B4) are HCC decision markers with superior capability to diagnose early-stage HCC in a large cohort of HCC patients, as compared to the currently popular trio of HCC diagnostic markers: glypican 3, glutamine synthetase, and heat-shock protein 70. Targeted inactivation of BANF1, PLOD3, and SF3B4 inhibits in vitro and in vivo liver tumorigenesis by selectively modulating epithelial-mesenchymal transition and cell-cycle proteins. Treatment of nanoparticles containing small-interfering RNAs of the three genes suppressed liver tumor incidence as well as tumor growth rates in a spontaneous mouse HCC model. We also demonstrated that SF3B4 overexpression triggers SF3b complex to splice tumor suppressor KLF4 transcript to nonfunctional skipped exon transcripts. This contributes to malignant transformation and growth of hepatocyte through transcriptional inactivation of p27Kip1 and simultaneously activation of Slug genes. CONCLUSION: The findings suggest molecular markers of BANF1, PLOD3, and SF3B4 indicating early-stage HCC in precancerous lesion, and also suggest drivers for understanding the development of hepatocarcinogenesis. (Hepatology 2018;67:1360-1377).

Pro-tumorigenic roles of TGF-ß signaling during the early stages of liver tumorigenesis through upregulation of Snail.

Many studies have focused on the tumor suppressive role of TGF-ß signaling during the early stages of tumorigenesis by activating the target genes involved in cytostasis and apoptosis. We investigated the effects of TGF-ß inhibition on early tumorigenesis in the liver, by employing diverse inhibitory methods. Strikingly, TGF-ß inhibition consistently suppressed hepatic tumorigenesis that was induced either by activated RAS plus p53 downregulation or by the co-activation of RAS and TAZ signaling; this demonstrates the requirements for canonical TGF-ß signaling in tumorigenesis. Moreover, we found that Snail is the target gene of the TGF-ß signaling pathway that promotes hepatic carcinogenesis. The knockdown of Snail suppressed the early tumorigenesis in the liver, as did the TGF-ß inhibition, while the ectopic expression of Snail restored tumorigenesis that was suppressed by the TGF-ß inhibition. Our findings establish the oncogenic TGF-ß-Smad- Snail signaling axis during the early tumorigenesis in the liver. [BMB Reports 2017; 50(12): 599-600].

Transforming Growth Factor-ß Promotes Liver Tumorigenesis in Mice via Up-regulation of Snail.

BACKGROUND & AIMS: Transforming growth factor beta (TGF-ß) suppresses early stages of tumorigenesis, but also contributes to migration and metastasis of cancer cells. A large number of human tumors contain mutations that inactivate its receptors, or downstream proteins such as Smad transcription factors, indicating that the TGF-ß signaling pathway prevents tumor growth. We investigated the effects of TGF-ß inhibition on liver tumorigenesis in mice. METHODS: C57BL/6 mice received hydrodynamic tail-vein injections of transposons encoding HRASG12V and a short hairpin RNA (shRNA) to down-regulate p53, or those encoding HRASG12V and MYC, or those encoding HRASG12V and TAZS89A, to induce liver tumor formation; mice were also given injections of transposons encoding SMAD7 or shRNA against SMAD2, SMAD3, SMAD4, or SNAI1 (Snail), with or without ectopic expression of Snail. Survival times were compared, and livers were weighted and examined for tumors. Liver tumor tissues were analyzed by quantitative reverse-transcription PCR, RNA sequencing, immunoblots, and immunohistochemistry. We analyzed gene expression levels in human hepatocellular carcinoma samples deposited in The Cancer Genome Atlas. A cell proliferation assay was performed using human liver cancer cell lines (HepG2 and Huh7) stably expressing Snail or shRNA against Snail. RESULTS: TGF-ß inhibition via overexpression of SMAD7 (or knockdown of SMAD2, SMAD3, or SMAD4) consistently reduced formation and growth of liver tumors in mice that expressed activated RAS plus shRNA against p53, or in mice that expressed activated RAS and TAZ. TGF-ß signaling activated transcription of the Snail gene in liver tumors induced by HRASG12V and shRNA against p53, and by activated RAS and TAZ. Knockdown of Snail reduced liver tumor formation in both tumor models. Ectopic expression of Snail restored liver tumorigenesis suppressed by disruption of TGF-ß signaling. In human hepatocellular carcinoma, Snail expression correlated with TGF-ß activation. Ectopic expression of Snail increased cellular proliferation, whereas Snail knockdown led to reduced proliferation in human hepatocellular carcinoma cells. CONCLUSIONS: In analyses of transgenic mice, we found TGF-ß signaling to be required for formation of liver tumors upon expression of activated RAS and shRNA down-regulating p53, and upon expression of activated RAS and TAZ. Snail is the TGF-ß target that is required for hepatic tumorigenesis in these models.

Deubiquitinase YOD1 potentiates YAP/TAZ activities through enhancing ITCH stability.

Hippo signaling controls the expression of genes regulating cell proliferation and survival and organ size. The regulation of core components in the Hippo pathway by phosphorylation has been extensively investigated, but the roles of ubiquitination-deubiquitination processes are largely unknown. To identify deubiquitinase(s) that regulates Hippo signaling, we performed unbiased siRNA screening and found that YOD1 controls biological responses mediated by YAP/TAZ. Mechanistically, YOD1 deubiquitinates ITCH, an E3 ligase of LATS, and enhances the stability of ITCH, which leads to reduced levels of LATS and a subsequent increase in the YAP/TAZ level. Furthermore, we show that the miR-21-mediated regulation of YOD1 is responsible for the cell-density-dependent changes in YAP/TAZ levels. Using a transgenic mouse model, we demonstrate that the inducible expression of YOD1 enhances the proliferation of hepatocytes and leads to hepatomegaly in a YAP/TAZ-activity-dependent manner. Moreover, we find a strong correlation between YOD1 and YAP expression in liver cancer patients. Overall, our data strongly suggest that YOD1 is a regulator of the Hippo pathway and would be a therapeutic target to treat liver cancer.