Stella Chai

Octamer 4/microRNA-1246 signaling axis drives Wnt/β-catenin activation in liver cancer stem cells

Wnt/β‐catenin signaling is activated in CD133 liver cancer stem cells (CSCs), a subset of cells known to be a root of tumor recurrence and therapy resistance in hepatocellular carcinoma (HCC). However, the regulatory mechanism of this pathway in CSCs remains unclear. Here, we show that human microRNA (miRNA), miR‐1246, promotes cancer stemness, including self‐renewal, drug resistance, tumorigencity, and metastasis, by activation of the Wnt/β‐catenin pathway through suppressing the expression of AXIN2 and glycogen synthase kinase 3β (GSK3β), two key members of the β‐catenin destruction complex. Clinically, high endogenous and circulating miR‐1246 was identified in HCC clinical samples and correlated with a worse prognosis. Further functional analysis identified octamer 4 (Oct4) to be the direct upstream regulator of miR‐1246, which cooperatively drive β‐catenin activation in liver CSCs. Conclusion: These findings uncover the noncanonical regulation of Wnt/β‐catenin in liver CSCs by the Oct4/miR‐1246 signaling axis, and also provide a novel diagnostic marker as well as therapeutic intervention for HCC. (Hepatology 2016;64:2062‐2076).

Clinical implications of microRNAs in liver cancer stem cells.

The prognosis of patients diagnosed with hepatocellular carcinoma (HCC) is often dismal, mainly due to late presentation, high recurrence rate, and frequent resistance to chemotherapy and radiotherapy. Accumulating evidence on the differential microRNA (miRNA) expression patterns between non-tumor and HCC tissues or between liver cancer stem cells (CSCs) and non-CSC subsets and the significant clinical implications of these differences suggest that miRNAs are a promising, non-invasive marker for the prognosis and diagnosis of the disease. This perspective article summarizes the current knowledge of miRNAs in liver CSCs and highlights the need for further investigations of the role of miRNAs in regulating liver CSC subsets for possible future clinical applications.

C-terminal truncated hepatitis B virus X protein promotes hepatocellular carcinogenesis through induction of cancer and stem cell-like properties.

Tumor relapse after chemotherapy typifies hepatocellular carcinoma (HCC) and is believed to be attributable to residual cancer stem cells (CSCs) that survive initial treatment. Chronic infection with hepatitis B virus (HBV) has long been linked to the development of HCC. Upon infection, random HBV genome integration can lead to truncation of hepatitis B virus X (HBx) protein at the C-terminus. The resulting C-terminal-truncated HBx (HBx-ΔC) was previously shown to confer enhanced invasiveness and diminished apoptotic response in HCC cells. Here, we found HBx-ΔC to promote the appearance of a CD133 liver CSC subset and confer cancer and stem cell-like features in HCC. HBx-ΔC was exclusively detected in HCC cell lines that were raised from patients presented with a HBV background with concomitant CD133 expression. Stable overexpression of the naturally occurring HBx-ΔC mutants, HBx-Δ14 or HBx-Δ35, in HCC cells Huh7 and immortalized normal liver cells MIHA resulted in a significant increase in the cells ability to self-renew, resist chemotherapy and targeted therapy, migrate and induce angiogenesis. MIHA cells with the mutants stably overexpressed also resulted in the induction of CD133, mediated through STAT3 activation. RNA sequencing profiling of MIHA cells with or without HBx-ΔC mutants stably overexpressed identified altered FXR activation. This, together with rescue experiments using a selective FXR inhibitor suggested that C-terminal truncated HBx can mediate cancer stemness via FXR activation. Collectively, we find C-terminal truncated HBx mutants to confer cancer and stem cell-like features in vitro and to play an important role in driving tumor relapse in HCC.

Neuropilin-2 promotes tumourigenicity and metastasis in oesophageal squamous cell carcinoma through ERK-MAPK-ETV4-MMP-E-cadherin deregulation.

Oesophageal squamous cell carcinoma (ESCC) is the most common histological subtype of oesophageal cancer. The disease is particularly prevalent in southern China. The incidence of the disease is on the rise and its overall survival rate remains dismal. Identification and characterization of better molecular markers for early detection and therapeutic targeting are urgently needed. Here, we report levels of transmembrane and soluble neuropilin‐2 (NRP2) to be significantly up‐regulated in ESCC, and to correlate positively with advanced tumour stage, lymph node metastasis, less favourable R category and worse overall patient survival. NRP2 up‐regulation in ESCC was in part a result of gene amplification at chromosome 2q. NRP2 overexpression promoted clonogenicity, angiogenesis and metastasis in ESCC in vitro, while NRP2 silencing by lentiviral knockdown or neutralizing antibody resulted in a contrary effect. This observation was extended in vivo in animal models of subcutaneous tumourigenicity and tail vein metastasis. Mechanistically, overexpression of NRP2 induced expression of ERK MAP kinase and the transcription factor ETV4, leading to enhanced MMP‐2 and MMP‐9 activity and, as a consequence, suppression of E‐cadherin. In summary, NRP2 promotes tumourigenesis and metastasis in ESCC through deregulation of ERK–MAPK–ETV4–MMP–E‐cadherin signalling. NRP2 represents a potential diagnostic or prognostic biomarker and therapeutic target for ESCC. Copyright

TP53INP1 Downregulation Activates a p73-Dependent DUSP10/ERK Signaling Pathway to Promote Metastasis of Hepatocellular Carcinoma

Identifying critical factors involved in the metastatic progression of hepatocellular carcinoma (HCC) may offer important therapeutic opportunities. Here, we report that the proapoptotic stress response factor TP53INP1 is often selectively downregulated in advanced stage IV and metastatic human HCC tumors. Mechanistic investigations revealed that TP53INP1 downregulation in early-stage HCC cells promoted metastasis via DUSP10 phosphatase-mediated activation of the ERK pathway. The DUSP10 promoter included putative binding sites for p73 directly implicated in modulation by TP53INP1. Overall, our findings show how TP53INP1 plays a critical role in limiting the progression of early-stage HCC, with implications for developing new therapeutic strategies to attack metastatic HCC. Cancer Res; 77(17); 4602-12. ©2017 AACR.

Abstract 2223: C-terminal truncated hepatitis B virus X protein promotes hepatocarcinogenesis through enhanced stemness and resistance to therapy

This journal suppl. entitled: Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA

Efficacy of annexin A3 blockade in sensitizing hepatocellular carcinoma to sorafenib and regorafenib

BACKGROUND & AIMS Advanced hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. Sorafenib is the only FDA-approved first-line targeted drug for advanced HCC, but its effect on patient survival is limited. Further, patients ultimately present with disease progression. A better understanding of the causes of sorafenib resistance, enhancing the efficacy of sorafenib and finding a reliable predictive biomarker are crucial to achieve efficient control of HCC. METHODS The functional effects of ANXA3 in conferring sorafenib resistance to HCC cells were analyzed in apoptotic and tumorigenicity assays. The role of ANXA3/PKCδ-mediated p38 signaling, and subsequently altered autophagic and apoptotic events, was assessed by immunoprecipitation, immunoblotting, immunofluorescence and transmission electron microscopy assays. The prognostic value of ANXA3 in predicting response to sorafenib was evaluated by immunohistochemistry. The therapeutic value of targeting ANXA3 to combat HCC with anti-ANXA3 monoclonal antibody alone or in combination with sorafenib/regorafenib was investigated ex vivo and in vivo. RESULTS ANXA3 conferred HCC cells with resistance to sorafenib. ANXA3 was found enriched in sorafenib-resistant HCC cells and patient-derived xenografts. Mechanistically, overexpression of ANXA3 in sorafenib-resistant HCC cells suppressed PKCδ/p38 associated apoptosis and activated autophagy for cell survival. Clinically, ANXA3 expression correlated positively with the autophagic marker LC3B in HCC and was associated with a worse overall survival in patients who went on to receive sorafenib treatment. Anti-ANXA3 monoclonal antibody therapy combined with sorafenib/regorafenib impaired tumor growth in vivo and significantly increased survival. CONCLUSION Anti-ANXA3 therapy in combination with sorafenib/regorafenib represents a novel therapeutic strategy for HCC treatment. ANXA3 represents a useful predictive biomarker to stratify patients with HCC for sorafenib treatment. LAY SUMMARY This study represents the most extensive pre-clinical characterization of anti-ANXA3 monoclonal antibodies for the treatment of hepatocellular carcinoma to date. These results support the clinical trial development of anti-ANXA3 antibodies in combination with sorafenib/regorafenib. Further studies will optimize patient target selection and identify the best treatment combinations.

Abstract 4842: TP53INP1 negatively regulates ERK1/2 via p73-dependent DUSP10 expression to promote metastasis in hepatocellular carcinoma

The recurrence and metastasis of hepatocellular carcinoma (HCC) portends a poor prognosis and represents important clinical challenges. There is a great need to identify critical factors involved in HCC metastasis that will facilitate the development of new therapeutic strategies. We have previously found that the initiation, growth and self-renewal of CD133 + liver tumors to be fine-tuned by a balance of miR-130b overexpression and tumor protein 53 inducible nuclear protein 1 (TP53INP1) down-regulation, suggesting that TP53INP1 is a critical effector driving hepatocarcinogenesis. In this study, we aimed to further investigate the role of TP53INP1 in HCC metastasis. We showed TP53INP1 to be frequently down-regulated in advanced stage IV and metastatic human HCC tumors as compared with early stage (I-III) and primary tumors. Functional studies in immortalized normal liver cell line MIHA and HCC cell line MHCC-97L found TP53INP1 suppression in HCC to promote metastasis in vitro and in vivo. To elucidate the downstream signaling mechanism by which TP53INP1 regulates HCC metastasis, a Proteome Profiler Human Phospho-Kinase Array was utilized. Phospho-ERK was found to be consistently up-regulated in HCC cell lines with TP53INP1 knocked down, with its involvement in TP53INP1-mediated metastasis subsequently validated by rescue experiments using an ERK inhibitor U0126 or shERK1/2 knockdown approach. ERK1/2 is known to be negatively regulated by a family of dual-specificity MAPK phosphatase called DUSP/MKP. Screening of a panel of DUSP/MKP family members by qPCR identified DUSP10 to be commonly down-regulated in both MIHA and MHCC-97L cells with TP53INP1 suppressed as compared to controls. The importance of DUSP10 inactivation on ERK in TP53INP1-mediated HCC was substantiated by rescue experiments whereby DUSP10 ectopic expression in TP53INP1 suppressed cells reversed up-regulated phospho-ERK expression and enhanced HCC aggressiveness. Subsequent analysis of the DUSP10 promoter region by open-access database revealed four putative binding sites for p73, of which transcriptional activity has previously been found to be modulated by TP53INP1. Chromatin immunoprecipitation and luciferase reporter assays collectively demonstrated that TP53INP1 is vital for p73 transcriptional activity of DUSP10. Taken together, TP53INP1 down-regulation promotes HCC metastasis through a p73-dependent DUSP10/ERK signaling pathway. Note: This abstract was not presented at the meeting. Citation Format: Kai Yu Ng, Lok Hei Chan, Stella Chai, Man Tong, Xin Yuan Guan, Nikki P Lee, Yun Fei Yuan, Dan Xie, Terence K Lee, Alice Carrier, Nelson J Dusetti, Stephanie Ma. TP53INP1 negatively regulates ERK1/2 via p73-dependent DUSP10 expression to promote metastasis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4842. doi:10.1158/1538-7445.AM2017-4842

Abstract 2895: Octamer-4/microRNA-1246 signaling axis drives Wnt/β-catenin activation in liver cancer stem cells

Hepatocellular carcinoma (HCC), the main type of liver cancer in human, is one of the most prevalent and deadly malignancies in the world. Despite advances in therapy, prognosis remains dismal, largely attributed by our limited understanding on information related to the progressive development of the disease, particularly in their cancer-initiating and stem cell-like properties. Wnt/β-catenin signaling is activated in CD133 liver cancer stem cells (CSC), a subset of cells known to be a root of tumor recurrence and therapy resistance in HCC. However, the regulatory mechanism of this pathway in CSCs remains unclear. Here, we show that human miRNA, miR-1246, promotes cancer stemness including self-renewal, drug resistance, tumorigencity and metastasis by activation of Wnt/β-catenin pathway via suppressing the expression of AXIN2 and GSK3β, two key members of the β-catenin destruction complex. This observation was validated by both in vitro and in vivo functional / cell biological studies on HCC cell lines with or without miR-1246 expression modulated by lentiviral based knockdown and overexpression strategies as well as in miR-1246 repressed HCC cells with concomitant expression of wild-type or constitutively active β-catenin. Clinically, high endogenous and circulating miR-1246 was identified in HCC clinical samples and correlated with a worse prognosis. Further functional analysis identified Oct4 to be the direct upstream regulator of miR-1246, which cooperatively drive β-catenin activation in liver CSCs. In conclusion, our findings not only uncover the non-canonical regulation of Wnt/β-catenin in liver CSCs by Oct4/miR-1246 signaling axis, but also provide a novel diagnostic marker as well as therapeutic intervention for HCC. Citation Format: Stella Chai, Kai-Yu Ng, Man Tong, Eunice Y. Lau, Terence K. Lee, Kwok Wah Chan, Yun Fei Yuan, Tan To Cheung, Siu Tim Cheung, Xiao Qi Wang, Nathalie Wong, Chung Mau Lo, Kwan Man, Xin Yuan Guan, Stephanie K. Ma. Octamer-4/microRNA-1246 signaling axis drives Wnt/β-catenin activation in liver cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2895. doi:10.1158/1538-7445.AM2017-2895

Abstract LB-53: Regulatory role of miR-142-3p on the functional hepatic cancer stem cell marker CD133

Tumor relapse after therapy typifies hepatocellular carcinoma (HCC) and is believed to be attributable to residual cancer stem cells (CSCs) that survive treatment. We have previously identified a CSC population derived from HCC that is characterized by CD133. Despite our growing knowledge of the importance of this subset of cells in driving HCC, the regulatory mechanism of CD133 is not known. Epigenetic changes are believed to be essential in the control of cancer and stem cells. Here, we report the epigenetic regulation of CD133 by miR-142-3p. The interaction between CD133 and miR-142-3p was identified by in silico prediction and substantiated by luciferase reporter analysis. Expression of CD133 was found to be inversely correlated with miR-142-3p in HCC clinical samples as well as in cell lines. Importantly, lower miR-142-3p expression in HCC was significantly associated with worst survival. Functional studies with miR-142-3p stably transduced in HCC cells demonstrated a diminished ability to self-renew, initiate tumor growth, invade, migrate, induce angiogenesis and resist chemotherapy. Rescue experiments whereby CD133 and miR-142-3p is simultaneously overexpressed compensated the deregulated ability of the cells to confer these features. Thus, miR-142-3p directly targets CD133 to regulate its ability to confer cancer and stem cell-like features in HCC.