Kai Yu Ng

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).

ANXA3/JNK signaling promotes self-renewal and tumor growth, and its blockade provides a therapeutic target for hepatocellular carcinoma

Summary Frequent tumor relapse in hepatocellular carcinoma (HCC) has been commonly attributed to the presence of residual cancer stem cells (CSCs) after conventional treatments. We have previously identified and characterized CD133 to mark a specific CSC subset in HCC. In the present study, we found endogenous and secretory annexin A3 (ANXA3) to play pivotal roles in promoting cancer and stem cell-like features in CD133+ liver CSCs through a dysregulated JNK pathway. Blockade of ANXA3 with an anti-ANXA3 monoclonal antibody in vitro as well as in human HCC xenograft models resulted in a significant reduction in tumor growth and self-renewal. Clinically, ANXA3 expression in HCC patient sera closely associated with aggressive clinical features. Our results suggest that ANXA3 can serve as a novel diagnostic biomarker and that the inhibition of ANXA3 may be a viable therapeutic option for the treatment of CD133+ liver-CSC-driven HCC.

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

FSTL1 promotes metastasis and chemoresistance in esophageal squamous cell carcinoma through NFκB-BMP signaling cross-talk

Esophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, and molecular markers to improve early detection and predict outcomes are greatly needed. Here, we report that the BMP-binding follistatin-like protein FSTL1 is overexpressed in ESCCs, where it correlates with poor overall survival. Genetic amplification of FSTL1 or chromosome 3q, where it is located, occurred frequently in ESCC, where FSTL1 copy number correlated positively with higher FSTL1 protein expression. Elevating FSTL1 levels by various means was sufficient to drive ESCC cell proliferation, clonogenicity, migration, invasion, self-renewal, and cisplatin resistance in vitro and tumorigenicity and distant metastasis in vivo Conversely, FSTL1 attenuation by shRNA or neutralizing antibody elicited the opposite effects in ESCC cells. mRNA profiling analyses suggested that FSTL1 drives ESCC oncogenesis and metastasis through various pathways, with deregulation of NFκB and BMP signaling figuring prominently. Cross-talk between the NFκB and BMP pathways was evidenced by functional rescue experiments using inhibitors of NFκB and TLR4. Our results establish the significance of FSTL1 in driving oncogenesis and metastasis in ESCC by coordinating NFκB and BMP pathway control, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in this disease setting. Cancer Res; 77(21); 5886-99. ©2017 AACR.

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

PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation

Arginine methylation is a post-translational modification that plays pivotal roles in signal transduction and gene transcription during cell fate determination. We found protein methyltransferase 6 (PRMT6) to be frequently downregulated in hepatocellular carcinoma (HCC) and its expression to negatively correlate with aggressive cancer features in HCC patients. Silencing of PRMT6 promoted the tumor-initiating, metastasis, and therapy resistance potential of HCC cell lines and patient-derived organoids. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a chemical-induced HCC PRMT6 knockout (PRMT6-/-) mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and showed that PRMT6 interacted with CRAF on arginine 100, which decreased its RAS binding potential and altered its downstream MEK/ERK signaling. Our work describes a critical repressive function for PRMT6 in maintenance of HCC cells by regulating RAS binding and MEK/ERK signaling via methylation of CRAF on arginine 100.

Abstract 1349: Functional and mechanistic characterization of PRMT6 regulated autophagy in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a major health burden worldwide. Tumor recurrence and therapy resistance represent major obstacles in the treatment of the disease, with their mechanism of action largely uncharacterized. Autophagy is a critical survival factor for cancer cells, whereby it maintains cellular homeostasis including degradation of damaged organelles and unwanted proteins as well as the support of cellular biosynthesis in response to environmental stress, preventing cells from undergoing apoptosis. Our group has recently reported on the regulatory role of protein arginine methyltransferase 6 (PRMT6) down-regulation in maintenance of HCC, particularly potentiating resistance to sorafenib and chemotherapy. By tandem affinity purification/mass spectrometry analysis of cells with PRMT6 stably overexpressed, we identified a number of autophagy related proteins as potential interacting partners of PRMT6, suggesting a possible role of PRMT6 in modulating autophagy in HCC. Treatment of HCC cells with stress induced conditions, including sorafenib, nutrient deprivation and hypoxic condition, resulted in a marked reduction of PRMT6 expression, concomitant with elevated levels of LC3BII. Further, under these various stimuli, knockdown of PRMT6 in HCC cells would also result in enhanced expression of LC3BII, accumulation of GFP-LC3B positive puncta staining as well as an increase in autophagosome formation. Consistently, treatment of HCC cells with PRMT6 overexpressed would leading to an opposing effect. Further studies by immunoprecipitation analysis validated PRMT6 to physically interact with AMBRA1 (activating molecule in BECN1-regulated autophagy protein 1), a well-known autophagy initiative component, which takes part in autophagy vesicle nucleation, a critical step for autophagic initiation. Our findings suggest PRMT6 down-regulation in HCC tumors to promote tumorigenicity through autophagic flux de-regulation. Targeting the mechanisms of stress response may provide novel therapeutic insights for the disease. Citation Format: Noelia Che, Man Tong, Kai Yu Ng, Phillis WF Kau, Xin Yuan Guan, Michael SY Huen, Stephanie Ma. Functional and mechanistic characterization of PRMT6 regulated autophagy in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1349.

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