ObjectiveWe investigated the mutational landscape of mammalian target of rapamycin (mTOR) signalling cascade in hepatocellular carcinomas (HCCs) with chronic HBV background, aiming to evaluate and delineate mutation-dependent mechanism of mTOR hyperactivation in hepatocarcinogenesis.DesignWe performed next-generation sequencing on human HCC samples and cell line panel. Systematic mutational screening of mTOR pathway-related genes was undertaken and mutant genes were evaluated based on their recurrence. Protein expressions of tuberous sclerosis complex (TSC)1, TSC2 and pRPS6 were assessed by immunohistochemistry in human HCC samples. Rapamycin sensitivity was estimated by colony-formation assay in HCC cell lines and the treatment was further tested using our patient-derived tumour xenograft (PDTX) models.ResultsWe identified and confirmed multiple mTOR components as recurrently mutated in HBV-associated HCCs. Of significance, we detected frequent (16.2%, n=18/111) mutations of TSC1 and TSC2 genes in the HCC samples. The spectrum of TSC1/2 mutations likely disrupts the endogenous gene functions in suppressing the downstream mTOR activity through different mechanisms and leads to more aggressive tumour behaviour. Mutational disruption of TSC1 and TSC2 was also observed in HCC cell lines and our PDTX models. TSC-mutant cells exhibited reduced colony-forming ability on rapamycin treatment. With the use of biologically relevant TSC2-mutant PDTXs, we demonstrated the therapeutic benefits of the hypersensitivity towards rapamycin treatment.ConclusionsTaken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyperactivation and frequent TSC1/2 mutations in HBV-associated HCCs. They define a molecular subset of HCC having genetic aberrations in mTOR signalling, with potential significance of effective specific drug therapy.
Mitotic progression of mammalian cells is tightly regulated by the E3 ubiquitin ligase anaphase promoting complex (APC)/C. Deregulation of APC/C is frequently observed in cancer cells and is suggested to contribute to chromosome instability and cancer predisposition. In this study, we identified Daxx as a novel APC/C inhibitor frequently overexpressed in prostate cancer. Daxx interacts with the APC/C coactivators Cdc20 and Cdh1 in vivo, with the binding of Cdc20 dependent on the consensus destruction boxes near the N-terminal of the Daxx protein. Ectopic expression of Daxx, but not the D-box deleted mutant (DaxxΔD-box), inhibited the degradation of APC/Cdc20 and APC/Cdh1 substrates, leading to a transient delay in mitotic progression. Daxx is frequently upregulated in prostate cancer tissues; the expression level positively correlated with the Gleason score and disease metastasis (P = 0.027 and 0.032, respectively). Furthermore, ectopic expression of Daxx in a non-malignant prostate epithelial cell line induced polyploidy under mitotic stress. Our data suggest that Daxx may function as a novel APC/C inhibitor, which promotes chromosome instability during prostate cancer development.
The recent introduction of docetaxel in the treatment of hormone refractory prostate cancer (HRPC) has made a small but significant impact on patient survival. However, its effect is limited by intolerance and resistance. The aim of our study was to investigate if the garlic-derived compound, S-allylmercaptocysteine (SAMC), was able to act as a docetaxel sensitizing agent. First, the effect of SAMC on docetaxel sensitivity was examined on 3 HRPC cell lines by colony forming assay. We found that SAMC increased the efficacy of docetaxel on colony forming inhibition by 9-50% compared to single agent treatment. Second, using the HRPC CWR22R nude mice model, we found that the combination of SAMC and docetaxel was 53% more potent than docetaxel alone (p 5 0.037). In addition, there was no additive toxicity in the mice treated with the combination therapy evidenced by histological and functional analysis of liver, kidney and bone marrow. These results suggest that SAMC is able to increase the anticancer effect of docetaxel without causing additional toxic effect in vivo. Third, flow cytometry and Western blotting analysis on HRPC cell lines demonstrated that SAMC promoted docetaxel-induced G2/M phase cell cycle arrest and apoptotic induction. In addition, immunohistochemistry on CWR22R xenograft revealed a suppression of Bcl-2 expression and upregulation of E-cadherin in the SAMC and docetaxel treated animals. These results suggest that SAMC may promote docetaxel-induced cell death through promoting G2/ M cell cycle arrest and apoptosis. Our study implies a potential role for SAMC in improving docetaxel based chemotherapy for the treatment of HRPC.
We investigated the mutational landscape of mTOR signaling cascade in hepatocellular carcinomas (HCCs) with chronic hepatitis B (HBV) background, aiming to evaluate and delineate mutation-dependent mechanism of mTOR hyper-activation in hepatocarcinogenesis. We performed next-generation sequencing on human HCC samples and cell line panel. Systematic mutational screening of mTOR pathway-related genes was undertaken and mutant genes were evaluated based on their recurrence. Protein expressions of TSC1, TSC2 and pRPS6 were assessed by immunohistochemistry in human HCC samples. Rapamycin sensitivity was estimated by colony formation assay in HCC cell lines and the treatment was further tested using our patient derived tumor xenograft (PDTX) models. Results: We identified and confirmed multiple mTOR components as recurrently mutated in HBV-associated HCCs. Of significance, we detected frequent (16.2%, n = 18/111) mutations of TSC1 and TSC2 genes in the HCC samples. The spectrum of TSC1/2 mutations likely disrupts the endogenous gene functions in suppressing the downstream mTOR activity through different mechanisms and leading to more aggressive tumor behavior. Mutational disruption of TSC1 and TSC2 was also observed in HCC cell lines and our PDTX models. TSC-mutant cells exhibited reduced colony forming ability upon Rapamycin treatment. With the use of the biologically relevant TSC2-mutant PDTXs, we demonstrated the therapeutic benefits of the hyper-sensitivity towards Rapamycin treatment. Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyper-activation and frequent TSC1/2 mutations in HBV-associated HCCs. They define a molecular subset of HCC having genetic aberrations in mTOR signaling, with potential significance of effective specific drug therapy. Citation Format: Daniel Wai Ho, Lo Kong Chan, Yung Tuen Chiu, Iris Ming Xu, Ronnie Poon, Tan To Cheung, Chung Ngai Tang, Victor Tang, Irene Lo, Polly Lam, Derek Yau, Miao Xin Li, Chun Ming Wong, Irene O. L. Ng. TSC1/2 mutations define a molecular subset of HCC with aggressive behavior and treatment implication [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 4389. doi:10.1158/1538-7445.AM2017-4389
Hepatocellular carcinoma (HCC) is the third leading cause of all cancer-related deaths. Its rapid proliferative nature and HCC specific therapy such as transarterial chemoembolization (TACE) that block arterial blood supply to the tumor frequently induce hypoxic microenvironments i.e. regions with oxygen deprivation. Hypoxia has emerged as a major driving force to promote aggressive phenotypes and stemness of cancer cells in solid tumors. We aimed to identify the key contributors to HCC progression in hypoxic microenvironment. From the analysis of transcriptomic profiles of human HCCs in both our in-house RNA-sequencing (RNA-seq) data and TCGA data as well as HCC cells treated with hypoxia and normoxia, we identified Ephrin-A3 (EFNA3) as a potential hypoxia-inducible oncogene. EFNA3 is a membrane-bound ligand which can activate multiple receptor tyrosine kinases of the Eph family. This family of ligands and receptors are frequently dysregulated in multiple cancer types and have implications in stem cell plasticity. We further validated that EFNA3 was significantly overexpressed in HCCs as compared with their non-tumorous liver samples using qRT-PCR in a separate cohort of HCCs (p<0.0001, n=97). Upon clinicopathological correlation, overexpression of EFNA3 was significantly correlated with the presence of venous invasion (p=0.004) and more advanced tumor stage (p=0.026). In concordance, TCGA-LIHC dataset revealed significantly higher expression of EFNA3 in HCC samples with vascular invasion (p=0.0094) and advanced tumor stage (p=0.0002). Furthermore, we confirmed the upregulation of EFNA3 upon hypoxia treatment in multiple HCC cell lines. Functionally, stable knockdown of EFNA3 using short hairpin RNA approach significantly reduced proliferation and migratory rates of HCC cells. In addition, orthotopic liver injection model of the EFNA3 knockdown HCC cells demonstrated a significant reduction of the growth of the primary tumors. There was also a reduced incidence of distant metastasis to lungs in vivo. Using sphere formation assay to test self-renewal ability in vitro, EFNA3-knockdown HCC cells showed significantly reduced self-renewal ability. In addition, HCC cells positive/high for stemness markers (EpCAM, CD13, CD24, CD44, CD47, CD133) isolated from our PDTX model showed a consistently higher EFNA3 mRNA expression as compared with the negative/low cells. By flow cytometric analysis, the EFNA3 KD MHCC-97L demonstrated a decreased expression of CD47. Conclusion: We identified EFNA3 as a potential hypoxia-inducible oncogene in HCC and demonstrated its critical role in tumor progression, metastasis and cancer stemness of HCC. Based on our results, EFNA3 may be an attractive therapeutic target to counteract hypoxia-specific disease progression. Citation Format: Abdullah Husain, Yung Tuen Chiu, Daniel Wai Ho, Karen Man Sze, Lo Kong Chan, Yu Man Tsui, Carmen Chak Wong, Irene Oi Ng. EFNA3, a key functional mediator of hypoxic microenvironment 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 2431.
Hepatocellular carcinoma (HCC) is a leading cancer worldwide and especially prevalent in Southeast Asia, due to prevalent hepatitis B virus (HBV) infection. HBV-associated HCC has a male predominance, with a male-to-female ratio of about 4-5:1. HBV is a partially double-stranded DNA virus containing four overlapping open reading frames. Among them, X gene is most frequently integrated into host genome naturally and plays an important role in hepatocarcinogenesis. In male HBV carriers, the risk of HCC development is significantly increased in those with higher serum androgen and androgen receptor (AR) activity. HBV infection may, therefore, particularly cooperate with male-specific AR signalling to accelerate hepatocarcinogenesis. In this study, we hypothesized that HBx cooperated with male-specific AR signalling to enhance hepatocarcinogenesis, and natural HBx truncated mutants exerted different effects on this AR signalling. Firstly, we demonstrated that the expression of androgen receptor was significantly higher in the HCC cells when compared to the immortalized non-tumorigenic normal liver cell lines (LO2 and MIHA). Moreover, the HCC cell lines with higher metastatic potential (MHCC-97L, MHCC-97M and LM3) had a higher AR protein expression level, suggesting a role of AR in HCC development and progression. In human HBV-associated HCC tissues, we found that the natural COOH-terminal truncated HBx was more frequently detectable in HCC tumors from male than female patients (43% and 11%, respectively). Here, we also showed that the natural COOH-terminal HBx truncated mutants HBx ΔC3 (1-95 a.a) and HBx ΔC4 (1-89a.a) physically interacted with AR, indicating the possibility of HBx ΔC3 and HBx ΔC4 in regulating AR activity and the binding of HBx with AR at its N-terminal part. Furthermore, we demonstrated that HBx truncated mutants localized in the nuclear fraction, unlike the full-length HBx protein which localized in the cytoplasm. The capability of the nuclear translocation may suggest a role of the HBx truncated mutants in regulating AR transcriptional activity. In addition, androgen induced the expression of GADD45, a DNA damage-inducible gene, in HCC cells. The androgen-induced GADD45 expression was even higher with the co-expression of full-lengthen HBx protein, demonstrating a synergistic effect between HBx and AR in inducing DNA damage and genetic instability in HCC cells. Taken altogether, these findings may provide a linkage between HBx, AR signaling, genomic instability and HCC carcinogenesis Citation Format: Yung Tuen Chiu, Irene Oi Lin Ng. Dissecting the gender disparity of hepatocellular carcinoma: roles of HBV X protein and androgen receptor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4778. doi:10.1158/1538-7445.AM2013-4778
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