Ct Lam

Abstract 5346: Identification of essential genes for the development of hepatitis B virus-associated hepatocellular carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background and Aim: Hepatocellular carcinoma (HCC) is a highly lethal and prevalent cancer, posing a grave threat to human health globally. Hepatitis B virus (HBV) infection is considered as a major risk factor for this cancer, especially in the Asia-Pacific region. Unfortunately, the molecular mechanisms of hepatocarcinogenesis remain obscure, which hinders the development of effective therapies for the disease. In the present study, we attempted to elucidate the molecular details of HBV-induced hepatocarcinogenesis by investigating differentially regulated genes at multiple developmental stages of HCC in a HBV transgenic mouse model. Materials and Methods: The transgenic mice which overproduced HBV large envelope polypeptide in hepatocytes and developed liver tumors spontaneously were used in this study. To unravel transcriptomics dynamics underlying hepatocarcinogenesis, RNA prepared from livers of both transgenic and wild type mice of different ages (at months 2, 12, 18 and 19) were subjected to RNA sequencing. Selected target genes were first validated by quantitative PCR (qPCR) using a larger set of mouse liver tissues (n=96) collected from 8 time points. Clinical implications of the selected genes were then explored in a set of human liver samples comprising 18 normal, 29 cirrhosis and 96 pairs of HCC. RNA and protein expression levels were determined by qPCR, immunohistochemical staining and Western blotting, respectively. Results: Upon analysis of 20,209 gene transcripts, 2574 and 1035 transcripts were found to be up-regulated (≥2 folds) and down-regulated (≤2 folds) in tumors, respectively, when compared with the wild type controls. Among these, 133 most prominent genes that exhibited concordant differential expression throughout the stages of tumor progression were chosen for validation in mouse liver tissues. Correlation analysis showed a high correlation between RNA sequencing and qPCR data (r=0.7495; P<0.0001), indicating a high validity of the data. Forty-six biologically informative genes were further validated in human liver samples. By Gene Ontology analysis, the target genes were revealed to play roles in a variety of biological processes including stress and inflammation responses, metabolic and apoptotic processes. Immunohistochemical staining and Western blotting demonstrated significant differential expression of these genes between HCC and non-tumorous livers. Statistical analyses revealed their significant correlation with clinicopathological parameters including venous infiltration, tumor size and overall survival, implicating their roles in hepatocarcinogenesis. Conclusion: This study has demonstrated a systematic strategy for identifying crucial genes for HBV-associated HCC, which may have profound implications in combating this deadly cancer. Citation Format: CT Lam, ZF Yang, JC Lau, MN Ng, WC Yu, DW Ho, ST Fan. Identification of essential genes for the development of hepatitis B virus-associated hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2014-5346

Abstract 3862: The potential role of CD44 in liver regeneration

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background and Aim: Liver regeneration occurs after liver damage by ischaemia, hepatitis, or in hepatocellular carcinoma (HCC) patients receiving hepatic resection or liver transplantation. Understanding the molecular bases of this process will provide novel therapeutic approaches for patients with various liver diseases. Previous studies suggested the linkage between CD44, a multifunctional cell surface receptor, and liver regeneration. The detailed role of this receptor in liver regeneration, however, remained unclear. The present study aimed at elucidating the role of CD44 in liver regeneration. Materials and Methods: A mouse model of liver regeneration induced by partial hepatectomy (PH) was employed. Serum and liver tissues were collected at different time points after PH. ELISA was performed to measure hyaluronic acid (ligand for CD44) level in serum. Flow cytometry was done to characterize liver cell populations after PH. Liver regeneration was quantified by measurement of liver mass and by immunohistochemical staining for PCNA (a proliferation marker). Quantitative PCR was performed to compare gene expression pattern of 46 genes between CD45− CD44+ cells and their CD44− counterparts. Results: Firstly, serum hyaluronic acid level was elevated at the early stage of liver regeneration. Secondly, by flow cytometry, an increase in CD45− CD44+ cell population was found. Thirdly, immunohistochemical analysis revealed active hepatocyte proliferation during this period. Interestingly, the hepatectomy-induced cell proliferation was suppressed after treating mice with anti-CD44 antibody, suggesting a functional role of CD44 in liver regeneration. Lastly, dysregulation of genes involving in cell proliferation, angiogenesis, and metabolism of hyaluronic acid was disclosed in CD45− CD44+ cells. Conclusion: The present study indicated that CD44-mediated pathways are required for liver regeneration, which may have therapeutic implications in treating liver diseases, including HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3862. doi:1538-7445.AM2012-3862

Abstract 2554: Synergistic antitumor effect of AUY922 in combination with sorafenib in hepatocellular carcinoma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL PURPOSE: Molecularly targeted therapy is of particular importance for HCC due to a lack of efficacy with currently approved chemo-toxic therapies. It is of interest to investigate the combined effect of two molecular targeted drugs, sorafenib (a multi-kinase inhibitor with anti-angiogenic, pro-apoptotic and Raf kinase inhibitory activity) and a highly potent non-geldamycin HSP90 inhibitor, NVP-AUY922 on HCC treatment. EXPERIMENTAL DESIGNS: Cell viability after different treatments was measured by both 3,[4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide (MTT) assay and cytofluorometric annexin-V apoptotic assay to compare the apoptotic cells induced by single versus combined treatment. Cell cycle analysis was also performed, and Western blotting was employed to determine the effects on MEK signaling, and in vivo efficacy was determined in nude mice with PLC/PRF/5 xenografts. RESULTS: Combined drugs treatment produced a synergistic effect on decreased HCC cell viability shown by MTT assay. An increase in the number of apoptotic cells were observed by apoptotic assay in combined drugs group when compared with single agents sorafenib or NVP-AUY922. Also, sorafenib combined with NVP-AUY922 induced S-phase arrest which was demonstrated by cell cycle analysis. Sorafenib alone decreases phospho-Erk1/2 expression, and this effect was further enhanced in combination treatment with NVP-AUY922. Finally, sorafenib plus NVP-AUY922 significantly suppressed PLC/PRF/5 xenograft tumor growth when compared with single (NVP-AUY922 or Sorafenib) treatment alone. CONCLUSION: The combination therapy of sorafenib and NVP-AUY922 can be a new and promising therapeutic approach to the treatment of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2554. doi:10.1158/1538-7445.AM2011-2554

Abstract 1305: The proangiogenic role of brain-derived neurotrophic factor in tumor development

Background and Aim: Hepatocellular carcinoma is a hypervascularized solid tumor which requires angiogenesis for its growth. Previous studies suggested that neurotrophins, such as brain-derived neurotrophic factor (BDNF), may serve as angiogenic factors. The involvement of BDNF in tumor angiogenesis, however, remains unclear. The present study aimed at elucidating the role of BDNF in regulating angiogenesis and tumor development. Materials and Methods: BDNF was overexpressed in a normal mouse endothelial cell line by transfection. The angiogenic properties, including proliferation, cell motility, invasiveness and cell survival, of the transfectants were subsequently assessed by MTT, cell migration, invasion and Annexin V labeling assays, respectively. Microarray analysis was performed to explore the BDNF-mediated angiogenic pathway. An in vivo cell co-injection model, using a mouse transformed hepatocyte cell line and BDNF transfectants, was used to study the role of BDNF in tumor development. Results: Firstly, overexpression of BDNF could promote endothelial cell proliferation, migration, invasion and survival. Secondly, the gene expression profiling data suggested that the BDNF-induced angiogenic effects could be attributed to a dozen of genes including transcription factors, cell adhesion molecules, chemokines and growth factors. Thirdly, the in vivo cell co-injection experiment showed that high BDNF-expressing endothelial cells are able to promote growth of tumors with significantly higher number of microvessels. On the other hand, knock-down of BDNF in a tumor-derived endothelial cell line by shRNAs impairs such tumor promoting effect. Conclusion: The present study showed that BDNF is crucial for tumor angiogenesis and may serve as a potential target for anti-angiogenic treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1305.

Short-term cardiotrophin-1 (CT-1) rescues marginalcirrhotic liver remnant after major hepatectomywithout increasing the risk of tumor recurrence

Background and Aims Asian patients have traditionally been considered to respond poorly to interferon-based treatment. We explored the effect of peginterferon alfa-2a (PEGASYS) in Asian patients with HBeAg-negative CHB enrolled in a large randomized clinical trial investigating the response to PEGASYS +/− lamivudine versus lamivudine (Marcellin et al. NEJM 2004). Patients and Methods 177 patients with HBeAg-negative CHB received 48 weeks of PEGASYS (180 μg once-weekly) + placebo. Patients were assessed 24 weeks post-treatment. Results Of the 177 patients, 108 (61%) were of Asian origin. Almost all the Asian patients (97%) were infected with genotypes B or C (42 patients with B, 63 with C, one with D and two other).The rate of combined response (ALT normalization and HBV DNA <20,000cp/ml) in Asian patients treated with PEGASYS was 45% (49/108), which was higher than that observed in the overall ITT population (36%). Response to PEGASYS was equally good in Asian patients infected with HBV genotype B (43%) and genotype C (49%). HBV DNA suppression of <400cp/ml was achieved in 26% of Asian patients compared with 19% in the overall ITT population. A total of three (3%) Asian patients treated with PEGASYS achieved HBsAg seroconversion after only 6 months of follow-up. Conclusions Asian patients responded favorably to PEGASYS treatment; 45% of Asian patients had a virological and biochemical response which was sustained 24 weeks after the end of treatment. Response rates were similar in patients infected with genotypes B or C.