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Cellular & Molecular Research
The Division of Cellular & Molecular Research is an active research department within the National Cancer Centre, Singapore. The close proximity of the research laboratories to the clinics within the National Cancer Centre and the Duke-NUS Graduate Medical School allows a collegial, collaborative and productive scientific and clinical research environment to forge research in translational biomedical science. The strategy is to focus on testing the applicability of scientific discoveries to solve clinical problems. The Division engages in vigorous basic clinical & translational research leading to novel clinical applications in oncology including innovative diagnostic tests, novel cancer prevention and treatment protocols, and genomic-based personalized treatment in oncology. The Division’s comprises The The Bek Chai Heah Laboratory of Cancer Genomics, headed by Prof. Kam M. Hui (also Head of the Division); Laboratory of Molecular Carcinogenesis, headed by Prof. Kanaga Sabapathy; Laboratory of Cancer Gene Therapy, headed by Assoc Prof. Paula Lam; Laboratory of Molecular Endocrinology, headed by Prof. Huynh The Hung; Laboratory of Genome Maintenance, headed by Dr. Susan Loong; and The Laboratory of Mammary Gland Biology headed by Dr. Alexandra Pietersen. The specific focuses of these laboratories are as follows:
The Bek Chai Heah Laboratory of Cancer Genomics has invovled in the generation and analysis of large-scale genomic data generated from expression profling studies and integrate with clinical information to identify novel genes that are cruccial for the devlopment of human cancers. Human cancers are characterized by genetic aberrations accompanied by altered expression and function of numerous genes. Applying genome-wide, microarray gene expression analysis to identify deregulated genes in different tumour types can provide potential gene candidates as diagnostic and prognostic tools and promising targets for drug development. In collaboration with different clinicial departments, the laboratory has characterized tumour-specific genes for many human cancers that are common in Singapore. The human cancers being studied include hepatocellular carcinoma (HCC), and lung carcinoma. The malfunction of some of these genes might be responsible for the development and metastasis of cancer. The group is investigating the potentials of some of these genes for the early diagnosis and treatment of these cancers.
Most recently, this laboratory has focused on studying vascular invasion in the context of HCC. Vascular invasion is one of the clinicopathologic features that is associated with early recurrent of HCC. Previously, this laboratory has identified a gene signature that could accurately predict the early recurrence of HCC and one of these genes is the human leukocyte cell-derived chemotaxin 2 (LECT-2). LECT-2 is only expressed in fetal liver tissues, adult liver and testis tissues. Expression of LECT-2 in human HCC biopsies was significantly reduced (fold change >-7.2, p < 0.0001) when compared with non-tumorous adjacent liver tissues. Furthermore, the reduction of LECT-2 expression was highly correlated with the early recurrent and poor prognosis of the patient. To study the possibility that LECT-2 is a bona fide tumor suppressor in human HCC and to validate the ability of LECT-2 to repress the growth of HCC, this group has designed an adenoviral vector encoding the secreted human LECT-2 (AdLECT-2) was introduced into the human HCC cell lines Hep3B and PLC/PRF/5 which do not express endogenous LECT-2. Over-expression of LECT-2 resulted in the significant reduction of in vitro migration and invasion of the AdLECT-2-transfected HCC cells. Additionally, over-expression of AdLECT-2 in subcutaneous Hep3B tumor xenografts in athymic nude mice resulted in significant suppression of tumor growth (p<0.05). In summary, besides being a candidate prognostic marker of human HCC, LECT-2 appears to be a novel tumor suppressor and as such a potential therapeutic target for HCC.
The tumour suppressor gene p53 is considered to be one of the most significant of all genes in cancer because mutations of this gene are found in more than half of all human cancers. In normal cells, p53 regulates cell growth by controlling cell proliferation and cell death. Mutations in p53 lead to the loss of this growth-suppressive function, thus leading to uncontrolled growth. The main objective of the Laboratory of Molecular Carcinogenesis is to elucidate the biochemical and biological processes that underlie the ability of p53 and its family members to act as tumour suppressors. In this respect, this laboratory has been continuing to focus on understanding the molecular mechanisms regulating carcinogenesis. The last year has been very productive and we have continued on well, with several publications. On the p53 front, we have continued our work on association studies, and have extended the previous work and have identified several novel SNPs in the p53 gene. Importantly, we have identified an intronic SNP that co-segregates with the common codon 72 polymorphism reported to regulate p53’s apoptotic and DNA-repair functions. We have now performed allele frequency studies, using the leukemia and lung cancer samples in collaboration with Dr Linn Yeh Ching (SGH) and Adeline Seow (NUH). This work is completed and is being submitted for publication. We have also been continuing to understand the molecular significance of the intronic SNP, and have identified transcriptional regulatory functions from this site. Work is ongoing to characterize the promoter activity herein. On the p73 aspect, we have published 2 high profile papers in PNAS and Cell, Death and Differentiation. These are on the role of p73 in mitotic cell death, as well as on the regulation of p73 stability. Part of the work on p73 stability was performed in collaboration with Dr Gerry Melino (Leicesheter Univ and Univ Rome). On the c-Jun/JNK front, we have characterized
the role of tyrosine 170 phosphorylation and its effects on c-Jun function. This work has been completed and been published in Cell Signaling. In addition, we have continued our efforts to identify JNK-interacting proteins, and have shortlisted three candidates that have been found to interact with JNKs in cell culture by immunoprecipitation experiments. We are now focusing on one major candidate that has a role in cell death and repair, and work has been going on well. We have completed the biochemical characterization of this interaction, and hope to report on the
complete story in the next round.
The Cancer Gene Therapy Laboratory is interested in the generation of novel therapeutic vectors, either viral or cellular-based system, to target primary human-tumor derived xenograft mouse models. At present, viral vectors remained the most efficient means by which therapeutic genes can be introduced into mammalian cells. One of our primary objectives is to improve the current vector system with respect to its ability to confer specific; regulatable and
stable transgene expression only in tumor cells. Further, the group is keen to understand the molecular mechanism of how human bone marrow-derived mesenchymal stem cells (MSC) track and engraft into the tumor cells. In particular, we have isolated MSC that exhibited high migratory potential toward human tumor cells versus those that do not appear to migrate well. Our previous studies have shown that matrix metalloproteinase 1 (MMP1) is critical in the migratory acitivities of MSC. Furthermore, the overexpression of MMP1 in MSC correlates with correspondingly higher level of SDF-1∝. This has led us to hypothesize that MMP1 could either directly or indirectly regulate the expression of SDF-1∝, which could subsequently influence the migration activities of MSC. The MSC isolates with different migration abilities will be employed to facilitate the understanding of MSC migration toward tumor cells or stromal microenvironment. In parallel, their ability to serve as therapeutic gene carriers will also be evaluated in vivo. The clinical implications of these studies is that the understanding of how MSC homes to the tumor microenvironment may, in future, provide the means for therapy of metastatic tumors and assist in the development of improved therapeutic vectors against human cancers.
To identify effective therapies, the National Xenograft Therapeutic Program (NXTP) for HCC and other solid tumors was established in April 2006. The Molecular Endocrinology Laboratory has developed ectopic and orthotopic xenograft lines in the SCID mouse models from resected tumor specimens. NXTP is fast becoming a recognized industry and academic leader in the area of drug development. The patient-derived xenografts appear to be the only
currently available means that will permit the propagation of a significant proportion of these primary carcinomas. TNXTP aims to identify novel clinically-relevant molecular signatures and develop a system to predict optimal treatments for cancer patients. NXTP presently serves as a repository of living tumor samples. The tumors are efficiently maintained to ensure that tumor samples remain viable and characterized for as long as they are required either for improved treatment regimens for the patients or for in-house or third party pharmaceutical research. We are currently collaborating with Novartis, AstraZeneca, Genzyme, GSK, BMS, Bayer Schering Pharma AG, and Roche to develop new therapies as well as to improve our current therapies for various cancer types. AstraZeneca signed an alliance between our National Cancer Centre and Singaporean institutions both on a pre-clinical and clinical level. AstraZeneca agreed to test 6 to 8 pipeline compounds against HCC in pre-clinical assessment, phase I and II through Singapore institutions, both at the level of the HCC xenograft models and also in first-in-man or first-in-class clinical trials. Recently, Roche has collaborated with NCCS and SingHealth to perform some preclinical studies of HCC and gastric cancer.
To translate our discovery to the clinics (Bench to Bedside), Dr. Choo Su Pin designed A Phase I Study of Rapamycin in combination with Bevacizumab in Patients with Unresectable Hepatocellular Carcinoma. This clinical trial steams from pre-clinical work on tumor vasculature and the mTOR signaling pathway in our laboratory (Patent: P029082GB SingHealth HCC Combination Therapy). A Phase I clinical trial of BEV/RAPA showed promising results with complete response was observed in one patient (5.6%) and 9 patients underwent stable disease (50%). Median overall survival was 17.5 months (Childs-Pugh A) and 3.1 months (Childs-Pugh B) (p=0.0005). Median PFS was 6.6 months. Using similar HCC models, we also demonstrated sunitinib was more toxic than sorafenib and the antitumor effect of 50 mg/kg sorafenib was greater than that of 40 mg/kg sunitinib. Our preclinical studies of BEV/RAPA and sorafenib versus sunitinib in HCC are recapitulated in Phase I of BEV/RAPA and the Phase III of study of sunitinib in advanced HCC, respectively. As a result, a phase II study of combination of bevacizumab with rapamycin analog RAD001 (Novartis pharmaceuticals) in patients with unresectable hepatocellular carcinoma will start in late year 2010. Based on the preclinical study reported in J. of Hepatology (2010) 52:79-87, Bayer and Astrazeneca agreed to provide AZD6224 and sorafenib respectively to run a phase I/II study of sorafenib and in combination with MEK inhibitor AZD6244 in advanced HCC in February 2010. Through collaboration with Novartis, we recently discovered that TKI258 potently inhibited tumor growth of HCC xenografts. The anti-tumor effect of TKI258 appeared superior to sorafenib, which is a current standard drug for HCC treatment. This study led to a phase II study of TKI258 (Novartis pharmaceuticals) in patients with unresectable hepatocellular carcinoma (start at the end of year 2010).
Cancer, at its very core, is a genetic disease. Recently, its molecular underpinnings have been traced to a group of genes coding for proteins that mediate cell cycle progression, DNA repair, programmed suicide (apoptosis) and the ‘upstream’ cell membrane signalling and transduction regulatory cascades. Collectively, these systems form a complex, interconnected homeostatic circuitry - denoted genome maintenance network (GMN) - that enables a human cell to sense and respond to different types of genotoxic stress (e.g. solar UV rays or toxic chemicals). The primary aim of the Laboratory of Genome Maintenance is to gain mechanistic insights into the genome maintenance network, i.e., the complex homeostatic circuitry comprising interconnected cell cycle control, DNA repair and apoptotic processes that enable a human cell to sense and respond to DNA damage. To this end, the Laboratory has made considerable progress in the identification of novel candidate substrates of ATM protein kinase, the DNA damage-sensing product of the ATM gene mutated in the rare cancer-predisposition and radiotherapy-sensitivity syndrome ataxia-telangiectasia (AT). In related work, the Laboratory has continued to investigate the molecular basis of the severe, late side effects routinely encountered by a small percentage (5%) of cancer patients who have undergone curative radiotherapy. Recently, in vitro error prone non-homologous end-joining (NHEJ) of non-compatible DNA ends was found to be a feature of these cell lines. In one of the cell lines, preliminary findings suggest this is due to defective interaction between proteins involved in the classic NHEJ pathway. The work is now focused on delineating the mutation(s) that has caused this defective interaction.
The cancer stem cell theory has attracted considerable attention in recent years, however it remains unclear to what extend processes that regulate normal stem cells contribute to cancer. This is addressed by the Laboratory of Mammary Gland Biology, which is part of both the Division and the cancer and stem cell biology program at Duke-NUS, and headed by Dr. Alexandra Pietersen. Since the start of the lab in July 2008, essential techniques to study mammary stem cells have been set up, such as the use of multiparameter flow cytometry to isolate the stem cells and limiting dilution transplantation for functional in vivo validation. The laboratory is currently using these techniques to study the role of transcriptional repressors Tbx2 and Tbx3. Both genes are found overexpressed in breast cancer and correlate with resistance to chemotherapy. Tbx3 is required to keep embryonic stem cells in an undifferentiated state, so we hypothesized that it may play a similar role in mammary stem cells. However, in contrast to our expectations, we demonstrated that Tbx3 is specifically expressed in a small subset of the luminal population. Interestingly, there is recent data to suggest that several subtypes of breast cancer in fact arise from luminal progenitors rather than stem cells. We are currently characterizing the potential of the Tbx3-GFP+ cells in colony forming assays and cleared fat pad reconstitution transplantations, and analyzing the effects of Tbx3 overexpression on both stem and progenitor cell function. Based on our investment in setting up state-of-the-art stem cell techniques in the last two years, the laboratory was able to obtain funding to identify novel stem-cell specific microRNAs and investigate the role of Wip1 phosphatase in mammary stem cells and radiation-resistance in breast cancer.
Kam M Hui, Ph.D., FRCPath
Director
Cellular and Molecular Research
