Research
Cancer is a complex disease and typically involves multiple gene-specific and global genetic and epigenetic changes in human genome. The major class of genes that has been causally linked to the process of tumorigensis is tumor suppressors, which acts as a “breaks” and oncogenes, which functions as “accelerators” and typically promotes cancer. Recently, microRNAs are also linked to cancer wherein they can function either as tumor suppressors or oncogenes. The focus of our lab is to understand the mechanisms of genetic and epigenetic regulation of cancer causing genes and translating this understanding for early detection and treatment of human cancers. More specifically the lab has following major ongoing projects-
Role of Epigenetic Regulation in Cancer Progression and Identifying Cancer Stage Specific Epigenetic Signature
Although cancer cells have a globally hypomethylated genome, it is now well established that in almost all cancers a subset of tumor suppressors or pro-apoptotic genes become silenced by DNA methylation or oncogenes get activated by DNA demethylation. These changes in the promoter methylation state of cancer causing genes may contribute to cancer progression. There are two unanswered questions, which our lab is working on
1. Does activation of oncogenes and/or loss of tumor suppressors change the epigenetic state of normal cells to favor cancer progression? If so, do these genetic events operate through regulating the epigenetic architecture of cells to make them cancerous?
2. Which genes that are epigenetically silenced in a given cancer contribute to cancer initiation and progression?
As a first step towards answering these questions, we are using following well-established cancer progression models.
1. Hereditary colorectal cancer
Hereditary colorectal cancer is an excellent model that provides the opportunity to study the progressive onset of cancer in two different syndromes, familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPPC). Although major genetic changes such as APC mutation, K-Ras activation and loss of p53 function has been previously implicated and their contributions are understood in this cancer, very little if anything, is known about the epigenetic modifications that take place dependent or independent of these genetic mutations. To understand the hereditary colorectal cancer we will are using APCmin mouse model (min, multiple intestinal neoplasia) and colon cancer samples from human patient, which are being analyzed by ChIP-seq (SOLEXA and 454 platforms).
2. Melanoma
Similar to hereditary colon cancer, melanoma also provides an opportunity to study the contribution of epigenetic changes from early stages to advanced stages of melanoma genesis. Many important pathways such as Ras, Raf and PI3K are deregulated in melanoma. Another advantage of studying melanoma is that human samples of normal skin, benign and dysplastic nevi as well as malignant melanoma are readily available. This again, similar to hereditary colon cancer, allows the study of stepwise progression of melanoma.
Based on some of the preliminary results from these cancer models, we are now employing genome wide RNAi screens and small molecule library screens to understand – and possibly therapeutically target – important epigenetic changes.
Identification of Gene Specific Regulators of Epigenetic Silencing
DNA methylation-mediated inactivation of tumor suppressor genes is a common event in neoplastic transformation. Many important tumor suppressors such as p16, p14, BRCA1, and RASSF1A are subject to promoter hypermethylation in various cancers and their inactivation contributes to disease development. Currently, inhibitors of DNA methylation are being used for cancer treatment, suggesting that a better understanding of the DNA methylation process in breast cancer cells will have direct implications for the treatment of the disease.
Recently, we performed a genome-wide RNAi screens to identify factors involved in the maintenance of epigenetic silencing of RASSF1A, we first generated a reporter construct in which the RASSF1A promoter was used to direct expression of a gene encoding red fluorescent protein (RFP) fused to the blasticidin-resistance (BlastR) gene. This RASSF1A-RFP-BlastR reporter construct was stably transduced into MDA-MB-231 cells, in which the endogenous RASSF1A gene is epigenetically silenced. We then selected cells in which the reporter gene had been silenced as evidenced by loss of RFP expression and acquisition of blasticidin sensitivity. Transcriptional repression of the reporter gene was due to DNA methylation of the RASSF1A promoter as evidenced by the appearance of blasticidine-resistant colonies following treatment with the DNA methyltransferase inhibitor 5-aza-2’-deoxydytidine. A human shRNA library comprising ~62,400 shRNAs directed against ~28,000 genes was divided into 10 pools, which were packaged into retrovirus particles and used to stably transduce the MDA-MB-231/RASSF1A-RFP-BlastR reporter cell line. Blasticidin-resistant colonies, indicative of de-repression of the epigenetically silenced reporter gene, were selected and the shRNAs identified by sequence analysis. We identified 11 genes in this screen. One of the genes identified was HOXB3, an oncogene, for which the mechanism of action was not known. We found that HOXB3 functions as an oncogene, at least in part, by transcriptionally repressing tumor suppressor RASSF1A by DNA methylation.
This screen has provided us with a platform, which now can be used to understand the mechanism of epigenetic silencing of any tumor suppressor or oncogene. The long-term goal for similar screens will be to identify cancer specific epigenetic regulation signature for a given tumor suppressor and utilize that for selectively reexpressing a tumor suppressor of choice, with a possible therapeutic outcome.
Understanding the mechanism of Cellular Senescence
Cellular senescence refer to a cell state where cells are “irreversibly” arrested and even upon treatment with mitogenic agents fail to enter into cell cycle and divide. In recent years, cellular senescence has emerged as an important regulatory mechanism, which can modulate the initiation, progression and maintenance of a tumor in a host. Our lab is studying the mechanism and regulation of three major forms of cellular senescence namely; replicative senescence (RS), oncogene-induced senescence (OIS) and accelerated cellular senescence (ACS). Replicative senescence refers to the loss of human cells ability to divide due to telomere attrition. While in case of oncogene-induced senescence (OIS) an activating oncogenic event leads to an “irreversible” cell cycle arrest independent of telomere length. Recent studied have signified OIS as a tumor suppression mechanism. Finally, ACS refers to the senescence induced by genotoxic stress such a irradiation, which in principle is similar to OIS. Specific projects in the area of cellular senescence that are underway in the lab are following:
1. Identification of the Regulators of Cellular Senescence using Genome Wide-RNAi Technology.
2. Identification of New Senescence-based Early Detection Cancer Biomarkers and Therapeutic Targets
3. Small Molecule Library Screen (both DOS libraries and Bioactive compound library) to Identify Potential Small Molecules that can be used to Treat Cancer Cells that Originate due to bypass of ACS and Cause Recurrence and Drug resistant in Cancer.
Role of microRNAs in Senescence and Cancer
Recently, we have identified multiple miRNAs using Illumina-based SOLEXA deep sequencing from a human senescent cell. Currently, we are working on understanding the transcription regulation of these miRNAs and their role in cancer. We are also employing Ago2 immunoprecipitation based HITS-CLIP protocol for identifying the targets of these miRNAs. Importantly, with the development of new techniques in RNAi delivery and miRNA silencing, our long-term goal will be to target oncogenic miRNAs identified from the SOLEXA deep sequencing for therapeutic benefits.
The lab also have multiple rotation projects for undergraduate, masters, medical and graduate students, which involves use of (but not limited to) high-throughput genome-wide RNAi technologies, genome-wide deep sequencing technology and nude mice xenograft cancer models for studying the role of oncogenes and tumor suppressors in epigenetic regulation, senescence and DNA damage response pathways. Our lab also has a dedicated interest in developing new methodology for RNAi and protein delivery to specific cancer cells to achieve therapeutic efficacy.
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Publications
Selected Publications:
- Paul M Lizardi, Qin Yan and Narendra Wajapeyee 2010, (In press). Analysis of DNA methylation in Mammalian Cells. Molecular Cloning, Ed. 4 Cold Spring Harbor Laboratory Press. ;
- Narendra Wajapeyee#, Shu-Zong Wang, Ryan W. Serra, Peter D. Solomon, Arvindhan Nagarajan, Xiaochun Zhu and Michael R. Green#. Senescence induction in human fibroblasts and hematopoietic progenitors by leukemogenic fusion-proteins. Blood, April, 2010 (in press). # Joint corresponding authors
- Qin Yan# and Narendra Wajapeyee#. Exploiting Cellular Senescence to Treat Cancer and Circumvent Drug Resistance. Cancer Biology and Therapy (in press) Jan, 2010. # Joint corresponding author
- Rajendra K. Palakurthy#, Narendra Wajapeyee#, Manas Kumar Santra, Ling Lin, Stephane Gobeil, Claude Gazin, and Michael R. Green. Epigenetic Silencing of RASSF1A tumor suppressor Genethrough HOXB3-Mediated Induction of DNMT3B Expression. Molecular Cell (Oct 23, 2009). # Co-first author
- Manas K. Santra#, Narendra Wajapeyee# and Michael R. Green. F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage. Nature, 4th June, 2009.459: 722-5. # Co-first author
- Narendra Wajapeyee, Ryan Serra, XiaChun Zhu, Meera Mahalingam and Michael R Green. Oncogenic BRAF induces Senesences through an Autocrine/Paracrine pathway by the Secreted Protein IGFBP7. Cell, 8th Feb 2008. 132; 363-74
- Claude Gazin#, Narendra Wajapeyee#, Stephan Gobeil, Amy Virbasius and Michael R Green. An Elaborate Pathway Required for Ras-Mediated Epigenetic Silencing. Nature 25th October, 2007. 449; 1073-7. # Co-first author
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