Cell cycle regulation, DNA damage and DNA repair pathways and their role in cancer initiation and maintenance

A typical cell cycle of a mammalian cell can be divided into four phases. These include G1 phase, S-phase, G2-phase and M phase. Dividing cells spend a certain amount of time each cell cycle stage, G1 phase being the longest. Primary human cells under genotoxic conditions undergo a state of cell cycle arrest, which is referred to as Cell cycle checkpoints. During this phase of cell cycle arrest primary human cells repair the DNA damage and then re-enter the cell cycle. However, in case of cancer cells these DNA damage checkpoints are defective leading to cycling of cells even in the presence of DNA damage stimulus. These defects in cell cycle checkpoints contributes to accumulation of mutations, which eventually with other genetic changes can lead to neoplastic transformation of human cells. One of the ongoing efforts in the lab is to identify and characterize new DNA damage checkpoint proteins and study their role in cancer. In addition to the cell cycle defects, cancer cells show defects in DNA damage response pathway and DNA repair pathway. Our lab is also working on identifying genes that regulate DNA damage response and DNA repair pathway, with the aim of developing new methods to targets these specific defects in cancer cells.

the cell cycle and the checkpoints

DNA methylation regulation in cancer and identifying cancer-driving DNA methylation changes in human genome

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 re-expressing a tumor suppressor of choice, with a possible therapeutic outcome.

Regulation of cellular senescence and its role in immortalization and cellular transformation

Cellular senescence is a form a irreversible cell cycle arrest. Depending upon the senescence inducing stimuli cellular senescence can be divided into three forms: Replicative senescence (due to telomere shortening), Oncogene-induced senescence ( due to introduction of a activated oncogene in a primary cells) or Accelerated cellular senescence (due to exposure to DNA damage inducing chemicals such as DNA damage inducing drugs). p53 and RB pathways plays a very important role in the regulation of both Replicative senescence and oncogene-induce senescence. We have also performed a large-scale RNAi screen to identify regulators of BRAF-induced senescence. Our lab now working on studying other protein coding genes and non-coding RNAs (specifically miRNAs) for their role in the regulation of cellular senescence. We use melanoma and lung cancer as systems to study the early cellular senescence events and understand their implication in primary cell immortalization and eventually transformation.

Identification of regulators of cellular senescence in Primary Human and cancer cells and targeting them for Cancer treatment