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MRJ: A Molecular Switch for Cell Cycle and Cancer Metastasis
The breast cancer metastasis suppressor gene, BRMS1, significantly suppresses metastasis of multiple mammary cell lines of human and mouse origin. We discovered that this nuclear protein binds to MRJ (a member of Hsp40 family, with J domain). Previously we have demonstrated that BRMS1 is a part of mSin3-HDAC complex that is implicated in RB-E2F regulations and cell cycle arrest. SV40 T antigen's J domain is involved in abrogating RB function. Thus we hypothesize that BRMS1 containing regulatory protein complex binds to MRJ to stop metastasis of breast cancer cells by restoration RB-E2F check point.
Objective: Elucidate the members of the MRJ-BRMS1 complex and investigate its role in metastasis suppression.
1. Perform molecular dissection of MRJ-BRMS1 interaction to address which amino acids of each protein are involved in the interaction.
2. Determine the members of RB-E2F pathway that bind to MRJ
3. Perform transcriptional gene silencing of MRJ and analyze the effects on cell cycle and apoptosis.
Aim 1: Approach
• C-terminal and internal domain deletion of MRJ to check for loss of interaction with BRMS1.
• Generate point mutations in MRJ that lose the BRMS1 interaction by site directed mutagenesis or yeast reverse two hybrid screens.
• Identify point mutations in BRMS1 (BRMS1mut) that lose interaction with MRJ.
• Generating stable cell lines (from highly metastatic human breast cancer cell lines i.e. MDA-MB-231 and MDA-MB435) that express BRMS1mut protein.
• Evaluating these cell lines for alteration of metastatic potential using in vitro metastasis assays.
• Mutants altered for metastasis suppression phenotype, will be tested using in vivo metastasis models.
Aim 2. Approach
• Immunoprecipitation of the complex(es) from the fraction using anti-MRJ antibody.
• Separation of the complex using SDS-PAGE or 2-D gel electrophoresis.
• Analyze novel bands (or spots) compared to the control by electrospray based mass spectrometry assay (LC-MS-MS analysis using Q-TofTM Ultima) and further confirm their identified members using Western blots.
Aim 3. Approach
• MRJ expression from metastatic human breast cancer cell lines will be abolished using RNA interference (pSuper based stable cloning).
• Cells that have been transcriptionally silenced for MRJ will be compared with the parental lines for cell cycle using propidium iodide staining followed by flow cytometric analysis.
• The apoptosis will also be evaluated by AnnexinV staining and TUNEL.
Cancer cells are capable of spreading from the primary tumor to distant sites. This phenomenon is known as metastasis. The survival rates for breast cancer are high when there is no dissemination, but when the tumor cells leave the primary site and colonize distant tissues such as bones or lungs, cure rates drop significantly. Thus, decreased morbidity and mortality can be achieved by effective prevention and/or treatment of metastatic disease. Metastasis involves interplay of many genes. Metastasis-regulatory genes can be recognized as metastasis-promoting or metastasis-suppressing. Though metastasis requires a coordinated expression of many genes, it takes only one gene to inhibit metastasis (a block in any one step of metastasis blocks the conversion of non metastatic tumor to metastatic). Thus, understanding the mechanism of action of a metastasis suppressor gene offers a handle to block the metastatic process in patients and also will prove helpful in identification of patient population(s) likely to suffer due to the spread of a primary tumor. It is intriguing to note that there are very few such metastasis suppressor genes identified to date (NM 23, Kai1, KiSS1, etc.), and the precise mechanism of action of many of them is just beginning to be revealed.
We recently discovered a gene with great potentials, BRMS1, which suppresses metastasis of human breast carcinoma cell lines. While unraveling the mechanism this suppression phenomenon, we found that MRJ (a heat shock protein/chaperone with J domain) binds to with BRMS1. “J” domain is implicated in regulation of the tumor suppressor gene retinoblastoma (RB). It stops RB function leading to uncontrolled cell division and formation of tumor. Since BRMS1 binds to MRJ, we propose that it stops MRJ from inhibiting RB function, thus allowing proper cell cycle control. This may activate the apoptosis (cell death) machinery, leading to destruction of cancer cells.
To facilitate the transfer of the benefit of a discovery of interaction of potentially clinically important genes (such as MRJ and BRMS1) to actual therapeutic use, it is necessary to understand the mechanism by which they function in the cell. The outcome of this work will not only help in understanding the mechanism of action but also help to get clues about important steps in the metastasis suppression of breast cancer. In the long-term, the outcome of the proposed work will aid identification of novel therapeutic targets and knowledge based mimetic design and/or gene therapy for suppressing the metastasis of breast cancer.