The broad goal of this application is to understand the mechanisms by which RNA binding protein CUGBP2 regulates gene expression at the posttranscriptional level of mRNA stability and translation in intestinal epithelial cells. Overexpression of the protein in cancer cells results in cell death. We have determined that CUGBP2 interacts with AU-rich sequences in the 3'untranslated region of cyclooxygenase-2 and Mcl-1 mRNAs and upon binding downregulates the translation of both mRNAs. We have also determined that CUGBP2 levels are decreased in cancer cells but its expression is elevated in cells undergoing mitotic catastrophe. In addition, CUGBP2 is regulated at the levels of transcription by three different promoters. Activation from promoters located upstream from the commonly used proximal promoter results in the inclusion of additional amino acids in the N-terminus of the protein. Our studies suggest that the consequence of this addition is differences in cellular localization of the protein, but more importantly in the activity of the protein. The variants with the added amino acids do not affect cell viability. In addition, there are differential effects on target gene splicing with the three variants. Based on these observations, we have proposed to determine three aims.
In Aim 1, we will determine the mechanism(s) by which the three CUGBP2 promoters are regulated. In addition, CUGBP2 is regulated at the posttranscriptional level of alternative splicing resulting in the novel inclusion of one intron in the 5'untranslated region. We will identify the cis-acting sequences and trans-acting factors regulating this process.
In Aim 2, we will determine the cellular functions of CUGBP2. We have identified cellular factors that bind to CUGBP2. We will perform systematic deletion and fine mutagenesis to identify the domains in CUGBP2 that are involved in RNA:protein and protein:protein interactions. In addition, we will determine the CUGBP2 domains that regulate CUGBP2 localization under basal conditions and when the cells are exposed to 3-irradiation. We will also determine the effect of these interactions on CUGBP2 mediated RNA splicing, mRNA stability and translation regulation.
In Aim 3, we will determine whether the CUGBP2 gene is both a novel tumor suppressor and a protooncogene. We will determine the expression levels of the three isoforms in a panel of human colon tumors. Furthermore, we will determine whether the different CUGBP2 isoforms affect tumor behavior in xenograft models. Completion of these experiments should give us a better understanding of how the RNA binding protein CUGBP2 functions in normal epithelial cells, and whether changes in the CUGBP2 expression that is observed in tumor cells is responsible for tumor behavior.
Colorectal cancer is the leading in cancer related deaths in the United States. Understanding how the normal cell progresses to a cancer will aid in our developing novel therapies for both diseases. We have identified a protein, CUGBP2 whose expression is lost in cancer cells. Restoring the protein into the cancer cell kills the cells. We are in the process of identifying how the gene expressing CUGBP2 is silenced in cancer cells so that we can determine ways to reverse the process. This might stop or slow down the tumorigenesis.
| Kwatra, Deep; Subramaniam, Dharmalingam; Ramamoorthy, Prabhu et al. (2013) Methanolic extracts of bitter melon inhibit colon cancer stem cells by affecting energy homeostasis and autophagy. Evid Based Complement Alternat Med 2013:702869 |
| He, Zhiyun; Subramaniam, Dharmalingam; Zhang, Zhongtao et al. (2013) Honokiol as a Radiosensitizing Agent for Colorectal cancers. Curr Colorectal Cancer Rep 9: |
| Subramaniam, Dharmalingam; Periyasamy, Giridharan; Ponnurangam, Sivapriya et al. (2012) CDK-4 inhibitor P276 sensitizes pancreatic cancer cells to gemcitabine-induced apoptosis. Mol Cancer Ther 11:1598-608 |
| Sahoo, Kaustuv; Dozmorov, Mikhail G; Anant, Shrikant et al. (2012) The curcuminoid CLEFMA selectively induces cell death in H441 lung adenocarcinoma cells via oxidative stress. Invest New Drugs 30:558-67 |
| Gutheil, William G; Reed, Gregory; Ray, Amitabha et al. (2012) Crocetin: an agent derived from saffron for prevention and therapy for cancer. Curr Pharm Biotechnol 13:173-9 |
| Ponnurangam, Sivapriya; Mammen, Joshua M V; Ramalingam, Satish et al. (2012) Honokiol in combination with radiation targets notch signaling to inhibit colon cancer stem cells. Mol Cancer Ther 11:963-72 |
| Ramalingam, Satish; Ramamoorthy, Prabhu; Subramaniam, Dharmalingam et al. (2012) Reduced Expression of RNA Binding Protein CELF2, a Putative Tumor Suppressor Gene in Colon Cancer. Immunogastroenterology 1:27-33 |
| Subramaniam, Dharmalingam; Nicholes, Nathan D; Dhar, Animesh et al. (2011) 3,5-bis(2,4-difluorobenzylidene)-4-piperidone, a novel compound that affects pancreatic cancer growth and angiogenesis. Mol Cancer Ther 10:2146-56 |
| He, Zhiyun; Subramaniam, Dharmalingam; Ramalingam, Satish et al. (2011) Honokiol radiosensitizes colorectal cancer cells: enhanced activity in cells with mismatch repair defects. Am J Physiol Gastrointest Liver Physiol 301:G929-37 |
| Subramaniam, Dharmalingam; Ramalingam, Satish; Linehan, David C et al. (2011) RNA binding protein CUGBP2/CELF2 mediates curcumin-induced mitotic catastrophe of pancreatic cancer cells. PLoS One 6:e16958 |
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