Survivin and its expression-correlated genes: a possible regulatory mechanism involved in colon cancer development

Date
2010
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Publisher
University of Delaware
Abstract
Survivin (BIRC5), a member of the inhibitor of apoptosis family of proteins (IAP) is a 16.5kDa protein that is highly over-expressed in many types of cancer including colorectal cancer (CRC), but is almost undetectable in normal tissue. It has been considered a biomarker for tumor progression. Over-expression of survivin predicts poor patient survival and high tumor recurrence rates (Sarela, Macadam et al. 2000). Preliminary large scale expresseion analysis between healthy and diseased colon revealed that most of the genes that were expression-correlated with survivin were classified as cell-cycle and mitosis genes. Survivin itself is expressed preferentially during mitosis and is needed for spindle assembly checkpoint (Mita, Mita et al. 2008). Survivin is known to interact with a high number of signaling molecules, regulators, transcriptional networks that, are involved in its functions directly or indirectly (Altieri 2008). To appreciate the complexity it is not enough to think of survivin alone, but it becomes apparent by performing network analyses that link survivin to multiple signaling circuits. This led us to predict that there may be many genes that are simultaneously regulated with survivin by a network of transcriptional regulators. As the expression correlation analysis showed that there is a specific cohort of genes that are expression-correlated with survivin, I proposed that the expression of this set of genes can be co-regulated by a common set of transcription factors (TFs). To identify enriched regulatory elements supporting my hypothesis of co-regulation, the promoter regions of these genes were analyzed to identify shared transcriptional regulatory elements (TREs), which provide information on cognate TFs that may be involved in this co-regulation. Eleven TFs were selected corresponding to those TREs that showed statistically significant over-representation in the set of expression-correlated genes. The panel included TP53, MYB, E2F4, E2F1, PAX6, NKX2-5, USF1, NFE2, NFYA, ELK1 and TDP2. Nothing much is known about the TFs that co-regulate survivin and its group of expression-correlated gene especially in CRC. This led to my hypothesis that specific TFs corresponding to common TREs in promoter regions of survivin and its expressioncorrelated genes are differentially expressed in normal versus malignant colon tissues at the mRNA as well as the protein level. For this thesis research project, differential expression of the candidate TFs was first investigated at the mRNA level using quantitative real-time polymerase chain reaction (Q-RT-PCR), to analyze the mRNA levels between human normal and tumor colon tissues. The results demonstrate that there is differential expression of mRNA between CRC versus normal colonic epithelium in most of the TFs analyzed. Many of the TFs showed increased expression, which was statistically significant, in tumor tissue compared to adjacent normal colonic epithelium. To analyze whether changes at the mRNA level correspond to changes at the translational level, protein levels were analyzed using immunohistochemistry. Among the six Q-RT-PCR validated TFs tested by immunostaining, five (TP53, MYB, E2F4, PAX6 and NKX2-5) showed differential expression in tumor compared to the normal colonic tissue. All these TFs showed increased expression in tumor tissue compared to normal except E2F1, which showed no change in expression in normal compared to tumor tissue consistent with mRNA quantification results. In conclusion, my results indicated that out of the ten TFs tested for, more than six TFs showed increased expression in colon cancer tissue compared to normal colonic epithelium in mRNA as well as protein level. Even though, NKX2-5 showed increased expression of protein in tumor tissue compared to the normal colonic epithelium at the protein level, its mRNA levels showed negligible change between normal and tumor. E2F1 showed no change in expression at either the mRNA or the protein level. Survivin has been known to be over-expressed in many cancers including CRC (Altieri 2003). By large scale network analysis, it was shown that along with survivin many other genes are expression-correlated. My results indicate that TFs involved in the regulation of survivin and its expression-correlated genes that showed enrichment of TREs using computational analysis tool promoter analysis and interaction network toolset (PAINT), shows an increased expression in tumor tissue compared to the normal colonic mucosa in the mRNA as well as the protein level. This shows that cancer has a dysregulated transcriptional network, which might play a part in the dysregulation of survivin and its expression-correlated genes. Consequently this change in expression pattern of transcriptional regulatory molecules might play a role in changing the expression of survivin and its expression-correlated genes which may contribute to the progression and development of CRC. These results might be used towards the development of a diagnostic test or a survivin cancer network-targeted antagonist based on the expression pattern and transcriptional regulatory network involved with survivin and its expression-correlated genes. Targeting survivin and its expression-correlated genes can be considered as a step in that direction with a CRC perspective. The study of survivin and its expressioncorrelated genes can be considered an invaluable study that sheds light on many pathways that are involved in CRC which can lead to the development of powerful drugs that help to conquer CRC.
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