Browsing by Author "Viswanathan, Vignesh"
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Item MicroRNAs in normal and malignant colon stem cells and their possible role in stem cell origin of colon cancer(University of Delaware, 2014) Viswanathan, VigneshThe role of miRNAs in colon cancer development pertaining specifically to the stem cell origin of cancer is yet to be elucidated. We hypothesized that perturbation of miR expression levels contributes to changes in target gene belonging to self-renewal pathways, which initiate colon tumorigenesis. Our miR profiling studies to identify miRs that regulate colon cancer stem cells (CSCs) were broadly divided in two parts. In one study we profiled the normal crypt bottom subsection that is enriched in stem cells and identified miR23b to be one of the miRs to have a significant differential expression. MiR23b has already been identified as having a role in renal, prostate, bladder, breast and colon cancers involving cell migration, invasion and apoptosis. Recently, a role for miR23b in ovarian CSCs and response to chemotherapy has also been elucidated. Consequently, I postulated that miR23b regulates colon CSCs. My results showed that miR23b is overexpressed in the ALDEFLUOR high sub-population of HT29 and SW480 colon cancer cells. It was shown to control the colon CSC phenotype and significantly affects proliferation, cell cycle, self-renewal, EMT, invasion and chemoresistance to the anti-cancer drug 5- FU. I validated that the colon CSC marker LGR5 is a target of miR23b, showing its role in modulating Wnt signaling. MiR23b also influences the transcription of multiple gene targets such as ATF2 and AKT2, which were identified by our extensive RNA SEQ analysis. The other aspect of the study was to identify miRs, which are differentially expressed in the CSCs as compared to the normal SCs from fresh patient samples. Normal and tumor tissue were initially screened for the expression of multiple putative CSC markers (ALDH1, LRIG1, CD166, ABCG2, BMI1, Telomerase) with an aim to decide which markers should be used alone or in combination to isolate SCs. My results indicated for the first time that SC markers ALDH1, LRIG1 and CD166 do not co-stain cells in tumors suggesting a co-existence of subpopulations of CSCs in tumor. MiR profiling identified miRs such as miR200c, miR92a, miR20a and miR93 that had a significant differential expression in ALDEFLUOR high tumor cells as compared to ALDEFLUOR high normal cells. MiR92a was also significantly upregulated in ALDEFLUOR high cells of colon cancer cell lines HT29 and regulated its proliferation. Future studies have to be done on the other newly recognized candidate miRs that show differential expression in primary tumor SCs. Understanding the role of miRs in CSCs could contribute to identification of new targets for therapeutic intervention.Item The v8-10 variant isoform of CD44 is selectively expressed in the normal human colonic stem cell niche and frequently is overexpressed in colon carcinomas during tumor development(Cancer Biology & Therapy, 2023-04-02) Boman, Bruce M.; Viswanathan, Vignesh; Faceya, Caroline O. B.; Fields, Jeremy Z.; Stave, James W.CD44 protein and its variant isoforms are expressed in cancer stem cells (CSCs), and various CD44 isoforms can have different functional roles in cells. Our goal was to investigate how different CD44 isoforms contribute to the emergence of stem cell (SC) overpopulation that drives colorectal cancer (CRC) development. Specific CD44 variant isoforms are selectively expressed in normal colonic SCs and become overexpressed in CRCs during tumor development. We created a unique panel of anti-CD44 rabbit genomic antibodies to 16 specific epitopes that span the entire length of the CD44 molecule. Our panel was used to comprehensively investigate the expression of different CD44 isoforms in matched pairs (n = 10) of malignant colonic tissue and adjacent normal mucosa, using two (IHC & IF) immunostaining approaches. We found that: i) CD44v8–10 is selectively expressed in the normal human colonic SC niche; ii) CD44v8–10 is co-expressed with the SC markers ALDH1 and LGR5 in normal and malignant colon tissues; iii) colon carcinoma tissues frequently (80%) stain for CD44v8–10 while staining for CD44v6 was less frequent (40%). Given that CD44v8–10 expression is restricted to cells in the normal human colonic SC niche and CD44v8–10 expression progressively increases during CRC development, CD44v8–10 expression likely contributes to the SC overpopulation that drives the development and growth of colon cancers. Since the CD44 variant v8–10 epitope is located on CD44’s extracellular region, it offers great promise for targeted anti-CSC treatment approaches.