Cell cycle regulators and transcriptional targeting in Medullary Thyroid Cancer
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The Ohio State University / OhioLINK
2018
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=osu1523891651533415 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu1523891651533415 |
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English |
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Biology |
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Biology Valenciaga, Anisley Cell cycle regulators and transcriptional targeting in Medullary Thyroid Cancer |
| author |
Valenciaga, Anisley |
| author_facet |
Valenciaga, Anisley |
| author_sort |
Valenciaga, Anisley |
| title |
Cell cycle regulators and transcriptional targeting in Medullary Thyroid Cancer |
| title_short |
Cell cycle regulators and transcriptional targeting in Medullary Thyroid Cancer |
| title_full |
Cell cycle regulators and transcriptional targeting in Medullary Thyroid Cancer |
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Cell cycle regulators and transcriptional targeting in Medullary Thyroid Cancer |
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Cell cycle regulators and transcriptional targeting in Medullary Thyroid Cancer |
| title_sort |
cell cycle regulators and transcriptional targeting in medullary thyroid cancer |
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The Ohio State University / OhioLINK |
| publishDate |
2018 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1523891651533415 |
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AT valenciagaanisley cellcycleregulatorsandtranscriptionaltargetinginmedullarythyroidcancer |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu15238916515334152021-08-03T07:06:15Z Cell cycle regulators and transcriptional targeting in Medullary Thyroid Cancer Valenciaga, Anisley Biology Medullary thyroid cancer is a currently incurable disease. Most patients present with invasive or distant disease, which carries a 10-year survival rate of 21%. While some advances in therapy have been developed, particularly the approval of multiple kinase inhibitors (MKIs) that target RET and other receptor tyrosine kinases, more research in this area is needed. Current MKIs do not offer complete responses and patients develop resistance over time. Almost all hereditary and 50% of sporadic MTC cases are caused by RET mutations, with some mutations correlated clinically with more aggressive disease in some studies. However, other alternative pathways have been studied. The CDK/RB cell cycle pathway shows potential as a different target for MTC given that loss of rb and other regulators of the pathway lead to MTC development in mice, and loss of some regulatory factors cooperate with ret in vivo, implicating this pathway in MTC biological behavior and therapeutic targeting. Loss of heterozygosity at the p18 and E2F2 loci has been found in a small cohort of human MTCs, but RB expression levels and cell cycle regulating pathway activation in MTC has been evaluated only in a small number of tumors with differing results. In addition to the “classical” CDK pathway, targeting transcription regulation using CDK9 and CDK7 inhibitors have been shown to reduce tumor burden in pre-clinical studies of difficult-to-treat tumors, but not MTC. The objective of this study was to determine if cell cycle pathways represent predictors of poor clinical outcome and/or are therapeutic targets for MTC, including treatment-resistant disease.In Chapter 2, we tested whether reduced expression of RB and/or overexpression of hyperphosphorylated RB (pRB) as biomarkers of CDK activation predict MTC aggressive behavior in 56 MTC human cases. We found that on univariate analysis, reduced RB (<75% tumor cell staining) trended with lower MTC-specific survival for primary tumor and metastatic nodes (primary tumor HR 3.54 (95%CI: 0.81, 15.47), p=0.08 and lymph node HR 4.35 (95%CI: 0.87, 21.83), p=0.05). For primary tumors, multivariable analysis showed that low nuclear RB expression independently associated with worse disease-specific (p=0.01) and overall (p=0.02) survival. pRB levels were not associated with survival for either primary tumor or lymph node metastases. Thus, activation of this pathway appears to predict aggressive MTC behavior.In Chapter 3, we explored targeting of the CDK/RB pathway in vitro using two MTC cell lines TT and MZ-CRC-1 with different RET driver mutations. CDK4/6 inhibition resulted in cell growth arrest but did not affect cell metabolic activity as a measure of cell viability or induce apoptosis. By contrast, targeting cyclin dependent kinases 1, 2, 5, and 9 using Dinaciclib was much more effective. Both cell lines were sensitive to Dinaciclib (IC50 at 72h: 4.6nM for TT and 13.9nM for MZ-CRC-1) and TT and MZ-CRC-1 cells with acquired resistance to Vandetanib retained sensitivity to Dinaciclib. With Dinaciclib treatment at the IC50 concentrations, CDK1/2/5 protein levels were maintained but unexpectedly, CDK9 protein and mRNA levels markedly decreased and CDK9 activity was reduced. RNA sequencing showed a global reduction of gene transcription in both cell lines after Dinaciclib treatment, particularly genes with RNA polymerase II-dependent transcription consistent with loss of CDK9 function. To further analyze this mechanism, the MTC cells were treated with THZ1, a CDK7 inhibitor. The cells were sensitive to this compound (IC50 at 72h: 26.68nM for TT and 87.97nM for MZ-CRC-1) and treatment resulted in loss of CDK9 expression. The specific reduction in CDK9 protein seen with Dinaciclib treatment was not due to changes in protein stability or proteosomal degradation, indicating a transcriptional regulatory mechanism. At the same time, in silico findings predict a super-enhancer regulating CDK9.In Chapter 4, we describe further analysis of Dinaciclib and THZ1 treated cells, which revealed that these treatments also decreased RET protein and mRNA expression, similar to CDK9. RET is also predicted to have a super-enhancer motif in silico, making this type of regulation a possible explanation for the specific effect of these treatments on RET as well. These treatments enhanced Ret activation in the surviving cells, consistent with oncogene addiction. This activation showed potential for combination therapies. Exploring this possibility, we treated MTC cells with Dinaciclib and Vandetanib, and found synergistic effects on cell metabolic activity in addition to reduction of RET activation with Vandetanib treatment after Dinaciclib exposure.In summary, this document describes experiments elucidating a potential biomarker for MTC clinical prognosis, and new therapeutic targets for MTC in vitro. We showed that reduced RB expression is associated with decreased patient survival in univariate and multivariable analysis, independent from patient age at surgery or cancer stage. Future studies involving larger MTC patient populations are needed to determine if lower RB expression levels may serve as a biomarker for aggressive disease in patients with MTC. We also determined that Dinaciclib and the CDK7 inhibitor THZ1 are active against naive and Vandetanib-resistant MTC cells in vitro, alone and in combination with Vandetanib. Inhibiting CDK9 or CDK7 affected CDK9 and RET transcription and protein levels, indicating a potential role for transcriptional targeting in MTC treatment and suggesting the possibility of a super-enhancer mechanism regulating CDK9 and RET. 2018-07-26 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1523891651533415 http://rave.ohiolink.edu/etdc/view?acc_num=osu1523891651533415 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |