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Date of Award
Histone Deacetylase 6 is a class IIb HDAC, a deacetylase of α-tubulin that regulates cell migration and motility. Over the last decade, HDAC6 has been characterized as an oncogenic protein in a variety of tumor types. We have since found that HDAC6 is upregulated across all three subtypes of non-small cell lung cancer (NSCLC), and that HDAC6 depletion can sensitize NSCLC cell lines to DNA damage. Our initial radiotherapy trials confirmed that HDAC6 knockdown can sensitize cells via PARP-1 cleavage, γ-H2AX foci persistence, and trypan blue exclusion. Interestingly, these trials also revealed that total Chk1 protein levels failed to resolve in HDAC6-knockdown cells. Chk1 is a Ser/Thr kinase activated by ATR in response to a variety of DNA aberrations, including the resected ends of a DNA double-strand break. Active Chk1 prevents the cell from progressing through S and G2 phase, and resolution of Chk1 is essential for cell cycle progression. Further analysis revealed that the stable Chk1 is active in HDAC6 knockdown cells, in stark contrast to our control cells where both total and active Chk1 resolve shortly after γ-H2AX foci become undetectable. Using pharmacological and genetic inhibition of Chk1, we found that eliminating Chk1 kinase activity had a protective effect on the ability of cells to survive post-IR, suggesting that the mechanism behind sensitization of HDAC6 knockdown cells to IR involves Chk1 activity. Examination of overexpressed and purified Chk1 and HDAC6 confirmed that these two proteins interact, that Chk1 specifically interacts with the DAC1 domain of HDAC6, and that HDAC6 can promote ubiquitination of Chk1 in a cellular system. Furthermore, we have functionally verified that Lysine 436, contained within the C-terminus of Chk1, is essential for optimal ubiquitination. While our previous report determined that HDAC6 functions by sequentially deacetylating and then ubiquitinating its substrate protein, we found that pharmacological inhibition of only HDAC6’s deacetylase activity did not influence the resolution of Chk1 protein levels post-IR. Here, we propose that HDAC6 regulates Chk1 protein levels via ubiquitination, and that elimination of HDAC6 prevents cells from escaping S/G2 and subsequently disables the cells’ ability to resolve radiationinduced DNA damage, promoting cell death.
Moses, Niko, "A Novel Target Of Hdac6’s E3 Ubiquitin Ligase Activity, Chk1, Is Responsible For The Sensitization Of Non-Small Cell Lung Cancer Cells To Ionizing Radiation" (2020). Wayne State University Dissertations. 2473.