Research Proposal on "Deubiquitinases in Cancer and Health"

Research Proposal 4 pages (1433 words) Sources: 4

[EXCERPT] . . . .

, 2015). Several small molecule inhibitors of DUBs are available (D'Arcy, Wang, & Linder, 2015) and treatment of breast and ovarian cancer cells with two DUB inhibitors induced autophagy (Vogel et al., 2015). Treating breast and ovarian cancer cells with a small molecule inhibitor for USP14 and Chloriquine induced caspase-independent programmed cells death in a synergistic manner. The effect of these treatments on DSB repair by non-homologous end-joining (NHEJ) can be determined by measuring the levels of ?-H2AX and polyubiquitylated ?-H2AX (Ikura et al., 2007). These experiments will help reveal whether the effects of DUB inhibition in prostate cancer cells not only affects autophagy, but also DSB repair.

The second aim of this proposal will involve elucidating the role of USP14 in autophagy-deficient cells. As discussed above, autophagy can be inhibited by treating cells with the lysosome inhibitor Chloriquine (Vogel et al., 2015). The small molecule inhibitors specific for USP14 are IU1, b-AP15, and AC17 (D'Arcy, Wang, & Linder, 2015) and these can be used to treat prostate cancer and control cells to evaluate what roles this DUB may be playing in authophagy-deficient cells. The main variables of interest are proteosome activity, autophagy, and DSB repair activity. The expectation would be that inhibition of USP14 activity would increase proteosome activity, interact synergistically with Chloriquine to induce cell death (Vogel et al., 2015), and possibly result in the accumulation of ?-H2AX (Nijnik et al., 2012). The results of these experiments will determine the extent to which USP14 inhibition will have an impact on these processes in prostate cancer cells.

The third aim of this proposal will examine the mechanism of USP14 deubiquitination of ?-H2AX. If USP14 is involved in deubiquitinating ?-H2AX then its inhibition may result in the accumulation of polyubiquitinated ?-H2AX and the induction of programmed cell death and necrosis (Nijnik et al., 2012). Prostate cancer cells will be irradiated to induce DSBs and the accumulation of polyubiquitinated ?-H2AX will be quantified, in the presence or absence of an USP14 inhibitor. The results of these experiments will determine whether USP14 plays a role in DSB repair and is therefore a viable therapeutic target for selectively enhancing the efficacy of radiation-induced DNA damage in prostate tumors.

The fourth and final aim of this proposal will involve experiments designed to evaluate whether USP14 inhibition can restore DSB repair in synthetic lethal for homologous recombination (HR) and NHEJ. Synthetic lethality due to defective HR and NHEJ activities can be induced a number of ways, including treatment with small molecule inhibitors of APE1, ATM kinase, DNA-PKs, and DSB repair (Kinner, Wu, Staudt, & Iliakis, 2008). Treating cells with an ATM inhibitor, with or without an USP14 inhibitor should reveal whether DUB inhibition will restore the DSB repair synthetic lethal phenotype.

References

D'Arcy, P., Wang, X., & Linder, S. (2015). Deubiquitinase inhibition as a cancer therapeutic strategy. Pharmacology & Therapeutics, 147, 32-54.

Downing, S.R., Russell, P.J., & Jackson, P. (2003). Alterations of p53 are common in early stage prostate cancer. Canadian Journal of Urology, 10(4), 1924-33.

Glick, D., Barth, S., & Macleod, K.F. (2010). Atophagy: Cellular and molecular mechanisms. Journal of Pathology, 221(1), 3-12.

Human Protein Atlas. (n.d.). USP14. Retrieved from http://www.proteinatlas.org/ENSG00000101557-USP14/cancer/tissue/prostate+cancer.

Ikura, T., Tashiro, S., Kakino, A., Shima, H., Jacob, N., Amunugama, R. et al. (2007). DNA damage-dependent acetylation and ubiquitination of H2AX enhances chromatin dynamics. Molecular and Cellular Biology, 27(20), 7028-40.

Kinner, A., Wu, W., Staudt, C., & Iliakis, G. (2008). -H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Research, 36(17), 5678-94.

Liu, E.Y., Xu, N., O'Prey, J., Lao, L.Y., Joshi, S., Long, J.S. et al. (2015). Loss of autophagy causes a synthetic lethal deficiency in DNA repair. Proceedings of the National Academy of Sciences, U.S.A., 112(3), 773-8.

Nijnik, A., Clare, S., Hale, C., Raisen, C., McIntyre, R.E., Yusa, K. et al. (2012). The critical role of histone H2A-deubiquitinase Mysm1 in hematopoiesis and lymphocyte differentiation. Blood, 119(6), 1370-9.

U.S. Centers for Disease Control and Prevention. (2014). Prostate cancer statistics. Retrieved from http://www.cdc.gov/cancer/prostate/statistics/.

Vogel, R.I., Coughlin, K., Scotti, A., Iizuka, Y., Anchoori, R., Roden, R.B.S. et al. (2015). Simultaneous inhibition of deubiquitinating enzymes (DUBs) and autophagy synergistically kills breast cancer cells. Oncotarget, 6(6), 4159-70. READ MORE

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