Date of Graduation
8-2016
Document Type
Dissertation (PhD)
Program Affiliation
Experimental Therapeutics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Zahid H. Siddik, Ph.D.
Committee Member
Gary Gallick, Ph.D.
Committee Member
Peng Huang, MD, Ph.D.
Committee Member
Shuxing Zhang, Ph.D.
Committee Member
Bulent Ozpolat, MD, Ph.D.
Committee Member
Sonal Gupta, Ph.D.
Abstract
Abstract
CIRCUMVENTING CISPLATIN RESISTANCE IN OVARIAN CANCERS THROUGH REACTIVATION OF P53 BY NON-CROSS-RESISTANT PLATINUM ANALOGS
Michelle Martinez-Rivera, B.S.
Advisory Professor: Zahid H. Siddik, Ph.D.
Cisplatin (cis-Pt), an anticancer platinum (Pt) drug, is used widely in the treatment of several malignancies, such as ovarian cancer. This Pt compound induces DNA damage, which results in p53 activation through post-translational modifications, mainly phosphorylation, culminating in execution of programmed cell-death. However, despite initial therapeutic response to cis-Pt, clinical resistance to this drug emerges leading to disease progression. Pt-resistance phenotypes have been associated with dysfunction in the p53 signaling pathway. Therefore, an effort to understand molecular mechanisms that prevent p53 activity and induce cis-Pt resistance becomes vital for designing Pt-based drugs able to re-activate p53 and improve clinical management of ovarian cancer patients. To investigate the mechanism responsible for p53 inactivation, an ovarian tumor panel composed of cis-Pt sensitive (A2780) and resistant (2780CP/Cl-16, OVCAR-10, HEY and OVCA 433) cell lines was established, with two (2780CP/Cl-16 and OVCAR-10) harboring missense mutant p53. The data obtained from these cancer cell lines have identified a correlation between cis-Pt resistance, regardless of p53 status (wild-type vs. mutant), and lack of phosphorylation of p53 at Ser20 after cis-Pt treatment. Cis-Pt resistant cell lines expressed low levels of Chk2, a kinase responsible to phosphorylate p53 at Ser20, as a common feature. It was confirmed, through the generation of Chk2 knock-out clones from A2780 cells using the CRISPR/Cas9 system, that Chk2 is essential for cis-Pt to mediate phosphorylation of p53 at Ser20 and induce p53 transcriptional activity. As validation of its critical role, Chk2 knock-out in these cells leads to cis-Pt resistance. However, cis-Pt resistance was circumvented by a number of cis-Pt analogs. In this regard, oxaliplatin (oxali-Pt), a non-cross-resistant Pt analog currently used in colon cancer but not ovarian cancer, was the most effective. Interestingly, the mechanism for oxali-Pt involved restoration of p53 phosphorylation at Ser20 through a Chk2 independent pathway. RPPA analysis has identified the MAPK pathway as a possible target of activation by oxali-Pt to phosphorylate p53 at Ser20. Systematic studies using targeted inhibitors have identified MEK1/2, but not ERK1/2, as a novel biomarker important to mediate p53-Ser20 phosphorylation by oxali-Pt. Overall, the findings gathered in this research project have revealed Ser20 of p53 as a key site that induces cis-Pt resistance when its phosphorylation is not induced by cis-Pt due to loss of Chk2, whereas its phosphorylation by oxali-Pt via MEK1/2 leads to circumvention of this resistance. This knowledge may lead to repurposing oxali-Pt in ovarian cancer and improve survival of cancer patients.
Keywords
cisplatin, platinum resistance, oxaliplatin, p53, ovarian cancer, Chk2, MEK1/2