CCSG Pilot: The Role of Calpain-5 in Endometrial Cancer

Abstract Calpains are calcium-activated thiol proteases that catalyze limited proteolysis of their substrates. Calpain- 5/CAPN5 is an understudied member of the calpain family, and members of the calpain family, including CAPN5, were reported to regulate cancer formation and progression. In endometrial cancer (EC), high levels of CAPN5 messenger RNA strongly correlated with poor prognosis. However, the mechanism by which CAPN5 influences EC prognosis is unknown, and the substrate spectrum of CAPN5 has yet to be fully uncovered. Defects in DNA repair pathways have also been implicated in EC development and progression. The loss of MLH1 function caused by either mutations or epigenetic inactivation is a prevalent cause of EC. Our exciting preliminary results identified and confirmed two CAPN5 interaction partners that are critical regulators of DNA repair: the mismatch DNA repair regulator MLH1 and the key Non-Homologous End Joining (NHEJ) DNA double strand break repair enzyme DNA-dependent protein kinase DNAPKcs/PRKDC. We also found that overexpression of CAPN5 in Ishikawa EC cells caused significant elevation in the level of Ser139- phosphorylated histone H2AX (γH2AX), a marker of unrepaired dsDNA breaks, and Ser2056-phosphorylated DNAPKcs, the active form of the kinase. We therefore hypothesize that elevated CAPN5 expression results in the dysregulation of the MMR and NHEJ DNA repair pathways, leading to high levels of genome instability and poor prognosis in endometrial cancer. We also predict that high CAPN5 expression will alter the sensitivity of EC to radiation and chemotherapy. To test these hypotheses, we will in Aim 1, determine the correlation of the CAPN5 protein levels with that of γH2AX, total and active DNAPKcs, and MLH1 detected in EC tissue microarrays with immunohistochemistry. We will also determine the correlation between the CAPN5 protein levels and pathologic and clinical staging, subsequent recurrence, metastasis, survival case annotation data and patient demographic information from the Markey Cancer Research Informatics SRF. This will assess the potential correlation in a unique patient population residing in an underserved rural community that may differ from large public databases. In Aim 2, we will determine the effect of CAPN5 overexpression in endometrial cancer cells on MMR function and their sensitivity to X-Ray irradiation. We will also determine the effect of CAPN5 overexpression on cell proliferation and viability. Our ultimate goals are to establish the effect of CAPN5 overexpression on EC tumor aggressiveness (invasion) and growth, and to determine whether targeting CAPN5 will enhance EC sensitivity to radiation therapy or DNAPK inhibition. Success of the proposed studies will generate critical preliminary data for an NIH R01 grant application to study the role of CAPN5 in endometrial cancer and provide important pre-clinical/translational data supporting the further investigation of CAPN5 in modulating radiation and drug sensitivity of EC. The PI of this application, Dr. Eddy Yang has a track record in the translation of basic research findings into pre-clinical and clinical studies. Dr. Yang is a Member of the Translational Oncology (TO) program. Dr. Eva Goellner and Dr. Chi Wang are Members of the Molecular and Cellular Oncology (MCO) program. Aim 1 will utilize the tissue microarrays and IHC service provided by the Biospecimen Procurement and Translational Pathology Shared Resource Facility (BPTP SRF). Dr. Dava Piecoro (UK Department of Pathology) will ensure that the IHC protocols are appropriate, and the IHC data are properly interpreted. Dr. Chi Wang is also Assistant Director of the Biostatistics and Bioinformatics Shared Resource Facility (BB SRF). Dr. Wang will participate in the statistical analysis of the TMA data in Aim 1 and provide statistical support for the entire project.