Functional and molecular study of human kinase NEK6 as a target of drugs against prostate cancer

Author(s)

Isadora Carolina Betim Pavan

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Farmacêuticas
• Defense date: 2023-05-24

Examining board members

Jörg Kobarg; Clarissa Ribeiro Reily Rocha; Ana Lucia Tasca Gois Ruiz; Marcos Rodrigo Alborghetti; Natássia Elena Búfalo

• Advisor: Jörg Kobarg

Abstract

Introduction: NIMA (Never In Mitosis, gene A)-related kinase-6 (NEK6) belongs to a superfamily of 11 members of NIMA-related kinases. The functions of NEK6 are little studied, except for its already evidenced participation in the cell cycle. The literature has shown its relevance in castration-resistant prostate cancer (CRPC), evidencing its central role in androgen-independent tumor growth, suggesting that NEK6 regulates cell survival pathways. However, it is still unclear which these pathways and mechanisms regulated by NEK6 make it participate in the CPRC. Aim: This study aimed to explore NEK6 in CRPC, evaluating the cellular effects in the absence of its expression and inhibition, as well as identifying the molecular survival mechanisms that NEK6 regulates in this cancer. In addition, we also evaluated the relevance of NEK6 depletion and/or inhibition in the chemotherapy treatment used in CPRC, such as cisplatin and docetaxel, and the natural anti-tumor compound, resveratrol. Material and methods: NEK6 knockout cells were generated in DU-145 and PC-3 cell lines using the CRISPR/Cas9 system. The use of a specific NEK6 inhibitor was also tested on DU-145 and PC-3 cells. Functional assays to evaluate viability, clonogenic capacity, cell death, migration, generation of Reactive Oxygen Species (ROS), and permeability of the mitochondrial membrane were performed in these cells. Evaluation of expression at the level of messenger and protein RNA of genes related to progression and chemoresistance in prostate cancer cells were performed. Results: NEK6 knockouts decreased clonogenic capacity, proliferation, cell viability, and mitochondrial activity. NEK6 depletion increased the level of intracellular ROS, decreased expression of antioxidant defenses (SOD1, SOD2, and PRDX3), increased phosphorylation of JNK, a stress-responsive kinase, and increased DNA damage markers (p-ATM and ?H2AX). Overexpression of NEK6 also increased the antioxidant defenses and decreased DNA damage. NEK6 lack induced apoptosis reduced Bcl-2 protein. DU-145 cells lacking NEK6 are more sensitive to cisplatin treatment. NEK6 modulated the nuclear localization of NF-?B2. Therefore, NEK6 alters redox balance, regulates antioxidant protein expression and DNA damage, and its absence induces DU-145 cell death. We observed that NEK6 inhibition sensitized DU-145 cells to docetaxel treatment, induced significant markers of apoptosis (PARP1 cleavage, BAK), reduced survival (phosphorylated p65) and cell migration (TJP1) pathways. The NEK6 inhibitor, individually, was able to significantly reduce Bcl-xL, phosphorylated Bad, and TJP1 8 expressions. The NEK6 inhibitor exhibited an attractive IC50 in DU-145 and PC-3 and could be an interesting compound to be explored in prostate cancer. Co-treatment with docetaxel and NEK6 inhibitor significantly reduced colony formation and cell migration. Knockout of NEK6 in DU-145 and PC-3 cells sensitizes the cells to docetaxel treatment. Resveratrol was also able to inhibit the expression of NEK6 and HK2. It was observed that NEK6 regulates the expression of the glycolytic enzyme HK2, connecting NEK6 with metabolic pathways in CRPC. It is suggested that the NEK6- HK2 signaling axis may be a possible resveratrol regulation mechanism. Conclusion: NEK6 may be a strategic target in prostate cancer and NEK6 inhibitors should be explored, as well as their use in combination with chemotherapy drugs (AU)

• FAPESP's process: 18/08391-2 - Functional and molecular studies of the human protein kinase Nek6 as a target candidate for drug design in Prostate Cancer
• Grantee: Isadora Carolina Betim Pavan
• Support Opportunities: Scholarships in Brazil - Doctorate