Serrano, Samuel

AbstractBackgroundThe E‐cadherin, α‐ and β‐Catenin interaction at the cell adherens junction plays a key role in cell adhesion; alteration in the expression and function of these genes are associated with disease progression in several solid tumors including prostate cancer. The membranous β‐Catenin is dynamically linked to the cellular cytoskeleton through interaction with α‐Catenin at amino acid positions threonine 120 (T120) to 151 of β‐Catenin. Nuclear presence of α‐Catenin modulates the sensitivity of cells to DNA damage. The objective of this study is to determine the role of α‐Catenin and protein kinase D1 (PrKD1) in DNA damage response.MethodsProstate cancer cells; LNCaP, LNCaP (Sh‐PrKD1; silenced PrKD1), C4‐2 and C4‐2 PrKD1 were used for various sets of experiments to determine the role of DNA damage in PrKD1 overexpression and silencing cells. These cells were treated with compound‐10 (100 nM) and Etoposide (30 µM), total cell lysates, cytosolic and nuclear fractions were prepared to observe various protein expressions. We performed single cell gel electrophoresis (COMET assay) to determine the etoposide induce DNA damage in C4‐2 and C4‐2 PrKD1 cells. The animal experiments were carried out to determine the tolerability of compound‐10 by mice and generate preliminary data on efficacy of compound‐10 in modulating the α‐Catenin and PrKD1 expressions in inhibiting tumor progression.ResultsPrKD1, a novel serine threonine kinase, phosphorylates β‐Catenin T120. In silico analysis, confirmed that T120 phosphorylation alters β‐ to α‐Catenin binding. Forced expression of PrKD1 in prostate cancer cells increased β‐ and α‐Catenin protein levels associated with reduced etoposide induced DNA damage. Downregulation of α‐Catenin abrogates the PrKD1 mitigation of DNA damage. The in vitro results were corroborated in vivo using mouse prostate cancer patient derived xenograft model by inhibition of PrKD1 kinase activity with compound‐10, a selective PrKD inhibitor, demonstrating decreased total β‐ and α‐Catenin protein levels, and β‐Catenin T120 phosphorylation.ConclusionsAlteration in DNA damage response pathways play major role in prostate cancer progression. The study identifies a novel mechanism of α‐Catenin dependent DNA damage mitigation role for PrKD1 in prostate cancer.

Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of several genes including protooncogene cellular myelocytomatosis (c-Myc). c-Myc is difficult to target directly by agents due to its disordered alpha helical protein structure and predominant nuclear localization. The epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. MT-1 is a bivalent BD inhibitor that is 100-fold more potent than the first-in-class BD inhibitor JQ1. MT-1 decreased cell viability and causes cell cycle arrest in G0/G1 phase in castration-sensitive and resistant PC cell lines in a dose-dependent fashion. The inhibition of c-Myc function by MT-1 was molecularly corroborated by the de-repression of Protein Kinase D1 (PrKD) and increased phosphorylation of PrKD substrate proteins: threonine 120, serine 11, and serine 216 amino acid residues in β-Catenin, snail, and cell division cycle 25c (CDC25c) proteins, respectively. The treatment of 3D cell cultures derived from three unique clinically annotated heavily pretreated patient-derived PC xenografts (PDX) mice models with increasing doses of MT-1 demonstrated the lowest IC50 in tumors with c-Myc amplification and clinically resistant to Docetaxel, Cabazitaxel, Abiraterone, and Enzalutamide. An intraperitoneal injection of either MT-1 or in combination with 3jc48-3, an inhibitor of obligate heterodimerization with MYC-associated protein X (MAX), in mice implanted with orthotopic PC PDX, decreased tumor growth. This is the first pre-clinical study demonstrating potential utility of MT-1 in the treatment of PC with c-Myc dysregulation.