IDENTIFICATION OF PKC-IOTA AS POTENT BIOMARKER AGAINST HUMAN BREAST CANCER

Abstract

Breast cancer is one of the most widespread cancers and is the leading cause of fatalities in women around the globe with incidence and risk rate being elevated with age. The advancements in diagnosis and treatment have increased the overall chances of survival and the currently offered therapies for breast cancer are helpful but there are various limitations and considerable side effects. Emerging cases of drug resistance to the prevailing chemotherapy and other therapeutic options are great of concern. That is why, the utmost requirement is to find the appropriate biomarkers that could help in the early detection of cancer, as well in the design and selection of appropriate drug against the specific biomarker. Protein Kinase C iota (PKCɩ), a member of PKC family has an oncogenic role in various cancers and its SNPs, and expression have been reported to be linked with the development of cancer. Single nucleotide polymorphisms (SNPs) are found to have a significant role in the causation different types of cancers. Our research highlights the association of non-coding and coding SNPs of PKCɩ with various diseases including cancer computationally and the experimental validation of most deleterious SNPs, the assessment of expression of PKCɩ and related genes in breast cancer patients and the role of taxifolin in the treatment and management of breast cancer. The non-coding UTR SNPs in PKCɩ were assessed for involvement with miRNA and effect on transcription factors. The deleteriousness of missense variants on the structure and function of protein was assessed through nine web-based tools and molecular dynamic simulations. The association of most deleterious PB1 domain SNPs (G34W, F66W) was validated using Tetra primer ARMS PCR. The expression pattern of PKCɩ along with related genes was analyzed via real time PCR and Western blotting. The study also involves assessment of xxii drug likeness and binding affinities of taxifolin (drug) with protein PKCɩ by employing molecular docking, simulations and pharmacophore analysis. The results were validated on breast cancer cell lines (MDA-MD-231 and MCF-07) using various cell culture assays such as cell viability assay, colony formation migration assay, PRKCI expression was inspected in untreated and treated cell lines and a comparison of metabolites was performed using Gas Chromatography Mass Spectroscopy (GC-MS). From Insilico analysis of non coding SNPs, the SNPs from 3ʹ UTR region, rs115170199 from 5ʹ UTR region 750297755, rs968409340 were destabilizing secondary structure of mRNA with substantial ∆G. The 5ʹ UTR variant 750297755 was studied to cause maximum impact on PRKCI transcription factor binding sites. Nine variants were predicted to manifest noteworthy alteration in the structural and functional dynamics of PRKCI, which can endorse its malfunctioning and can lead towards various diseases. In the experimental validation, significant association of SNP G34W that resides in the PB1 domain of PKCɩ with breast cancer was found. The general expression levels of PKCɩ, TPD52 AKT, VEGF, SOC3, and HIF1α were analyzed to be elevated in breast patients when compared to control subjects. In assessment of drug on breast cancer cell lines, the decline in number of viable cells, migrating capability and colony formation in a dose-dependent approach was observed. The IC50 values of taxifolin was found to be 300nM and 180nm for MCF and MDA-MB-231 respectively. The PRKCI was downregulated in cell lines after taxifolin treatment. GC-MS analysis revealed that the drug is interrupting EGFR and FAK signaling pathway mainly in MCF 07 while G-coupled protein receptor signaling in MDA-MB-231 cell lines. The findings of the study suggest that PKCɩ can possibly serve as potential biomarkers for diagnosis, prognosis and therapeutic purposes of breast cancer.