Effect of EBV-LMP1 mutations (24bp and 30bp) on the frequency of PIK3CA mutation with and without PI3K-inhibitors

Abstract

The study was designed to elucidate the presence of Epstein –Bar virus (EBV) in invasive ductal carcinoma (IDC) by detection of latent membrane protein 1(EBV-LMP1) by conventional polymerase chain reaction (PCR). The ability of wild type EBV-LMP1 and its isolated mutations to the disrupt the signaling pathway of phosphoinositol 3 kinase catalytic subunit A (PI3KCA). One hundred fifty-six paraffin-fixed embedded blocks of breast tumor samples which were taken from women in Basrah province were included in the study; 84 samples were benign of fibroadenoma and 72 were malignant of invasive ductal carcinoma.

EBV-LMP1 and PI3KCA DNA sequencing was confirmed with polymorphisms and two EBV-LMP1 mutations; deletion of 30bp at (213-243) and 24bp at (544-568) were successfully cloned in peGFP-C1 plasmid. Immunoprecipitation assay revealed the interaction of wild type LMP1 and its mutant 24bp deletion with PI3KCA through transfection of mammalian MCF7 cell line. On the contrary, LMP1 30bp deletion was failed to interact with PI3KCA in immunoprecipitation assay and decreased the acceleration time of proliferation in wound healing assay, which may indicate the importance of this deletion in linking with PI3KCA and activation of cell proliferation. Immunoprecipitation and wound healing assay were repeated with the presence of alpelisib drug as PI3KCA inhibitor. The result confirmed that these 30bp deletion was important to interact with PI3KCA and during inhibition of PI3KCA, the EBV-LMP1-30bp was failed to bind to PI3KCA and lose the ability of LMP1 to accelerate the artificial wound healing in MCF7 cell culture. These finding stress the importance of those 30bp deletions, which may require to stop proliferation and their role in PI3KCA mutations that detected in this study in association with presence of EBV-LMP1. PI3KCA mutations of E542K or H1047R exhibited more aggressive phenotype in breast carcinoma that could assist the constitutive activation of PI3K and cell growth, especially when coupled to EBV-LMP1 oncogenes.

In conclusion, the large variability of PI3K signaling pathway and several EBV-LMP1 mutations may contribute in the emergence of therapeutic resistance, disease relapses and activation of cells proliferation.