Development of 3D model and understanding translational regulation of breast cancer

Abstract

Breast cancer is the most common cancer among women in India and worldwide. It is a highly heterogeneous disease caused by several genetic and epigenetic alterations in mammary epithelial cells. Both inter- and intra-patient heterogeneity exists at various levels including genetic, cellular, and physiological. Due to this heterogeneity, each patient’s response to anti-neoplastic drugs varies. A major reason behind this is the use of essentially homogenous cancer cell lines in the initial drug screening process that does not adequately capture the heterogeneity. Therefore, it is important to develop an in vitro cell culture model that mimics breast tumour microenvironment. Our laboratory recently developed a 3D porous polycaprolactone (PCL) scaffold that better mimics breast tumour microenvironment and validated the same using metastatic breast cancer cell line MDA-MB-231. The current study aimed to further develop this scaffold-based culture system for the growth of patient-derived cancer cells. Multiple tissue processing strategies that perturb the tumour tissue microenvironment to varying levels, and deposition of extracellular matrix, was employed. These studies led to the development of a personalized 3D culture model for patient-derived breast cancer cells that is also amenable for screening drugs. Normal epithelial cells die by apoptosis upon detachment from the extra-cellular matrix, which is known as anoikis. However, cancer cells of solid tumours acquire anoikis resistance – one of the pre-requisite features for successful metastasis. Most of the cancer-related deaths are due to the metastasis of cancer cells to other organs via circulation. Our laboratory has identified AMP-activated protein kinase (AMPK) as a key player in the acquisition of anoikis resistance; yet, its downstream actions are largely unknown. Once active, AMPK brings about energy homeostasis by inhibiting energy-consuming pathways and promoting energy-producing pathways. Protein synthesis is a major energy-consuming pathway that is inhibited by AMPK. In a mass-spectrometric analysis of the phospho-proteome of matrix-deprived breast cancer cells, a novel AMPK-dependent phosphorylation of EIF4GII (Eukaryotic translation initiation factor 4 gamma II) on Serine 889 was identified, suggesting possible regulation of mRNA translation initiation. Immunohistochemical analysis revealed elevated EIF4GII protein expression in breast cancer tissue compared to adjacent normal tissue. Site-directed mutagenesis of EIF4GII on Serine 889 was conducted to create phospho-dead (S889A) and phospho-mimetic (S889E) mutants to study the effect on protein translation. Additionally, analysis of EIF4GII-interactome in matrix-deprived cells revealed reduced interaction with multiple cellular phosphatases. Specifically, DUSP6 (dual-specific phosphatase-6) interaction with EIF4GII was compromised. Inhibition of AMPK restored the interaction of DUSP6 with EIF4GII. Taken together, these data begin to suggest a novel role for AMPK in anoikis resistance via regulating the initiation of mRNA translation.