Abstract A68: Integrin α3β1-dependent regulation of Cox2 in human breast cancer

Abstract

Abstract During the process of gene transcription, inclusion or exclusion of exons can have profound effects on the stability of messenger RNA and affect many aspects of tumorigenesis such as growth, migration and invasion. Changes in cell adhesion are important during cancer progression and metastasis. In this process, integrins are implicated in tumor growth and cancer cell invasion from the primary tumor as well as tumor cell attachment at distant sites of metastasis. Integrins are heterodimeric cell surface receptors consisting of an α and a β subunit which interact with the extracellular matrix (ECM) and mediate inside-out and outside-in signaling. Integrin α3β1 is expressed in a variety of different cell types and its expression is enhanced in breast cancer. Experiments performed with breast cancer cell lines suggests that laminin-332, which is a major ligand for α3β1, is secreted from mammary epithelial cells and enhances the motility and aggressive features of breast cancer cells indicating the importance of α3β1 signaling in invasion. Using MDA-MB-231 breast cancer cell line, which was stably transduced with lentivirus encoding either control shRNA or α3-targeting shRNA, we performed microarray analysis using the Affymetrix GeneChip® Human Exon 1.0 ST Array platform. We identified 72 genes that were upregulated at least 2-fold and 329 genes that were downregulated at least 2-fold, when α3β1 was suppressed. Cox2 mRNA expression levels as well as differential exon inclusion in the mRNA was affected by the presence or absence of α3β1. We were able to identify and validate differential exon inclusion in the mRNA of Cox2 using RT-PCR and real time PCR assays. In addition, stability of Cox2 mRNA was shown to be regulated by α3β1 expression in a mRNA decay assay using RT-PCR as an end-point readout. The presence of differing exons as well as extended 3'-UTR has been shown to confer Cox2 mRNA susceptibility to nonsense-mediated mRNA decay (NMD). Knocking down UPF1, a key regulator of NMD, stabilized Cox2 mRNA in an α3β1-dependent manner as determined by both RT-PCR and RNAse protection assays. In addition, the NMD pathway appears to be repressed in the presence of α3β1 as determined by phosphorylation status of eIF2α. Taken together, α3β1 is a key factor that determines the splice variant of Cox2 mRNA that is generated in MDA-MB-231 cells. It also affects the process of NMD thereby affecting Cox2 mRNA turnover and protein expression thus affecting angiogenesis and tumor progression. Understanding the role that α3β1 plays in various molecular events leading to differential exon usage and mRNA stability of specific breast cancer genes such as Cox2 could be vital to the development of therapies to target metastatic breast cancer. Citation Format: Sita Subbaram, Kimberly K. Svenson, Sean L. Hammond, Lorena Craig, Sridar V. Chittur, C. Michael DiPersio. Integrin α3β1-dependent regulation of Cox2 in human breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A68.