Investigating the Phosphoinositide 3-Kinase (PI3K) Pathway for Therapeutic Strategies for Breast Cancer

Abstract

Despite major advances in our understanding of the etiology of breast cancer, it remains a leading cause of cancer death in women worldwide. This warrants the search for novel alternatives for diagnosis and therapeutic intervention. The phosphoinositide 3-kinase (PI3K) pathway regulates all aspects of breast cancer development, from initiation to metastatic dissemination. This is underscored by the high prevalence of somatic mutations in PIK3CA, the gene encoding the catalytic subunit of PI3K, in breast cancer. Although targeting the PI3K pathway is a viable therapeutic approach, many PI3K pathway inhibitors have yet to show significant clinical efficacy. Here we examine the potential chemotherapeutic benefit of aspirin in PI3K-driven breast cancer. We demonstrate that mutant PIK3CA breast cancer cells show a dose-dependent decrease in cell viability and anchorage-independent growth in soft agar, when treated with increasing concentrations of aspirin/salicylate. Co-treatment of aspirin sensitizes mutant PIK3CA breast cancer cells to PI3K inhibitors to enhance suppression of proliferation. Additionally, immortalized mammary epithelial cells expressing mutant PIK3CA H1047R show greater sensitivity to aspirin when compared to cells expressing wild-type PIK3CA grown in three-dimensional (3D) Matrigel culture. Mechanistic studies indicate that the growth inhibitory effect of aspirin are due to activation of AMP-activated protein kinase (AMPK), inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling and induction of autophagy. Importantly, this growth suppression is independent of the activities of cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB). We have also investigated the transcriptional responses to PI3K activation in breast cancer cells. By performing microarray and gene ontology analysis, we show that constitutive PI3K pathway activation preferentially affects the gene expression of cytokines, chemokines and other secreted factors. We identify a novel PI3K-regulated gene termed long Pentraxin 3 (PTX3), whose functional role in cancer is largely unknown. We demonstrate that the PI3K pathway regulates the expression of PTX3 in an NF-κB dependent manner. Using functional cell-based assays, we demonstrate a role for PTX3 in PI3K-driven proliferation and survival in two-dimensional (2D) monolayer and three-dimensional (3D) Matrigel culture. Additionally, we identify a novel large intergenic noncoding RNA (lincRNA) transcript termed c14orf34, which is upregulated in both mutant PIK3CA and Src-transformed MCF10A cells. By performing gene expression profiling, we show that this transcript is associated with basal-like breast cancer cells and genomically altered in a subset of invasive breast carcinomas from human patients. By employing ChIP-seq analysis, we demonstrate that c14orf34 is regulated by the transcription factor STAT3. Loss-of-function studies suggest a role for this lincRNA in cell invasion and migration. We also identify several genes that are differentially expressed upon overexpression of c14orf34 in MCF10A cells, indicative of a potential role in regulating global gene expression.