Pilot and feasibility study: prospective proteomic profiling of mammary epithelial cells from high-risk women provides evidence of activation of pro-survival pathways

Abstract

Normal mammary gland homeostasis requires the coordinated regulation of protein signaling networks. However, we have little prospective information on whether activation of protein signaling occurs in premalignant mammary epithelial cells, as represented by cells with cytological atypia from women who are at high risk for breast cancer. This information is critical for understanding the role of deregulated signaling pathways in the initiation of breast cancer and for developing targeted prevention and/or treatment strategies for breast cancer in the future. In this pilot and feasibility study, we examined the expression of 52 phosphorylated, total, and cleaved proteins in 31 microdissected Random Periareolar Fine Needle Aspiration (RPFNA) samples by high-throughput Reverse Phase Protein Microarray. Unsupervised hierarchical clustering analysis indicated the presence of four clusters of proteins that represent the following signaling pathways: (1) receptor tyrosine kinase/Akt/mammalian target of rapamycin (RTK/Akt/mTOR), (2) RTK/Akt/extracellular signal-regulated kinase (RTK/Akt/ERK), (3) mitochondrial apoptosis, and (4) indeterminate. Clusters 1 through 3 comprised moderately to highly expressed proteins, while Cluster 4 comprised proteins that are lowly expressed in a majority of RPFNA samples. Our exploratory study showed that the interlinked components of mitochondrial apoptosis pathway are highly expressed in all mammary epithelial cells obtained from high-risk women. In particular, the expression levels of anti-apoptotic Bcl-xL and pro-apoptotic Bad are positively correlated in both non-atypical and atypical samples (unadjusted P < 0.0001), suggesting a delicate balance between the pro-apoptotic and anti-apoptotic regulation of cell proliferation during the early steps of mammary carcinogenesis. Our feasibility study suggests that the activation of key proteins along the RTK/Akt pathway may tip this balance to cell survival. Taken together, our results demonstrate the feasibility of mapping proteomic signaling networks in limited RPFNA samples obtained from high-risk women and the promise of developing rational drug targets or preventative strategies for breast cancer in future proteomic studies with a larger cohort of high-risk women.

Elevated leptin disrupts epithelial polarity and promotes premalignant alterations in the mammary gland

“…However, leptin’s oncogenic effects in the breast are likely determined by the local interstitial concentration of the adipokine in the mammary epithelial microenvironment, rather than circulating levels. We therefore quantified breast tissue levels from a panel of adipokines, cytokines, and growth factors in surgical excision specimens from women at high risk for breast cancer [26] (Table 1). We did not detect changes in levels of adiponectin, insulin, MCP1, and IL-8, previously reported to be associated with obesity [9, 27, 28].…”

Rapid Estrogen Signaling Negatively Regulates PTEN Activity Through Phosphorylation in Endometrial Cancer Cells

“…These data support observations in the Pten hyper mice as a reduction in PTEN correlated with an increase in phospho-AKT (S473) and Ki-67 in mammory tumors [3]. Additionally, pilot studies using microdissected epithelial cells from random periareolar fine needle aspiration samples have shown that women at risk for breast cancer, have elevated levels of phosphorylated PTEN (S380), total PTEN and active phospho-AKT (S473) [20]. These studies suggest that E2 regulation of PTEN may be an important mechanism in a number of steroid responsive tissues.…”

Tumor mechanics and metabolic dysfunction

“…Given the key role that matrix stiffness plays in regulating PI3K/AKT-signaling, these studies also provide evidence for the convergence of mechanosignaling and redox activation in aggressive cancers. Since AKT-network signaling is active in premalignant tissue from women at high-risk for breast cancer prior to the development of an invasive phenotype (Pilie, Ibarra-Drendall et al 2011; Ibarra-Drendall, Troch et al 2012), this opens the possibility that combination therapies which target both cellular metabolism and tissue stiffness may serve to more effectively halt disease progression.…”