Knockdown of IGF‐IR by siRNA Mechanisms: Effect on Post‐ confluent Growth and Cellular Endpoints in MCF‐7 Cells

Abstract

Breast cancer is the most common cancer among women worldwide. 17β-estradiol (E2) plays a major role in normal breast growth and development, and is implicated in breast cancer. High circulating insulin-like growth factor (IGF) levels are also linked to increased breast cancer risk. Data indicate interaction between the estrogen receptor (ER)-and IGF-signaling pathways culminating in a synergistic increase in proliferation. Foci are an in vitro cancer phenotype. We previously established that E2 exposure causes the development of multilayered cellular aggregates (foci) in MCF-7 cell cultures which occurs only after a confluent monolayer has been established, and is inhibited by co-exposure to nontoxic concentrations of antiestrogens. The necessity of IGF-IR and its signaling pathways for formation of post-confluent foci was determined through generation of stable IGF-IR siRNA clones. Expression constructs for siRNA against IGF-IR were stably transfected into MCF-7 cells and clones screened for IGF-IR knockdown. Clones with substantially reduced IGF-IR levels also showed ER loss, although E2 still positively regulated IGF-IR. IGF-IR knockdown significantly reduced foci numbers; this was not reversed by adding IGF. The studies indicate the integral relationship between ER and IGF-IR. Gene array analysis indicated changes in both expected and novel cellular processes and pathways. These included alterations in pathways relating to apoptisis and steroid metabolism. Current studies address the molecular basis underlying the changes in the physiological endpoint, focal growth.