Role of IGF-1 in the CCN6-dependent regulation of ZEB1 in benign and breast cancer cells

CABANILLAS, ANA MARIA; LORENZATTI, GUADALUPE; PAL, A; KLEER, CELINA

Ann Arbor, MI. USA.

Simposio; University of Michigan Cancer Center Fall Research Symposium; 2009

UNIVERSITY OF MICHIGAN COMPREHENSIVE CANCER CENTER

Background. Our lab has shown that CCN6 has tumor inhibitory functions in breast cancer and functions as a negative modulator of the insulin-like growth factor 1 (IGF-1) signaling pathway. CCN6 knockdown in breast epithelial cells triggers an EMT (epithelial to mesenchymal transition) and promotes invasion through up-regulation of the transcription factor ZEB1 and down-regulation of E-cadherin. It has been shown that IGF-1 can up-regulate ZEB1 in a prostate cancer cell line. We hypothesize that CCN6 regulates ZEB1 expression and EMT in breast epithelial cells through activation of IGF-1 signaling. Methods. HME cells with short hairpin inhibition of CCN6 in a lentivirus vector (HME/shCCN6) and HME/shControl cells, and MDA-MB-231 breast cancer cells were employed. Protein and mRNA expressions were assessed by quantitative RT-PCR and Western blots. Results. HME/shCCN6 cells exhibited up-regulation of ZEB1 and IGF-1 m RNA and protein expression. We found that HME/shCCN6 cells grown in serum free conditions secrete IGF-1 protein into the conditioned media at higher levels than MDA-MB-231 breast cancer cells. This was accompanied by activation of IGF-1 signaling pathway activation evidenced by increased phosphorylation of IGF-IR and IRS1.  Treatment of HME/shCCN6 cells with 500 ng/mL of human recombinant CCN6 protein (rhCCN6) reverted the effect of CCN6 knockdown on ZEB1 and IGF-1 transcript and protein levels, and rescued the activation of IGF-1R and IRS1. rhCCN6 treated cells showed downregulation of the mesenchymal marker vimentin (WB and IF) and decreased invasive abilities. Conclusions. Under serum starvation, CCN6 knockdown in HME cells induces the secretion of IGF-1 which leads to activation of IGF-1R and IRS1.  Our results suggest that CCN6 may regulate ZEB1- dependent EMT and cancer invasion through IGF-1 signaling in breast epithelial cells. Background. Our lab has shown that CCN6 has tumor inhibitory functions in breast cancer and functions as a negative modulator of the insulin-like growth factor 1 (IGF-1) signaling pathway. CCN6 knockdown in breast epithelial cells triggers an EMT (epithelial to mesenchymal transition) and promotes invasion through up-regulation of the transcription factor ZEB1 and down-regulation of E-cadherin. It has been shown that IGF-1 can up-regulate ZEB1 in a prostate cancer cell line. We hypothesize that CCN6 regulates ZEB1 expression and EMT in breast epithelial cells through activation of IGF-1 signaling. Methods. HME cells with short hairpin inhibition of CCN6 in a lentivirus vector (HME/shCCN6) and HME/shControl cells, and MDA-MB-231 breast cancer cells were employed. Protein and mRNA expressions were assessed by quantitative RT-PCR and Western blots. Results. HME/shCCN6 cells exhibited up-regulation of ZEB1 and IGF-1 m RNA and protein expression. We found that HME/shCCN6 cells grown in serum free conditions secrete IGF-1 protein into the conditioned media at higher levels than MDA-MB-231 breast cancer cells. This was accompanied by activation of IGF-1 signaling pathway activation evidenced by increased phosphorylation of IGF-IR and IRS1.  Treatment of HME/shCCN6 cells with 500 ng/mL of human recombinant CCN6 protein (rhCCN6) reverted the effect of CCN6 knockdown on ZEB1 and IGF-1 transcript and protein levels, and rescued the activation of IGF-1R and IRS1. rhCCN6 treated cells showed downregulation of the mesenchymal marker vimentin (WB and IF) and decreased invasive abilities. Conclusions. Under serum starvation, CCN6 knockdown in HME cells induces the secretion of IGF-1 which leads to activation of IGF-1R and IRS1.  Our results suggest that CCN6 may regulate ZEB1- dependent EMT and cancer invasion through IGF-1 signaling in breast epithelial cells.