[1726] Blockade of CCN6 Activates Growth Factor-Independent Survival of Breast Cells through the Phosphatidylinositol 3-Kinase/Akt Pathway

W Huang, ME Gonzalez, K Toy, CG Kleer. University of Michigan, Ann Arbor, MI

Background: CCN6 is a secreted cysteine rich matricellular protein (36.9 kDa) that modulates IGF-1 signaling in breast cells and exerts tumor suppressor functions in inflammatory breast cancers. CCN6 loss has been associated significantly with decreased breast cancer cell growth and proliferation, and with the development of metastasis. However, the details of the survival mechanism remain undefined.
Design: We developed shRNA knockdown of CCN6 using lentiviral vectors in two benign human mammary epithelial cells: HME and MCF10A. We studied cell proliferation in complete medium and under serum starvation by WST-1 and trypan blue assays. Apoptosis was studied using flow cytometry. Protein and mRNA expression were measured by Western blot and real time PCR. Anchorage independent growth was evaluated using soft agar, and anoikis (detachment-induced cell death) was investigated using poly-HEMA assays.
Results: We found that CCN6 blockade protects breast cells from apoptosis and activates growth factor-independent survival. In the absence of serum, CCN6 knockdown allowed MCF10A and HME cells to grow in anchorage independent conditions in soft agar and protected the cells from anoikis. Upon serum starvation, CCN6 knockdown in MCF10A and HME cells induced Akt phosphorylation at serine 473. Both cell survival and Akt phosphorylation were stunted in CCN6 knockdown cells when treated with either recombinant human CCN6 protein or the phosphatidylinositol 3-kinase inhibitor LY294002. We further identified that the survival and apoptotic effects of CCN6 knockdown were reverted by specific shRNA downregulation of Akt-1.
Conclusions: Our data show that CCN6 knockdown promotes anchorage independent growth, proliferation and resistance to detachment induced cell death (anoikis), all features of malignant conversion. Furthermore, we pinpoint one mechanism by which CCN6 controls survival of breast cells, implicating the PI3K/Akt-1 pathway.
Category: Pathobiology

Wednesday, March 24, 2010 9:30 AM

Poster Session V # 233, Wednesday Morning

 

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