Molecular Characterization of the Tumor-Suppressive Function of Nischarin in Breast Cancer

Abstract

Nischarin (encoded by NISCH), an α5 integrin-binding protein, has been identified as a regulator of breast cancer cell invasion. We hypothesized that it might be a tumor suppressor and were interested in its regulation. We examined nischarin expression in approximately 300 human breast cancer and normal tissues using quantitative polymerase chain reaction and immunohistochemistry. Loss of heterozygosity analysis was performed by examining three microsatellite markers located near the NISCH locus in normal and tumor tissues. We generated derivatives of MDA-MB-231 human metastatic breast cancer cells that overexpressed nischarin and measured tumor growth from these cells as xenografts in mice; metastasis by these cells after tail vein injection; and α5 integrin expression, Rac, and focal adhesion kinase (FAK) signaling using western blotting. We also generated clones of MCF-7 human breast cancer cells in which nischarin expression was silenced and measured tumor growth in mouse xenograft models (n = 5 for all mouse experiments). P values were from two-sided Student t tests in pairwise comparisons. Normal human breast tissue samples had statistically significantly higher expression of nischarin mRNA compared with tumor tissue samples (mean level in normal breast = 50.7 [arbitrary units], in breast tumor = 16.49 [arbitrary units], difference = 34.21, 95% confidence interval [CI] = 11.63 to 56.79, P = .003), and loss of heterozygosity was associated with loss of nischarin expression. MDA-MB-231 cells in which nischarin was overexpressed had statistically significantly reduced tumor growth and metastasis compared with parental MDA-MB-231 cells (mean volume at day 40, control vs nischarin-expressing tumors, 1977 vs 42.27 mm(3), difference = 1935 mm(3), 95% CI = 395 to 3475 mm(3), P = .025). Moreover, MCF-7 tumor xenografts in which nischarin expression was silenced grew statistically significantly faster than parental cells (mean volume at day 63, tumors with scrambled short hairpin RNA [shRNA] vs with nischarin shRNA, 224 vs 1262 mm(3), difference = 1038 mm(3), 95% CI = 899.6 to 1176 mm(3), P < .001). Overexpression of nischarin was associated with decreased α5 integrin expression, FAK phosphorylation, and Rac activation. Nischarin may be a novel tumor suppressor that limits breast cancer progression by regulating α5 integrin expression and subsequently α5 integrin-, FAK-, and Rac-mediated signaling.