Metastasis from breast cancer remains the major stumbling block in disease control: Specific treatment is limited. We have characterized a novel gene/protein that plays a critical role in breast cancer dissemination. Using a PCR Subtraction Hybridization approach, we have cloned a gene encoding a 150 kDa protein in concanavalin A-stimulated human HT1080 fibrosarcoma cells. Based on upregulation in cancer and enhanced cell migration/invasion, we have named this gene, Cancer-Related Invasive Gene (CRIG). CRIG is identical to the KIAA1199 gene reported in the HUGE database. CRIG/KIAA1199 has been reported to be essential in development of auditory function since mutations are associated with non-syndromic hearing loss. Employing dot blot analysis of the human mRNA master blot, we demonstrated that CRIG is expressed in the central nervous system including brain and spinal cord; limited expression in other normal tissues was noted. Transfection of COS-1 cells (a monkey kidney epithelial cell line) or weakly aggressive human MCF-7 breast cancer cells with CRIG-GFP chimeric cDNA followed by fluorescence microscopy resulted in localization of CRIG to the endoplasmic reticulum (ER); endogenous localization in the ER was confirmed using specific antibodies. By mining DNA microarray databases at Gene Expression Omnibus (NCBI) and Oncomine (Cancer Profiling Database), we identified upregulation of CRIG in human breast cancer tissues; correlation with cancer grade, stage, recurrence, HER-2, and estrogen receptor negative status was noted. The CRIG gene is highly expressed in aggressive, estrogen independent human breast cancer cell lines (MDA-MB-435 and MDA-MB-231), but not in estrogen-dependent cancer cells (MCF-7). Overexpression of CRIG by cDNA transfection into non malignant COS-1 cells or MCF-7 breast cancer cells enhanced cell migration. Down-regulation of CRIG expression in MDA-MB-435 breast cancer cells using short hairpin RNA (shRNA) resulted in: 1) marked inhibition of cancer cell invasion in 3 dimensional type I collagen, 2) morphologic transition from fibroblast-like to epithelial-like cells (mesenchymal-to-epithelial transition), and 3) no effect on cell doubling time in vitro. These data support a cell invasion-dependent, but cell proliferation-independent effect of CRIG in cancer. Tumor growth and spontaneous metastasis to the lungs were markedly diminished (>90%) by shRNA knockdown of CRIG in MDA-MB-435 cancer cells orthotopically transplanted into the mammary fat pad of immunodeficient mice: Correlation between degree of CRIG mRNA down-regulation and both size of primary tumor and number of metastases was demonstrated. These data establish a unique role for CRIG in breast cancer progression. The low expression of CRIG in most normal tissues and critical function in breast cancer, suggest that CRIG is a suitable therapeutic target.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2227.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO