Recently, we reported an association of sperm-associated antigen 9 (SPAG9) expression, a new member of CT antigen family, in various types of cancers [9]. Using plasmid-based small interfering RNA (siRNA) approach to knockdown SPAG9, find more we demonstrated significant reduction in cellular proliferation, colony forming ability, cellular migration, invasion and wound healing capacity in different types of cancers [11–13]. Interestingly, we also demonstrated an association of SPAG9 immuno-reactivity score (IRS) in early grades of breast AZD6244 datasheet cancer patients. In addition, 88% breast cancer

specimens showed SPAG9 expression independent of tumor stages and grades [14]. Collectively, our data suggested that SPAG9 could be playing a potential role

in various malignant properties of breast tumorigenesis. In the present study, we investigated the SPAG9 expression Selleckchem JNJ-64619178 in different breast cancer cell line models of different hormone receptor status and different subtypes. Further, involvement of SPAG9 was investigated for various malignant properties in triple-negative MDA-MB-231 cells, employing siRNA approach. Our data revealed that SPAG9 mRNA and protein expression was detected in all breast cancer cells. In addition, relative qPCR data demonstrated 20 to 52 folds higher expression of SPAG9 mRNA in MCF-7, MDA-MB-231, BT-474 and SK-BR-3 breast cancer cells as compared to normal mammary epithelial cells. SPAG9 was also shown to be anchored on the plasma membrane of breast cancer cells. Employing gene silencing approach, knockdown of SPAG9 gene revealed that SPAG9 plays an important role in cellular proliferation, colony forming ability, migration and invasion. Furthermore,

in vivo breast xenograft studies in nude mice revealed that Bumetanide SPAG9 siRNA plasmid injected mice showed significant reduction in tumor growth. Collectively, our data has laid foundation for SPAG9 to be used as a potential therapeutic target for triple-negative breast cancer. Material and method Breast cancer cell lines Four breast cancer cell lines of various subtypes, harboring different hormone receptors, such as MCF-7 (luminal-A, ER+ PR+ Her2-), BT-474 (luminal-B, ER+ PR+ Her2+), SK-BR-3 (HER2 overexpressing, ER- PR- Her2+) and MDA-MB-231 (highly metastatic basal, triple-negative ER- PR- Her2-) were used in the study and were procured from American Type Culture Collection (ATCC, Manassas, VA). All the cells were cultured in recommended medium under standard conditions. Human normal mammary epithelial cells were purchased and maintained according to manufacturer’s directions (Gibco, Life Technologies Corporation, Carlbad, CA).