Sperm associated antigen 9 (SPAG9) a promising therapeutic target of ovarian carcinoma.

SPAG9 is a novel tumor associated antigen, expressed in variety of malignancies. However, its role in ovarian cancer remains unexplored. SPAG9 expression was validated in ovarian cancer cells by real time PCR and Western blot. SPAG9 involvement in cell cycle, DNA damage, apoptosis, paclitaxel sensitivity and epithelial- mesenchymal transition (EMT) was investigated employing RNA interference approach. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro. Quantitative PCR and Western blot analysis revealed SPAG9 expression in A10, SKOV-3 and Caov3 compared to normal ovarian epithelial cells. SPAG9 ablation resulted in reduced cellular proliferation, colony forming ability and enhanced cytotoxicity of chemotherapeutic agent paclitaxel. Effect of ablation of SPAG9 on cell cycle revealed S phase arrest and showed decreased expression of CDK1, CDK2, CDK4, CDK6, cyclin B1, cyclin D1, cyclin E and increased expression of tumor suppressor p21. Ablation of SPAG9 also resulted in increased apoptosis with increased expression of various pro- apoptotic molecules including BAD, BID, PUMA, caspase 3, caspase 7, caspase 8 and cytochrome C. Decreased expression of mesenchymal markers and increased expression of epithelial markers was found in SPAG9 ablated cells. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro assays which showed that ablation of SPAG9 resulted in increased paclitaxel sensitivity and caused enhanced cell death. In vivo ovarian cancer xenograft studies showed that ablation of SPAG9 resulted in significant reduction in tumor growth. Present study revealed therapeutic potential of SPAG9 in ovarian cancer.

Heat shock protein 70-2 (HSP70-2) overexpression in breast cancer.

BACKGROUND: Breast cancer is one of the leading cause of cancer-related deaths in women worldwide and increasing rapidly in developing countries. In the present study, we investigated the potential role and association of HSP70-2 with breast cancer. METHODS: HSP70-2 expression was examined in 154 tumor and 103 adjacent non-cancerous tissue (ANCT) specimens and breast cancer cell lines (MCF7, BT-474, SK-BR-3 and MDA-MB-231) by RT-PCR, quantitative-PCR, immunohistochemistry, Western blotting, flow cytometry and indirect immunofluorescence. Plasmid driven short hairpin RNA approach was employed to validate the role of HSP70-2 in cellular proliferation, senescence, migration, invasion and tumor growth. Further, we studied the effect of HSP70-2 protein ablation on signaling cascades involved in apoptosis, cell cycle and Epithelial-Mesenchymal-Transition both in culture as well as in-vivo human breast xenograft mouse model. RESULTS: HSP70-2 expression was detected in majority of breast cancer patients (83 %) irrespective of various histotypes, stages and grades. HSP70-2 expression was also observed in all breast cancer cells (BT-474, MCF7, MDA-MB-231 and SK-BR-3) used in this study. Depletion of HSP70-2 in MDA-MB-231 and MCF7 cells resulted in a significant reduction in cellular growth, motility, onset of apoptosis, senescence, cell cycle arrest as well as reduction of tumor growth in the xenograft model. At molecular level, down-regulation of HSP70-2 resulted in reduced expression of cyclins, cyclin dependent kinases, anti-apoptotic molecules and mesenchymal markers and enhanced expression of CDK inhibitors, caspases, pro-apoptotic molecules and epithelial markers. CONCLUSIONS: HSP70-2 is over expressed in breast cancer patients and was involved in malignant properties of breast cancer. This suggests HSP70-2 may be potential candidate molecule for development of better breast cancer treatment.

Sperm-associated antigen 9 (SPAG9) promotes the survival and tumor growth of triple-negative breast cancer cells.

Recently, we demonstrated the association of sperm-associated antigen 9 (SPAG9) expression with breast cancer. Among breast cancer, 15 % of the cancers are diagnosed as triple-negative breast cancers (TNBC) based on hormone receptor status and represent an important clinical challenge because of lack of effective available targeted therapy. Therefore, in the present investigation, plasmid-based small hairpin (small hairpin RNA (shRNA)) approach was used to ablate SPAG9 in aggressive breast cancer cell line model (MDA-MB-231) in order to understand the role of SPAG9 at molecular level in apoptosis, cell cycle, and epithelial-to-mesenchymal transition (EMT) signaling. Our data in MDA-MB-231 cells showed that ablation of SPAG9 resulted in membrane blebbing, increased mitochondrial membrane potential, DNA fragmentation, phosphatidyl serine surface expression, and caspase activation. SPAG9 depletion also resulted in cell cycle arrest in G0-G1 phase and induced cellular senescence. In addition, in in vitro and in vivo xenograft studies, ablation of SPAG9 resulted in upregulation of p21 along with pro-apoptotic molecules such as BAK, BAX, BIM, BID, NOXA, AIF, Cyto-C, PARP1, APAF1, Caspase 3, and Caspase 9 and epithelial marker, E-cadherin. Also, SPAG9-depleted cells showed downregulation of cyclin B1, cyclin D1, cyclin E, CDK1, CDK4, CDK6, BCL2, Bcl-xL, XIAP, cIAP2, MCL1, GRP78, SLUG, SNAIL, TWIST, vimentin, N-cadherin, MMP2, MMP3, MMP9, SMA, and ß-catenin. Collectively, our data suggests that SPAG9 promotes tumor growth by inhibiting apoptosis, altering cell cycle, and enhancing EMT signaling in in vitro cells and in vivo mouse model. Hence, SPAG9 may be a potential novel target for therapeutic use in TNBC treatment.

A novel cancer testis antigen target A-kinase anchor protein (AKAP4) for the early diagnosis and immunotherapy of colon cancer.

Colorectal cancer (CRC) is mainly a disease of developed countries and a major cause of death worldwide. The present study was undertaken to investigate the association of novel cancer testis (CT) antigen, A-kinase anchor protein (AKAP4) with CRC. AKAP4 gene and protein was examined by RT-PCR, in situ hybridization and immunohistochemistry (IHC) in 200 clinical specimens of different stages and grades. In addition, humoral response against AKAP4 was detected by enzyme-linked immunosorbent assay and Western blotting in 172 available sera samples of CRC patients. We observed that majority of CRC patients demonstrated AKAP4 expression and elicited immune response. AKAP4 protein expression, based on immunoreactivity score (IRS) predicted presence of CRC with 84% sensitivity, 100% specificity, 100% of positive predictive value (PPV) and 83.33% negative predictive value (NPV). Humoral response against AKAP4 protein was generated in 82% of the CRC patients. Further, statistical analysis revealed that antibodies found against AKAP4 in CRC patients predicted presence of malignancy with 81.98% sensitivity, 100% specificity, 100% PPV, and 63.53% NPV. Collectively, our data suggests that the majority of CRC cases show significant difference of AKAP4 expression among stages and grades and also generated antibodies against AKAP4 protein. Therefore, AKAP4 may be potential candidate molecule for developing as a biomarker for early diagnosis and immunotherapy of CRC.

A-kinase anchor protein 4 (AKAP4) a promising therapeutic target of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) ranks third among the estimated cancer cases and cancer related mortalities in the Western world. Early detection and efficient therapy of CRC remains a major health challenge. Therefore, there is a need to identify novel tumor markers for early diagnosis and treatment of CRC. METHODS: A-kinase anchor protein 4 (AKAP4) gene and protein expression was monitored by quantitative polymerase chain reaction (qPCR), reverse transcription (RT)-PCR and Western blotting in normal colon tissue lysate, normal colon epithelial cells and in colon cancer cell lines viz., Caco-2, COLO205, COLO320DM, HCT-15, HCT116, HT-29, SW480, and SW620. The effect of AKAP4 on cellular growth, migration and invasion abilities was studied using gene silencing approach. The role of AKAP4 in various pathways involved in cell cycle, apoptosis, senescence was investigated in in vitro and in human xenograft mouse model. RESULTS: Our studies showed that AKAP4 gene and protein expression was expressed in all colon cancer cells while no expression was detectable in normal colon cells. Ablation of AKAP4 led to reduced cellular growth, migration, invasion and increased apoptosis and senescence of CRC cells in in vitro assays and tumor growth in human xenograft mouse. Human colon xenograft studies showed a significant decrease in the levels of cyclins B1, D and E and cyclin dependent kinases such as CDK1, CDK2, CDK4 and CDK6. Interestingly, an up-regulation in the levels of p16 and p21 was also observed. Besides, an increase in the levels of pro-apoptotic molecules AIF, APAF1, BAD, BID, BAK, BAX, PARP1, NOXA, PUMA and cyt-C and Caspase 3, 7, 8 and 9 was also found in cancer cells as well as in xenograft tissue sections. However, anti-apoptotic molecules BCL2, Bcl-xL, cIAP2, XIAP, Axin2 and Survivin were down regulated in these samples. Our data also revealed elevated expression of epithelial marker E-cadherin and down regulation of EMT markers N-cadherin, P-cadherin, SLUG, α-SMA, SNAIL, TWIST and Vimentin. Further ablation of AKAP4 resulted in the down regulation of invasion molecules matrix metalloproteinase MMP2, MMP3 and MMP9. CONCLUSION: AKAP4 appears to be a novel CRC-associated antigen with a potential for developing as a new clinical therapeutic target.