Renal cell carcinoma (RCC) represents one of the most resistant tumors to radiation and chemotherapy. Current therapies for RCC patients are inefficient due to the lack of diagnostic and therapeutic markers. Our recent studies have suggested an association of sperm-associated antigen 9 (SPAG9) with ovarian carcinomas. In the present study, we investigated the clinical relevance of SPAG9 in RCC patients. RT-PCR analysis showed expression of SPAG9 transcript in RCC tissues and RCC cell lines. In situ RNA hybridization and immunohistochemistry analyses confirmed the expression of SPAG9 in 88% of cancer patients, suggesting that SPAG9 participates in renal cancer. In addition, immunoblotting and ELISA analyses revealed a humoral immune response against SPAG9 in the sera of RCC patients but not in healthy individuals. Consistent with the clinical findings, knockdown of SPAG9 expression in RCC cells with specific siRNA significantly reduced cell growth and colony formation. Using in vitro wound healing and Matrigel invasion assays, we found that cell migration and invasive ability were also significantly inhibited. Furthermore, in vivo xenograft studies in nude mice revealed that administration of a SPAG9 siRNA plasmid significantly inhibited tumor growth. In conclusion, SPAG9 expression is associated with clinicopathologic features of tumors, suggesting that SPAG9 could contribute to the early spread of cancer. These results indicate that SPAG9 may have a role in tumor development and metastasis and thus could serve as a novel target for early detection and treatment of RCC. [Cancer Res 2008;68(20):8240-8]

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

Purpose: Cancer testis antigens are a group of tumor antigens with gene expression restricted to male germ cells in the testis and in various cancerous tissues. Recently, we reported a novel testisspecific sperm-associated antigen 9 (SPAG9) gene, a new member of the c-Jun NH 2 -terminal kinase^interacting protein family, having functional role in sperm-egg fusion and mitogenactivated protein kinase signaling pathway. National Center for Biotechnology Information Blast searches revealed SPAG9 nucleotide sequence similarities with expressed sequence tags of various cancerous tissues. In an effort to examine the clinical utility of SPAG9, we investigated the SPAG9 mRNA and protein expression in epithelial ovarian cancer (EOC). Humoral immune response to SPAG9 was also evaluated in EOC patients. Experimental Design: We determined the expression profile of SPAG9 transcript by reverse transcription-PCR and RNA in situ hybridization and SPAG9 protein expression by immunohistochemistry in EOC specimens and human ovarian cancer cell lines. Using ELISA and Western blotting, we analyzed specific antibodies for SPAG9 in sera from patients with EOC. Results: SPAG9 mRNA and protein expression was detected in 90% of EOC tissues and in all three human ovarian cancer cell lines. Specific SPAG9 antibodies were detected in 67% of EOC patients and not in sera from healthy individuals. Conclusions: Our findings indicate that SPAG9 is highly expressed in EOC and immunogenic in patients. Humoral immune response against SPAG9 in early stages of EOC suggests its important role in early diagnostics.These results collectively suggest that SPAG9, a novel member of cancer testis antigen family, could be a potential target for the development of diagnostic and therapeutic methods in EOC.

BackgroundBreast 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.MethodsHSP70-2 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.ResultsHSP70-2 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.ConclusionsHSP70-2 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0425-9) contains supplementary material, which is available to authorized users.

The predominant view of pluripotency regulation proposes a stable ground state with coordinated expression of key transcription factors (TFs) that prohibit differentiation. Another perspective suggests a more complexly regulated state involving competition between multiple lineage-specifying TFs that define pluripotency. These contrasting views were developed from extensive analyses of TFs in pluripotent cells in vitro. An experimentally-validated, genome-wide repertoire of the regulatory interactions that control pluripotency within the in vivo cellular contexts is yet to be developed. To address this limitation, we assembled a TF interactome of adult human male germ cell tumors (GCTs) using the Algorithm for the Accurate Reconstruction of Cellular Pathways (ARACNe) to analyze gene expression profiles of 141 tumors comprising pluripotent and differentiated subsets. The network (GCTNet) comprised 1305 TFs, and its Ingenuity Pathway analysis identified pluripotency and embryonal development as the top functional pathways. We experimentally validated GCTNet by functional (silencing) and biochemical (ChIP-seq) analysis of the core pluripotency regulatory TFs POU5F1, NANOG, and SOX2 in relation to their targets predicted by ARACNe. To define the extent of the in vivo pluripotency network in this system, we ranked all TFs in the GCTNet according to sharing of ARACNe-predicted targets with those of POU5F1 and NANOG using an Odds-Ratio analysis method. To validate this network, we silenced the top 10 TFs in the network in H9 ES cells. Silencing of each led to downregulation of pluripotency and induction of lineage; 7 of the 10 TFs were identified as pluripotency regulators for the first time.