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Supplementary Information from Sperm-Associated Antigen 9 Is Associated With Tumor Growth, Migration, and Invasion in Renal Cell Carcinoma

Background Human chorionic gonadotropin (hCG), critical to the success of pregnancy, is also made by many cancers. The presence of tumour-derived hCG (or its β subunit) is associated with poor patient prognosis, the reasons for which remain largely obscure; similar links with the closely-related gonadotropins luteinizing hormone (LH) or follicle stimulating hormone (FSH) have not been described. This study was prompted by the fact that chemoresistance is associated with poor patient prognosis in a variety of cancers. Methods The ability of αhCG, βhCG, hCG, LH and FSH to protect tumor cells (of three different lineages) from the detrimental effects of six different cytotoxic drugs (in terms of viability, proliferation, and apoptosis) was assessed, and potential LH-mediated antagonisms on hCG- and βhCG-mediated chemoprotection evaluated; associated signalling events were delineated employing phosphoblots and inhibitors. Potential TLR agonist- and apoptotic bleb-additivity in chemoprotection was determined. The ability of hormone-induced, tumor cell-elicited exosomes to horizontally transfer chemoprotection to gonadotropin-naïve tumor cells was assessed. The unpaired Student’s t-test, or one-way ANOVA (Holm-Sidak) were used to calculate statistical significance. Results hCG as well as its β subunit (but its α subunit, nor LH or FSH) significantly reduced the viability-decreasing, anti-proliferative and apoptosis-inducing effects of six chemotherapeutic drugs, and of UV radiation, on the tumour cells of three distinct lineages; LH and (but not FSH) antagonised the chemo-protective action of hCG and βhCG, a finding indicative of overlapping binding sites but differential signalling. Indeed, hCG and βhCG triggered signalling pathways that were distinct from those triggered by LH and FSH in tumor cells, and specific signalling pathways drove hCG-mediated chemoprotection against specific drugs. Co-incubation of hCG with agonists for TLR-7, TLR-8, TLR-9, or with specific apoptotic blebs (components of which include endogenous TLR agonists), resulted in additive amelioration of drug action. Further, hCG-elicited (but not LH- or FSH-elicited) tumour exosomes mediated the horizontal transfer of chemoresistance to gonadotropin-naïve tumor cells. Conclusion Anti-hCG immunization (already demonstrated to have anti-tumor effects) coupled with specific drug-signalling inhibitor combinations, and/or LH supplementation, could be beneficial in subjects harbouring gonadotropin-responsive tumours.

Video head impulse test (vHIT) is widely accepted as a test for the assessment of functional integrity of semicircular canals (SCCs). It allows for the evaluation of the functioning of all six SCCs independent of each other. It works on the principle of the vestibulo-ocular reflex (VOR). In individuals with vestibular pathologies, the VOR is impaired, and hence, the use of vHIT may provide vital information about the functional status of SCCs and the VOR pathway originating from them.In the recent past, studies reported excellent test–retest reliability of vHIT in healthy individuals. However, these studies used analysis of variance or the nonparametric counterpart Wilcoxon signed-rank test, which are insufficient statistical methods for conclusions about test–retest reliability. Further, because vHIT assesses VOR function in individuals with vestibular pathologies, it is important to assess test–retest reliability in the pathological group as well. Therefore, the present study aimed to evaluate test–retest reliability of vHIT in healthy individuals and individuals with vestibular pathology.Repeated measures.Twenty healthy individuals with no history of vestibular pathology and 20 individuals with known vestibular pathology were included.Each participant underwent vHIT testing for all three SCCs of both sides on four different occasions. VOR gain and the presence of pathological saccades were noted and analyzed for each recording.Intraclass correlation coefficient (ICC) revealed excellent test–retest reliability for VOR gain in both groups (ICC ≥ 0.76). Kappa coefficient analysis for the presence of refixation saccades demonstrated moderate to excellent agreement between test sessions (K ≥ 0.63) for the lateral canal. For the anterior and posterior SCC, there was large variability between sessions for refixation saccades.This study provides evidence about test–retest reliability of VOR gain and refixation saccades assessed using vHIT in healthy individuals and individuals with vestibulopathies. These findings suggest that both measures are highly reliable and replicable across test sessions, except refixation saccades in vertical canals which varied between sessions in some individuals.

Background Dendritic cell (DC)-based immunotherapy is capable of activating the immune system and in particular tumor-specific cytotoxic T lymphocytes (CTLs) to eradicate the tumor. However, major limitations are the availability of autologous tumor cells as antigenic source and the selection of antigen that may have potential to activate both CD4+ and CD8+ T cells in immune-specific manner. Recently, we reported the expression of sperm associated antigen 9 (SPAG9) that is associated with various types of malignancies including cervical cancer. We examined the recombinant human SPAG9 (rhSPAG9) as an antigenic source for generating efficient DCs to stimulate CD4+ and CD8+ T cell responses for future DCs-based vaccine trials in cervical cancer patients. Methods Human monocytes derived DCs were pulsed with different concentrations (250 ng/ml to 1000 ng/ml) of recombinant human SPAG9 (rhSPAG9) and evaluated for their phenotypic and functional ability. The efficacy of DCs primed with 750 ng/ml of rhSPAG9 (SPDCs) was compared with DCs primed with autologous tumor lysates (TLDCs), to induce CD4+, CD8+ T cells and activating NK cells. In addition, we investigated the effect of the chemotherapeutic drug cisplatin on phenotypic and functional potential of SPDCs. Results Phenotypic and functional characterization of DCs pulsed with 750 ng/ml rhSPAG9 was found to be optimal and effective for priming DCs. SPDCs were also capable of stimulating allogeneic T cells similar to TLDCs. SPDCs showed a statistically insignificant increase in the expression of maturation marker CD83 and migration towards CCL19 and CCL21 compared with TLDCs (CD83; P = 0.4; migration; P = 0.2). In contrast, although TLDCs showed better proliferation and secretion of Th1 cytokines (IL12p40, IL12p70 and IFNγ) compared to SPDCs, this difference was not statistically significant (IL12p40, P = 0.06). Further we also observed that clinical dose of cisplatin (200 µM) treated SPDCs were able to stimulate the proliferation of cytotoxic T lymphocytes without increasing the FOXP3+ Tregs in autologous co-cultures. Conclusions In summary, in order to overcome the limitation of the availability of autologous tumor cells as antigenic sources, our present strategy provides an insight to consider rhSPAG9 as a strong immunogen for DC-based immunotherapy for cervical cancer trials and warrants further studies. This is the first report to suggest that rhSPAG9 is an effective antigen for pulsing DCs that are capable of eliciting a potent Th1 response which, in turn, may help in decreasing the tumor burden when used along with a cisplatin based combinatorial regimen for therapeutic intervention. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-021-01951-7.

Heat shock protein 70-2 (HSP70-2) is known to be involved in tumor progression. However, its molecular role and mechanism in epithelial ovarian cancer (EOC) remains unknown. In the present investigation, we examined the role of HSP70-2 in cell cycle, apoptosis and epithelial mesenchymal transition pathways in EOC cells in in vitro and in-vivo xenograft mouse model. To investigate the role of HSP70-2 in ovarian cancer, plasmid driven short hairpin RNA approach was used to examine HSP70-2 gene and protein expression in ovarian cancer cell line A-10 (origin: serous papillary cystadenocarcinoma), Caov-3 (origin: adenocarcinoma) and SKOV3 (origin: adenocarcinoma; derived from metastatic site: ascites) by RT-PCR, quantitative-PCR, immunohistochemistry and Western blotting. Light microscopy, scanning electron microscopy, viability tests, and flow cytometry were used to study the cellular proliferation, onset of senescence, colony forming ability and morphological features of cancer cells. Cell migration and invasion ability was evaluated by wound healing and Boyden chamber assays. Further, we studied the effect of HSP70-2 protein ablation on human ovarian xenograft mice model. At molecular level, various molecules involved in apoptosis, cell cycle and epithelial-mesenchymal-transition were also examined both in in-vitro and in-vivo xenograft mouse model. The knockdown of HSP70-2 expression by gene silencing resulted in the onset of apoptosis, senescence, reduced cellular growth and colony forming ability of EOC cells. Interestingly, the migration, invasion and wound healing abilities of cells were also significantly inhibited. In addition, the ablation of HSP70-2 resulted in the upregulation of cytochrome-C, caspase 3, caspase 7, caspase 9, APAF1, BAX, BIM, BAK, BAD, BID, PUMA, NOXA, p16, p21, Rb, E-cadherin, cytokeratin 18, EMA in these cells as well as in the xenograft tumor specimens. However, there was downregulation of PARP1, BCL-2, Bcl-xL, MCL-1, Survivin, XIAP, cIAP2, CDK1, CDK2, CDK4, CDK6, cyclin D1, cyclin E, cyclin A2, cyclin B1, p-Rb, N-cadherin, SNAIL, SLUG, VIMENTIN, SMA, MMP2, MMP3, MMP9 and TWIST in these samples. Furthermore, the xenograft studies showed significant reduction in the tumor growth. Our results suggest that HSP70-2 can promote cellular growth and invasion of EOC cells and therefore may be a potential therapeutic target in EOC.

// Vikash Kumar 1 , Nirmala Jagadish 1 and Anil Suri 1 1 Cancer Microarray, Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, 110067, New Delhi, India Correspondence to: Anil Suri, email: anil@nii.res.in Keywords: AKAP4, PKA, reactive oxygen species, s-phase arrest, apoptosis Received: December 29, 2016 Accepted: May 10, 2017 Published: May 24, 2017 ABSTRACT Ovarian cancer represents one of the most common malignancies among women with very high mortality rate worldwide. A-kinase anchor protein 4 (AKAP4), a unique cancer testis (CT) antigen has been shown to be associated with various malignant properties of cancer cells. However, its involvement in various molecular pathways in ovarian cancer remains unknown. In present investigation, employing gene silencing approach, we examined the role of AKAP4 in cell cycle, apoptosis and epithelial-mesenchymal transition (EMT). Further, we also investigated the effect of ablation of AKAP4 on tumor growth in SCID mice ovarian cancer xenograft mouse model. Our results showed that ablation of AKAP4 resulted in increased reactive oxygen species (ROS) generation, DNA damage, cell cycle arrest and apoptosis in ovarian cancer cells. AKAP4 knockdown lead to degradation of protien kinase A (PKA) which was rescued by proteosome inhibitor MG-132. ROS quencher N-acetyl cysteine (NAC) treatment rescued cell cycle arrest and resumed cell division. Subsequently, increased expression of pro-apoptotic molecules and decreased expression of pro-survival/anti-apoptotic factors was observed. As a result of AKAP4 depletion, DNA damage response proteins p-γH2AX, p-ATM and p21 were upregulated. Also, knockdown of CREB resulted in similar findings. Further, PKA inhibitor (H89) and oxidative stress resulted in similar phenotype of ovarian cancer cells as observed in AKAP4 ablated cells. Collectively, for the first time our data showed the involvement of AKAP4 in PKA degradation and perturbed signaling through PKA-CREB axis in AKAP4 ablated ovarian 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 β-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.

Summary Human male germ cell tumors (GCTs) are derived from primordial germ cells (PGCs). The master pluripotency regulator and neuroectodermal lineage effector transcription factor SOX2 is repressed in PGCs and the seminoma (SEM) subset of GCTs. The mechanism of SOX2 repression and its significance to GC and GCT development currently are not understood. Here, we show that SOX2 repression in SEM-derived TCam-2 cells is mediated by the Polycomb repressive complex (PcG) and the repressive H3K27me3 chromatin mark that are enriched at its promoter. Furthermore, SOX2 repression in TCam-2 cells can be abrogated by recruitment of the constitutively expressed H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling, leading to expression of neuronal and other lineage genes. SOX17 has been shown to initiate human PGC specification, with its target PRDM1 suppressing mesendodermal genes. Our results are consistent with a role for SOX2 repression in normal germline development by suppressing neuroectodermal genes., Graphical Abstract, Highlights • SOX2 is repressed in hPGC, germ cell neoplasia in situ, and seminoma • SOX2 repression is mediated by PcG and H3K27me3 enrichment at its promoter • Retinoid signaling recruits UTX to SOX2 promoter leading to reactivation of SOX2 • These studies shed light on the role of SOX2 in germline development, In this article, Chaganti and colleagues show that SOX2 repression in human germ cell tumors and possibly also their precursor hPGC is mediated by PcG and H3Kme27 at its promoter. The repression can be reversed by recruitment of UTX, a H3K27 demethylase, leading to upregulation of neuronal lineage genes.

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) is known to be involved in tumor progression. However, its molecular role and mechanism in epithelial ovarian cancer (EOC) remains unknown. In the present investigation, we examined the role of HSP70-2 in cell cycle, apoptosis and epithelial mesenchymal transition pathways in EOC cells in

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

Background Colorectal cancer (CRC) is the third leading cause of cancer related deaths worldwide both in men and women. Our recent studies have indicated an association of heat shock protein 70–2 (HSP70-2) with bladder urothelial carcinoma. In the present study, we investigated the association of HSP70-2 with various malignant properties of colorectal cancer cells and clinic-pathological features of CRC in clinical specimens. Methods HSP70-2 mRNA and protein was investigated expression by RT-PCR, immunohistochemistry, immunofluorescence, flow cytometry and Western blotting in CRC clinical specimens and COLO205 and HCT116 cell lines. Plasmid-based gene silencing approach was employed to study the association of HSP70-2 with various malignant properties of COLO205 and HCT116 cells in in vitro and with tumor progression in in vivo COLO205 human xenograft mice model. Results HSP70-2 expression was detected in 78 % of CRC patients irrespective of various stages and grades by RT-PCR and IHC. Our analysis further revealed that HSP70-2 expression was detected in both COLO205 and HCT116 cell lines. Ablation of HSP70-2 expression resulted in reduced cellular growth, colony forming ability, migratory and invasive ability of CRC cells. In addition, ablation of HSP70-2 expression showed significant reduction in tumor growth in COLO205 human xenograft in in vivo mouse model. Conclusion Collectively, our results indicate that HSP70-2 is associated with CRC clinical specimens. In addition, down regulation of HSP70-2 expression reduces cellular proliferation and tumor growth indicating that HSP70-2 may be a potential therapeutic target for CRC treatment.

Background: Tympanometry is a non-invasive test clinical test for the assessment of middle ear status/functioning. Several studies over the past few years have produced conflicting results regarding changes in the middle ear function with advancing age. Hence, the present study is aimed to give evidences on distributions of type of tympanograms in males and females under various age groups.Methods: The study followed retrospective design, where data collected from January 2017 to October 2017 were used. A total of 2,292 tympanograms of the 1146 patients were analysed. Descriptive statistics and Wilcoxon’s signed rank test were used. A p-value Results: It was documented that higher occurrence of ‘B’ type and ‘Ad’ type of tympanogram in paediatric group and geriatric group respectively. As it is expected, ‘A’ type of tympanogram is predominant in adult and older adult group. Also, there was no gender difference seen in type of tympanograms.Conclusions: Higher occurrence of ‘B’ type of tympanogram in paediatric group warrants medical line of treatment and educating on ear-hygiene in them.

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]

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 testis-specific sperm-associated antigen 9 (SPAG9) gene, a new member of the c-Jun NH2-terminal kinase–interacting protein family, having functional role in sperm-egg fusion and mitogen-activated 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.

Colorectal cancer ranks third among the estimated cancer cases and cancer related mortalities in United States in 2014. Early detection and efficient therapy remains a significant clinical challenge for this disease. Therefore, there is a need to identify novel tumor associated molecules to target for biomarker development and immunotherapy. In this regard, cancer testis antigens have emerged as a potential targets for developing novel clinical biomarkers and immunotherapy for various malignancies. These germ cell specific proteins exhibit aberrant expression in cancer cells and contribute in tumorigenesis. Owing to their unique expression profile and immunogenicity in cancer patients, cancer testis antigens are clinically referred as the most promising tumor associated antigens. Several cancer testis antigens have been studied in colorectal cancer but none of them could be used in clinical practice. This review is an attempt to address the promising cancer testis antigens in colorectal cancer and their possible clinical implications as biomarkers and immunotherapeutic targets with particular focus on challenges and future interventions.

To examine the immunogenicity of the plasmid DNA encoding human sperm associated antigen 9 (hSPAG9), the cDNA corresponding to hSPAG9 was cloned in mammalian expression vector pcDNA 3.1 down stream of cytomegalovirus promoter. Immunization of female BALB/cJ mice with pcDNA-hSPAG9 plasmid DNA in saline by intramuscular (i.m.), by adsorbing onto gold microcarriers (delivered by gene gun) and by recombinant hSPAG9 (r-hSPAG9) protein generated antibody response against Escherichia coli expressed r-hSPAG9 protein and native SPAG9 in human sperm. Although mice immunized with r-hSPAG9 protein exhibited highest antibodies titres (P0.001), the difference in the antibody titres seen by the two modes of plasmid DNA delivery were not significant (P0.05). A dominant IgG1 isotype response was observed in mice immunized with pcDNA-hSPAG9 plasmid DNA delivered by gene gun as compared to a mixed IgG1-IgG2a isotype response in mice immunized with r-hSPAG9 protein and pcDNA-hSPAG9 plasmid DNA delivered by i.m. Further, antibodies generated by pcDNA-hSPAG9 plasmid DNA localized acrosomal compartment of human sperm and inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. These studies for the first time, demonstrate the feasibility of generating an immune response to sperm specific hSPAG9 protein by DNA vaccine and that antibodies thus generated recognize native SPAG9 in human sperm.

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 α-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 p38α 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.

The present study was conducted to isolate macaque proteomic homologue of human SPAG9 (EMBL nomenclature human sperm associated antigen 9: hSPAG9; Shankar et al., 1998: Biochem Biophys Res Commun 243:561-565) in order to find out whether the macaque can provide a suitable model for examining its immunocontraception effects. Macaque SPAG9 was cloned and sequenced from the macaque testis cDNA library. The macaque cDNA contained open reading frame encoding 712 amino acids. A 84.9% and 94% homology between macaque and human SPAG9 was found at protein and DNA levels. Northern analysis and RNA in situ hybridization experiments revealed testis- and stage-specific expressions of macaque SPAG9 mRNA, mainly confined to round spermatid suggesting haploid germ cell expression. Anti-human SPAG9 antibodies recognized native SPAG9 in macaque sperm extract in Western blotting and the acrosomal compartment region of macaque sperm in indirect immunofluorescence. Flow cytometry analysis further revealed surface localization of macaque SPAG9 in live macaque sperm. The amino acid sequence data for nonhuman primate SPAG9 suggest that antibodies generated by vaccinating macaque with hSPAG9 will recognize nonhuman primate SPAG9, supporting the testing of SPAG9 contraceptive vaccine based on hSPAG9 in the nonhuman primate model.

Problem: It has been well documented that antisperm antibodies can be causative factors for infertility. In this report we have identified a protein on human sperm referred as human sperm-associated protein (HSAP) using serum of an immunoinfertile woman; it is thus a sperm-specific protein – a candidate molecule for control of fertility. Method of study: An immunoinfertile woman serum showing head–head sperm agglutination and acrosomal localization, reacted with human sperm protein of apparent molecular weight of 48 kDa on Western blot. Anti-48 kDa antiserum was raised in rabbit by eluting 48 kDa protein and was used to screen the human testis cDNA expression library. A putative positive hsap cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting. The cell type-specific expression was done using in situ RNA hybridization studies. To obtain recombinant HSAP (r-HSAP), hsap cDNA was cloned in pET 22b(+) expression vector. r-HSAP was expressed as polyhistidine fusion protein in Escherichia coli and purified. Rabbits were immunized with the purified r-HSAP, which led to generation of antibodies. In order to evaluate in vitro immunocontraceptive potential, the anti-r-HSAP antibodies were characterized by agglutination assay, zona-free hamster egg penetration assay, indirect immunofluorescence (IIF) assay, and by flow cytometry analysis. Results: We have cloned a human testis gene encoding a protein (HSAP) of 328 amino acids. Antibodies against the purified recombinant protein specifically recognized approximately 40 kDa r-HSAP, and a cognate 48 kDa protein band in human sperm extract in Western blot procedure. The anti-r-HSAP antibodies localized acrosomal compartment, inhibited sperm binding/attachment in zona-free hamster penetration assay and revealed surface binding with human live sperm by flow cytometry. The cDNA sequence has been submitted to EMBL and has been given the accession number Y16676. Conclusion: This study has put in evidence that novel sperm-specific r-HSAP has role in sperm function and may have application in the development of a contraceptive vaccine. The availability of the recombinant protein will facilitate studies on the assessment of its potential as a contraceptive immunogen.

Previously, we cloned and sequenced a sperm specific antigen, designated as HSS (EMBL nomenclature human sperm associated antigen 9: hSPAG9) from human testis (Shankar et al.: Biochem Biophys Res Commun 243:561-565, 1998). The present study was conducted to isolate baboon proteomic homologue in order to find out whether the baboon can provide a suitable model for examining its immunocontraception effects. Baboon SPAG9 (bSPAG9) was cloned and sequenced from the baboon testis cDNA library. The baboon cDNA contained open reading frame encoding 760 amino acids. A 90.6 and 96.8% homology between baboon and human SPAG9 was found at protein and DNA levels. Analysis for tissue specificity by Northern blot procedure using various baboon tissues indicated that bSPAG9 was specifically expressed only in the baboon testis. Further, cell type expression analysis by in situ hybridization in baboon testis demonstrated the expression of bSPAG9 mRNA transcript only in the round spermatid suggesting haploid germ cell expression. Anti-human SPAG9 antibodies recognized the acrosomal compartment region of baboon sperm in indirect immunofluorescence (IIF). Flow cytometry analysis showed surface localization of bSPAG9 in live baboon sperm. The amino acid sequence data for nonhuman primate SPAG9 suggest that antibodies generated by vaccinating baboon with hSPAG9 will recognize nonhuman primate SPAG9, supporting the testing of SPAG9 contraceptive vaccine based on hSPAG9 in the nonhuman primate model.

Cancer immunotherapy is a promising field with limited success, also due to lack of tumor-specific targets. In our attempt of exploring novel biomarkers and immunotherapeutic targets against cancer, we have discovered a novel cancer testis antigen, SPAG9, in cancers of different histological origin and demonstrated its potential role in oncogenesis.

Aim: Video head impulse test is a clinical tool found useful for assessment of each of the 6 semicircular canals independent of each other. However there is little known about relative efficacy of the two measures, vestibulo-ocular reflex gain and refixation saccade, in identifying the presence of a vestibular pathology. The present study therefore aimed at investigating the utility of each of these measures in identifying vestibular pathology. Materials and Methods: Twenty subjects with normal audio-vestibular system and 20 subjects with vestibulopathies underwent video head impulse testing. Results: The VOR gain was significantly higher in lateral and posterior canals and presence of refixation saccades was in significantly higher proportion of individuals with vestibulopathies than the controls (p

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 1,305 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 embryonic stem 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. Stem Cells 2015;33:367–377

BackgroundMajority of bladder cancer deaths are caused due to transitional cell carcinoma (TCC) which is the most prevalent and chemoresistant malignancy of urinary bladder. Therefore, we analyzed the role of Sperm associated antigen 9 (SPAG9) in bladder TCC.Methodology and findingsWe examined SPAG9 expression and humoral response in 125 bladder TCC patients. Four bladder cancer cell lines were assessed for SPAG9 expression. In addition, we investigated the effect of SPAG9 ablation on cellular proliferation, cell cycle, migration and invasion in UM-UC-3 bladder cancer cells by employing gene silencing approach. Our SPAG9 gene and protein expression analysis revealed SPAG9 expression in 81% of bladder TCC tissue specimens. High SPAG9 expression (>60% SPAG9 positive cells) was found to be significantly associated with superficial non-muscle invasive stage (P = 0.042) and low grade tumors (P = 0.002) suggesting SPAG9 putative role in early spread and tumorigenesis. Humoral response against SPAG9 was observed in 95% of patients found positive for SPAG9 expression. All four bladder cancer cell lines revealed SPAG9 expression. In addition, SPAG9 gene silencing in UM-UC-3 cells resulted in induction of G0-G1 arrest characterized by up-regulation of p16 and p21 and consequent down-regulation of cyclin E, cyclin D and cyclin B, CDK4 and CDK1. Further, SPAG9 gene silencing also resulted in reduction in cellular growth, and migration and invasion ability of cancer cells in vitro.ConclusionsCollectively, our data in clinical specimens indicated that SPAG9 is potential biomarker and therapeutic target for bladder TCC.

Earlier, we reported an association of A-kinase anchor protein 4 (AKAP4) expression in cervical cancer patient specimens, indicating its implications as an immunotherapeutic target. In this study, we investigated the possible role of AKAP4 in cervical carcinogenesis. AKAP4 messenger RNA and protein expression was assessed in four cervical cancer cell line models, C-33A, CaSki, HeLa and SiHa. Gene silencing approach was employed to investigate the potential role of AKAP4 in cellular growth, proliferation, colony-forming ability, migration and invasion in aggressive squamous cell carcinoma cells (SiHa). Further, the effect of downregulation of AKAP4 on tumor growth was examined in the cervical cancer xenograft model in nude mice. Our data clearly indicated that AKAP4 was expressed in all cervical cancer cells at the gene and protein level. We also observed distinct cytoplasmic and surface localization by indirect immunofluorescence and flow cytometry, respectively. Ablation of AKAP4 protein caused significant inhibition in cellular proliferation, colony-forming ability, migration and invasion ability of SiHa cells. Further, gene silencing of AKAP4 also resulted in reduced tumor growth in nude mice in vivo. Collectively, AKAP4 surface localization and its significant association with malignant properties of cervical cancer cells imply its clinical utility as an immunotherapeutic target.

Background Cervical cancer is one of the major gynecologic cancers. In developing countries, because of a lack of medical support and infrastructure, cervical cancer is the leading cause of cancer-related deaths. Therefore, there is a need to identify novel biomarkers for cervical cancers. In this context, cancer-testis (CT) antigens represent a unique class of tumor antigens that have been shown to be associated with various solid tumors. These antigens have restricted expression in testis and no expression in somatic tissues. Because of their restricted expression, CT antigens are novel candidate molecules for early detection and diagnosis and immunotherapy. In the present study, novel CT antigen A-kinase anchor protein 4 (AKAP4) expression and humoral response were investigated in patients with cervical cancer. Methods In this study, 74 cervical cancer tissue specimens, which included different tumor stages (stage I [n = 35], stage II [n = 39]) and histologic grades (grade 1 [n = 17], grade 2 [n = 46], and grade 3[n = 11]) and 62 adjacent noncancerous tissue specimens were investigated for AKAP4 gene expression by using reverse transcriptase polymerase chain reaction and in situ RNA hybridization. Furthermore, AKAP4 protein expression was determined by immunohistochemistry. In addition, humoral response against purified recombinant AKAP4 protein was determined in available sera of 70 patients with cervical cancer by enzyme-linked immuno assay (ELISA). Findings Our study revealed that AKAP4 gene and protein expression was detected in 86% of total patients with cervical cancer. Based on the AKAP4 immunoreactivity score, most of stage I (n = 22/29) and stage II (n = 30/35) specimens revealed high AKAP4 expression (≥50% AKAP4-positive cells). A-kinase anchor protein 4 expression was significantly associated with early grades tumor specimens (P = 0.023). In addition, humoral response was detected in 53% of patients irrespective of stages, lymph node positivity, and grades. Conclusions Collectively, our data indicate the putative role of AKAP4 in early tumorigenesis and may be implicated as a biomarker and immunotherapeutic target for cervical cancer.

Background Breast cancer is the second leading cause of cancer related deaths in women worldwide. Reports about the early diagnosis of breast cancer are suggestive of an improved clinical outcome and overall survival rate in cancer patients. Therefore, cancer screening biomarker for early detection and diagnosis is urgently required for timely treatment and better cancer management. In this context, we investigated an association of cancer testis antigen, A-Kinase anchor protein 4 (AKAP4) with breast carcinoma. Methodology/Findings We first compared the AKAP4 gene and protein expression in four breast cancer cells (MCF7, MDA-MB-231, SK-BR3 and BT474) and normal human mammary epithelial cells. In addition, 91 clinical specimens of breast cancer patients of various histotypes including ductal carcinoma in situ, infiltrating ductal carcinoma and infiltrating lobular carcinoma and 83 available matched adjacent non-cancerous tissues were examined for AKAP4 gene and protein expression by employing in situ RNA hybridization and immunohistochemistry respectively. Humoral response against AKAP4 was also investigated in breast cancer patients employing ELISA. Our in vitro studies in all breast cancer cells revealed AKAP4 gene and protein expression whereas, normal human mammary epithelial cells failed to show any expression. Using in situ RNA hybridization and immunohistochemistry, 85% (77/91) tissue specimens irrespective of histotypes, stages and grades of breast cancer clinical specimens revealed AKAP4 gene and protein expression. However, matched adjacent non-cancerous tissues failed to display any AKAP4 gene and protein expression. Furthermore, humoral response was observed in 79% (72/91) of total breast cancer patients. Interestingly, we observed that 94% (72/77) of breast cancer patients found positive for AKAP4 protein expression generated humoral response against AKAP4 protein. Conclusions Collectively, our data suggests that AKAP4 may be used as serum based diagnostic test for an early detection and diagnosis of breast cancer and may be a potential target for immunotherapeutic use.

The core pluripotency regulatory master transcription factor SOX2 is repressed in human primordial germ cells (PGCs) and in the seminoma (SEM) subset of human germ cell tumors (GCTs), which arises by transformation of PGCs. Neither the mechanism of this repression not its significance to GC and GCT development have so far been clarified. Here we show that SOX2 repression in TCam-2 SEM cells is mediated by the presence of the Polycomb repressive complex (PcG) and by the H3K27me3 chromatin mark at the SOX2 transcription start site. Moreover, repression can be abrogated by recruitment of the H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling. SOX2-activated TCam-2 cells express neuronal and other lineage genes, consistent with the gene's function as a neurectodermal effector. Based on the recent discovery that SOX17 initiates human PGC specification, with its downstream target PRDM1 acting to suppress mesendodermal genes, we propose that SOX2 repression is required for PGC specification to suppress neuroectodermal genes. Overt pluripotency is initiated in latently pluripotent transformed PGCs presenting as the SOX2 upregulated embryonal carcinoma (EC) subset of GCTs; by gene expression profiling analysis we characterize the functional pathways that distinguish PGC-like unipotent SEM from blastocyst-like pluripotent EC. Citation Format: Kushwaha Ritu, Nirmala Jagadish, George Bosl, Raju Chaganti. Mechanism and role of SOX2 repression in human germline specification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2528.

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.

Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of D-glucopyranose-6-phosphate to D-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 A and belonged to the orthorhombic space group I2(1)2(1)2(1), with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 A.

BACKGROUND: The human sperm-associated antigen 9 (hSPAG9) is of special interest attributing to the findings indicating that SPAG9 is an acrosomal molecule. SPAG9 is not only restricted to acrosomal compartment but also persists in equatorial segment post-acrosome reaction, which is a key location in sperm-egg interaction. METHODS AND RESULTS: Immunogenicity studies in macaques were carried out with recombinant hSPAG9 (rhSPAG9) adsorbed on alum, which resulted in high titres of anti-rhSPAG9 antibodies as determined by enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis employing anti-rhSPAG9 antibodies generated in monkeys indicated that antibodies specifically reacted with native SPAG9 from macaque and human sperm and rhSPAG9 protein. Furthermore, indirect immunofluorescence experiments demonstrated SPAG9 localization in the acrosomal compartment of macaque and human sperm. In addition, monkey antibodies against rhSPAG9 significantly inhibited the human spermatozoa adherence or penetration in zona-free hamster oocytes. CONCLUSION: These results demonstrate that rhSPAG9 adsorbed on alum is highly immunogenic in subhuman primate model and therefore represents a suitable sperm-based vaccine immunogen for fertility trials in macaque.

Salivary gland cancers are highly aggressive epithelial tumor associated with metastatic potential and high mortality. The tumors are biologically diverse and are of various histotypes. Besides, the detection and diagnosis is a major problem of salivary gland cancer for available treatment modalities. In the present study, we have investigated the association of sperm associated antigen 9 (SPAG9) expression with salivary gland tumor (SGT). Clinical specimens of benign (n = 16) and malignant tumors (n = 86) were examined for the SPAG9 expression. In addition, the sera and adjacent non-cancerous tissues (n = 72) from available patients were obtained. Our in situ RNA hybridization and immunohistochemistry (IHC) analysis revealed significant difference (p = 0.0001) in SPAG9 gene and protein expression in benign (63%) and malignant tumor (84%) specimens. Further, significant association was also observed between SPAG9 expression and malignant tumors (P = 0.05). A cut-off value of >10% cells expressing SPAG9 protein designated as positive in IHC, predicted presence of malignant SGT with 83.72% sensitivity, 100% specificity, 100% PPV and 83.72% NPV. Humoral response against SPAG9 protein was generated in 68% of SGT patients. A cut-off value of 0.212 OD for anti-SPAG9 antibodies in ELISA predicted presence of malignant SGT with 69.23% sensitivity, 100% specificity, 100% PPV and 78.94% NPV. Collectively, our data suggests that the majority of SGT show significant difference and association among benign and malignant tumors for SPAG9 gene and protein expression and also exhibit humoral response against SPAG9 protein. Hence, SPAG9 may be developed as a biomarker for detection and diagnosis of salivary gland tumors.

Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.

Human sperm-associated antigen 9 (hSPAG9) is a potential target for sperm-based contraceptive vaccine in lieu of its location on the sperm acrosomal compartment and its implication in sperm-egg interaction. SPAG9 is an acrosomal molecule which is not only restricted to a specific region (domain) of the acrosome but also undergoes relocation to the equatorial region in a stage-specific manner during acrosome reaction, demonstrating its potential role in sperm-egg binding. Human SPAG9 nucleotide sequence revealed 94% identity with macaque SPAG9 and 96.8% with baboon SPAG9 over the entire sequence. The amino acid sequence comparison of human SPAG9 with macaque and baboon revealed an overall homology of 84.9% and 90.6%, respectively. The presence of a high level of homology at the amino acid and nucleotide levels indicates that SPAG9 is conserved in macaque, baboon and human sharing common function and common origin in the biological past. Immunogenicity studies were carried in rats, which demonstrated that recombinant hSPAG9 protein adsorbed on alum is highly immunogenic. Antibodies thus generated after immunization reacted with recombinant human SPAG9 (rhSPAG9) and native SPAG9 protein from human sperm in Western blot analysis. In an in vitro assay, anti-rhSPAG9 antibodies inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. Further, anti-SPAG9 antibodies inhibited the binding of human sperm to intact human oocyte as well as to matched hemi-zonae, indicating that the recombinant protein is a suitable contraceptive vaccinogen. Together these results demonstrate that the rhSPAG9 adsorbed on alum is immunogenic in nature, which is a permissible adjuvant for immunogenicity and fertility trials in non-human primates.

Previously, we cloned and sequenced a novel human sperm associated antigen 9 (SPAG9). Northern blot analysis and RNA in situ hybridization experiments revealed testis- and stage-specific expression of SPAG9 mRNA, mainly confined to round spermatid suggesting haploid germ cell expression Studies on the human and non-human primates (macaque and baboon) have shown a homology of 84.9% and 90.6% at amino acid level and 94% and 96.8% at DNA level, respectively. The presence of high level of homology at amino acid and DNA level indicates that SPAG9 is conserved in human, baboon and macaque sharing common function and common origin in the biological past. In addition, SPAG9 protein revealed structural homology with c-Jun NH2-terminal kinase (JNK) interacting protein (JIP). The amino acid sequence analysis of SPAG9 predicted coiled coil, leucine zipper and transmembrane domain, speculating the involvement of SPAG9 mediated signal transduction pathways in reproductive processes.

It has been well documented that antisperm antibodies can be causative factors for infertility. In this report we have identified a protein on human sperm referred as human sperm-associated protein (HSAP) using serum of an immunoinfertile woman; it is thus a sperm-specific protein--a candidate molecule for control of fertility.An immunoinfertile woman serum showing head-head sperm agglutination and acrosomal localization, reacted with human sperm protein of apparent molecular weight of 48 kDa on Western blot. Anti-48 kDa antiserum was raised in rabbit by eluting 48 kDa protein and was used to screen the human testis cDNA expression library. A putative positive hsap cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting. The cell type-specific expression was done using in situ RNA hybridization studies. To obtain recombinant HSAP (r-HSAP), hsap cDNA was cloned in pET 22b(+) expression vector. r-HSAP was expressed as polyhistidine fusion protein in Escherichia coli and purified. Rabbits were immunized with the purified r-HSAP, which led to generation of antibodies. In order to evaluate in vitro immunocontraceptive potential, the anti-r-HSAP antibodies were characterized by agglutination assay, zona-free hamster egg penetration assay, indirect immunofluorescence (IIF) assay, and by flow cytometry analysis.We have cloned a human testis gene encoding a protein (HSAP) of 328 amino acids. Antibodies against the purified recombinant protein specifically recognized approximately 40 kDa r-HSAP, and a cognate 48 kDa protein band in human sperm extract in Western blot procedure. The anti-r-HSAP antibodies localized acrosomal compartment, inhibited sperm binding/attachment in zona-free hamster penetration assay and revealed surface binding with human live sperm by flow cytometry. The cDNA sequence has been submitted to EMBL and has been given the accession number Y16676.This study has put in evidence that novel sperm-specific r-HSAP has role in sperm function and may have application in the development of a contraceptive vaccine. The availability of the recombinant protein will facilitate studies on the assessment of its potential as a contraceptive immunogen.

Ovarian cancer is one of the neoplasms affecting the reproductive tract associated with high mortality rate because of limited therapeutic options and an elevated incidence of chemoresistance and recurrence. In this context, immunotherapy may constitute a promising approach to improve survival rates and clinical outcome, raising the need for specific target antigens. Cancer-testis antigens (CTAs) are considered promising candidates in this sense because they are aberrant expressed by various malignancies but not by non-transformed tissue, with the exception of testes. Here, we examined the expression and potential to promote humoral immune responses of a novel CTA, A-kinase anchor protein 4 (AKAP4), among 38 ovarian carcinoma patients. Our results reveal that AKAP4 was expressed at both the mRNA and protein levels in 89% (34/38) of ovarian carcinoma tissue specimens but not in 21 matched adjacent non-cancerous tissues. In addition, a humoral response against AKAP4 was detected in 58% (22/38) of ovarian carcinoma patients by ELISA. In particular, 65% (22/34) patients bearing an AKAP4-expressing tumor exhibited circulating anti-AKAP4 antibodies. Interestingly, the majority of specimens were categorized as ovarian serous adenocarcinoma and serous papillary carcinoma, of which 93% (28/30) and 100% (6/6), respectively, expressed AKAP4. A humoral response against AKAP4 was detected in 79% (19/24) and 67% (4/6) of ovarian serous adenocarcinoma and serous papillary carcinoma patients, respectively. The presence of circulating anti-AKAP4 antibodies suggests the AKAP4 is highly immunogenic in ovarian serous carcinoma patients. Our study lays the foundations for exploring AKAP4 as a potential target for the immunotherapy of ovarian cancer.

Pluripotent embryonal carcinoma (EC) cells derived from germ cell tumors (GCTs) are an excellent model system to study the interface between malignancy, pluripotency, and lineage differentiation. We previously characterized the gene expression profile (GEP) of a panel of 135 germ cell tumor (GCT) biopsies comprising all described differentiation lineages and 6 normal testes as controls, using the Affymetrix U133A+B arrays (Korkola et al. Cancer Res., 66: 820-7, 2006 and J. Clin. Oncol., 27: 5240-7, 2009). We applied the ARACNE reverse engineering algorithm to this GEP to obtain the first de novo and context-specific transcriptional interactome (GCTNet) comprising 1,310 transcription factors (TFs). GCTnet recapitulated 830 regulatory targets together of POU5F1, NANOG, and SOX2, the core pluripotency TFs. ChIP-seq and ChIP-PCR analysis of NT2/D1 embryonal carcinoma and H9 ES cells provided significant validation of ARACNe-inferred targets, besides providing the first detailed mapping of DNA binding sites of these 3 genes in human cells. Functional validation of ARACNe-inferred targets by stable knockdown of the POU5F1, NANOG and SOX2 in NT2/D1 cells also showed a significant enrichment of ARACNe targets. Analysis of GEP in a time-course of SOX2 knockdown by principal component analysis (PCA) identified 3 orthogonal gene expression programs: (1) genes with monotonic change (PC1), (2) genes showing a strong and focal gene expression at day 5 (PC2), and (3) genes which show oscillatory pattern of expression (PC3). Ingenuity and GO annotation analysis of PCA provided new insights into the pathways regulated by this TF. Our results provide a novel, comprehensive, and accurate regulatory networks responsible for pluripotency and lineage differentiation in GCTs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3410. doi:10.1158/1538-7445.AM2011-3410

Recent interest and effort have been focused on identification of transcription factors (TFs) and TF networks that regulate pluripotency and lineage differentiation in human and murine cells. Yet, a complete repertoire of regulatory interactions supporting these processes remains elusive. Embryonic stem (ES) cells, induced pluripotent (iPS) cells, and embryonal carcinoma (EC) cells derived from germ cell tumors (GCTs) comprise the three classic pluripotent cell types. EC cells exhibit, in vivo and in vitro, many properties in common with ES cells, making them an excellent system to study pluripotency and lineage differentiation. We previously characterized the gene expression profile (GEP) of a panel of 135 GCT biopsies comprising all described in vivo differentiation lineages and 6 normal testes as controls, using the Affymetrix U133A+B arrays (Korkola et al. Cancer Res., 66: 820-7, 2006 and J. Clin. Oncol., 27: 5240-7, 2009). These GEP data were analyzed using the ARACNe reverse engineering algorithm (Basso et al., Nat. Genet., 37: 382-90, 2005) to reconstruct a global transcriptional interaction network regulating pluripotency and lineage differentiation in these tumors. The ensuing interactome, comprising 1312 TFs, displayed three strongly intra-connected modules: Module A, including 187 genes associated with regulation of development and differentiation, Module B, including 89 genes associated with regulation of immune response and B-cell development, and Module C, including 80 genes associated with regulation of transcription. Furthermore, the interactome recapitulated 830 regulatory targets of the core pluripotency TFs, POU5F1, NANOG, and SOX2 (369 of POU5F1, 375 of NANOG, and 306 of SOX2). Validation of these targets by ChIP-PCR and ChIP-seq in NT2/D1 (EC) and H9 (ES) cells revealed that all of the top 10 target genes of POU5F1 (NANOG, FAM46B, L1TD1, DND1, ZYG11, FRAT2, DPPA4, ARGEF1, KLRG2 and CBR3) and SOX2 (SEPHS1, PLCB2, ZNF518, ECE2, SLC26A3, GPM6B, PHLDA2, PATZ1, ADRB3 and MAP1A) could specifically bind the corresponding TF in their promoter regions, within both cell types. For NANOG, 9 of the top 10 targets (POU5F1, FAM46B, DPPA4, L1TD1, UTF1, FRAT2, RAB15, TEAD4 and ZYG11A) showed specific promoter binding of the TF in both cell types, while one target (DND1) showed specific binding only in H9 cells. ChIP-seq analysis was consistent with the results of ChIP-PCR. We sought functional validation of the targets by performing lentivirus-mediated shRNA silencing of POU5F1 in NT2/D1 cells. Results showed that the ARACNe-inferred targets were both positively and negatively regulated, consistently with regulation of pluripotency and/or differentiation. Taken together, these results define a novel and highly accurate regulatory model for the systems biology study of TFs involved in regulation of pluripotency and lineage differentiation in mammalian cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4240.