Sperm-associated Antigen 9 Expression Research Articles

Sperm-Associated Antigen 9: A Novel Diagnostic Marker for Thyroid cancer

Cancer-testis antigens are the unique class of testis proteins expressed in tumor but not healthy tissue except testis and might represent ideal targets for the development of novel diagnostics and therapeutic methods in thyroid cancer, which is the most common malignancy of the endocrine system. Our objective was to investigate the clinical relevance of cancer-testis antigen sperm-associated antigen 9 (SPAG9) as early diagnostic and therapeutic target in thyroid cancer. SPAG9 gene and protein expression was determined in thyroid cancer cell lines in 138 thyroid tumor specimens, 60 adjacent noncancerous tissues (ANCT), 22 multinodular goiters (nonneoplastic hyperplasia), and 20 follicular adenoma tissue samples by RT-PCR, in situ RNA hybridization, and immunohistochemistry. Gene silencing approach was used to examine the effects of suppression of SPAG9 protein on cellular growth and colony formation. Humoral immune response against SPAG9 in thyroid cancer patients was analyzed using ELISA. SPAG9 mRNA and protein expression was detected in 78% of the thyroid cancer patients but not multiple goiters and follicular adenoma disease patients. It is interesting to note that majority of early-stage (T1) thyroid cancer patients exhibited higher antibody response against SPAG9. Small interfering RNA-mediated knockdown of SPAG9 expression in thyroid cancer cell significantly reduced cellular growth and colony formation. SPAG9 expression may play a role in cellular growth and thyroid carcinogenesis. These findings support a potential role for SPAG9 as diagnostic biomarker as well as a possible therapeutic target in thyroid cancer treatment.

Sperm‐associated antigen 9 is a biomarker for early cervical carcinoma

Cervical cancer is the second most common malignancy in women, with nearly half a million new cases diagnosed each year worldwide. The authors' recent studies have suggested an association of the cancer testis antigen sperm-associated antigen 9 (SPAG9) in ovarian carcinomas. The aim of the current study was to evaluate the clinical utility of SPAG9 expression and humoral immune response in cervical carcinomas. SPAG9 mRNA expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ RNA hybridization. In addition, the authors investigated SPAG9 protein expression by immunohistochemistry and analyzed its association with various stages and grades of cervical cancer patients. They also tested the humoral immune response against SPAG9 in cervical cancer patients. RT-PCR, in situ RNA hybridization, and immunohistochemical analyses revealed that SPAG9 expression was significantly associated with tumor grades in 82% of early stage cervical cancer specimens. SPAG9 antibodies were detected in approximately 80% of cervical cancer patients, but not in healthy controls. Statistical analysis revealed that a significant proportion of early stage cancer patients with a high SPAG9 immunoreactivity score (IRS) exhibited significantly higher antibody response against SPAG9 compared with moderate SPAG9 IRSs, suggesting a close relation between SPAG9 protein expression and humoral immune response. The current study findings revealed that in early stage cervical cancer, a substantial number of patients exhibited SPAG9 expression and generated SPAG9 antibodies, supporting its potential role in early detection and diagnosis in cervical cancer management. Furthermore, these findings provide leads for future development of noninvasive serologic biomarkers for the early detection, diagnosis, and treatment of cervical cancer.

Sperm-Associated Antigen 9, a Novel Biomarker for Early Detection of Breast Cancer

To date, there have been no tumor biomarkers validated and incorporated into oncologic practice for the early diagnosis of breast cancer. Recently, we showed that sperm-associated antigen 9 (SPAG9), a member of cancer testis (CT) antigen family, is associated with ovarian carcinomas. In the present study, we investigated SPAG9 expression and humoral immune response in breast cancer. We further evaluated the diagnostic potential of autoantibodies to SPAG9 protein in various stages, grades, and histotypes of breast cancer. We analyzed the association of SPAG9 immunoreactivity score (IRS) with predicted risk of breast cancer recurrence over 10 years. Our reverse transcription-PCR and immunohistochemical analyses revealed SPAG9 expression in 88% breast cancer specimens independent of tumor stages and grades. Further, the humoral immune response against SPAG9 was detected in 80% breast cancer patients with SPAG9-expressing tumors. The linear regression modeling predicted a direct relationship between presence of lymphovascular invasion and high SPAG9 IRS, whereas the univariate and multivariate logistic regression models predicted a strong association of SPAG9 IRS with tumor grade. Further, our data indicated a significant higher trend of SPAG9 IRS with the predicted high risk of breast cancer recurrence. The present investigation reports for the first time SPAG9 expression and humoral immune response in early stages and low-grade breast cancer. Although our data indicated that autoantibodies against SPAG9 represent a promising approach for the development of biomarker, further large-scale validation studies are required to establish its potential use in early diagnosis and monitoring of breast cancer recurrence.

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

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.

Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

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.

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein

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.