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• W1972254353 abstract "Colorectal cancer (CRC) is the second most common tumor in developed countries. The present study was undertaken to determine the expression of the sperm-associated antigen 9 gene (SPAG9) as a possible biomarker in CRC, to investigate its correlation with humoral immune response and different stages and grades in CRC patients, and to explore its possible role in colon tumorigenesis in vitro and in an in vivo mouse model. SPAG9 expression was determined by RT-PCR, in situ RNA hybridization, and immunohistochemistry. Humoral response against SPAG9 was detected by enzyme-linked immunosorbent assay and Western blotting. SPAG9 gene silencing was performed using plasmid-based small interfering RNA to study various malignant properties of colon cancer cells in vitro and in vivo. The majority of CRC patients showed SPAG9 expression and generated humoral response. There was a close relationship between SPAG9 protein expression and humoral immune response in the majority of early-stage CRC patients, indicating that anti-SPAG9 antibodies could be a novel serum biomarker for early diagnosis. The down-regulation of SPAG9 (mediated by small interfering RNA) inhibited malignant properties in in vitro and significantly suppressed tumor growth in vivo. These findings collectively suggest that SPAG9 may have a role in tumor development and early spread and thus could serve as a novel target for early detection and for cancer immunotherapy. Colorectal cancer (CRC) is the second most common tumor in developed countries. The present study was undertaken to determine the expression of the sperm-associated antigen 9 gene (SPAG9) as a possible biomarker in CRC, to investigate its correlation with humoral immune response and different stages and grades in CRC patients, and to explore its possible role in colon tumorigenesis in vitro and in an in vivo mouse model. SPAG9 expression was determined by RT-PCR, in situ RNA hybridization, and immunohistochemistry. Humoral response against SPAG9 was detected by enzyme-linked immunosorbent assay and Western blotting. SPAG9 gene silencing was performed using plasmid-based small interfering RNA to study various malignant properties of colon cancer cells in vitro and in vivo. The majority of CRC patients showed SPAG9 expression and generated humoral response. There was a close relationship between SPAG9 protein expression and humoral immune response in the majority of early-stage CRC patients, indicating that anti-SPAG9 antibodies could be a novel serum biomarker for early diagnosis. The down-regulation of SPAG9 (mediated by small interfering RNA) inhibited malignant properties in in vitro and significantly suppressed tumor growth in vivo. These findings collectively suggest that SPAG9 may have a role in tumor development and early spread and thus could serve as a novel target for early detection and for cancer immunotherapy. Colorectal carcinoma (CRC) is the third most common cause of cancer and is the second leading cause of cancer-related death.1Jemal A. Siegel R. Ward E. Hao Y. Xu J. Murray T. Thun M.J. Cancer statistics, 2008.CA Cancer J Clin. 2008; 58: 71-96Crossref PubMed Scopus (10196) Google Scholar Surgical resection is the primary treatment modality for CRC. Chemotherapy and/or radiotherapy may be recommended, depending on the individual patient's staging and other medical factors. Current chemotherapy for advanced CRC yields only a modest improvement in survival.2Gill S. Thomas R.R. Goldberg R.M. New targeted therapies in gastrointestinal cancers.Curr Treat Options Oncol. 2003; 4: 393-403Crossref PubMed Scopus (23) Google Scholar It has been reported, however, that CRC patients have a higher survival rate if the cancer is detected at early stages, before lymphovascular invasion.3Compton C.C. Colorectal carcinoma: diagnostic, prognostic, and molecular features.Mod Pathol. 2003; 16: 376-388Crossref PubMed Scopus (306) Google Scholar Thus, there is an urgent need to identify tumor biomarkers that may be used for early detection and diagnosis of CRC. The underlying principle for pursuing colon cancer screening is that detection of early-stage cancer may help reduce the risk of the disease. Because CRC develops slowly from precancerous lesions that can be removed, and because it is curable at early stages, screening for CRC has the potential to reduce both incidence and mortality of the disease.4Walsh J.M.E. Terdiman J.P. Colorectal cancer screening.JAMA. 2003; 289: 1288-1296Crossref PubMed Scopus (369) Google Scholar However, testing for fecal occult blood and colonoscopy (the primary tools in current use) have the defects of poor compliance and lack of widespread access. A noninvasive biomarker would therefore be of great clinical benefit and could be useful in the early diagnosis of CRC patients. A unique class of testis proteins known as cancer/testis (CT) antigens has been found to be expressed in various malignancies.5Suri A. Cancer testis antigens-their importance in immunotherapy and in the early detection of cancer.Expert Opin Biol Ther. 2006; 6: 379-389Crossref PubMed Scopus (67) Google Scholar Based on their restricted expression in cancerous tissues and in view of their immunogenicity in cancer patients, CT antigens are the ideal targets for cancer immunotherapy and serum-based biomarkers.5Suri A. Cancer testis antigens-their importance in immunotherapy and in the early detection of cancer.Expert Opin Biol Ther. 2006; 6: 379-389Crossref PubMed Scopus (67) Google Scholar Recently, we characterized the sperm-associated antigen 9 gene (SPAG9; HGNC: 14524), newly identified as a member of the CT antigen family, as associated with various malignancies.6Jagadish N. Rana R. Selvi R. Mishra D. Garg M. Yadav S. Herr J.C. Okumura K. Hasegawa A. Koyama K. Suri A. Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.Biochem J. 2005; 389: 73-82Crossref PubMed Scopus (77) Google Scholar The SPAG9 gene generates eight protein isoforms by alternative splicing, and all differ from the canonical sequence of the full-length protein c-Jun-amino-terminal kinase-interacting protein 4 (JIP4 isoform 1; UniProt O60271-1). It has been reported that approximately three-quarters of all human genes undergo alternative splicing,7Johnson J.M. Castle J. Garrett-Engele P. Kan Z. Loerch P.M. Armour C.D. Santos R. Schadt E.E. Stoughton R. Shoemaker D.D. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.Science. 2003; 302: 2141-2144Crossref PubMed Scopus (1192) Google Scholar, 8Clark T.A. Schweitzer A.C. Chen T.X. Staples M.K. Lu G. Wang H. Williams A. Blume J.E. Discovery of tissue-specific exons using comprehensive human exon microarrays.Genome Biol. 2007; 8: R64Crossref PubMed Scopus (231) Google Scholar, 9Kampa D. Cheng J. Kapranov P. Yamanaka M. Brubaker S. Cawley S. Drenkow J. Piccolboni A. Bekiranov S. Helt G. Tammana H. Gingeras T.R. Novel RNAs identified from an in-depth analysis of the transcriptome of human chromosomes 21 and 22.Genome Res. 2004; 14: 331-342Crossref PubMed Scopus (435) Google Scholar which may affect functional role, cellular localization, binding and interacting properties, and stability of the encoded proteins.10Stamm S. Ben-Ari S. Rafalska I. Tang Y. Zhang Z. Toiber D. Thanaraj T.A. Soreq H. Function of alternative splicing.Gene. 2005; 344: 1-20Crossref PubMed Scopus (685) Google Scholar In earlier studies, we found that SPAG9 protein (JIP4 isoform 5; UniProt O60271-5) is involved in the c-Jun N-terminal kinase (JNK) signaling module6Jagadish N. Rana R. Selvi R. Mishra D. Garg M. Yadav S. Herr J.C. Okumura K. Hasegawa A. Koyama K. Suri A. Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.Biochem J. 2005; 389: 73-82Crossref PubMed Scopus (77) Google Scholar, 11Shankar S. Mohapatra B. Suri A. Cloning of a novel human testis mRNA specifically expressed in testicular haploid germ cells, having unique palindromic sequences and encoding a leucine zipper dimerization motif.Biochem Biophys Res Commun. 1998; 243: 561-565Crossref PubMed Scopus (43) Google Scholar and functions as a scaffolding protein for binding to JNKs that play an important regulatory role in several physiological processes, including cell survival, proliferation, apoptosis, and tumor development.6Jagadish N. Rana R. Selvi R. Mishra D. Garg M. Yadav S. Herr J.C. Okumura K. Hasegawa A. Koyama K. Suri A. Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.Biochem J. 2005; 389: 73-82Crossref PubMed Scopus (77) Google Scholar, 12Qi M. Elion E.A. MAP kinase pathways.J Cell Sci. 2005; 118: 3569-3572Crossref PubMed Scopus (415) Google Scholar Cancer-specific alternatively spliced mRNAs and protein isoforms may be used as cancer biomarkers. Our recent study demonstrated SPAG9 expression and its association with clinicopathological characteristics of tumors in renal cell carcinoma,13Garg M. Kanojia D. Khosla A. Dudha N. Sati S. Chaurasiya D. Jagadish N. Seth A. Kumar R. Gupta S. Gupta A. Lohiya N.K. Suri A. Sperm-associated antigen 9 is associated with tumor growth, migration, and invasion in renal cell carcinoma.Cancer Res. 2008; 68: 8240-8248Crossref PubMed Scopus (68) Google Scholar epithelial ovarian cancer,14Garg M. Chaurasiya D. Rana R. Jagadish N. Kanojia D. Dudha N. Kamran N. Salhan S. Bhatnagar A. Suri S. Gupta A. Suri A. Sperm-associated antigen 9, a novel cancer testis antigen, is a potential target for immunotherapy in epithelial ovarian cancer.Clin Cancer Res. 2007; 13: 1421-1428Crossref PubMed Scopus (63) Google Scholar breast cancer,15Kanojia D. Garg M. Gupta S. Gupta A. Suri A. Sperm-associated antigen 9, a novel biomarker for early detection of breast cancer.Cancer Epidemiol Biomarkers Prev. 2009; 18: 630-639Crossref PubMed Scopus (73) Google Scholar cervical carcinoma,16Garg M. Kanojia D. Salhan S. Suri S. Gupta A. Lohiya N.K. Suri A. Sperm-associated antigen 9 is a biomarker for early cervical carcinoma.Cancer. 2009; 115: 2671-2683Crossref PubMed Scopus (55) Google Scholar and thyroid cancer.17Garg M. Kanojia D. Suri S. Gupta S. Gupta A. Suri A. Sperm-associated antigen 9: a novel diagnostic marker for thyroid cancer.J Clin Endocrinol Metab. 2009; 94: 4613-4618Crossref PubMed Scopus (41) Google Scholar These findings suggest that SPAG9 may have a role in early spread of disease. Furthermore, SPAG9 expression was also found to be associated with circulating anti-SPAG9 antibodies in early stages and in low grade of breast cancer15Kanojia D. Garg M. Gupta S. Gupta A. Suri A. Sperm-associated antigen 9, a novel biomarker for early detection of breast cancer.Cancer Epidemiol Biomarkers Prev. 2009; 18: 630-639Crossref PubMed Scopus (73) Google Scholar and cervical cancer patients,16Garg M. Kanojia D. Salhan S. Suri S. Gupta A. Lohiya N.K. Suri A. Sperm-associated antigen 9 is a biomarker for early cervical carcinoma.Cancer. 2009; 115: 2671-2683Crossref PubMed Scopus (55) Google Scholar suggesting its potential use in early detection of disease. In the present study, we systematically investigated SPAG9 mRNA and protein expression, as well as in vivo immunogenicity, in human CRC patients and their role in the tumorigenicity of colon cancer. The findings demonstrated a close relationship between SPAG9 expression and humoral immune response in early stages, pointing to a functional role in tumorigenesis of colon cancer. SPAG9 could be considered as a potential noninvasive biomarker and target molecule for developing antigen-based vaccine and immunotherapeutic approaches for treatment of CRC. The present investigation was conducted with clinical specimens obtained from CRC patients who underwent surgical resection of the tumor, in accordance with the Institutional Ethics Committee and after obtaining informed consent. SPAG9 gene and protein expression was analyzed in 78 CRC tissues, and humoral response against SPAG9 was investigated in the 54 blood samples available from these patients. The study included 52 men and 26 women, with a median age of 54 ± 15.90 years (range, 25 to 86 years). In 26 cases, paired adjacent noncancerous tissue specimens were collected. We also investigated SPAG9 protein expression in 40 control colon tissue samples, obtained from the archives of the Department of Pathology. Clinicopathological characteristics are given in Table 1. Blood samples were also obtained from 50 normal healthy donors.Table 1Clinicopathological Characteristics of Colorectal Carcinoma Patients and SPAG9 ExpressionClinicopathological characteristicsSPAG9 expression⁎The ELISA analysis detected circulating autoantibodies against SPAG9. The RT-PCR and IHC analyses detected SPAG9 mRNA expression.ELISA, n/N (%)RT-PCR/IHC, n/N (%)Tumor, total38/54 (70)58/78 (74)ANCT, total0/26 (0)Normal colon tissue, total0/40 (0)Tumor stages Stage I1/1 (100)7/10 (70) Stage II11/11 (100)16/16 (100) Early stages (I + II)12/12 (100)23/26 (88) Stage III20/33 (61)29/43 (67) Stage IV6/9 (67)6/9 (67) Late stages (III + IV)26/42 (62)35/52 (67)Histologic grades Well differentiated17/23 (74)28/37 (76) Moderately differentiated17/23 (74)26/33 (79) Mucinous4/8 (50)4/8 (50)Lymph node involvement Positive26/41 (63)30/45 (67) Negative12/13 (92)28/33 (85)Metastasis Positive7/10 (70)11/14 (79) Negative31/44 (70)47/64 (73)ANCT, adjacent noncancerous tissue; ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry. The ELISA analysis detected circulating autoantibodies against SPAG9. The RT-PCR and IHC analyses detected SPAG9 mRNA expression. Open table in a new tab ANCT, adjacent noncancerous tissue; ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry. Human colon cancer cell lines COLO 205 and HCT 116 were procured from the American Type Culture Collection (ATCC, Manassas, VA) and were maintained according to standard procedures. COLO 205 or HCT 116 cells (1 × 105) were plated in 6-well plates, allowed to attach, and were transiently transfected using Lipofectamine reagent (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacturer's instructions and as described previously.13Garg M. Kanojia D. Khosla A. Dudha N. Sati S. Chaurasiya D. Jagadish N. Seth A. Kumar R. Gupta S. Gupta A. Lohiya N.K. Suri A. Sperm-associated antigen 9 is associated with tumor growth, migration, and invasion in renal cell carcinoma.Cancer Res. 2008; 68: 8240-8248Crossref PubMed Scopus (68) Google Scholar RT-PCR was performed to investigate the expression of SPAG9 mRNA in colon cancer cells and in CRC tissues. RNA was isolated from colon cancer cells and from 78 colon cancer tissues and 26 paired adjacent noncancerous tissue specimens. RT-PCR was performed using SPAG9 gene-specific primers, as described previously.14Garg M. Chaurasiya D. Rana R. Jagadish N. Kanojia D. Dudha N. Kamran N. Salhan S. Bhatnagar A. Suri S. Gupta A. Suri A. Sperm-associated antigen 9, a novel cancer testis antigen, is a potential target for immunotherapy in epithelial ovarian cancer.Clin Cancer Res. 2007; 13: 1421-1428Crossref PubMed Scopus (63) Google Scholar The PCR product thus obtained was cloned into TOPO vector (Invitrogen, Life Technologies) for confirming the SPAG9 DNA sequence. SPAG9 protein expression in colon cancer cells was examined using indirect immunofluorescence microscopy and flow cytometric analysis using anti-SPAG9 antibody, as described previously.13Garg M. Kanojia D. Khosla A. Dudha N. Sati S. Chaurasiya D. Jagadish N. Seth A. Kumar R. Gupta S. Gupta A. Lohiya N.K. Suri A. Sperm-associated antigen 9 is associated with tumor growth, migration, and invasion in renal cell carcinoma.Cancer Res. 2008; 68: 8240-8248Crossref PubMed Scopus (68) Google Scholar Antibodies to pan cadherin (CH-19, ab6528; Abcam, Cambridge, MA), calnexin (6D195, sc-70481; Santa Cruz Biotechnology, Santa Cruz, CA), GM130 (B-10, sc-55591; Santa Cruz Biotechnology), mitochondria (MTCO2 mitochondrial marker, ab3298; Abcam), and lamin A/C (636, sc-7292; Santa Cruz Biotechnology) were used for colocalization studies. Fluorescence microscopy was performed with a Zeiss confocal microscope. Sectioning of paraffin-embedded CRC tissue was performed in RNase-free water (water treated with diethyl pyrocarbonate) for probing SPAG9 mRNA expression, as described previously.14Garg M. Chaurasiya D. Rana R. Jagadish N. Kanojia D. Dudha N. Kamran N. Salhan S. Bhatnagar A. Suri S. Gupta A. Suri A. Sperm-associated antigen 9, a novel cancer testis antigen, is a potential target for immunotherapy in epithelial ovarian cancer.Clin Cancer Res. 2007; 13: 1421-1428Crossref PubMed Scopus (63) Google Scholar Paraffin-embedded serial sections of CRC specimens were analyzed for the localization of SPAG9 protein using anti-SPAG9 antibody, as described previously.15Kanojia D. Garg M. Gupta S. Gupta A. Suri A. Sperm-associated antigen 9, a novel biomarker for early detection of breast cancer.Cancer Epidemiol Biomarkers Prev. 2009; 18: 630-639Crossref PubMed Scopus (73) Google Scholar We assessed the immunostaining of SPAG9 by counting >500 cells from each CRC specimen in five random fields under ×400 magnification in each tissue section. The SPAG9 immunoreactivity score was defined as the percentage of CRC cells expressing SPAG9 protein. We considered a distinct positive immunoreactivity in a specimen as showing >10% of cancer cells stained for SPAG9 protein. To confirm SPAG9 immunoreactivity, a neutralization experiment was performed by including recombinant SPAG9 protein (15 μg/ml) in the incubation with primary antibody, as described previously.13Garg M. Kanojia D. Khosla A. Dudha N. Sati S. Chaurasiya D. Jagadish N. Seth A. Kumar R. Gupta S. Gupta A. Lohiya N.K. Suri A. Sperm-associated antigen 9 is associated with tumor growth, migration, and invasion in renal cell carcinoma.Cancer Res. 2008; 68: 8240-8248Crossref PubMed Scopus (68) Google Scholar In gene silencing experiments, two independent siRNA target sequences directed against SPAG9 were used (designated SPAG9 siRNA-I and SPAG9 siRNA), along with control siRNA (scrambled SPAG9), as described previously.13Garg M. Kanojia D. Khosla A. Dudha N. Sati S. Chaurasiya D. Jagadish N. Seth A. Kumar R. Gupta S. Gupta A. Lohiya N.K. Suri A. Sperm-associated antigen 9 is associated with tumor growth, migration, and invasion in renal cell carcinoma.Cancer Res. 2008; 68: 8240-8248Crossref PubMed Scopus (68) Google Scholar COLO 205 or HCT 116 cells were transfected with SPAG9 siRNA-I, SPAG9 siRNA, or control siRNA using Invitrogen Lipofectamine transfection reagent according to the manufacturer's instructions. After 48 hours, cells were harvested and processed for immunoblotting to determine SPAG9 protein expression. The effects of down-regulation of SPAG9 expression using SPAG9 siRNA on cellular growth and colony forming ability in COLO 205 or HCT 116 cells were investigated as described previously.18Garg M. Kanojia D. Suri S. Suri A. Small interfering RNA-mediated down-regulation of SPAG9 inhibits cervical tumor growth.Cancer. 2009; 115: 5688-5699Crossref PubMed Scopus (27) Google Scholar The effects of SPAG9 siRNA on cell migration, invasion, and wound healing were assessed as described previously.18Garg M. Kanojia D. Suri S. Suri A. Small interfering RNA-mediated down-regulation of SPAG9 inhibits cervical tumor growth.Cancer. 2009; 115: 5688-5699Crossref PubMed Scopus (27) Google Scholar Male Nii/NIH (S) (nu/nu) nude mice (5 to 6 weeks old) were used and maintained in pathogen-free conditions. Exponentially growing COLO 205 cells (1 × 107) were injected subcutaneously. Once the mice attained a tumor volume of 50 to 100 mm3, SPAG9 siRNA or control siRNA (50 μg) was administered intratumorally, followed by a booster injection with 25 μg of plasmid twice a week for 6 weeks. Kinetics of tumor formation was estimated by measuring tumor size and volume at 3-day intervals. Tumor size was measured with calipers, and tumor volume was determined using the following formula: volume = 0.5 × width2 × length. At the end of the experiment, animals were sacrificed and tumors were dissected, weighed, and processed for immunohistochemical analysis. To detect in vivo inhibition of cellular proliferation by an siRNA approach, murine tumor tissues were processed for histological H&E staining and for immunohistochemistry using anti-Ki-67 and anti-SPAG9 antibody, as described previously.18Garg M. Kanojia D. Suri S. Suri A. Small interfering RNA-mediated down-regulation of SPAG9 inhibits cervical tumor growth.Cancer. 2009; 115: 5688-5699Crossref PubMed Scopus (27) Google Scholar The anti-SPAG9 antibody in CRC patients and in normal healthy donors was detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting using recombinant SPAG9 protein, as described previously.13Garg M. Kanojia D. Khosla A. Dudha N. Sati S. Chaurasiya D. Jagadish N. Seth A. Kumar R. Gupta S. Gupta A. Lohiya N.K. Suri A. Sperm-associated antigen 9 is associated with tumor growth, migration, and invasion in renal cell carcinoma.Cancer Res. 2008; 68: 8240-8248Crossref PubMed Scopus (68) Google Scholar Western blot analysis for detecting circulating anti-SPAG9 antibodies was also performed by resolving recombinant SPAG9 protein (0.5 μg/lane) in SDS-polyacrylamide gel electrophoresis and transferring onto polyvinylidene fluoride membrane, as described previously.13Garg M. Kanojia D. Khosla A. Dudha N. Sati S. Chaurasiya D. Jagadish N. Seth A. Kumar R. Gupta S. Gupta A. Lohiya N.K. Suri A. Sperm-associated antigen 9 is associated with tumor growth, migration, and invasion in renal cell carcinoma.Cancer Res. 2008; 68: 8240-8248Crossref PubMed Scopus (68) Google Scholar Statistical analyses were performed using Student's t-test, Mann-Whitney U-test and Pearson's χ2 test (Table 2). Results are expressed as means ± SE and represent the results of at least three experiments. Statistical calculations were performed using SPSS version 16.0 (SPSS, Chicago, IL). A two-sided significance level of 0.05 was used for all statistical tests.Table 2Statistical AnalysisClinicopathologic featuresMann-Whitney U-test (P value)Pearson's χ2 test (P value)RT-PCR/IHCELISART-PCR/IHCELISATumor stage I + II0.3490.008⁎P < 0.05 (statistically significant).0.020⁎P < 0.05 (statistically significant).0.020⁎P < 0.05 (statistically significant). II + III0.9810.2810.009⁎P < 0.05 (statistically significant).0.013⁎P < 0.05 (statistically significant). III + IV0.3580.0510.7400.740 I + II & III + IV0.3320.5720.044⁎P < 0.05 (statistically significant).0.011⁎P < 0.05 (statistically significant).Histological grade WD, MD + mucinous——0.2390.393 WD + MD0.6170.011⁎P < 0.05 (statistically significant).0.7570.900 WD + mucinous0.9120.0910.1460.213 MD + mucinous0.3400.1350.0990.213Lymph node positivity0.4930.3380.0690.047⁎P < 0.05 (statistically significant).Metastasis positivity0.6910.018⁎P < 0.05 (statistically significant).0.6900.977ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry; MD, moderately differentiated; WD, well differentiated. P < 0.05 (statistically significant). Open table in a new tab ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry; MD, moderately differentiated; WD, well differentiated. Our RT-PCR analysis showed that 58 of 78 (74%) CRC patients expressed SPAG9 mRNA. Both COLO 205 and HCT 116 colon cancer cells also expressed SPAG9 mRNA. No SPAG9 expression was detected in adjacent noncancerous tissue specimens. The size of the PCR product in tumor tissues, colon cancer cells, and in testis was the same (Figure 1A) and showed no mutations, which was confirmed by DNA sequence analysis. The relationship between SPAG9 mRNA expression and clinicopathological features is summarized in Table 1. SPAG9 expression was detected in 23 of 26 (88%) early stage (I and II) and in 35 of 52 (67%) late stage (III and IV) categories of CRC specimens. Based on histopathological grading categories, SPAG9 mRNA expression was detected in 28 of 37 (76%) well differentiated, 26 of 33 (79%) moderately differentiated, and 4 of 8 (50%) mucinous type of CRC specimens. Further, SPAG9 expression was detected in 30 of 45 (67%) CRC specimens with positive lymph node involvement as compared to 28 of 33 (85%) CRC specimens with negative lymph node involvement. In addition, our data indicated that 11 of 14 (79%) metastatic CRC patients revealed SPAG9 expression as compared to 47 of 64 (73%) negative metastatic CRC patients. Statistical analysis using Pearson's χ2 test revealed a significant association between SPAG9 expression and various tumor stages of CRC patients, (early stages I + II, P = 0.020; late stages II + III, P = 0.009; and both early and late stages (P = 0.044) (Table 2), suggesting a close relationship between SPAG9 expression and tumor stage. However, SPAG9 mRNA expression was independent of tumor histological grade, lymph node, and metastasis positivity, indicating no correlation between SPAG9 expression and tumor grade (P = 0.239), lymph node positivity (P = 0.069) and metastasis (P = 0.690). These results suggest that SPAG9 may participate in early cancer growth. Indirect immunofluorescence assays showed strong SPAG9 immunoreactivity in virtually all of the cancer cells, with predominant expression in cytoplasm in fixed and permeabilized cells (Figure 1B) and surface localization in live cells (Figure 1B). To further examine the SPAG9 colocalization with various cell organelles, multiple markers were used, including a plasma membrane marker (pan cadherin), Golgi marker (GM130), an endoplasmic reticulum marker (calnexin), a mitochondrial marker (MTCO2), and nuclear marker (lamin A/C). SPAG9 colocalized with the plasma membrane, Golgi marker, and endoplasmic reticulum markers (Figure 1C), but did not appear to colocalize directly with the mitochondria marker or with nuclear lamin (Figure 1C). Thus, SPAG9 has distinct cytoplasmic and surface localization and colocalization with Golgi, endoplasmic reticulum, and plasma membrane. The surface localization was further confirmed by flow cytometric analysis in COLO 205 and HCT 116 cells (Figure 1D), indicating that SPAG9 may be a potential target molecule for antigen-based vaccine and immunotherapy. Our in situ RNA hybridization studies using antisense riboprobe further confirmed the expression of SPAG9 mRNA in all stages (Figure 2), whereas sense riboprobe failed to show SPAG9 expression. Subsequently, SPAG9 protein expression was validated by immunohistochemistry in serial tissue sections of CRC and adjacent noncancerous tissue specimens (Figure 3). Notably, cytoplasmic localization of SPAG9 protein expression was observed in 58 of 78 (74%) CRC specimens expressing SPAG9 mRNA, but no expression was detected in 26 paired adjacent noncancerous tissue specimens or in 40 control colon tissue specimens.Figure 3Immunohistochemical staining of CRC specimens for SPAG9 protein expression. Representative H&E- stained sections of stage I, II, III, and IV are shown. Serial tissue sections at different stages revealed SPAG9 cytoplasmic localization; however, no SPAG9 immunoreactivity was observed in ANCT and control colon specimens or with control IgG. The H&E-stained sections for stages I-IV are identical to those shown in Figure 2. Original magnification, ×200; objective, 20×.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A significant association was found between SPAG9 expression and various stages [stage I + II (P = 0.020), stages II + III (P = 0.009), and stages I + II & III + IV (P = 0.044)], using Pearson's χ2 test, indicating that SPAG9 expression is strongly associated with tumor stage (Table 2). However, no significant association was found between SPAG9 expression and histological grades (P = 0.239) using the Pearson's χ2 test. Furthermore, based on SPAG9 immunoreactivity score in colorectal tumors, two groupings were analyzed: i) a moderate-score group, with ≤50% tumor cells expressing SPAG9 protein; and ii) a high-score group, with >50% tumor cells expressing SPAG9 protein. Our results indicated that 46 of 58 (79%) CRC patients had high SPAG9 immunoreactivity scores (70.48 ± 1.8), the remainder (12 of 58) having moderate SPAG9 immunoreactivity scores (38.58 + 2.28). When we compared the moderate and high SPAG9 immunoreactivity score groups in terms of stage of disease, using the Mann-Whitney U-test, a significant difference (P < 0.0001) was found, with a greater number of CRC patients demonstrating high SPAG9 expression at early than at late stages. This finding suggests that a majority of CRC patients exhibited elevated SPAG9 protein expression during early stages of the disease. Thus, our analysis on SPAG9 immunoreactivity scores in tumors of early stages indicates that this biomarker may have good predictive value for the early detection of cancer. The circulating anti-SPAG9 antibodies were determined in sera of 54 CRC patients, using an enzyme-linked immunosorbent assay (Figure 4A). The enzyme-linked immunosorbent assay titers greater than the mean + 2 SD (0.240 + 0.06) of the 50 normal healthy sera were considered positive. Antibodies against SPAG9 were detected in 70% (38 of 54) in various stages and histological grades of CRC patients (Table 1). The presence of autoantibodies against SPAG9 in the sera of CRC patients found positive in enzyme-linked immunosorbent assay was further examined by Western blotting, which exhibited immunoreactivity against SPAG9 pr" @default.
• W1972254353 created "2016-06-24" @default.
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• W1972254353 date "2011-03-01" @default.
• W1972254353 modified "2023-10-16" @default.
• W1972254353 title "Sperm-Associated Antigen 9 Is a Novel Biomarker for Colorectal Cancer and Is Involved in Tumor Growth and Tumorigenicity" @default.
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• W1972254353 doi "https://doi.org/10.1016/j.ajpath.2010.11.047" @default.
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