Siddartha Datta Gupta

Discovery and Verification of Head-and-neck Cancer Biomarkers by Differential Protein Expression Analysis Using iTRAQ Labeling, Multidimensional Liquid Chromatography, and Tandem Mass Spectrometry

Multidimensional LC-MS/MS has been used for the analysis of biological samples labeled with isobaric mass tags for relative and absolute quantitation (iTRAQ) to identify proteins that are differentially expressed in human head-and-neck squamous cell carcinomas (HNSCCs) in relation to non-cancerous head-and-neck tissues (controls) for cancer biomarker discovery. Fifteen individual samples (cancer and non-cancerous tissues) were compared against a pooled non-cancerous control (prepared by pooling equal amounts of proteins from six non-cancerous tissues) in five sets by on-line and off-line separation. We identified 811 non-redundant proteins in HNSCCs, including structural proteins, signaling components, enzymes, receptors, transcription factors, and chaperones. A panel of proteins showing consistent differential expression in HNSCC relative to the non-cancerous controls was discovered. Some of the proteins include stratifin (14-3-3σ); YWHAZ (14-3-3ζ); three calcium-binding proteins of the S100 family, S100-A2, S100-A7 (psoriasin), and S100-A11 (calgizarrin); prothymosin α (PTHA); l-lactate dehydrogenase A chain; glutathione S-transferase Pi; APC-binding protein EB1; and fascin. Peroxiredoxin2, carbonic anhydrase I, flavin reductase, histone H3, and polybromo-1D (BAF180) were underexpressed in HNSCCs. A panel of the three best performing biomarkers, YWHAZ, stratifin, and S100-A7, achieved a sensitivity of 0.92 and a specificity of 0.91 in discriminating cancerous from non-cancerous head-and-neck tissues. Verification of differential expression of YWHAZ, stratifin, and S100-A7 proteins in clinical samples of HNSCCs and paired and non-paired non-cancerous tissues by immunohistochemistry, immunoblotting, and RT-PCR confirmed their overexpression in head-and-neck cancer. Verification of YWHAZ, stratifin, and S100-A7 in an independent set of HNSCCs achieved a sensitivity of 0.92 and a specificity of 0.87 in discriminating cancerous from non-cancerous head-and-neck tissues, thereby confirming their overexpressions and utility as credible cancer biomarkers.

Clinical significance of promoter hypermethylation of DNA repair genes in tumor and serum DNA in invasive ductal breast carcinoma patients

AIMS The clinical relevance of frequent methylation of CpG islands of key cancer genes in breast cancer is being increasingly recognized. Our study aimed to evaluate the promoter methylation status of DNA repair genes-BRCA1MGMT and GSTP1 in tumor and circulating DNA of invasive ductal breast carcinoma patients. MAIN METHODS Methylation-specific PCR was carried out to investigate the promoter methylation status of genes in tumor and circulating DNA of 100 breast cancer patients in a prospective study. The effect of promoter methylation on protein expression was evaluated by immunohistochemistry. KEY FINDINGS The frequency of tumor hypermethylation was 27% in BRCA1, 32% in MGMT and 25% in GSTP1 and correlated with methylation of these genes in paired serum DNA. Immunohistochemical analysis showed no detectable expression of BRCA1 and MGMT in 51/89 (57%) and 35/89 (39%) tumors, respectively. MGMT promoter methylation mediated gene silencing was associated with loss of its protein expression (p=0.002, O.R.=4.5, 95% C.I.=1.7-12.0). BRCA1 promoter methylation was not associated with loss of its protein expression, indicating that methylation is not the sole mechanism accounting for the loss/reduced BRCA1 protein expression. Importantly, GSTP1 and BRCA1 hypermethylation were found to be independent of other prognostic factors in predicting disease recurrence (p=0.02, HR=7.6, 95% C.I.=1.4-44.1; p=0.04, HR=6.2, 95% C.I.=1.1-35.7). SIGNIFICANCE Our study underscores the potential utility of DNA methylation of these genes in serum as a promising biomarker and can serve as a surrogate for tumor DNA methylation for diagnosis and prognosis of breast cancer.

CpG hypomethylation of MDR1 gene in tumor and serum of invasive ductal breast carcinoma patients.

OBJECTIVES Multidrug resistance 1 (MDR1) gene encodes P-glycoprotein (P-gp), a transmembrane calcium-dependent efflux pump, implicated in drug resistance. In this prospective study, methylation status of MDR1 promoter and its correlation with clinicopathological parameters were evaluated in tumor and serum of breast cancer patients. DESIGN AND METHODS Methylation-specific PCR was carried out to investigate the promoter methylation status of MDR1 in tumor and serum of 100 patients with invasive ductal carcinomas of breast (IDCs). The effect of promoter methylation on protein expression was evaluated by immunohistochemistry. RESULTS MDR1 was hypomethylated in 47% tumors and 44% paired sera of IDC patients and correlated significantly with increased tumor size and advanced tumor stage. Promoter hypomethylation of MDR1 in serum DNA showed 98% specificity and 50% sensitivity. CONCLUSIONS Hypomethylation of MDR1 promoter in IDCs accounted for P-gp overexpression and aggressive biologic behavior in a subset of patients. Detection of these epigenetic changes in circulating DNA may not only enhance insight into the biological behavior of the primary tumor of an individual but may also provide valuable information regarding prognosis that can be readily monitored throughout the disease course.

Prognostic relevance of promoter hypermethylation of multiple genes in breast cancer patients

Background: Methylation-mediated suppression of detoxification, DNA repair and tumor suppressor genes has been implicated in cancer development. This study was designed to investigate the impact of concurrent methylation of multiple genes in breast tumors on disease prognosis. Methods: Methylation specific PCR was carried out to analyze the methylation status of seven genes in archived breast tissues and determine the effect of aberrant methylation of multiple genes on disease prognosis and patients’ survival. Results: Promoter hypermethylation was observed in PRB 67%, ERα 64%, RASSF1A 63%, p16INK4A 51%, PRBβ2 22%, GSTP1 25% and BRCA1 27% of the breast cancers, respectively. Concurrent methylation of BRCA1, ERα, GSTP1 and RARβ2, was observed in a large proportion of breast cancers analyzed, suggesting that these genes do not appear to be methylated alone. Patients with high methylation indices had poor prognosis (p < 0.001, Hazards ratio = 14.58). Cox regression analysis showed RARβ2 promoter methylation to be an independent important determinant of breast cancer prognosis. Conclusions: Our results suggest that methylation of multiple genes plays an important role in prognosis of breast cancer. Our study not only describes the association of methylation mediated silencing of multiple genes with the severity of disease, but also drives to speculate the molecular crosstalk between genes or genetic pathways regulated by them individually.

Clinical significance of Stratifin, ERα and PR promoter methylation in tumor and serum DNA in Indian breast cancer patients

OBJECTIVES The objective of this study was to determine the concordance of promoter methylation of stratifin, ERalpha and PR in tumor and circulating DNA in breast cancer patients and their association with clinicopathological parameters and disease prognosis. DESIGN AND METHODS Methylation specific PCR were carried out to investigate the promoter methylation status of stratifin, ERalpha and PR in tumor and circulating DNA in 100 breast cancer patients in a prospective study. The effect of promoter methylation on protein expression was evaluated by immunohistochemistry. RESULTS Significant association was observed between promoter methylation of stratifin in tumors (61%) and paired sera (56%) (r=0.78; p < or = 0.001). Loss of stratifin expression was observed in 47% tumors and was associated with poor overall survival (p=0.05). Significant correlation was observed between methylation status of ERalpha with PRB (p<0.0001, OR=20.8, 95% CI=7.4-58.0) and stratifin (p=0.003, OR=2.0, 95% CI=0.8-4.4). CONCLUSION This study underscores the potential utility of serum DNA methylation of these genes as surrogate for tumor DNA methylation as a promising tool for cancer diagnosis.

Clinical significance of Maspin promoter methylation and loss of its protein expression in invasive ductal breast carcinoma: correlation with VEGF-A and MTA1 expression

Maspin is a serine protease inhibitor with tumor-suppressor activity. Maspin can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. Previous studies indicate that the loss of Maspin expression is closely linked to aberrant methylation of the Maspin promoter. We examined the promoter methylation status of Maspin in tumor and corresponding serum of breast cancer patients. In addition, protein expression of this gene was also assessed to determine possible correlation between promoter hypermethylation and gene silencing. Further, we investigated the correlation of Maspin expression with vascular endothelial growth factor (VEGF-A) and MTA1 expression. Maspin methylation was analyzed by methylation-specific PCR in 100 invasive ductal breast carcinoma patients’ tumors and circulating DNA in a prospective study. Promoter hypermethylation was correlated with expression of the encoded protein in tumors by immunohistochemistry. Significant correlation was observed between promoter hypermethylation of Maspin (r = +0.88; p ≤ 0.0001) in tumors and paired sera. Significant association was found between Maspin promoter hypermethylation and loss of its protein expression (p = 0.01, OR = 3.1, 95% CI = 1.3–7.4). The expression of VEGF-A and MTA1 was lower in tumors with high Maspin expression compared to tumors with loss of Maspin expression. Our results indicate that aberrant promoter methylation is associated with loss of Maspin immunoreactivity in breast cancer tissues. Further, loss of Maspin expression is significantly correlated with increased expression of VEGF-A and MTA1.

DNA methylation of circulating DNA: a marker for monitoring efficacy of neoadjuvant chemotherapy in breast cancer patients

Identification of biomarkers for monitoring efficacy of neoadjuvant chemotherapy in breast cancer patients is of utmost importance in individual tailoring of treatment and save from toxicity due to non-effective drugs. We hypothesized that methylation of circulating tumor-specific DNA may reflect changes in tumor burden in response to chemotherapy and help stratify responders from non-responders. The aim of this study was to evaluate the potential of methylation changes in circulating DNA to monitor treatment response of breast cancer patients. Six consecutive sera samples collected from 30 breast cancer patients undergoing neoadjuvant chemotherapy were analyzed for methylation status of a panel of five genes namely, BRCA1, MGMT, GSTP1, Stratifin, and MDR1. Among these five genes, BRCA1 methylation frequency was different among responders and non-responders groups. The correlation coefficients between total gene methylation with initial chemotherapy and tumor volume reduction were R2 = 0.8 and R2 = 0.05 in the responders and non-responders groups, respectively. Our findings warrant further development of this approach for monitoring response in patients undergoing neoadjuvant chemotherapy.

Clinical significance of GPR56, transglutaminase 2, and NF-κB in esophageal squamous cell carcinoma.

Proteins do not operate as individual units, and components of intracellular canonical pathways often cross talk in tumor genesis. We hypothesized that G-protein-coupled receptor 56 (GPR56), transglutaminase (TG2), and nuclear factor-κB (NF-κB) may collaborate in interconnected pathways and contribute to the aggressive behavior of esophageal squamous cell carcinoma (ESCC). Immunohistochemical analysis of GPR56, TG2, and NF-κB was carried out using ESCC tissue microarrays. Immunostaining of all the three proteins revealed a significant increase in their expression in ESCCs as compared with normal epithelia and correlated with their concomitant expression. A significant correlation between GPR56, TG2, and NF-κB was observed that correlated with nodal metastasis and tumor invasion in ESCCs.

Clinical significance of promoter hypermethylation of RASSF1A, RARβ2, BRCA1 and HOXA5 in breast cancers of Indian patients

Promoter hypermethylation of genes is implicated in the pathogenesis of many cancers, including breast cancer. Herein, we analyzed the promoter methylation status of a panel of critical growth regulatory genes, RASSF1A, RARbeta2, BRCA1 and HOXA5, in 54 breast cancers and 5 distant normal breast tissues of Indian patients. The methylation data were correlated with clinicopathological characteristics and hormone receptor status to determine the impact of methylation in breast carcinogenesis. Promoter hypermethylation of RASSF1A was observed in 39/54 (72%), HOXA5 in 36/54 (67%), BRCA1 in 15/54 (28%) and RARbeta2 in 8/54 (15%) breast cancers. Our most significant findings were the association of RASSF1A methylation with nodal metastasis (p=0.05); and RARbeta2 methylation with age (all tumors in patients in the older age group were methylated, p=0.04). Further, the interactions between DNA methylation and hormone receptor biology in breast cancer cells are beginning to be clearly understood. In this context the association of HOXA5 methylation with loss of ERalpha (p=0.009) is noteworthy.

Overexpression of a splice variant of oncostatin M receptor beta in human esophageal squamous carcinoma

BackgroundExpression of oncostatin M receptor beta (OSMRβ) has been reported in human cancers, however its role in esophageal squamous cell carcinoma (ESCC) remains unknown. Using differential display, earlier we reported the identification of an alternatively spliced variant of OSMRβ in ESCC. Here in we characterized this novel variant encoding a soluble form of this receptor (sOSMRβ) and determined its clinical significance and correlation with the expression of oncostatin (OSM) and leukemia inhibitory factor receptor beta (LIFR β) in ESCC.Materials and MethodsIn silico analysis was carried out to characterize the differentially expressed transcript of OSMRβ and its expression was determined in ESCCs and matched normal esophageal tissues using semiquantitative RT-PCR. The expressions of both truncated and full length OSMRβ proteins were analyzed in ESCC tissues and patients’ sera using western blotting and immunoprecipitation. By immunoprecipitation we have also shown direct interaction between sOSMRB and OSM. We also explored the relationship between expression of OSM and its receptors, OSMRβ and LIFRβ, in primary human ESCCs and normal epithelia using immunohistochemistry.ResultsOverexpression of alternatively spliced OSMR β transcript was detected by RT-PCR in 9 of 11 ESCCs. Analysis of the soluble receptor revealed absence of sOSMRβ protein in esophageal tissues, however, immunoprecipitation and western blot analysis showed its presence in sera of ESCC patients further confirming expression of the alternatively spliced OSMR β in ESCC patients. Immunohistochemical analysis in tissue microarray (TMA) format showed expression of OSMR β, LIFR and OSM in 11/50 (23%), 47/50 (94%) and 47/50 (94%) ESCCs, respectively. Strong correlation was observed between cytoplasmic expression of LIFRβ and OSM in tumor cells (p = 0.000, O.R = 50, 95%CI = 8–31.9), and nuclear expression of LIFRβ and OSM (p = 0.039 OR = 3.1, 95% CI = 1.1–8.2), suggesting that LIFRβ serves as the major receptor in ESCCs.ConclusionAn alternatively spliced variant of OSMR transcribing a soluble form of this receptor has been characterized in ESCC. We speculate that the truncated OSMR characterized here in may act as a neutralizing receptor for OSM. Our immunohistochemical study showed that OSMRβ and its pathway is not activated in ESCCs.