SP17 is a mammalian protein found in the testis and spermatozoa that have been identified as a tu... more SP17 is a mammalian protein found in the testis and spermatozoa that have been identified as a tumor-associated antigen in a range of human cancers. A unique method for fabricating the first ultrasensitive, selective, and label-free immunosensor for the detection of SP17, a new cancer biomarker in complicated serum samples, is presented in this paper. This immunosensor was also the first biosensor built using a disposable ITO sheet modified with an aminosilane known as APTMS as an immobilization platform for fabricating the SP17 biosensor. The immobilization of chemical and biological species onto the electrode surface was cross-verified by various analytical and morphological techniques. Stepwise modifications done on the immunoelectrodes were also studied using electrochemical techniques. Selective interaction between anti-SP17 and SP17 with varying concentrations (100-5000 pg mL-1) was measured with the DPV technique. The immunosensor exhibited low LOD and LOQ of 70.07 and 233.57 pg mL-1, respectively, with a sensitivity of 0.013 μA mL pg-1 cm-2. The fabricated immunosensor performance was analyzed by quantifying the SP17 concentrations in patient serum samples. The data obtained from the developed immunosensor demonstrated excellent reproducibility, repeatability, and selectivity among various interferants, including cancer biomarkers. Further, the observed results have been validated via ELISA, which showed good agreement with the electrochemical results. This could establish a new platform for detecting other cancer biomarkers and can be employed for clinical diagnostics applications.
MARCH8 is an E3 ligase, primarily involved in immune-modulation. Recently, we reported its aberra... more MARCH8 is an E3 ligase, primarily involved in immune-modulation. Recently, we reported its aberrant expression in human esophageal squamous cell carcinoma. However, exact mechanisms by which it regulates cancer have been poorly understood. We applied high-throughput quantitative proteomics approach to identify downstream protein targets of MARCH8. Silencing of endogenous MARCH8 in ESCC cells followed by LC-MS/MS analysis led to identification of 1029 unique proteins showing altered expression post MARCH8 knockdown. Several previously reported MARCH8 target proteins viz. TFR1, syntaxin-4, e-cadherin and CD44 were found to be upregulated. Furthermore, new putative targets of MARCH8, including β2M, were identified in the present study. We demonstrated that MARCH8 interacts with and ubiquitinates CDH1 and β2M. Inhibiting proteasome activity with MG132 prevented CDH1 and β2M degradation, indicating that MARCH8 might be targeting CDH1 and β2M for proteasomal degradation. Further, loss of β2M and CDH1 expression significantly and inversely correlated with MARCH8 expression in ESCC tissues (r = -0.737 and - 0.651, respectively; p < 0.01). In conclusion, our present study has led to identification of new targets of MARCH8 and suggests the role of MARCH8 in regulating CDH1 and β2M turnover in esophageal cancer cells. SIGNIFICANCE: The use of quantitative proteomics carried out has led to the recognition of new targets of MARCH8. The present study gives a broad understanding of the molecular remodeling arising in the ESCC after MARCH8 knockdown. The study also solidifies the idea that role of MARCH8 is not just limited to immunomodulation as silencing of MARCH8 affects various other processes such as protein processing and localization. This study might help in understanding the regulation of MARCH8 in ESCCs and the mechanism by which MARCH8 might be facilitating cancer cells to evade immune surveillance.
Esophageal cancer (EC) is a highly complex disease with high incidence and mortality rates. Recen... more Esophageal cancer (EC) is a highly complex disease with high incidence and mortality rates. Recent studies have shown that miRNAs play critical roles in diverse biological processes including oncogenesis, and we previously reported significantly increased expression of tissue and circulating miR-144 in EC. This study evaluates the functional significance of miR-144 in esophageal squamous cell carcinoma. Herein, we analysed the role of miR-144 in ESCC by silencing it in KYSE-410 cells, and followed this with cell cycle analysis and the following assays; MTT, annexin, colony formation, scratch and matrigel invasion assay. The miR-144 knockdown significantly suppressed ESCC cell proliferation by 72 hours post transfection (p=0.029). This significantly decreased the migration, invasion and colony formation potential of KYSE-410 cells compared to cells treated with negative control (NC). Potential targets of miR-144 were predicted by the in-silico approach followed by in-vitro validation...
Background: Growing evidence suggests importance of aberrant microRNA (miRNA) expression in devel... more Background: Growing evidence suggests importance of aberrant microRNA (miRNA) expression in development and progression of cancer. Recent studies have demonstrated the potential of miRNA panels as promising diagnostic, prognostic and predictive biomarkers. Objective: The purpose of the present study was to evaluate the clinical and functional significance of miRNAs in esophageal cancer (EC). Methods: Herein, we evaluated the diagnostic potential of a five miRNA panel (consisting of miR-21, miR-144, miR-107, miR-93 and miR-342) in EC detection using quantitative real time PCR (qRTPCR). To evaluate the discriminatory power of miRNA panel, receiver operating characteristic curve (ROC) curves were generated for each of the miRNAs followed by risk score analysis. Briefly, linear regression models were fitted using the cancer status and each of the risk score as input. ROC curves were then used to evaluate the diagnostic potential of the panel and to find out the appropriate cut-off point...
The International Journal of Biological Markers, 2008
We recently found 14-3-3 zeta to be overexpressed in esophageal squamous cell carcinomas (ESCCs) ... more We recently found 14-3-3 zeta to be overexpressed in esophageal squamous cell carcinomas (ESCCs) by differential display. In the present study we determined the clinical significance of 14-3-3 zeta in esophageal tumorigenesis. Immunohistochemical analysis was carried out in 61 ESCCs, 33 dysplasia samples, 14 hyperplasia samples and 7 matched histologically normal esophageal tissues and correlated with clinicopathological parameters. Cytoplasmic expression of 14-3-3 zeta protein was observed in 95% of ESCCs; 63% of tumors also showed nuclear localization. All hyperplastic and dysplastic tissues distant from ESCCs as well as dysplastic endoscopic biopsies showed cytoplasmic immunopositivity for 14-3-3 zeta, while nuclear localization was observed in 58% of dysplasia and 36% of hyperplasia samples. Matched distant histologically normal epithelia either showed basal cytoplasmic expression of 14-3-3 zeta or no detectable nuclear expression of the protein. Interestingly, immunopositivity observed in normal esophageal tissues and early hyperplasia was confined to cytoplasm only, though significant nuclear expression was detected in dysplasia and ESCC. Immunoblotting and RT-PCR analyses further confirmed 14-3-3 zeta expression in dysplasia and ESCC. To our knowledge, this is the first report demonstrating overexpression of 14-3-3 zeta in esophageal hyperplasia, dysplasia and squamous cell carcinoma, suggesting that alteration in its expression occurs in early stages and is associated with esophageal tumorigenesis.
Journal of Cancer Research and Clinical Oncology, 2001
The p16/cyclin D1/pRb pathway plays a critical role in tumourigenesis. We recently reported alter... more The p16/cyclin D1/pRb pathway plays a critical role in tumourigenesis. We recently reported alterations in expression of tumour suppressor gene products, p16 and pRb in esophageal cancer. Knowledge of alterations in cyclin D1, a vital component of this pathway in esophageal carcinomas from the Indian subcontinent, where the etiology and pathogenesis may be confounded by various unique dietary and environmental factors, is presently scanty. In order to bridge the gap between the accentuating incidence of esophageal cancer and aberrations in the components of this vital pathway, we analysed cyclin D1 expression in esophageal squamous cell carcinoma in the Indian population. Immunohistochemical analysis of cyclin D1 expression was carried out in paraffin-embedded sections of surgically resected esophageal squamous cell carcinomas (ESCC) (70 patients) and matched with histopathologically normal esophageal tissues from a distant site. The findings were correlated with clinicopathological parameters. Overexpression of cyclin D1 was observed in the tumour nuclei in 41 out of 70 (59%) patients. We found concomitant alterations in 16 and cyclin D1 (p16-/CycD1+ phenotype) in 16 of the 70 patients (23%), while alterations of pRb and cyclin D1 (pRb-/CycD1+) were observed in 36 of the 70 (51%) patients of ESCCs. Cyclin D1 overexpression was significantly associated with the loss of p16 immunoreactivity (P = 0.005). The pRb- and p16-/pRb-/Cyc D+ phenotypes showed significant association with differentiation of the tumour (P = 0.005, 0.05, respectively). Kaplan-Meier analysis for disease recurrence showed increased disease recurrence in cyclin D1 overexpressed patients. Median time to disease recurrence in the cyclin D1+ group was 15 months as against 18 months observed in the cyclin D1- patients (P = 0.067; log-rank test). Alterations in at least one of the components of the p16/cyclin D1/pRb pathway in majority of the 70 patients analysed herein, and concomitant alterations in all the three proteins in 19 patients (35%) underscore the critical role of this pathway in esophageal tumourigenesis. The results of the present study taken together with our previous findings on p16 and pRb alterations in ESCCs suggest that these alterations are not mutually exclusive and may cooperatively provide greater tumour growth advantage. The prognostic significance of alterations in the expression of these components cyclin D1, p16, and pRb remains to be established in a larger cohort.
Journal of Cancer Research and Clinical Oncology, 2004
Matrix metalloproteinases (MMPs) are known to play an important role in extracellular matrix remo... more Matrix metalloproteinases (MMPs) are known to play an important role in extracellular matrix remodeling during the process of tumor invasion and metastasis. However, little is known about their role in preinvasive lesions and early esophageal carcinomas. Method: Immunohistochemical analysis of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression was carried out in paraffin-embedded sections of surgically resected esophageal squamous cell carcinoma (ESCC) (58 cases) and paired distal normal esophageal tissues (44 cases) and correlated with clinicopathological parameters. Result: Overexpression of MMP-2 and MMP-9 proteins was observed in 39 (67%) and 32 (55%) of the 58 ESCCs, respectively localized in tumor cell cytoplasm and stromal elements. Histological evaluation of hematoxylin-and eosin-stained 44 matched distal normal esophageal tissue sections revealed that 26 comprised of normal epithelium, while 15 tissues showed evidence of dysplasia and three tissues showed hyperplasia. Interestingly, 12 (80%) and 13 (87%) of these 15 dysplasias showed immunostaining for MMP-2 and MMP-9 proteins, respectively. Low levels of MMP-2 and MMP-9 were observed in 10 (38%) and 6 (23%) of 26 matched histologically normal esophageal tissues, respectively. Higher MMP-2 immunopositivity was observed in well and moderately differentiated SCCs in comparison with poorly differentiated tumors. The expression of MMP-2 was significantly reduced with the progressive de-differentiation of esophageal SCCs (P =0.03). Overexpression of MMP-2 and MMP-9 in dysplasia as well as SCC suggests that these alterations occur in early stages of esophageal tumorigenesis. Conclusion: Increased levels of MMP-2 and MMP-9 proteins in ESCCs as compared to normal esophageal tissues suggest their association with esophageal tumorigenesis. Increased levels of these MMPs are observed in majority of dysplasias analyzed herein, indicating that these alterations may be early events in esophageal tumorigenesis. In-depth studies are warranted to determine their role in development and progression of esophageal cancer.
The pRb (p16-pRb-cyclin D1) and p53 (p53-MDM2-p21) pathways play a critical role in tumorigenesis... more The pRb (p16-pRb-cyclin D1) and p53 (p53-MDM2-p21) pathways play a critical role in tumorigenesis. To evaluate which of these cell cycle regulatory proteins are related to patients' prognosis, a comprehensive analysis of alterations in these components was carried out in 100 ESCCs (oesophageal squamous cell carcinoma) using immunohistochemistry and correlated with clinicopathological parameters by univariate analysis. Overexpression of p53, MDM2 and cyclin D1 proteins was observed in 73, 42 and 67% of the cases, respectively, while loss of expression of p21, p16 and pRb was observed in 36, 45 and 75% of the cases, respectively. Multiple logistic regression analysis revealed that loss of p16 immunoreactivity was a significant risk factor for tumour stage (pT) (Odds Ratio (OR)=3.3), whereas the loss of pRb was a significant risk factor for nodal metastasis (pN) (OR=8.8). MDM2 overexpression emerged as the most significant risk factor for distant organ metastasis (pM) (OR=4.6). Of the ESCC patients who underwent oesophagectomy, 50 cases were followed-up for a maximum period of 44 months and median of 16 months. Survival analysis revealed that Cyclin D1 overexpression is an adverse prognosticator for disease-free survival, as well as overall survival, and tumour stage (pT) is an adverse prognosticator for disease-free survival. In conclusion, these data support a model of oesophageal cancer pathogenesis in which both the pRb and p53 pathways are inactivated and suggests an in-depth evaluation of the clinical utility of these putative markers is warranted.
The degradation of the extracellular matrix (ECM) is a major step in the process of tumor invasio... more The degradation of the extracellular matrix (ECM) is a major step in the process of tumor invasion and metastasis, mediated by matrix metalloproteinases (MMPs). Knowledge of alterations in the expression of stromelysin-2 (ST-2) or MMP-10 in human esophageal squamous cell carcinoma (ESCC) is meager. Immunohistochemical analysis of ST-2 expression was carried out in surgically resected ESCCs (50 cases) and paired distal histologically normal esophageal tissues (50 cases), correlated with clinicopathological parameters. Overexpression of ST-2 protein, in tumor cell cytoplasm and stromal elements, was observed in 37 of the 50 (74%) ESCCs localized in tumor cell cytoplasm and stromal elements. Low levels of ST-2 were observed in 8 of the 50 (16%) matched histologically normal esophageal tissues. Significant associations were observed between ST-2 overexpression and tumor size (r = 0.02, P = 0.04), local invasiveness of the tumor (r = -0.30, P = 0.002) and distant organ metastasis (r = -0.227, P = 0.02), suggestive of its involvement in development and progression of ESCCs. The data underscore the significance of ST-2 expression in context to the aggressive tumor characteristics observed in ESCCs in the Indian population, wherein extremely poor prognosis has been ascribed to extensive local invasion and metastasis.
MARCH7 is an E3 ubiquitin ligase known to regulate neuronal development, T-cell proliferation, an... more MARCH7 is an E3 ubiquitin ligase known to regulate neuronal development, T-cell proliferation, and cell and tissue differentiation. But, the altered expression of MARCH7 has been observed in various malignancies. Herein, the cellular localization and role of MARCH7have been elucidated in esophageal squamous cell carcinoma (ESCC), the information regarding which is currently limited. To check the expression of MARCH7 and its correlation with immune cells infiltration in ESCC, immunohistochemical analysis was performed. RNAi approach was used to investigate the role of MARCH7 in esophagealcancer cells. Interestingly, we found a significantly higher expression of MARCH7 protein in 84% of ESCC tissues than in distant matched non-malignant tissues (p ≤ 0.001). In addition to this, immunohistochemistry results have shown a negative correlation between MARCH7 protein expression and tumor-infiltrating immune cells such as CD8 + T cells (r=-0.667, p = 0.035) and PD1 + T cells (r=-0.509, p = ...
Background: Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exert... more Background: Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of cancer. However, little is known about the expression and function of FGFRL1 in esophageal cancer (EC). Methods: We systematically evaluated the expression of FGFRL1 in TCGA and GETex datasets followed by expression analysis in EC cell lines and clinical specimens using immunofluorescence (IF) and immunohistochemistry (IHC) respectively. Results: GEPIA analysis on TCGA and GETex datasets identified significant upregulation of FGFRL1 in EC patients (n=182) compared to normal controls (n=286, p<0.05). IHC analysis showed significantly higher FGFRL1 expression in EC tissues as compared to the distant matched non-malignant tissues (p<0.001). Immunoflourescence in EC cells suggested increased expression of FGFRL1 from WDSCC (KYSE30) to MDSCC (KYSE140) and finally to PDSCC (KYSE410). In-silico tools predicted miR-107 as most significant miRNA regulating FGFRL1 expression. qRT-PCR revealed miR-107 expression to be significantly and inversely correlated with FGFRL1 expression in 73% (22/30) EC tissues (p=0.015) and over-expression of miR-107 resulted in significantly decreased expression of FGFRL1 at mRNA (fold change=0.11, p=0.0016) as well as protein level in miR-107 versus NC treated cells. Luciferase reporter assay using FGFRL1-3'UTR further confirmed it to be a direct target of miR-107. Conclusion: Our results herein document clinical as well as functional relevance of FGFRL1 in EC and its regulation by miR-107.
Atlas of Genetics and Cytogenetics in Oncology and Haematology, 2014
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 ... more This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence.
Asia-Pacific journal of clinical oncology, Jan 30, 2018
To evaluate the diagnostic potential of a six microRNAs (miRNAs) panel consisting of miR-21, miR-... more To evaluate the diagnostic potential of a six microRNAs (miRNAs) panel consisting of miR-21, miR-144, miR-107, miR-342, miR-93 and miR-152 for esophageal cancer (EC) detection. The expression of miRNAs was analyzed in EC sera samples using quantitative real-time PCR. Risk score analysis was performed and linear regression models were then fitted to generate the six-miRNA panel. In addition, we made an effort to identify significantly dysregulated miRNAs and mRNAs in EC using the Cancer Genome Atlas (TCGA) miRNAseq and RNAseq datasets, respectively. Further, we identified significantly correlated miRNA-mRNA target pairs by integrating TCGA EC miRNAseq dataset with RNAseq dataset. The panel of circulating miRNAs showed enhanced sensitivity (87.5%) and specificity (90.48%) in terms of discriminating EC patients from normal subjects (area under the curve [AUC] = 0.968). Pathway enrichment analysis for potential targets of six miRNAs revealed 48 significant (P < 0.05) pathways, viz. p...
Herein, for the first time, we report aberrant expression of membrane-associated RING-CH8 (MARCH8... more Herein, for the first time, we report aberrant expression of membrane-associated RING-CH8 (MARCH8) in human esophageal squamous cell carcinoma. MARCH8 is a member of the recently discovered MARCH family of really interesting new genes (RING) E3 ligases. Though initial studies primarily focused on its immunomodulatory role, the newly discovered targets of this E3 ligase point towards its possible role in other biological processes such as embryogenesis and inhibition of apoptosis. However, its relevance in cancers is yet to be elucidated. We carried out quantitative real time PCR and immunohistochemistry to examine the levels of MARCH8 mRNA and protein in esophageal squamous cell carcinoma tissues. The role of MARCH8 in esophageal cancer cells was evaluated by cell proliferation, clonogenic and migration/invasion assays and flow cytometry with MARCH8 gene knockdown. Significantly increased expression of MARCH8 mRNA was found in esophageal squamous cell carcinoma as compared to distan...
Previously, we reported significantly decreased expression of tissue and circulating miR-107 in e... more Previously, we reported significantly decreased expression of tissue and circulating miR-107 in esophageal cancer (EC). However, its role in esophageal tumorigenesis still remains elusive. Therefore, the aim of the present study was to analyze the role of miR-107 in esophageal squamous cell carcinoma (ESCC). The role of miR-107 in ESCC was evaluated using MTT assay, cell cycle analysis by flow cytometry, annexin assay, colony formation assay and scratch assay. Overexpression of miR-107 in KYSE-410 cells suppressed cell proliferation at 72 h post-transfection (P=0.0001). Moreover, a significant increase in the G0/G1 population (P<0.001) and a significant decrease in the G2/M (P=0.032) population was also observed in the miR-107-treated cells as compared to the negative control (NC). Notably, miR-107 overexpression attenuated the colony formation potential of ESCC cells by 41.83% as compared to the NC (P=0.007). miR-107 mimic inhibited ESCC cell migration in a time-dependent manner, reducing the wound closure to only 50.41±7.23% at 72 h posttransfection (P=0.041). Further analysis by Matrigel invasion assay revealed a significant decrease in the migratory and invasive abilities of the KYSE-410 cells at 72 h post miR-107 transfection. qRT-PCR analysis showed decreased expression of one of the newly identified targets of miR-107, Cdc42, at the mRNA level. Further validation by western blotting confirmed a significant reduction in the identified target at the protein level. In addition, the relative luciferase activity of the reporter containing Cdc42 3'UTR was significantly decreased upon miR-107 co-transfection, indicating it to be a direct target of miR-107. Our results herein document that miR-107 functions as a tumor suppressor and inhibits the proliferation, migration and invasion of ESCC cells. Moreover, this is the first report showing Cdc42 as a downstream target of miR-107.
Background & objectives: Insidious symptomatology, late clinical presentation and poor prognosis ... more Background & objectives: Insidious symptomatology, late clinical presentation and poor prognosis of oesophageal cancer (EC) highlight the pressing need for novel non-invasive biomarkers for early tumour diagnosis and better prognosis. The present study was carried out to evaluate the clinical significance of circulating and tissue miR-144 expression in oesophageal cancer. Methods: Clinical significance of miR-144 expression was evaluated in preneoplastic (12) and neoplastic (35) oesophageal cancer tissues as well as matched distant non-malignant tissues using real-time PCR (qPCR). Circulating levels of miR-144 were also analyzed in serum samples of EC patients as well as normal individuals to determine the diagnostic potential of miR-144. Further, targets of miR-144 were predicted using bioinformatic tools and their gene ontology (GO) terms were assigned. Results: Real-time PCR analysis revealed significant upregulation of miR-144 in 29 of 35 (83%) EC tissues as compared to matched distant non-malignant tissues (P=0.010). All the dysplastic tissues showed upregulation of miR-144 as compared to their matched distant non-malignant tissues. Relative levels of circulating miR-144 in serum significantly distinguished EC patients from normal controls (P=0.015; AUC = 0.731) with high sensitivity of 94.7 per cent. Bioinformatically predicted target, PURaplha (PURA) was found to be significantly (P=0.018) downregulated in 81 per cent (26/32) EC patients and its expression was found to be significantly and negatively correlated with miR-144 expression at mRNA level. Interpretation & conclusions: Our findings showed significant upregulation of miR-144 in serum samples of EC patients indicating its potential as minimally invasive marker. Further studies need to be done to understand the role of miR-144 in the pathogenesis of EC.
Background: Biomarkers to predict the risk of disease recurrence in Esophageal squamous cell carc... more Background: Biomarkers to predict the risk of disease recurrence in Esophageal squamous cell carcinoma (ESCC) patients are urgently needed to improve treatment. We developed proteins expression-based risk model to predict recurrence free survival for ESCC patients. Methods: Alterations in Wnt pathway components expression and subcellular localization were analyzed by immunohistochemistry in 80 ESCCs, 61 esophageal dysplastic and 47 normal tissues; correlated with clinicopathological parameters and clinical outcome over 86 months by survival analysis. Significant prognostic factors were identified by multivariable Cox regression analysis. Results: Biomarker signature score based on cytoplasmic β-catenin, nuclear c-Myc, nuclear DVL and membrane α-catenin was associated with recurrence free survival [Hazard ratio = 1.11 (95% CI = 1.05, 1.17), p < 0.001, C-index = 0.68] and added significant prognostic value over clinical parameters (p < 0.001). The inclusion of Slug further improved prognostic utility (p < 0.001, C-index = 0.71). Biomarker Signature Score slug improved risk classification abilities for clinical outcomes at 3 years, accurately predicting recurrence in 79% patients in 1 year and 97% in 3 years in high risk group; 73% patients within low risk group did not have recurrence in 1 year, with AUC of 0.76. Conclusions: Our comprehensive risk model predictive for recurrence allowed us to determine the robustness of our biomarker panel in stratification of ESCC patients Research Paper Oncotarget 1021 www.oncotarget.com at high or low risk of disease recurrence; high risk patients are stratified for more rigorous personalized treatment while the low risk patients may be spared from harmful side effects of toxic therapy.
SP17 is a mammalian protein found in the testis and spermatozoa that have been identified as a tu... more SP17 is a mammalian protein found in the testis and spermatozoa that have been identified as a tumor-associated antigen in a range of human cancers. A unique method for fabricating the first ultrasensitive, selective, and label-free immunosensor for the detection of SP17, a new cancer biomarker in complicated serum samples, is presented in this paper. This immunosensor was also the first biosensor built using a disposable ITO sheet modified with an aminosilane known as APTMS as an immobilization platform for fabricating the SP17 biosensor. The immobilization of chemical and biological species onto the electrode surface was cross-verified by various analytical and morphological techniques. Stepwise modifications done on the immunoelectrodes were also studied using electrochemical techniques. Selective interaction between anti-SP17 and SP17 with varying concentrations (100-5000 pg mL-1) was measured with the DPV technique. The immunosensor exhibited low LOD and LOQ of 70.07 and 233.57 pg mL-1, respectively, with a sensitivity of 0.013 μA mL pg-1 cm-2. The fabricated immunosensor performance was analyzed by quantifying the SP17 concentrations in patient serum samples. The data obtained from the developed immunosensor demonstrated excellent reproducibility, repeatability, and selectivity among various interferants, including cancer biomarkers. Further, the observed results have been validated via ELISA, which showed good agreement with the electrochemical results. This could establish a new platform for detecting other cancer biomarkers and can be employed for clinical diagnostics applications.
MARCH8 is an E3 ligase, primarily involved in immune-modulation. Recently, we reported its aberra... more MARCH8 is an E3 ligase, primarily involved in immune-modulation. Recently, we reported its aberrant expression in human esophageal squamous cell carcinoma. However, exact mechanisms by which it regulates cancer have been poorly understood. We applied high-throughput quantitative proteomics approach to identify downstream protein targets of MARCH8. Silencing of endogenous MARCH8 in ESCC cells followed by LC-MS/MS analysis led to identification of 1029 unique proteins showing altered expression post MARCH8 knockdown. Several previously reported MARCH8 target proteins viz. TFR1, syntaxin-4, e-cadherin and CD44 were found to be upregulated. Furthermore, new putative targets of MARCH8, including β2M, were identified in the present study. We demonstrated that MARCH8 interacts with and ubiquitinates CDH1 and β2M. Inhibiting proteasome activity with MG132 prevented CDH1 and β2M degradation, indicating that MARCH8 might be targeting CDH1 and β2M for proteasomal degradation. Further, loss of β2M and CDH1 expression significantly and inversely correlated with MARCH8 expression in ESCC tissues (r = -0.737 and - 0.651, respectively; p < 0.01). In conclusion, our present study has led to identification of new targets of MARCH8 and suggests the role of MARCH8 in regulating CDH1 and β2M turnover in esophageal cancer cells. SIGNIFICANCE: The use of quantitative proteomics carried out has led to the recognition of new targets of MARCH8. The present study gives a broad understanding of the molecular remodeling arising in the ESCC after MARCH8 knockdown. The study also solidifies the idea that role of MARCH8 is not just limited to immunomodulation as silencing of MARCH8 affects various other processes such as protein processing and localization. This study might help in understanding the regulation of MARCH8 in ESCCs and the mechanism by which MARCH8 might be facilitating cancer cells to evade immune surveillance.
Esophageal cancer (EC) is a highly complex disease with high incidence and mortality rates. Recen... more Esophageal cancer (EC) is a highly complex disease with high incidence and mortality rates. Recent studies have shown that miRNAs play critical roles in diverse biological processes including oncogenesis, and we previously reported significantly increased expression of tissue and circulating miR-144 in EC. This study evaluates the functional significance of miR-144 in esophageal squamous cell carcinoma. Herein, we analysed the role of miR-144 in ESCC by silencing it in KYSE-410 cells, and followed this with cell cycle analysis and the following assays; MTT, annexin, colony formation, scratch and matrigel invasion assay. The miR-144 knockdown significantly suppressed ESCC cell proliferation by 72 hours post transfection (p=0.029). This significantly decreased the migration, invasion and colony formation potential of KYSE-410 cells compared to cells treated with negative control (NC). Potential targets of miR-144 were predicted by the in-silico approach followed by in-vitro validation...
Background: Growing evidence suggests importance of aberrant microRNA (miRNA) expression in devel... more Background: Growing evidence suggests importance of aberrant microRNA (miRNA) expression in development and progression of cancer. Recent studies have demonstrated the potential of miRNA panels as promising diagnostic, prognostic and predictive biomarkers. Objective: The purpose of the present study was to evaluate the clinical and functional significance of miRNAs in esophageal cancer (EC). Methods: Herein, we evaluated the diagnostic potential of a five miRNA panel (consisting of miR-21, miR-144, miR-107, miR-93 and miR-342) in EC detection using quantitative real time PCR (qRTPCR). To evaluate the discriminatory power of miRNA panel, receiver operating characteristic curve (ROC) curves were generated for each of the miRNAs followed by risk score analysis. Briefly, linear regression models were fitted using the cancer status and each of the risk score as input. ROC curves were then used to evaluate the diagnostic potential of the panel and to find out the appropriate cut-off point...
The International Journal of Biological Markers, 2008
We recently found 14-3-3 zeta to be overexpressed in esophageal squamous cell carcinomas (ESCCs) ... more We recently found 14-3-3 zeta to be overexpressed in esophageal squamous cell carcinomas (ESCCs) by differential display. In the present study we determined the clinical significance of 14-3-3 zeta in esophageal tumorigenesis. Immunohistochemical analysis was carried out in 61 ESCCs, 33 dysplasia samples, 14 hyperplasia samples and 7 matched histologically normal esophageal tissues and correlated with clinicopathological parameters. Cytoplasmic expression of 14-3-3 zeta protein was observed in 95% of ESCCs; 63% of tumors also showed nuclear localization. All hyperplastic and dysplastic tissues distant from ESCCs as well as dysplastic endoscopic biopsies showed cytoplasmic immunopositivity for 14-3-3 zeta, while nuclear localization was observed in 58% of dysplasia and 36% of hyperplasia samples. Matched distant histologically normal epithelia either showed basal cytoplasmic expression of 14-3-3 zeta or no detectable nuclear expression of the protein. Interestingly, immunopositivity observed in normal esophageal tissues and early hyperplasia was confined to cytoplasm only, though significant nuclear expression was detected in dysplasia and ESCC. Immunoblotting and RT-PCR analyses further confirmed 14-3-3 zeta expression in dysplasia and ESCC. To our knowledge, this is the first report demonstrating overexpression of 14-3-3 zeta in esophageal hyperplasia, dysplasia and squamous cell carcinoma, suggesting that alteration in its expression occurs in early stages and is associated with esophageal tumorigenesis.
Journal of Cancer Research and Clinical Oncology, 2001
The p16/cyclin D1/pRb pathway plays a critical role in tumourigenesis. We recently reported alter... more The p16/cyclin D1/pRb pathway plays a critical role in tumourigenesis. We recently reported alterations in expression of tumour suppressor gene products, p16 and pRb in esophageal cancer. Knowledge of alterations in cyclin D1, a vital component of this pathway in esophageal carcinomas from the Indian subcontinent, where the etiology and pathogenesis may be confounded by various unique dietary and environmental factors, is presently scanty. In order to bridge the gap between the accentuating incidence of esophageal cancer and aberrations in the components of this vital pathway, we analysed cyclin D1 expression in esophageal squamous cell carcinoma in the Indian population. Immunohistochemical analysis of cyclin D1 expression was carried out in paraffin-embedded sections of surgically resected esophageal squamous cell carcinomas (ESCC) (70 patients) and matched with histopathologically normal esophageal tissues from a distant site. The findings were correlated with clinicopathological parameters. Overexpression of cyclin D1 was observed in the tumour nuclei in 41 out of 70 (59%) patients. We found concomitant alterations in 16 and cyclin D1 (p16-/CycD1+ phenotype) in 16 of the 70 patients (23%), while alterations of pRb and cyclin D1 (pRb-/CycD1+) were observed in 36 of the 70 (51%) patients of ESCCs. Cyclin D1 overexpression was significantly associated with the loss of p16 immunoreactivity (P = 0.005). The pRb- and p16-/pRb-/Cyc D+ phenotypes showed significant association with differentiation of the tumour (P = 0.005, 0.05, respectively). Kaplan-Meier analysis for disease recurrence showed increased disease recurrence in cyclin D1 overexpressed patients. Median time to disease recurrence in the cyclin D1+ group was 15 months as against 18 months observed in the cyclin D1- patients (P = 0.067; log-rank test). Alterations in at least one of the components of the p16/cyclin D1/pRb pathway in majority of the 70 patients analysed herein, and concomitant alterations in all the three proteins in 19 patients (35%) underscore the critical role of this pathway in esophageal tumourigenesis. The results of the present study taken together with our previous findings on p16 and pRb alterations in ESCCs suggest that these alterations are not mutually exclusive and may cooperatively provide greater tumour growth advantage. The prognostic significance of alterations in the expression of these components cyclin D1, p16, and pRb remains to be established in a larger cohort.
Journal of Cancer Research and Clinical Oncology, 2004
Matrix metalloproteinases (MMPs) are known to play an important role in extracellular matrix remo... more Matrix metalloproteinases (MMPs) are known to play an important role in extracellular matrix remodeling during the process of tumor invasion and metastasis. However, little is known about their role in preinvasive lesions and early esophageal carcinomas. Method: Immunohistochemical analysis of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression was carried out in paraffin-embedded sections of surgically resected esophageal squamous cell carcinoma (ESCC) (58 cases) and paired distal normal esophageal tissues (44 cases) and correlated with clinicopathological parameters. Result: Overexpression of MMP-2 and MMP-9 proteins was observed in 39 (67%) and 32 (55%) of the 58 ESCCs, respectively localized in tumor cell cytoplasm and stromal elements. Histological evaluation of hematoxylin-and eosin-stained 44 matched distal normal esophageal tissue sections revealed that 26 comprised of normal epithelium, while 15 tissues showed evidence of dysplasia and three tissues showed hyperplasia. Interestingly, 12 (80%) and 13 (87%) of these 15 dysplasias showed immunostaining for MMP-2 and MMP-9 proteins, respectively. Low levels of MMP-2 and MMP-9 were observed in 10 (38%) and 6 (23%) of 26 matched histologically normal esophageal tissues, respectively. Higher MMP-2 immunopositivity was observed in well and moderately differentiated SCCs in comparison with poorly differentiated tumors. The expression of MMP-2 was significantly reduced with the progressive de-differentiation of esophageal SCCs (P =0.03). Overexpression of MMP-2 and MMP-9 in dysplasia as well as SCC suggests that these alterations occur in early stages of esophageal tumorigenesis. Conclusion: Increased levels of MMP-2 and MMP-9 proteins in ESCCs as compared to normal esophageal tissues suggest their association with esophageal tumorigenesis. Increased levels of these MMPs are observed in majority of dysplasias analyzed herein, indicating that these alterations may be early events in esophageal tumorigenesis. In-depth studies are warranted to determine their role in development and progression of esophageal cancer.
The pRb (p16-pRb-cyclin D1) and p53 (p53-MDM2-p21) pathways play a critical role in tumorigenesis... more The pRb (p16-pRb-cyclin D1) and p53 (p53-MDM2-p21) pathways play a critical role in tumorigenesis. To evaluate which of these cell cycle regulatory proteins are related to patients' prognosis, a comprehensive analysis of alterations in these components was carried out in 100 ESCCs (oesophageal squamous cell carcinoma) using immunohistochemistry and correlated with clinicopathological parameters by univariate analysis. Overexpression of p53, MDM2 and cyclin D1 proteins was observed in 73, 42 and 67% of the cases, respectively, while loss of expression of p21, p16 and pRb was observed in 36, 45 and 75% of the cases, respectively. Multiple logistic regression analysis revealed that loss of p16 immunoreactivity was a significant risk factor for tumour stage (pT) (Odds Ratio (OR)=3.3), whereas the loss of pRb was a significant risk factor for nodal metastasis (pN) (OR=8.8). MDM2 overexpression emerged as the most significant risk factor for distant organ metastasis (pM) (OR=4.6). Of the ESCC patients who underwent oesophagectomy, 50 cases were followed-up for a maximum period of 44 months and median of 16 months. Survival analysis revealed that Cyclin D1 overexpression is an adverse prognosticator for disease-free survival, as well as overall survival, and tumour stage (pT) is an adverse prognosticator for disease-free survival. In conclusion, these data support a model of oesophageal cancer pathogenesis in which both the pRb and p53 pathways are inactivated and suggests an in-depth evaluation of the clinical utility of these putative markers is warranted.
The degradation of the extracellular matrix (ECM) is a major step in the process of tumor invasio... more The degradation of the extracellular matrix (ECM) is a major step in the process of tumor invasion and metastasis, mediated by matrix metalloproteinases (MMPs). Knowledge of alterations in the expression of stromelysin-2 (ST-2) or MMP-10 in human esophageal squamous cell carcinoma (ESCC) is meager. Immunohistochemical analysis of ST-2 expression was carried out in surgically resected ESCCs (50 cases) and paired distal histologically normal esophageal tissues (50 cases), correlated with clinicopathological parameters. Overexpression of ST-2 protein, in tumor cell cytoplasm and stromal elements, was observed in 37 of the 50 (74%) ESCCs localized in tumor cell cytoplasm and stromal elements. Low levels of ST-2 were observed in 8 of the 50 (16%) matched histologically normal esophageal tissues. Significant associations were observed between ST-2 overexpression and tumor size (r = 0.02, P = 0.04), local invasiveness of the tumor (r = -0.30, P = 0.002) and distant organ metastasis (r = -0.227, P = 0.02), suggestive of its involvement in development and progression of ESCCs. The data underscore the significance of ST-2 expression in context to the aggressive tumor characteristics observed in ESCCs in the Indian population, wherein extremely poor prognosis has been ascribed to extensive local invasion and metastasis.
MARCH7 is an E3 ubiquitin ligase known to regulate neuronal development, T-cell proliferation, an... more MARCH7 is an E3 ubiquitin ligase known to regulate neuronal development, T-cell proliferation, and cell and tissue differentiation. But, the altered expression of MARCH7 has been observed in various malignancies. Herein, the cellular localization and role of MARCH7have been elucidated in esophageal squamous cell carcinoma (ESCC), the information regarding which is currently limited. To check the expression of MARCH7 and its correlation with immune cells infiltration in ESCC, immunohistochemical analysis was performed. RNAi approach was used to investigate the role of MARCH7 in esophagealcancer cells. Interestingly, we found a significantly higher expression of MARCH7 protein in 84% of ESCC tissues than in distant matched non-malignant tissues (p ≤ 0.001). In addition to this, immunohistochemistry results have shown a negative correlation between MARCH7 protein expression and tumor-infiltrating immune cells such as CD8 + T cells (r=-0.667, p = 0.035) and PD1 + T cells (r=-0.509, p = ...
Background: Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exert... more Background: Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of cancer. However, little is known about the expression and function of FGFRL1 in esophageal cancer (EC). Methods: We systematically evaluated the expression of FGFRL1 in TCGA and GETex datasets followed by expression analysis in EC cell lines and clinical specimens using immunofluorescence (IF) and immunohistochemistry (IHC) respectively. Results: GEPIA analysis on TCGA and GETex datasets identified significant upregulation of FGFRL1 in EC patients (n=182) compared to normal controls (n=286, p<0.05). IHC analysis showed significantly higher FGFRL1 expression in EC tissues as compared to the distant matched non-malignant tissues (p<0.001). Immunoflourescence in EC cells suggested increased expression of FGFRL1 from WDSCC (KYSE30) to MDSCC (KYSE140) and finally to PDSCC (KYSE410). In-silico tools predicted miR-107 as most significant miRNA regulating FGFRL1 expression. qRT-PCR revealed miR-107 expression to be significantly and inversely correlated with FGFRL1 expression in 73% (22/30) EC tissues (p=0.015) and over-expression of miR-107 resulted in significantly decreased expression of FGFRL1 at mRNA (fold change=0.11, p=0.0016) as well as protein level in miR-107 versus NC treated cells. Luciferase reporter assay using FGFRL1-3'UTR further confirmed it to be a direct target of miR-107. Conclusion: Our results herein document clinical as well as functional relevance of FGFRL1 in EC and its regulation by miR-107.
Atlas of Genetics and Cytogenetics in Oncology and Haematology, 2014
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 ... more This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence.
Asia-Pacific journal of clinical oncology, Jan 30, 2018
To evaluate the diagnostic potential of a six microRNAs (miRNAs) panel consisting of miR-21, miR-... more To evaluate the diagnostic potential of a six microRNAs (miRNAs) panel consisting of miR-21, miR-144, miR-107, miR-342, miR-93 and miR-152 for esophageal cancer (EC) detection. The expression of miRNAs was analyzed in EC sera samples using quantitative real-time PCR. Risk score analysis was performed and linear regression models were then fitted to generate the six-miRNA panel. In addition, we made an effort to identify significantly dysregulated miRNAs and mRNAs in EC using the Cancer Genome Atlas (TCGA) miRNAseq and RNAseq datasets, respectively. Further, we identified significantly correlated miRNA-mRNA target pairs by integrating TCGA EC miRNAseq dataset with RNAseq dataset. The panel of circulating miRNAs showed enhanced sensitivity (87.5%) and specificity (90.48%) in terms of discriminating EC patients from normal subjects (area under the curve [AUC] = 0.968). Pathway enrichment analysis for potential targets of six miRNAs revealed 48 significant (P < 0.05) pathways, viz. p...
Herein, for the first time, we report aberrant expression of membrane-associated RING-CH8 (MARCH8... more Herein, for the first time, we report aberrant expression of membrane-associated RING-CH8 (MARCH8) in human esophageal squamous cell carcinoma. MARCH8 is a member of the recently discovered MARCH family of really interesting new genes (RING) E3 ligases. Though initial studies primarily focused on its immunomodulatory role, the newly discovered targets of this E3 ligase point towards its possible role in other biological processes such as embryogenesis and inhibition of apoptosis. However, its relevance in cancers is yet to be elucidated. We carried out quantitative real time PCR and immunohistochemistry to examine the levels of MARCH8 mRNA and protein in esophageal squamous cell carcinoma tissues. The role of MARCH8 in esophageal cancer cells was evaluated by cell proliferation, clonogenic and migration/invasion assays and flow cytometry with MARCH8 gene knockdown. Significantly increased expression of MARCH8 mRNA was found in esophageal squamous cell carcinoma as compared to distan...
Previously, we reported significantly decreased expression of tissue and circulating miR-107 in e... more Previously, we reported significantly decreased expression of tissue and circulating miR-107 in esophageal cancer (EC). However, its role in esophageal tumorigenesis still remains elusive. Therefore, the aim of the present study was to analyze the role of miR-107 in esophageal squamous cell carcinoma (ESCC). The role of miR-107 in ESCC was evaluated using MTT assay, cell cycle analysis by flow cytometry, annexin assay, colony formation assay and scratch assay. Overexpression of miR-107 in KYSE-410 cells suppressed cell proliferation at 72 h post-transfection (P=0.0001). Moreover, a significant increase in the G0/G1 population (P<0.001) and a significant decrease in the G2/M (P=0.032) population was also observed in the miR-107-treated cells as compared to the negative control (NC). Notably, miR-107 overexpression attenuated the colony formation potential of ESCC cells by 41.83% as compared to the NC (P=0.007). miR-107 mimic inhibited ESCC cell migration in a time-dependent manner, reducing the wound closure to only 50.41±7.23% at 72 h posttransfection (P=0.041). Further analysis by Matrigel invasion assay revealed a significant decrease in the migratory and invasive abilities of the KYSE-410 cells at 72 h post miR-107 transfection. qRT-PCR analysis showed decreased expression of one of the newly identified targets of miR-107, Cdc42, at the mRNA level. Further validation by western blotting confirmed a significant reduction in the identified target at the protein level. In addition, the relative luciferase activity of the reporter containing Cdc42 3'UTR was significantly decreased upon miR-107 co-transfection, indicating it to be a direct target of miR-107. Our results herein document that miR-107 functions as a tumor suppressor and inhibits the proliferation, migration and invasion of ESCC cells. Moreover, this is the first report showing Cdc42 as a downstream target of miR-107.
Background & objectives: Insidious symptomatology, late clinical presentation and poor prognosis ... more Background & objectives: Insidious symptomatology, late clinical presentation and poor prognosis of oesophageal cancer (EC) highlight the pressing need for novel non-invasive biomarkers for early tumour diagnosis and better prognosis. The present study was carried out to evaluate the clinical significance of circulating and tissue miR-144 expression in oesophageal cancer. Methods: Clinical significance of miR-144 expression was evaluated in preneoplastic (12) and neoplastic (35) oesophageal cancer tissues as well as matched distant non-malignant tissues using real-time PCR (qPCR). Circulating levels of miR-144 were also analyzed in serum samples of EC patients as well as normal individuals to determine the diagnostic potential of miR-144. Further, targets of miR-144 were predicted using bioinformatic tools and their gene ontology (GO) terms were assigned. Results: Real-time PCR analysis revealed significant upregulation of miR-144 in 29 of 35 (83%) EC tissues as compared to matched distant non-malignant tissues (P=0.010). All the dysplastic tissues showed upregulation of miR-144 as compared to their matched distant non-malignant tissues. Relative levels of circulating miR-144 in serum significantly distinguished EC patients from normal controls (P=0.015; AUC = 0.731) with high sensitivity of 94.7 per cent. Bioinformatically predicted target, PURaplha (PURA) was found to be significantly (P=0.018) downregulated in 81 per cent (26/32) EC patients and its expression was found to be significantly and negatively correlated with miR-144 expression at mRNA level. Interpretation & conclusions: Our findings showed significant upregulation of miR-144 in serum samples of EC patients indicating its potential as minimally invasive marker. Further studies need to be done to understand the role of miR-144 in the pathogenesis of EC.
Background: Biomarkers to predict the risk of disease recurrence in Esophageal squamous cell carc... more Background: Biomarkers to predict the risk of disease recurrence in Esophageal squamous cell carcinoma (ESCC) patients are urgently needed to improve treatment. We developed proteins expression-based risk model to predict recurrence free survival for ESCC patients. Methods: Alterations in Wnt pathway components expression and subcellular localization were analyzed by immunohistochemistry in 80 ESCCs, 61 esophageal dysplastic and 47 normal tissues; correlated with clinicopathological parameters and clinical outcome over 86 months by survival analysis. Significant prognostic factors were identified by multivariable Cox regression analysis. Results: Biomarker signature score based on cytoplasmic β-catenin, nuclear c-Myc, nuclear DVL and membrane α-catenin was associated with recurrence free survival [Hazard ratio = 1.11 (95% CI = 1.05, 1.17), p < 0.001, C-index = 0.68] and added significant prognostic value over clinical parameters (p < 0.001). The inclusion of Slug further improved prognostic utility (p < 0.001, C-index = 0.71). Biomarker Signature Score slug improved risk classification abilities for clinical outcomes at 3 years, accurately predicting recurrence in 79% patients in 1 year and 97% in 3 years in high risk group; 73% patients within low risk group did not have recurrence in 1 year, with AUC of 0.76. Conclusions: Our comprehensive risk model predictive for recurrence allowed us to determine the robustness of our biomarker panel in stratification of ESCC patients Research Paper Oncotarget 1021 www.oncotarget.com at high or low risk of disease recurrence; high risk patients are stratified for more rigorous personalized treatment while the low risk patients may be spared from harmful side effects of toxic therapy.
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