Rinu Sharma

Prognostic Significance of Stromelysin-3 and Tissue Inhibitor of Matrix Metalloproteinase-2 in Esophageal Cancer

Objective: Stromelysins (matrix metalloproteinases: MMP-10 or ST-2 and MMP-11 or ST-3) and tissue inhibitors of matrix metalloproteinases (TIMP-1 and 2) have been shown to be associated with human tumor progression, invasion and metastasis. The aim of the present study was to determine the prognostic significance of these proteins in esophageal squamous cell carcinoma (ESCC). Methods: Immunohistochemical analysis was carried out in 65 surgically resected ESCCs and 49 distant histologically normal esophageal tissues and 16 cases of dysplasias. Statistical analyses were performed to determine the associations between the protein expression and clinicopathological parameters and survival of esophageal cancer patients. Results: Expression of ST-2, ST-3, TIMP-1 and TIMP-2 was observed in 43/65 (66%), 51/65 (78%), 43/65 (66%) and 47/65 (72%) ESCC cases, respectively. Univariate analysis showed that TIMP-2 expression was associated with tumor site (OR = 2.63, p = 0.017). TIMP-1+/TIMP-2+ phenotype was inversely correlated with nodal invasiveness of the tumor (OR = 0.4, p = 0.04). Interestingly, p53 expression was associated with increased levels of ST-3 (OR = 0.11, p = 0.02) and TIMP-1 (OR = 3.2, p = 0.007) suggesting possible involvement of p53 in the regulation of these proteins. An increased expression of ST-2, ST-3, TIMP-1 and TIMP-2 was observed in 11/16 (69%), 7/17 (44%), 11/16 (69%) and 8/16 (50%) dysplasias also suggesting that these alterations are early events in esophageal tumorigenesis. All the ESCC patients were followed up postesophagectomy for a maximum period of 59 months (mean disease-free survival = 12 months). Kaplan-Meier survival analysis showed that patients with ST-3-positive and TIMP-2-negative carcinoma had a significantly shorter disease-free survival (median disease-free survival time of 4 months) as compared to patients in the other groups (median disease-free survival time of 20 months; p = 0.0016). To our knowledge this is the first report showing that ST-3+/TIMP-2– phenotype remained of significant predictive value for disease-free survival (p = 0.0007) in multivariate analysis including a conventional clinicopathological factor, tumor stage (p = 0.051). Conclusion: Our results suggest that ST-3+/TIMP-2– phenotype is an adverse prognosticator in esophageal cancer patients.

Clinical significance of sperm protein 17 expression and immunogenicity in esophageal cancer.

We recently identified sperm protein 17 (Sp17) transcripts in esophageal squamous cell carcinomas (ESCCs) by differential display. This study was designed to determine the clinical significance of Sp17 protein in different stages of esophageal tumorigenesis and to test the hypothesis that aberrant localization of Sp17 protein to immunosurveillant site may lead to production of anti‐Sp17 antibodies in serum, which may be of clinical relevance in ESCCs. Sp17 transcripts were detected by RT‐PCR in 26 of 30 (86%) ESCCs, while no transcripts were detected in normal esophageal tissues. Rabbit polyclonal antibody was raised against an immunogenic peptide of Sp17 and used to evaluate protein expression by immunohistochemistry. Expression of Sp17 protein was observed in 60/80 (75%) of ESCCs and 27/30 (90%) dysplastic tissues, while no detectable Sp17 expression was observed in 13 distant histologically normal epithelia. Sixteen of the 60 immunopositive ESCCs showed nuclear expression in addition to cytoplasmic localization of the protein. The circulating levels of anti‐Sp17 antibodies, determined by ELISA, were significantly elevated in ESCC patients when compared with normal subjects (p < 0.001). Increasing Sp17 antibody titers were observed to be associated with the progressive disease in 4 patients. In conclusion, the study demonstrates expression of Sp17 protein in esophageal tumor as well as dysplastic tissues, suggesting it to be an early event in the development of ESCC. To our knowledge, this is the first report showing elevated levels of anti‐Sp17 antibodies in ESCC patients.

TC21/R-Ras2 Upregulation in Esophageal Tumorigenesis: Potential Diagnostic Implications

Objectives: Early detection of esophageal cancer is hampered by paucity of molecular markers for diagnosis of this aggressive gastrointestinal malignancy in early stages. We recently identified TC21/R-Ras2, a small GTP-binding protein (SMG) in esophageal squamous cell carcinomas (ESCCs) by differential display. This study was designed to test the hypothesis that differential expression of TC21 in normal, dysplastic and malignant esophageal tissues may be of clinical relevance in esophageal tumorigenesis. Methods:Immunohistochemical analysis of TC21 was carried out in 83 ESCCs, 37 dysplasias and 29 matched histologically normal esophageal tissues and correlated with clinicopathological parameters. The cellularlocalization of TC21 was determined by confocal microscopy. Results: Expression of TC21 protein was observed in 60/83 (73%) ESCCs predominantly localized in tumor nuclei. Intriguingly, intense TC21 immunoreactivity was observed in all endoscopic biopsies with histological evidence of dysplasia (16 cases) as well as in dysplastic areas distant to ESCCs (21 cases), while matched distant histologically normal epithelia did not show detectable TC21 expression. Immunoblotting and semi-quantitative RT-PCR confirmed TC21 expression in dysplastias and ESCCs. Confocal microscopy showed nuclear as well as cytoplasmic TC21 expression in ESCCs and TE13 cells. Conclusions: To our knowledge, this is the first report demonstrating differential expression of TC21 in normal, dysplastic and ESCC tissues, suggesting that TC21 expression is associated with early stages of esophageal tumorigenesis. Nuclear localization of TC21 makes it the third of over 100 small SMGs identified to be localized in the nucleus.

Clinical significance of 14-3-3 zeta in human esophageal cancer

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.

Decreased levels of circulating and tissue miR-107 in human esophageal cancer

Abstract Context: Aberrant expression of miRNAs has emerged as an important hallmark of cancer. Objectives: We evaluated the clinical significance of circulating and tissue miR-107 expression in esophageal squamous cell carcinoma (ESCC) patients. Materials and methods: Quantitative real-time PCR was used to analyze the expression of miR-107 and its bioinformatically identified targets, PIM-1 and CDC42. Results: Significant downregulation of miR-107 was observed in neoplastic and preneoplastic esophageal tissues (p = 0.004). Relative levels of circulating miR-107 significantly distinguished ESCC patients from normal subjects (p = 0.007). Significant inverse correlation was observed between miR-107 and PIM1 expression (r = −0.398; p = 0.015) suggesting PIM1 to be the downstream target of miR-107. Discussion and conclusion: Our results, for the first time, document that the miR-107 levels may discriminate ESCC patients from healthy individuals emphasizing its diagnostic potential.

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.

Slug Is a Predictor of Poor Prognosis in Esophageal Squamous Cell Carcinoma Patients

Background Slug, a regulator of epithelial mesenchymal transition, was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. This study aimed to determine the clinical significance of Slug overexpression in ESCC and determine its correlation with clinicopathological parameters and disease prognosis for ESCC patients. Methods Immunohistochemical analysis of Slug expression was carried out in archived tissue sections from 91 ESCCs, 61 dysplastic and 47 histologically normal esophageal tissues. Slug immunopositivity in epithelial cells was correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients. Results Increased expression of Slug was observed in esophageal dysplasia [cytoplasmic, 24/61 (39.3%) cases, p = 0.001, odd’s ratio (OR) = 4.7; nuclear, 11/61 (18%) cases, p < 0.001, OR = 1.36] in comparison with normal esophageal tissues. The Slug expression was further increased in ESCCs [cytoplasmic, 64/91 (70.3%) p < 0.001, OR = 10.0; nuclear, 27/91 (29.7%) p < 0.001, OR = 1.42]. Kaplan Meier survival analysis showed significant association of nuclear Slug accumulation with reduced disease free survival of ESCC patients (median disease free survival (DFS) = 6 months, as compared to those that did not show overexpression, DFS = 18 months; p = 0.006). In multivariate Cox regression analysis nuclear Slug expression [p= 0.005, Hazard’s ratio (HR) = 2.269, 95% CI = 1.289 - 3.996] emerged as the most significant independent predictor of poor prognosis for ESCC patients. Conclusions Alterations in Slug expression occur in early stages of development of ESCC and are sustained during disease progression. Slug may serve as a diagnostic biomarker and as a predictor of poor disease prognosis to identify ESCC patients that are likely to show recurrence of the disease.

miRNA–mRNA crosstalk in esophageal cancer: From diagnosis to therapy

The asymptomatic nature of esophageal cancer (EC) at early stages results in late clinical presentation leading to poor prognosis and limited success of therapeutic modalities. Efforts to identify diagnostic/prognostic markers have proven to be unsuccessful for translation into clinics. Hence, there is a pressing need for establishment of novel non-invasive biomarker for early diagnosis/better prognosis of EC. Recently, alteration in microRNA (miRNA) expression has emerged as an important hallmark of cancer. This review summarizes the differential expression of miRNAs in EC and addresses how their aberrant expression influences crucial biological processes such as apoptosis, cell proliferation, invasion and metastasis. Additionally, this review highlights the current status of circulating miRNA based diagnostic/prognostic markers. An effort has been made to find a connection between different miRNAs involved in EC and a detailed analysis has been done to screen out micoRNAs involved in prognosis and multidrug resistance. Further, investigation of these miRNAs would not only provide a gene therapy based strategy to prevent/treat cancer but also to reverse multidrug resistance leading to decreased requirement of harmful chemotherapeutic drugs.

Increased Levels of Circulating and Tissue mRNAs of Oct-4, Sox-2, Bmi-1 and Nanog is ESCC Patients: Potential Tool for Minimally Invasive Cancer Diagnosis

Background Early stages of esophageal cancer lack a specific symptom, a reliable biomarker and accurate non-invasive diagnostic modalities prompting the pressing need for identification of a marker for early diagnosis of this disease. Methods In the present study we investigated the levels of circulating and tissue mRNAs of Oct-3/4, Sox-2, Nanog and Bmi-1 in esophageal cancer patients using Reverse-Transcription Polymerase Chain Reaction (RT-PCR) with the aim of evaluating their potential as minimally invasive diagnostic markers. Result Increased transcript levels of Oct-4, Sox-2, Bmi-1 and Nanog were detected in (92%), (95%), (75%) and (67%) of the esophageal cancer tissues, respectively as compared with the matched distant normals. Conclusion Interestingly, most of the preneoplastic tissues exhibited increased transcript levels of these stemness markers suggesting their role in early stages of esophageal tumorigenesis. Furthermore, the detection of elevated levels of circulating mRNAs of Oct-4 and Nanog in sera of esophageal cancer patients emphasizes their potential as minimally invasive diagnostic markers for esophageal cancer.

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.