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Perturbations in cell adhesion molecules are linked to alterations in cadherin-catenin complexes and likely play major roles in invasion and metastasis; their impact on early precancerous stages remains yet unknown. We showed ALCAM overexpression in early oral lesions and its cytoplasmic accumulation in oral squamous cell carcinoma (OSCC) to be a predictor of disease progression and poor prognosis. This study tested the hypothesis that alterations in E-cadherin and β -catenin expressions are early events in oral tumorigenesis, associated with disease prognosis, and correlate with perturbations in ALCAM expression. Expressions of E-cadherin and β-catenin were analyzed in the same cohort of 105 OSCCs, 76 oral lesions and 30 normal oral tissues by immunohistochemistry and correlated with clinicopathological parameters and prognosis. The effect of siRNA mediated ALCAM knockdown on E-cadherin and β -catenin was determined using western blot, confocal microscopy and RT-PCR analysis in oral cancer cells. Significant loss of membranous E-cadherin and β-catenin expression was observed from normal, hyperplasia, dysplasia to OSCCs (p(trend) <0.001); and correlated with cytoplasmic ALCAM accumulation in OSCCs (p  = 0.006). Multivariate analysis revealed β-catenin membrane loss and ALCAM/β-catenin(nuclear/cytoplasmic) accumulation to be significant predictors for late clinical stage (p<0.001, OR = 8.7; p = 0.006, OR = 9.9, respectively) and nodal metastasis (p = 0.003, OR = 3.8; p = 0.025, OR = 3.4 respectively). Cox's regression showed E-cadherin membrane loss/ALCAM cytoplasmic expression [p<0.001; HR = 4.8] to be independent adverse prognosticators in OSCCs. siRNA mediated silencing of ALCAM resulted in concurrent increase in E-cadherin and β-catenin both at the transcript and protein levels. Losses of E-cadherin and β-catenin expressions are early events in oral tumorigenesis; their associations with aggressive tumor behavior and disease recurrence underscore their potential as prognostic markers. Correlation of loss of E-cadherin and β-catenin with cytoplasmic ALCAM accumulation both in vitro and in in vivo suggests that these dynamic changes in cell adhesion system may play pivotal role in oral cancer.

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. 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. 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. 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.

Molecular markers for predicting breast cancer patients at high risk of recurrence are urgently needed for more effective disease management. The impact of alterations in extracellular matrix components on tumor aggressiveness is under intense investigation. Overexpression of Transglutaminase 2 (TG2), a multifunctional enzyme, in cancer cells impacts epithelial mesenchymal transition, growth, invasion and interactions with tumor microenvironment. The objective of our study is to determine the clinical relevance of stromal TG2 overexpression and explore its potential to identify breast cancers at high risk of recurrence. This retrospective study is based on immunohistochemical analysis of TG2 expression in normal breast tissues (n = 40) and breast cancers (n = 253) with clinical, pathological and follow-up data available for up to 12 years. TG2 expression was correlated with clinical and pathological parameters as well as disease free survival (DFS) of breast cancer patients. Stromal TG2 overexpression was observed in 114/253 (45.0%) breast cancer tissues as compared to breast normal tissues. Among invasive ductal carcinomas (IDC) of the breast, 97/168 (57.7%) showed strong TG2 expression in tumor stroma. Importantly, IDC patients showing stromal TG2 accumulation had significantly reduced DFS (mean DFS = 110 months) in comparison with patients showing low expression (mean DFS = 130 months) in Kaplan-Meier survival analysis (p<0.001). In Cox multivariate regression analysis, stromal TG2 accumulation was an independent risk factor for recurrence (p = 0.006, Hazard's ratio, H.R. = 3.79). Notably, these breast cancer patients also showed immunostaining of N-epsilon gamma-glutamyl lysine amino residues in tumor stroma demonstrating the transamidating activity of TG2. Accumulation of TG2 in tumor stroma is an independent risk factor for identifying breast cancer patients at high risk of recurrence. TG2 overexpression in tumor stroma may serve as a predictor of poor prognosis for IDC of the breast.

Oral squamous cell carcinoma (OSCC) patients diagnosed in late stages have limited chemotherapeutic options underscoring the great need for development of new anticancer agents for more effective disease management. We aimed to investigate the anticancer potential of Apaziquone, [EOquin, USAN, E09, 3-hydroxy-5- aziridinyl-1-methyl-2(1H-indole-4,7-dione)-prop-β-en-α-ol], a pro-drug belonging to a class of anti-cancer agents called bioreductive alkylating agents, for OSCC. Apaziquone treatment inhibited cell proliferation and induced apoptosis in OSCC cells in vitro. Apaziquone treated OSCC cells showed increased activation of Caspase 9 and Caspase 3, and Poly (ADP ribose) polymerase (PARP) cleavage suggesting induction of apoptosis by apaziquone in oral cancer cells. Importantly, apaziquone treatment significantly reduced oral tumor xenograft volume in immunocompromised NOD/SCID/Crl mice without causing apparent toxicity to normal tissues. In conclusion, our in vitro and in vivo studies identified and demonstrated the pre-clinical efficacy of Apaziquone, as a potential novel anti-cancer therapeutic candidate for oral cancer management.

Background Proteolytic cleavage of the extracellular domain (EpEx) of Epithelial cell adhesion molecule (EpCAM) and nuclear signaling by its intracellular oncogenic domain Ep-ICD has recently been implicated in increased proliferation of cancer cells. The clinical significance of Ep-ICD in human tumors remains an enigma. Methods EpEx, Ep-ICD and β-catenin immunohistochemistry using specific antibodies was conducted on 58 archived thyroid cancer (TC) tissue blocks from 34 patients and correlated with survival analysis of these patients for up to 17 years. Results The anaplastic (ATC) and aggressive thyroid cancers showed loss of EpEx and increased nuclear and cytoplasmic accumulation of Ep-ICD. In contrast, the low grade papillary thyroid cancers (PTC) showed membranous EpEx and no detectable nuclear Ep-ICD. The ATC also showed concomitant nuclear expression of Ep-ICD and β-catenin. Kaplan-Meier Survival analysis revealed reduced overall survival (OS) for TC patients showing nuclear Ep-ICD expression or loss of membranous EpEx (p < 0.0004), median OS = 5 months as compared to 198 months for patients who did not show nuclear Ep-ICD or demonstrated only membranous EpE. Conclusion We report reciprocal loss of membrane EpEx but increased nuclear and cytoplasmic accumulation of Ep-ICD in aggressive TC; nuclear Ep-ICD correlated with poor OS of TC patients. Thus nuclear Ep-ICD localization may serve as a useful biomarker for aggressive TC and may represent a novel diagnostic, prognostic and therapeutic target for aggressive TC.

We previously demonstrated that nuclear and cytoplasmic accumulation of the intracellular domain (Ep-ICD) of epithelial cell adhesion molecule (EpCAM) accompanied by a reciprocal reduction of its extracellular domain (EpEx), occurs in aggressive thyroid cancers. This study was designed to determine whether similar accumulation of Ep-ICD is a common event in other epithelial cancers. Ten epithelial cancers were immunohistochemically analyzed using Ep-ICD and EpEx domain-specific antibodies. The subcellular localization of EpEx and Ep-ICD in the human colon adenocarcinoma cell line CX-1 was observed using immunofluorescence. Nuclear and cytoplasmic Ep-ICD expression was increased in cancers of the breast (31 of 38 tissues, 82%), prostate (40 of 49 tissues, 82%), head and neck (37 of 57 tissues, 65%) and esophagus (17 of 46 tissues, 37%) compared to their corresponding normal tissues that showed membrane localization of the protein. Importantly, Ep-ICD was not detected in the nuclei of epithelial cells in most normal tissues. High nuclear and cytoplasmic Ep-ICD accumulation also occurred in the other six epithelial cancer types analyzed - lung, colon, liver, bladder, pancreatic, and ovarian. A concomitant reduction in membrane EpEx expression was observed in a subset of all cancer types. Receiver operating characteristic curve analysis revealed nuclear Ep-ICD distinguished breast cancers with 82% sensitivity and 100% specificity and prostate cancers with 82% sensitivity and 78% specificity. Similar findings were observed for cytoplasmic accumulation of Ep-ICD in these cancers. We provide clinical evidence of increased nuclear and cytoplasmic Ep-ICD accumulation and a reduction in membranous EpEx in these cancers. Increased nuclear and cytoplasmic Ep-ICD was observed in all epithelial cancers analyzed and distinguished them from normal tissues with high-sensitivity, specificity, and AUC. Development of a robust high throughput assay for Ep-ICD will facilitate the determination of its diagnostic, prognostic and therapeutic relevance in epithelial cancers.

Background Thyroid cancer is among the fastest growing malignancies; almost fifty-percent of these rapidly increasing incidence tumors are less than or equal to 1cm in size, termed papillary thyroid microcarcinoma (PTMC). The management of PTMC remains a controversy due to differing natural history of these patients. Epithelial cell adhesion molecule (EpCAM) is comprised of an extracellular domain (EpEx), a single transmembrane domain and an intracellular domain (Ep-ICD). Our group reported nuclear Ep-ICD correlated with poor prognosis in thyroid cancer (Ralhan et al., BMC Cancer 2010,10:331). Here in, we hypothesized nuclear and cytoplasmic accumulation of Ep-ICD and loss of membranous EpEx may aid in distinguishing metastatic from non-metastatic PTMC, which is an important current clinical challenge. To test our hypothesis, Ep-ICD and EpEx expression levels were analyzed in PTMC and the staining was correlated with metastatic potential of these carcinomas. Methods Thirty-six PTMC patients (tumor size 0.5 - 1cm; metastatic 8 cases and non-metastatic 28 cases) who underwent total thyroidectomy were selected. The metastatic group consisted of patients who developed lymph node or distant metastasis at diagnosis or during follow up. The patients’ tissues were stained for Ep-ICD and EpEx using domain specific antibodies by immunohistochemistry and evaluated. Results PTMC patients with metastasis had higher scores for nuclear and cytoplasmic Ep-ICD immunostaining than the patients without metastasis (1.96 ± 0.86 vs. 1.22 ± 0.45; p = 0.007 and 5.37 ± 0.33 vs. 4.72 ± 1.07; p = 0.016, respectively). Concomitantly, the former had lower scores for membrane EpEx than the non-metastatic group (4.64 ± 1.08 vs. 5.64 ± 1.51; p = 0.026). An index of aggressiveness, Ep-ICD subcellular localization index (ESLI), was defined as sum of the IHC scores for accumulation of nuclear and cytoplasmic Ep-ICD and loss of membranous EpEx; ESLI = [Ep − ICD nuc  + Ep − ICD cyt  + loss of membranous EpEx]. Notably, ESLI correlated significantly with lymph node metastasis in PTMC (p = 0.008). Conclusion Nuclear and cytoplasmic Ep-ICD expression and loss of membranous EpEx were found to correlate positively with metastasis in PTMC patients. In addition, ESLI had the potential to identify metastatic behavior in PTMC which could serve as a valuable tool for solving a current dilemma in clinical practice.

Molecular markers for predicting prostate cancer (PCa) that would have poor prognosis are urgently needed for a more personalized treatment for patients. Regulated intramembrane proteolysis of Epithelial cell adhesion molecule results in shedding of the extracellular domain (EpEx) and release of its intracellular domain (Ep-ICD) which triggers oncogenic signaling and might correlate to tumor aggressiveness. This study aimed to explore the potential of Ep-ICD and EpEx to identify PCa that have poor prognosis. Immunohistochemical analysis of Ep-ICD and EpEx was carried out in normal prostate tissues (n = 100), benign prostate hyperplasia (BPH, n = 83), and prostate cancer (n = 249) using domain specific antibodies. The expression of Ep-ICD and EpEx was correlated with clinico- pathological parameters and disease free survival (DFS). Reduced expression of nuclear Ep-ICD and membrane EpEx was observed in PCa in comparison with BPH and normal prostate tissues (p = 0.006, p < 0.001 respectively). For patients who had PCa with Gleason Score less than 7, preserved nuclear Ep-ICD emerged as the most significant marker in multivariate analysis for prolonged DFS, where these patients did not have recurrence during follow up of up to 12 years (p = 0.001). Reduced expression of nuclear Ep-ICD was associated with shorter disease free survival in patients with a Gleason Score less than 7 and may be useful in identifying patients likely to have aggressive tumors with poor prognosis. Furthermore, nuclear Ep-ICD can differentiate between normal and prostate cancer tissues for ambiguous cases.

Nuclear accumulation of the intracellular domain of epithelial cell adhesion molecule (Ep-ICD) in tumor cells was demonstrated to predict poor prognosis in thyroid carcinoma patients in our earlier study. Here, we investigated the clinical significance of Ep-ICD subcellular localization index (ESLI) in distinguishing aggressive papillary thyroid carcinoma (PTC) from non-aggressive cases. Using domain specific antibodies against the intracellular (Ep-ICD) and extracellular (EpEx) domains of epithelial cell adhesion molecule, 200 archived tissues from a new cohort of patients with benign thyroid disease as well as malignant aggressive and non aggressive PTC were analyzed by immunohistochemistry (IHC). ESLI was defined as sum of the IHC scores for accumulation of nuclear and cytoplasmic Ep-ICD and loss of membranous EpEx; ESLI = [Ep-ICD(nuc) + Ep-ICD(cyt) + loss of membranous EpEx]. For the benign thyroid tissues, non-aggressive PTC and aggressive PTC, the mean ESLI scores were 4.5, 6.7 and 11 respectively. Immunofluorescence double staining confirmed increased nuclear Ep-ICD accumulation and decreased membrane EpEx expression in aggressive PTC. Receiver-operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.841, 70.2% sensitivity and 83.9% specificity for nuclear Ep-ICD for differentiating aggressive PTC from non-aggressive PTC. ESLI distinguished aggressive PTC from non-aggressive cases with improved AUC of 0.924, 88.4% sensitivity and 85.5% specificity. Our study confirms nuclear accumulation of Ep-ICD and loss of membranous EpEx occurs in aggressive PTC underscoring the potential of Ep-ICD and ESLI to serve as diagnostic markers for aggressive PTC. Kaplan Meier survival analysis revealed significantly reduced disease free survival (DFS) for ESLI positive (cutoff >10) PTC (p<0.05), mean DFS=133 months as compared to 210 months for patients who did not show positive ESLI. ESLI scoring improves the identification of aggressive PTC and thereby may serve as a useful index for defining aggressiveness and poor prognosis among PTC patients.

Background Oral squamous cell carcinoma (OSCC) patients are at high risk of loco-regional recurrence and 5-year survival rates are about 50%. Identification of patients at high risk of recurrence will enable rigorous personalized post-treatment management. Most novel biomarkers have failed translation for clinical use because of their limited successful validation in external patient cohorts. The aim of this study was to determine the prognostic significance of alterations in sub-cellular expression of S100A2, a pro-tumorigenic calcium binding protein, identified as a candidate biomarker in our proteomic analysis in OSCC and validation of its clinical utility in an external cohort. Methods In a retrospective study, immunohistochemical analysis of S100A2 was carried out in 235 Indian OSCC (Test set) and 129 normal oral tissues, correlated with clinicopathological parameters and disease outcome over 122 months for OSCC patients following the REMARK criteria. The findings were validated in an external cohort (Validation set 115 Canadian OSCC and 51 normal tissues) and data analyzed using the R package. Results Significant increase in cytoplasmic and decrease in nuclear S100A2 expression was observed in OSCC in comparison with normal tissues. Cox multivariable regression analysis internally and externally validated cytoplasmic S100A2 association with tumor recurrence. Kaplan Meier analysis of patients stratified to high and low risk groups showed significantly different recurrence free survival (Test set- log rank test, p = 0.005, median survival 16 and 69 months respectively and Validation set - p < 0.00001, median survival 9.4 and 59.9 months respectively); 86% and 81% of patients who had recurrence were correctly stratified into the high risk group. Seventy percent and 81% patients stratified into low risk group did not show cancer recurrence within 1 year in Test and Validation sets. Conclusions Our study provided clinical evidence for the potential of cytoplasmic S100A2 overexpression as a predictor of recurrence risk in OSCC patients. A unique translational aspect of our study is validation of S100A2 as prognostic marker in two independent cohorts (Canadian and Indian) suggesting this protein is likely to find widespread utility in clinical practice for identifying oral cancer patients at high risk of disease recurrence.