Clinical significance of altered expression of ß-catenin and E-cadherin in oral dysplasia and cancer: potential link with ALCAM expression.

BACKGROUND: 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 ß -catenin expressions are early events in oral tumorigenesis, associated with disease prognosis, and correlate with perturbations in ALCAM expression. METHODS: Expressions of E-cadherin and ß-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 ß -catenin was determined using western blot, confocal microscopy and RT-PCR analysis in oral cancer cells. RESULTS: Significant loss of membranous E-cadherin and ß-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 ß-catenin membrane loss and ALCAM/ß-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 ß-catenin both at the transcript and protein levels. CONCLUSIONS: Losses of E-cadherin and ß-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 ß-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.

Clinical significance of phosphatidyl inositol synthase overexpression in oral cancer.

BACKGROUND: We reported increased levels of phosphatidyl inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST) exposure. METHODS: Tissue microarray (TMA) Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. RESULTS: Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000). Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005) and tobacco consumption (p = 0.03, OR = 9.0). Exposure of oral cell systems to smokeless tobacco (ST) in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K) and cyclin D1 levels. CONCLUSION: Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco.

Cytoplasmic accumulation of activated leukocyte cell adhesion molecule is a predictor of disease progression and reduced survival in oral cancer patients.

Activated leukocyte cell adhesion molecule (ALCAM) has been proposed to function as a cell surface sensor for cell density, controlling the transition between local cell proliferation and tissue invasion in cancer progression. Herein, we determined ALCAM expression in 107 oral squamous cell carcinomas (OSCCs), 78 oral lesions (58 hyperplasias and 20 dysplasias) and 30 histologically normal oral tissues using immunohistochemistry and correlated with clinicopathological parameters. Significant increase in ALCAM immunopositivity was observed from normal oral mucosa, hyperplasia, dysplasia to OSCCs (p(trend) < 0.001). Increased ALCAM expression was observed in cytoplasm of epithelial cells as early as in hyperplasia (p = 0.001, OR = 3.8). Sixty-five of 107 (61%) OSCCs showed significant overexpression of ALCAM protein in cytoplasm/membrane of tumor cells (p = 0.043; OR = 3.3) in comparison with the normal oral tissues. Among OSCCs, cytoplasmic ALCAM was associated with advanced tumor size, tumor stage and tobacco consumption. Importantly, cytoplasmic ALCAM was an independent predictor of poor prognosis of OSCCs in multivariate analysis (p = 0.012, OR = 6.2). In an attempt to understand the molecular basis of cytoplasmic localization of ALCAM, 14-3-3 zeta and 14-3-3 sigma were identified as its novel binding partners in oral cancer cells. In conclusion, increased expression of ALCAM is an early event in oral tumorigenesis; its cytoplasmic accumulation in tumor cells is a predictor of poor prognosis of OSCCs, underscoring its potential as a candidate prognostic marker for oral cancer.

Expression of NF-kappaB parallels COX-2 expression in oral precancer and cancer: association with smokeless tobacco.

Nuclear Factor-kappaB (NF-kappaB) activation and COX-2 overexpression have been reported in head and neck cancer, but the relationship between these proteins remains to be investigated. To determine the relationship between NF-kappaB and COX-2 in Smokeless Tobacco (ST) associated oral tumorigenesis, we performed immunohistochemistry in serial sections from 107 OSCCs, 78 oral precancerous lesions (OPLs) (58 hyperplasias, 20 dysplasias) and 15 histologically normal oral tissues and correlated with clinicopathological data. Significant increase in NF-kappaB and COX-2 immunopositivity was observed from normal oral mucosa to OPLs to OSCCs (p = 0.009 and p = 0.002 respectively). Upregulation of NF-kappaB and COX-2 was observed as early as in hyperplasia [p = 0.006; OR = 6.1 and p = 0.003; OR = 7.6, respectively]. Expression of both proteins was found to be significantly associated in OPLs (p = 0.000; OR = 12.6) and OSCCs (p = 0.001; OR = 4.0). Intriguingly, khaini consumption correlated with NF-kappaB immunopositivity in OPLs (p = 0.05, OR = 3.8) and OSCCs (p = 0.01, OR = 3.4) and with COX-2 expression in OPLs (p = 0.03; OR = 4.3). In vitro experimental system of ST associated oral carcinogenesis was used to demonstrate ST (khaini) and NNK mediated activation of NF-kappaB and COX-2, supporting the clinical data. In conclusion, this study demonstrates correlation between over expression of NF-kappaB and COX-2 in early precancerous stages of development of oral cancer and sustained elevation down the tumorigenic pathway, underscoring their potential as targets for early intervention. In vitro studies demonstrated that NNK may be one of the carcinogenic components of ST (khaini) inducing activation of NF-kappaB and COX-2 in oral precancer and cancer cells, suggesting plausible role in ST-induced oral carcinogenesis.

MGMT expression in oral precancerous and cancerous lesions: correlation with progression, nodal metastasis and poor prognosis.

Alkylation of DNA at the O(6) position of guanine is a critical step in the induction of mutations by carcinogenic and chemotherapeutic alkylating agents. O(6)-methylguanine-DNA methyltransferase (MGMT) is an enzyme that removes mutagenic adducts from the O(6) position of guanine, thereby protecting the genome against guanine to adenine transitions. We hypothesized that alteration in MGMT expression might occur in early stages of development of oral cancer and be associated with disease progression. Immunohistochemical analysis of MGMT expression was carried out in 107 oral squamous cell carcinomas (OSCCs), 78 oral precancerous lesions (OPLs) (58 hyperplasias and 20 dysplasias) and 30 histologically normal oral tissues and correlated with clinicopathological parameters as well as major risk factors. Decreased MGMT expression was observed as early as in hyperplasia (p=0.003; Odd's Ratio (OR)=5.0). Significant loss of MGMT expression was observed from hyperplasia to dysplasia (p=0.034; OR=4.0). Loss of MGMT expression was associated with late clinical stage of OSCCs (p=0.027, OR=2.0) and nodal metastasis (p=0.031, OR=2.5). Decreased MGMT expression was associated with smokeless tobacco (ST) consumption in patients with OPLs (p=0.017, OR=3.6) and OSCCs (p=0.031, OR=2.8). Significant association was also observed between loss of MGMT expression and poor prognosis of OSCC patients (p=0.02; OR=5.2). The decreased MGMT expression in OPLs suggested that deregulation of MGMT expression is an early event in the development of oral cancer. In OSCCs, its correlation with late clinical stage, and nodal metastasis suggests association with aggressive tumor behavior and cancer progression, underscoring its potential as a candidate predictive marker for nodal metastasis and disease prognosis. Correlation of loss of MGMT expression with ST consumption underscored its significance in ST-associated oral carcinogenesis.

Age related changes in Fas (CD95) and Fas ligand gene expression and cytokine profiles in healthy Indians.

Ageing in human and animal models show changes in many aspects of protective immunity, particularly lymphopoenia and progressive decline in immune functions leading to increased frequency of infection and neoplasia. However, the exact mechanism of these defects is still unclear. In this study, elderly subjects showed a decline in CD3+ and CD4+ T-cell subsets as well as serum IL-2 levels. Serum IL-6 was significantly raised while expression of its signaling receptor gp130 was significantly impaired in elderly as compared to the younger ones. Additionally, all the elderly individuals showed constitutive expression of Fas and FasL mRNA; however, none of the younger individuals expressed mRNA transcripts constitutively although induced expression was seen in both the groups. Similarly, frequency of Fas and FasL expressing CD4+ and CD8+ T-cell subsets were significantly (p < 0.001) higher in elderly subjects as compared to the younger ones. Elderly individuals also showed a significantly (p < 0.001) higher frequency of activation induced cell death (AICD). Since interaction of Fas with its cognate ligand (FasL) activates death inducing caspases leading to apoptosis, and gp130 induces anti-apoptotic signal through STAT-3 pathway, these results suggest that the decline in protective immune functions in aged individuals may be related to Fas and FasL mediated apoptosis of peripheral T-cell subsets.

Novel germline mutations in the BRCA1 and BRCA2 genes in Indian breast and breast-ovarian cancer families.

The two major hereditary breast/ovarian cancer predisposition tumor suppressor genes, BRCA1 and BRCA2 that perform apparently generic cellular functions nonetheless cause tissue-specific syndromes in the human population when they are altered, or mutated in the germline. However, little is known about the contribution of BRCA1 and BRCA2 mutations to breast and/or ovarian cancers in the Indian population. We have screened for mutations the entire BRCA1 and BRCA2 coding sequences, and intron-exon boundaries, as well as their flanking intronic regions in sixteen breast or breast and ovarian cancer families of Indian origin. We have also analyzed 20 female patients with sporadic breast cancer regardless of age and family history, and 69 unrelated normal individuals as control. Thus a total of 154 samples were screened for BRCA1 and BRCA2 mutations using a combination of polymerase chain reaction-mediated site directed mutagenesis (PSM), polymerase chain reaction-single stranded conformation polymorphism assay (PCR-SSCP) and direct DNA sequencing of PCR products (DS). Twenty-one sequence variants including fifteen point mutations were identified. Five deleterious pathogenic, protein truncating frameshift and non-sense mutations were detected in exon 2 (c.187_188delAG); and exon 11 (c.3672G>T) [p.Glu1185X] of BRCA1 and in exon 11 (c.5227dupT, c.5242dupT, c.6180dupA) of BRCA2 (putative mutations - four novel) as well as fourteen amino acid substitutions were identified. Twelve BRCA1 and BRCA2 missense variants were identified as unique and novel. In the cohort of 20 sporadic female patients no mutations were found.