Abdelmajid Khabir

Inactivation of RASSF1A, RARβ2 and DAP-kinase by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinoma

Epigenetic modification is one of the mechanisms leading to gene silencing in neoplastic cells. By methylation-specific PCR, we analyzed the promoter methylation of three cancer-related genes: Ras Association domain Family 1A (RASSF1A), Death Associated Protein kinase (DAP-kinase) and Retinoic Acid Receptor β2 (RARβ2) in two NPC xenografts (C15 and C17), 68 primary NPC tumors, and nine normal nasopharyngeal epithelia. We showed that C15 and C17 displayed a complete promoter methylation of RASSF1A, RARβ2 and DAP-kinase genes. In primary NPC tumors, the incidence of promoter methylation was very high for all three tested genes: 91% for RASSF1A, 88% for both RARβ2 and DAP-kinase whereas all normal nasopharyngeal epithelia were unmethylated. Interestingly, our study revealed that aberrant promoter methylation of the three genes were statistically associated with the lymph node involvement (p < 0.0001). In addition, hypermethylation of RASSF1A was correlated with age at diagnosis (p = 0.047) and T stage (p = 0.037) while the RARβ2 hypermethylation was associated with histological type (p = 0.011). Taken together, our results demonstrate that silencing of RASSF1A and RARβ2 expression by promoter hypermethylation is associated with highly differentiated tumors, advanced tumor stage and the presence of lymph node metastasis. To assess the functional significance of the epigenetic silencing of RARβ2 and DAP-kinase in NPC, we analysed the expression of two downstream target genes COX-2 and p53 by reverse PCR (RT-PCR) and immunohistochemistry (IHC). We revealed a significant association between expression of COX-2 and loss of RARβ2 through aberrant methylation (p = 0.003) in NPC biopsies. We concluded that the inactivation of RASSF1A, RARβ2 and DAP-Kinase by hypermethylation is a key step in NPC tumorigenesis and progression.

PIK3CA amplification is predictive of poor prognosis in Tunisian patients with nasopharyngeal carcinoma

PI3Ks (phosphatidylinositol 3‐kinases) are lipid kinases that regulate signalling pathways involved in cell proliferation, motility, and adhesion. Somatic mutations and amplification of the PIK3CA gene have been reported in various types of human cancers. However, little is known about the frequency and prognosis role of PIK3CA activation in nasopharyngeal carcinoma (NPC). This study was conducted with the aim to screen for PIK3CA mutations in the two hot spot regions (exons 9 and 20) and to investigate for the PIK3CA gene amplification combined with the expression analysis of the phosphorylated Akt (pAkt). We showed that among 88 specimens, none had mutation in the helical domain (exon 9) and only one (1.13%) had mutation in the kinase domain (exon 20). On the other hand, PIK3CA gene amplification was found in 21.6% of cases and was strongly associated with distant metastasis (P = 0.002), lymph node involvement (P = 0.032), and advanced tumor stage (P < 0.001). Moreover, patients with PIK3CA copy number gain have a significant reduced overall survival time (P log rank = 0.02). We concluded that PIK3CA gene amplification is frequent in NPC and occurs in the advanced stage of NPC. Moreover, our finding emphasizes the association of PIK3CA gene amplification with worse prognosis in nasopharyngeal carcinoma. (Cancer Sci 2009); 00: 000–000)

Hypermethylation of tumor-related genes in Tunisian patients with gastric carcinoma: clinical and biological significance.

Promoter hypermethylation is an alternative mechanism of gene silencing in cancers including gastric carcinoma (GC). Its affects genes with crucial functions as tumor suppressor.

BCL2L12 is a Novel Biomarker for the Prediction of Short-Term Relapse in Nasopharyngeal Carcinoma

BCL2-like 12 (BCL2L12) is a new member of the apoptosis-related BCL2 gene family, members of which are implicated in various malignancies. Nasopharyngeal carcinoma is a highly metastatic, malignant epithelial tumor, with a high prevalence in South-east Asia and North Africa. The purpose of the current study was to quantify and investigate the expression levels of the BCL2L12 gene in nasopharyngeal carcinoma biopsies and to assess its prognostic value. Total RNA was isolated from 89 malignant and hyperplastic nasopharyngeal biopsies from Tunisian patients. After testing the quality of the extracted RNA, cDNA was prepared by reverse transcription. A highly sensitive real-time polymerase chain reaction (PCR) method for BCL2L12 mRNA quantification was developed using SYBR® Green chemistry. GAPDH served as a reference gene. Relative quantification analysis was performed using the comparative CT (2−ΔΔCT) method. Higher BCL2L12 mRNA levels were detected in undifferentiated carcinomas of the nasopharynx, rather than in nonkeratinizing nasopharyngeal tumors (P = 0.045). BCL2L12 expression status was also found to be positively associated with the presence of distant metastases (P = 0.014). Kaplan-Meier survival analysis demonstrated that patients with BCL2L12-positive nasopharyngeal tumors have significantly shorter disease-free survival (P = 0.020). Cox regression analysis showed BCL2L12 expression to be an unfavorable and independent prognostic indicator of short-term relapse in nasopharyngeal carcinoma (P = 0.042). Our results suggest that mRNA expression of BCL2L12 may constitute a novel biomarker for the prediction of short-term relapse in nasopharyngeal carcinoma.

Genetic Polymorphisms in the EGFR (R521K) and Estrogen Receptor (T594T) Genes, EGFR and ErbB-2 Protein Expression, and Breast Cancer Risk in Tunisia

We evaluated the association of epidermal growth factor receptor (EGFR) 142285G>A (R521K) and estrogen receptor alpha (ESR1) 2014G>A (T594T) single nucleotide polymorphisms with breast cancer risk and prognosis in Tunisian patients. EGFR 142285G>A and ESR1 2014G>A were genotyped in a sample of 148 Tunisian breast cancer patients and 303 controls using PCR-RFLP method. Immunohistochemitsry was used to evaluate the expression levels of EGFR, HER2, ESR1, progesterone receptor and BCL2 in tumors. We found no evidence for an association between EGFR R521K polymorphism and breast cancer risk. However, we found that the homozygous GG (Arg) genotype was more prevalent in patients with lymph node metastasis (P = .03) and high grade tumors (P = .011). The ESR1 2014G allele showed significant association with breast cancer risk (P = .025). The GG genotype was associated with HER2 overexpression and this association withstood univariate and multivariate analyses (P = .009; P = .021, resp.). These data suggest that the R521K might be a prognostic factor, because it correlates with both tumor grade and nodule status. The higher expression of HER2 in ESR1 T594T GG patients suggests the possibility that ESR1 gene polymorphisms accompanied by HER2 expression might influence the pathogenesis of breast cancers.

Clinical Significance of Epigenetic Inactivation of hMLH1 and BRCA1 in Tunisian Patients with Invasive Breast Carcinoma

Aberrant hypermethylation of gene promoter regions is one of the mechanisms for inactivation of tumour suppressor genes in many human cancers including breast carcinoma. In the current study, we aimed to assess by MSP, the methylation pattern of two cancer-related genes involved in DNA repair: hMLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) and BRCA1 (breast cancer 1, early onset) in 78 primary breast cancers from Tunisian patients. The methylation frequencies were 24.36% for hMLH1 and 46% for BRCA1. BRCA1 methylation correlated with age at diagnosis (P = .015) and 5-years disease free survival (P = .016) while hMLH1 methylation was more frequent in larger tumors (P = .002) and in presence of distant metastasis (P = .004). Furthermore, methylation of hMLH1 significantly correlated with high level of P53 expression (P = .006) and with overall survival (P = .015) suggesting that silencing of hMLH1 through aberrant promoter methylation could be used as a poor prognosis indicator in breast cancer.

Hypermethylation of RARβ2 correlates with high COX-2 expression and poor prognosis in patients with colorectal carcinoma

Silencing of gene expression by aberrant methylation at the CpG islands is common in human tumors, including colorectal cancer. This epigenetic alteration affects promoter of genes having crucial cellular functions such as tumor suppressor, DNA repair, apoptosis, cell adhesion, etc. We investigated the methylation status in the promoter regions of the RARβ2, RASSF1A, DAPKinase, and CDH1 genes in 73 colorectal carcinoma and 43 paired normal tissues of Tunisian patients using methylation-specific PCR assays. The association between methylation status and the clinicopathological features was evaluated. To determine whether aberrant methylation affects gene expression, we performed immunohistochemistry analysis for E-cadherin and COX-2, a target gene of RARβ2. The methylation frequencies vary from 80.8% for RARβ2 to 35.6% for RASSF1A while in non-tumor-paired samples; the frequencies of methylation are significantly lower for all the fourth genes tested. The methylation status did not correlate with any of the clinical features considered; however, aberrant methylation of RARβ2 was associated with a shortened overall patients’ survival (p logrank = 0.026); nevertheless, it needs to be confirmed on larger sample size. Moreover, a significant inverse association was observed between methylation status of RARβ2 and COX-2 protein expression in tumor specimen (p = 0.014). On the other hand, we found that loss of E-cadherin expression was significantly associated with aberrant methylation of the CDH1 promoter (p = 0.005). Our findings showed that RARβ2 was frequently methylated in colorectal cancer and correlated with a worse prognosis and high expression of COX-2 suggesting a link between these two proteins in colorectal carcinogenesis. We also showed that epigenetic alteration of CDH1 is a major mechanism of the loss of E-cadherin protein expression in primary colorectal tumors.

Apoptosis and TRAF-1 cleavage in Epstein-Barr virus-positive nasopharyngeal carcinoma cells treated with doxorubicin combined with a farnesyl-transferase inhibitor

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinomas (NPC) are much more sensitive to chemotherapy than other head and neck carcinomas. Spectacular regressions are frequently observed after induction chemotherapy. However, these favorable responses are difficult to predict and often of short duration. So far there have been only few experiments to investigate the mechanisms which underline the cytotoxic effects of anti-neoplastic drugs against NPC cells. In addition, these studies were performed almost entirely on EBV-negative cell lines therefore not truly representative of NPC cells. For the first time, we have used two EBV-positive NPC tumor lines derived from a North African (C15) and a Chinese (C666-1) patient as in vitro targets for a panel of anti-neoplastic agents. Doxorubicin, taxol and in a lesser extent cis-platinum efficiently inhibited NPC cell proliferation at clinically relevant concentrations, but all three agents failed to induce apoptosis. However, massive apoptosis of C15 cells was achieved when doxorubicin (1 microM) was combined with a farnesyl-transferase inhibitor, BIM 2001 (5 microM). Moreover, this apoptotic process was associated with a caspase-dependent early cleavage of the TNF-receptor associated factor 1 (TRAF-1) molecule, a signaling adaptor which is specifically expressed in latently EBV-infected cells. TRAF-1 cleavage might become a useful indicator of chemo-induced apoptosis in EBV-associated NPCs.

Quantitative analysis of BCL2 mRNA expression in nasopharyngeal carcinoma: an unfavorable and independent prognostic factor

Programmed cell death plays a vital role in a wide variety of physiological processes. Defects in apoptotic cell death contribute to neoplastic diseases by preventing or delaying normal cell death. BCL2 (Bcl-2) is an anti-apoptotic gene with marked up-regulation in various malignancies, such as breast cancer, in which expression of the BCL2 protein has been proposed as a prognostic tumor biomarker. The purpose of the current study was to investigate mRNA expression of the BCL2 gene in nasopharyngeal carcinoma (NPC) biopsies and assess its prognostic value. For this purpose, total RNA was isolated from 89 malignant and hyperplastic nasopharyngeal biopsies from Tunisian patients. After testing the quality of the extracted RNA, cDNA was prepared by reverse transcription. A highly sensitive real-time PCR methodology for BCL2 mRNA quantification was developed using SYBR® Green chemistry. GAPDH served as an endogenous control gene. Relative quantification analysis was performed using the comparative CT (2−∆∆CT) method. High BCL2 mRNA levels were detected in advanced-stage nasopharyngeal tumors (p = 0.030). Furthermore, BCL2 mRNA expression was strongly associated with lymph node involvement (p = 0.009) and presence of distal metastases (p = 0.013). Survival analysis demonstrated that patients with BCL2-positive nasopharyngeal tumors have significantly shorter disease-free and overall survival (p = 0.011 and p = 0.028, respectively). The major contribution of the current study is the quantification and evaluation, for the first time, of the prognostic significance of the BCL2 mRNA expression in nasopharyngeal carcinoma (NPC) patients. Our results suggest that mRNA expression levels of BCL2 may represent a novel unfavorable and independent tumor biomarker for nasopharyngeal carcinoma.

HER2 Polymorphisms and Breast Cancer in Tunisian Women

HER2 has been thought to play a critical role in both breast cancer development and progression. Any functional polymorphisms can potentially affect breast cancer risk as well as cancer phenotype and outcome. In our study, we analyzed three polymorphisms in the HER2 gene: the single-nucleotide polymorphism (SNP) HER2 Ile(655)Val as well as another SNP (rs903506) close to it and a new screened dinucleotide repeat H(AC)I4 in intron 4, in a sample of 148 cases and 290 controls from the Tunisian population and investigated their association with breast cancer risk. For the HER2 Ile(655)Val, we found similar allele frequencies between cases and controls (frequency of I allele was 0.92 and 0.91, respectively). The same was observed for the noncoding SNP (rs903506). These two SNPs also showed no association with any clinical parameters, except the association of HER2 Ile(655)Val with tumor size (p = 0.002). But, a significant association was found between the short tandem repeat (STR) [H(AC)I4] and breast cancer risk at both genotypic and allelic levels (p = 0.0004 and p = 0.0001, respectively). Multivariate analysis with binary logistic regression of disease status on genotypes of the three polymorphisms confirmed the association of STR with breast cancer risk (p = 0.016). Therefore, this STR seems to be a promising biomarker in breast cancer and deserves further investigation.