Novel and recurrent BRCA1/BRCA2 germline mutations in patients with breast/ovarian cancer: a series from the south of Tunisia.

BACKGROUND: The incidence of breast cancer (BC) and/or ovarian cancer (OC) is increasing in Tunisia especially in young women and mostly those with family history. However, the spectrum of BRCA mutations remains little explored in Tunisian patients in particular in the southern region. METHODS: We sequenced the entire coding regions of BRCA1and BRCA2 genes using next generation sequencing (NGS) in 134 selected patients with BC and/or OC. RESULTS: Among the 134 patients, 19 (14.17%) carried pathogenic mutations (10 are BRCA1 mutation carriers and 9 are BRCA2 mutation carriers) that are mainly frameshift index (76.9%). Interestingly, 5 out of the 13 variants (38.46%) were found at least twice in unrelated patients, as the c.1310-1313 delAAGA in BRCA2 and the c.5030_5033 delCTAA that has been identified in 4/98 BC patients and in 3/15 OC patients from unrelated families with strong history of cancer. Besides recurrent mutations, 6 variant (4 in BRCA1 and 2 in BRCA2) were not reported previously. Furthermore, 3 unrelated patients carried the VUS c.9976A > T, (K3326*) in BRCA2 exon 27. BRCA carriers correlated significantly with tumor site (p = 0.029) and TNBC cases (p = 0.008). In the groups of patients aged between 31 and 40, and 41-50 years, BRCA1 mutations occurred more frequently in patients with OC than those with BC, and conversely BRCA2 carriers are mostly affected with BC (p = 0.001, and p = 0.044 respectively). CONCLUSIONS: The overall frequency of the BRCA germline mutations was 14.17% in patients with high risk of breast/ovarian cancer. We identified recurrent mutations as the c.1310_1313 delAAGA in BRCA2 gene and the c.5030_5033 delCTAA in BRCA1 gene that were found in 4% and 20% of familial BC and OC respectively. Our data will contribute in the implementation of genetic counseling and testing for families with high-risk of BC and/or OC.

Identification of novel candidate genes by exome sequencing in Tunisian familial male breast cancer patients.

Male Breast Cancer (MBC) is a rare and aggressive disease that is associated with genetic factors. Mutations in BRCA1 and BRCA2 account for 10% of all MBC cases suggesting that other genetic factors are involved. The aim of the present study is to screen whole BRCA1 and BRCA2 exons using the Ampliseq BRCA panel in Tunisian MBC patients with family history. Furthermore, we performed exome sequencing using the TruSight One sequencing panel on an early onset BRCA negative patient. We showed that among the 6 MBC patients, only one (MBC-F1) harbored a novel frameshift mutation in exon 2 of the BRCA2 gene (c.17-20delAAGA, p.Lys6Xfs) resulting in a short BRCA2 protein of only 6 amino-acids. We selected 9 rare variants after applying several filter steps on the exome sequencing data. Among these variants, and based on their role in breast carcinogenesis, we retained 6 candidate genes (MSH5, DCC, ERBB3, NOTCH3, DIAPH1, and DNAH11). Further studies are needed to confirm the association of the selected genes with family MBC.

Association of FOXA1 and EMT markers (Twist1 and E-cadherin) in breast cancer.

The transcription factor FOXA1 (forkhead box A1) plays key roles in tumor development and progression. In the present study, we analyzed the expression of FOXA1 in 52 breast tumors and 10 normal tissues, and investigated the relationship between FOXA1 and two EMT markers, namely Twist1 and E-cadherin by RT-PCR and IHC respectively. The expression level of FOXA1 was higher in tumor compared to normal tissues but the difference was not statistically significant (P = 0.138). FOXA1 expression correlated with less aggressive behavior as SBR grade I (P = 0.04), small tumors size (P = 0.05), and longer survival (P = 0.001). Furthermore, estrogen and progesterone positive tumors exhibit high level of FOXA1 (P = 0.002 and P = 0.038 respectively). Survival analysis showed that patients with ER positive/FOXA1 positive (P log rank = 0.001), PR positive/FOXA1 positive (P log rank = 0.044) and HER-2 negative/FOXA1 positive (P log rank = 0.002) tumors have a significant prolonged overall survival. On the other hand, the expression of E-cadherin positively correlated with FOXA1 (P = 0.028), whereas negative association was seen between the expression of Twist1 and FOXA1 (P = 0.016). Kaplan-Meier plots showed that patients with Twist1negative/FOXA1positive tumors have a significant prolonged overall survival (P log rank = 0.001) and FOXA1 appeared as independent predictors of patient survival in multivariate analyses. Overall, our results indicate that FOXA1 could be a useful biomarker to predict prognosis in breast cancer patients.

Anti-melanogenesis potential of a new series of Morita-Baylis-Hillman adducts in B16F10 melanoma cell line.

Melanin is a natural polymer pigment which provides skin photoprotection against ultraviolet radiation. An excessive synthesis of melanin leads to hyperpigmentation disorders. Tyrosinase catalyzes the rate limiting steps on melanogenesis. Therefore, tyrosinase inhibitors have potential applications in medicine and cosmetic fields. We carried out herein the screening of a family of cyclic Morita-Baylis-Hillman adducts (MBH) to find out their effects on tyrosinase activity and on melanogenesis in murine melanoma B16F10 cell line. Kinetic analysis of tyrosinase inhibition showed that compounds 1a (2-hydroxymethyl) cyclohex-2-enone) and 3f (diethyl (1-(6-oxocyclohex-1-en-1-yl) ethyl-phosphonate) were competitive inhibitors, whereas the compound 2b (6-oxocyclohex-1-en-1-yl) ethyl acetate) was a non-competitive one. Additionally we have found that (1a, 2b and 3f) compounds had a strong melanogenesis inhibition effect in isobutylmethylxanthine (IBMX)-treated murine melanoma B16F10 cells when tested at low and non cytotoxic dose (10-50 µM), by attenuating the melanin production, intracellular tyrosinase activity and tyrosinase expression. Thus, we suggest that these compounds could be used as effective skin-whitening agents.

Overexpression of miR-10b in colorectal cancer patients: Correlation with TWIST-1 and E-cadherin expression.

MicroRNAs are emergent players of epigenetics that function as oncogenes or tumor suppressors and that have been implicated in regulating diverse cellular pathways. MiR-10b is an oncogenic microRNA involved in tumor invasion and metastasis in various cancers. Our data have shown that miR-10b is overexpressed in colorectal cancer samples in comparison with non-tumorous adjacent mucosa (p = 0.0025) and that it is associated with severe features such as tumor size >5 cm (p = 0.023), distant metastasis (p = 0.0022), non-differentiated tumors (p = 0.016), and vascular invasion (p = 0.01). Regarding the regulation of its expression, positive correlation between the loss of miR-10b and aberrant DNA methylation (p = 0.02) as well as a loss of TWIST-1 messenger RNA (p = 0.018) have been observed. Furthermore, expression analysis of the downstream miR-10b targets has shown that there are associations between low HOXD10 messenger RNA and E-cadherin protein levels (p < 0.0001, p = 0.0008, respectively) and overexpression of miR-10b. Our data suggests that overexpression of miR-10b results from high levels of TWIST-1 and may induce a decrease of E-cadherin membranous protein levels, thus contributing to the acquisition of metastatic phenotypes in colorectal cancer.

Quantitative measurement of iNOS expression in melanoma, nasopharyngeal, colorectal, and breast tumors of Tunisian patients: comparative study and clinical significance.

Chronic inflammation increases the risk of development of human malignancies. iNOS is an enzyme dominantly expressed during inflammatory reactions and seems to play a critical role in tumorigenesis. Our aim was to assess the iNOS expression in four types of human tumors: breast, colorectal, nasopharyngeal, and melanoma, of Tunisian patients. The level of iNOS was measured by RT-QPCR in tumor specimens. We showed that the expression of iNOS was higher in breast compared to colorectal and nasopharyngeal tumors, whereas in melanoma, the level of iNOS expression was low. Significant associations were found when comparing the iNOS expression in cancers pairs such as melanoma versus colorectal (p < 0.0001), colorectal versus nasopharyngeal (p = 0.0072), and melanoma versus breast (p < 0.0001). Furthermore, iNOS expression correlated with the Breslow thickness, Clark level, and histological subtype in melanoma, while in nasopharyngeal carcinoma, significant association was seen with age at diagnosis, TNM, metastasis, response to treatment, and expression of COX-2. Furthermore, the expression of iNOS correlated with tumor size, TNM, tumor location, and histological type in colorectal cancer, and with tumor size, tumor stage, SBR grade, and triple negative cases in breast cancer. On the other hand, immunohistochemistry analysis shows that the expression of iNOS is observed in the stroma and tumor cells as well. Overall, our results highlight that iNOS is a reliable marker for advanced stage and aggressive behavior for the four types of cancer and might be a potential promising therapeutic target.

Over-expression of miR-10b in NPC patients: correlation with LMP1 and Twist1.

Aberrant expression of miR-10b has been described in many cancers but remains unexplored in nasopharyngeal carcinoma (NPC). Therefore, we aimed to study the miR-10b expression level in 43 NPC biopsies collected from Tunisian patients and three NPC xenografts. Then, we investigated the correlation between miR-10b expression and its upstream regulators LMP1/Twist1 as well as its adjacent gene HoxD4. We showed that miR-10b was significantly up-regulated in NPC biopsies compared to non-tumor nasopharyngeal tissues (fold change 153; p = 0.004) and associated with advanced clinical stage and young age at diagnosis (p = 0.005 and p = 0.011, respectively). In addition, over-expression of miR-10b was positively associated with the transcription factor Twist1 as well as the EBV oncoprotein LMP1 (fold change 6.32; p = 0.014, fold change 6.58; p = 0.01 respectively). Furthermore, higher level of miR-10b was observed in tumors with simultaneous expression of LMP1 and Twist1, compared to those expressing only Twist1 (fold change 2.49; p = 0.033). Meanwhile, the analysis of the link between miR-10b and its neighbor gene HoxD4 did not show any significant correlation (Fisher test p = 0.205; Mann-Whitney test p = 0.676). This study reports the first evidence of miR-10b over-expression in NPC patients. Furthermore, our findings can support hsa-miR-10b gene regulation through LMP1/Twist1 in NPC malignancy.

Combined effects of α-amylase, xylanase, and cellulase coproduced by Stachybotrys microspora on dough properties and bread quality as a bread improver.

This study aims to explore the feasibility of introducing, during the manufacture of bakery bread, an enzymatic cocktail coproduced by the fungus Stachybotrys microspora: α-amylases, xylanases and cellulases, using wheat bran as a nutrient source. Among the characteristics of the alveograph (dough tenacity "P" and dough extensibility "L"), the addition of a cocktail of enzymes at a concentration of 2 %, to weak wheat flour, has made it possible to significantly reduce its P/L ratio from 2.45 to 1.41. Furthermore, the use of enzyme cocktails at 2 %, 4 %, and 6 % concentrations increases the brown color of the bread crust. The great reduction in the rate of bread firmness, during storage over 5 days, was obtained in the presence of an enzyme cocktail in comparison with bread control (65.13 N for the control and 22.99 N, 23.24 N, and 18.24 N for bread enriched with enzyme cocktail at 2 %, 4 % and 6 % concentrations, respectively). In conclusion, the enzyme cocktail added can synergistically improve bread dough rheology and bread properties.

A novel PIK3CA hot-spot mutation in breast cancer patients detected by HRM-COLD-PCR analysis.

BACKGROUND: The PI3K protein is involved in the PI3K/AKT/mTOR pathway. Deregulation of this pathway through PIK3CA mutation is common in various tumors. The aim of this work is to identify hotspot mutation at exons 9 and 20 in Tunisian patients with sporadic or hereditary breast cancer. METHODS: Hotspot mutations in exon 9 and exon 20 of the PIK3CA gene were identified by QPCR-High Resolution Melting followed by COLD-PCR and sequencing in 63 (42 sporadic cases and 21 hereditary cases) tumor tissues collected from Tunisian patient with breast cancer. MCF7, and BT20 breast cancer cell lines harboring the PIK3CA hotspot mutations E545K and H1047R in exon 9 and exon 20 respectively, were used as controls in HRM experiments. RESULTS: PIK3CA hotspot mutations were detected in 66.7% (28 out of 42) of sporadic BC cases, and in 14.3% (3 out of 21) of hereditary BC. The E545K and the H1048Y were the most prevalent mutations identified in patients with sporadic and hereditary BC, whereas the H1047R hotspot mutation was not found in our patients. Statistical analysis showed that PIK3CA mutation associated with an aggressive behavior in patients with sporadic BC, while it's correlated with age, tumor stage and tumor size in the group patients with hereditary breast cancer. CONCLUSIONS: Our results showed a novel PIK3CA hotspot mutation in Tunisian breast cancer patients detected by HRM-COLD-PCR. Moreover, the absence of PIK3CA hotspot mutation associated with good prognosis.

Development of a simple genotyping method based on indel mutations to rapidly screen SARS-CoV-2 circulating variants: Delta, Omicron BA.1 and BA.2.

The high need of rapid and flexible tools that facilitate the identification of circulating SARS-CoV-2 Variants of Concern (VOCs) remains crucial for public health system monitoring. Here, we develop allele-specific (AS)-qPCR assays targeting three recurrent indel mutations, ΔEF156-157, Ins214EPE and ΔLPP24-26, in spike (S) gene to identify the Delta VOC and the Omicron sublineages BA.1 and BA.2, respectively. After verification of the analytical specificity of each primer set, two duplex qPCR assays with melting curve analysis were performed to screen 129 COVID-19 cases confirmed between December 31, 2021 and February 01, 2022 in Sfax, Tunisia. The first duplex assay targeting ΔEF156-157 and Ins214EPE mutations successfully detected the Delta VOC in 39 cases and Omicron BA.1 in 83 cases. All the remaining cases (n = 7) were identified as Omicron BA.2, by the second duplex assay targeting Ins214EPE and ΔLPP24-26 mutations. The results of the screening method were in perfect concordance with those of S gene partial sequencing. In conclusion, our findings provide a simple and flexible screening method for more rapid and reliable monitoring of circulating VOCs. We highly recommend its implementation to guide public health policies.

Characteristics of epstein barr virus variants associated with gastric carcinoma in Southern Tunisia.

BACKGROUND: EBV-associated Gastric Carcinoma (EBVaGC) has a distinct clinical features and its prevalence is variable worldwide. RESULTS: To determine the prevalence of EBVaGC in Tunisia, EBV-encoded small RNA (EBER) expression was assessed in 81 gastric carcinoma (GC) specimens. The nuclear EBER expression was detected in 12 out of 81 GC cases (14.81%) and concordance between the score range of EBER staining and the number of EBV DNA copies as estimate by QPCR is observed. On the other hand, we found that EBVaGC strongly correlated with age at diagnosis, and weakly with tumor differentiation and venous invasion.Furthermore, the EBVaGC specimens were subjected to determine the EBV DNA polymorphisms. Our results show a unique genetic profile of the EBV strains regarding the A and D types, the F prototype, the retention of XhoI restriction site and the 30 bp del-LMP1 variant. According to our previous studies on nasopharyngeal carcinoma (NPC), we suggested that EBV strains associated to GC and NPC shared some similarities in Tunisian patients. CONCLUSION: The prevalence of EBVaGC is of 14.81% in the southern Tunisia and that common EBV strain are associated with both NPC and GC which are likely to differ from Asian strains. Our findings support therefore a certain geographical distribution of EBV strains which is not restricted to EBV-associated malignancies.

Establishment of primary cell culture of Ruditapes decussatus haemocytes for metal toxicity assessment.

In ecotoxicology, in vitro testing on cell cultures represents an ideal alternative to in vivo strategies for emerging contaminants. These tests have limited use particularly with marine invertebrates like the clams Ruditapes decussatus. In the present study, a primary culture of R. decussatus haemocytes was realized for the first time in order to determine the effect of metals (copper, zinc, and cobalt) on haemocyte parameters like viability and phagocytosis. Results showed that (i) among the studied medium, the modified Leibovitz (L-15) is the best for R. decussatus haemocytes primary culture. (ii) The primary culture system used here represents a suitable in vitro model for assessing cytotoxic responses, (iii) a decrease of cell viability and phagocytosis after 24 h exposure to 100 µg mL-1 CoSO4 and an increase of phagocytosis after 24 h exposure to 50 µg mL-1CuSO4.

FOXA1 Expression in Nasopharyngeal Carcinoma: Association with Clinicopathological Characteristics and EMT Markers.

The forkhead box (FOXA) family of transcription factors regulates gene expression and chromatin structure during tumorigenesis and embryonic development. Until now, the relationship between FOXA1 and the nasopharyngeal carcinoma (NPC) has not yet been reported. Therefore, our purpose is to analyze the expression of FOXA1 in 56 NPC patients compared to 10 normal nasopharyngeal mucosae and to correlate the expression with the clinicopathological features. Besides, we investigated the association between FOXA1 and LMP1 gene expression, as well as the EMT markers namely the E-cadherin and Twist1. Among 56 NPC tissues, 34 (60.7%) cases were positive for FOXA1. Furthermore, we noticed that FOXA1 expression correlated with TNM (p = 0.037), and age at diagnosis (p = 0.05). Moreover, positive expression of FOXA1 is likely to be associated with prolonged disease-free survival and overall survival rates. On the other hand, we observed a positive association between the expression of E-cadherin and FOXA1 (p = 0.0051) whereas Twist1 correlated negatively with FOXA1 (p = 0.004). Furthermore, knowing that LMP1 plays a key role in the pathogenesis of NPC, we explored the association of FOXA1 with the LMP1 gene expression in both NPC cell lines and tissues. We found that, in the C666-1 which displays low levels of LMP1, the expression of FOXA1 is high, and inversely in the C15 cell line that expresses a high level of LMP1, the level of FOXA1 is low. Besides, in accordance to our results, we found that in NPC tissues there is a negative association between LMP1 and FOXA1. In conclusion, our results suggest that the overexpression of FOXA1 is associated with a nonaggressive behavior and favorable prognosis in NPC patients. FOXA1 could contribute in the EMT process through key factors as E-cadherin, Twist1, and LMP1.

IgA antibodies against the Epstein-Barr nuclear antigen1 as a valuable biomarker for the diagnosis of nasopharyngeal carcinoma in Tunisian patients.

Serological tests for Epstein-Barr virus (EBV) have been used for many years as diagnostic predictors of nasopharyngeal carcinoma. It has been shown previously that the conventional immunofluorescence assay has a limited diagnostic value, especially in young patients from North African area. In the search for more reliable immunoglobulin (Ig) G or IgA antibody markers for the diagnosis of nasopharyngeal carcinoma, immunoblot analysis was performed using a full spectrum of EBV proteins. Sera were collected from 108 patients with nasopharyngeal carcinoma and three control groups composed of 18 patients with lymphoma, 18 other patients with autoimmune diseases and 55 healthy EBV carriers. It was observed that the IgA Epstein-Barr nuclear antigen 1 (EBNA1), IgA early antigen (EA)-p138 and IgG EA-p138 antibodies represent the most specific anti-EBV responses in either young or older patients with nasopharyngeal carcinoma which yield higher positive rates compared to the three control groups. Since the IgA EBNA1 response showed the highest sensitivity value for the detection of nasopharyngeal carcinoma, a novel enzyme-linked immunosorbent assay (ELISA) was established using a GST-EBNA1 protein expressed in bacteria, containing the P-threonine EBNA1 subtype cloned from DNA EBV sequence of C15 xenograft cells. Detection rates were 85.7% and 94.9% in young and older patients with nasopharyngeal carcinoma respectively, while only 3.6%, 11.1%, and 16.6% in healthy EBV carriers, patients with lymphoma and patients with autoimmune diseases, respectively. Thus, IgA EBNA1 ELISA may be useful for early diagnosis and mass screening of nasopharyngeal carcinoma in Tunisia even in young patients.

High levels of autoantibodies against catalase and superoxide dismutase in nasopharyngeal carcinoma.

INTRODUCTION: Autoantibodies against the antioxidant enzymes have been described in Epstein-Barr virus-associated diseases. Here, we hypothesize that Epstein-Barr virus (EBV), which is associated with nasopharyngeal carcinoma (NPC), induces anticatalase and/or antisuperoxide dismutase autoantibodies that inhibit catalase and/or superoxide dismutase activities and thereby contribute to the oxidative stress status described in this pathology. METHODS: Using a standard enzyme-linked immunosorbent assay (ELISA), the levels of immunoglobulin G (IgG), and M (IgM) directed against catalase and superoxide dismutase in the sera of 30 NPC patients and 30 healthy control individuals were evaluated. The antioxidative profile was tested among the same patients by measuring serum catalase and superoxide dismutase activities. To investigate the implication of EBV in the establishment of autoantibody production in NPC patients, a correlation study between serological testing for EBV viral capsid antigen immunoglobulin G (VCA IgG) and autoantibodies against both enzymes was undertaken. FINDINGS: The levels of IgG against superoxide dismutase and catalase were found to be increased in sera patients compared to controls (P<0.001). NPC patients exhibited decreased catalase (P<0.001) and superoxide dismutase activities (P<0.001) in their sera. However, a positive correlation between superoxide dismutase IgM antibody and IgG antibody titers to VCA (P<0.05, r=0.483, n=21) was found. A positive correlation between catalase (IgM) antibodies and IgG antibody titers to VCA (P<0.05, r=0.546, n=30) was also found. CONCLUSION: High levels of anticatalase and antisuperoxide dismutase antibodies in the sera of NPC patients were found.

Triacylglycerols accumulation and glycolipids secretion by the oleaginous yeast Rhodotorula babjevae Y-SL7: Structural identification and biotechnological applications.

The newly isolated oleaginous yeast, Rhodotorula babjevae Y-SL7, was shown to accumulate high intracellular content of microbial oil (mainly triacylglycerols) and to secret, under the same culture conditions, an atypical glycolipid. This unusual behavior was induced when the strain was subjected to nitrogen limitation and high amount of carbon. A series of analytical methods was adopted in order to structurally characterize the secreted glycolipid. The latter consists of a mixture of 9 molecules formed by a polyol head group, bound through the carboxyl end of an acetylated 3-hydroxy fatty acid with C18 or C16 chain length. In addition of their physicochemical properties such as emulsifying activity on hydrophobic substrates, Y-SL7 glycolipids have shown a therapeutically promising cytotoxic effect against different cancer cell lines. In fact, Y-SL7 strain can be used for the production of triacylglycerols as energetic molecules and for the secretion of a biosurfactant of therapeutic and environmental interest.

B1.12: a novel peptide interacting with the extracellular loop of the EBV oncoprotein LMP1.

Latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus (EBV) plays an important role in EBV-induced cell transformation. Down-regulation of the LMP1 expression had shown promising results on cancer cell therapy. In this study, we identified by Phage display a novel peptide called B1.12 (ACPLDLRSPCG) which selectively binds to the extracellular loop (B1) of the LMP1 oncoprotein as demonstrated by molecular docking, NMR and ITC. Using an LMP1 expressing cell line, we showed that B1.12 decreased cell viability, and induced G0/G1 cell cycle arrest. In addition, the expression of A20, pAkt, and pNFkb (pRelA536) in C666-1 cells treated with B1.12 decreased compared to the untreated cells. In conclusion, we selected a novel peptide able to bind specifically to the extracellular loop of LMP1 and thus modulate its oncogenic properties.

Aberrant methylation of p16, DLEC1, BLU and E-cadherin gene promoters in nasopharyngeal carcinoma biopsies from Tunisian patients.

BACKGROUND: Aberrant methylation of tumor suppressor gene (TSG) promoters has been extensively investigated in nasopharyngeal carcinomas (NPC) from South East Asia but not from North Africa. PATIENTS AND METHODS: The methylation status of p16, deleted in lung and esophageal cancer (DLEC1), zinc finger, MYND-type containing 10 (BLU) and E-cadherin gene promoters was investigated in 44 Tunisian NPC biopsies and three NPC xenografts, by methylation-specific PCR (MSP) combined with a quantitative assessment of some of the samples. RESULTS: The frequencies of aberrant promoter methylation were similar to previous figures reported for Asian series: p16 27/44 (65%), DLEC1 38/44 (86.3%), BLU 15/44 (34.1%) and E-cadherin 35/44 (79.5%). Although in other malignancies, aberrant promoter hypermethylation increases with patient age, it was at the same high frequency in the juvenile and adult forms of Tunisian NPCs. However, there was a strong association between aberrant methylation of E-cadherin promoter and lymph node invasion (p < 0.01). In addition, aberrant methylation of the BLU promoter was significantly correlated with an undifferentiated histological type (p = 0.03). CONCLUSION: Aberrant methylation of tumor suppressor genes occurs with the same high frequency in NPCs from North Africa as in South East Asia, regardless of patient age.

Phage-display screening identifies LMP1-binding peptides targeting the C-terminus region of the EBV oncoprotein.

Latent membrane protein 1 (LMP1), a major oncoprotein of Epstein Barr Virus (EBV) is responsible for transforming B lymphocytes in vitro. LMP1 is overexpressed in several EBV-associated malignancies, and different approaches have been developed to reduce its level and accordingly its oncogenic function in tumor tissues. This study aimed to use phage display peptide library to obtain peptides which could specifically bind to the cytoplasmic region of LMP1 to prevent its interaction with signaling proteins. The LMP1 C-terminus region was produced in bacterial E. coli and used as target for the phage library panning. After 3 rounds, 20 phage clones were randomly selected and 8 showed high binding affinity to the recombinant C-terminus LMP1 protein. The most interesting candidates are the FO5 "QPTKDSSPPLRV" and NO4 "STTSPPAVPHNN" peptides since both bind the C-terminus LMP1 as showed by molecular docking. Furthermore, sequence alignment revealed that the FO5 peptide shared sequence similarity with the Death Receptor 4 which belongs to the tumor necrosis factor-related apoptosis-inducing receptor which plays key role in anti-tumor immunity.

Identification of a novel methylated gene in nasopharyngeal carcinoma: TTC40.

To further explore the epigenetic changes in nasopharyngeal carcinoma (NPC), methylation-sensitive arbitrarily primed PCR was performed on NPC biopsies and nontumor nasopharyngeal samples. We have shown mainly two DNA fragments that appeared to be differentially methylated in NPCs versus nontumors. The first, defined as hypermethylated, corresponds to a CpG island at the 5'-end of the tetratricopeptide repeat domain 40 (TTC40) gene, whereas the second, defined as hypo-methylated, is located on repetitive sequences at chromosomes 16p11.1 and 13.1. Thereafter, the epigenetic alteration on the 5'-TTC40 gene was confirmed by methylation-specific PCR, showing a significant aberrant methylation in NPCs, compared to nontumors. In addition, the bisulfite sequencing analysis has shown a very high density of methylated cytosines in C15, C17, and X666 NPC xenografts. To assess whether TTC40 gene is silenced by aberrant methylation, we examined the gene expression by reverse transcription-PCR. Our analysis showed that the mRNA expression was significantly lower in tumors than in nontumors, which is associated with 5'-TTC40 gene hypermethylation. In conclusion, we found that the 5'-TTC40 gene is frequently methylated and is associated with the loss of mRNA expression in NPCs. Hypermethylation of 5'-TTC40 gene might play a role in NPC development; nevertheless, other studies are needed.