Hagit Schayek

MiR-377 targets E2F3 and alters the NF-kB signaling pathway through MAP3K7 in malignant melanoma

BackgroundThe incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We recently reported that a large miRNAs cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma. miR-377, one of the miRNAs located within this cluster, was studied here.MethodsqRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection, mRNA expression was assessed using mRNA arrays and protein expression was assessed by Western blot analysis. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques.ResultsmiR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 3′UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377.E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription.MAP3K7 (known as TAK1), a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-kB signaling pathway.ConclusionOur results suggest that miR-377 is an important negative regulator of E2F and MAP3K7/NF-kB signaling pathway in melanoma cells; it is tempting to speculate that its silencing in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways.

High-level expression of receptor tyrosine kinase Ret and responsiveness to Ret-activating ligands in pheochromocytoma cell lines from neurofibromatosis knockout mice.

Pheochromocytoma cell lines derived from neurofibromatosis knockout mice express high levels of the receptor tyrosine kinase Ret, which is involved in the pathogenesis of human pheochromocytomas in hereditary multiple endocrine neoplasia syndrome type 2 (MEN2). Mouse pheochromocytoma (MPC) cells respond to the Ret-activating ligand GDNF by exhibiting Ret phosphorylation, neurite outgrowth, decreased proliferation, and altered expression of catecholamine biosynthetic enzymes. GDNF exerts similar effects on human pheochromocytoma cells in primary cultures. Ret is minimally expressed by normal mouse chromaffin cells, from which pheochromocytomas are derived. Its expression at high levels by MPC cells suggests possible relationships between two previously unrelated tumor syndromes, neurofibromatosis, and MEN2. The responsiveness of these cells to GDNF suggests that they may be a valuable new model for neurobiology.

The rate of recurrent BRCA1, BRCA2, and TP53 mutations in the general population, and unselected ovarian cancer cases, in Belo Horizonte, Brazil

In Brazil, several recurring mutations in BRCA1 and BRCA2 and a TP53 mutation (R337H) have been reported in high risk breast cancer cases. We hypothesized that these recurring mutations may also be detected in the general population and ovarian cancer cases in the state of Minas Gerais. To test this notion, participants were recruited from the outpatient and the Gynecological clinic in the UFMG Medical Center in Belo Horizonte, Minas Gerais, Brazil. BRCA1 (c.68_69delAG, c.5266dupC, c.181T>G, c.4034delA, c.5123C>A), BRCA2 (c.5946delT, c.8537_8538delAG, 4936_4939delGAAA), the c.156_157insAlu* BRCA2 and the c.1010G>A *TP53 mutation were genotyped using validated techniques. Overall, 513 cancer free participants (273 men) (mean age 47.7 ± 15.1 years) and 103 ovarian cancer cases (mean age at diagnosis 58.7 ± 9.6 years) were studied. None of the participants were found to carry any of the genotyped mutations. We conclude that the recurring mutations in BRCA1, BRCA2 and TP53 cannot be detected in the general population or consecutive ovarian cancer cases in this geographical region in Brazil.

The yield of targeted genotyping for the recurring mutations in BRCA1 / 2 in Israel

BackgroundHereditary breast cancer is predominantly associated with germline mutations in the BRCA1 or BRCA2 genes. A few recurring mutations in these genes were reported in ethnically diverse Jewish populations. Since 2013, most oncogenetic laboratories in Israel adopted a two-step approach for BRCA1/2 genotyping, where the first step is genotyping for 14 seemingly recurring mutations—first-pass genotyping. The aim of this study was to assess the yield of this targeted BRCA sequencing.MethodsClinical and genotyping data of all individuals who underwent oncogenetic counseling and first-pass BRCA genotyping at the Oncogenetic Service Sheba and Assaf Harofeh Medical Centers from 1 February 2013 to 30 June 2017 were reviewed. All study participants were unrelated to each other.ResultsOverall, 5152 oncogenetic tests were reviewed in the present study, of which 4452 had no a priori known familial mutation. The majority of participants (68.6%) were genotyped because of personal history of cancer; 20.6% were tested because of family history of cancer, and details for the remaining 10.7% were missing. Overall, 256/4452 (5.8%) carriers were detected, 141 BRCA1 and 115 BRCA2 mutation carriers. In 54% of cancer-free carriers, no clinically suspicious family history of cancer was ascertained.ConclusionsThe currently used scheme of first-pass genotyping in Israel seems to have a high yield of mutation detection even in the absence of a significant family history of cancer. The challenge is to optimize the currently used targeted panel of common mutations and adjust it to the accumulating new data in the Israeli population.

Accuracy of Risk Prediction Models for Breast Cancer andBRCA1/BRCA2Mutation Carrier Probabilities in Israel

Background/Aim: Several algorithms have been developed to assess the risk of predicting BRCA mutation and breast cancer (BC) risk. The aim of this study was to evaluate the accuracy of these prediction algorithms in the Israeli population. Patients and Methods: Risk for developing breast cancer and the probability for carrying BRCA1/2 mutations using BOADICEA, BRCAPRO, IBIS, MYRIAD and PENN2 models were computed for individuals counseled and genotyped at the Oncogenetics unit in 2000 and 2005. The predicted mutation carriers and BC risks were compared with actual carrier rates by genotyping and BC diagnoses derived from the Israeli National Cancer Registry database. Results: Overall, 65/648 (10%) study participants were BRCA1/2 mutation carriers. Of 373 cancer-free participants at counseling, 25 had breast cancer by 2016. BOADICEA and BRCAPRO performed best for predicting BRCA mutation (AUC=0.741, 0.738, respectively). No model was clinically useful in predicting breast cancer risk. Conclusion: BOADICEA and BRCAPRO outperformed the other tested algorithms in BRCA mutation prediction in Israeli women, but none was valuable in breast cancer risk prediction.

De novo mutation in MEN1 is not associated with parental somatic mosaicism.

Multiple endocrine neoplasia type 1 (MEN1-OMIM #131100) is characterized by the co-occurrence of tumors in at least two of the following three endocrine tissues: parathyroid, endocrine pancreas and anterior pituitary (Thakker 2014). Familial MEN1 follows an autosomal dominant mode of inheritance, and germline mutations in the MEN1 gene at 11q13 can be detected in affected family members (Chandrasekharappa et al. 1997). Mutations in the CDKN1B gene (CDKN1B, KIP1 and OMIM #600778) have been also reported in MEN1 families but are far less frequent (Pellegata et al. 2006). Germline mutations in the MEN1 gene have also been detected in seemingly sporadic MEN1 cases and have been referred to as de novo mutations. Indeed, an often quoted number in MEN1-focused reports is 10% of the cases showing a de novo mutation (Bassett et al. 1998). The occurrence of a mutation in an affected individual with none of the parents exhibiting MEN1-suggestive features may be attributed to non-paternity, non-penetrant mutant allele, gonadal mosaicism or parental germline/somatic mosaicism (Koper & Lamberts 2000). The assignment of a de novo MEN1 gene mutation in previous studies was based on lack of a parental phenotype (Peppa et al. 2009), inferred haplotype (Bassett et al. 1998) or lack of the proband’s mutation in either parent using Sanger sequencing (Teh et al. 1998, Rix et al. 2004). No published study used deep sequencing to assess the possibility of parental, low-level mosaicism. The diagnostic criteria of MEN1 include having at least two of the three predominant neoplasms in at least one case (Thakker 2014). Clinical associations other than benign and malignant tumors are scarce in MEN1. Notably, there is a single report of an MEN1-affected individual who also exhibited a distinct phenotype that includes autism and dysmorphic features and harbors a 570 kb deletion at 11q13 encompassing the MEN1 gene (Mohrmann et al. 2011). Here we evaluated the possibility of low-level germline parental mosaicism in a Jewish Ashkenazi family where the proband also exhibits autism.

Spectrum of germline mutations in smokers and non-smokers in Brazilian non-small-cell lung cancer (NSCLC) patients

Lung cancer (LC) is a leading cause of cancer-related mortality. Although smoking is the major risk factor, ~15% of all cases occur in never-smokers, suggesting that genetic factors play a role in LC predisposition. Indeed, germline mutations in the TP53 gene predispose to multiple cancer types, including LC. To date, few studies compared the somatic and germline mutational profiles of LC cases by smoking status, and none was reported in Brazilians. Whole-exome sequencing (WES) was performed on two pools (seven smokers and six non-smokers) of tumor-derived DNA using the Illumina HiSeq2000 platform. Files from pools were analyzed separately using Ingenuity®Variant AnalysisTM and Mendel,MD. Validation of all candidate variants was performed by Sanger sequencing. Subsequently, validated mutations were analyzed in germline DNA from the same patients and in ethnically matched controls. In addition, a single recurring Brazilian TP53 germline mutation (R337H) was genotyped in 45 non-small-cell lung cancer patients.Four novel germline variants in the ATAD2, AURKA, PTPRD and THBS1 genes were identified exclusively in smoker patients, and four germline missense variants in PLCD1, RAD52, CP and CDC6 genes were identified solely in non-smokers. There were 4/45 (8.9%) germline carriers of the R337H TP53 mutation. In conclusion, the recurring Brazilian TP53 mutation should be genotyped in all non-small-cell lung cancer in Brazil, regardless of smoking status. Distinct pathogenic mutations and novel sequence variants are detected in Brazilian non-small-cell lung cancer patients, by smoking status. The contribution of these sequence variants to LC pathogenesis remains to be further explored.