miR-514a regulates the tumour suppressor NF1 and modulates BRAFi sensitivity in melanoma

To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E-targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.

Presence of the R1748X mutation in the NF1 gene in a Brazilian patient with ectropion uveae

Congenital ectropion uveae is a rare, nonprogressive anomaly characterized by the presence of iris pigment epithelium on the anterior surface of the iris stroma and is occasionally associated with Rieger's anomaly, Prader-Willi syndrome and neurofibromatosis type 1 (NF1). The most important complication of ectropion uveae is congenital or juvenile glaucoma. We described a patient with ectropion and the mutation R1748X in the NF1 gene. This is the third report in the literature describing ectropion associated with neurofibromatosis. If this association is confirmed by other authors, the NF1 patients should be examined for the presence of ectropion and, consequently, for the development of glaucoma. Copyright (C) 2004 S. Karger AG, Basel.

Mutational analysis of the GAP-related domain of the neurofibromatosis type 1 gene in Brazilian NF1 patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Germline NF1 mutational spectra and loss-of-heterozygosity analyses in patients with pheochromocytoma and neurofibromatosis type 1

BACKGROUND: Neurofibromatosis type 1 (NF1) is a pheochromocytoma-associated syndrome. Because of the low prevalence of pheochromocytoma in NF1, we ascertained subjects by pheochromocytoma that also had NF1 in the hope of describing the germline NF1 mutational spectra of NF1-related pheochromocytoma. MATERIALS AND METHODS: An international registry for NF1-pheochromocytomas was established. Mutation scanning was performed using denaturing HPLC for intragenic variation and quantitative PCR for large deletions. Loss-of-heterozygosity analysis using markers in and around NF1 was performed. RESULTS: There were 37 eligible subjects (ages 14-70 yr). Of 21 patients with corresponding tumor available, 67% showed somatic loss of the nonmutated allele at the NF1 locus vs. 0 of 12 sporadic tumors (P = 0.0002). Overall, 86% of the 37 patients had exonic or splice site mutations, 14% large deletions or duplications; 79% of the mutations are novel. The cysteine-serine rich domain (CSR) was affected in 35% but the RAS GTPase activating protein domain (RGD) in only 13%. There did not appear to be an association between any clinical features, particularly pheochromocytoma presentation and severity, and NF1 mutation genotype. CONCLUSIONS: The germline NF1 mutational spectra comprise intragenic mutations and deletions in individuals with pheochromocytoma and NF1. NF1 mutations tended to cluster in the CSR over the RAS-GAP domain, suggesting that CSR plays a more prominent role in individuals with NF1-pheochromocytoma than in NF1 individuals without this tumor. Loss-of-heterozygosity of NF1 markers in NF1-related pheochromocytoma was significantly more frequent than in sporadic pheochromocytoma, providing further molecular evidence that pheochromocytoma is a true component of NF1.

Peptides containing a consensus Ras binding sequence from Raf-1 and theGTPase activating protein NF1 inhibit Ras function.

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. We have now identified a consensus Ras binding sequence shared among a subset of Ras effectors. We have also shown that peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, our demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents.

Assessment of tumor prevention in Type 1 Neurofibromatosis using a nitroxide compound

Background Type 1 Neurofibromatosis (NF1) is a genetic disorder linked to mutations of the NF1 gene. Clinical symptoms are varied, but hallmark features of the disease include skin pigmentation anomalies (café au lait macules, skinfold freckling) and dermal neurofibromas. Method These dermal manifestations of NF1 have previously been reported in a mouse model where Nf1+/− mice are topically treated with dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). We adopted this mouse model to test the protective effects of a nitroxide antioxidant, 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO). Antioxidants have previously been shown to increase longevity in nf1-deficient fruitflies. Doses of 4 μM and 40 μM CTMIO provided ad libitum in drinking water were given to Nf1-deficient mice. Results Consistent with previous reports, Nf1-deficient mice showed a 4.7-fold increase in papilloma formation (P < 0.036). However, neither dose of CTMIO had any significant affect on papilloma formation. A non-significant decrease in skin pigmentation abnormalities was seen with 4 μM but not 40 μM CTMIO. Subsequent analysis of genomic DNA isolated from papillomas indicated that DMBA/TPA induced tumors did not exhibit a local loss of heterozygosity (LOH) at the Nf1 locus. Conclusion These data reveal that oral antioxidant therapy with CTMIO does not reduce tumor formation in a multistage cancer model, but also that this model does not feature LOH for Nf1.

The identification of therapeutic targets in metastatic melanoma

Metastatic melanoma, a cancer historically refractory to chemotherapeutic strategies, has a poor prognosis and accounts for the majority of skin cancer related mortality. Although the recent approval of two new drugs combating this disease, Ipilimumab and Vemurafenib (PLX4032), has demonstrated for the first time in decades an improvement in overall survival; the clinical efficacy of these drugs has been marred by severe adverse immune reactions and acquired drug resistance in patients, respectively. Thus, understanding the etiology of metastatic melanoma will contribute to the improvement of current therapeutic strategies while leading to the development of novel drug approaches. In order to identify recurrently mutated genes of therapeutic relevance in metastatic melanoma, a panel of stage III local lymph node melanomas were extensively characterised using high-throughput genomic technologies. This led to the identification of mutations in TFG in 5% of melanomas from a candidate gene sequencing approach using SNP array analysis, 24% of melanomas with mutations in MAP3K5 or MAP3K9 though unbiased whole-exome sequencing strategies, and inactivating mutations in NF1 in BRAF/NRAS wild type tumours though pathway analysis. Lastly, this thesis describes the development of a melanoma specific mutation panel that can rapidly identify clinically relevant mutation profiles that could guide effective treatment strategies through a personalised therapeutic approach. These findings are discussed in respect to a number of important issues raised by this study including the current limitation of next-generation sequencing technology, the difficulty in identifying ‘driver’ mutations critical to the development of melanoma due to high carcinogenic exposure by UV radiation, and the ultimate application of mutation screening in a personalised therapeutic setting. In summary, a number novel genes involved in metastatic melanoma have been identified that may have relevance for current therapeutic strategies in treating this disease.

Whole exome sequencing is an efficient and sensitive method for detection of germline mutations in patients with phaeochromcytomas and paragangliomas

Background Genetic testing is recommended when the probability of a disease-associated germline mutation exceeds 10%. Germline mutations are found in approximately 25% of individuals with phaeochromcytoma (PCC) or paraganglioma (PGL); however, genetic heterogeneity for PCC/PGL means many genes may require sequencing. A phenotype-directed iterative approach may limit costs but may also delay diagnosis, and will not detect mutations in genes not previously associated with PCC/PGL. Objective To assess whether whole exome sequencing (WES) was efficient and sensitive for mutation detection in PCC/PGL. Methods Whole exome sequencing was performed on blinded samples from eleven individuals with PCC/PGL and known mutations. Illumina TruSeq™ (Illumina Inc, San Diego, CA, USA) was used for exome capture of seven samples, and NimbleGen SeqCap EZ v3.0 (Roche NimbleGen Inc, Basel, Switzerland) for five samples (one sample was repeated). Massive parallel sequencing was performed on multiplexed samples. Sequencing data were called using Genome Analysis Toolkit and annotated using annovar. Data were assessed for coding variants in RET, NF1, VHL, SDHD, SDHB, SDHC, SDHA, SDHAF2, KIF1B, TMEM127, EGLN1 and MAX. Target capture of five exome capture platforms was compared. Results Six of seven mutations were detected using Illumina TruSeq™ exome capture. All five mutations were detected using NimbleGen SeqCap EZ v3.0 platform, including the mutation missed using Illumina TruSeq™ capture. Target capture for exons in known PCC/PGL genes differs substantially between platforms. Exome sequencing was inexpensive (<$A800 per sample for reagents) and rapid (results <5 weeks from sample reception). Conclusion Whole exome sequencing is sensitive, rapid and efficient for detection of PCC/PGL germline mutations. However, capture platform selection is critical to maximize sensitivity.

Early detection of melanoma metastases using microRNAs as novel biomarkers

Early detection of melanoma skin cancer, prior to metastatic spread, is critical to improve survival outcomes in patients. This study identified a melanoma-related panel of blood markers that can detect the presence of melanoma with high sensitivity and accuracy which is superior to currently used markers for melanoma progression, recurrence, and survival. Overall, the findings discussed in this thesis may lead to more precise measurement of disease progression allowing for better treatments and an increase in overall survival.

Pheo-type: A diagnostic gene-expression assay for the classification of pheochromocytoma and paraganglioma

Context: Pheochromocytomas and paragangliomas (PPGLs) are heritable neoplasms that can be classified into gene-expression subtypes corresponding to their underlying specific genetic drivers. Objective: This study aimed to develop a diagnostic and research tool (Pheo-type) capable of classifying PPGL tumors into gene-expression subtypes that could be used to guide and interpret genetic testing, determine surveillance programs, and aid in elucidation of PPGL biology. Design: A compendium of published microarray data representing 205 PPGL tumors was used for the selection of subtype-specific genes that were then translated to the Nanostring gene-expression platform. A support vector machine was trained on the microarray dataset and then tested on an independent Nanostring dataset representing 38 familial and sporadic cases of PPGL of known genotype (RET, NF1, TMEM127, MAX, HRAS, VHL, and SDHx). Different classifier models involving between three and six subtypes were compared for their discrimination potential. Results: A gene set of 46 genes and six endogenous controls was selected representing six known PPGL subtypes; RTK1–3 (RET, NF1, TMEM127, and HRAS), MAX-like, VHL, and SDHx. Of 38 test cases, 34 (90%) were correctly predicted to six subtypes based on the known genotype to gene-expression subtype association. Removal of the RTK2 subtype from training, characterized by an admixture of tumor and normal adrenal cortex, improved the classification accuracy (35/38). Consolidation of RTK and pseudohypoxic PPGL subtypes to four- and then three-class architectures improved the classification accuracy for clinical application. Conclusions: The Pheo-type gene-expression assay is a reliable method for predicting PPGL genotype using routine diagnostic tumor samples.

Elucidating the cancer-specific genetic alteration spectrum of glioblastoma derived cell lines from whole exome and RNA sequencing

Cell lines derived from tumor tissues have been used as a valuable system to study gene regulation and cancer development. Comprehensive characterization of the genetic background of cell lines could provide clues on novel genes responsible for carcinogenesis and help in choosing cell lines for particular studies. Here, we have carried out whole exome and RNA sequencing of commonly used glioblastoma (GBM) cell lines (U87, T98G, LN229, U343, U373 and LN18) to unearth single nucleotide variations (SNVs), indels, differential gene expression, gene fusions and RNA editing events. We obtained an average of 41,071 SNVs out of which 1,594 (3.88%) were potentially cancer-specific. The cell lines showed frequent SNVs and indels in some of the genes that are known to be altered in GBM-EGFR, TP53, PTEN, SPTA1 and NF1. Chromatin modifying genes-ATRX, MLL3, MLL4, SETD2 and SRCAP also showed alterations. While no cell line carried IDH1 mutations, five cell lines showed hTERT promoter activating mutations with a concomitant increase in hTERT transcript levels. Five significant gene fusions were found of which NUP93-CYB5B was validated. An average of 18,949 RNA editing events was also obtained. Thus we have generated a comprehensive catalogue of genetic alterations for six GBM cell lines.

Elucidating the cancer-specific genetic alteration spectrum of glioblastoma derived cell lines from whole exome and RNA sequencing

Cell lines derived from tumor tissues have been used as a valuable system to study gene regulation and cancer development. Comprehensive characterization of the genetic background of cell lines could provide clues on novel genes responsible for carcinogenesis and help in choosing cell lines for particular studies. Here, we have carried out whole exome and RNA sequencing of commonly used glioblastoma (GBM) cell lines (U87, T98G, LN229, U343, U373 and LN18) to unearth single nucleotide variations (SNVs), indels, differential gene expression, gene fusions and RNA editing events. We obtained an average of 41,071 SNVs out of which 1,594 (3.88%) were potentially cancer-specific. The cell lines showed frequent SNVs and indels in some of the genes that are known to be altered in GBM-EGFR, TP53, PTEN, SPTA1 and NF1. Chromatin modifying genes-ATRX, MLL3, MLL4, SETD2 and SRCAP also showed alterations. While no cell line carried IDH1 mutations, five cell lines showed hTERT promoter activating mutations with a concomitant increase in hTERT transcript levels. Five significant gene fusions were found of which NUP93-CYB5B was validated. An average of 18,949 RNA editing events was also obtained. Thus we have generated a comprehensive catalogue of genetic alterations for six GBM cell lines.

High frequencies of plexiform neurofibromas, mental retardation, learning difficulties, and scoliosis in Brazilian patients with neurofibromatosis type 1

A clinical study of Brazilian patients with neurofibromatosis type 1 (NF1) was performed in a multidisciplinary Neurofibromatosis Program called CEPAN (Center of Research and Service in Neurofibromatosis). Among 55 patients (60% females, 40% males) who met the NIH criteria for the diagnosis of NF1, 98% had more than six café-au-lait patches, 94.5% had axillary freckling, 45% had inguinal freckling, and 87.5% had Lisch nodules. Cutaneous neurofibromas were observed in 96%, and 40% presented plexiform neurofibromas. A positive family history of NF1 was found in 60%, and mental retardation occurred in 35%. Some degree of scoliosis was noted in 49%, 51% had macrocephaly, 40% had short stature, 76% had learning difficulties, and 2% had optic gliomas. Unexpectedly high frequencies of plexiform neurofibromas, mental retardation, learning difficulties, and scoliosis were observed, probably reflecting the detailed clinical analysis methods adopted by the Neurofibromatosis Program. These same patients were screened for mutations in the GAP-related domain/GRD (exons 20-27a) by single-strand conformation polymorphism. Four different mutations (Q1189X, 3525-3526delAA, E1356G, c.4111-1G>A) and four polymorphisms (c.3315-27G>A, V1146I, V1317A, c.4514+11C>G) were identified. These data were recently published.

Neurofibromin: a general outlook

Neurofibromin is a cytoplasmic protein that is predominantly expressed in neurons, Schwann cells, oligodendrocytes, astrocytes and leukocytes. It is encoded by the gene NF1, located on chromosome 17, at q11.2, and has different biochemical functions, including association to microtubules and participation in several signaling pathways. Alterations in this protein are responsible for a phacomatosis named neurofibromatosis type 1.