Mutations in the RAS-RAF-MEK-ERK (MAPK) pathway give rise to a range of developmental disorders collectively referred to as the RASopathies. De novo germline mutations in patients suffering from these syndromes promote similar phenotypes, which include heart abnormalities, characteristic facial features, cutaneous malformations, gastrointestinal malfunctions, failure to thrive and a spectrum of mental retardation. Although many RASopathies patients show a propensity to develop early-onset benign and malignant tumours, Cardio-faciocutaneous (CFC) syndrome patients do not seem to share this predisposition, with the exception of an increased number of naevi. CFC syndrome is caused by mutations in BRAF, MEK1 or MEK2, with the majority of patients harbouring BRAF mutations. Intriguingly, both kinase-activating and kinase-impaired mutations have been identified in CFC patients. Here, I use the zebrafish system to address the activity of the CFC syndrome alleles and the MAPK pathway in a developmental context and test the potential of small molecule inhibitors to restore normal development. I established an assay for the activity of CFC, melanoma and engineered BRAF and MEK human mutated alleles in vivo. Using zebrafish as an animal model organism, a panel of 31 mutant and wild-type BRAF, MEK1 and MEK2 alleles were expressed in early zebrafish embryos to assess their role in development. Irrespective of the predicted kinase activity, all embryos expressing BRAF and MEK mutant alleles reproducibly manifested the cell movement phenotype during gastrulation. Consistent with aberrant fibroblast growth factor (FGF) signalling and defective gastrulation, in situ hybridisation against convergence-extension markers showed misregulated convergence-extension movement patterns in CFC zebrafish embryos. Finally, I performed whole embryo RNA expression microarrays to identify genes regulated downstream of the CFC mutations, and I discuss the potential for a possible link to some of the phenotypes associated with a CFC zebrafish model. I established that the CFC, BRAF and MEK mutant embryos are sensitive to inhibition of MEK signalling by small molecules. Importantly, a time-window of treatment was identified which was sufficient to restore normal gastrulation movements and to prevent the developmental side effects promoted by the inhibitors at later stages of development. In order to begin considering the therapeutic potential of small molecules in developmental disorders (at least in our model system), the effect of low concentrations of the inhibitors in the normal formation of diverse tissues was thoroughly examined during zebrafish development. From these studies, I identified a concentration of MEK inhibitor that could be administered in a continuous fashion to prevent CFC-associated cell movement defects during gastrulation, without additional later developmental defects. Finally, I addressed the role of MEK-ERK signalling in a specific behavioural phenotype in zebrafish. Many RASopathies patients suffer from mental retardation and experience learning and attention difficulties. Research in our laboratory has identified a novel zebrafish behaviour induced by enhanced cAMP signalling, where the zebrafish seek shaded areas in their environment and exhibit frequent defensive shoaling behaviour. I used western blotting to establish that enhanced cAMP signalling activates the MAPK signalling pathway and, in collaboration with members our laboratory, that this phenotype can be suppressed by administration of the PD325901 MEK inhibitor. While we do not yet know the effect of CFC syndrome mutations on this behaviour, we suggest that altered MEK-ERK signalling may underlie important features of vertebrate behaviour.

There will be an estimated 60,000 new cases and nearly 8000 deaths in the US this year due to malignant melanoma. People living in the US are expected to have a 1 in 71 lifetime risk of developing the disease. Activating mutations in BRAF occur in approximately 60% of melanomas and in 80% of benign melanocytic nevi. PTEN is a tumor suppressor that has been shown to be deleted or epigenetically silenced in approximately 30% of melanomas. Cdkn2a is a locus encoding 2 tumor suppressors in alternate reading frames that has been found to be mutated in up to 40% of familial melanomas and is near universally lost in human melanoma cell lines. We used these data to generate novel mouse models of metastatic melanoma involving an inducible Cre transgenic mouse (Tyr::CreER-T2). We demonstrate that Pten loss, Cdkn2a loss or Braf activation in isolation does not induce melanoma. In contrast, when Braf activation is combined with Pten loss, mice develop aggressive pigmented melanomas with 100% penetrance and a mean tumor free survival of 19.5 days. Melanocytic proliferation occurs immediately following induction with virtually no latency. Expansile metastases are observed in lymph nodes and isolated tumor cells are present in the lungs and brain. Both incipient and established melanomas are sensitive to the mTOR inhibitor rapamycin. Furthermore, mTORC1 signaling is prevented upon rapamycin treatment, but mTORC2, MAPK, and Akt signaling appear to be unaffected. Additionally, when Cdkn2a loss is combined with Braf activation, the mice develop a nevic phenotype with complete penetrance and stochastic progression to melanoma. Median melanoma free survival is 85.5 days and tumors are metastatic to lymph nodes in 100% of mice. These novel mouse models of melanoma will likely be useful in the study of the biology of metastasis, in tumor immunology, and in new models of preclinical testing.

Un naevus congénital mélanocytique (CMN) est une prolifération bénigne de mélanocytes cutanés, cliniquement apparente à la naissance. On en retrouve chez approximativement 1% des nouveau-nés. Les CMN de taille large (LCMN), définis par un diamètre le plus large de plus de 20 cm, affectent environ 1 nouveau-né sur 20.000. Puisque les LCMN ont une propension à dégénérer en tumeur mélanomateuse (dans 2 à 5% des cas) et que leur grande taille apporte un contingent cellulaire abondant, ils représentent un bon modèle d’étude des étapes initiales de la mélanotumorigénèse. Nous disposions précisément de cultures primaires de mélanocytes établies à partir de vingt-sept cas de naevi congénitaux (24 de taille large et 3 de taille moyenne (MCMN)) curetés pour la plupart chez des enfants en période néonatale. La première phase de ce projet a permis d’identifier un mécanisme alternatif d’activation de l’oncogène BRAF dans deux cas de LCMN. Nous avons en effet mis en évidence dans ces deux cas de LCMN une translocation chromosomique entrainant une activation constitutive de BRAF par le biais d’une perte de son domaine auto-inhibiteur. Si des mutations activatrices de BRAF sont fréquemment rencontrées dans les tumeurs mélanocytiques, elles restent rares dans les naevi congénitaux et les mélanomes survenant dans des zones non exposées au soleil. Les translocations chromosomiques décrites ici pourraient représenter un mécanisme moléculaire récurrent d’activation de l’oncogène BRAF dans ces groupes de tumeurs mélanocytiques. La seconde phase du projet consistait à réaliser sur notre série de 27 L/MCMN des analyses caryotypiques, des analyses « mutationnelles » (pour rechercher la présence de mutations activatrices des oncogènes BRAF et NRAS) et des études d’expression différentielle. A l’inverse de ce que l’on observe dans le mélanome malin, les anomalies chromosomiques sont rares et isolées, reflétant très probablement le caractère bénin de ces lésions. La mutation BRAFV600E a été mise en évidence pour 4/27 CMN (15%), des mutations du gène NRAS pour 19/27 (70%), soit une situation en miroir de ce que l’on observe dans les CMN de petite taille (SCMN) et dans les naevi acquis bénins, confirmant les résultats obtenus par d’autres. L’étude du profil d’expression transcriptionnelle révèle des dysrégulations communes à l’ensemble des échantillons naeviques, comme une franche augmentation d’expression du transcrit de l’Ostéopontine. Le profil d’expression des échantillons de CMN BRAFV600E semble refléter une réduction de la synthèse et de la distribution de pigment et une activation de gènes impliqués dans la réponse cellulaire aux dommages à l’ADN. Comme des altérations des mécanismes de la pigmentation peuvent générer des dommages oxydatifs au niveau de l’ADN, l’activation de la réponse cellulaire aux dommages à l’ADN pourrait refléter la capacité des cellules naeviques à se protéger contre le stress cellulaire. Enfin, on observait aussi une expression élevée de gènes médiant la chimiorésistance dans différents cancers, ce qui pourrait éventuellement jouer un rôle dans l’inefficacité caractéristique et bien connue de la chimiothérapie dans le mélanome malin.

Activating mutations of genes coding for two different tyrosine kinase receptor, either RET or NTRK1 (also named TRKA), as well as of RAS or BRAF gene are associated with human thyroid papillary carcinoma (PTC). RET or NTRK1 protooncogene encodes a cell-surface transmembrane tyrosine kinase receptor with nerve growth factor as its lignand. Oncogenic potential of these two genes in thyrocytes results from replacement of their 5' portion by regulatory parts of other genes, leading to constitutive activation of their tyrosine kinase activity. The four reported oncogenic rearrangements of NTRK1 (TRK) are the consequences of fusion of its tyrosine kinase domain with one of the three genes (TPM3 gene, TPR gene, TFG gene). In our previous study, a PTC sample was found to express the NTRK1 tyrosine kinase domain without harboring NTRK1 rearrangement. We, therefore, assumed that there might have a novel NTRK1 rearrangement in this sample. 5¡¦RACE strategy was employed to clone the unknown 5¡¦end. Sequence of the cloned DNA fragment demonstrated that it is an aberrant transcription product containing an unspliced intron 9. In addition, the variant of NTRK1 wild type termed TRKA¢¹, which lacks exon 9, was also detected in this particular specimen. We conclude that amplification of TK domain of NTRK1 may serve as a rapid screening method for the presence of NTRK1-related transcript in PTCs. Mutations of the BRAF protein serine/threonine kinase gene have recently been identified in a variety of human cancers, especially in melanoma and papillary thyroid carcinomas. Among benign and malignant thyroid tumors, BRAF V599E mutations were reported to be restricted to papillary carcinomas. In this study, we analyzed mutations of BRAF in conjunction with our previous studies on RAS, RET rearrangement and NTRK1 rearrangement in PTCs to investigate genetic alterations in the RAS/RAF/MEK/MAPK kinase pathway. BRAF V599E mutations were detected in 49 of 105 (47%) PTCs but not in other type of thyroid tumor. There was no overlap between papillary carcinomas harboring RET rearrangement, NTRK1 rearrangement and BRAF mutations. Correlation between BRAF mutations and various clinicopathological parameters in 101 papillary carcinomas did not reveal any association with age, sex, tumor size, cervical lymph node metastasis, extrathyroidal extension, distant metastases and clinical stage. We conclude that BRAF mutations are restricted to papillary carcinomas in thyroid tumor. The overall frequencies in our study are in line with data previously reported. In Taiwan, BRAF mutation is the most prevalent oncogene in papillary thyroid carcinomas so far identified.

The RAF-MEK-ERK is an important signaling pathway that controls cellular proliferation, differentiation and survival. Recent reports indicate that R-RAF is mutated at a high frequency in human cancer. The mutations are clustered in the glycine-rich loop and activation segment which are encoded by exon 11 and exon 15, respectively. Among these mutations, V600E is the most prevalent found in varieties of human cancers, include melanoma and thyroid carcinomas. In this thesis, we analyzed 86 human cancer specimens, including 62 lung cancers and 24 esophageal cancers, for the mutation of exons 11 and 15 by PCR and direct DNA sequencing. However, we can not detect any mutation in these two exons in these clinical samples, these results suggest indicating that BRAF mutation might be rare and analysis of larger sample size is needed to confirmed this conclusion.

Reactivation of master regulators of epithelial to mesenchymal transition (MR-EMT) represents the molecular basis for tumour cell plasticity, malignant transformation and metastases. However, the current evidence on the specific role of MR-EMT in melanomagenesis has not been fully addressed. The purpose of this investigation was to assess the expression and regulation of these factors in malignant melanoma and to evaluate their prognostic and clinical significance. In vitro experiments indicated that a switch in MR-EMT protein expression ZEB2/SNAI2 to ZEB1/TWIST1 is RAS-RAF-MAPK signalling dependent. In addition, evidence supported a MR-EMT interactome, in which transcriptional repression of ZEB2 by Fra-1 resulted in upregulation of ZEB1, independently of miR-200 family. Further in vitro and immunohistochemical (IHC-P) analyses showed that E-cadherin and VDR protein levels were significantly reduced by the presence of ZEB1 in melanoma cells and archive tissues. Motility assays demonstrated that ZEB1 but not ZEB2 enhances cell migration. IHC-P analyses of ZEB2/SNAI2 (n=142/28) showed a statistically significant gradient of stronger staining at superficial sites compared to the deep sites in a select cohort of independent and matched melanoma tumour samples. In contrast, ZEB1 (n=142) and TWIST1 (n=133) showed higher staining in deep sites of primary melanomas and metastases. Trend analyses showed a significant MR-EMT switch in this progression series from high levels of ZEB2/SNAI2 in naevi towards high ZEB1/TWIST1 expression in melanomas. In primary melanomas these factors were also significant in Kaplan Meier survival curves and after two step cluster analysis the combined profile of ZEB1[superscript high]/TWIST1[superscript high]/ZEB2[superscript low] predicted the worse prognosis (P=0.001). Multivariate Cox regression analyses of IHC-P staining indicated that only the gain of ZEB1 (P<0.002, n=98) and superficial TWIST1 (P=0.012) were associated with poor metastasis-free survival and independent of breslow depth. In conclusion, the reversible switch between ZEB1/TWIST1 and ZEB2/SNAI2 is controlled by RAS-RAF-MAPK pathway activity and constitutes an independent factor of poor prognosis in patients with malignant melanoma.

Melanoma is the most lethal type of skin cancer with over 132,000 cases occurring globally each year and continually rising incidence. BRAFV600E inhibitors have led to clinically significant improvements in outcomes for melanoma patients, yet many patients with metastatic melanoma rapidly succumb to the disease due to eventual chemoresistance or insensitivity to the drug. Thus, it is critical to identify new therapies that can act alone, or be combined with available treatments for enhanced efficacy and/or to overcome drug resistance. Evidence from human melanoma indicates that the melanocyte lineage is critical for melanoma survival and contributes to therapeutic resistance. MITF is a highly conserved “master melanocyte transcription factor” with a complex role in melanoma. Our lab has previously developed a temperature sensitive BRAFV600E mitfavc7 zebrafish melanoma model carrying a human oncogene and mitfavc7 splice site mutation that enables the conditional control of its endogenous activity by changes to water temperature. As part of my PhD project, I characterized and compared two new models developed since then: a very aggressive BRAFV600E mitfavc7p53M214K melanoma model with three driving mutations and a slower developing BRAF-independent mitfavc7p53M214K. I showed that the MITF activity is crucial for melanocyte survival in both models and that both mutated BRAF and p53 deficiency are oncogenic with low levels of MITF, and result in fish nevi and melanoma resembling the pathology of human disease. Both models are also relevant to a low-MITF subclass of human melanomas that emerged from a recent classification by The Cancer Genome Atlas Network. In addition, I established that, similarly to the BRAFV600Emitfavc7, complete inhibition of MITF activity leads to rapid tumour regression, but once its activity is restored the melanomas recur at the same site as the original tumour. I used histopathology studies and melanocyte lineage transgenes to identify and visualize subpopulations of cells remaining at the site of regression in these new zebrafish melanoma models. I hypothesised that these are the cells of origin for tumour recurrence (melanoma stem or progenitor cells), showed that some of them express a cancer stem cell marker aldehyde dehydrogenase, and attempted to target these subpopulations using 5-nitrofurans (a prodrug NFN1, shown previously by our lab to target ALDHhigh subpopulations in context of melanoma) in fish after melanoma regression. Finally, I also developed and described a new primary zebrafish melanoma cell line that I derived from one of these zebrafish tumours. This study is still in progress, but the cell line will be a useful tool for further investigation of these proposed melanoma progenitor cells in vitro, with potential applications for lineage tracing and transplantations.

Dysregulation of the chemokine receptor 4 (CXCR4) and its primary ligand CXCL12 (SDF-1, stromal cell-derived factor-1), has been implicated in the progression of melanoma and the mechanisms by which the CXCR4/CXCL12 axis has been shown to activate cell cycle progression is via stimulation of the mitogen-activated protein kinase (MAPK) pathway. Given this, we sought to ascertain the potential cooperativity of CXCR4 with established histopathologic prognosticators including the BRAF status in primary cutaneous melanoma. In this IRB approved study, archived tissue samples with diagnosis of primary cutaneous melanoma were retrieved from the Skin Pathology Laboratory at BUSM, Boston, MA and a total of 107 cases identified as meeting criteria for inclusion. Protein expression of CXCR4 and CXCL12 were assessed using commercially available rabbit polyclonal antibodies (Ab2074 and, ab9797 respectively, Abcam, Cambridge, MA, USA). CXCR4 gene expression (mRNA) was measured by semiquantitative RT-PCR with appropriate controls. For IHC, a semi-quantitative scoring (ranging from 0-3) was used and cases with a score of ≥2 (>10%) were considered positive. Molecular analysis for CXCR4 gene expression and BRAF exon 15 mutation status was performed using mRNA semi-quantitative RT-PCR and DNA Sanger sequencing respectively. Univariate analyses of CXCR4 mRNA expression revealed a statistically significant correlation between elevated CXCR4 expression (low ΔCt value) and presence of the BRAF mutation and absence of a host response (p=0.03 and p=0.0003 respectively). Univariate analyses revealed a significant correlation between elevated CXCR4 mRNA (low ΔCt value) and the following: presence of BRAF mutation and absence of a host response (p=0.03 and 0.0003 respectively). CXCR4 mRNA was significantly higher among both AJCC stage 1 and stage 3 compared to stage 2 (p=0.01). Compared with CXCR4 negative samples, univariate analyses of CXCR4 protein showed that the proportion of CXCR4 positives was significantly greater in melanomas with absence of mitoses (p<0.0001), ulceration (p=0.0008) and regression (p=0.02). Patients presenting at shallower stages (AJCC 1-2) exhibited a larger proportion of CXCR4 positives (76.9%, p<0.0001 and 69.0%, p=0.004), while those at deeper stages (AJCC 3-4) exhibited a larger proportion of negatives (75.0%, p=0.002 and 66.7%, p=0.10). In a multivariable analysis, lower odds of CXCR4 protein expression were associated with AJCC stage-3 (OR=0.16, p=0.01), stage-4 (OR=0.17, p=0.04), and mitoses (OR=0.21, p=0.01). Lack of correlation between CXCR4 mRNA and protein expression suggests that further study is required for a more precise understanding of mRNA-protein interaction for CXCR4 in order to identify factors contributing to the lack of concordance. CXCR4 protein appears to be associated with established prognosticators of good clinical outcome as its expression is less frequently observed in melanomas with mitoses, ulceration and depth >2 mm. The association between CXCR4 mRNA and a brisk host response suggests that it may serve as a biomarker for recruiting melanoma patients for immunotherapy. Higher CXCR4 mRNA in patients with a BRAF mutation suggests its utility as a putative therapeutic target.

No description available.

Neste estudo foram avaliados os efeitos farmacológicos do vemurafenibe (inibidor BRAFV600E) e da cloroquina (inibidor de autofagia) na viabilidade celular e crescimento tumoral das sublinhagens de melanoma:G361 pLKO que expressa dermicidina e G361 IBC I com silenciamento da expressão. As células G-361 responderam a vemurafenibe (2 &#956;M) e cloroquina (100 &#956;M), isoladamente ou combinadas, com aumento apoptose, e redução das taxas de senescência. Vemurafenibe (50 mg/kg / 21 dias) inibiu o crescimento tumoral em camundongos imunodeficientes independente da expressão da DCD. A combinação com Cloroquina (30 mg/kg) a cada 24 horas, acelerou, enquanto a cada 72 horas, reduziu o crescimento tumoral. Os tumores apresentaram alterações morfológicas e núcleos atípicos; e não expressaram os marcadores S100, HMB-45, Mela-A ou citoqueratinas. Este trabalho confirmar a eficácia do vemurafenibe e sugere o potencial adjuvante da cloroquina no tratamento de melanomas. O estudo também confirma o papel da dermcidina como oncogne e fator de crescimento de células de melanoma maligno. / In this study we evaluated the pharmacological effects of vemurafenib ( inhibitor BRAFV600E) and chloroquine (autophagy inhibitor) in cell viability and tumor growth of two melanoma cell lines identified as G-361 pLKO, which expresses dermcidin, and G361 IBC I which silenced DCD expression. G-361 melanoma cells responded to vemurafenib (1-2 &#956;M) and chloroquine (50-100 &#956;M) alone or combined, with increased apoptosis rates, while decreasing senescent cells. Vemurafenib (50 mg/kg / 21 days) inhibited melanoma growth in immunodeficient mice independent of dermicidin. Chloroquine (30 mg/kg) in combination with vemurafenib, accelerated (at 24 hour interval), and reduced (at 72 hours interval), melanoma growth. Tumor tissues showed atypical cell morphology and nuclear histological patterns and melanocytic differentiation biomarkers S100, HMB-45, Melan-A or pancytokeratins were not. This work confirms the efficacy of vemurafenib and suggests potential adjuvant effect of chloroquine. It also confirms the role of dermcidin as growth factor and oncogene for melanoma cells.