Simon Saule

A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma

So far, no common environmental and/or phenotypic factor has been associated with melanoma and renal cell carcinoma (RCC). The known risk factors for melanoma include sun exposure, pigmentation and nevus phenotypes; risk factors associated with RCC include smoking, obesity and hypertension. A recent study of coexisting melanoma and RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia-associated transcription factor (MITF) has been proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), the pathway of which is targeted by kidney cancer susceptibility genes. We therefore proposed that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. Here we identify a germline missense substitution in MITF (Mi-E318K) that occurred at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers, when compared with controls. Overall, Mi-E318K carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers. Codon 318 is located in a small-ubiquitin-like modifier (SUMO) consensus site (ΨKXE) and Mi-E318K severely impaired SUMOylation of MITF. Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity compared to wild-type MITF. Further, we observed a global increase in Mi-E318K-occupied loci. In an RCC cell line, gene expression profiling identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Lastly, the mutant protein enhanced melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide insights into the link between SUMOylation, transcription and cancer.

GLI2-Mediated Melanoma Invasion and Metastasis

BACKGROUNDnThe transforming growth factor-beta (TGF-beta) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-beta signaling.nnnMETHODSnWe used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided.nnnRESULTSnAmong melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm(2), difference = 1.88 mm(2), 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-beta-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-beta vs shCtrl + TGF-beta, 31.0 vs 161.9, difference = -130.9, 95% CI = -96.2 to -165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors.nnnCONCLUSIONnGLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.

GLI2 and M‐MITF transcription factors control exclusive gene expression programs and inversely regulate invasion in human melanoma cells

We recently identified GLI2, the most active of GLI transcription factors, as a direct TGF‐β/SMAD target, whose expression in melanoma cells is associated with increased invasiveness and metastatic capacity. In this work, we provide evidence that high GLI2 expression is inversely correlated with that of the melanocyte‐specific transcription factor M‐microphthalmia transcription factor (M‐MITF) and associated transcriptional program. GLI2‐expressing cell lines were characterized by the loss of M‐MITF‐dependent melanocytic differentiation markers and reduced pigmentation. The balance between M‐MITF and GLI2 expression did not correlate with the presence or absence of BRAF‐activating mutations, but rather was controlled by two distinct pathways: the TGF‐β pathway, which favors GLI2 expression, and the protein kinase A (PKA)/cAMP pathway, which pushes the balance toward high M‐MITF expression. Furthermore, overexpression and knockdown experiments demonstrated that GLI2 and M‐MITF reciprocally repress each other’s expression and control melanoma cell invasion in an opposite manner. These findings thus identify GLI2 as a critical transcription factor antagonizing M‐MITF function to promote melanoma cell phenotypic plasticity and invasive behavior.

Patient-derived xenografts recapitulate molecular features of human uveal melanomas.

We have previously developed a new method for the development and maintenance of uveal melanoma (UM) xenografts in immunodeficient mice. Here, we compare the genetic profiles of the primary tumors to their corresponding xenografts that have been passaged over time. The study included sixteen primary UMs and corresponding xenografts at very early (P1), early (P4), and late (P9) in vivo passages. The tumors were analyzed for mutation status of GNAQ, GNA11, GNAS, GNA15, BAP1, and BRAF, chromosomal copy number alterations using Affymetrix GeneChip® Genome‐Wide Human SNP6.0 arrays, gene expression profiles using GeneChip® Human Exon 1.0 ST arrays, BAP1 mRNA and protein expression, and MAPK pathway status using Reverse Phase Protein Arrays (RPPA). The UM xenografts accurately recapitulated the genetic features of primary human UMs and they exhibited genetic stability over the course of their in vivo maintenance. Our technique for establishing and maintaining primary UMs as xenograft tumors in immunodeficient mice exhibit a high degree of genetic conservation between the primary tumors and the xenograft tumors over multiple passages in vivo. These models therefore constitute valuable preclinical tool for drug screening in UM.

PRL-3/PTP4A3 phosphatase regulates integrin β1 in adhesion structures during migration of human ocular melanoma cells

ABSTRACT In a previous transcriptomic analysis of 63 ocular melanomas of the uvea, we found that expression of the PRL‐3/PTP4A3 gene, encoding a phosphatase that is anchored to the plasma membrane, was associated with the risk of metastasis, and a poor prognosis. We also showed that PRL‐3 overexpression in OCM‐1 ocular melanoma cells significantly increased cell migration in vitro and invasiveness in vivo, suggesting a direct role for PRL‐3 in the metastatic spreading of uveal melanoma. Here, we aimed to identify PRL‐3 substrates at the plasma membrane involved in adhesion to the extracellular matrix. We focused on integrin &bgr;1, which is the most highly expressed integrin in our cohort of uveal melanomas. We show that preventing PRL‐3 anchorage to the plasma membrane i) abolishes PRL‐3‐induced migration in OCM‐1 cells, ii) specifically enhances the spreading of OCM‐1 cells overexpressing PRL‐3, and iii) favors the maturation of large focal adhesions (FAs) containing integrin &bgr;1 on collagen I. Knockdown experiments confirmed integrin &bgr;1 involvement in PRL3‐induced migration. We identified interactions between PRL‐3 and integrin &bgr;1, as well as with FAK P‐Y397, an auto‐activated form of Focal Adhesion Kinase found in FAs. We also show that integrin &bgr;1 may be dephosphorylated by PRL‐3 in its intracytoplasmic S/T region, an important motif for integrin‐mediated cell adhesion. Finally, we observed that PRL‐3 regulated the clustering of integrin &bgr;1 in FAs on collagen I but not on fibronectin. This work identifies PRL‐3 as a new regulator of cell adhesion structures to the extracellular matrix, and further supports PRL‐3 as a key actor of metastasis in uveal melanoma, of which molecular mechanisms are still poorly understood. Graphical abstract Figure. No Caption available. HighlightsMembrane phosphatase PRL‐3 promotes cell migration of ocular melanoma cells.PRL‐3‐induced migration involves integrin &bgr;1.PRL‐3 regulates integrin &bgr;1 clustering in focal adhesions (FAs).PRL‐3 may dephosphorylate FAK P‐Y397 and integrin &bgr;1 T788/T789 in FAs.PRL‐3 regulates integrin &bgr;1 clustering on collagen I but not on fibronectin.

Erratum: A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma (Nature (2011) 480:94-98 doi:10.1038/nature10539)

This corrects the article DOI: 10.1038/nature10539

Erratum: Corrigendum: A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma

This corrects the article DOI: 10.1038/nature10539

Corrigendum: A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma

This corrects the article DOI: 10.1038/nature10539

Both PAX6 and MITF are required for pigment epithelium development in vivo

a compelling explanation as to why pigmented and dysplastic nevi are particularly vulnerable to sporadic melanoma; and (ii) artificial (as opposed to indoor) tanning strategies that increase melanin production via tyrosine-based or synthetic melanotropin products may not be safe alternatives to UV tanning. However, melanin content, melanin composition, and the size and distribution of melanosomes in the human epidermis vary considerably with both ethnicity and chronic sun exposure. Thus, extrapolating the HGF-pigmented mouse findings to humans is conditional on understanding the differences between facultative UV-inducible pigmentation in the melanoma-sensitive mouse model and pigment variation in the human population, particularly the continuous constitutive pigmentation in dark-skinned human populations that show low risk of melanoma. This study serves to enrich our understanding of the complex etiology of melanoma and directs our attention to other challenging questions still outstanding. For instance, what are the primary events that result in melanoma initiation after a single neonatal dose of UVA or UVB and are these changes associated with genetic or non-genetic mechanisms? The high incidence of C fi T mutations associated with direct damage in DNA (Pleasance et al., 2010; Wei et al., 2011) and the less consistent occurrence of transversion mutations associated with indirect free radical damage would suggest that, as in carcinoma, direct damage and C fi T mutations may be the dominant initiating events. However, a direct connection between C fi T transitions and melanoma initiation has not been shown. These mutations may arise in sun-exposed tumors during progression and have nothing to do with initiation. It is possible that UVA-melanin free radicals or up-regulated melanin synthesis could produce as yet unidentified DNA lesions responsible for as yet unidentified melanoma signature mutations. Careful analysis of mutation spectra in melanomas with known exposure histories (e.g., sporadic vs. sun-exposed; primary vs. metastatic) is warranted. The observation that neonatal exposure is obligatory for melanoma genesis in animal models, suggests that epigenetics may play a significant, if not essential, role in melanoma initiation. The occurrence of a common acquired mutation in the TRRAP gene (transformation ⁄ transcription domain-associated protein) in a significant number of human melanomas (Wei et al., 2011) supports this idea. TRRAP functions as a scaffolding protein in chromatin accessibility and is associated with histone acetylation complexes. Parallels to epigenetic events associated with exposure to endocrine disrupting chemicals during early development and increased risk of prostate and breast cancer come to mind. There is obviously much left to be learned about melanoma. However, the elucidation of distinct UV pathways in melanoma and the importance of melanin in UVA-induced melanoma is a significant step in our understanding of this disease and will facilitate future studies on its prevention and treatment.

PTP4A3, a Signal Molecule Deregulated in Uveal Melanoma Metastasis

Despite improvements in primary treatment protocols, more than 50% of the patients with uveal melanoma die of late-occurring metastases located in the liver. After diagnosis of metastases the average life expectancy is 6 months and no effective treatment is available at this stage of the disease. Transcriptome analysis linked to comparative genomic hybridization have been used for this particular melanoma to identify a set of genes linked to metastasis that may represent valuable future targets for specific treatments.