Multi-omics comparison of malignant and normal uveal melanocytes reveals molecular features of uveal melanoma.

Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.

Exportin 1-mediated nuclear/cytoplasmic trafficking controls drug sensitivity of classical Hodgkin's lymphoma.

Exportin 1 (XPO1) is the main nuclear export receptor that controls the subcellular trafficking and the functions of major regulatory proteins. XPO1 is overexpressed in various cancers and small inhibitors of nuclear export (SINEs) have been developed to inhibit XPO1. In primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin's lymphoma (cHL), the XPO1 gene may be mutated on one nucleotide and encodes the mutant XPO1E571K . To understand the impact of mutation on protein function, we studied the response of PMBL and cHL cells to selinexor, a SINE, and ibrutinib, an inhibitor of Bruton tyrosine kinase. XPO1 mutation renders lymphoma cells more sensitive to selinexor due to a faster degradation of mutant XPO1 compared to the wild-type. We further showed that a mistrafficking of p65 (RELA) and p52 (NFκB2) transcription factors between the nuclear and cytoplasmic compartments accounts for the response toward ibrutinib. XPO1 mutation may be envisaged as a biomarker of the response of PMBL and cHL cells and other B-cell hemopathies to SINEs and drugs that target even indirectly the NFκB signaling pathway.

A high-risk retinoblastoma subtype with stemness features, dedifferentiated cone states and neuronal/ganglion cell gene expression.

Retinoblastoma is the most frequent intraocular malignancy in children, originating from a maturing cone precursor in the developing retina. Little is known on the molecular basis underlying the biological and clinical behavior of this cancer. Here, using multi-omics data, we demonstrate the existence of two retinoblastoma subtypes. Subtype 1, of earlier onset, includes most of the heritable forms. It harbors few genetic alterations other than the initiating RB1 inactivation and corresponds to differentiated tumors expressing mature cone markers. By contrast, subtype 2 tumors harbor frequent recurrent genetic alterations including MYCN-amplification. They express markers of less differentiated cone together with neuronal/ganglion cell markers with marked inter- and intra-tumor heterogeneity. The cone dedifferentiation in subtype 2 is associated with stemness features including low immune and interferon response, E2F and MYC/MYCN activation and a higher propensity for metastasis. The recognition of these two subtypes, one maintaining a cone-differentiated state, and the other, more aggressive, associated with cone dedifferentiation and expression of neuronal markers, opens up important biological and clinical perspectives for retinoblastomas.

The Phosphatase PRL-3 Is Involved in Key Steps of Cancer Metastasis.

PRL-3 belongs to the PRL phosphatase family. Its physiological role remains unclear, but many studies have identified that PRL-3 is a marker of cancer progression and shown it to be associated with metastasis. Evidence implicating PRL-3 in various elements of the metastatic process, such as the cell cycle, survival, angiogenesis, adhesion, cytoskeleton remodeling, EMT, motility and invasion, has been reported. Furthermore, several molecules acting as direct or indirect substrates have been identified. However, this information was obtained in many different studies, and it remains difficult to see the larger picture. We therefore systematically collected the published information together and used it to develop a comprehensive signaling network map. By analyzing this network map, we were able to retrieve the signaling pathways via which PRL-3 governs the key steps of the metastatic process in cancer. In this review, we summarize current knowledge of the role of PRL-3 in cancer and the molecular mechanisms involved. We also provide the web-based open-source PRL-3 signaling network map, for use in further studies.

Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2.

Uveal melanoma (UM) is an aggressive tumor in which approximately 50% of patients develop metastasis. Expression of the PTP4A3 gene, encoding a phosphatase, is predictive of poor patient survival. PTP4A3 expression in UM cells increases their migration in vitro and invasiveness in vivo. Here, we show that CRMP2 is mostly dephosphorylated on T514 in PTP4A3 expressing cells. We also demonstrate that inhibition of CRMP2 expression in UM cells expressing PTP4A3 increases their migration in vitro and invasiveness in vivo. This phenotype is accompanied by modifications of the actin microfilament network, with shortened filaments, whereas cells with a inactive mutant of the phosphatase do not show the same behavior. In addition, we showed that the cell cytoplasm becomes stiffer when CRMP2 is downregulated or PTP4A3 is expressed. Our results suggest that PTP4A3 acts upstream of CRMP2 in UM cells to enhance their migration and invasiveness and that a low level of CRMP2 in tumors is predictive of poor patient survival.

Evaluation of Tumor Cell Invasiveness In Vivo: The Chick Chorioallantoic Membrane Assay.

Metastases is largely responsible for the mortality among cancer patients. Metastasis formation is a complex multistep process, which results from the propagation of cancer cells from the primary tumor to distant sites of the body. Research on cancer metastasis aims to understand the mechanisms involved in the spread of cancer cells through the development of in vivo assays that assess cell invasion. Here we describe the use of the chick chorioallantoic membrane to evaluate cancer cell invasiveness in vivo. The chick chorioallantoic membrane assay is based on the detection and quantification of disseminated human tumor cells in the chick embryo femurs by real-time PCR amplification of human Alu sequences.

PRL-3/PTP4A3 phosphatase regulates integrin ß1 in adhesion structures during migration of human ocular melanoma cells.

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 ß1, 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 ß1 on collagen I. Knockdown experiments confirmed integrin ß1 involvement in PRL3-induced migration. We identified interactions between PRL-3 and integrin ß1, as well as with FAK P-Y397, an auto-activated form of Focal Adhesion Kinase found in FAs. We also show that integrin ß1 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 ß1 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.

Protein Tyrosine Phosphatase 4A3 (PTP4A3) Promotes Human Uveal Melanoma Aggressiveness Through Membrane Accumulation of Matrix Metalloproteinase 14 (MMP14).

PURPOSE: To study PTP4A3 phosphatase and MMP14 metalloprotease synergy in uveal melanoma aggressiveness. METHODS: Cell membrane localization of matrix metalloprotease 14 (MMP14) in uveal melanoma cells expressing protein tyrosine phosphatase A3 (PTP4A3) was assessed by flow cytometry or immunohistochemistry. The vesicular trafficking of MMP14 in the presence of PTP4A3 was evaluated in OCM-1 cells expressing either the wild-type or mutated phosphatase. Finally, MMP14 localization at the cell membrane of OCM-1 cells was impaired using RNA interference, and the PTP4A3-related migration in vitro and invasiveness in vivo of the treated cells were evaluated. RESULTS: We found that the membrane-anchored MMP14 is enriched at the cell surface of OCM-1 cells, patient-derived xenograft cells, and human primary uveal melanoma tumors expressing PTP4A3. Moreover, we show that PTP4A3 and MMP14 colocalize and that the vesicular trafficking of MMP14 is faster in the presence of active PTP4A3. Finally, we demonstrate that inhibition of MMP14 expression in uveal melanoma cells expressing PTP4A3 impairs their migration in vitro and invasiveness in vivo. CONCLUSIONS: Our observations indicate that PTP4A3 increases cell membrane accumulation of MMP14 as a result of increased cellular trafficking of the metalloprotease. We also show that downregulation of MMP14 expression reduced PTP4A3-induced cell migration and invasiveness. Taken together, our findings suggest that PTP4A3-related subcellular localization of MMP14 is an important event in metastasis induction.

DNA-PKcs plays role in cancer metastasis through regulation of secreted proteins involved in migration and invasion.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a major role in DNA damage signaling and repair and is also frequently overexpressed in tumor metastasis. We used isogenic cell lines expressing different levels of DNA-PKcs to investigate the role of DNA-PKcs in metastatic development. We found that DNA-PKcs participates in melanoma primary tumor and metastasis development by stimulating angiogenesis, migration and invasion. Comparison of conditioned medium content from DNA-PKcs-proficient and deficient cells reveals that DNA-PKcs controls secretion of at least 103 proteins (including 44 metastasis-associated with FBLN1, SERPINA3, MMP-8, HSPG2 and the inhibitors of matrix metalloproteinases, such as α-2M and TIMP-2). High throughput analysis of secretomes, proteomes and transcriptomes, indicate that DNA-PKcs regulates the secretion of 85 proteins without affecting their gene expression. Our data demonstrate that DNA-PKcs has a pro-metastatic activity via the modification of the tumor microenvironment. This study shows for the first time a direct link between DNA damage repair and cancer metastasis and highlights the importance of DNA-PKcs as a potential target for anti-metastatic treatment.

Involvement of Bcl-2-associated transcription factor 1 in the differentiation of early-born retinal cells.

Retinal progenitor proliferation and differentiation are tightly controlled by extrinsic cues and distinctive combinations of transcription factors leading to the generation of retinal cell type diversity. In this context, we have characterized Bcl-2-associated transcription factor (Bclaf1) during rodent retinogenesis. Bclaf1 expression is restricted to early-born cell types, such as ganglion, amacrine, and horizontal cells. Analysis of developing retinas in Bclaf1-deficient mice revealed a reduction in the numbers of retinal ganglion cells, amacrine cells and horizontal cells and an increase in the numbers of cone photoreceptor precursors. Silencing of Bclaf1expression by in vitro electroporation of shRNA in embryonic retina confirmed that Bclaf1 serves to promote amacrine and horizontal cell differentiation. Misexpression of Bclaf1 in late retinal progenitors was not sufficient to directly induce the generation of amacrine and horizontal cells. Domain deletion analysis indicated that the N-terminal domain of Bclaf1 containing an arginine-serine-rich and a bZip domain is required for its effects on retinal cell differentiation. In addition, analysis revealed that Bclaf1 function occurs independently of its interaction with endogenous Bcl-2-related proteins. Altogether, our data demonstrates that Bclaf1expression in postmitotic early-born cells facilitates the differentiation of early retinal precursors into retinal ganglion cells, amacrine cells, and horizontal cells rather than into cone photoreceptors.

Protein tyrosine phosphatase 4A3 (PTP4A3) is required for Xenopus laevis cranial neural crest migration in vivo.

Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.

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.

Vimentin and the K-Ras-induced actin-binding protein control inositol-(1,4,5)-trisphosphate receptor redistribution during MDCK cell differentiation.

Inositol-(1,4,5)-triphosphate receptors (InsP(3)Rs) are ligand-gated Ca(2+) channels that control Ca(2+) release from intracellular stores and play a central role in a wide range of cellular responses. In most epithelial cells, InsP(3)Rs are not uniformly distributed within the endoplasmic reticulum (ER) membrane, with the consequence that agonist stimulation results in compartmentalized Ca(2+) signals. Despite these observations, little is known about the mechanisms that regulate the intracellular localization of InsP(3)Rs. Here, we report that exogenously expressed InsP(3)R1-GFP and endogenous InsP(3)R3 interact with the K-Ras-induced actin-binding protein (KRAP) in both differentiated and undifferentiated Madin-Darby canine kidney (MDCK) cells. KRAP mediates InsP(3)R clustering in confluent MDCK cells and functions as an adapter, linking InsP(3)Rs to vimentin intermediate filaments. Upon epithelial differentiation, KRAP and vimentin are both required for InsP(3)R accumulation at the periphery of MDCK cells. Finally, KRAP associates with vimentin in chicken B lymphocytes and with keratins in a breast cancer cell line devoid of vimentin. Collectively, our data suggest that intermediate filaments in conjunction with KRAP may govern the localization of InsP(3)Rs in a large number of cell types (including epithelial cells) and in various physiological or pathological contexts.

Genetic determinants of uveal melanoma.

Uveal melanoma (UM) arises from neural crest-derived melanocytes of the choroid and the ciliary body. About 50% of patients develop metastatic disease despite efficient control of the primary tumor. For about 15 years, cytogenetic and, recently, genome-wide analysis techniques have shown that UM can be classified into 2 genomic groups correlating with prognostic clinicopathologic features: class 1 tumors, with a low risk of metastases, typically characterized by a gain of the 6p chromosome arm, often associated with a gain of the distal part of the 8q chromosome arm, and class 2 tumors, with a high metastatic risk, presenting loss of the entire chromosome 3 and gain of the entire 8q, related to the formation of isochromosomes. Genome-wide expression profiling has proved to be a powerful tool for separating these 2 classes. However, despite advances in the genomic and prognostic characterization of UM, the knowledge of pathways deregulated in these tumors is just emerging and, in contrast to cutaneous melanoma, no major predisposing genes are known. Altered or deregulated genes are reviewed in this chapter. Inactivating mutations have recently been identified by exome sequencing in gene BAP1, mapping to 3p21.1, in class 2 tumors. Among other discriminant genes identified from genome-wide expression profiling, PTP4A3, mapping to 8q24.3, coding for a protein promoting cell migration, is highly overexpressed in class 2 tumors. The overall expression signature of class 2 tumors suggests they may originate from neuroectodermal stem cells.

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 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-ß/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-ß 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.

High PTP4A3 phosphatase expression correlates with metastatic risk in uveal melanoma patients.

A high percentage of uveal melanoma patients develop metastatic tumors predominantly in the liver. We studied the molecular profiles derived from gene expression microarrays and comparative genomic hybridization microarrays, to identify genes associated with metastasis in this aggressive cancer. We compared 28 uveal melanomas from patients who developed liver metastases within three years of enucleation with 35 tumors from patients without metastases or who developed metastases more than 3 years after enucleation. Protein tyrosine phosphatase type IV A member 3 (PTP4A3/PRL3), was identified as a strong predictor of metastasis occurrence. We demonstrated that the differential expression of this gene, which maps to 8q24.3, was not merely a consequence of 8q chromosome overrepresentation. PTP4A3 overexpression in uveal melanoma cell lines significantly increased cell migration and invasiveness in vivo, suggesting a direct role for this protein in metastasis. Our findings suggest that PTP4A3 or its cellular substrates could constitute attractive therapeutic targets to treat metastatic uveal melanomas.

GLI2-mediated melanoma invasion and metastasis.

BACKGROUND: The 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. METHODS: We 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. RESULTS: Among 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. CONCLUSION: GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.