EMMPRIN/CD147 is an independent prognostic biomarker in cutaneous melanoma.

CD147 has been implicated in melanoma invasion and metastasis mainly through increasing metalloproteinase synthesis and regulating VEGF/VEGFR signalling. In this study, the prognostic value of CD147 expression was investigated in a cohort of 196 cutaneous melanomas including 136 consecutive primary malignant melanomas, 30 lymph nodes, 16 in-transit and 14 visceral metastases. A series of 10 normal skin, 10 blue nevi and 10 dermal nevi was used as control. CD147 expression was assessed by immunohistochemistry, and the association of its expression with the clinicopathological characteristics of patients and survival was evaluated using univariate and multivariate statistical analyses. Univariate analysis showed that high CD147 expression was significantly associated with metastatic potential and with a reduced overall survival (P < 0.05 for both) in primary melanoma patients. CD147 expression level was correlated with histological factors which were associated with prognosis: Clark level, ulceration status and more particularly with Breslow index (r = 0.7, P < 10(-8) ). Multivariate analysis retained CD147 expression level and ulceration status as predicting factors for metastasis and overall survival (P < 0.05 for both). CD147 emerges as an important factor in the aggressive behaviour of melanoma and deserves further evaluation as an independent prognostic biomarker.

EMMPRIN regulates ß1 integrin-mediated adhesion through Kindlin-3 in human melanoma cells.

EMMPRIN is known to promote tumor invasion through extracellular matrix (ECM) degradation. Here we report that EMMPRIN can regulate melanoma cell adhesion to the ECM through an interaction with ß1 integrin involving kindlin-3. In this study, EMMPRIN knockdown in the human melanoma cell line M10 using siRNA decreased cell invasion and significantly increased cell adhesion and spreading. A morphological change from a round to a spread shape was observed associated with enhanced phalloidin-labelled actin staining. In situ proximity ligation assay and co-immunoprecipitation revealed that EMMPRIN silencing increased the interaction of ß1 integrin with kindlin-3, a focal adhesion protein. This was associated with an increase in ß1 integrin activation and a decrease in the phosphorylation of the downstream integrin kinase FAK. Moreover, the expression at both the transcript and protein level of kindlin-3 and of ß1 integrin was inversely regulated by EMMPRIN. EMMPRIN did not regulate either talin expression or its interaction with ß1 integrin. These results are consistent with our in vivo demonstration that EMMPRIN inhibition increased ß1 integrin activation and its interaction with kindlin-3. To conclude, these findings reveal a new role of EMMPRIN in tumor cell migration through ß1 integrin/kindlin-3-mediated adhesion pathway.

EMMPRIN/CD147 up-regulates urokinase-type plasminogen activator: implications in oral tumor progression.

BACKGROUNDS: An elevated level of EMMPRIN in cancer tissues have been correlated with tumor invasion in numerous cancers including oral cavity and larynx. Although EMMPRIN's effect has been generally attributed to its MMP inducing activity, we have previously demonstrated in breast cancer model that EMMPRIN can also enhance invasion by upregulating uPA. In this study, the role of EMMPRIN in regulating uPA and invasion was investigated in oral squamous cell carcinoma (OSCC) progression. METHODS: Precancerous and invasive oral tumoral tissues were used as well as the corresponding cell lines, DOK and SCC-9 respectively. The paracrine regulation of uPA by EMMPRIN was investigated by treating culture cells with EMMPRIN-enriched membrane vesicles. UPA expression was analyzed by qPCR and immunostaining and the consequence on the invasion capacity was studied using modified Boyden chamber assay, in the presence or absence of EMMPRIN blocking antibody, the uPA inhibitor amiloride or the MMP inhibitor marimastat. RESULTS: OSCC tumors were shown to express more EMMPRIN and uPA compared to dysplastic lesions. The corresponding cell models, SCC-9 and DOK cells, displayed similar expression pattern. In both cell types EMMPRIN upregulated the expression of uPA as well as that of MMP-2 and MMP-9. EMMPRIN treatment led to a significant increase in cell invasion both in the invasive SCC-9 and in the less invasive dysplastic DOK cells, in an MMP and uPA dependent manner. CONCLUSIONS: Our results suggest that the upregulation of uPA contributes to EMMPRIN's effect in promoting oral tumor invasion.

EMMPRIN promotes angiogenesis through hypoxia-inducible factor-2alpha-mediated regulation of soluble VEGF isoforms and their receptor VEGFR-2.

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is thought to promote tumor angiogenesis mostly through its protease-inducing function and more recently by its ability to increase tumor cell expression of vascular endothelial growth factor (VEGF). In this study, we present evidence that EMMPRIN can promote angiogenesis by a direct effect on endothelial cells through a paracrine regulation of the VEGF/VEGF-receptor (VEGFR) system. Using human microvascular endothelial cell line-1 endothelial cells, we show that EMMPRIN selectively increased the soluble VEGF isoforms (121 and 165), but not the matrix-bound VEGF 189 form. In addition, EMMPRIN up-regulated the expression of VEGFR-2 without an effect on VEGFR-1. This increase in VEGFR-2 was responsible for the observed EMMPRIN stimulation of the migratory and tube formation capacity of endothelial cells. EMMPRIN's effects, which were matrix metalloproteinase and urokinase-type plasminogen activator independent, were mediated primarily through hypoxia-inducible factor-2alpha expression, also up-regulated by EMMPRIN. VEGFR-2 increase was also observed in vivo in a mouse model of xenograph tumors overexpressing EMMPRIN. These results suggest that in addition to increasing protease production, EMMPRIN may contribute to the formation of a reactive stroma also through the up-regulation of hypoxia-inducible factor-2alpha, VEGFR-2, and the soluble forms of VEGF in endothelial cells, thus directly regulating the angiogenic process.

Identification of a novel biomarker signature associated with risk for bone metastasis in patients with renal cell carcinoma.

When renal cell carcinoma (RCC) metastasizes to bone (a frequent site of systemic spread of this cancer) it becomes highly resistant to radiation therapy and chemotherapy. A better understanding of the biology of bone metastasis in RCC may permit to identify biomarkers for early detection of subclinical disease and better stratification of patients prior to treatment. We therefore investigated in this study, using a multiplex real-time RT-PCR assay, the expression of a panel of 16 biomarkers involved in angiogenesis and tumor invasion; the panel was applied to primary tumors and normal tissues obtained from clear-cell RCC patients with and without bone metastases. We identified a novel combination of biomarkers associated with the risk of bone metastasis. Among the transcripts of the genes studied, VEGFR-1, VEGFR-2, HIF-1alpha, uPA , and PA I-1 overexpression in tumor tissues was significantly associated with the presence of bone metastasis (p=0.02, p=0.02, p<0.0001, p=0.04, and p=0.03, respectively). No differences were found in the expression of these transcripts in the corresponding normal tissues. This preliminary study provides a promising tool that may help in the management of RCC patients with bone metastasis. Indeed, these predictive markers could be useful to identify subclinical disease, improve staging, and guide treatment decisions.

Quantification of VEGF isoforms and VEGFR transcripts by qRT-PCR and their significance in acute myeloid leukemia.

Vascular endothelial growth factor (VEGF) and its receptors are known to play an important role in normal and pathological hematopoiesis but the prognostic impact of VEGF isoform transcripts in acute myeloid leukemia (AML) has not been addressed. We conducted a single-institution prospective study to analyze the impact of these angiogenic factors and the expression of their receptors on the survival of adult patients newly diagnosed with AML. We investigated the levels of VEGF transcript isoforms VEGF121, -145, -165, -189 and -206 and their receptors, VEGFR-1 and VEGFR-2, using quantitative reverse transcriptase polymerase chain reaction assays in peripheral blood mononuclear cells (PBMCs) of 67 consecutive AML patients at diagnosis. VEGF total protein was measured for comparison with mRNA levels in PBMCs. The VEGF121 splice variant transcript in AML PBMCs was significantly higher than in the normal controls. VEGF transcripts were quantified in all samples while its protein was detected in 42/67 (63%) of AML samples. High levels of VEGF121, VEGF165 transcripts and VEGF protein in AML were significantly related to a worse prognosis when analyzing overall survival (p<0.0001, p=0.019 and p=0.012, respectively) or event-free survival (p<0.0001, p=0.010 and p=0.047) using univariate analysis. In multivariable analysis only VEGF121 expression remained an independent prognostic factor for either event-free survival or overall survival [aHR=8.83 (3.48-22.4), p<0.0001, and aHR=9.52 (3.41-26.6), p<0.0001]. No prognostic value was observed for the other isoforms and the two receptors. Our findings show that the level of VEGF121 mRNA in circulating cells from AML patients is a strong independent prognostic parameter, which could be useful in the management of unselected AML patients.

Extracellular matrix metalloproteinase inducer up-regulates the urokinase-type plasminogen activator system promoting tumor cell invasion.

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a membrane glycoprotein overexpressed in many cancer tissues and is known for its ability to stimulate MMP expression. In this work, we show that EMMPRIN is also a regulator of the urokinase-type plasminogen activation (uPA) system of serine proteases, thus participating to the increase of the overall proteolytic function of the cancer cells. Enhanced EMMPRIN expression in a tumorigenic breast epithelial cell line NS2T2A increased the levels of uPA, uPA receptor, and the uPA inhibitor plasminogen activator inhibitor-1 (PAI-1), as measured by quantitative reverse transcription-PCR, Western blot, and plasminogen-casein zymography. This response was down-regulated by either EMMPRIN small interfering RNA or a blocking antibody to EMMPRIN. EMMPRIN-containing purified membrane fraction from Chinese hamster ovary cells when added exogenously to NS2T2A cells induced a similar activation of the uPA/PAI-1 system. Additionally, overexpression of EMMPRIN in NS2T2A cells increased uPA levels in cocultured endothelial cells, showing a paracrine regulation loop involving a tumor-stroma interaction. EMMPRIN-expressing cells also exhibited enhanced invasive potential in vitro, and the use of amiloride (uPA inhibitor) and marimastat (MMP inhibitor) showed that the two proteolytic systems reduced alone and in combination the invasive potential mediated through EMMPRIN. These data show a novel regulatory pathway for uPA activity and suggest that EMMPRIN is involved in uPA dysregulation observed in cancer.

A targeted genomic alteration analysis predicts survival of melanoma patients under BRAF inhibitors.

Several mechanisms have been described to elucidate the emergence of resistance to MAPK inhibitors in melanoma and there is a crucial need for biomarkers to identify patients who are likely to achieve a better and long-lasting response to BRAF inhibitors therapy. In this study, we developed a targeted approach combining both mRNA and DNA alterations analysis focusing on relevant gene alterations involved in acquired BRAF inhibitor resistance. We collected baseline tumor samples from 64 melanoma patients at BRAF inhibitor treatment initiation and showed that the presence, prior to treatment, of mRNA over-expression of genes' subset was significantly associated with improved progression free survival and overall survival. The presence of DNA alterations was in favor of better overall survival. The genomic analysis of relapsed-matched tumor samples from 20 patients allowed us to uncover the largest landscape of resistance mechanisms reported to date as at least one resistance mechanism was identified for each patient studied. Alterations in RB1 have been most frequent and hence represent an important additional acquired resistance mechanism. Our targeted genomic analysis emerges as a relevant tool in clinical practice to identify those patients who are more likely to achieve durable response to targeted therapies and to exhaustively describe the spectrum of resistance mechanisms. Our approach can be adapted to new targeted therapies by including newly identified genetic alterations.

Discoidin domain receptors: A promising target in melanoma.

The discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family that signals in response to collagen and that has been implicated in cancer progression. In the present study, we investigated the expression and role of DDR1 in human melanoma progression. Immunohistochemical staining of human melanoma specimens (n = 52) shows high DDR1 expression in melanoma lesions that correlates with poor prognosis. DDR1 expression was associated with the clinical characteristics of Clark level and ulceration and with BRAF mutations. Downregulation of DDR1 by small interfering RNA (siRNA) in vitro inhibited melanoma cells malignant properties, migration, invasion, and survival in several human melanoma cell lines. A DDR tyrosine kinase inhibitor (DDR1-IN-1) significantly inhibited melanoma cell proliferation in vitro, and ex vivo and in tumor xenografts, underlining the promising potential of DDR1 inhibition in melanoma.

Kaposi's sarcoma-associated herpesvirus viremia is associated with the progression of classic and endemic Kaposi's sarcoma.

In order to gain further insight on the role of Kaposi's sarcoma-associated herpesvirus (KSHV) in classic and endemic Kaposi's sarcoma (KS) pathogenesis, we aimed to determine (i) whether KSHV is detectable in peripheral blood mononuclear cells (PBMCs), (ii) which PBMCs subpopulation harbor the virus, (iii) which clinical, histologic, and immunologic parameters are associated with KSHV viremia in a population of classic and endemic KS. KSHV viremia and various immunologic parameters were screened on 81 patients. KSHV viremia was positive in 58% of the patients. KSHV was detected in B cells, T cells, and monocytes. CD34+ cells depleted in circulating endothelial cells (CECs) were never infected and 50% of the patients tested had CECs infected by KSHV. We observed a significant increase of IL-2 and IFN-gamma production by CD4 T cells and an increase of IFN-gamma production by CD8 T cells compared to control patients. KS progression (P = 0.001) and KS staging (P = 0.03) were significantly and independently associated with positive KSHV viremia. Our results show that there is no specific immunosuppression in classic or endemic KS. We showed that KSHV can be detected within CECs and that KSHV viremia could be an indicator of circulating mature or precursor spindle cells.

Validation of a preclinical model for assessment of drug efficacy in melanoma.

The aim of personalized medicine is to improve our understanding of the disease at molecular level and to optimize therapeutic management. In this context, we have developed in vivo and ex vivo preclinical strategies evaluating the efficacy of innovative drugs in melanomas. Human melanomas (n = 17) of different genotypes (mutated BRAF, NRAS, amplified cKIT and wild type) were successfully engrafted in mice then amplified by successive transplantations. The exhaustive characterization of patient-derived xenografts (PDX) at genomic level (transcriptomic and CGH arrays) revealed a similar distribution pattern of genetic abnormalities throughout the successive transplantations compared to the initial patient tumor, enabling their use for mutation-specific therapy strategies. The reproducibility of their spontaneous metastatic potential in mice was assessed in 8 models. These PDXs were used for the development of histoculture drug response assays (ex vivo) for the evaluation of innovative drug efficacy (BRAF and MEK inhibitors). The pharmacological effects of BRAF and MEK inhibitors were similar between PDX-derived histocultures and their corresponding PDX, on 2 models of BRAF and NRAS-mutated melanomas. These models constitute a validated, effective tool for preclinical investigation of new therapeutic agents, and improve therapeutic strategies in the treatment of metastatic melanoma.

SIAH-1 interacts with CtIP and promotes its degradation by the proteasome pathway.

SIAH-1 and SIAH-2 are the human members of an evolutionary highly conserved E3 ligase family. SIAH-1 is a p53 and p21(Waf-1/Cip-1) induced gene during apoptosis and tumor suppression. In stable-transfected clones of MCF-7 cells, SIAH-1 overexpression was associated with apoptosis, mitotic alterations and p21(Waf-1/Cip-1) induction of expression. Using a two-hybrid screening, we identified here the transcriptional corepressor CtBP-interacting protein (CtIP) as a SIAH-1-interacting protein. CtIP has been proposed as a regulator of p21(Waf-1/Cip-1) gene transcription through a protein complex involving BRCA1. We demonstrate that SIAH-1 associates with CtIP both in vitro and in vivo. This interaction led to CtIP degradation by the ubiquitin-proteasome pathway. As expected, SIAH-1 induced p21(Waf-1/Cip-1) transcription in Jurkat-T cell. Surprisingly, a SIAH protein deleted of its RING finger, SIAH-1DeltaN, which is able to interact with CtIP but does not promote its degradation, also induced transcription from the p21(Waf-1) promoter in a similar extent as did SIAH-1. Our results suggest that p21(Waf-1/Cip-1) induction by SIAH-1 could not be mediated by CtIP degradation.

[Amphiregulin antisense RNA expression inhibits angiogenesis of human breast cancer in nude mice].

OBJECTIVE: To investigate the anti-angiogenic effect of amphiregulin (AR) antisense RNA expression in breast cancer. METHODS: Human AR cDNA antisense plasmid was transfected into NS2T2A1 cells (a human breast cancer cell line). Two selected clones expressed AR antisense RNA (AR AS1 and AR AS3 cell lines) in which AR protein expression was reduced. Control cell line NS2T2A1 V was obtained by empty vector transfection. These cells were injected subcutaneously into nude mice. The effects of conditioned media on proliferation of human microvascular endothelial cells (HMEC) were evaluated and VEGF secreted by the cells was measured by ELISA method. In tumor tissues, VEGF expression levels were measured by quantitative RT-PCR, and CD31-immunostaining was used for intra-tumoral vascular quantification. RESULTS: The proliferation index of HMEC cells grown in conditioned media with AR AS1 and AR AS3 was significantly reduced in comparison with that of control cells, accompanied by a decreased VEGF secretion. In tumors derived from AR AS1 and AR AS3 cells, intra-tumoral vascularization was reduced to about 50% of that derived from control cell line, accompanied with a decrease of VEGF expression. CONCLUSION: Amphiregulin antisense RNA expression inhibits efficiently the angiogenesis in breast cancer, suggesting this growth factor could represent a novel therapeutic target in breast cancer.

Driver KIT mutations in melanoma cluster in four hotspots.

There has been a great deal of interest in understanding the role of KIT in melanoma since the discovery of KIT mutations in a subset of melanoma. Although a significant proportion of these melanomas respond to KIT inhibitors, the presence of a KIT mutation does not guarantee a response to KIT inhibitors. Because recent data seem to indicate that only melanoma with specific KIT mutations respond to KIT inhibitors, we investigated which KIT mutations are driver mutations in melanoma and are therefore therapeutically relevant. We established that 70% of KIT mutations in melanoma are located in four hotspots (L576, K642, W557-V560, and D816-A829) and that these mutations are oncogenic in melanocytes and are bona-fide driver mutations. Testing for KIT mutations should therefore concentrate on these four hotspots, which can be targeted therapeutically.

EMMPRIN/CD147 is a novel coreceptor of VEGFR-2 mediating its activation by VEGF.

EMMPRIN/CD147 is mainly known for its protease inducing function but a role in promoting tumor angiogenesis has also been demonstrated. This study provides evidence that EMMPRIN is a new coreceptor for the VEGFR-2 tyrosine kinase receptor in both endothelial and tumor cells, as it directly interacts with it and regulates its activation by its VEGF ligand, signalling and functional consequences both in vitro and in vivo. Computational docking analyses and mutagenesis studies identified a molecular binding site in the extracellular domain of EMMPRIN located close to the cell membrane and containing the amino acids 195/199. EMMPRIN is overexpressed in cancer and hence is able to further potentiate VEGFR-2 activation, suggesting that a combinatory therapy of an antiangiogenic drug together with an inhibitor of EMMPRIN/VEGFR-2 interaction may have a greater impact on inhibiting angiogenesis and malignancy.

A novel tumor suppressor function of Kindlin-3 in solid cancer.

Kindlin-3 (FERMT-3) is known to be central in hemostasis and thrombosis control and its deficiency disrupts platelet aggregation and causes Leukocyte Adhesion Deficiency disease. Here we report that Kindlin-3 has a tumor suppressive role in solid cancer. Our present genetic and functional data show that Kindlin-3 is downregulated in several solid tumors by a mechanism involving gene hypermethylation and deletions. In vivo experiments demonstrated that Kindlin-3 knockdown in 2 tumor cell models (breast cancer and melanoma) markedly increases metastasis formation, in accord with the in vitro increase of tumor cell malignant properties. The metastatic phenotype was supported by a mechanism involving alteration in ß3-integrin activation including decreased phosphorylation, interaction with talin and the internalization of its active form leading to less cell attachment and more migration/invasion. These data uncover a novel and unexpected tumor suppressor role of Kindlin-3 which can influence integrins targeted therapies development.

A reliable method for the selection of exploitable melanoma archival paraffin embedded tissues for transcript biomarker profiling.

The source tissue for biomarkers mRNA expression profiling of tumors has traditionally been fresh-frozen tissue. The adaptation of formalin-fixed, paraffin-embedded (FFPE) tissues for routine mRNA profiling would however be invaluable in view of their abundance and the clinical information related to them. However, their use in the clinic remains a challenge due to the poor quality of RNA extracted from such tissues. Here, we developed a method for the selection of melanoma archival paraffin-embedded tissues that can be reliably used for transcript biomarker profiling. For that, we used qRT-PCR to conduct a comparative study in matched pairs of frozen and FFPE melanoma tissues of the expression of 25 genes involved in angiogenesis/tumor invasion and 15 housekeeping genes. A classification method was developed that can select the samples with a good frozen/FFPE correlation and identify those that should be discarded on the basis of paraffin data for four reference genes only. We propose therefore a simple and inexpensive assay which improves reliability of mRNA profiling in FFPE samples by allowing the identification and analysis of "good" samples only. This assay which can be extended to other genes would however need validation at the clinical level and on independent tumor series.

Invading basement membrane matrix is sufficient for MDA-MB-231 breast cancer cells to develop a stable in vivo metastatic phenotype.

INTRODUCTION: The poor efficacy of various anti-cancer treatments against metastatic cells has focused attention on the role of tumor microenvironment in cancer progression. To understand the contribution of the extracellular matrix (ECM) environment to this phenomenon, we isolated ECM surrogate invading cell populations from MDA-MB-231 breast cancer cells and studied their genotype and malignant phenotype. METHODS: We isolated invasive subpopulations (INV) from non invasive populations (REF) using a 2D-Matrigel assay, a surrogate of basal membrane passage. INV and REF populations were investigated by microarray assay and for their capacities to adhere, invade and transmigrate in vitro, and to form metastases in nude mice. RESULTS: REF and INV subpopulations were stable in culture and present different transcriptome profiles. INV cells were characterized by reduced expression of cell adhesion and cell-cell junction genes (44% of down regulated genes) and by a gain in expression of anti-apoptotic and pro-angiogenic gene sets. In line with this observation, in vitro INV cells showed reduced adhesion and increased motility through endothelial monolayers and fibronectin. When injected into the circulation, INV cells induced metastases formation, and reduced injected mice survival by up to 80% as compared to REF cells. In nude mice, INV xenografts grew rapidly inducing vessel formation and displaying resistance to apoptosis. CONCLUSION: Our findings reveal that the in vitro ECM microenvironment per se was sufficient to select for tumor cells with a stable metastatic phenotype in vivo characterized by loss of adhesion molecules expression and induction of pro-angiogenic and survival factors.