Carine Nyalendo

Src-dependent Phosphorylation of Membrane Type I Matrix Metalloproteinase on Cytoplasmic Tyrosine 573 ROLE IN ENDOTHELIAL AND TUMOR CELL MIGRATION

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr573) and that this phosphorylation requires the kinase Src. Using phosphospecific antibodies recognizing MT1-MMP phosphorylated on Tyr573, we observed that tyrosine phosphorylation of the enzyme is rapidly induced upon stimulation of tumor and endothelial cells with the platelet-derived chemoattractant sphingosine-1-phosphate, suggesting a role in migration triggered by this lysophospholipid. Accordingly, overexpression of a nonphosphorylable MT1-MMP mutant (Y573F) blocked sphingosine-1-phosphate-induced migration of Human umbilical vein endothelial cells and HT-1080 (human fibrosarcoma) cells and failed to stimulate migration of cells lacking the enzyme (bovine aortic endothelial cells). Altogether, these findings strongly suggest that the Src-dependent tyrosine phosphorylation of MT1-MMP plays a key role in cell migration and further emphasize the importance of the cytoplasmic domain of the enzyme in this process.

Impaired tyrosine phosphorylation of membrane type 1-matrix metalloproteinase reduces tumor cell proliferation in three-dimensional matrices and abrogates tumor growth in mice

Pericellular proteolysis of the extracellular matrix by membrane type 1-matrix metalloproteinase (MT1-MMP) confers tumor cells with the ability to proliferate within three-dimensional (3D) matrices and sustains tumor growth in mice. In this study, we show that in addition to its matrix-degrading activity, phosphorylation of MT1-MMP on its unique tyrosine residue located within its cytoplasmic sequence (Tyr573) may also participate to these processes. Fibrosarcoma cells expressing a proteolytically active but non-phosphorylable mutant of MT1-MMP showed a markedly reduced proliferation rate when embedded within 3D type I collagen matrices, this antiproliferative effect being correlated with arrest in the G(0)/G(1) phase of the cell cycle. Impaired tyrosine phosphorylation of MT1-MMP also inhibits anchorage-independent growth of HT-1080 cells in soft agar as well as their invasion of collagen barriers, two prominent attributes of tumor cells, suggesting a broad inhibitory effect of the MT1-MMP mutant on tumorigenesis. Accordingly, whereas HT-1080 cells formed well-vascularized tumors containing tyrosine-phosphorylated MT1-MMP, tumor growth was completely abolished by expression of the non-phosphorylable MT1-MMP mutant. These findings thus indicate a close co-operation between the matrix-degrading activity of MT1-MMP and tyrosine phosphorylation of its intracellular domain for tumor cell invasion and proliferation and suggest that the targeting of the intracellular signaling pathways leading to tyrosine phosphorylation of MT1-MMP may represent an unexpected alternative strategy for the inhibition of this enzyme.

Involvement of the CXCR7/CXCR4/CXCL12 Axis in the Malignant Progression of Human Neuroblastoma

Neuroblastoma (NB) is a typical childhood and heterogeneous neoplasm for which efficient targeted therapies for high-risk tumors are not yet identified. The chemokine CXCL12, and its receptors CXCR4 and CXCR7 have been involved in tumor progression and dissemination. While CXCR4 expression is associated to undifferentiated tumors and poor prognosis, the role of CXCR7, the recently identified second CXCL12 receptor, has not yet been elucidated in NB. In this report, CXCR7 and CXCL12 expressions were evaluated using a tissue micro-array including 156 primary and 56 metastatic NB tissues. CXCL12 was found to be highly associated to NB vascular and stromal structures. In contrast to CXCR4, CXCR7 expression was low in undifferentiated tumors, while its expression was stronger in matured tissues and specifically associated to differentiated neural tumor cells. As determined by RT-PCR, CXCR7 expression was mainly detected in N-and S-type NB cell lines, and was slightly induced upon NB cell differentiation in vitro. The relative roles of the two CXCL12 receptors were further assessed by overexpressing CXCR7 or CXCR4 receptor alone, or in combination, in the IGR-NB8 and the SH-SY5Y NB cell lines. In vitro functional analyses indicated that, in response to their common ligand, both receptors induced activation of ERK1/2 cascade, but not Akt pathway. CXCR7 strongly reduced in vitro growth, in contrast to CXCR4, and impaired CXCR4/CXCL12-mediated chemotaxis. Subcutaneous implantation of CXCR7-expressing NB cells showed that CXCR7 also significantly reduced in vivo growth. Moreover, CXCR7 affected CXCR4-mediated orthotopic growth in a CXCL12-producing environment. In such model, CXCR7, in association with CXCR4, did not induce NB cell metastatic dissemination. In conclusion, the CXCR7 and CXCR4 receptors revealed specific expression patterns and distinct functional roles in NB. Our data suggest that CXCR7 elicits anti-tumorigenic functions, and may act as a regulator of CXCR4/CXCL12-mediated signaling in NB.

CD133 expression is associated with poor outcome in neuroblastoma via chemoresistance mediated by the AKT pathway

Sartelet H, Imbriglio T, Nyalendo C, Haddad E, Annabi B, Duval M, Fetni R, Victor K, Alexendrov L, Sinnett D, Fabre M & Vassal G 
(2012) Histopathology 60, 1144–1155

Sphingosine-1-phosphate induces the association of membrane-type 1 matrix metalloproteinase with p130Cas in endothelial cells.

Membrane‐type 1 matrix metalloproteinase (MT1‐MMP) plays an important role in sphingosine‐1‐phosphate(S1P)‐dependent migration of endothelial cells but the underlying mechanisms remain largely unknown. Herein, we show that S1P promotes the relocalization of MT1‐MMP to peripheral actin‐rich membrane ruffles that is coincident with its association with the adaptor protein p130Cas at the leading edge of migrating cells. Immunoprecipitation and confocal microscopy analyses suggest that this interaction required the tyrosine phosphorylation of p130Cas and also involves S1P‐dependent phosphorylation of MT1‐MMP within its cytoplasmic sequence. The interaction of MT1‐MMP with p130Cas at the cell periphery suggests the existence of a close interplay between pericellular proteolysis and signaling pathways involved in EC migration.

Membrane-type 1 matrix metalloproteinase stimulates cell migration through epidermal growth factor receptor transactivation

Proteolysis of extracellular matrix proteins by membrane-type 1 matrix metalloproteinase (MT1-MMP) plays a pivotal role in tumor and endothelial cell migration. In addition to its proteolytic activity, several studies indicate that the proinvasive properties of MT1-MMP also involve its short cytoplasmic domain, but the specific mechanisms mediating this function have yet to be fully elucidated. Having previously shown that the serum factor sphingosine 1-phosphate stimulates MT1-MMP promigratory function through a process that involves its cytoplasmic domain, we now extend these findings to show that this cooperative interaction is permissive to cellular migration through MT1-MMP–dependent transactivation of the epidermal growth factor receptor (EGFR). In the presence of sphingosine 1-phosphate, MT1-MMP stimulates EGFR transactivation through a process that is dependent upon the cytoplasmic domain of the enzyme but not its catalytic activity. The MT1-MMP–induced EGFR transactivation also involves Gi protein signaling and Src activities and leads to enhanced cellular migration through downstream extracellular signal-regulated kinase activation. The present study, thus, elucidates a novel role of MT1-MMP in signaling events mediating EGFR transactivation and provides the first evidence of a crucial role of this receptor activity in MT1-MMP promigratory function. Taken together, our results suggest that the inhibition of EGFR may represent a novel target to inhibit MT1-MMP–dependent processes associated with tumor cell invasion and angiogenesis. (Mol Cancer Res 2007;5(6):569–83)

The Antiangiogenic Agent Neovastat (Æ-941) Stimulates Tissue Plasminogen Activator Activity

The plasminogen activator/plasmin system represents a key component of the proteolytic machinery underlying angiogenesis. In this work, we investigated the effect of Neovastat (Æ-941), a naturally occurring multifunctional antiangiogenic agent that is currently in Phase III clinical trials, on tissue and urokinase plasminogen activator activities. We found that in vitro, Neovastat at 100μg/ml markedly stimulates t-PA-mediated plasmin generation, while it slightly inhibits the generation of plasmin mediated by uPA. The stimulatory effect of Neovastat on t-PA activity was markedly increased by a heat treatment, resulting in a 15-fold increase in the rate of activation of plasminogen. Neovastat did not directly stimulate the activity of t-PA or plasmin towards exogenous substrates, suggesting that its effect requires the presence of plasminogen. Accordingly, kinetic analysis showed that Neovastat increases both the kcat of t-PA as well as its affinity for plasminogen by 10-fold. The stimulation of t-PA activity by Neovastat was also correlated with a direct interaction of Neovastat with plasminogen as monitored by the surface plasmon resonance technology. Overall, these results identify Neovastat as a potent stimulator of t-PA-dependent activation of plasminogen, further emphasizing its pleiotropic mechanism of action on several molecular events involved in angiogenesis.

Autophagy is associated with chemoresistance in neuroblastoma

BackgroundNeuroblastoma (NB) is a frequent pediatric tumor characterized by a poor prognosis where a majority of tumors progress despite intensive multimodality treatments. Autophagy, a self-degradative process in cells, could be induced by chemotherapy and be associated with chemoresistance. The aim of this study was to determine whether: 1) autophagy is present in NB, 2) chemotherapy modified its levels, and 3) its inhibition decreased chemoresistance.MethodsImmunohistochemical stainings were performed on samples from 184 NB patients in order to verify the expression of LC3B, a specific marker for autophagy, and Beclin 1, a positive regulator of autophagy. In addition, we performed an in vitro study with six NB cell lines and six drugs (vincristine, doxorubicin, cisplatin temozolomide, LY294002 and syrolimus). Inhibition of autophagy was performed using ATG5 knockdown cells or hydroxychloroquine (HCQ). Cell survival was measured using the MTT cell proliferation assay. Autophagy was detected by monodansylcadaverine, confocal microscopy and Western blot. In vivo study with tumor xenografts in NSG mice was performed.ResultsOur results have indicated that autophagy was present at low levels in NB and was not a prognostic factor, while Beclin 1 was highly expressed in children with poor NB prognosis. However, autophagy levels increased after chemotherapy in vitro and in vivo. Tumor progression was significantly decreased in mice treated with a combination of HCQ and vincristine.ConclusionsTaken together, autophagy is present in NB, induced by chemotherapy and associated with chemoresistance, which is significantly reduced by its inhibition. Therefore, targeting autophagy represents a very attractive approach to develop new therapeutic strategies in NB.

Genotype analysis of tumor-initiating cells expressing CD133 in neuroblastoma.

Neuroblastoma (NB) is the most common and lethal extracranial solid tumor of childhood. Despite aggressive therapy, more than half of the children with advanced NB will die of uncontrolled metastatic disease. After chemotherapy, tumor‐initiating cells (TICs) could persist, cause relapses and metastasis. The aim of this study is to demonstrate the tumor‐initiating properties of CD133high NB cells and to identify new specific genetic abnormalities. Isolation of the CD133high cell population from NB cell lines was followed by neurosphere formation, soft agar assays, and orthotopic injections in NOD/SCID/IL2Rγc‐null mice. A differential genotyping analysis was performed with Affymetrix SNP 6.0 arrays on CD133low and CD133high populations and the frequency of the abnormalities of 36 NB tumors was determined. Our results show that CD133high NB cells possess tumor‐initiating properties, as CD133high cells formed significantly more neurospheres and produced significantly more colonies in soft agar than CD133low. Injection of 500 CD133high cells was sufficient to generate primary tumors and frequent metastases in mice. Differential genotyping analysis demonstrated two common regions with gains (16p13.3 and 19p13.3) including the gene EFNA2 in the CD133high population, and two with loss of heterozygosity (16q12.1 and 21q21.3) in the CD133low population. The gain of EFNA2 correlated with increased expression of the corresponding protein. These abnormalities were found in NB samples and some were significantly correlated with CD133 expression. Our results show that CD133high NB cells have TICs properties and present different genotyping characteristics compared to CD133low cells. Our findings reveal insights into new therapeutic targets in NB TICs.

Identification of membrane-type 1 matrix metalloproteinase tyrosine phosphorylation in association with neuroblastoma progression

BackgroundNeuroblastoma is a pediatric tumor of neural crest cells that is clinically characterized by its variable evolution, from spontaneous regression to malignancy. Despite many advances in neuroblastoma research, 60% of neuroblastoma, which are essentially metastatic cases, are associated with poor clinical outcome due to the lack of effectiveness of current therapeutic strategies. Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), an enzyme involved in several steps in tumor progression, has previously been shown to be associated with poor clinical outcome for neuroblastoma. Based on our recent demonstration that MT1-MMP phosphorylation is involved in the growth of fibrosarcoma tumors, we examined the potential role of phosphorylated MT1-MMP in neuroblastoma progression.MethodsTyrosine phosphorylated MT1-MMP was immunostained on tissue microarray samples from 55 patients with neuroblastoma detected by mass screening (known to be predominantly associated with favourable outcome), and from 234 patients with standard diagnosed neuroblastoma. In addition, the effects of a non phosphorylable version of MT1-MMP on neuroblastoma cell migration and proliferation were investigated within three-dimensional collagen matrices.ResultsAlthough there is no correlation between the extent of tyrosine phosphorylation of MT1-MMP (pMT1-MMP) and MYCN amplification or clinical stage, we observed greater phosphorylation of pMT1-MMP in standard neuroblastoma, while it is less evident in neuroblastoma from mass screening samples (P = 0.0006) or in neuroblastoma samples from patients younger than one year (P = 0.0002). In vitro experiments showed that overexpression of a non-phosphorylable version of MT1-MMP reduced MT1-MMP-mediated neuroblastoma cell migration and proliferation within a three-dimensional type I collagen matrix, suggesting a role for the phosphorylated enzyme in the invasive properties of neuroblastoma cells.ConclusionOverall, these results suggest that tyrosine phosphorylated MT1-MMP plays an important role in neuroblastoma progression and that its expression is preferentially observed in tumor specimens from neuroblastoma patients showing poor clinical outcome.