Kidins220/ARMS interacts with Pdzrn3, a protein containing multiple binding domains.

We report the identification of a novel partner of Kidins220/ARMS (Kinase D-interacting substrate of 220 kDa/Ankyrin Repeat-rich Membrane Spanning) an adaptor of neurotrophin receptors playing crucial roles during neurogenesis. Screening a phage display library of brain cDNA products we found that D. rerio Pdzrn3, a protein containing RING-finger and PDZ-domains, interacts with Kidins220/ARMS through its first PDZ-domain. Both zebrafish proteins share high homology with the corresponding mammalian proteins and both genes are developmentally expressed in neural districts where early neurogenesis occurs. The interaction was also confirmed by biochemical assays and by co-localization at the tips of growing neurites of PC12 cells induced with nerve growth factor.

Role of PTPRJ genotype in papillary thyroid carcinoma risk.

The strong genetic predisposition to papillary thyroid carcinoma (PTC) might be due to a combination of low-penetrance susceptibility variants. Thus, the research into gene variants involved in the increase of susceptibility to PTC is a relevant field of investigation. The gene coding for the receptor-type tyrosine phosphatase PTPRJ has been proposed as a cancer susceptibility gene, and its role as a tumor suppressor gene is well established in thyroid carcinogenesis. In this study, we want to ascertain the role of PTPRJ genotype in the risk for PTC. We performed a case-control study in which we determined the PTPRJ genotype for the non-synonymous Gln276Pro and Asp872Glu polymorphisms by PCR amplification and sequencing. We calculated allele and genotype frequencies for the considered polymorphisms of PTPRJ in a total sample of 299 cases (PTC patients) and 339 controls (healthy subjects) selected from Caucasian populations. We observed a significantly higher frequency of homozygotes for the Asp872 allele in the group of PTC patients than in the control group (odds ratio=1.61, 95% confidence interval 1.15-2.25, P=0.0053). We observed a non-significant increased frequency of homozygotes for Gln276Pro polymorphism in PTC cases in two distinct Caucasian populations. Therefore, the results reported here show that the homozygous genotype for Asp872 of PTPRJ is associated with an increased risk to develop PTC.

The receptor-type protein tyrosine phosphatase J antagonizes the biochemical and biological effects of RET-derived oncoproteins.

Thyroid cancer is frequently associated with the oncogenic conversion of the RET receptor tyrosine kinase. RET gene rearrangements, which lead to the generation of chimeric RET/papillary thyroid carcinoma (PTC) oncogenes, occur in PTC, whereas RET point mutations occur in familial multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinomas (MTC). We showed previously that the expression of the receptor-type protein tyrosine phosphatase J (PTPRJ) is suppressed in neoplastically transformed follicular thyroid cells. We now report that PTPRJ coimmunoprecipitates with wild-type RET and with the MEN2A-associated RET(C634R) oncoprotein but not with the RET/PTC1 and RET-MEN2B isoforms. Using mutated forms of PTPRJ and RET-MEN2A, we show that the integrity of the respective catalytic domains is required for the PTPRJ/RET-MEN2A interaction. PTPRJ expression induces dephosphorylation of the RET(C634R) and, probably via an indirect mechanism, RET/PTC1 oncoproteins on two key RET autophosphorylation sites (Tyr1062 and Tyr905). This results in a significant decrease of RET-induced Shc and extracellular signal-regulated kinase 1/2 phosphorylation levels. In line with this finding, adoptive PTPRJ expression reduced the oncogenic activity of RET(C634R) in an in vitro focus formation assay of NIH3T3 cells. As expected from the coimmunoprecipitation results, the RET(M918T) oncoprotein, which is associated to MEN2B and sporadic MTC, was resistant to the dephosphorylating activity of PTPRJ. Taken together, these findings identify RET as a novel substrate of PTPRJ and suggest that PTPRJ expression levels may affect tumor phenotype associated with RET/PTC1 and RET(C634R) mutants. On the other hand, resistance to PTPRJ may be part of the mechanism of RET oncogenic conversion secondary to the M918T mutation.

Involvement of PI3K and MAPK signaling in bcl-2-induced vascular endothelial growth factor expression in melanoma cells.

We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1alpha expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways.

Lonidamine causes inhibition of angiogenesis-related endothelial cell functions.

The aim of this study was to assess whether lonidamine (LND) interferes with some steps in angiogenesis progression. We report here, for the first time, that LND inhibited angiogenic-related endothelial cell functions in a dose-dependent manner (1-50 microg/ml). In particular, LND decreased proliferation, migration, invasion, and morphogenesis on matrigel of different endothelial cell lines. Zymographic and Western blot analysis assays showed that LND treatment produced a reduction in the secretion of matrix metalloproteinase-2 and metalloproteinase-9 by endothelial cells. Vessel formation in a matrigel plug was also reduced by LND. The viability, migration, invasion, and matrix metalloproteinase production of different tumor cell lines were not affected by low doses of LND (1-10 microg/ml), whereas 50 microg/ml LND, which corresponds to the dose used in clinical management of tumors, triggered apoptosis both in endothelial and tumor cells. Together, these data demonstrate that LND is a compound that interferes with endothelial cell functions, both at low and high doses. Thus, the effect of LND on endothelial cell functions, previously undescribed, may be a significant contributor to the antitumor effect of LND observed for clinical management of solid tumors.

bcl-2 induction of urokinase plasminogen activator receptor expression in human cancer cells through Sp1 activation: involvement of ERK1/ERK2 activity.

We have previously demonstrated that Bcl-2 overexpression in human breast carcinoma and melanoma cells synergizes with hypoxia to increase angiogenesis through up-regulation of vascular endothelial growth factor. In this work we demonstrated, for the first time, that Bcl-2 overexpression in cancer cells exposed to hypoxia modulates urokinase plasminogen activator receptor (uPAR) expression through Sp1 transcription factor and that the extracellular signal-regulated kinase (ERK) pathway plays a role in Sp1 transcriptional activity. In particular, an increase in uPAR protein and mRNA expression was found in melanoma bcl-2 transfectants grown under hypoxia when compared with control cells, and a decrease of uPAR protein expression was induced by treatment of cells with specific bcl-2 antisense oligonucleotides. Up-regulation of uPAR expression was accompanied by increased Sp1 protein expression, stability, serine phosphorylation, and DNA binding activity. Treatment of cells with mitramycin A, an inhibitor of Sp1 activity, confirmed the role of Sp1 transcriptional activity in uPAR induction by Bcl-2. The contribution of the ERK pathway in Sp1-increased transcriptional activity was demonstrated by the use of chemical inhibition. In fact, ERK kinase activation was induced in Bcl-2-overexpressing cells exposed to hypoxia, and the ERK kinase inhibitor UO126 was able to down-regulate Sp1 phosphorylation and DNA binding activity. Using a human breast carcinoma line, we obtained data supporting our findings with melanoma cells and identified a link between the induction of Sp1 and uPAR expression as a common bcl-2-controlled phenomenon in human tumors. In conclusion, our results strongly indicate that up-regulation of uPAR expression by Bcl-2 in hypoxia is modulated by Sp1 DNA binding activity through the ERK signaling pathway.

Bcl-2 overexpression in human melanoma cells increases angiogenesis through VEGF mRNA stabilization and HIF-1-mediated transcriptional activity.

The aim of this paper was to study the molecular mechanisms by which bcl-2 increases hypoxia-induced vascular endothelial growth factor (VEGF) expression. We demonstrated that bcl-2 overexpression in M14 human melanoma cell line enhances hypoxia-induced VEGF mRNA stability and promoter activation. In particular, the half-life of the message was longer in bcl-2 transfectants (approximately 330 min) than in control cells (approximately 180 min). In addition, bcl-2 overexpression increased VEGF promoter activity through the hypoxia-inducible factor-1 (HIF-1) transcription factor. Increased HIF-1a protein expression and DNA binding activity were detected in bcl-2 overexpressing cells compared with control cells. An enhanced functional activity of secreted VEGF was found both in in vitro and in vivo angiogenic assays, and the use of VEGF specific antibodies validated the role of VEGF on bcl-2-induced angiogenesis. Taken together our results indicate that bcl-2 plays an important role in melanoma angiogenesis, and that VEGF mRNA stabilization and HIF-1-mediated transcriptional activity are two important control points in bcl-2/hypoxia-induced VEGF expression.

hnRNP A1 nucleocytoplasmic shuttling activity is required for normal myelopoiesis and BCR/ABL leukemogenesis.

hnRNP A1 is a nucleocytoplasmic shuttling heterogeneous nuclear ribonucleoprotein that accompanies eukaryotic mRNAs from the active site of transcription to that of translation. Although the importance of hnRNP A1 as a regulator of nuclear pre-mRNA and mRNA processing and export is well established, it is unknown whether this is relevant for the control of proliferation, survival, and differentiation of normal and transformed cells. We show here that hnRNP A1 levels are increased in myeloid progenitor cells expressing the p210(BCR/ABL) oncoprotein, in mononuclear cells from chronic myelogenous leukemia (CML) blast crisis patients, and during disease progression. In addition, in myeloid progenitor 32Dcl3 cells, BCR/ABL stabilizes hnRNP A1 by preventing its ubiquitin/proteasome-dependent degradation. To assess the potential role of hnRNP A1 nucleocytoplasmic shuttling activity in normal and leukemic myelopoiesis, a mutant defective in nuclear export was ectopically expressed in parental and BCR/ABL-transformed myeloid precursor 32Dcl3 cells, in normal murine marrow cells, and in mononuclear cells from a CML patient in accelerated phase. In normal cells, expression of this mutant enhanced the susceptibility to apoptosis induced by interleukin-3 deprivation, suppressed granulocytic differentiation, and induced massive cell death of granulocyte colony-stimulating factor-treated cultures. In BCR/ABL-transformed cells, its expression was associated with suppression of colony formation and reduced tumorigenic potential in vivo. Moreover, interference with hnRNP A1 shuttling activity resulted in downmodulation of C/EBPalpha, the major regulator of granulocytic differentiation, and Bcl-X(L), an important survival factor for hematopoietic cells. Together, these results suggest that the shuttling activity of hnRNP A1 is important for the nucleocytoplasmic trafficking of mRNAs that encode proteins influencing the phenotype of normal and BCR/ABL-transformed myeloid progenitors.

BCR-ABL suppresses C/EBPalpha expression through inhibitory action of hnRNP E2.

The arrest of differentiation is a feature of both chronic myelogenous leukemia cells in myeloid blast crisis and myeloid precursors that ectopically express the p210BCR-ABL oncoprotein; however, its underlying mechanisms remain poorly understood. Here we show that expression of BCR-ABL in myeloid precursor cells leads to transcriptional suppression of the granulocyte colony-stimulating factor receptor G-CSF-R (encoded by CSF3R), possibly through down-modulation of C/EBPalpha-the principal regulator of granulocytic differentiation. Expression of C/EBPalpha protein is barely detectable in primary marrow cells taken from individuals affected with chronic myeloid leukemia in blast crisis. In contrast, CEBPA RNA is clearly present. Ectopic expression of C/EBPalpha induces granulocytic differentiation of myeloid precursor cells expressing BCR-ABL. Expression of C/EBPalpha is suppressed at the translational level by interaction of the poly(rC)-binding protein hnRNP E2 with CEBPA mRNA, and ectopic expression of hnRNP E2 in myeloid precursor cells down-regulates both C/EBPalpha and G-CSF-R and leads to rapid cell death on treatment with G-CSF (encoded by CSF3). Our results indicate that BCR-ABL regulates the expression of C/EBPalpha by inducing hnRNP E2-which inhibits the translation of CEBPA mRNA.