New Method for Sorting Endothelial and Neural Progenitors from Human Induced Pluripotent Stem Cells by Sedimentation Field Flow Fractionation.

Human induced pluripotent stem cells (hiPSc) are a very useful solution to create and observe the behavior of specific and usually inaccessible cells, such as human motor neurons. Obtained from a patient biopsy by reprograming dermal fibroblasts (DF), hiPSc present the same properties as embryonic stem cells and can generate any cell type after several weeks of differentiation. Today, there are numerus protocols which aim to control hiPSC differentiation. The principal challenge is to obtain a sufficiently enriched specific cell population to study disease pathophysiology and to provide a good model for further investigation and drug screening. The differentiation process is very costly and time-consuming, because many specific factors and different culture media must be used. In this study, we used Sedimentation Field Flow Fractionation (SdFFF) to prepare enriched populations derived from hiPSc after only 10 days of culture in a classical medium. Based on phenotypic and proteomic characterization, "hyperlayer" elution resulted in a fraction expressing markers of endothelial progenitors while another fraction expressed markers of neural progenitors. The isolation of subpopulations representing various differentiation lineages is of major interest for the production of specialized, cell-enriched fractions and in the preparation of increasingly complex models for the development of new therapeutic tools.

p75 neurotrophin receptor and pro-BDNF promote cell survival and migration in clear cell renal cell carcinoma.

p75NTR, a member of TNF receptor family, is the low affinity receptor common to several mature neurotrophins and the high affinity receptor for pro-neurotrophins. Brain-Derived Neurotrophic Factor (BDNF), a member of neurotrophin family has been described to play an important role in development and progression of several cancers, through its binding to a high affinity tyrosine kinase receptor B (TrkB) and/or p75NTR. However, the functions of these two receptors in renal cell carcinoma (RCC) have never been investigated. An overexpression of p75NTR, pro-BDNF, and to a lesser extent for TrkB and sortilin, was detected by immunohistochemistry in a cohort of 83 clear cell RCC tumors. p75NTR, mainly expressed in tumor tissues, was significantly associated with higher Fuhrman grade in multivariate analysis. In two derived-RCC lines, 786-O and ACHN cells, we demonstrated that pro-BDNF induced cell survival and migration, through p75NTR as provided by p75NTR RNA silencing or blocking anti-p75NTR antibody. This mechanism is independent of TrkB activation as demonstrated by k252a, a tyrosine kinase inhibitor for Trk neurotrophin receptors. Taken together, these data highlight for the first time an important role for p75NTR in renal cancer and indicate a putative novel target therapy in RCC.

MKP1 mediates chemosensitizer effects of E1a in response to cisplatin in non-small cell lung carcinoma cells.

The adenoviral gene E1a is known to enhance the antitumor effect of cisplatin, one of the cornerstones of the current cancer chemotherapy. Here we study the molecular basis of E1a mediated sensitivity to cisplatin in an experimental model of Non-small cell lung cancer. Our data show how E1a blocks the induction of autophagy triggered by cisplatin and promotes the apoptotic response in resistant cells. Interestingly, at the molecular level, we present evidences showing how the phosphatase MKP1 is a major determinant of cisplatin sensitivity and its upregulation is strictly required for the induction of chemosensitivity mediated by E1a. Indeed, E1a is almost unable to promote sensitivity in H460, in which the high expression of MKP1 remains unaffected by E1a. However, in resistant cell as H1299, H23 or H661, which display low levels of MKP1, E1a expression promotes a dramatic increase in the amount of MKP1 correlating with cisplatin sensitivity. Furthermore, effective knock down of MKP1 in H1299 E1a expressing cells restores resistance to a similar extent than parental cells.  In summary, the present work reinforce the critical role of MKP1 in the cellular response to cisplatin highlighting the importance of this phosphatase in future gene therapy approach based on E1a gene.

Anti-apoptotic role and clinical relevance of neurotrophins in diffuse large B-cell lymphomas.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a fatal malignancy that needs to identify new targets for additional therapeutic options. This study aimed to clarify the clinical and biological significance of endogenous neurotrophin (nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF)) in DLBCL biopsy samples and cell lines. METHODS: We analysed expression of NGF, BDNF, and their receptors (Trk, p75(NTR)) in 51 biopsies and cell lines by immunohistochemistry, immunofluorescence, and western blotting. To investigate the biological role of BDNF/TrkB/p75(NTR) axis, effects of neurotrophin signalling inhibition were determined on tumour cell survival and vascular endothelial growth factor (VEGF) secretion. The pharmacological pan-Trk inhibitor K252a was used for in vitro and in vivo studies. RESULTS: A BDNF/TrkB axis was expressed in all biopsies, which was independent of the germinal centre B-cell (GCB)/non-GCB profile. p75(NTR), TrkB, and BDNF tumour scores were significantly correlated and high NGF expression was significantly associated with MUM1/IRF4, and the non-GCB subtype. Diffuse large B-cell lymphoma cell lines co-expressed neurotrophins and their receptors. The full-length TrkB receptor was found in all cell lines, which was also phosphorylated at Tyr-817. p75(NTR) was associated to Trk and not to its cell death co-receptor sortilin. In vitro, inhibition of neurotrophin signalling induced cell apoptosis. K252a caused cell apoptosis, decreased VEGF secretion, and potentiated rituximab effect, notably in less rituximab-sensitive cells. In vivo, K252a significantly reduced tumour growth and potentiated the effects of rituximab in a GCB-DLBCL xenograft model. CONCLUSIONS: This work argues for a pro-survival role of endogenous neurotrophins in DLBCLs and inhibition of Trk signalling might be a potential treatment strategy for rituximab resistant subgroups.

Abrogation of the p38 MAPK α signaling pathway does not promote radioresistance but its activity is required for 5-Fluorouracil-associated radiosensitivity.

The p38 Mitogen Activated Protein Kinase (MAPK) signaling pathway has become a major player in the response to DNA-damage. A growing body of evidences has been relating this signaling pathway to the cellular response to ionizing radiation (IR), suggesting a role in radioresistance. Here, we study the implication of this signaling pathway in the response to IR in terms of radioresistance. To this end we used 10 different cell lines derived from several types of tumors (colorectal, non-small cell lung cancer -NSCLC-, renal and glioblastoma). Although p38 MAPK is transiently activated by IR, our data, obtained by genetic and chemical approaches, showed that this signaling pathway is not implicated in cellular viability after IR exposure. Indeed, down-modulation of this signaling pathway promotes a mild radiosensitivity depending on the cell line. However, it is remarkable that lack of p38 MAPK α abrogates the radiosensitizing effect of 5-Fluorouracil (5-FU) in HCT116 cell line, supporting the role of this MAPK in the radiosensitizing action of 5-FU.

Balance between MKK6 and MKK3 mediates p38 MAPK associated resistance to cisplatin in NSCLC.

The p38 MAPK signaling pathway has been proposed as a critical mediator of the therapeutic effect of several antitumor agents, including cisplatin. Here, we found that sensitivity to cisplatin, in a system of 7 non-small cell lung carcinoma derived cell lines, correlated with high levels of MKK6 and marked activation of p38 MAPK. However, knockdown of MKK6 modified neither the response to cisplatin nor the activation of p38 MAPK. Deeper studies showed that resistant cell lines also displayed higher basal levels of MKK3. Interestingly, MKK3 knockdown significantly decreased p38 phosphorylation upon cisplatin exposure and consequently reduced the response to the drug. Indeed, cisplatin poorly activated MKK3 in resistant cells, while in sensitive cell lines MKK3 showed the opposite pattern in response to the drug. Our data also demonstrate that the low levels of MKK6 expressed in resistant cell lines are the consequence of high basal activity of p38 MAPK mediated by the elevated levels of MKK3. This finding supports the existence of a regulatory mechanism between both MAPK kinases through their MAPK. Furthermore, our results were also mirrored in head and neck carcinoma derived cell lines, suggesting our observations boast a potential universal characteristic in cancer resistance of cisplatin. Altogether, our work provides evidence that MKK3 is the major determinant of p38 MAPK activation in response to cisplatin and, hence, the resistance associated with this MAPK. Therefore, these data suggest that the balance between both MKK3 and MKK6 could be a novel mechanism which explains the cellular response to cisplatin.

ERK2, but not ERK1, mediates acquired and "de novo" resistance to imatinib mesylate: implication for CML therapy.

Resistance to Imatinib Mesylate (IM) is a major problem in Chronic Myelogenous Leukaemia management. Most of the studies about resistance have focused on point mutations on BCR/ABL. However, other types of resistance that do not imply mutations in BCR/ABL have been also described. In the present report we aim to study the role of several MAPK in IM resistance not associate to BCR/ABL mutations. Therefore we used an experimental system of resistant cell lines generated by co-culturing with IM (K562, Lama 84) as well as primary material from resistant and responder patient without BCR/ABL mutations. Here we demonstrate that Erk5 and p38MAPK signaling pathways are not implicated in the acquired resistance phenotype. However, Erk2, but not Erk1, is critical for the acquired resistance to IM. In fact, Bcr/Abl activates preferentially Erk2 in transient transfection in a dose dependent fashion through the c-Abl part of the chimeric protein. Finally, we present evidences demonstrating how constitutive activation of Erk2 is a de novo mechanism of resistance to IM. In summary our data support the use of therapeutic approaches based on Erk2 inhibition, which could be added to the therapeutic armamentarium to fight CML, especially when IM resistance develops secondary to Erk2 activation.

c-Abl activates p38 MAPK independently of its tyrosine kinase activity: Implications in cisplatin-based therapy.

Activation of p38 MAPK is a critical requisite for the therapeutics activity of the antitumor agent cisplatin. In this sense, a growing body of evidences supports the role of c-Abl as a major determinant of p38 MAPK activation, especially in response to genotoxic stress when triggered by cisplatin. Here, we demonstrate that p38 MAPK activation in response to cisplatin does not require the tyrosine kinase activity of c-Abl. Indeed, c-Abl can activate the p38 MAPK signaling pathway by a mechanism that is independent of its tyrosine kinase activity, but that instead involves the ability of c-Abl to increase the stability of MKK6. Similar results were obtained in chronic myeloid leukemia-derived cell lines, in which a chimeric Bcr/Abl protein mimics the effects of c-Abl overexpression on p38 MAPK activation. These findings may explain why a clinically used c-Abl inhibitor, imatinib mesylate, fails to inhibit the p38 MAPK pathway alone or in combination with cisplatin, and provide evidence of a novel signaling mechanism in which these antitumor agents act.