[Clinical research activity of the French cancer cooperative network: Overview and perspectives]. / Activité de recherche clinique académique des investigateurs du réseau des Groupes coopérateurs en oncologie (GCO) : bilan et perspectives.

INTRODUCTION: The French Cancer Plan 2014-2019 stresses the importance of strengthening collaboration between all stakeholders involved in the fight against cancer, including cancer cooperative groups and intergroups. This survey aimed to describe the basics characteristics and clinical research activity among the Cancer Cooperative Groups (Groupes coopérateurs en oncologie). The second objective was to identify facilitators and barriers to their research activity. METHODS: A questionnaire was sent to all the clinicians involved in 2014 as investigators in a clinical trial sponsored by one of the ten members of the Cancer Cooperative Groups network. The questions were related to their profile, research activity and the infrastructure existing within their healthcare center to support clinical research and related compliance activities. RESULTS: In total, 366 investigators responded to our survey. The academic clinical trials sponsored by the Cancer Cooperative Groups represented an important part of the research activity of the investigators in France in 2014. These academic groups contributed to the opening of many research sites throughout all regions in France. Factors associated with a higher participation of investigators (more than 10 patients enrolled in a trial over a year) include the existing support of healthcare professionals (more than 2 clinical research associate (CRA) OR=11.16 [3.82-32.6] compared to none) and the practice of their research activity in a University Hospital Center (CHU) rather than a Hospital Center (CH) (OR=2.15 [1.20-3.83]). CONCLUSION: This study highlighted factors that can strengthen investigator clinical research activities and subsequently improve patient access to evidence-based new cancer therapies in France.

Transcriptome analysis and in vivo activity of fluvastatin versus zoledronic acid in a murine breast cancer metastasis model.

Statins and bisphosphonates are two distinct classes of isoprenoid pathway inhibitors targeting downstream enzyme to HMG-CoA reductase (upstream enzyme) and farnesyl-pyrophosphate synthase, respectively. Here, we studied fluvastatin (Fluva) and zoledronate (Zol), representative molecules of each class, respectively. In vivo metastatic potentials of both molecules were assessed. For the first time, we observed a significant reduction in progression of established metastases with Fluva treatment. Treatment with both Zol at 100 µg/kg and Fluva at 15 mg/kg inhibited 80% of the metastasis bioluminescence signal and increased survival of mice. The Zol and Fluva transcriptomic profiles of treated MDA-MB-231 cells revealed analogous patterns of affected genes, but each of them reached with different kinetics. The observable changes in gene expression started after 24 h for Fluva IC(50 72 h) and only after 48 h for Zol IC(50 72 h). To obtain early changes in gene expression of Zol-treated cells, a 3 times higher dose of Zol IC(50 72 h) had to be applied. Combining Fluva and Zol in vivo showed no synergy, but a benefit of several days in survival of mice. This study demonstrated that Zol or Fluva is of potential clinical use for the treatment of established metastasis.

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions.

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones which overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knockdown NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared to V189shCtl, under stress conditions. Unexpectedly, NRP1 knock-down had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity towards NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.

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.

New symmetrically esterified m-bromobenzyl non-aminobisphosphonates inhibited breast cancer growth and metastases.

BACKGROUND: Although there was growing evidence in the potential use of Bisphosphonates (BPs) in cancer therapy, their strong osseous affinities that contrast their poor soft tissue uptake limited their use. Here, we developed a new strategy to overcome BPs hydrophilicity by masking the phosphonic acid through organic protecting groups and introducing hydrophobic functions in the side chain. METHODOLOGY/PRINCIPAL FINDINGS: We synthesized non-nitrogen BPs (non N-BPs) containing bromobenzyl group (BP7033Br) in their side chain that were symmetrically esterified with hydrophobic 4-methoxphenyl (BP7033BrALK) and assessed their effects on breast cancer estrogen-responsive cells (T47D, MCF-7) as well as on non responsive ones (SKBR3, MDA-MB-231 and its highly metastatic derived D3H2LN subclone). BP7033Br ALK was more efficient in inhibiting tumor cell proliferation, migration and survival when compared to BP7033Br. Although both compounds inhibited tumor growth without side effects, only BP7033Br ALK abrogated tumor angiogenesis and D3H2LN cells-induced metastases formation. CONCLUSION/SIGNIFICANCE: Taken together these data suggest the potential therapeutic use of this new class of esterified Bisphosphonates (BPs) in the treatment of tumor progression and metastasis without toxic adverse effects.

Mutagenesis of prochlorothrix plastocyanin reveals additional features in photosystem I interactions.

Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.

Plastocyanin-cytochrome f interactions: the influence of hydrophobic patch mutations studied by NMR spectroscopy.

Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived from NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant of 6 (+/-2) x 10(3) M(-1) for complex formation, 1 order of magnitude larger than that for the physiological plastocyanin-cytochrome f complex from Ph. laminosum. Chemical-shift perturbation mapping indicates that the hydrophobic patch of plastocyanin is involved in the complex interface. When the unusual hydrophobic patch residues of P. hollandica plastocyanin were reverted to the conserved residues found in most other plastocyanins (Y12G/P14L), the binding constant for the interaction with cytochrome f was unaffected. However, the chemical shift perturbation map was considerably different, and the size of the average perturbation decreased by 40%. The complexes of both the wild-type and double mutant plastocyanin with cytochrome f were sensitive to ionic strength, contrary to the physiological complex. The possible implications of these findings for the mechanism of transient complex formation are discussed.