Metabolic Rewiring toward Oxidative Phosphorylation Disrupts Intrinsic Resistance to Ferroptosis of the Colon Adenocarcinoma Cells

Glutathione peroxidase 4 (GPX4) has been reported as one of the major targets for ferroptosis induction, due to its pivotal role in lipid hydroperoxide removal. However, recent studies pointed toward alternative antioxidant systems in this context, such as the Coenzyme Q-FSP1 pathway. To investigate how effective these alternative pathways are in different cellular contexts, we used human colon adenocarcinoma (CRC) cells, highly resistant to GPX4 inhibition. Data obtained in the study showed that simultaneous pharmacological inhibition of GPX4 and FSP1 strongly compromised the survival of the CRC cells, which was prevented by the ferroptosis inhibitor, ferrostatin-1. Nonetheless, this could not be phenocopied by genetic deletion of FSP1, suggesting the development of resistance to ferroptosis in FSP1-KO CRC cells. Considering that CRC cells are highly glycolytic, we used CRC Warburg-incompetent cells, to investigate the role metabolism plays in this phenomenon. Indeed, the sensitivity to inhibition of both anti-ferroptotic axes (GPx4 and FSP1) was fully revealed in these cells, showing typical features of ferroptosis. Collectively, data indicate that two independent anti-ferroptotic pathways (GPX4-GSH and CoQ10-FSP1) operate within the overall physiological context of cancer cells and in some instances, their inhibition should be coupled with other metabolic modulators, such as inhibitors of glycolysis/Warburg effect

INTRINSIC OR INDUCED AGGRESSIVENESS-LINKED REGULATION OF VEGFC IN MEDULLOBLASTOMA CELL LINES

International audienceMedulloblastoma (MDB) is the most common pediatric malignant brain tumor. MDB is a very heterogeneous disease that consists of four subgroups, each of them with different molecular profiles, metastasis status and clinical outcomes. Treatment of MDB includes surgery, radiotherapy and chemotherapy. It cures 70% of the pa'ents nevertheless with many side effects. Relapse is always fatal. Understanding the mechanisms of this relapse might lead to the development of new targeted treatments. VEGFC and lympha'c markers are the main actors of the metasta'c process in many tumors. We thus meant to determine their role and regula'on in MDB-derived cells. We demonstrated that low-aggressiveness MDB-derived cells, DAOY, expressed high basal amounts of lympha'c marker mRNAs and proteins (VEGFC, PROX1, NRP2). Conversely, highly aggressive cells, HD-MB03, presented low amounts of these markers. X-ray irradia'on treatment of the cells induced a rise in VEGFC, at the mRNA and protein levels. We thus ques'oned the regula'on of lympha'c markers in MDB cells. We observed that in the highly aggressive HD-MB03 cells, VEGFC mRNA amount relies upon a very ac've NF-κB-dependent promoter, but that the mRNA gets intensely degraded, hence the low level of VEGFC mRNA in these cells. Conversely, DAOY cells present low ac'vity of VEGFC promoter and high stability of the synthesized mRNA. The promoter ac'vity is independent from NF-κB. Moreover, while VEGFC is secreted in DAOY cells, it is retained inside the cytoplasm of HD-MB03 cells. Since VEGFC is so precisely regulated in MDB cells, it may play an important part in MDB aggressiveness. HSP 90 VEGFC Claudin DAOY Clone 1 HD-MB03 DAPI DAPI DAPI VEGFC VEGFC VEGFC Merge Merge Merge Intrinsic characterisOcs of MB cell lines DAOY (SHH): High expression of lymphaOc markers and VEGFC > Low proliferaOon and high migraOon rates HDMB-03 (Grp3): High expression of lymphaOc markers and VEGFC > High proliferaOon and low migraOon rates VEGFC has a impact on the proliferaOon of HD-MB03 cells. VEGFC DAOY VEGFC promoter VEGFC VEGFC mRNA HD-MB0

Bevacizumab/docetaxel association is more efficient than docetaxel alone in reducing breast and prostate cancer cell growth: a new paradigm for understanding the therapeutic effect of combined treatment.

Bevacizumab (Bvz), a Vascular Endothelial Growth Factor (VEGF)-targeted humanised monoclonal antibody, provides clinical benefit in combination with docetaxel (DXL), a microtubule-stabilising agent, in the treatment of metastatic breast and prostate cancers. Since VEGF and their receptors are expressed by tumour cells, we hypothesised that Bvz, in addition to its impact on neo-vascularisation, could have an impact on tumour cells and enhance the DXL activity. Hence, we studied the effect of DXL and Bvz on metastatic breast (MDA MB-231) and prostate (PC3) cancer cells lines. Bvz alone did not decrease cell proliferation but in combination with DXL, Bvz enhanced the anti-proliferative activity of DXL. Other anti-angiogenic factors Sunitinib, Sorafenib and Gefitinib enhanced the anti-proliferative effect of DXL. qPCR experiments showed that DXL significantly increased the VEGF and VEGF receptor 2 (VEGF-R2) mRNA levels. Activation of VEGF and VEGF-R2 promoters demonstrated that enhanced mRNA levels are partly due to transcriptional activation. ELISA assays showed that DXL induced accumulation of cytoplasmic VEGF but decreased extracellular levels by 39% (MDA) and 48% (PC3). Cell surface localisation of VEGF-R2 was increased by DXL alone, but decreased after combined treatment of DXL plus Bvz. Abnormal expression of VEGF-R2 was also shown on breast and prostate tumour samples reinforcing the results obtained on cellular models. In conclusion, DXL and Bvz in combination decreased extracellular VEGF and VEGF-R2 levels at the plasma membrane thereby blocking an important growth/survival loop. Thus, the combined therapeutic impact of Bvz and DXL observed in clinical trials is associated with enhanced anti-proliferative activity and inhibition of the vascular network

“The anti-angiogenic compound axitinib demonstrates low toxicity and anti-tumoral effects against medulloblastoma”

Evolution of medulloblastoma (MB) treatments has increased the 5-year overall survival of to more than 70%. However, an increasing number of survivors face severe long-term adverse effects and associated morbidity due to multimodal treatments particularly harsh for the younger patients. Chemotherapeutic compounds inducing less adverse effects are key to improving the care of MB patients. The preclinical relevance of last generation anti-angiogenic compounds deserves to be fully assessed. Among these, axitinib showed the highest selectivity index for MB cells, efficiently reduced the growth rate of experimental tumors and led to less toxicity towards normal cells than did a reference treatment. In vivo, axitinib did not lead to acute toxicity in very young rats and was able to cross the blood brain barrier. Analysis of public databases shows that high expression of axitinib targets are of poor prognosis. Altogether, our results suggest that axitinib is a compelling candidate for MB treatment

Antiangiogenic Compound Axitinib Demonstrates Low Toxicity and Antitumoral Effects against Medulloblastoma

International audienceBackground: Despite the improvement of medulloblastoma (MB) treatments, survivors face severe long-term adverse effects and associated morbidity following multimodal treatments. Moreover, relapses are fatal within a few months. Therefore, chemotherapies inducing fewer adverse effects and/or improving survival at relapse are key for MB patients. Our purpose was to evaluate the last-generation antiangiogenic drugs for their relevance in the therapeutic arsenal of MB. Methods: We screened three EMA- and FDA-approved antiangiogenic compounds (axitinib, cabozantinib and sunitinib) for their ability to reduce cell viability of five MB cell lines and their low toxicity towards two normal cell lines in vitro. Based on this screening, single-agent and combination therapies were designed for in vivo validation. Results: Axitinib, cabozantinib and sunitinib decreased viability of all the tested tumor cells. Although sunitinib was the most efficient in tumor cells, it also impacted normal cells. Therefore, axitinib showed the highest selectivity index for MB cells as compared to normal cells. The compound did not lead to acute toxicity in juvenile rats and crossed the blood–brain barrier. Moreover, axitinib efficiently reduced the growth rate of experimental brain tumors. Analysis of public databases showed that high expression of axitinib targets correlates with poor prognosis. Conclusion: Our results suggest that axitinib is a compelling candidate for MB treatment

Additional file 9: of AKT1 restricts the invasive capacity of head and neck carcinoma cells harboring a constitutively active PI3 kinase activity

Electrical data used to generate the figures. The ECIS measurements of resistance in MΩ at a frequency of 4000 Hz were normalized to the first measurement and plotted in the Graphpad Prism software to generate the traces shown in Figs. 3a-c and 4a. The quantification data were obtained by measuring the mean resistance increase during the cell attachment phase (from 4 to 8 h after cell spreading). (XLSX 140 kb

Additional file 13: of AKT1 restricts the invasive capacity of head and neck carcinoma cells harboring a constitutively active PI3 kinase activity

Figure S4 Analysis of e-cadherin expression and localization by immunofluorescence in CAL27 and Detroit562 cells. Immunostaining of e-cadherin (green) and Alexa555-phalloidin (red) staining of the actin cytoskeleton (F-actin) in CAL27 and Detroit562 cells treated with the pan-AKT inhibitor MK-2206 (MK), Rapamycin (Rapa) or Erlotinib (Erlo). Nuclear DNA was counterstained with Hoechst 33,342 (blue). (PDF 247 kb

A group of novel VEGF splice variants as alternative therapeutic targets in renal cell carcinoma

The efficacy of anti‐angiogenic treatment by targeting VEGF/VEGF receptors in metastatic clear cell renal cell carcinoma (ccRCC) varies from patient to patient. Discovering the reasons behind this variability could lead to the identification of relevant therapeutic targets. Thus, we investigated the novel splice variants of VEGF that are less efficiently inhibited by anti‐VEGF/VEGFR targeting than the conventional isoforms. By in silico analysis, we identified a novel splice acceptor in the last intron of the VEGF gene resulting in an insertion of 23 bp in VEGF mRNA. Such an insertion can shift the open‐reading frame in previously described splice variants of VEGF (VEGFXXX), leading to a change in the C‐terminal part of the VEGF protein. Next, we analysed the expression of these alternatively spliced VEGF new isoforms (VEGFXXX/NF) in normal tissues and in RCC cell lines by qPCR and ELISA, and we investigated the role of VEGF222/NF (equivalent to VEGF165) in physiological and pathological angiogenesis. Our in vitro data demonstrated that recombinant VEGF222/NF stimulated endothelial cell proliferation and vascular permeability by activating VEGFR2. In addition, VEGF222/NF overexpression enhanced proliferation and metastatic properties of RCC cells, whereas downregulation of VEGF222/NF resulted in cell death. We also generated an in vivo model of RCC by implanting RCC cells overexpressing VEGF222/NF in mice, which we treated with polyclonal anti‐VEGFXXX/NF antibodies. VEGF222/NF overexpression enhanced tumour formation with aggressive properties and a fully functional vasculature, while treatment with anti‐VEGFXXX/NF antibodies slowed tumour growth by inhibiting tumour cell proliferation and angiogenesis. In a patient cohort from the NCT00943839 clinical trial, we investigated the relationship between plasmatic VEGFXXX/NF levels, resistance to anti‐VEGFR therapy and survival. High plasmatic VEGFXXX/NF levels correlated with shorter survival and lower efficacy of anti‐angiogenic drugs. Our data confirmed the existence of new VEGF isoforms that could serve as novel therapeutic targets in patients with RCC that are resistant to anti‐VEGFR therapy

Additional file 4: of AKT1 restricts the invasive capacity of head and neck carcinoma cells harboring a constitutively active PI3 kinase activity

CAL33-shControl cells untreated or treated with MK-2206 and CAL33-shAKT1.1 and 1.2 cells electrical resistance measurements. Raw output file of the ECIS measurement of resistance in MΩ at a frequency of 4000 Hz. (XLS 220 kb