4 research outputs found
Genetic Disruption of the γ-Glutamylcysteine Ligase in PDAC Cells Induces Ferroptosis-Independent Cell Death In Vitro without Affecting In Vivo Tumor Growth
The conceptualization of a novel type of cell death, called ferroptosis, opens new avenues for the development of more efficient anti-cancer therapeutics. In this context, a full understanding of the ferroptotic pathways, the players involved, their precise role, and dispensability is prerequisite. Here, we focused on the importance of glutathione (GSH) for ferroptosis prevention in pancreatic ductal adenocarcinoma (PDAC) cells. We genetically deleted a unique, rate-limiting enzyme for GSH biosynthesis, γ-glutamylcysteine ligase (GCL), which plays a key role in tumor cell proliferation and survival. Surprisingly, although glutathione peroxidase 4 (GPx4) has been described as a guardian of ferroptosis, depletion of its substrate (GSH) led preferentially to apoptotic cell death, while classical ferroptotic markers (lipid hydroperoxides) have not been observed. Furthermore, the sensitivity of PDAC cells to the pharmacological/genetic inhibition of GPx4 revealed GSH dispensability in this context. To the best of our knowledge, this is the first time that the complete dissection of the xCT-GSH-GPx4 axis in PDAC cells has been investigated in great detail. Collectively, our results revealed the necessary role of GSH in the overall redox homeostasis of PDAC cells, as well as the dispensability of this redox-active molecule for a specific, antioxidant branch dedicated to ferroptosis prevention
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
An Evolution-Guided Analysis Reveals a Multi-Signaling Regulation of Fas by Tyrosine Phosphorylation and its Implication in Human Cancers
International audienceDemonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive ``Fas apoptosis'' to ``Fas multisignals'' paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolutionguided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases