Yann Cheli

Microphthalmia-Associated Transcription Factor Controls the DNA Damage Response and a Lineage-Specific Senescence Program in Melanomas

Apoptosis and senescence are cellular failsafe programs that counteract excessive mitogenic signaling observed in cancer cells. Melanoma is known for its notorious resistance to apoptotic processes; therefore, senescence, which remains poorly understood in melanomas, can be viewed as a therapeutic alternative. Microphthalmia-associated transcription factor (MITF), in which its M transcript is specifically expressed in melanocyte cells, plays a critical role in melanoma proliferation, and its specific inhibition is associated with G(0)-G(1) growth arrest. Interestingly, decreased MITF expression has been described in senescent melanocytes, and we have observed an inhibition of MITF expression in melanoma cells exposed to chemotherapeutic drugs that induce their senescence. All these observations thereby question the role of MITF in controlling senescence in melanoma cells. Here, we report that long-term depletion of MITF in melanoma cells triggers a senescence program characterized by typical morphologic and biochemical changes associated with a sustained growth arrest. Further, we show that MITF-silenced cells engage a DNA damage response (DDR) signaling pathway, leading to p53 upregulation, which is critically required for senescence entry. This study uncovers the existence of a lineage-restricted DDR/p53 signaling pathway that is inhibited by MITF to prevent senescence and favor melanoma cell proliferation.

αMSH and Cyclic AMP Elevating Agents Control Melanosome pH through a Protein Kinase A-independent Mechanism

Melanins are synthesized in melanocytes within specialized organelles called melanosomes. Numerous studies have shown that the pH of melanosome plays a key role in the regulation of melanin synthesis. However, until now, acute regulation of melanosome pH by a physiological stimulus has never been demonstrated. In the present study, we show that the activation of the cAMP pathway by αMSH or forskolin leads to an alkalinization of melanosomes and a concomitant regulation of vacuolar ATPases and ion transporters of the solute carrier family. The solute carrier family members include SLC45A2, which is mutated in oculocutaneous albinism type IV, SLC24A4 and SLC24A5, proteins implicated in the control of eye, hair, and skin pigmentation, and the P protein, encoded by the oculocutaneous albinism type II locus. Interestingly, H89, a pharmacological inhibitor of protein kinase A (PKA), prevents the cAMP-induced pigmentation and induces acidification of melanosomes. The drastic depigmenting effect of H89 is not due to an inhibition of tyrosinase expression. Indeed, H89 blocks the induction of melanogenesis induced by LY294002, a potent inhibitor of the PI 3-kinase pathway, without any effect on tyrosinase expression. Furthermore, PKA is not involved in the inhibition of pigmentation promoted by H89 because LY294002 induces pigmentation independently of PKA. Also, other PKA inhibitors do not affect pigmentation. Taken together, our results strengthen the support for a key role of melanosome pH in the regulation of melanin synthesis and, for the first time, demonstrate that melanosome pH is regulated by cAMP and αMSH. Notably, these are both mediators of the response to solar UV radiation, the main physiological stimulus of skin pigmentation.

Alpha-MSH and cyclic-AMP elevating agents control melanosome pH through a PKA-independent mechanism.

Melanins are synthesized in melanocytes within specialized organelles called melanosomes. Numerous studies have shown that the pH of melanosome plays a key role in the regulation of melanin synthesis. However, until now, acute regulation of melanosome pH by a physiological stimulus has never been demonstrated. In the present study, we show that the activation of the cAMP pathway by αMSH or forskolin leads to an alkalinization of melanosomes and a concomitant regulation of vacuolar ATPases and ion transporters of the solute carrier family. The solute carrier family members include SLC45A2, which is mutated in oculocutaneous albinism type IV, SLC24A4 and SLC24A5, proteins implicated in the control of eye, hair, and skin pigmentation, and the P protein, encoded by the oculocutaneous albinism type II locus. Interestingly, H89, a pharmacological inhibitor of protein kinase A (PKA), prevents the cAMP-induced pigmentation and induces acidification of melanosomes. The drastic depigmenting effect of H89 is not due to an inhibition of tyrosinase expression. Indeed, H89 blocks the induction of melanogenesis induced by LY294002, a potent inhibitor of the PI 3-kinase pathway, without any effect on tyrosinase expression. Furthermore, PKA is not involved in the inhibition of pigmentation promoted by H89 because LY294002 induces pigmentation independently of PKA. Also, other PKA inhibitors do not affect pigmentation. Taken together, our results strengthen the support for a key role of melanosome pH in the regulation of melanin synthesis and, for the first time, demonstrate that melanosome pH is regulated by cAMP and αMSH. Notably, these are both mediators of the response to solar UV radiation, the main physiological stimulus of skin pigmentation.

The low-frequency isoform of platelet glycoprotein VIb attenuates ligand-mediated signal transduction but not receptor expression or ligand binding

The 2 most common haplotypes of human GP6, GP6a and GP6b, generate the allelic isoforms glycoprotein VI (GPVI)a and GPVIb that differ by 5 amino acids: S219P, K237E, and T249A in the ectodomains, and Q317L and H322N in the cytoplasmic domain. By quantitative Western blot, we found no association between GP6 genotype and total platelet GPVI content among 132 normal subjects. When expressed as soluble products or as membrane-associated receptors, GPVIa and GPVIb have identical affinities for type I collagen, collagen-related peptide, or convulxin. However, the cytoplasmic domain substitutions in GPVIb have a significant effect on GPVI-dependent subcellular associations and ligand-induced signal transduction. L317 increases binding to calmodulin, whereas N322 attenuates binding to Fyn/Lyn. Consistent with the latter finding, convulxin-induced Syk phosphorylation is significantly attenuated in Dami cells stably transfected with GPVIb, relative to GPVIa. This represents direct evidence that haplotype-related GPVI functional differences are inherent in the cytoplasmic domain substitutions, whereby GPVIb binds less strongly to Fyn/Lyn and attenuates the rate and extent of Syk phosphorylation. These allelic differences in GP6a and GP6b explain functional differences in the respective isoforms, but the molecular basis for the several-fold range in GPVI levels of human platelets remains to be determined.

Increased CD271 expression by the NF-kB pathway promotes melanoma cell survival and drives acquired resistance to BRAF inhibitor vemurafenib

Specific BRAFV600E inhibitors (BRAFi) are highly effective in the treatment of melanoma. However, acquired drug resistances invariably develop after the initial response. Therefore, the identification of new mechanisms of acquired resistance gives important clues towards the development of therapies that could elicit long lasting responses. Here we report that CD271 confers resistance to BRAFi in melanoma cells. The expression of CD271 is increased by BRAFi through a stimulation of tumor necrosis factor-alpha (TNFα) secretion that leads to NF-κB signaling pathway activation. CD271 is upregulated in a subset of BRAFi-resistant melanoma cells. The inhibition of TNFα/NF-κB pathway and CD271 silencing restore the BRAFi sensitivity of resistant melanoma cells. Finally, increase of CD271 expression is validated in BRAFi-resistant xenografts tumors and also in tumors from the patients who relapsed under BRAFi. In summary, these results reveal a novel TNFα/NF-κB/CD271 axis whose activation contributes to the acquisition of resistance to BRAFi and therefore may represent a novel therapeutic target to improve the efficacy of therapy in melanoma.

Effect of multimer size and a natural dimorphism on the binding of convulxin to platelet glycoprotein (GP)VI.

Summary.  Background: Convulxin (CVX), a C‐type lectin from the venom of Crotalus durissusterrificus, is a potent activator of human platelets, binding predominantly to glycoprotein (GP)VI. Native CVX is an octamer composed of four αβ‐heterodimers [(αβ)4]. Two different native sequences have been reported, one bearing lysine (K), the other glutamic acid (E), at β chain residue 89, but the physiological relevance of this difference is unknown. Objective: We used the Drosophila S2 system to express recombinant CVX (rCVX) heterodimers (αβ) and site‐directed mutagenesis to evaluate the influence of multimer size and the substitution βK89E on CVX function. Methods: By flow cytometry, native CVX and both recombinant forms bind to human platelets in whole blood. By surface plasmon resonance (BIAcore, Piscataway, NJ, USA), the calculated equilibrium dissociation constants (KD) were: rCVX αβ89K, 11.3 × 10−8 m; rCVX αβ89E, 9 × 10−8 m; and native CVX, 2.8 × 10−8 m. Results: Thus, the affinities of the two rCVX forms for human, recombinant GPVI are essentially the same, but the relative affinity of native CVX is about 3‐fold higher. The minimum concentration of native CVX that induces maximal human platelet aggregation (70 pm) is roughly 400‐fold lower than that of either rCVX (29 nm). Conclusions: These results are consistent with the hypothesis that the ability of the native CVX octamer to cluster mobile GPVI molecules within the platelet membrane may be the single most important factor that contributes to the efficiency with which CVX is able to induce platelet activation.

Conditional Knockout of Integrin α2β1 in Murine Megakaryocytes Leads to Reduced Mean Platelet Volume

We have engineered a transgenic mouse on a C57BL/6 background that bears a floxed Itga2 gene. The crossing of this mouse strain to transgenic mice expressing Cre recombinase driven by the megakaryocyte (MK)-specific Pf4 promoter permits the conditional knockout of Itga2 in the MK/platelet lineage. Mice lacking MK α2β1 develop normally, are fertile, and like their systemic α2β1 knockout counterparts, exhibit defective adhesion to and aggregation induced by soluble type I collagen and a delayed onset to low dose fibrillar collagen-induced aggregation, results consistent with blockade or loss of platelet α2β1. At the same time, we observed a significant reduction in mean platelet volume, which is consistent with the reported role of α2β1 in MK maturation and proplatelet formation in vivo. This transgenic mouse strain bearing a floxed Itga2 gene will prove valuable to distinguish in vivo the temporal and spatial contributions of α2 integrin in selected cell types.

Enhanced binding of poly(ADP-ribose)polymerase-1 and Ku80/70 to the ITGA2 promoter via an extended cytosine-adenosine repeat.

Background We have identified a cytosine-adenosine (CA) repeat length polymorphism in the 5′-regulatory region of the human integrin α2 gene ITGA2 that begins at −605. Our objective was to establish the contribution of this polymorphism to the regulation of integrin α2β1 expression, which is known to vary several-fold among normal individuals, and to investigate the underlying mechanism(s). Methodology/Principal Findings In combination with the SNP C-52T, previously identified by us as a binding site for the transcription factor Sp1, four ITGA2 haplotypes can be distinguished, in the order in which they enhance ITGA2 transcription: (CA)12/-52C>(CA)11/-52C>(CA)11/-52T>(CA)10/-52T. By DNA affinity chromatography and chromatin immunoprecipitation (ChIP) assays, we show that poly (ADP-ribose)polymerase-1 (PARP-1) and Ku80/70 bind specifically and with enhanced affinity to the longer (CA)12 repeat alleles. Conclusions/Significance The increased binding of PARP-1 and Ku80/70, known components of transcription co-activator complexes, to the longer (CA)12 alleles of ITGA2 coincides with enhanced α2β1 expression. The most likely explanation for these findings is that PARP-1 and Ku80/70 contribute to the transcriptional regulation of ITGA2. These observations provide new insight into the mechanisms(s) underlying haplotype-dependent variability in integrin α2β1 expression in human platelets and other cells.