Corine Bertolotto

Targeting Cancer Cell Metabolism: The Combination of Metformin and 2-Deoxyglucose Induces p53-Dependent Apoptosis in Prostate Cancer Cells

Targeting cancer cell metabolism is a new promising strategy to fight cancer. Metformin, a widely used antidiabetic agent, exerts antitumoral and antiproliferative action. In this study, the addition of metformin to 2-deoxyglucose (2DG) inhibited mitochondrial respiration and glycolysis in prostate cancer cells leading to a severe depletion in ATP. The combination of the two drugs was much more harmful for cancer cells than the treatment with metformin or 2DG alone, leading to 96% inhibition of cell viability in LNCaP prostate cancer cells. In contrast, a moderate effect on cell viability was observed in normal prostate epithelial cells. At the cellular level, the combination of metformin and 2DG induced p53-dependent apoptosis via the energy sensor pathway AMP kinase, and the reexpression of a functional p53 in p53-deficient prostate cancer cells restored caspase-3 activity. In addition to apoptosis, the combination of metformin and 2DG arrested prostate cancer cells in G(2)-M. This G(2)-M arrest was independent of p53 and correlated with a stronger decrease in cell viability than obtained with either drug. Finally, metformin inhibited 2DG-induced autophagy, decreased beclin 1 expression, and triggered a switch from a survival process to cell death. Our study reinforces the growing interest of metabolic perturbators in cancer therapy and highlights the potential use of the combination of metformin and 2DG as an anticancerous treatment.

Up-regulation of MET Expression by α-Melanocyte-stimulating Hormone and MITF Allows Hepatocyte Growth Factor to Protect Melanocytes and Melanoma Cells from Apoptosis

The MET proto-oncogene encodes for the hepatocyte growth factor (HGF) receptor, a plasma membrane tyrosine kinase that is involved in melanocyte growth and melanoma development. In mouse melanoma cells, Met expression is increased by αMSH via the activation of the cAMP pathway. However, the mechanism by which cAMP regulates MET and the biological consequences of this increase were not known. In the present report, we show that αMSH regulates MET expression in both human melanocytes and mouse melanoma cells through a transcriptional mechanism that requires MITF. Furthermore, the adenovirus driven expression of MITF is sufficient to increase MET in melanoma cells. Functional analysis of the MET promoter allows us to identify an E-box motif conserved in both human and mouse promoter that mediates the effect of MITF. Interestingly, up-regulation of MET expression by cAMP leads to an exacerbated HGF signaling and allows HGF to protect melanocytes and melanoma cells from apoptosis. Thus, physiological stimuli or pathological events that would induce MITF expression may lead to increased MET expression thereby favoring melanoma survival. These observations strengthen the roles of MITF and MET in melanoma development.

In Vitro and In Vivo Anti-Melanoma Effects of Ciglitazone

Activation of PPARgamma by synthetic ligands, thiazolidinediones, inhibits the proliferation of cancer cells. In this report, focusing our attention on ciglitazone, we show that ciglitazone inhibits melanoma growth by inducing apoptosis and cell-cycle arrest, whereas normal melanocytes are resistant to ciglitazone. In melanoma cells, ciglitazone-induced apoptosis is associated with caspase activations and a loss of mitochondrial membrane potential. Induction of cell-cycle arrest by ciglitazone is associated with changes in expression of key cell-cycle regulators such as p21, cyclin D1, and pRB hypophosphorylation. Cell-cycle arrest occurs at low ciglitazone concentrations and through a PPARgamma-dependent pathway, whereas the induction of apoptosis is caused by higher ciglitazone concentrations and independently of PPARgamma. These results allow an effective molecular dissociation between proapoptotic effects and growth inhibition evoked by ciglitazone in melanoma cells. Finally, we show that in vivo treatment of nude mice by ciglitazone dramatically inhibits human melanoma xenograft development. The data presented suggest that ciglitazone might be a better candidate for clinical trials in melanoma treatment than the thiazolidinediones currently used in the treatment of type 2 diabetes, such as rosiglitazone, which is devoid of a proapoptotic PPARgamma-independent function.

Aurora B Is Regulated by the Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase (MAPK/ERK) Signaling Pathway and Is a Valuable Potential Target in Melanoma Cells

Background: BRAFV600E melanoma cells develop resistance to vemurafenib. The BRAF/ERK axis controls melanoma cell proliferation. Aurora B is a key actor of mitosis. Results: The BRAF/ERK axis regulates Aurora B. Vemurafenib-resistant melanoma cells are sensitive to Aurora B inhibition. Conclusion: Aurora B is a valuable target in melanoma cells. Significance: Our findings provide insights into Aurora B regulation and on new druggable targets to overcome vemurafenib resistance. Metastatic melanoma is a deadly skin cancer and is resistant to almost all existing treatment. Vemurafenib, which targets the BRAFV600E mutation, is one of the drugs that improves patient outcome, but the patients next develop secondary resistance and a return to cancer. Thus, new therapeutic strategies are needed to treat melanomas and to increase the duration of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitor response. The ERK pathway controls cell proliferation, and Aurora B plays a pivotal role in cell division. Here, we confirm that Aurora B is highly expressed in metastatic melanoma cells and that Aurora B inhibition triggers both senescence-like phenotypes and cell death in melanoma cells. Furthermore, we show that the BRAF/ERK axis controls Aurora B expression at the transcriptional level, likely through the transcription factor FOXM1. Our results provide insight into the mechanism of Aurora B regulation and the first molecular basis of Aurora B regulation in melanoma cells. The inhibition of Aurora B expression that we observed in vemurafenib-sensitive melanoma cells was rescued in cells resistant to this drug. Consistently, these latter cells remain sensitive to the effect of the Aurora B inhibitor. Noteworthy, wild-type BRAF melanoma cells are also sensitive to Aurora B inhibition. Collectively, our findings, showing that Aurora B is a potential target in melanoma cells, particularly in those vemurafenib-resistant, may open new avenues to improve the treatment of metastatic melanoma.

Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance

We have discovered and developed a series of molecules (thiazole benzenesulfonamides). HA15, the lead compound of this series, displayed anti-cancerous activity on all melanoma cells tested, including cells isolated from patients and cells that developed resistance to BRAF inhibitors. Our molecule displayed activity against other liquid and solid tumors. HA15 also exhibited strong efficacy in xenograft mouse models with melanoma cells either sensitive or resistant to BRAF inhibitors. Transcriptomic, proteomic, and biochemical studies identified the chaperone BiP/GRP78/HSPA5 as the specific target of HA15 and demonstrated that the interaction increases ER stress, leading to melanoma cell death by concomitant induction of autophagic and apoptotic mechanisms.

Melanoma: From Melanocyte to Genetic Alterations and Clinical Options

Metastatic melanoma remained for decades without any effective treatment and was thus considered as a paradigm of cancer resistance. Recent progress with understanding of the molecular mechanisms underlying melanoma initiation and progression revealed that melanomas are genetically and phenotypically heterogeneous tumors. This recent progress has allowed for the development of treatment able to improve for the first time the overall disease-free survival of metastatic melanoma patients. However, clinical responses are still either too transient or limited to restricted patient subsets. The complete cure of metastatic melanoma therefore remains a challenge in the clinic. This review aims to present the recent knowledge and discoveries of the molecular mechanisms involved in melanoma pathogenesis and their exploitation into clinic that have recently facilitated bench to bedside advances.

Involvement of FKHRL1 in melanoma cell survival and death

Melanoma is a highly aggressive tumour characterized by a strong resistance to apoptotic stimuli that give rise to a selective advantage for tumour progression and metastasis formation. Therefore, it is of paramount importance to better understand the mechanisms involved in this resistance to apoptosis. In this report, we focused our attention on FKHRL1, a member of the forkhead family of transcription factors, which controls expression of genes involved in cell cycle progression and apoptosis. In melanoma cells, we show that IGF1, which exerts pro‐survival properties, induces the phosphorylation and nuclear exclusion of FKHRL1 in a PI3K/AKT‐dependent pathway. Moreover, we observe that over‐expression of a non‐phosphorylable mutant of FKHRL1 (FKHRL1‐TM), constitutively localized to the nucleus, promotes apoptotic cell death of melanoma cells. Finally, we find that FKHRL1‐TM decreases the expression of survivin, a member of the inhibitor of apoptosis protein and that survivin re‐expression partially rescues the deleterious effects of FKHRL1. Taken together, these findings reveal, in melanoma cells, that endogenous FKHRL1 is a downstream target of the PI3K/AKT pathway and suggest that the phosphorylation of this transcription factor may be involved in the pro‐survival effects of growth factors such as IGF1. On the other hand, forced nuclear localization of FKHRL1 decreases melanoma cell growth and may serve as a therapeutic strategy against melanoma.

Inhibition of Melanogenesis by the Antidiabetic Metformin

Several reports have demonstrated the inhibitory effect of metformin, a widely used drug in the treatment of type 2 diabetes, on the proliferation of many cancers including melanoma. Recently, it has been shown that metformin is able to modulate the cAMP level in the liver. As cAMP has a crucial role in melanin synthesis and skin pigmentation, we investigated the effect of metformin on melanogenesis both in vitro and in vivo. We showed that metformin led to reduced melanin content in melanoma cells and in normal human melanocytes by decreasing cAMP accumulation and cAMP-responsive element-binding protein phosphorylation. This inhibitory effect is correlated with decreased expression of master genes of melanogenesis, microphthalmia-associated transcription factor, tyrosinase, dopachrome tautomerase, and tyrosinase-related protein 1. Furthermore, we demonstrated that the antimelanogenic effect of metformin is independent of the AMPK pathway. Interestingly, topical application of metformin induced tail whitening in mice. Finally, we confirmed the antimelanogenic effect of metformin on reconstituted human epidermis and on human skin biopsies. These data emphasize the depigmenting effect of metformin and suggest a clinical strategy for using metformin in the topical treatment of hyperpigmentation disorders.

A germline oncogenic MITF mutation and tumor susceptibility.

MITF (Microphthalmia-associated transcription factor) is a lineage specific transcription factor that plays a critical role in melanocyte homeostasis and whose deregulation has been shown to contribute to melanoma disease. A germline mutation in MITF, impairing SUMOylation and predisposing to cutaneous malignant melanoma, was recently identified. Interestingly, an association of the MITF mutation with coexisting melanoma and renal cell carcinoma was also shown. Collectively, these data suggest that MITF has an important oncogenic function in tumorigenesis of multiple tissues/melanocytes and kidney cells.