Farnesyl transferase inhibitors (FTIs) inhibit the farnesylation of proteins, including RAS and RHEB (Ras homolog enriched in brain). RAS signals to the RAF–MEK–ERK (MAPK) and PI3K–AKT–mTOR (AKT) ...signaling pathways, which have a major role in melanoma progression. RHEB positively regulates mammalian target of rapamycin (mTOR). We investigated the effects of the FTI lonafarnib alone and in combination with MAPK (mitogen-activated protein kinase) or AKT (acutely transforming retrovirus AKT8 in rodent T-cell lymphoma) pathway inhibitors on proliferation, survival, and invasive tumor growth of melanoma cells. Lonafarnib alone did not sufficiently inhibit melanoma cell growth. Combinations of lonafarnib with AKT pathway inhibitors did not significantly increase melanoma cell growth inhibition. In contrast, combinations of lonafarnib with MAPK pathway inhibitors yielded additional growth-inhibiting effects. In particular, the combination of the FTI lonafarnib with the pan-RAF inhibitor sorafenib synergistically inhibited melanoma cell growth, significantly enhanced sorafenib-induced apoptosis, and completely suppressed invasive tumor growth in monolayer and organotypic cultures, respectively. Apoptosis induction was associated with upregulation of the endoplasmic reticulum stress-related transcription factors p8 and CHOP (CAAT/enhancer binding protein (C/EBP) homologous protein), and downregulation of the antiapoptotic Bcl-2 (B-cell lymphoma-2) family protein Mcl-1(myeloid cell leukemia 1). Lonafarnib did not affect MAPK and AKT but did affect mTOR signaling. Together, these findings suggest that the FTI lonafarnib inhibits mTOR signaling and enforces sorafenib-induced apoptosis in melanoma cells and may therefore represent an effective alternative for melanoma treatment.
High-dose ascorbate paradoxically acts as a pro-oxidant causing the formation of hydrogen peroxide in an oxygen dependent manner. Tumor cells (in particular melanoma cells) show an increased ...vulnerability to ascorbate induced reactive oxygen species (ROS). Therefore, high-dose ascorbate is a promising pharmacological approach to treating refractory melanomas, e.g., with secondary resistance to targeted BRAF inhibitor therapy. BRAF mutated melanoma cells were treated with ascorbate alone or in combination with the BRAF inhibitor vemurafenib. Viability, cell cycle, ROS production, and the protein levels of phospho-ERK1/2, GLUT-1 and HIF-1α were analyzed. To investigate the treatment in vivo, C57BL/6NCrl mice were subcutaneously injected with D4M.3A (Braf
) melanoma cells and treated with intraperitoneal injections of ascorbate with or without vemurafenib. BRAF mutated melanoma cell lines either sensitive or resistant to vemurafenib were susceptible to the induction of cell death by pharmacological ascorbate. Treatment of Braf
melanoma bearing mice with ascorbate resulted in plasma levels in the pharmacologically active range and significantly improved the therapeutic effect of vemurafenib. We conclude that intravenous high-dose ascorbate will be beneficial for melanoma patients by interfering with the tumor's energy metabolism and can be safely combined with standard melanoma therapies such as BRAF inhibitors without pharmacological interference.
Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 ...mutations. Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK- or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes. Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS.
In this study we show that β-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi. We further demonstrate that β-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo. Unexpectedly, β-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of β-catenin in melanoma progression and therapy resistance. We further demonstrate that β-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi.
Scheme of the change of interacting proteins with β-catenin during the acquisition of resistance to BRAFi from TCF/LEF family members to novel interactants like Stat3. Binding of ligands to different receptor tyrosine kinases like c-met and Egfr family members activate the MAPK signaling pathway and PI3K signaling pathway independently of mutated BRAF. Additional MAPK signaling reactivating mechanisms like the acquisition of novel mutations (asterisk), the formation of Raf heterodimers to the BRAFi. In this environment a switch in the interaction partners of β-catenin to Stat3 occurs further enhancing resistance and contributing to the relapse of the tumor. Dotted lines indicated weak activating potential or indirect mechanisms. Display omitted
•Melanoma cells with acquired resistance frequently express high amounts of β-catenin.•In these cells a hyper-activation of Stat3 co-occurs leading to an interaction with β-catenin.•Such BRAFi resistant melanoma cells are sensitive to knockdown of β-catenin and Stat3 or pharmacologic Stat3 inhibition.
Treatment with BRAF inhibitors is the basis of the standard therapy for melanoma patients with BRAFV600 mutated metastases. Although this therapy achieves impressive short-term benefit, many patients suffer from relapse after several months of treatment even if the therapy is combined with MEK inhibitors. The development of therapy resistant tumor cells is a multifactorial transformation and several cellular mechanisms are already described. Here, we found an interaction of two well-known tumor-associated proteins, namely β-catenin and Stat3 in resistant metastatic melanoma cells. Patient data support that these proteins are involved in the resistance to BRAF inhibitor therapy.
Despite recent progress in melanoma therapy via inhibition of activated oncogenes or immune stimulation, most stage IV melanoma patients still have limited survival times. Existing therapeutic ...approaches eventually fail to prevent further invasion and metastasis, which is driven by a morphological process termed epithelial-mesenchymal transition (EMT). We previously demonstrated that inhibition of EMT in melanoma cells via antagonizing the bone morphogenetic protein (BMP)-pathway abrogated EMT and neural crest migration of melanoma cells in chick embryos. Here, we show that BMP-2 is highly expressed in invasive melanoma cells and is elevated in the serum of stage IV melanoma patients compared to stage IB-IIC patients and healthy controls. Highly BMP-2-expressing melanoma cells display enhanced invasion in the rhombencephalon of the chick embryo. In addition to driving neural crest migration in the zebrafish embryo, the agonists BMP-2, BMP-7 and nodal induce EMT/invasion in radial growth phase melanoma cells and in human melanocytes in skin reconstructs. Blocking either BMP or nodal signaling by antagonists (noggin, lefty), or the Alk4/5/7-receptor inhibitor SB431542, decreases EMT and invasion of melanoma cells in human epidermal skin reconstructs. Together, our data suggest that inhibition of EMT-inducing pathways in melanoma might be a therapeutic approach to attenuate melanoma cell invasiveness.
Y-box binding protein 1 (YB-1) is a multifunctional protein involved in various cellular processes including both transcriptional and translational regulation of target gene expression. Significantly ...increased YB-1 levels have been reported in a number of human malignancies and shown to be associated with poor prognosis and disease recurrence. Indeed, YB-1 can act as a versatile oncoprotein playing an important role in tumour cell proliferation and progression. Consequently, YB-1 not only proves to be a good prognostic tumour marker, but also may be a promising emerging molecular target for the development of new therapeutical strategies. In this review, we discuss both the role of YB-1 in cancer and specifically in malignant melanoma as well as possible translations into the clinics derived thereof.
Abstract Mitogen-activated protein kinase (MAPK) pathway has an important role in normal cells and can be activated under physiological conditions. MAPK pathway activation is a fundamental step in ...several intracellular processes requiring a sequential phosphorylation of the different pathway components. In normal cells, when MAPK pathway activation occurs, it leads to cell growth and differentiation. In order to prevent persistent MAPK pathway activation, physiological upstream negative feedback also takes place. In cells harbouring BRAFV600 mutations, the process leading to MAPK pathway activation is different, and the negative physiological feedback does not exist thus leading to permanent MAPK pathway activation, which ultimately can lead to uncontrolled proliferation. Targeted therapy with rapidly accelerated fibrosarcoma – B (BRAF) and/or mitogen-activated extracellular signal-regulated kinase kinase (MEK) inhibitors is indicated in patients with metastatic melanoma harboring BRAFV600 mutations. However, several different resistance mechanisms to this therapy were identified. In this review, we focus on primary or intrinsic resistance mechanisms to BRAF and MEK inhibition. In this setting, although a BRAF mutation is identified, there is no response to treatment with either BRAF or MEK inhibitor.
Abstract BRAF mutation can be identified in about 45% of the patients with metastatic melanoma. In these patients, BRAF and MEK inhibitors are able to induce rapid responses and to prolong survival. ...However, a significant percentage of patients will develop resistance to targeted therapy and will have progressive disease. MAPK pathway is the most important pathway involved in BRAF/MEK inhibition resistance, particularly MAPK pathway reactivation. Resistance mechanisms can be classified as 1) primary or intrinsic characterised by no response to therapy, 2) secondary or acquired with MAPK pathway reactivation after a time of tumour regression and 3) as adaptive with initial response and early resistance. BRAF inhibition also alters the immune response. Several publications have described immune effects of BRAF inhibition in melanoma tumours, showing that combining targeted and immunotherapy can improve response, despite a possible cross-resistance. Here, we continue the review on resistance mechanisms to BRAF/MEK inhibition and focus on the secondary and adaptive mechanisms.
Immune checkpoint inhibitors (ICIs) like cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA4) and programmed death cell protein 1 (anti-PD1) have revolutionized cancer treatment. As ICI use ...becomes widespread, more immune-related adverse events (irAE's) are being reported. Our aim was to investigate the frequency and nature of new irAE's as well as report the frequency of flare-ups of pre-existing autoimmune conditions occurring after ICI therapy.We performed a retrospective chart review of all patients treated for cancer with anti-PD1 or anti-CTLA4 or combination therapy at our tertiary care center from January 2014 to April 2016. Demographic data, cancer type and stage, irAE's (new immune disorders and disease flares of pre-existing autoimmune disorders on ICI therapy), and drug treatment information were extracted.We identified 220 patients treated with ICI therapy during the study period out of which 27% (60/220) developed irAE's. 11% in anti-CTLA4 group and 16% among anti-PD1 treated patients developed irAE's. IrAE's resulted in discontinuation of cancer therapy in 28% of those who developed irAE's. 21.4% had a flare of their autoimmune disease but only 1 required discontinuation of immunotherapy.IrAE's are an important emerging clinical disease entity for specialists to be aware of. Our study shows that ICI's can be safely used in patients with pre-existing autoimmune conditions with close monitoring. However, there is still a large unmet need to have a better understanding of how to systematically evaluate and manage patients with irAE's as well as for identifying the predictors of irAE's.
Recent advances in melanoma therapy have significantly improved the prognosis of metastasized melanoma. However, large therapeutic gaps remain that need to be closed by new strategies. Antiapoptotic ...Bcl-2 proteins critically contribute to apoptosis deficiency and therapy resistance. They can be targeted by BH3 mimetics, small molecule antagonists that mimic the Bcl-2 homology domain 3 (BH3) of proapoptotic BH3-only proteins. By applying in vitro experiments, we aimed to obtain an overview of the possible suitability of BH3 mimetics for future melanoma therapy. Thus, we investigated the effects of ABT-737 and ABT-263, which target Bcl-2, Bcl-xsub.L and Bcl-w as well as the Bcl-2-selective ABT-199 and the Mcl-1-selective S63845, in a panel of four BRAF-mutated and BRAF-WT melanoma cell lines. None of the inhibitors showed significant effectiveness when used alone; however, combination of S63845 with each one of the three ABTs almost completely abolished melanoma cell survival and induced apoptosis in up to 50–90% of the cells. Special emphasis was placed here on the understanding of the downstream pathways involved, which may allow improved applications of these strategies. Thus, cell death induction was correlated with caspase activation, loss of mitochondrial membrane potential, phosphorylation of histone H2AX, and ROS production. Caspase dependency was demonstrated by a caspase inhibitor, which blocked all effects. Upregulation of Mcl-1, induced by S63845 itself, as reported previously, was blocked by the combinations. Indeed, Mcl-1, as well as XIAP (X-linked inhibitor of apoptosis), were strongly downregulated by combination treatments. These findings demonstrate that melanoma cells can be efficiently targeted by BH3 mimetics, but the right combinations have to be selected. The observed pronounced activation of apoptosis pathways demonstrates the decisive role of apoptosis in the loss of cell viability by BH3 mimetics.
PDF
