<p>Insulin Reduces the Efficacy of Vemurafenib and Trametinib in Melanoma Cells</p>

Abstract

Background: Despite the progress made in the clinical management of metastatic melanoma, a patient's response to treatment cannot be fully predicted, and intrinsic or acquired resistance that is developed in most melanoma patients warrants further research efforts. In addition to genetic factors, microenvironmental input should be considered to explain the diversity of response to treatment among melanoma patients. In this study, we evaluated the impact of insulin on patient-derived BRAF V600E melanoma cells, either untreated or treated with vemurafenib or trametinib, inhibitors of BRAF V600 and MEK1/2, respectively. Methods: Cells were cultured in serum-free conditions, either with or without insulin. The activity of the MAPK/ERK and PI3K/AKT pathways was assessed by Western blotting, cell viability, and percentages of Ki-67-and NGFR-positive cells by flow cytometry. Transcript levels were analyzed using qRT-PCR, and γ-H2AX levels by immunoblotting and confocal microscopy. A luminescence-based assay was used to measure glutathione content. Results: While insulin did not influence the MAPK/ERK pathway activity, it had a strong influence on melanoma cells, in which this pathway was suppressed by either vemurafenib or trametinib. In the presence of insulin, both drugs were much less efficient in 1) inhibiting proliferation and reducing the percentage of Ki-67-positive cells, and 2) inducing apoptosis and phosphorylation of histone H2AX in melanoma cells. Changes induced by vemurafenib and trametinib in glutathione homeostasis and DNA repair gene expression were also attenuated by insulin. Moreover, insulin impaired the combined effects of targeted drugs and doxorubicin in melanoma cells. In addition to insulin-induced PI3K/AKT activity, which was either transient or sustainable depending on the cell line, an insulin-triggered increase in the percentage of cells expressing NGFR, a marker of neural crest stem-like cells, may contribute to the reduced drug efficacy. Conclusion: Our results demonstrate the role of insulin in reducing the efficacy of vemurafenib and trametinib. This needs clinical assessment.

Targeting Genome Stability in Melanoma—A New Approach to an Old Field

“…Clinical reports indicate that MGMT promoter is methylated, and thus suppressed, in about 21.5–35% of metastatic melanomas [ 119 , 121 , 122 , 123 ], which entails that inhibition of BRAF, MEK and HDAC could potentially benefit a large number of melanoma patients. Compatible with findings by Maertens and colleagues, our group has recently reported that vemurafenib and trametinib reduced the expression of BRIP1, BRCA1 and BRCA2 in 4 out of 5 patient-derived BRAF V600E cell lines [ 42 ]. In addition, we found that insulin modulated the response of melanoma cells to MAPK inhibition by attenuating drug-induced reduction in the expression of DNA repair genes, deregulation of glutathione homeostasis and increase in DNA damage, which altogether led to substantially reduced cytotoxicity of vemurafenib and trametinib [ 42 ].…”

“…Compatible with findings by Maertens and colleagues, our group has recently reported that vemurafenib and trametinib reduced the expression of BRIP1, BRCA1 and BRCA2 in 4 out of 5 patient-derived BRAF V600E cell lines [ 42 ]. In addition, we found that insulin modulated the response of melanoma cells to MAPK inhibition by attenuating drug-induced reduction in the expression of DNA repair genes, deregulation of glutathione homeostasis and increase in DNA damage, which altogether led to substantially reduced cytotoxicity of vemurafenib and trametinib [ 42 ]. The role of MAPK-targeted therapeutics in deregulation of DNA repair was also confirmed in other cancer types: BRAF V600E inhibition using vemurafenib was shown to suppress double strand break repair and sensitize thyroid cancer cells to ionizing radiation [ 124 ], and MEK inhibition using trametinib induced similar effects in pancreatic cancer cells [ 125 ].…”

“…While treatment of melanoma patients has been revolutionized with molecularly targeted therapeutics against the most frequently altered signaling cascade in melanoma—the RAS/RAF/MEK/ERK (MAPK) pathway, the majority of patients relapse within months [ 35 , 36 , 37 , 38 ]. Several lines of investigation indicate that treatment of melanoma cells with MAPK inhibitors uncovers DNA damage-associated vulnerabilities in melanoma cells that could be exploited therapeutically [ 39 , 40 , 41 , 42 ]. Furthermore, it is becoming increasingly recognized that the efficacy of immunotherapy can be substantially improved by disrupting genome integrity in melanoma cells [ 43 , 44 , 45 , 46 ].…”

Mechanisms of Melanoma Progression and Treatment Resistance: Role of Cancer Stem-like Cells

“…The main cause of this noted poor prognosis is attributed to BRAF inhibitor-mediated compensatory mechanisms. These compensatory mechanisms include elevation of ARAF and CRAF proteins, a mechanism through which melanoma cells sustain the activation of MAPK activity during BRAF inhibitors treatment [206][207][208]. Although BRAF inhibitor-resistant cells remain sensitive to MEK inhibitors, the resistance of these melanoma cells to various structurally different MEK inhibitors can be mediated by the generation of the compensatory mechanism via activation of RTKs such as IGF-1R [205][206][207][208].…”

“…Like other cells with a melanocyte origin, melanoma cells express IGF-1R. Importantly, MEK inhibitor-resistant melanoma cells demonstrate higher surface levels of IGF-1R when compared to BRAF-sensitive melanoma cells [205][206][207][208]. As such, IGF-1R is known to play an important role in tumor progression and drug resistance [209].…”