Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance

Abstract

Regardless of the significant improvements in treatment of melanoma, the majority of patients develop resistance whose mechanisms are still not completely understood. Hence, we generated and characterized two melanoma-derived cell lines, primary WM793B and metastatic A375M, with acquired resistance to the RAF inhibitor vemurafenib. The morphology of the resistant primary WM793B melanoma cells showed EMT-like features and exhibited a hybrid phenotype with both epithelial and mesenchymal characteristics. Surprisingly, the vemurafenib-resistant melanoma cells showed a decreased migration ability but also displayed a tendency to collective migration. Signaling pathway analysis revealed the reactivation of MAPK and the activation of the PI3K/AKT pathway depending on the vemurafenib-resistant cell line. The acquired resistance to vemurafenib caused resistance to chemotherapy in primary WM793B melanoma cells. Furthermore, the cell-cycle analysis and altered levels of cell-cycle regulators revealed that resistant cells likely transiently enter into cell cycle arrest at the G0/G1 phase and gain slow-cycling cell features. A decreased level of NME1 and NME2 metastasis suppressor proteins were found in WM793B-resistant primary melanoma, which is possibly the result of vemurafenib-acquired resistance and is one of the causes of increased PI3K/AKT signaling. Further studies are needed to reveal the vemurafenib-dependent negative regulators of NME proteins, their role in PI3K/AKT signaling, and their influence on vemurafenib-resistant melanoma cell characteristics.

Ezrin Inhibition Overcomes Acquired Resistance to Vemurafenib in BRAFV600E-Mutated Colon Cancer and Melanoma Cells In Vitro

“…In vitro observation of the involvement of ezrin in vemurafenib resistance in BRAFV600E-mutated RKO colon cancer cells prompted us to examine whether ezrin plays an important role in acquired resistance to BRAFV600E inhibition in another BRAFV600E-mutated cancer such as melanoma. Previous literature data already provide an evidence of the role of the actin cytoskeleton in mediating adaptive resistance to vemurafenib in BRAFV600E-mutant melanoma cells [ 20 , 21 ]. Knowing that ezrin is involved in the regulation of the actin cytoskeleton organisation, we analysed the expression levels of total ezrin and phospho-ezrin (T567) in vemurafenib-sensitive and vemurafenib-resistant A375 melanoma cells harbouring the BRAFV600E mutation under the baseline culture conditions and after exposure to 0.8 µM vemurafenib (corresponding to the IC50 concentration of vemurafenib measured in sensitive melanoma cells) for 72 h ( Figure 6 ).…”

p53 Family in Resistance to Targeted Therapy of Melanoma

“…Furthermore, reduced levels of TAp73 and ∆Np73 were observed in BRAFi-resistant primary melanoma cells with the activation of the PI3K/AKT pathway, while increased levels of TAp73 and ∆Np73 were detected in resistant metastatic melanoma cells with the re-activation of the MAPK pathway [79]. The BRAFi-resistant primary melanoma cells show features of slow-cycling cells, e.g., mesenchymal morphology, reduced proliferation and migration, increased resistance to chemotherapeutic agents, i.e., cisplatin and etoposide, as well as altered cell cycle profile and levels of cell cycle regulators [106], implying that the specific p53 isoform expression pattern could correlate with specific features of BRAFi-resistant melanoma cells. Interestingly, the slow-cycling cell phenotype, known to be a feature of a targeted therapy-resistant melanoma cells [28,106], can be prevented by the inhibition of p53, thus sensitizing melanoma cells to BRAFi/MEKitargeted therapy [28].…”

“…The BRAFi-resistant primary melanoma cells show features of slow-cycling cells, e.g., mesenchymal morphology, reduced proliferation and migration, increased resistance to chemotherapeutic agents, i.e., cisplatin and etoposide, as well as altered cell cycle profile and levels of cell cycle regulators [106], implying that the specific p53 isoform expression pattern could correlate with specific features of BRAFi-resistant melanoma cells. Interestingly, the slow-cycling cell phenotype, known to be a feature of a targeted therapy-resistant melanoma cells [28,106], can be prevented by the inhibition of p53, thus sensitizing melanoma cells to BRAFi/MEKitargeted therapy [28]. In more detail, it has been shown that the slow-cycling phenotype is driven by non-canonical Wnt signaling via the Wnt5A protein, which stabilizes and utilizes p53.…”

Kisspeptin-mediated improvement of sensitivity to BRAF inhibitors in vemurafenib-resistant melanoma cells

“…Pre-clinical studies carried out in cell lines with primary or acquired resistance as model systems have enabled the dissection of molecular mechanisms that act by sustaining MAPK signaling or parallel signaling networks despite BRAF inhibition. In vitro studies identified different epigenetic, metabolic, and phenotypic reprogramming events associated to resistance, contributing to the definition of the heterogeneous alterations associated with the reactivation of MAPK signaling ( 21 ). In addition, these model systems represent a tool to develop novel drug combinations to improve precision medicine strategies.…”