Original ArticleEverolimus selectively targets vemurafenib resistant BRAFV600E melanoma cells adapted to low pH
Introduction
Melanoma is a very aggressive skin cancer and its incidence has dramatically increased during the last decades. More than ∼50% of melanoma is characterized by point mutations of BRAF (V-raf murine sarcoma viral oncogene homolog B1) that is a regulator of the MAPK pathway and the most common mutation displays a valine to glutamic acid substitution (V600E) causing constitutive kinase activation [1]. The discovery of V600E point mutation encouraged the generation of compounds specifically targeting this hyperactive mutated kinase. One of these compounds, vemurafenib (also known as PLX4032) an orally ATP-competitive small-molecule inhibitor, received FDA approval for the treatment of late-stage melanoma on August 17, 2011.
Vemurafenib selectively inhibits the ATP-binding site of BRAFV600E kinase resulting in an inhibition of over-activated MAPK signaling with reduction of tumor cell proliferation. Despite the initial benefits due to the treatment with vemurafenib, almost all patients develop drug resistance after weeks to months of therapy [2], [3], [4], [5]. Documented mechanisms of BRAF inhibitor resistance are under study and up-to-now include MAPK signaling reactivation through expression of alternative splicing forms of BRAFV600E, amplification of BRAFV600E, mutations in NRAS or MEK (MAP2K1), or loss of NF1 [6], [7], [8], [9], [10]. Another mechanism of resistance includes the over expression of COT (MAP3K8) that leads to ERK activation regardless of RAF signaling [11]. Also, the activation of the PI3K/AKT pathway in presence or in absence of MAPK reactivation drives vemurafenib resistance and this activation can be mediated by phosphatase and PTEN loss [12], or by the increase of RTK signaling [8]. Lastly platelet-derived growth factor receptor beta (PDGFRβ) and the insulin-like growth factor receptor (IGF1R) can be involved in the development of drug resistance [8], [13].
Here, we proposed that a particular aspect of tumor microenvironment, that is a low extracellular pH, might participate to promote vemurafenib resistance in BRAFV600E melanoma cells.
Indeed solid tumors express acidic regions and that acidosis correlates with a poor prognosis and recurrence [14]. We know that tumor cells, even when there is enough O2 to support mitochondrial function, use the so-called “aerobic glycolysis” which leads to the conversion of one molecule of glucose into 2 molecules of lactic acid and 2 H+ to produce 2 ATP, compared to the 36 ATP produced by aerobic metabolism [15], [16]. Aerobic glycolysis up-regulation in malignant phenotype, that is mostly due to stable genetic or epigenetic changes [15], is important for proliferating tumor cells since glucose can be converted to macromolecules (acetyl-CoA, glycolytic intermediates and ribose, fatty acids, nonessential amino acids and nucleotide biosynthesis) to be used for proliferation [17]. When oxygen tension reduces and a hypoxic microenvironment develops, stabilization of HIF-1α may regulate an elevated number of metabolic genes driving in tumor cells an anaerobic glycolysis pathway. So lactate and protons are produced in large excess either by aerobic and anaerobic glycolysis, and transported outside by a redundant families of lactate and H+ transporters in order to maintain an intracellular pH compatible with survival and/or proliferation. Therefore, the extracellular pH of tumors turns to acidic and this acidity is also sustained by a poor blood perfusion and limited lymphatic vessels [18].
Acidosis is also linked with a multidrug resistance phenotype, so here we show that an acid microenvironment (pH 6.7 ± 0.1) may drive a vemurafenib resistance in melanoma cells carrying V600E mutation, disclosing a new subset of melanoma cells able to undergo tumor relapse after drug treatment. In addition, we have identified that resistance of acidic BRAFV600E melanoma cells may be overcome by everolimus treatment, a drug acting on mTOR activity.
Section snippets
Cell lines and culture conditions
In this study, we used the melanoma cell line A375M6, isolated in our laboratory from lung metastasis of SCID bg/bg mice i.v. injected with A375 human melanoma cell lines, obtained from American Type Culture Collection (ATCC, Rockville, MD). In some experiments we used also the human melanoma cell lines WM266-4 (from ATCC), SkMel28 (from ATCC) and M21 (kindly provided by Dr. Antony Montgomery, The Scripps Research Institute, La Jolla, CA). Melanoma cells were cultivated in Dulbecco's Modified
Features acquired by BRAFV600E melanoma cells exposed to a transient or chronic low pH that are relevant for a resistant phenotype
Vemurafenib is a RAF inhibitor approved by the US Food and Drug Administration for the treatment of BRAF-mutant melanoma. Before determining whether transient- or chronic-exposed melanoma cells express a resistance to vemurafenib, we evaluated some characteristics of their phenotype conferring a profile compatible with an higher resistance. This study was also helpful to verify differences in behavior between melanoma cells exposed for 24 h to an acidic medium (pH 6.7 ± 0.1) (transient
Discussion
Acidosis of tumor microenvironment is a common characteristic of most solid tumors as a consequence of an altered metabolism, reduced vasculogenesis and high interstitial pressure. Tumor cells exposed to low pH develop a phenotype conferring additional advantages over tumor cells grown in standard pH, such as an EMT program characterized by a higher level of invasiveness, resistance to apoptosis and organ colonization [28]. In addition, an acidic microenvironment stimulates in melanoma cells a
Funding
This work was supported by the “Istituto Toscano Tumori” (ITT) and “Ente Cassa di Risparmio Firenze” (ECRF).
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RJ and PS contributed equally to this manuscript.