Melanoma is one of the most aggressive cancers, with increasing incidence worldwide [
1,
2]. Currently available cytotoxic treatment options produce low rates of patient response and have modest survival impact. Therefore, there is an urgent need for development of more effective therapies that may rely on molecularly targeted individualized treatments. One of the key oncogenic pathways most frequently altered in melanoma is the RAS/BRAF/MEK pathway, thus providing potential promising therapeutic targets [
3‐
7]. Specific inhibitors have been developed, partially investigated
in vitro and some of them entered clinical trials [
8‐
10]. Recent melanoma patient improvement has been observed using targeted therapy or immunotherapy. Indeed, the BRAF inhibitor, vemurafenib, and anti cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody, ipilimumab, demonstrated a survival benefit [
11,
12]. Despite the success of these treatments, most patients eventually progress. In addition, BRAF regulatory loops may circumvent its inhibition, thus Mek, being downstream of BRAF in this key molecular pathway, may represent a highly relevant clinical target [
10,
13,
14]. Currently, thirteen MEK inhibitors, including trametinib, pimasertib, refametinib, PD-0325901, TAK733, MEK162 (ARRY 438162), RO5126766, WX-554, RO4987655 (CH4987655), GDC-0973 (XL518), and AZD8330 have been tested clinically but only trametinib (GSK1120212), a selective inhibitor of MEK 1 and 2, has emerged as the first MEK inhibitor to show favorable clinical efficacy in a phase III trial in BRAF mutated melanoma. It is being evaluated by FDA for the treatment of metastatic melanoma with BRAF V600 mutation. Finally, several clinical trials are currently ongoing using MEK inhibitors in combination with chemotherapeutic drugs (including dacarbazine or paclitaxel). However, schedules and doses of Mek inhibitors compatible with satisfactory antitumor efficacy associated with low systemic toxicity need to be further defined [
15‐
19]. On the other hand, it would be relevant to determine whether the pathway signature of the bulk tumor characterizes also the melanoma initiating cell (MIC) compartment in order to favor potentially more curative MIC-effective molecularly targeted approaches [
20‐
22]. In fact, increasing experimental evidence supports the assertion that many tumors including melanomas, contain Cancer Stem Cells (CSC) or Tumor-Initiating Cells (TIC) and that they affect tumor biology, thus acquiring dramatic clinical relevance [
4,
20,
23]. This course has triggered emerging interest and important studies have been performed in the attempt to understand the nature of MIC. Several putative MIC markers have been identified including CD20, CD133, ABCB5, CD271, JARIDB1, ALDH, however most of these markers have not yet been validated in independent studies [
24‐
35]. Intense debate in this field is on-going and, to date, several controversies surrounding this field remain unsolved, including those concerning the frequency of MIC. [
29,
30,
35‐
38]. Extending beyond the general view that CSC are static entities, recent evidence support a model of dynamic stemness in which tumor maintenance, in some solid tumors, may be a dynamic process mediated by a temporarily distinct sub-population of cells that may transiently acquire stemness properties and continually arise and disappear (“moving target”) depending on the tumor context, with consequent therapeutic implications [
30,
32,
37‐
39]. However, even though their frequency, phenotype and nature still remain controversial issues, the existence of a sub-population of cells with increased tumor-initiating potential in melanomas is not questioned [
40].
We investigated the activation and potential targeting of the MEK pathway, exploiting highly reliable
in vitro and
in vivo pre-clinical models of melanomas based on melanospheres. We isolated the highly tumorigenic cell sub-population from patient metastatic melanomas based on its functional ability to grow indefinitely as melanospheres. We previously proved that this approach efficiently enriches tumorigenic cells
in vitro[
41‐
44]. Given that this strategy did not rely on any prospective cell separation based on putative CSC-markers, it allowed us to overcome the possible bias of selecting cell populations based on the presence of transiently expressed antigens. The availability of exponentially growing melanospheres allowed us to obtain their deep
in vitro validation and develop preclinical therapeutic approaches to target both the more tumorigenic and bulk tumor cell populations
in vitro and
in vivo.