Introduction
Cutaneous melanoma is a tumor arising from epidermal melanocytes. It particularly affects young patients as it is the third most frequent cancer in the age range of 20–39 years (1). Although melanoma accounts for only 4% of all skin cancers, it is responsible for 80% of deaths (2). The survival rate at 10 years for patients with metastatic melanoma is <10% (2). During the two last decades, the incidence of melanoma as well as the associated-mortality have strongly increased (3), transforming this disease into a real public health problem.
With the increase in public awareness campaigns, the diagnosis of melanoma is being diagnosed at an earlier stage when the disease is still curable by surgery. At this stage, the survival rate at 5 years is 90% (4). On the other hand, metastatic melanoma is an incurable disease. Multimodality treatments are necessary. Historically, chemotherapy (dacarbazine, cisplatin) and immunotherapy (interferon α-2b and interleukin-2) were for a long time the mainstay of treatment. Dacarbazine was the only FDA approved cytotoxic agent even though it produced low response rates. Interferon and interleukin have showed impressive antitumoral activity with a number of complete responses but in a very small subset of patients. The landscape of the treatment of metastatic melanoma has changed recently with the emergence of new immuno-modulatory agents such as anti-CTLA-4, anti-PD1, anti-PD-L1 monoclonal antibodies (5,6) and pathway inhibitors such as BRAF and MEK inhibitors (7–9).
The description of activating BRAF gene mutations in 50–60% of melanomas (10) opened a new therapeutic perspective. Indeed, treatment with the specific V600EBRAF inhibitor vemurafenib resulted in spectacular tumor regressions (11), improved rates of overall and progression-free survival compared to dacarbazine (12) and induced clinical response in >50% of metastatic melanoma patients harbouring the BRAF mutation (median overall survival was ~16 months) (7). Nevertheless, in spite of impressive initial responses, selection and/or resistance developed in many cases due to exogenous growth factors and cytokines (13), switches between pathways (14), COT/MAP3K8 activation (15), appearance of new activation mutations in C121SMEK1 (16), dimerization of aberrantly spliced V600EBRAF (17) or prevalence of wild-type cells in the tumors (18).
Besides BRAF, several other mutated or upregulated proteins involved in major signalling pathways may be considered as additional potential targets in cutaneous melanoma. First, the RAS/RAF/MEK/ERK signalling pathway is a major target for therapy as it is hyper-activated in ~75% of metastatic tumors. Indeed, NRAS activated mutations are found in 15–25% of cases with the remaining being BRAF mutated, both mutations being mutually exclusive (10). Second, PI3K/AKT/mTOR signalling is another important pathway in melanoma because of its activation through the loss of the expression of the tumor suppressor PTEN (30–50%) or the amplification of AKT (60%) (19). Interestingly, genetic interaction between NRAS and BRAF mutations and PTEN inactivation has been reported, (20) suggesting possible cooperation between both MAPK and PI3K/AKT activations in melanoma development (21). Finally, STAT3 is a point of convergence for many tyrosine kinases such as JAK and SRC. It is constitutively activated in a majority of human melanoma cell lines and tumors where it plays a key role in growth and survival (22). Moreover, its activation has been reported as a compensatory mechanism allowing cell survival and contributing to resistance to targeted drugs such as SRC inhibitors (23).
In light of the recent data on targeted therapy and keeping in mind the identified crosstalk between different signalling pathways in melanoma, it was evident that more effective treatments need concomitant inhibition of multiple pathways. The choice and modalities of such combinations are currently investigated both in in vitro models and in clinical trials. Our working hypothesis is based on a particular approach by which a sustained inhibition of a given signalling pathway renders cells dependent on other compensatory pathways for their proliferation and survival. The identification and subsequent targeting of the latter as well could substantially potentiate cell toxicity. Hence, we induced changes in signalling profiles in three representative melanoma cell lines (wild-type or mutated in NRAS or BRAF) by using a sequential exposure first to non-toxic (<10% toxicity) but effective (substantial inhibition of targeted kinase) concentrations of various protein kinase inhibitors targeting MEK, PI3K, SRC or JAK and second, while maintaining these inhibitions, to adequate protein kinase inhibitors targeting the activated compensatory signalling pathways. The results have been systematically compared to each effector used alone or in simultaneous combination with the others.
Materials and methods
Inhibitors
Four specific inhibitors of protein kinases has been used: the SRC family inhibitor PP2 (IC50 = ~5 nM, 50% of kinase activity inhibition), the MEK1/2 inhibitor U0126 (IC50 = ~0.1 μM), the PI3K inhibitor LY294002 referred as LY29 (IC50 = ~1 μM) (all from Tocris Bioscience, Bristol, UK) and the JAK Inhibitor I pyridone 6 referred as PYR (IC50 = ~5 nM) (Calbiochem, Darmstadt, Germany).
Melanoma cell lines
Human melanoma cell lines were established in our laboratory from lymph node metastases. HBL cells (LOCE-MM001) are wild-type for NRAS and BRAF, LOCE-MM057 present the Q61L NRAS mutation and LOCE-MM074 are bearing the V600E BRAF mutation.
Cell culture
Cells were cultured at 37°C in a humidified 95% air and 5% carbon dioxide atmosphere. For routine maintenance, cells were propagated in 175 cm2-flasks containing HAM-F10 medium supplemented with 5% heat-inactivated fetal calf serum and 5% heat-inactivated newborn calf serum and with L-glutamine, penicillin and streptomycin at standard concentrations (all from Gibco, Invitrogen, UK). Cells were harvested by trypsinization (0.05% trypsin - EDTA) (Gibco) and subcultured twice weekly. One day after seeding, the culture medium was replaced by fresh medium. All lines are routinely checked for mycoplasma contamination using MycoAlert™ Mycoplasma Detection kit (Lonza, Basel, Switzerland).
Experimental conditions
Experimental conditions are presented in Fig. 1 as follows: condition 1 (cell exposure to one single inhibitor), cells were exposed to increasing concentrations (from 10−12 to 10−4 M) of each of the protein kinase inhibitors for 3 days; condition 2 (concomitant exposure to two inhibitors), cells were incubated with a given concentration of an inhibitor and co-treated with increasing concentrations of another for 3 days; condition 3 (sequential exposure), cells were exposed to each of the inhibitors for 7 days (medium and inhibitor renewal at days 1, 4 and 7) and, while maintaining this pre-treatment, cells were incubated for 3 additional days with increasing concentrations of each of the other inhibitors.
Proliferation assay
All cells were seeded in 96-well plates (8,000 cells/well) containing HAM-F10 medium supplemented or not with inhibitors. One day after plating, the culture medium was replaced by a fresh one containing inhibitors depending on experimental conditions (Fig. 1) and further cultured for 3 additional days. Cell proliferation was assessed by crystal violet assay. Briefly, culture medium was removed and cells were gently rinsed with phosphate-buffered saline (PBS), fixed with 1% glutaraldehyde/PBS for 15 min and stained with 0.1% (w/v in water) crystal violet for 30 min. Cells were destained under running tap water and subsequently lysed with 0.2% (v/v in water) Triton X-100 for 90 min. The absorbance was measured at 570 nm using a Multiskan EX Microplate Photometer (Thermo Scientific, Courtaboeuf Cedex, France). On each plate, blank wells containing medium alone were used for background subtraction and untreated (control) cells were cultured in parallel to treated cells.
Western blot analysis
Expression and/or phosphorylation levels of key proteins of targeted signalling pathways were determined by western blotting in untreated cells and cells exposed to inhibitors for 30 min or 10 days. All cells were plated in Petri dishes (3×106 cells/dish) containing HAM-F10 medium and cultured according to the experimental conditions outlined in Fig. 1. Cells were then lysed using detergent cocktail (M-PER mammalian extraction buffer) supplemented with protease inhibitors (Halt protease inhibitor cocktail) and phosphatase inhibitors (Halt phosphatase inhibitor cocktail) (all from Pierce, Rockford, IL, USA). Protein concentrations were determined by the BCA protein assay (Pierce) using bovine serum albumin as a standard. Equal amounts of cell proteins (30 μg) were subjected to 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes using iBlot® Dry Blotting System (Invitrogen, Life Technologies, Gent, Belgium). Immunodetection was performed using antibodies raised against pSRC (Tyr 416) (1/1,000), SRC (1/1,000), pAKT (Ser 473) (D9E, 1/500), AKT (40D4, 1/2,000), PTEN (138G6, 1/1,000), pSTAT3 (Tyr 705) (D3A7, 1/1,000) (all from Cell Signaling Technology, Danvers, MA, USA) and pERK (Tyr 204) (E-4, 1/1,000), ERK2 (C-14, 1/2,000), STAT3 (K-15, 1/200) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Peroxidase-labeled anti-rabbit IgG antibody (1/5,000) or peroxidase-labeled anti-mouse IgG antibody (1/5,000) (both from Amersham Pharmacia Biotech) were used as secondary reagents to detect corresponding primary antibodies. Bound peroxidase activity was revealed using the SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Immunostaining signals were digitalized with a PC-driven LAS-3000 CCD camera (Fujifilm, Tokyo, Japan), using a software specifically designed for image acquisition (Image Reader, Raytest®, Straubenhardt, Germany).
Statistical analysis
IC10 and IC50 values were calculated using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Data are reported as means ± SEM and significance was calculated by Student's t-test using SPSS software (SPSS Inc., Paris, France). *p<0.05, **p<0.01, ***p<0.001.
Results
Characteristics of the representative melanoma cell lines relevant for the study
HBL cells are wild-type for NRAS and BRAF, MM057 cells present the Q61L mutation in NRAS and MM074 cells bear the V600E mutation in BRAF. Analysis of constitutive levels of phosphorylation/expression of key proteins involved in the master signalling pathways (SRC, MAPK, PI3K/AKT, JAK/STAT) in melanoma cells showed that HBL cells exhibit low phosphorylation of ERK and high phosphorylation of AKT, MM057 cells have high phosphorylation of both ERK and AKT, while MM074 cells show high phosphorylation of ERK but low phosphorylation of AKT (Fig. 2A). PTEN expression is not different among lines. Interestingly, we observed an inverse relation between the phosphorylations of SRC and STAT3, with high SRC phosphorylation in HBL and MM074 cells and high STAT3 phosphorylation in MM057 cells. Of note, these phosphorylation profiles of MM057 and MM074 cells are compatible with the mutational status of NRAS and BRAF, respectively.
On the other hand, we examined the proliferation rate of each cell line (day-3/day-1 ratio, crystal violet assay) and found that HBL cells were highly proliferative, MM057 cells were moderately, while MM074 cells had the lowest proliferation rate (2-fold less compared to HBL), suggesting that activating mutations in NRAS or BRAF are not necessarily associated with a higher proliferation rate (Fig. 2B) as also suggested by others (22).
Cell exposure to each inhibitor alone (condition 1)
We evaluated the anti-proliferative effect of each inhibitor in all cell lines after 3 days of treatment (condition 1, see Materials and methods) (Fig. 3) and we determined the IC10 and IC50 in all cases (Table I). We found that wild-type BRAF cells (HBL, MM057) were more sensitive to the SRC inhibitor PP2 than V600EBRAF cells (MM074), whereas HBL and MM057 cells had a lower level of phosphorylation of SRC than MM074 cells (Fig. 2A). As expected, the V600EBRAF cells were much more responding to the MEK inhibitor U0126 than the wild-type BRAF ones (especially HBL cells), in agreement with the hyper-activation of the MAPK signalling pathway in the former cells. The PI3K inhibitor LY29 had comparable weak inhibitory effects in the 3 cell lines, while the PI3K/AKT signalling pathway appeared weakly activated in MM074 cells. Finally, the JAK inhibitor PYR was the less effective in the Q61LNRAS cells (MM057) which are the only ones to exhibit an activation of STAT3. Thus, because of probable activation of alternative signalling pathways in cells under treatments, inhibitors were not necessary more effective in lines exhibiting higher activation of their targeted pathways.
Concomitant cell exposure to two inhibitors (condition 2)
Based on the effect of each inhibitor in all lines (Table I), we selected the highest non-toxic concentrations (0.05 μM PP2, 0.5 μM U0126, 5 μM LY29 and 0.05 μM PYR) after one week exposure to be subsequently used for inhibitor combination experiments (condition 2, see Materials and methods). Importantly, using western blot analysis, they were effective in inhibiting targeted signalling pathways after 30-min exposure (Fig. 4). Indeed, PP2 inhibited SRC phosphorylation in both HBL and MM074 cells, U0126 affected ERK phosphorylation in all cell lines, LY29 interfered with AKT phosphorylation in HBL and MM057 lines and PYR decreased STAT3 phosphorylation in MM057 cells.
Sequential cell exposure to inhibitors (condition 3)
We first examined the anti-proliferative effect of each inhibitor alone at the above mentioned non-toxic concentrations after 10 days of continuous exposure. As documented in Fig. 5, we found that all cell lines were not or were only weakly affected by long-term treatment with each inhibitor (proliferation rate >1), except in the V600EBRAF cells (MM074) where 0.5 μM U0126 is highly toxic (proliferation rate ~0.4).
Then, we evaluated whether pre-treatment with a first inhibitor potentiated the anti-proliferative effect of a second inhibitor (condition 3), as compared to simultaneous treatment with both inhibitors (condition 2) and the effect of the second inhibitor alone (condition 1). By comparing IC50s, we found that pre-treatment (condition 3) with MEK, PI3K or JAK inhibitors showed synergistic effects when specimens were subsequently exposed to an SRC inhibitor (Table II, in italics) in both HBL and MM057 cell lines. These effects may be explained by the fact that pre-treatment with U0126, LY29 or PYR increased SRC expression/phosphorylation in these lines (Fig. 6). In addition, we also observed synergy between PP2 or U0126 pre-treatment and then exposure to PYR in HBL cells and between U0126 pre-treatment and PYR in MM074 cells (Table II, in bold). However, STAT3 was not necessary more phosphorylated after these pre-treatments, suggesting that other JAK downstream targets should have been activated. Furthermore, sequential exposure to U0126 and then LY29 showed synergy in inhibiting MM074 cell proliferation (Table II, underlined), in the line with the AKT higher phosphorylation due to the pre-treatment with a MEK inhibitor (Fig. 6). By contrast, some concomitant treatments (condition 2) resulted in antagonistic effects when SRC and JAK inhibitors are combined (Table II), as compared to each inhibitor alone in all three cell lines (Table I).
Discussion
Conventional chemotherapy with dacarbazine, approved by the FDA (Food Drugs Administration) in 1975, was the standard for treatment of melanoma patients until recently (24). Overall response rate is 15% (mostly partial responses) but with no evidence of survival benefit. Immunotherapy with high-dose interleukin-2 or interferon α-2b have been associated with relatively durable responses but only in a small subset of patients, with yet no factors predicting which patients will respond to this therapy (25). Although they showed limited efficacy, these various therapies allowed considerable progress in the understanding of melanoma biology and molecular mechanisms involved in melanomagenesis.
Recent advances in cell biology and the design of targeted therapies led to unprecedented response rates with the new targeted agents. A strategy aiming at restoring the immune system response to disease has been developed, in particular, with ipilumumab, an antibody raised against CTLA-4 (cytotoxic T-lymphocyte antigen 4), a protein receptor responsible for immune tolerance. It improved overall survival in patients within a trial randomizing patients with previously treated metastatic melanoma into 3 treatment arms. Moreover, 9 of 15 responders (60%) in the ipilimumab only group maintained an objective response for ≥2 years (5). Ipilimumab in combination with dacarbazine improved overall survival versus dacarbazine plus placebo in patients with previously untreated metastatic melanoma (26). A similar immune strategy with an anti-PD1 or anti-PD-L1 (programmed death 1, ligand) antibody produced objective responses in ~28% of patients with melanoma. Interestingly, preliminary data suggest a relationship between PD-L1 expression on tumor cells and objective responses (6).
Furthermore, recent clinical data provided a strong indication for the efficacy of single agent targeted therapy approaches in melanoma. First, vemurafenib has been reported to induce clinical responses in >50% of metastatic melanoma patients bearing V600EBRAF mutated tumors (7). Then, dabrafenib, another V600EBRAF inhibitor, produced promising tumor shrinkage in patients with brain metastases, a frequent complication of metastatic melanoma (8). Lastly, trametinib, a selective inhibitor of MEK1 and MEK2, downstream effectors within the MAP kinase pathway, improved progression-free survival and overall survival as compared to chemotherapy in metastatic melanoma patients with BRAF V600E/K mutations (9). Importantly, the combination of dabrafenib and trametinib had an acceptable safety profile with a lower incidence of MEK inhibitor-related rash and BRAF inhibitor-induced hyper-proliferative skin lesions compared to the single agents (27). Unfortunately, the impressive response rates with these agents are short-lived, prompting the research for combinatorial strategies and the discovery of research mechanisms. A discontinuous dosing regimen with vemurafenib could emerge as a strategy to overcome resistance because vemurafenib-resistant melanomas become drug-dependent for their continued proliferation (28). Although many studies both in vitro and in clinical trials have been devoted to search for the best targeted drug combinations, encouraging but modest results have been recorded most probably due to the heterogeneous biological behaviour of melanoma tumor cells. In a recent study, pairwise combinations of an array of small-molecule inhibitors on early-passage melanoma cultures using combinatorial drug screening revealed several inhibitor combinations effective for melanoma treatment (29). Such investigations have to be continued in order to identify the most effective inhibitor combinations. In our study, we aimed to design an alternative rationale to combine targeted drugs to potentiate anti-proliferative effect of protein kinase inhibitors in order to overcome one of the major resistance mechanism to such drugs in melanoma. We selected 3 representative cell lines based on their MAPK mutation status: V600EBRAF cells (MM074), Q61LNRAS cells (MM057) and wild-type cells (HBL). Of note, inverse correlations were observed between ERK phosphorylation and AKT phosphorylation in wild-type HBL cells and V600EBRAF MM074 cells, confirming crosstalk between MAPK and PI3K/AKT pathways (21), while ERK and AKT were highly phosphorylated in the Q61LNRAS MM057 cells, because NRAS is upstream of both signalling pathways. Previous studies reported that the consequence of a constitutive MAPK activation in melanoma includes the increase in cell proliferation and invasion (30) and that, whereas V600EBRAF stimulated cell proliferation in melanoma, it induced senescence in melanocytes (31–34). However, and in agreement with others (35), we observed that the V600EBRAF mutation was associated with significantly lower proliferation rates in many cell lines that we have examined up-to-now (data not shown) and in particular is true for MM074 cells used in the present study. Of note, we confirmed that the BRAF mutation makes cancer cells more dependent on the MAPK pathway for their survival (36).
We also examined if the phosphorylation level of key kinases (SRC, ERK, AKT, STAT3) could be predictive of the response to protein kinase inhibitors regarding cell proliferation. We found that: (I) although both wild-type (HBL) and V600EBRAF (MM074) cells exhibited a high phosphorylation level of SRC, the wild-type cells were 15-fold more sensitive to an SRC inhibitor, (II) while Q61LNRAS (MM057) and V600EBRAF (MM074) cells have a high phosphorylation level of ERK, V600EBRAF cells were 30-fold more sensitive to a MEK inhibitor, (III) whereas wild-type (HBL) and Q61LNRAS (MM057) cells showed higher AKT phosphorylation levels than V600EBRAF (MM074) cells, all cells had similar sensitivity to PI3K inhibitor and (IV) although Q61LNRAS cells are the only ones to exhibit STAT3 phosphorylation, they were the least sensitive to a JAK inhibitor. Altogether, our data indicate that even if the phosphorylation level of key kinases correlates with the activation of the corresponding signalling pathway, it cannot be used alone, as a marker to predict the response to corresponding up-stream specific inhibitors because similar level of phosphorylation could be caused by various alternative mechanisms present in a given cell line, mainly related to exogenous growth factors and cytokines stimulations, mutation status, or crosstalk between signalling pathways. For example, ERK phosphorylation may be induced by growth factor stimulation, RAS or SRC activation, BRAF mutation and AKT-mediated phosphatase inhibition.
Many studies have reported the efficacy of multiple targeted therapies against cancer (37,38) and suggested a benefit of simultaneous inhibitions of different signalling pathways (39). However, little attention has been paid to sequential treatment that might be more relevant to clinical situation. Our hypothesis was that a long-term inhibition of a given signalling pathway renders melanoma cells dependent on other compensatory pathways for their proliferation and survival that can be different among subgroups of tumors. This information is crucial to subsequently target the correct pathway to possibly potentiate the effect (24). Accordingly, we found that a 10-day cell exposure to a protein kinase inhibitor affected the signalling pathway profiles as documented by western blot analyses of the phosphorylation levels of the master signalling proteins. For example, in wild-type HBL and Q61LNRAS MM057 cell lines, MEK, PI3K and JAK inhibitors stimulated the phosphorylation of SRC, rendering both lines more sensitive to SRC inhibition. Moreover, in V600EBRAF MM074, MEK inhibitor increased the phosphorylation of AKT leading to cells more responsive to AKT inhibition. Thus, we found that long-term cell exposure to a specific protein kinase inhibitor may enhance the anti-proliferative effect of another protein kinase inhibitor, supporting that melanoma cells became dependent on an alternative pathway for survival after a pre-treatment (40). Most interesting is the fact that these pre-treatments were done at non-toxic but kinase inhibition effective concentrations. By contrast, simultaneous use of inhibitors may surprisingly yield antagonistic effects. Indeed, this was observed with a concomitant exposure to SRC and JAK inhibitors, both proteins can affect the STAT pathway (41).
In conclusion, the present study adds new insight into drug combination strategies by focusing on a sequential use of kinase targeted inhibitors and on long-term priming/sensitizing tumor cells with a first effector used at non-toxic but effective concentrations to potentiate the effect of a second inhibitor. Our data strongly support a strategy based on the identification of both mutation status and signalling pathway profiles of a given tumor to select melanoma patients and propose adequate drug combinations and most importantly sequential administration schedules.