Background
Cutaneous melanoma is a deadly form of skin cancer that develops from melanocytes, specialized pigmented cells that reside underneath the epidermis. 50% of melanomas harbour activating mutations in the kinase BRAF, the most common being a V600E substitution [
1], and 25% harbour mutations in the GTPase NRAS. Both oncogenes stimulate the MAP-kinase (MAPK)-pathway, which is found hyperactivated in 90% of all melanomas [
2]. Whereas BRAF only activates the MAPK-pathway, NRAS activates several other effectors including Ral-GDS or PI3-kinase, which is of special relevance for melanoma [
2].
It is now accepted that genetic lesions in BRAF and NRAS have different consequences in melanoma formation and it is becoming apparent that BRAF can regulate invasion and metastasis through mechanisms different to NRAS [
3].
Importantly, the genetic background of melanoma also impacts on the response to therapies targeting the MAPK-pathway. While there is no efficient targeted therapy against wild type BRAF melanomas, BRAF mutant (mutBRAF) melanomas are addicted to the MAPK-pathway and small molecule inhibitors targeting either mutBRAF or MEK have shown impressive clinical responses [
4‐
7]. Unfortunately, these responses are transient, and patient relapse due to acquired resistance [
8]. In contrast to mutBRAF melanomas, mutNRAS tumours are largely resistant to BRAF inhibitors [
9,
10], and moreover these drugs paradoxically stimulate the MAPK-pathway [
11]. Thus, despite the initial successes with BRAF targeted therapy, relapsed patients as well as the 50% of patients harbouring a wild type BRAF (including the 25% with NRAS mutations) will still require alternative treatment such as chemotherapy and/or immunotherapy.
In melanoma some of the most commonly used chemo-therapeutics are the monofunctional-alkylating agents dacarbazine (DTIC) and temozolomide (TMZ), the chloro-ethylating agents carmustine or the bifunctional alkylating agents like cisplatin [
12]. Also anti-mitotic drugs like paclitaxel and vinblastine are used in the treatment of melanoma patients (
http://www.cruk.org). Historically the prodrug DTIC has been the first line treament with an average overall response of 20% [
12]. In patients DTIC is metabolized in the microsomes of hepatocytes into MTIC, which undergoes spontaneous transformation into a toxic DNA methylating agent [
13]. More recently patients are being treated with Temozolomide (TMZ), which does not require metabolic activation, but spontaneously converts into MTIC and shows a clinical response almost identical to DTIC [
13,
14].
Notably, compared to brain tumors, melanoma responses to alkylating agents are poor. In patients with malignant melanoma, the overall response to temozolomide is around 15% compared to 47 and 61% in glioma and astrocytoma patients [
12,
15]. Interestingly mutBRAF melanoma patients have shown responses to DTIC of up to 23% [
16] but, on the other hand, recent reports state that activating mutations in BRAF have no impact on the response of stage IV melanoma patients to DTIC or TMZ [
17]. However, BRAF and NRAS mutation status has never been tested retrospectively for its potential as predictive marker for DTIC responses. Resistance to these alkylating agents is thought to be due to several factors, including the altered expression of components of the apoptotic and DNA damage repair machineries and to multi-drug resistance phenotype-associated proteins such as the ABC drug transporters [
18‐
20]. Pre-clinical studies using melanoma and glioma cells and xenografts have shown that expression of the DNA repair protein
O6-methylguanine-DNA-methyl-transferase (MGMT) confers resistance to mono-alkylating agents such as DTIC, TMZ and carmustine [
21]. However, this has not been successfully translated into the clinic and the use of MGMT inactivating agents to sensitise cancer cells to alkylating drugs has not provided any clinical benefit [
21]. Thus, in contrast to targeted therapy, and despite extensive studies into DNA repair mechanisms in relation to tumour response, there are no good markers to predict a patient’s response to chemotherapy. Since in melanoma the genetic background delineates specific mechanisms of proliferation, survival or invasion/migration and regulates the response of melanoma cells to targeted therapy, we hypothesized with the possibility that mutations in BRAF or NRAS might affect melanoma cell response to chemotherapeutic agents.
Discussion
The aim of this study was to determine whether the mutational status of melanoma cells would correlate with their response to chemotherapeutic agents. Our results demonstrate that in melanoma the presence of mutually exclusive BRAF and NRAS mutations has no influence on the response to DNA alkylating agents such as TMZ. Similar results considering BRAF or RAS mutation status are found in a large data set containing drug-treatment data from 732 cancer cell lines of different origin [
25]. Thus, it appears that mutBRAF and mutNRAS share common mechanisms of resistance to methylating agents such as drug efflux, or deregulation of pro-apoptotic or DNA repair pathways.
Surprisingly, we found that mutBRAF and mutNRAS cells respond very differently to light activated DTIC. Activation of DTIC by exposure to white light has been described to recapitulate its chemotherapeutic activity
in vitro[
28,
29] but our results provide experimental evidence that the toxic effect described for light activated DTIC is independent of DNA methylation. Through DNA alkylation assays and combinatorial treatments using DTIC and a MGMT inhibitor we provide clear evidence that light exposure does not transform DTIC into a DNA methylating agent, but rather an inhibitor of DNA synthesis. This finding is of major importance considering that light activation of DTIC has been extensively used to study the mechanisms underlying its cytotoxic effects as well as leading to acquired resistance in patients [
28‐
31]. In this context it is important to mention that wild type BRAF melanoma cells that had been selected for resistance to light activated DTIC
in vitro exhibited increased tumour growth
in vivo, a phenotype that correlates well with enhanced DNA synthesis activity [
32]. Most strikingly these resistant cells and tumours displayed hyper-activation of the MAP-kinase pathway, resulting in increased IL8 and VEGF expression [
28,
32]. The fact that we now show that light activated DTIC inhibits nucleotide synthesis, most probably by inhibiting IMPDH, suggests a novel link between DNA synthesis pathways and MAP-kinase signalling.
Our results indicate that non-metabolically activated DTIC mediates its effects through 2-AzaHX. Strikingly, there is evidence that metabolic activation of DTIC in patients is inefficient and that, shortly after DTIC isolated limb perfusion, significant amounts of 2-AzaHX can be detected in the bloodstream and urine of patients [
33]. Thus it is possible that 2-AzaHX could contribute to the DTIC-dependent toxicity, although it is well established that the anti-tumour activity of DTIC is mainly the result of DNA methylation [
13].
In this context it is important to mention that conversion of 2AzaHX by HGPRT to 2-AzaIMP is able to inhibit IMPDH [
34,
35]. We found that mutNRAS melanoma cells are significantly more resistant to two bona fide IMPDH inhibitors (MPA and AVN944), suggesting that in NRAS mutant cells IMPDH activity or its downstream signalling is elevated. This, together with the fact that mutNRAS cells express higher levels of TK1 and consequently are more effective in using thymidine for DNA synthesis, provides strong evidence that mutNRAS melanoma cells are significantly more efficient in nucleotide salvaging.
Increased
IMPDH2 expression in cancer cells has been linked to resistance to methotrexate in osteosarcoma, colorectal and erythroleukemia cells [
36‐
38]. However, although
IMPDH2 is overexpressed in melanoma compared to benign melanocytic lesions (not shown), its expression did not differ in mutNRAS and mutBRAF melanoma cells. Therefore, the difference in the response to IMPDH inhibitors rather suggests that IMPDH activity or its downstream signalling is regulated differently in mutNRAS compared to mutBRAF cells. Apart from IMPDH, we also show that thymidine can compensate for DHFR inhibition in resistant mutNRAS cells, which express higher levels of TK1. Whether elevated TK1 expression is directly regulated by NRAS is not yet known, but it will be crucial to identify the underlying mechanism. Importantly, we did not find differences in TK2 expression between mutNRAS and mutBRAF cells (not shown), which is maybe not surprising considering the more ubiquitous role of TK2 [
39].
Historically antifolate drugs such as methotrexate or edatrexate have shown very little activity in clinical trials with melanoma patients although these trials were performed before the discovery of BRAF and RAS as drivers of melanomagenesis [
40,
41]. The lack of response in melanoma patients can be explained by several mechanisms of resistance such as melanosomal sequestration of drugs, the upregulation of both DHFR and the pro-survival transcription factor MITF in response to MTX, or the E2F and Chk1 mediated effects, as recently described [
42‐
45]. Despite the inherent capacity to resist any chemotherapy our data suggest that stratifying patients according to their BRAF/RAS mutation status could lead to better responses to antifolate based therapies.
Importantly, our findings suggest that the correlation between NRAS and BRAF mutations and their differential response to antifolate drugs might apply to other cancer types. Therefore, in cancer types where antifolate based therapies contribute to achieve clinical responses in RAS patients (e.g. colorectal carcinoma) [
46], it would be interesting to assess whether mutBRAF patients show even improved responses. If that were the case it would open the possibility to use mutational status as a predictor of patient response. In summary, our findings identify the mutually exclusive NRAS and BRAF mutation status as possible predictive marker for the response to DNA synthesis inhibitors such as antifolate drugs in melanoma patients.
Materials and methods
Cell culture
Nine mutant BRAF cell lines and nine mutant NRAS cell lines were used in the study (Additional file
1: Table S1). These cells were a kind gift from Dr. Richard Marais and Dr. Adam Hurlstone. Cell stocks were expanded, frozen, and kept in liquid nitrogen. New aliquots were thawed every 5–7 weeks. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (SIGMA) or in RPMI-164 medium (RPMI) (SIGMA) as previously indicated, supplemented with 0.5% penicillin and streptomycin (SIGMA) and 10% bovine calf serum (PAA, Yeovil, UK). Cells were grown at 37°C in a 5% CO
2 environment.
Reagents
HAT supplement (50X) was from Sigma. Dacarbazine, carmustine, cisplatin and temozolomide and lomeguatrib were from SIGMA. Hypoxanthine, guanosine and 5′-guanosine monophosphate were from Sigma. AZD6244 was from Selleck Chemicals, Newmarket, UK. Mycophenolic Acid and AVN944 were from Sigma and ChemieTek respectively. Aminopterin, pyrimethamine and amethopterin (methotrexate) were from Sigma. All drugs were dissolved in dimethylsulfoxide (DMSO) and, apart from dacarbazine, directly added to cell in culture at the indicated concentrations. Prior to addition onto cells DTIC was exposed to white light for 1 h, as previously described [
28,
30].
Determination of MGMT activity
Melanoma cell free extracts prepared from 10
6 cells were analysed for MGMT activity using calf thymus DNA methylated in vitro with N-nitroso-N-[
3H]-methylurea (~20 Ci/mmol) as the substrate [
47]. MGMT activity was expressed as fmol/μg DNA to avoid the possible effect of variable protein content on apparent MGMT activity expressed per unit protein [
48]. No significant differences in the study results were noted when MGMT activity was expressed per unit protein. Results are the mean of quadruplicate determinations for each sample. Cell free extracts prepared from the human breast cancer cell line MCF-7 were assayed for MGMT activity as a positive control.
Determination of O6-methylguanine levels in DNA
O6-methylguanine (
O6-meG) in DNA was quantified using a modification of the standard MGMT activity assay procedure [
49]. Increasing amounts of the DNA samples were pre-incubated with a standard amount of purified recombinant human MGMT [
50] and residual activity was then determined.
O6-meG in DNA stoichiometrically inactivates MGMT. Thus the amount of
O6-meG in the DNA sample equals the amount of inactivation of the purified MGMT.
Determination of GI50
To determine the drug concentration necessary to inhibit cell growth by 50% (GI50), 2000 cells per well were plated in 96 well plates (Corning). After 24 hours, drugs were added in triplicates in serial 1:3 dilutions. In experiments where cells were co-treated with the MGMT inactivating agent lomeguatrib, the drug was added 1 hour before the addition of serial dilutions of DTIC or TMZ. After 3 or 5 days cells were washed with PBS and simultaneously fixed and stained for 1 hour with 4% Formaldehyde (Fisher Scientific) and 0.5% Toluidine Blue (Fluka Analytical) in PBS. Plates were washed, dried and the dye was solubilized with 1% Sodium dodecyl sulfate (SDS) (Fisher Scientific) in PBS. Finally, a spectrophotometer (BIO-TEK®, NorthStar Scientific) was used to measure the O.D. and GI50 values were calculated using the GraphPad Prism software (GraphPad Software, 4.0a).
Databases
To study the expression profile of APR1, HPRT1 and TK1 genes in human melanoma versus normal skin or benign nevus, and to compare TK1 expression between mutBRAF and mutRAS human melanoma cell lines we used Oncomine Cancer Microarray database (
http://www.oncomine.org/).
RNA isolation and qPCR analysis
RNA was isolated with TRIZOL® and selected genes were amplified by quantitative real time PCR using SYBR green (Qiagen, Valencia, CA, USA).
Primers sequences were:
TK1:
Forward: 5′-TGGCTGTCATAGGCATCGAC-3′,
Reverse: 5′-CCAGTGCAGCCACAATTACG-3′
BETA-ACTIN:
Forward: 5′-GCAAGCAGGAGTATGACGAG-3′,
Reverse: 5′-CAAATAAAGCCATGCCAATC-3′
EdU incorporation assays
Cells were labelled with 10 μM EdU (Invitrogen) for 4 h before they were formalin fixed and processed following the manufacturer’s instructions. Stained cells were analysed using a BDpathway 855 Bioimager.
FACS analysis
100000 cells were treated as indicated, fixed in ice-cold 80% ethanol. Cells were then washed in PBS and incubated in a solution containing PBS, RNase A and Propidium Iodide (SIGMA) at 37°C for 1 hour. The analysis was performed using FACS Calibur (Becton Dickinson).
Statistical analysis
Unless indicated otherwise, data are from assays performed in triplicate, with error bars to represent standard deviations or errors from the mean. Statistics used were: predominately Student t-test and One-way ANOVA with Dunnett’s Multiple Comparison Test performed using GraphPad Prism version 4.00 for Mac OS, GraphPad Software, San Diego California USA,
http://www.graphpad.com.
Acknowledgements
This study was supported in part by the National Science Foundation of China and Cancer Research UK (C11591/A10202). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests
The authors declare no conflict of interest.
Authors’ contributions
IA conceived the study, designed and carried out most of the experiments, analysed the data and wrote the manuscript. IG and OE carried out the experiments to assess the GI50 of every alkylating agent and the effect on the cell cycle. JF was in charge of q-RT-PCR experiments. GPM carried out DNA methylation and MGMT activity assays and helped write the manuscript. CW conceived and coordinated the study, and wrote the manuscript. All authors read and approved the final manuscript.