Melanoma Skin Cancer

Introduction

 Melanoma Skin Cancer

In the last 50 years the global incidence rate of melanoma increased more promptly than other cancer types. The estimated diagnosed cases of melanoma in 2018 was circa 90.000 for the United states alone, and the predicted deaths are around the number 9300. Melanoma is more often reported in Caucasian populations due to lower levels of melanin which is an internal photo-protector of the skin. Development of melanoma is associated with the exposure to ultraviolet rays, of which the sun is the main source; smoking, HPV infections, ionizing radiation, ulcers and the use of immunosuppressive medications.

Skin cancers can be divided in two subtypes: Non-malignant skin cancer – including basal-cell skin cancer and squamous-cell skin cancer – and melanoma. The latter is the most common cause of death by skin cancer although it represents less than 5% of skin related malignancies. The malignant skin tumor is the result of uncontrolled proliferation of pigment producing cells, also called melanocytes.

Signal Transduction Pathway in Health and Melanoma

Three-tiered mitogen-activated protein kinase (MAPK) cascade

The signal transduction pathway associated with melanoma is the MAPK/ERK cascade, as depicted in figure 1.

The most upstream protein MAP kinase kinase kinase (MAPKKK) can be activated by several stimuli, such as mitogens, osmotic stress, heat shock and cytokines, phosphorylate and thus activate MAP kinase kinase (MAPKK). Upon activation MAPKK can in turn phosphorylate the protein MAPK on two different positions in its activation loop. This successive phosphorylation and activation of the three kinases in the MAPK cascade is preceded by mitogens such as the epidermal growth factor (EGF) binding to its receptor, in this case transmembrane receptor EGFR.

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This ligand-receptor binding results in the activation of the tyrosine kinase activity by dimerization and phosphorylation of the receptor. Upon this phosphorylation, the receptor is able to recruit the protein Grb2, which in turn can bind the two SH3 domains of the guanine-nucleotide exchange factor (GEF) hSOS. Upon this binding, the small G protein Ras can be activated, by displacing GDP after which Ras can bind GTP and thus be activated. This activation can function as stimuli for the MAPKKK, such as BRAF, which can facilitate phosphorylation of the MAPKK, in this case MEK. MEK recognizes the amino acid residues tyrosine and serine/threonine of the MAPK enzyme Extracellular signal-Regulated kinase Kinase (ERK).

This dual phosphorylation is necessary for the activation of ERK. Activation of ERK results in a increased amount of transcription factors, resulting in a number of processes including proliferation, differentiation, survival, migration, stress responses and apoptosis. The MAPK cascade can be physically organized by scaffold proteins such as the Kinase Suppressor of RAS (KSR), which promotes efficient signaling by increasing the proximity of several proteins (BRAF, MEK and ERK) in the MAPK cascade to each other. Another function of the scaffold protein is the localization of the MAP kinases by association with nuclear and cytosolic binding partners. Another protein that is able to regulate the Raf kinase is the 14-3-3 enzyme, which works coordinately binding two phosphorylation sites.

BRAF Oncogene

The RAF Serine/Threonine family constitutes of three members: A-RAF, B-RAF and C-RAF. In approximately 40% of all melanomas, the MAPK pathway is activated in an constitutive manner by mutations in the BRAF oncogene. The most common mutation is V600E, in more than 90% of the BRAF mutations this occurs by a single base substitution.

Inhibitors and Resistance

The ERK/MAPK pathway is required in both physiological and pathological cell proliferation. Due to this combined with the fact that the BRAF oncogene mutations are the most common in melanoma, it comes as no surprise that this enzyme is an investigated target. Examples of inhibitors of the BRAF oncogene are: dacarbazine, vemurafenib (PLX4032), sorafenib, GDC-0879, PLX-4720, and LGX818. Of these inhibitors Vemurafenib was specifically approved by the FDA to treat melanoma. However, most patients treated with vemurafenib relapse after approximately one year. Possible pathways of resistance include the reactivation of the MAPK pathway by BRAF amplification, by expression of a BRAF splice variant, by activating mutations of the more upstream located NRAS or KRAS or the more downstream located MEK1 and MEK2 kinases, overexpression of the other RAFs, or via loss of kinase repressors such as NF1. Of course the exact pathway is more complicated than this linear approached pathway illustrated in figure 1, more mechanisms might be involved than proposed.

Aim

The aim of the research was to indicate collateral vulnerability of BRAF inhibitor-resistant melanoma due to increased levels of reactive oxygen species (ROS) in drug-resistant cells. Thereby exploring possibilities for new treatments targeting inhibitor-resistant melanoma.

Results

Vulnerability of MAPKi-resistant melanoma

First, MAPKi-resistant melanoma cells were cultured by exposing derivatives of BRAF mutant A375 human melanoma cells to the BRAF-inhibitor vemurafenib and to a combination of the BRAF-inhibitor dabrafenib and the MEK-inhibitor trametinib. The first one resulted in resistant cell line A375R and the second one in the double-resistant cell line A375DR. A short term proliferation assay was performed with A375, A375R and A375DR cells exposed to vemurafenib and a combination of dabrafenib and trametinib. Parental A375 cells were sensitive to the MAPK inhibition in contrast to the resistant cells. Also, absence of MAPK inhibitors induced the drug holiday effect, meaning the proliferation of the MAPKi-resistant cells was temporarily paused. These results are displayed in figure 2A. A cell quantification assay showed the sustained viability of A375R and A375DR cells in the presence of MAPK inhibitors. The viability of parental A375 cells was inhibited by the MAPK inhibitors. These results are illustrated in figure 2B.

To test if the increase in ROS levels may form a vulnerability for drug-resistant cells, the cell lines were exposed to the ROS inducer paraquat. The further increase of ROS levels inhibited the proliferation of A375R and A375DR cell colonies while the effect on parental cell colonies was minimal. Also, the sensitivity to paraquat was proportional to the elevation of the ROS levels. Furthermore, DNA damage was increased for A375R and A375DR cells. The addition of the ROS scavenger N-acetyl-L-cysteine (NAC) reduced ROS levels, apoptosis and DNA damage in all cell colonies. This supports the idea that the increased sensitivity of MAPKi-resistant cells to paraquat is mediated by an increase in ROS levels. These results are illustrated in figure 3B.

The clinically approved histone deacetylase inhibitor (HDACi) vorinostat also induces ROS. Long and short term assays were performed in the presence of vorinostat and/or NAC. Flow cytometry showed an increase in ROS levels in the short term assay that could be prevented by addition of NAC. This is illustrated in figure 4A. The long term assay in figure 4B revealed that the proliferation of drug-resistant cells was inhibited by vorinostat while the anti-proliferation effect on parental cells was minimal. This could be explained by the more elevated ROS levels in drug-resistant cells. Co-treatment with NAC prevented the decrease of proliferation in parental and drug-resistant cells. This is illustrated in figure 4C. Also, vorinostat induced DNA damage and apoptosis in drug-resistant cells and not in parental cells. Co-treatment with NAC also prevented these effects. These results are illustrated in figure 4D.

HDACi antagonizes MAPKi

Earlier research has shown that an increase in ROS levels can activate MAPK pathways and thereby counteract the effect of MAPK inhibitors. This was tested by treating cells with the HDACi vorinostat and/or with MAPK inhibitors dabrafenib and trametinib. MAPK signaling can be observed by measuring the phosphorylation of MEK (pMEK) and p-P90RSK. The results in figure 5A show that MAPK inhibitors reduce levels of pMEK and p-P90RSK in BRAFi/MAPKi-resistant cells A375DR and in parental cells. The addition of vorinostat reduced this effect significantly. Moreover, the cell proliferation in figure 5B of MAPKi-resistant cells was less prohibited when vorinostat and MAPK inhibitors were administered simultaneously. Similar results were obtained for A375 and A375R cells in the presence of vorinostat and/or vemurafenib. This effect can be explained by reduced MAPK signaling by the MAPKi’s, resulting in lower ROS levels and thus a lower effect of the ROS inducer vorinostat. Therefore, MAPK and HDAC inhibitors need to be administered sequentially instead of simultaneously.

HDACi Confers a Disadvantage to MAPKi-Resistant Melanoma

[bookmark: _gjdgxs]To prove that the treatment of HDACi is more effective in MAPKi resistant melanoma than in drug-sensitive cells, an assay was performed with a mixed population of drug-sensitive and drug-resistant cells. Drug-sensitive cells were labeled with green fluorescent proteins and drug-resistant cells with red fluorescent proteins, illustrated in figure 6A. These cells were cultured in the presence or absence of MAPKi’s or the HDACi vorinostat. Flow cytometry was used to determine the relative abundance of the two populations over 17 days. MAPK inhibition affected the depletion of the drug-sensitive cells and enriched the drug-resistant cells. On the other hand, vorinostat decreased the depletion of drug-resistant cells and enriched drug-sensitive cells. Also, the drug holiday had similar effects as the HDACi vorinostat. However, the effect of the drug holiday took longer to onset and was less pronounced, supporting that switching to HDACi treatment after the development of MAPKi resistance is more effective than a drug holiday. These results are illustrated in figure 6B and 6C.

HDACi induces ROS through suppression of SLC7A11

To elucidate how HDACi induced ROS in MAPKi resistant melanoma, transcriptome profiling by next-generation RNA sequencing (RNA seq) was performed. A375 parental and MAPKi-resistant derivatives A375R and A375DR were treated with and without HDAC inhibitor vorinostat. The result of RNA seq showed a total of 12 genes that were downregulated in these cell lines after treatment with vorinostat. SLC7A11 is one of the 12 genes that was downregulated and it encodes for a sodium-independent cystine-glutamate antiporter xCT. This antiporter is important for the intake of cystine, which is needed for the biosynthesis of antioxidant glutathione (GSH).

GSH is capable of preventing intracellular cell damage caused by ROS and therefore, suppressing SLC7A11 leads to increased cellular ROS. To confirm that vorinostat causes suppression of the SLC7A11 gene leading to reduced GSH concentration, qPCR was performed in parental and resistant melanoma cells treated with vorinostat. The results are shown in figure 7A and B. Vorinostat did suppress the expression of the SLC7A11 gene and reduced the GSH levels in melanoma cells. To prove that reduced SLC7A11 expression is correlated to increased ROS levels, short hairpin RNAs were used to target and silence the SLC7A11 gene. Silencing SLC7A11 indeed caused an increase in ROS levels (figure 7C). This only induced cell apoptosis in A375R and A375DR cells (figure 7D). However, overexpression of SLC7A11 in melanoma cells was able to rescue HDACi-mediated ROS and the anti-proliferation effect of vorinostat (figure 7E), meaning that SLC7A11 is an important gene for reducing ROS levels and cell survival.

Relative mRNA levels of SLC7A11 in parental and MAPKi-resistant cells. Cells were treated with DMSO or vorinostat. mRNA levels were analyzed with qRT-PCR. GSH concentration in parental and MAPKi-resistant cells after treatment with or without vorinostat. GSH levels was measured with colorimetric-based glutathione detection assay Relative ROS levels in parental and MAPKi-resistant cells upon SLC7A11 knockdown. CellROX-Green flow cytometry assay was used to measure ROS levels. Effects of SLC7A11 knockdown in parental and MAPKi-resistant cells. DNA damage marker (gamma-H2AX) and apoptosis marker (cleaved-PARP) were used to observe the effects of SLC7A11 knockdown in melanoma cells. Effect of overexpressed SLC7A11 mRNA levels on cell proliferation in melanoma cells in the presence or absence of vorinostat.

In vivo study of sequential drug treatment

The effectiveness of drug treatments was tested in melanoma animal model. Mice were injected subcutaneously with melanoma cells, A375 cells. Drugs (PLX4720 (analog of vemurafenib) and vorinostat) were administered in animals when tumors reached 500 mm³. (figure 8A and B) Treatment of mice with vorinostat did not lead to tumor regression whereas treatment with PLX4720 did. However, drug resistance against PLX4720 treatment developed after approximately 60 days which is caused by reactivation of MAPK signaling (figure 8A).

BRAFi-resistant melanomas in mice were investigated to see whether they were able to respond to vorinostat. In this experiment, mice were treated with no drug, vorinostat only, PLX4720 only or a combination of vorinostat and PLX4720. As shown in figure 8B, mice that were continuous treated with PLX4720 showed rapid tumor growth compared to the control group. MAPKi resistant melanoma cells treated with PLX4720, an analog of vemurafenib are still capable to activate MAPK pathway (figure 8C). This is consistent with the results from earlier in vitro experiments. Combination of vorinostat and PLX4720 treatment showed a slow tumor growth which could be explained by the antagonistic effect of these drugs. The tumor volume declined when mice were only treated with vorinostat which was expected due the cytotoxic effect of vorinostat in BRAFi-resistant melanoma cells.

Tumor growth of A375 cells after injection with or without compounds (PLX4720 or vorinostat) in mice. Post-injection of vorinostat, PLX4720 or vorinostat and PLX4720 in mice. Experiment was performed after drug resistance of PLX4720 was acquired. Western blot analysis of the phosphorylation of SHP2, BRAF, and MEK in parental and ex vivo clones. Ex vivo clones were isolated from A375 tumors that were continuously treated with PLX4720.Phosphorylation was compared between parental and ex vivo cells treated with or without vemurafenib.

Clinical validation of sequential drug treatment

The efficacy of sequential treatment of MAPKi and HDACi was tested in BRAFV600E mutated advanced melanoma patients. Biopsies (pre-, during, and post-vorinostat treatment) were taken from patients. Transcriptome sequencing was performed to measure SLC7A11 levels in pre-, during, and post-vorinostat-treated tumor biopsies. Figure 9C shows the SLC7A11 levels was decreased after treatment of vorinostat which is consistent with in vitro data. Additionally, vorinostat therapy was able to eradicate tumor cells that were resisted to BRAFi and MEKi (e.g. KRAS and NRAS mutations) therapy figure 9A and B. These results indicate that vorinostat treatment could be a potential therapy to eliminate melanoma cells that are resistant to BRAF and MAPK inhibitors.

Discussion and Conclusion

Overall, a detailed description was given of the experiments in this paper. However, the duration of the exposure of the cell colonies to MAPKi’s and/or vorinostat in figure 3C was not mentioned. Presumably, the duration was equal to the ten days in figure 3D but this is not clearly stated in the text or in the legend.

The aim of the research was to indicate the collateral acquired sensitivity of BRAF inhibitor-resistant melanoma, and prove the relation to increased levels of ROS. Resistance to MEK inhibitors in combination with BRAF inhibitors was correlated to an increase in ROS levels. This was also amplified by ROS inducers paraquat and the HDACi vorinostat. Additionally, the histone deacetylase inhibitor vorinostat was able to suppress SLC7A11 expression in an GFP assay using transcriptome profiling. This resulted in an even further increase of ROS levels, killing the combined MEK+BRAF inhibitor resistant cells in an selective apoptotic manner. In vivo studies showed that vorinostat is able to decrease tumor growth significantly. Moreover, the result of the clinical study showed that patients with certain mutations (e.g. KRAS and NRAS) responded to vorinostat leading to eradication of MAPKi resistant tumor cells. Also, it was clinically validated that MAPKi and HDACi have to be administered sequentially instead of simultaneously.

This research has a high impact on the investigation of resistant tumors. Similar experiments could be used to find acquired sensitivities in other types of resistant cancer, thereby creating new leads for treatments. For example, other increased values than ROS levels could be used to induce selective apoptosis of resistant cells.

Future Recommendations

The authors of this article state that it would be possible that other drug resistance mechanisms contribute to the resistance of MAPK inhibitors. Other mechanisms that are involved in MAPKi resistance may not respond to vorinostat therapy. Therefore it is necessary to investigate what and how other mechanisms could contribute to the resistance of MAPKi. Possible vulnerabilities of these mechanisms could then be investigated to develop treatments for melanoma that are not caused by the reactivation of MAPK pathway. Additionally, it would be interesting to test if the vulnerability to ROS of tumor cells that developed drug resistance can be exploited in other types of cancer. Further tests and expansion of the test group are needed for the validation and more complete elucidation of the drug resistance mechanism in tumor cells.

Also, treatment of resistant BRAF V600E mutated advanced melanoma with vorinostat is currently in the second phase of the clinical trials. If this trial fails, other HDACi’s could be investigated as treatment of resistant BRAF V600E melanoma.