Fluvastatin

Fluvastatin exerts an antitumor effect in vemurafenib-resistant melanoma cells Masao

Nishiyaa,b, Shinji Yasuhiraa, Masahiko Shibazakia, Hiroki Oikawab, Tomoyuki Masudab and Chihaya Maesawaa

Although vemurafenib has been shown to improve the overall survival of patients with metastatic melanoma harboring the BRAF V600E mutation, its efficacy is often hampered by drug resistance acquired within a relatively short period through several distinct mechanisms. In the present study, we investigated the effect of fluvastatin as a possible strategy to overcome such acquired resistance using a cultured cell line model. We established vemurafenib-resistant (VR) cells from three BRAF (V600E)- mutated melanoma lines (C32, HMY-1, and SK-MEL-28) and evaluated the mechanism of acquired resistance of VR cells by water-soluble tetrazolium salts assay, western blot, real- time quantitative PCR, and immunofluorescent microscopy. The efficacy of the combination of growth inhibitory effect of vemurafenib and fluvastatin on respective parental and VR cells were assessed by calculating combination index and western blot. IC50 values of three VR cells were ~ 5–100-fold higher than those for the respective parental cells. The VR cells derived from HMY-1 and SK-MEL-28 showed constitutive activation of AKT kinase, and the specific AKT inhibitor MK-2208 or the PI3K inhibitor wortmannin increased the cellular sensitivity to vemurafenib.
Intriguingly, application of a statin-related drug, fluvastatin, also resulted in a synergistic increase of sensitivity to vemurafenib in the VR cells (combination index: 0.73–0.86) probably by alleviating constitutive AKT activation, whereas the same treatment did not notably alter the vemurafenib sensitivity of the parental cells. Our results suggest the possible usefulness of statin-related drugs for overcoming vemurafenib resistance acquired through constitutive activation of the PI3K–AKT axis. Anti-Cancer Drugs

Keywords: AKT, BRAF, fluvastatin, melanoma, vemurafenib resistance

Introduction

Cutaneous malignant melanoma is a leading cause of death among the various skin cancers, exhibiting aggressive tumor behavior [1]. Whereas conventional chemotherapeutics and other strategies including classi- cal immunotherapies have had a limited impact on the course of this disease [2,3], the emergence of two new modalities – immune checkpoint blockade and signal transduction inhibition – has opened a new era in the treatment. Immune checkpoint blockade with monoclonal antibodies has achieved a durable long-term response after treatment despite a relatively low response rate in the short term; such treatment includes the antibody against cytotoxic T-lymphocyte antigen 4, ipilimumab [4], or those against programmed cell death ligand 1/receptor 1, nivolumab [5], and pembrolizumab [6]. National Comprehensive Cancer Network guidelines now recommend these antibodies for the first-line treatment of metastatic melanomas [7]. In Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website, www.anti-cancerdrugs.com contrast, signal transduction inhibitors (vemurafenib/dabra- fenib for BRAF and trametinib for MEK) have achieved rapid and high short-term response rates, whereas the dur- ability of the responses is limited [8,9].
To achieve greater clinical benefit for patients with advanced melanoma, overcoming resistance to therapy in both the early and late phases is inevitable for both types of therapeutic approach. While 35–60% of melanoma patients exhibit minimal or no response to programmed cell death receptor 1 and cytotoxic T-lymphocyte antigen-4 blockade at the outset (primary resistance) [5,10], half of all patients with metastatic melanoma who initially respond to immune-based treatments develop resistance within 3 years (acquired resistance). The mechanisms underlying both primary and acquired resistance to immune checkpoint blockade have been unclear. No precise biomarker that can predict a patient’s response has yet been developed, despite extensive investigation [11].

The mechanisms of primary/acquired resistance to signal transduction inhibitors have been relatively well clarified [12–16]. Although several molecular causes of vemur- afenib resistance (VR) have been proposed, either MAPK reactivation due to genetic alteration or activation of the PI3K/AKT/mTOR pathway appears to be most common. Mutations in NRAS [17] and MEK1 [18] that result in reactivation of the MAPK/ERK pathway are frequently detected in tumors with acquired VR. For these cases, combination therapy with dabrafenib and trametinib has been attempted to delay VR. For VR due to activation of the PI3K/AKT/mTOR pathway, the possible efficacy of AKT/mTOR inhibitors including approved drugs such as metformin, rapamycin, and resveratrol has been suggested [19–21]. Besides these cases, Lin and coleague- shave demonstrated that activation of YAP/TAZ protein, targets of the Hippo pathway, promotes resistance to BRAF and MEK inhibitors. This observation suggests that combined repression of YAP and BRAF/MEK could be a promising therapy for some melanoma patients [22,23].
We have recently demonstrated that treatment with fluvas- tatin (a small-molecule inhibitor of 3-hydroxy-3-methylglu- taryl coenzyme A, i.e. a statin) compromised nuclear translocation of YAP/TAZ proteins in malignant mesothe- lioma cells, thus, downregulating transcriptional targets of YAP/TAZ including RHAMM [24]. Wang et al. [25] have reported that simvastatin, another statin-related drug, also strongly perturbs the PI3K/AKT/mTOR pathway by enhancing the expression of PTEN. These results may suggest that statins might function as an efficient modulator for melanoma with acquired VR either through YAP/TAZ or through PI3K/AKT/mTOR pathways. In the present study, we established and characterized three VR melanoma cells initially focusing on the rela- tionship between YAP/TAZ activation and VR in these cell lines. We found that fluvastatin increased the vemurafenib sensitivities of the VR cells not through YAP/TAZ but PI3K/AKT/mTOR.

Materials and methods

Compounds

Vemurafenib (BRAF inhibitor, S1267), wortmannin (low- specificity, covalent inhibitor of phosphoinositide 3-kinases, S2758) and MK-2206 2HCl (high-specificity inhibitor of Akt1/2/3, S1078) were purchased from Selleckchem (Houston, Texas, USA), and fluvastatin (PHR1620) was purchased from Sigma-Aldrich (St Louis, Missouri, USA). Stock solutions were prepared in dimethyl sulfoxide at 10 mmol/l.

Cell culture and media

All cells were cultured at 37°C in RPMI 1640 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supple- mented with 10% fetal bovine serum in a humidified atmosphere with 5% CO2. The C32 and SK-MEL-28 human melanoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). The HMY-1 human melanoma cell line was purchased from the Japanese Collection of Research
Bioresources (JCRB, Tsukuba, Japan). To obtain VR cells, all cells were seeded and exposed to increasing concentra- tions of vemurafenib (1–4 µmol/l) for 4 weeks. These cell lines were named C32/VR, HMY-1/VR, and SK-MEL-28/ VR, respectively. This series of VR cells were maintained in medium containing 4 µmol/l vemurafenib.

Cell viability assay

Cell viability was determined by water-soluble tetra- zolium salts assay using a Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Cells were seeded into 96-well plates (3000 cells/well) and cultured for 24 h before treatment with each reagent. After 72 h of treat- ment, the medium in each well was replaced with 100 µl of drug-free fresh medium and 10 µl of Cell Counting Kit-8 solution, incubated for an additional 2–3 h, and the absorbance at 450 nm was measured using a Multiskan Spectrum spectrophotometer (Thermo Fisher Scientific).

Protein preparation and immunoblotting

Cells at 80–90% confluence were washed twice with ice- cold PBS, treated with PBS containing 10% TCA for 30 min on ice, and scraped off into a tube. The cell pellet was washed twice with deionized water, and then lysed in 9 mol/l urea, 2% Triton X-100 and 1% dithiothreitol. Protein concentration was measured with a BCA protein assay kit (Merck Millipore, Billerica, Massachusetts, USA) before the addition of dithiothreitol. Equal amounts of protein (10 µg) per lane were electrophoresed on 10% SDS-PAGE gel for 60 min at 200 V and then transferred to polyvinylidene fluoride transfer mem- branes (Pall Corporation, Port Washington, New York, USA). The membranes were blocked with 5% nonfat dried milk (Cell Signaling Technology, Danvers, Massachusetts, USA) or 5% BSA (Sigma-Aldrich) in 1 × TBS-T for 1 h at room temperature. Then the membranes were incubated with an appropriate primary antibody overnight at 4°C and with an HRP-conjugated secondary antibody (GE Healthcare Life Sciences, Buckinghamshire, UK) for 1 h at room temperature. Signals were identified with ECL prime detection reagents (GE Healthcare Life Sciences) and ChemiDoc XRS (Bio-Rad Laboratories, Hercules, California, USA).

Immunofluorescence microscopy

Two thousand cells were grown on a poly-L-lysine-coated 18 mm2 coverslip at 37°C overnight, and then fixed with 4% paraformaldehyde at 37°C for 30 min. The cells were blocked with PBS containing 5% goat serum and 0.3% Triton X-100 at room temperature for 60 min, treated with rabbit anti-YAP antibody (#14074; Cell Signaling Technology) and mouse anti-TAZ antibody (#560235; BD Pharmingen, Franklin Lakes, New Jersey, USA) at 1 : 200 dilution in PBS containing 1% BSA and 0.3% Triton X-100 for 2 h, and subsequently with Alexa Fluor 488-conjugated anti-rabbit IgG goat antibody and Alexa Fluor 546-conjugated anti-mouse IgG goat antibody (Thermo Fisher Scientific) at 1 : 1000 dilution for 2 h. After washing, the coverslips were mounted with ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific) and observed using a confocal micro- scope (C1 and EZ-C1; Nikon, Tokyo, Japan).

siRNA transfection

Silencer Select predesigned siRNAs against YAP1 (ID s20367), WWTR1 (ID s24787), and control nonspecific human siRNA, Lipofectamine RNAiMAX transfection reagent, and Opti-MEM were all obtained from Thermo Fisher Scientific. Cells were transfected with 10 nmol/l
siRNA using 7.5 μl of RNAiMAX diluted with Opti- MEM in a six-well plate format in accordance with the manufacturer’s instructions.

Real-time quantitative RT-PCR

Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. The RNA concentration was measured by NanoDrop (Thermo Fisher Scientific) and an equal amount of extracted RNA was reverse-transcribed using SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific). cDNAs of YAP1, WWTR1, CTGF, ANKRD1, and GAPDH were quantified by real-time PCR (7500 Real- Time PCR System; Thermo Fisher Scientific) using the TaqMan Gene Expression MasterMix and TaqMan Gene Expression Assays for YAP1 (Hs00902712_g1), WWTR1 (Hs00210007_m1), CTGF (Hs00170014_m1), ANKRD1 (Hs00173317_m1), and GAPDH (Hs02758991_g1).

Evaluation of drug combination effects

The combination indices and the drug reduction indices were calculated according to the method established by Chou and Talalay [26]. The mean values were calculated from three independent experiments.

Statistical analysis

Data are presented as the mean ± SEM unless stated otherwise. A logistic function was fitted to the cell sur- vival data points with the
least squares estimates of the parameters. IC50 values were calculated from the fitted function. The statistical software R (version 3.4.2) was used [27]. Confidence intervals for IC50 were calculated with the ‘confint()’ command.

Results

Establishment of vemurafenib-resistant cells
To obtain VR cells, we treated three malignant melanoma cell lines (C32, HMY-1, and SK-MEL-28) harboring the BRAF V600E mutation with step-wise increases in the concentration of vemurafenib (1–4 µmol/l) for 4 weeks. This successfully gave rise to VR cells from all three cell lines, whose IC50s were increased between 4.3- and 96.7-fold relative to the parental cells (Fig. 1 and Table 1). We examined the expression level and phosphorylation status of MAPK-related proteins, as well as AKT, in the presence or absence of vemurafenib using immunoblot- ting (Fig. 2). Because of the BRAF V600E mutation, phosphorylation of both MEK and ERK was con- stitutively high in all of the three parental lines, and vemurafenib markedly reduced it. In contrast, vemur- afenib barely affected the level of phosphorylation in the VR cells, suggesting that some bypass may operate at the level of or downstream of BRAF. Intriguingly, VR cells derived from two of the melanoma lines (HMY-1/VR and SK-MEL-28/VR) showed a dramatic increase of AKT phosphorylation (Thr308 and Ser473) regardless of vemurafenib treatment, in comparison with the respec- tive parental cells (Fig. 2). It is noteworthy that these VR cells also showed a higher increase of IC50 than C32/VR line, the latter not showing any increase of AKT phos- phorylation relative to the parental line. Although we examined the expression levels of COT and BRAF kinases as well as PTEN phosphatase as possible causes for the acquired vemurafenib resistance, neither showed any notable change [Fig. 2 and Supplementary Figs 1 (Supplemental digital content 1, http://links.lww.com/ACD/ A293) and 2 (Supplemental digital content 2, http://links. lww.com/ACD/A294)].

YAP/TAZ activity and drug resistance in VR cells Several recent studies have shown that overexpression of YAP/TAZ transcription factors might contribute to acquired vemurafenib resistance, possibly by the expression of their targets involved in cell growth or survival. To assess the activity of the YAP/TAZ transcriptional axis in VR cells, we examined the expression levels of YAP/TAZ and their targets using quantitative PCR. Although the mRNA for ANKRD1, an established target of YAP/TAZ, showed a dramatic increase in all of the VR cells (3.2–142.6-fold; Supplementary Fig. 3, Supplemental digital content 3, http://links.lww.com/ACD/A295), mRNAs for YAP and TAZ .Effect of fluvastatin on MEK/ERK/AKT phosphorylation It has been suggested that inhibition of prenylation of small G proteins such as RAS or RHO by statins could result in downregulation of the PI3K/AKT/mTOR pathway. We investigated how fluvastatin affects the phosphorylation status of MAPK-related proteins and AKT in both the parental and VR melanoma cells and its possible connection with acquired resistance. In the VR cells, fluvastatin did not markedly change the phos- phorylation status of MEK and ERK proteins, whereas it reduced the phosphorylation of AKT (Thr308 and Ser473) in a dose-dependent manner (Fig. 3). The reduction did not coincide with an increase in the phos- phorylation of PTEN protein, dismissing the involve- ment of PTEN in this context (Supplementary Fig. 2, Supplemental digital content 2, http://links.lww.com/ACD/ A294). These observations, together with the increase of basal AKT phosphorylation in two of the three VR lines, suggest a crucial role of AKT in acquired resistance. Consistent with the synergy of cell survival, the AKT activity was reduced further by a combination of vemurafenib with fluvastatin than by fluvastatin alone (Fig. 4).

Primary or acquired resistance to vemurafenib often involves reactivation of the MAPK pathway itself as well as constitutive activation of the PI3K/AKT pathway. Shi and colleagues reported that the MAPK and PI3K/ PTEN/AKT pathways were altered in 70 and 22% of 44 patients with progressive tumors, respectively, and that in 20% of the cases, both pathways were activated simul- taneously [16]. Consistent with these observations, in the present study, we identified vemurafenib-refractory reactivation of the MAPK pathway in VR cells derived from all of the three melanoma lines and an increased level of constitutive phosphorylation of AKT kinase in cells from two of them.
In tumor cells with the BRAF V600E-mutated allele, the MAPK pathway becomes vemurafenib-refractory either through alterations that activate the target kinase(s) positioned downstream of BRAF, or acquisition of the BRAF bypass pathway through ARAF and/or CRAF, often coupled with upstream alterations [28,29]. The present study revealed no association between the reac- tivation of the MAPK pathway and specific molecular changes. In our preliminary experiments, however, treatment with a pan-RAF kinase inhibitor (A/B/C RAF kinase inhibitor, LY3009120) or trametinib (MEK inhi- bitor) did not have a strong suppressive effect on ERK phosphorylation in VR cells. In addition, COT over- expression, a well-known bypass that activates ERK- independent of BRAF [13], was not evident in any of the VR cells. These observations suggest that genetic alteration at the level of MEK or downstream may be involved in the reactivation. This possibility awaits clar- ification in a future study.

Constitutive activation of the PI3K/AKT pathway has been shown to occur through several distinct mechanisms such as NRAS mutation, overexpression of PDGFR or depletion of PTEN [30]. While we observed no notable reduction of PTEN phosphorylation, other possible causes were not explored in the present study. Impairment of the Hippo signaling pathway or hyper- activation of its downstream effector YAP/TAZ has attracted attention as another mechanism for vemur- afenib resistance in a subset of malignant melanoma [23]. Lin et al. [22] have shown that YAP functions as a parallel survival input to promote resistance to RAF–MEK inhibitor therapy. On the basis of these findings, we treated VR cells with fluvastatin expecting that its inhibitory effect on YAP-dependent transcription would potentiate cell-killing by vemurafenib. A synergistic effect was evident, but it was found to occur through inhibition of PI3K/AKT. Although a connection between statin- related drugs and the PI3K/AKT pathway has been suggested previously [25,31–33], it has not been eval- uated in the context of treatment for vemurafenib resis- tance. Preclinical studies have showed synergy between inhibitors for PI3K/AKT and those for BRAF or MEK kinases [34–38]. Statin-related drugs may have an advantage over other PI3K/AKT inhibitors as their pharmacological safety has been well established.

Our findings appear to provide evidence for the possible use of statin-related drugs for overcoming resistance to vemurafenib.
It should be noted that apparent nonessentiality of YAP/ TAZ in the present study may not be general features of the acquired vemurafenib resistance. Nevertheless, statin-related drugs might also be effective to overcome YAP-dependent form of vemurafenib resistance by the action on the mevalonate pathway, as we initially surmised. A recent study has showed that the Hippo–YAP axis also contributes to the suppression of antitumor immunity in addition to the acquisition of VR [39]. In this study, inactivation of LATS1/2 in mouse melanoma cells resulted in an increase of tumor immunogenicity and enhancement of tumor vaccine efficacy. Fluvastatin is attractive from this viewpoint also, with an ability to suppress PI3K/AKT and YAP, both acting to overcome vemurafenib resistance and the latter enhancing the degree of sensitivity to immune checkpoint therapy. Further research will be required to investigate these possibilities.

Acknowledgements
Conflicts of interest
There are no conflicts of interest.

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