Background
Melanoma accounts for a large proportion of skin-related deaths and its incidence and mortality is on the rise [
1,
2]. Despite advances in treatment options, the 5-year survival for patients suffering from late stage disease is only 20% [
2]. The current therapeutic landscape encompasses surgery to remove early stage melanomas, traditional chemotherapy and radiation therapy for the more advanced stages, targeted therapies as well as immunotherapy. An increased understanding of the molecular landscape driving melanoma particularly activating mutations such as BRAF
V600E harbored by 50% of melanoma patients, has led to the development of small molecule inhibitors designed to specifically target multiple nodes of the MAPK pathway [
3]. The approval of the Anti-CTLA checkpoint inhibitor Ipilimumab [
4] in 2011 ushered immunotherapies focused on targeting the PD1/PD-L1 axis. This has had a tremendous impact on the therapy landscape in treating patients with advanced melanoma improving not only overall survival but leading to long-term survival in some patients. However, resistance to targeted therapies as well immunotherapy where bio-markers of response are not yet well-established [
5,
6], present challenges in the treatment of melanoma. Although combinatorial approaches of the various targeted therapies together with immunotherapies are underway [
7], the high-costs [
5] associated with immunotherapy highlights an urgent need for novel anti-melanoma therapeutic options. The application of drugs used for alternate diseases as novel anti-cancer therapeutics, known as drug repositioning, has been successfully implemented in the clinical setting [
8] and these compounds can be a rich potential source of novel, readily available anti-cancer therapeutics.
We conducted a pharmacologic screen composed of the NIH Clinical Collection (NCC) of 770 small molecules, FDA-approved or which have been previously used in human clinical trials to identify novel anti-melanoma agents. Each molecule was screened in the murine B16F10 cell line and its half maximal inhibitory concentrations (IC50) was determined. Amongst the compounds whose IC50 values were in the low micromolar range, Tegaserod (TM), a serotonin receptor 4 (HTR4) agonist, validated successfully in secondary screening approaches with BRAF WT and BRAF
V600E human melanoma cell lines and was pursued in further in vitro and in vivo studies. In melanoma, serotonin has been found to increase melanogenesis via HTR2A, an effect that was reversed by HTR antagonists [
9]. And while HTR2B-C antagonists have been shown to inhibit migration in uveal [
10] and metastatic melanoma [
11], little is known about serotonin agonists, particularly HTR4 agonists in the context of this tumor type.
TM induced apoptosis in the B16F10 murine melanoma cell line as well as several human melanoma cell lines. In vivo, TM was well tolerated and efficacy was demonstrated in a syngeneic melanoma model testing primary tumor growth and metastasis. Importantly, TM strongly synergized with the standard of care BRAFV600E targeting Vemurafenib in human melanoma cell lines harboring this mutation. Mechanistically, TM suppressed PI3K/Akt/mTOR signaling converging on the ribosomal protein S6 (S6) in vitro and in vivo. PI3K/Akt/mTOR inhibition was likely responsible for TM’s pro-apoptotic effects and anti-metastatic effects in melanoma cell lines as pharmacological inhibition of the pathway using specific inhibitors recapitulated the apoptotic phenotype confirming the sensitivity of melanoma cells to PI3K/Akt/mTOR pathway perturbation.
Discussion
Our screen identified several potential hits with anti-melanoma activity including serotonin agonists and other compounds, such as statins, antihelmintics and antifungals which are already being re-purposed as anti-cancer agents pre-clinically or in the clinical setting. The serotonin signaling class of compounds that were positive hits in the original screen included serotonin agonists as well as the anti-depressants indatraline and maprotiline. The latter two are multi-functional and not only prevent the re-uptake of serotonin but also dopamine and norepinephrine and did not have appreciable anti-melanoma activity when compared to the other compounds in the serotonin signaling class including TM. Serotonin signaling occurs when serotonin, a neurotransmitter present in the gut, blood platelets and the central nervous system (CNS), binds to serotonin receptors (5-HTRs) resulting in complex physiological and behavioral changes affecting mood, cognition, digestion, pain perception [
18,
36]. The pharmacological opportunities to modulate these physiological processes and impact human disease are vast and have resulted in a plethora of 5-HTR agonist and antagonist ligands. There are seven families of human serotonin receptors mostly part of the G-protein coupled receptor family differentially expressed throughout the CNS, liver, kidney, heart, gut [
18]. We were intrigued by the possibility of investigating TM because the role of serotonin signaling in cancer remains controversial. Serotonin and 5-HTR2A agonists were found to induce melanogenesis in melanoma cell lines [
9]. Jiang et al. reported increased levels of serotonin and 5-HTR2B in human pancreatic ductal adenocarcinomas which promoted pancreatic tumor growth in mice [
37]. Many other studies have similarly reported growth stimulatory effects of serotonin signaling through various 5-HTRs and inhibitory effects of 5-HTR antagonists in many tumor types [
38,
39]. However, there have also been reports, albeit much fewer, suggesting that treatment with serotonin agonists might also have anti-cancer effects in glioma [
40] and breast cancer cells [
41]. Involvement within the autocrine loops and activation of the MAPK, JNK, PI3K/Akt/mTOR [
37,
38] pathways has been implicated in serotonin’s mitogenic role.
We did not observe any pro or anti-mitogenic effects following treatment with serotonin (5-HT) in melanoma cells. Co-treatment of TM with 5-HT did not effect the compound’s ability to induce apoptosis in the melanoma cells. This suggests that the affinity of the synthetic ligand TM is stronger for the 5-HTR’s than for the natural ligand 5-HT, and/or that the pro-apoptotic effects of TM can be uncoupled from serotonin signaling. Treatment with 5-HTR ligands, agonists or antagonist presents a complex scenario. As previously reported [
38] treatment with one ligand can yield opposing concentration dependent results. Serotonin signaling following TM treatment might occurring through other 5-HTRs. TM has been reported to be an agonist for the 5-HTR1A-D and an antagonist for 5-HTR2A-B [
42]. In our case, we used doses in the low micromolar range, high enough to elicit tumor apoptosis inducing pleiotropic effects [
42,
43]. Although we did not observe significant changes in cAMP levels and 5-HT responsive genes following TM treatment in most of the cell lines, increased p-CREB levels were observed in the SH4 and MEL-JUSO cells suggesting a possible involvement of other serotonin receptors including ones previously unidentified as being targets for TM. However, other antagonists and agonists present in the screen including the 5-HTR4 agonists (Cisparide) that did not have any anti-cancer effects further suggesting that TM is uniquely acting to distinctly target other molecules, likely upstream receptors or kinases of the PI3K/Akt/mTOR pathways.
The current repertoire of clinically approved treatment options in melanoma encompasses agents that inhibit proliferation and induce cell death [
44]. This includes targeted inhibitors of the BRAF pathway and checkpoint inhibitors. The former class of agents such as Vemurafenib cause cell arrest and trigger apoptosis [
35,
45] while the checkpoint inhibitors cause immunogenic cell death through lytic and apoptotic cell death mediated by activated CD8
+ T and NK cells respectively [
6,
44]. Resistance to the targeted inhibitors and variable checkpoint inhibitor response rates has shifted the focus in recent years interest to finding novel combination treatments to overcome resistance and increase response rates [
7]. Strategies include targeting other forms of cell death such as necroptosis [
46], inhibiting MAPK reactivation that occurs following targeted therapy treatment, and concomitantly inhibiting other pathways including the PI3K/Akt/mTOR [
47,
48].
Recently, a report has shown phosphorylation of S6 to be marker of sensitivity to BRAF mutated melanoma and that suppression of S6 after MAPK treatment was a predictor of progression-free survival [
23]. In our investigation, TM’s suppression of p-S6 and its strong synergy with vemurafenib in BRAF mutated human melanoma cell lines is in accordance with the above report. Importantly, TM also suppressed S6 phosphorylation in non-BRAF mutated melanoma cell lines indicating a broader therapeutic potential of TM in patients without the BRAF mutation but where the PI3K/Akt/mTOR pathway is activated such as in patients harboring NRAS mutations [
48]. The suppression of S6 phosphorylation is likely mediated by decreased mTORC1 activity as phosphorylation of the direct upstream regulator of S6, p70 S6 kinase was also blunted. mTORC1 integrates several upstream pathways related to cellular growth and metabolism including MAPK through RSK [
22], PI3K/Akt [
25] as well as the liver kinase B1 (LKB1)-adenosine monophosphate-activated protein kinase (AMPK) [
49]. As TM did not perturb the MAPK pathway but decreased Akt phosphorylation at a residue known to be phosphorylated by mTORC2 [
27], it’s likely that S6 is affected through the PI3K/Akt pathway although the potential contribution of AMPK would also have to be explored. Interestingly, Yoon et al. found that dual mTORC1/2 inhibition following treatment with Torin1 in A375 melanoma cells induced focal adhesion re-organization, increased the size of focal adhesions and increased migration and invasion
in vitro [
29]. TM did not phenocopy Torin 1 using B16F10 cells, as treatment with TM decreased the number metastases in vivo in an immuno-competent murine model where the presence of tumor infiltrating lymphocytes was considered. The immunosuppressive and pro-tumorigenic contribution of regulatory CD4
+ T cells in the tumor microenvironment is well established [
50]. As the infiltration of FOXP3 expressing CD4
+ T cells and regulatory CD4
+ T cells in TM treated tumors was decreased, this likely contributes to TM’s anti-cancer effects in vivo
.
Tegaserod (Zelnorm, Zelmac) which is used for the treatment of irritable bowel syndrome (IBS) [
51,
52] was also shown to be effective against chronic constipation [
53]. Although Tegaserod was well-tolerated and effective, it was removed off the market in the Unites States in 2007 at the FDA’s request [
54] chiefly due cardiovascular (CV) safety concerns raised through retrospective clinical trial analysis. However, all adverse cardiovascular events occurred in patients with CV disease and/or CV risk factors. Furthermore, the link between Tegaserod and adverse CV outcomes was not recapitulated in subsequent epidemiological studies [
55,
56] which found no association between Tegaserod use and adverse CV’s. The tolerability and availability of the drug would likely outweigh the relatively low cardiovascular risk (0.1%) associated with Tegaserod usage especially in melanoma patients with few treatment options. In vivo
, TM retarded decreased metastatic and primary tumor growth, induced apoptosis and suppressed p-Akt and p-S6 in tumor cells. TM is available in generic form and has the potential to be re-purposed as an anti-melanoma agent. The dose we used in mice, 5 mg/kg, once daily, is roughly equivalent to a Human Equivalent Dose (HED) [
57] of 0.405 mg/kg. Given that TM is available as a 6 mg pill administered twice daily, the doses we used in our in vivo studies are within the physiological range. Furthermore, as the compound synergized with Vemurafenib and other kinase inhibitors currently used in melanoma patients with late-stage disease, this is likely a favorable point of clinical entry especially since most patients eventually develop resistance to Vemurafenib and other kinase inhibitors [
7,
47]. Furthermore, as the BRAF WT cohort of patients are a diverse group, treatment options are much less clear cut [
44,
58] although immunotherapies, as with BRAF
V600E melanoma are a promising albeit costly treatment approach [
59]. Currently there are a lot of different combinations in clinical trials using MEK in combination with inhibitors of the PI3K/AKT/mTOR axis [
58] (NCT01941927, NCT01363232, NCT01337765).
Materials and methods
Cell culture and compounds
B16F10, A375, SH4, RPMI-7951 and SK-MEL-24 melanoma cell lines were purchased from ATCC. MeWo and MEL-JUSO cell lines were kindly provided by Dr. A. Roesh (Universitätsklinikum Essen, Essen, Germany). The MEL-JUSO and MeWo cell lines were both originally purchased from ATCC. B16F10 murine cells, A375 and SH4 human malignant melanoma cell lines were maintained Dulbecco Modified Eagle’s Medium (DMEM). Human RPMI-7951 malignant melanoma cells were maintained in Eagle’s MEM. SK-MEL-24 were maintained in Eagle in Earle’s BSS with non-essential amino acids. MeWo and MEL-JUSO cell lines were maintained in Roswell Park Memorial Institute (RPMI) medium. All media were supplemented with 10% FCS (15% for SK-MEL-24) and penicillin streptomycin. Cells were incubated at 37 °C in 5% CO2, and all cell lines were routinely confirmed to be mycoplasma-free (MycoAlert Mycoplasma Detection Kit, Lonza). The NIH Clinical Collection (NCC) composed of 770 small molecules mainly dissolved in DMSO at a concentration of 10 μM was obtained from the NIH, Tegaserod (Sigma) was dissolved in DMSO, serotonin (Sigma) was dissolved in water. MK-2206, ZSTK474, KU-0063794, Vemurafenib, Cobimetinib (Selleckchem) were dissolved in DMSO.
MTT assays
For the MTT colorimetric assay, cells were seeded in 96 well plates and viability was assessed following addition of the MTT (Sigma) reagent. Half-maximal inhibitory concentrations (IC50) values were computed from dose–response curves using Prism (v5.0, GraphPad Software).
Flow Cytometry
For Annexin V/7AAD apoptosis assays, trypsinized cells were washed and stained in Annexin V binding buffer (BD Biosciences). Melanoma cells were treated at doses of 2 x – 4x IC50 values for TM and 2 x IC50 for PI3K/Akt/mTOR inhibitors. Stainings of CD4+ cells for FOXP3, RORγ and GATA3 and of CD8+ cells for Granzyme B, perforin and IFNγ were performed using the Foxp3 mouse Treg cell staining buffer kit (eBioscience). Cells were analyzed using FACS (FACS Fortessa, BD Biosciences).
Immunofluorescence
For TUNEL staining, cells were seeded on cover slips, treated and 48 h later fixed by 4% formaldehyde in PBS for 30 min, permeabilized with 0.1% Triton X-100, 0.1% sodium citrate in PBS for 2 min and stained using the TUNEL staining kit as per manufacturer’s protocol (Roche). For p-S6 staining, cells seeded on cover slips were stained with primary anti-p-S6 antibody (Ser 235/6, Cell Signaling) overnight, followed by incubation with secondary anti-Rabbit IgG Cy3 conjugate antibody. Cover slips were incubated with DAPI in PBS for 30 min. Images were taken with an Axiocam 503 color microscope (ZEISS).
Immunoblotting
Cells were lysed using boiling hot SDS lysis buffer (1.1% SDS, 11% glycerol, 0.1 mol/L Tris, pH 6.8) with 10% β-mercaptoethanol. Tumor tissue was crushed using a tissue lyser (TissueLyser II, QIAGEN) and cells were gently lysed using Triton X-100. Blots were probed with anti-α-tubulin (Merck), anti-HTR4 (ThermoFischer), anti-cleaved Caspase 8, anti-Akt, anti-p-Akt (Ser 473), anti-S6, anti-p-S6 (Ser235/6, Ser240/4), anti-p70 S6, anti-p-p70 S6 (Thr421/Ser424), anti-p-ERK1/2, anti-ERK1/2, anti-p-CREB (Ser133) and anti-CREB (all from Cell Signaling) and detected using the Odyssey infrared imaging system (Odyssey Fc, LI-COR Biosciences). Immunoblots were quantified using ImageJ.
Combination index (CI) determination
Synergy between TM and Vemurafenib, and Cobimetinib was evaluated by calculating the CI [
60]. Dose–response curves were generated for TM, Vemurafenib and Cobimetinib alone and each drug in combination with TM at a constant ratio following compound exposure for 72 h. Viability was assessed by the MTT assay. CompuSyn software was used to evaluate synergy using the median-effect model.
Histology
Histological analysis was performed on snap frozen tissue. Briefly, snap-frozen tissue sections fixed in acetone, blocked with 10% FCS and stained with anti-active Caspase 3 (BD Biosciences), cleaved Caspase 8 (Cell Signaling). For p-S6 (Cell Signaling) staining, snap-frozen tissue sections were fixed in 10% neutral buffered formalin and blocked with 5% FCS/ 0.3% Triton X-100 in PBS. Images were taken with an Axiocam 503 color microscope (ZEISS) and quantified using Image J. For conventional immunohistochemistry tumor slides, IHC profiler Image J plugin was used as previously described in detail [
32].
Serum biochemistry
Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and L-Lactatdehydrogenase (LDH) were measured using the automated biochemical analyser Spotchem EZ SP-4430 (Arkray, Amstelveen, Netherlands) and the Spotchem EZ Reagent Strips Liver-1.
Quantitative RT-PCR
RNA was isolated using Trizol (Invitrogen) and RT-PCR analyses were performed using the iTaq™ Universal SYBR® GreenOne-Step RT-qPCR Kit (Biorad) according to the manufacturer’s instructions. For analysis, expression levels were normalized to GADPH.
Intracellular CAMP assay
Intracellular CAMP levels were determined as per manfacturer’s instructions (Enzo Life Biosciences).
Mice and in vivo treatments
C57BL/6 J mice were maintained under specific pathogen-free conditions. Seven to nine week old C57BL/6 J mice were subcutaneously injected with 5 × 105 B16F10 cells. Seven days post injection, when tumor volume reached approximately 50 mm3, mice were randomized and treated daily for 5 consecutive days with 5 mg/kg Tegaserod or vehicle control (2.5% DMSO in PBS). Tegaserod and vehicle were administered intraperitoneally (i.p.). Tumors were measured using calipers and tumor volume was calculated using the following formula: (tumor length x width2)/2. For metastases quantification experiments, C57BL/6 J mice were intravenously injected with 2 × 105 B16F10 cells and treatment with Tegaserod and vehicle (administered i.p.) occurred 1 day post inoculation and continued three times weekly till day 14 post inoculation at which time mice were sacrificed. Metastases from lungs, stored in PBS for short term storage, were manually counted. For survival experiments, C57BL/6 J mice were intravenously injected with 105 B16F10 cells. Treatment with Tegaserod and vehicle (administered i.p.) occurred 1 day post inoculation and continued three times weekly till day 17 post inoculation. Experiments were performed under the authorization of LANUV in accordance with German law for animal protection.
Data mining using the CCLE
RNA-Seq expression data (Read Count) from the Cancer Cell Line Encyclopedia (CCLE) [
16] (Broad Institute and Genomics Institute of the Novartis Research Foundation) for the selected human melanoma cell lines was analyzed using Xena Functional Genomics Explorer [
61] and visualized using the MORPHEUS matrix visualization software (
https://software.broadinstitute.org/morpheus).
Statistical analyses
Data are expressed as mean ± S.E.M. Statistically significant differences between two groups were determined using the student’s t-test and between three or more groups, the one-way ANOVA was used with a post-hoc Dunnett test. To assess significance between Kaplan Meier survival curves, the log-rank test was used. Values of P < 0.05 were considered statistically significant.
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