Combination of eribulin plus AKT inhibitor evokes synergistic cytotoxicity in soft tissue sarcoma cells

An activated AKT pathway underlies the pathogenesis of soft tissue sarcoma (STS), with over-expressed phosphorylated AKT (p-AKT) correlating with a poor prognosis in a subset of STS cases. Recently, eribulin, a microtubule dynamics inhibitor, has demonstrated efficacy and is approved in patients with advanced/metastatic liposarcoma and breast cancer. However, mechanisms of eribulin resistance and/or insensitivity remain largely unknown. In this study, we demonstrated that an increased p-AKT level was associated with eribulin resistance in STS cells. We found a combination of eribulin with the AKT inhibitor, MK-2206, synergistically inhibited STS cell growth in vivo as well as in vitro. Mechanistically, eribulin plus MK-2206 induced G1 or G2/M arrest by down-regulating cyclin-dependent kinases, cyclins and cdc2, followed by caspase-dependent apoptosis in STS cells. Our findings demonstrate the significance of p-AKT signaling for eribulin-resistance in STS cells and provide a rationale for the development of an AKT inhibitor in combination with eribulin to treat patients with STS.

identified as a negative surrogate marker for the survival of patients with STS. Therefore, AKT may represent an attractive target for STS therapeutics.
In the current study, we performed phospho-protein arrays with HT1080 and eribulin resistant HT1080 cell lines to investigate the molecular mechanisms of eribulin resistance in STS cells. This assay revealed that p-AKT levels were up-regulated by eribulin treatment. These observations prompted us to assess the cytotoxicity of eribulin plus AKT inhibitor and whether this combination treatment can overcome eribulin resistance in STS cells. A combination of the AKT inhibitor, MK-2206, with eribulin resulted in synergistically induced G1 or G2/M arrest, followed by apoptosis in STS cells. Overall, our findings provide a clinical framework for using MK-2206 with eribulin to treat patients with UM-STS.
Growing evidence suggested oncogenes, tumor suppressor genes, and transporter pumps are linked to chemoresistance in numerous cancers 9 . In particular, the activation of oncogenes, such as PI3K/AKT 9 , ERK 14 , NF-κB 15 , EGFR 16 and PDGFR-β 17 , by phosphorylation induced chemoresistance in cancer cells. However, mechanisms of eribulin resistance are less well studied in STS cells. First, we directed our efforts toward screening the phosphorylation status of a panel of kinases, known as oncogenes, using a phospho-kinase array. As shown in Fig. 1B, the expression of PDGFR-β and p-AKT(S473) was increased in r2-HT1080 cells compared to parental cells. This data refers to biological triplicates. We analyzed the result of an array to select druggable targets that can be promptly applied to clinical studies. First, we focused on PDGFR-β, since this is one of the targets of pazopanib, a drug that has already been introduced to patients with STS 5 . We carried out an MTT assay to analyze the cytotoxicity of eribulin plus pazopanib ( Supplementary Fig. S1). Unfortunately, this combination did not trigger a synergistic effect in HT1080 cells. The change noted in p-AKT of r2-HT1080 cells was small. We also examined the expression of p-AKT(S473) protein levels in r1/r2-HT1080 and r1/r2-SK-LMS-1 cells by western blotting to confirm the array data. We observed the increased expression of p-AKT(S473) in all eribulin-resistant cell lines ( Fig. 2A). Moreover, p-AKT levels were up-regulated, in a dose-dependent fashion, by eribulin treatment for 48 h in parental HT1080, SK-LMS-1 and SW872 (liposarcoma) cell lines (Fig. 2B). These results indicated that eribulin resistance in STS cells may be associated with the phosphorylation of AKT(S473) triggered by eribulin.
The combination of eribulin and MK-2206 triggers synergistic anti-sarcoma activity. Based on our results, we expected that a combination of eribulin plus AKT inhibitor would be effective therapy to overcome eribulin resistance in STS. We chose MK-2206 as an AKT inhibitor in this study since this is an allosteric and www.nature.com/scientificreports www.nature.com/scientificreports/ highly selective inhibitor 18 , and has been evaluated in several clinical trials [19][20][21][22] . Initially, we assessed the cytotoxicity of eribulin and MK-2206, respectively. Eribulin and MK-2206 suppressed the growth of STS cell lines, with values for IC 50 shown for various cell lines (Fig. 3A,B). Subsequently, we conducted a study of combination treatment using an MTT assay to test whether MK-2206 with eribulin induced increased cytotoxicity compared with each monotherapy. A combination of eribulin plus MK-2206 induced synergistic cytotoxicity in parental STS cell lines at all indicated MK-2206 doses (Fig. 3C), accompanied by the inhibition of AKT phosphorylation ( Supplementary Fig. S2). Even in cells resistant to eribulin, a subset of combination doses triggered synergistic cytotoxicity (Fig. 3D). Of note, eribulin plus MK-2206 treatment at IC 50 values ( Fig. 1A and Supplementary  Table S1) suppressed cell growth in established STS cell lines with resistance to eribulin (Fig. 3D).

Eribulin plus MK-2206 promotes G1 or G2/M arrest in STS cells.
To elucidate the cytotoxic mechanisms of eribulin plus MK-2206 in STS cells, we examined the effects of combination treatment on the cell cycle. HT1080 and SK-LMS-1 cell lines were treated with eribulin with or without MK-2206 IC 25 concentrations, for 24 hours and analyzed by flow cytometry. Eribulin is known to induce G2/M arrest 23 , while MK-2206 promotes G1 arrest in cancer cells 18 . As expected, eribulin significantly induced G2/M arrest in STS cells ( Fig. 4A; P < 0.01), but its effect at the indicated concentrations in SK-LMS-1 cells was of a low power. MK-2206 treatment significantly increased the percentage of cells in the G1 phase and significantly decreased the percentage of cells in the S phase in both cell lines ( Fig. 4A; P < 0.05 for HT1080 and P < 0.01 for SK-LMS-1). Notably, eribulin plus MK-2206 treatment dramatically induced G1 arrest in HT1080 cells, and G2/M arrest in SK-LMS-1 cells ( Fig. 4A; P < 0.01 for both). To analyze the mechanism of G1 or G2/M arrest induced by eribulin plus MK-2206, we determined protein levels of several cell cycle-related proteins in STS cells using western blotting. The expression of the G1 arrest-associated proteins, CDK4/6 and cyclin D3, were apparently decreased, while p21 expression was increased (Fig. 4B). In terms of G2/M arrest, the expression of cdc2 and cyclin B1 was suppressed simultaneously. These results showed that combination treatment of eribulin plus the AKT inhibitor MK-2206 inhibited the proliferation of STS cells and this was accompanied by G1 or G2/M arrest with the down-regulation of CDK4/6, cdc2 and cyclin B1/D3.

Eribulin plus MK-2206 treatment induces caspase-dependent apoptosis.
To investigate whether eribulin plus MK-2206 might also evoke apoptosis in STS cells, we performed flow cytometry analysis. As shown in Fig. 5B, Annexin V/7-AAD staining showed a remarkably higher percentage of apoptosis among HT1080 and SK-LMS-1 cells. Treatment with the pan-caspase inhibitor, Q-VD-OPH, inhibited eribulin plus MK-2206-induced apoptosis. We next assessed whether the apoptosis was mediated via an intrinsic or extrinsic pathway using flow cytometry. The activities of caspases 8 and 9 were significantly up-regulated by eribulin plus MK-2206 treatment compared to each monotherapy cohort (Fig. 5B). These data suggested that eribulin plus MK-2206-induced apoptosis occurred medicated through intrinsic and extrinsic caspase-dependent pathways. Taken together, this combination therapy may be effective for UM-STS patients in a clinical setting.

Anti-tumor effect of eribulin plus MK-2206 in STS xenograft models.
To explore the clinical potential of eribulin plus MK-2206, we examined the ability of this combination therapy to inhibit STS tumor growth in vivo using a subcutaneous HT1080 murine xenograft model. Mice were treated with 0.25 mg/kg eribulin once a week in combination with, or without, 0.12 mg/kg MK-2206 three times per week for three weeks. Tumor burden was significantly suppressed by treatment with eribulin compared with vehicle, whereas mice treated with MK-2206 showed a progressive increase in tumor volume throughout the evaluation period (Fig. 6A,B). When www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ MK-2206 was administered in combination with eribulin, anti-tumor activity was dramatically enhanced as with in vitro studies. Strikingly, eribulin plus MK-2206 treatment led to complete responses in 4 of 5 mice, and was well tolerated, without causing significant body weight loss compared to the eribulin alone cohort in this experimental setting (Fig. 6C). No adverse effects in normal tissues were attributable to the administration of eribulin and/or MK-2206 upon necropsy during this study (data not shown). Collectively, these data suggested the possibility of applying this combination for UM-STS treatment.

Discussion
In the present study, our data demonstrated that AKT plays a key role in the resistance to eribulin by a subset of STS. These observations led us to evaluate combination therapy of eribulin with an AKT inhibitor to combat eribulin resistance and/or insensitivity in STS cells. Eribulin combined with MK-2206 dramatically inhibited STS cell growth in vivo as well as in vitro, suggesting its clinical application for patients with UM-STS whose prognosis is poor.
Eribulin is approved as a therapeutic for patients with UM-STS who have received a prior anthracycline-containing regimen, with liposarcoma as the indication in the United States and European Union 24 . In Japan, eribulin can be used for all histological types of STS. A phase III trial revealed eribulin treatment significantly prolonged OS for 2 months compared to dacarbazine treatment for L-sarcoma 7 . To further evaluate efficacy in a relapsed/refractory setting, it was reasonable to explore combination therapy of eribulin with other agents. Additionally, identifying mechanisms of eribulin resistance may help in choosing agents to combine with eribulin. We hypothesize that AKT may be an appropriate target that can be applied promptly in clinical trials, with reference to the results of a phospho-kinase array and of previous reports [19][20][21][22] . Therefore, we investigated the use of an AKT inhibitor in combination with eribulin to overcome eribulin resistance in STS cells. As expected, this combination therapy synergistically evoked cell-cycle arrest followed by apoptosis in vitro, and effectively exerted anti-tumor activity in vivo studies of STS cells. These proof-of-concept experiments highlight how eribulin plus an AKT inhibitor is a promising strategy for treating patients with UM-STS. Additionally, determining the expression level of p-AKT may be useful as a biological marker of eribulin responsiveness.
MK-2206 is a highly selective and orally active AKT inhibitor 18 . The efficacy and toxicity of MK-2206 for advanced solid tumors and hematological malignancies is under investigation in a clinical setting. A phase II study revealed that MK-2206 plus erlotinib was effective for advanced non-small cell lung cancer 20 . In addition, two phase II studies for lymphoma and chronic lymphocytic leukemia, respectively, yielded favorable results 19,21 . In terms of STS, one of three leiomyosarcoma patients showed stable disease for four months in a phase I study 22 . The doses of MK-2206 used in the current study were lower than those of previous studies in human cancers, which is encouraging for any future clinical trials.
It has been shown that breast cancer cells that harbor mutated PTEN or PIK3CA were sensitive to MK-2206 25 . HT1080 and SK-LMS-1 cells that harbor wild-type PTEN and PIK3CA were resistant to MK-2206 in its clinical therapeutic ranges. Therefore, mutations of PTEN and PIK3CA may be related to sensitivity to MK-2206 in HT1080 and SK-LMS-1 cell lines. Accumulating evidence demonstrated MK-2206 increased the cytotoxicity of chemotherapeutics among a subset of cancer types, not only in experimental setups 25,26 but in human studies also 19,20,27 . To our knowledge, this study was the first attempt to find an effective eribulin-based therapy combined with other agents for STS. MK-2206 was found to sensitize cancer cells to DNA-damaging agents 28 . Specifically, MK-2206 cytotoxicity was enhanced when combined with anti-microtubule inhibitors such as eribulin and paclitaxel 29 . As previously reported, our present studies demonstrated that MK-2206 enhanced the eribulin-induced anti-tumor effect. Eribulin combined with MK-2206 induced G1 or G2/M arrest in STS cells. Such different mechanisms of cytotoxicity toward STS cells may depend on the cell type. Notably, this combination therapy evoked an extensive anti-tumor effect in vivo compared to in vitro studies. One of the plausible mechanisms for the drastic responses observed in vivo may be immune responses activated by eribulin treatment 30 . However, further examination is required of eribulin in combination with MK-2206 in clinical studies of UM-STS patients.
In conclusion, this study demonstrated that activated AKT is associated with eribulin resistance in STS cells. Furthermore, eribulin in combination with MK-2206 synergistically suppresses STS growth through G1 or G2/M arrest, and, subsequently, caspase-dependent apoptosis. Our results highlight a potential clinical application for eribulin and MK-2206 combination therapy in the improvement of clinical outcomes in patients with UM-STS.

Materials and Methods
Reagents and human cell lines. Eribulin was kindly provided by Eisai Inc. (Tokyo, Japan) and Sapporo Medical University for in vitro and in vivo use, respectively. MK-2206 was obtained from ChemScene (Monmouth, NJ, USA). Pazopanib was obtained from AdooQ BioScience (Manassas, VA, USA). Stock solutions of these reagents were generated by dissolving the powder in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich St. Louis, MO, USA) at 10 mM. HT1080, SK-LMS-1 and SW872 cell lines were purchased from ATCC (Manassas, VA, USA). Both cell lines were maintained in DMEM (Sigma-Aldrich) containing 10% fetal bovine serum, 1% penicillin-streptomycin and 2 μM L-glutamine. establishment of eribulin-resistant sts cell lines. Eribulin-resistant STS cell lines were generated by culturing HT1080 and SK-LMS-1 cell lines in stepwise increasing doses (0.1-5.0 nM and 1.0-10 µM, respectively) of eribulin for more than 3 months. Resistant cell lines were named r1/r2-HT1080 and r1/r2-SK-LMS-1, and were continuously cultured in eribulin-containing media.
phospho-kinase assay. Alterations in the phosphorylation status of multiple kinases in eribulin resistance compared to parental cells were screened using a Human Phospho-Kinase Array Kit (Proteome Profiler TM Array; R&D Systems, Minneapolis, MD, USA), which contained 43 different kinases. Briefly, cell lysates were prepared and incubated with membranes. Phosphorylated kinases were determined with an anti-phospho antibody. The