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Dual inhibition of histone deacetylases and the mechanistic target of rapamycin promotes apoptosis in cell line models of uveal melanoma

ABSTRACT: We investigated the effects of HDACi romidepsin and mTORi AZD-8055 in uveal melanoma cell line Mel202. Uveal melanomas harbor mutations in the GNAQ or GNA11 genes that activate survival pathways. As previous studies found that Ras-mutated cell lines were uniquely vulnerable to a combination of inhibitors of Ras-activated survival pathways and the histone-deacetylase inhibitor (HDI) romidepsin, we investigated whether this combination would be effective in models of uveal melanoma. A small-scale screen of inhibitors of bromodomain-containing protein 4 (BRD4, OTX-015), extracellular signal-related kinase (ERK, ulixertinib), mammalian target of rapamycin (mTOR, AZD-8055) or phosphoinositide 3-kinase (PI3K, GDC-0941) combined with a clinically-relevant administration of romidepsin was performed on a panel of uveal melanoma cell lines (92-1, Mel202, MP38, and MP41) and apoptosis was quantified by flow cytometry after 48 hr. RNA sequencing analysis was performed on Mel202 cells treated with romidepsin alone, the mTOR inhibitor AZD-8055 or the combination, and protein changes were validated by immunoblot. We found combining AZD-8055 with romidepsin was the most effective combination in inducing apoptosis in the cell lines. Increased caspase-3 and PARP cleavage were noted in the MP41 and Mel202 cell lines when they were treated with romidepsin and mTOR inhibitors. RNA sequence analysis of Mel202 cells revealed that apoptosis was the most affected pathway in the romidepsin/AZD-8055 treated cells. Increases in pro-apoptotic BCL2L11 and decreases in anti-apoptotic BIRC5 and BCL2L1 proteins noted in the sequencing analysis were confirmed at the protein level. Our data suggest that romidepsin in combination with mTOR inhibition could be an effective treatment strategy against uveal melanoma due in part to changes in apoptotic proteins.

ORGANISM(S): Homo sapiens

PROVIDER: GSE155452 | GEO | 2020/12/30

REPOSITORIES: GEO

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Project description:Combining direct anti-tumor effects with targeting of tumor microenvironment is an attractive strategy that may lead to more effective therapies for advanced melanoma. Here, we tested whether association of TRAIL with co-targeting of MEK and PI3K/mTOR, or with MEK blockade, could have synergistic anti-melanoma activity mediated not only by induction of tumor cell death, but also by effects on the tumor microenvironment. Drug interaction analysis by the Chou-Talalay approach in a panel of 21 melanoma cell lines indicated that strong synergism could be achieved by association of the MEK1/2 inhibitor AZD6244, the PI3K/mTOR inhibitor NPV-BEZ235 and TRAIL, as well as by the AZD6244/TRAIL combination. Synergism was observed on most tumors including TRAIL- or inhibitor-resistant melanomas, irrespective of the BRAF, NRAS, p53 and PTEN status, and was explained by enhanced induction of caspase-dependent melanoma apoptosis. The AZD/BEZ/TRAIL and AZD/TRAIL combinatorial treatments induced strong modulation of key apoptosis regulators along the extrinsic and intrinsic cell death pathways, including c-FLIP down-regulation, caspase-8 cleavage, BIMs and BAXa upregulation, clusterin and Mcl-1 inhibition, enhanced mitochondrial depolarization, suppression of inhibitors of apoptosis c-IAP1, c-IAP2, XIAP and Apollon, and caspase 3/7 activation. In an in vivo model, the AZD6244/TRAIL combinatorial treatment induced highly significant growth inhibition of a TRAIL-resistant tumor associated not only with melanoma cell death, but even with suppression of pro-angiogenic molecules HIF1a, VEGFa, IL-8 and TGFb1, and with inhibition of tumor angiogenesis. These results provide a proof of principle supporting the rationale for combinatorial treatments with synergistic anti-melanoma activity based on direct and indirect anti-tumor effects. The human melanoma cell line Me13 was established in our laboratory from a surgical specimen. Cells were routinely maintained in RPMI medium 1640 (Lonza, Basel, Switzerland) supplemented with 10% FBS (Lonza) and 2 mM glutamine (Lonza). Cells were maintained at 37°C in a water-saturated atmosphere of 5% CO2 in air. 3x10^6 cells were seeded in 75 cm2 flasks; after 24 hours, cells were left untreated or treated with Selumetinib (Selleck Chemicals, Houston, TX) at 0.1 micromol/L, NVP-BEZ-235 (Selleck Chemicals, Houston, TX) at 0.1 micromol/L and sTRAIL (AdipoGen) at 25ng/ml as single drugs or in combination for 8 hours. Each treatment or combination was performed in triplicate. At the end of treatment, cells were collected and RNA extracted.

Project description:PURPOSE: To assess the role of Apollon in melanoma resistance to intrinsic and extrinsic pathways of apoptosis and to identify strategies to reduce its expression. EXPERIMENTAL DESIGN: Expression of Apollon was assessed in melanoma cell lines and in surgical specimens.Gene expression profiling, mitochondrial depolarization, caspases activation and apoptosis assays were used to test the effect of Apollon silencing, by siRNA, on melanoma response to different anti-tumor agents. Levels of expression of Apollon and melanoma apoptosis were assessed after tumor treatment with the combination of MEK- and mTOR- inhibitors, or of anti-HLA class II mAbs and anti-tumor agents. RESULTS: Melanoma cells constitutively expressed Apollon, in-vitro and in-vivo. Extent of melanoma apoptosis correlated significantly with Apollon downmodulation upon treatment with a MEK inhibitor, a BRAFV600E-specific inhibitor or the nitrosourea fotemustine. Apollon silencing enhanced melanoma apoptosis in response to the cytotoxic drugs camptothecin, celecoxib, fotemustine and temozolomide, to MEK- and BRAFV600E-specific inhibitors, and to soluble or membrane-bound TRAIL. Mechanistically, Apollon silencing in melanoma cells promoted mitochondrial depolarization, caspase-2, -8, -9 and -3 activation and alteration in the expression of genes related to different cellular functions in response to different anti-tumor agents. Treatment of melanoma cells with combination of MEK and of mTOR inhibitors, or of HLA class II antigen-specific mAb and anti-tumor agents, promoted Apollon downmodulation, associated with enhanced apoptotic responses. CONCLUSIONS: The results suggest that Apollon is a relevant molecule in melanoma resistance to apoptosis, and that strategies aimed at downmodulating Apollon protein may improve melanoma cell death in response to anti-tumor agents that trigger the intrinsic or the extrinsic apoptosis pathways. The human melanoma cell line Me23682 was established in our laboratory from a surgical specimen. Cells were routinely maintained in RPMI medium 1640 (Lonza, Basel, Switzerland) supplemented with 10% FBS (Lonza) and 2 mM glutamine (Lonza). Cells were maintained at 37°C in a water-saturated atmosphere of 5% CO2 in air. 3x106 cells were seeded in 10 cm dishes; after 24 hours, cells were transfected with Apollon-specific siRNA or its unrelated control (Stealth RNAi siRNA, Invitrogen Life Technologies, Camarillo, CA) at a final concentration of 75 nmol/L and then left untreated or treated with fotemustine (Muphoran, Italfarmaco, Milan, Italy) at 150 umol/L for 6 hours, or with the BRAFV600E-specific inhibitor PLX4720 (Selleck Chemicals, Houston, TX) at 500 nmol/L for 8 hours, or with the MEK inhibitor PD0325901 (Cayman Chemicals, Ann Arbor, MI) at 5 nmol/L for 8 hours. Each treatment or combination was performed in triplicate. At the end of treatment, cells were collected and RNA extracted.

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Dual inhibition of histone deacetylases and the mechanistic target of rapamycin promotes apoptosis in cell line models of uveal melanoma

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