Salubrinal in Combination With 4E1RCat Synergistically Impairs Melanoma Development by Disrupting the Protein Synthetic Machinery.

Increased protein synthesis is a key process in melanoma, which is regulated by the ALDH18A1 gene encoding pyrroline-5-carboxylate synthase (P5CS). P5CS is involved in proline biosynthesis and targeting ALDH18A1 has previously been shown to inhibit melanoma development by decreasing intracellular proline levels to increase the phosphorylation of eIF2α mediated by GCN2, which then impairs mRNA translation. Since there are no current inhibitors of P5CS, decreased eIF2α phosphorylation in melanoma was targeted using salubrinal (a specific inhibitor of eIF2α phosphatase enzymes). While salubrinal alone was ineffective, the combined use of salubrinal and 4E1RCat (a dual inhibitor of eIF4E:4E-BP1 and eIF4E:eIF4G interaction to prevent assembly of the eIF4F complex and inhibit cap-dependent translation) was found to be effective at decreasing protein synthesis, protein translation, and cell cycle progression to synergistically decrease melanoma cell viability and inhibited xenograft melanoma tumor development. The combination of these agents synergistically decreased melanoma cell viability while having minimal effect on normal cells. This is the first report demonstrating that it is possible to inhibit melanoma viability by targeting eIF2α signaling using salubrinal and 4E1RCat to disrupt assembly of the eIF4F complex.

Identification of WEE1 as a target to make AKT inhibition more effective in melanoma.

AKT3 is one of the major therapeutic targets in melanoma but clinically targeting AKT3 alone seems to be an ineffective therapeutic approach. To identify unique strategies to enhance the efficacy of targeting AKT3, a screen was undertaken where AKT3 was co-targeted with a panel of kinases important in melanoma development. The screen identified WEE1 as the most potent target that when inhibited along with AKT3 would enhance the efficacy of targeting AKT3 in melanoma. RNAi mediated inhibition of AKT3 and WEE1 synergistically inhibited the viability of melanoma cells leading to a 65-75% decrease in tumor development. This approach was effective by mechanistically modulating pathways associated with the transcription factors p53 and FOXM1. Simultaneously regulating the activity of these two transcriptionally driven pathways, cooperatively deregulated cell cycle control and DNA damage repair to synergistically kill melanoma cells. This study uniquely identifies a potential approach to improve the efficacy of targeting AKT3 in melanoma.

Improving pharmacological targeting of AKT in melanoma.

Targeting AKT with pharmacological agents inhibiting this protein in the melanoma clinic is ineffective. This is a major contradiction considering the substantial preclinical data suggesting AKT as an effective target. Various approaches have been undertaken to unravel this contradiction and drug combinations sought that could resolve this concern. We have shown that genetic targeting AKT3 or WEE1 can be effective for inhibiting tumor growth in preclinical animal models. However, no one has examined whether combining pharmacological agents targeting each of these enzymes could be more effective than inhibiting each alone and enhance the efficacy of targeting AKT in melanoma. This report shows that combining the AKT inhibitors (AZD5363 or MK1775) with the WEE1 inhibitor, AZD5363, can synergistically kill cultured melanoma cells and decrease melanoma tumor growth by greater than 90%. Co-targeting AKT and WEE1 led to enhanced deregulation of the cell cycle and DNA damage repair pathways by modulating the transcription factors p53 and FOXM1, as well as the proteins whose expression is regulated by these two proteins. Thus, this study identifies a unique combination of pharmacological agents and the ratio needed for efficacy that could be used to potentially improve the therapeutic effectiveness of targeting AKT in the clinic.

Nanoparticle-Based Celecoxib and Plumbagin for the Synergistic Treatment of Melanoma.

Using multiple drugs to kill cancer cells can decrease drug resistance development. However, this approach is frequently limited by the bioavailability and toxicity of the combined agents and delivery at ratios to specific locations that synergistically kill cancer cells. Loading the individual agents into a nanoparticle that releases the drugs at synergizing ratios at a single location is one approach to resolve this concern. Celecoxib and plumbagin are two drugs that were identified from a screen to synergistically kill melanoma cells compared with normal cells. Combined use of these agents by traditional approaches was not possible due to poor bioavailability and toxicologic concerns. This study details the development of a nanoliposomal-based agent containing celecoxib and plumbagin, called CelePlum-777, which is stable and releases these drugs at an optimal ratio for maximal synergistic killing efficacy. CelePlum-777 was more effective at killing melanoma than normal cells and inhibited xenograft melanoma tumor growth by up to 72% without apparent toxicity. Mechanistically, the drug combination in CelePlum-777 led to enhanced inhibition of melanoma cell proliferation mediated by decreasing levels of key cyclins important for cancer cell proliferation and survival, which was not observed with the individual agents. Thus, a novel nanoparticle-based drug has been developed containing celecoxib and plumbagin that lacks toxicity and delivers the agents at a synergistically killing drug ratio to kill cancer cells. Mol Cancer Ther; 16(3); 440-52. ©2016 AACR.

Therapeutic interventions to disrupt the protein synthetic machinery in melanoma.

Control of the protein synthetic machinery is deregulated in many cancers, including melanoma, to increase the protein production. Tumor suppressors and oncogenes play key roles in protein synthesis from the transcription of rRNA and ribosome biogenesis to mRNA translation initiation and protein synthesis. Major signaling pathways are altered in melanoma to modulate the protein synthetic machinery, thereby promoting tumor development. However, despite the importance of this process in melanoma development, involvement of the protein synthetic machinery in this cancer type is an underdeveloped area of study. Here, we review the coupling of melanoma development to deregulation of the protein synthetic machinery. We examine existing knowledge regarding RNA polymerase I inhibition and mRNA translation focusing on their inhibition for therapeutic applications in melanoma. Furthermore, the contribution of amino acid biosynthesis and involvement of ribosomal proteins are also reviewed as future therapeutic strategies to target deregulated protein production in melanoma.

Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.

UNLABELLED: Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway. IMPLICATIONS: This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy.

Growth inhibitory effects of large subunit ribosomal proteins in melanoma.

Ribosome biogenesis can modulate protein synthesis, a process heavily relied upon for cancer cell proliferation. In this study, involvement of large subunit ribosomal proteins (RPLs) in melanoma has been dissected and RPLs categorized based on modulation of cell proliferation and therapeutic targeting potential. Based on these results, two categories of RPLs were identified: the first causing negligible effects on cell viability, p53 expression, and protein translation, while the second category decreased cell viability and inhibited protein synthesis mediated with or without p53 protein stabilization. RPL13 represents the second category, where siRNA-mediated targeting inhibited tumor development through decreased cellular proliferation. Mechanistically, decreased RPL13 levels increased p53 stability mediated by RPL5 and RPL11 binding to and preventing MDM2 from targeting p53 for degradation. The consequence was p53-dependent cell cycle arrest and decreased protein translation. Thus, targeting certain category 2 RPL proteins can inhibit melanoma tumor development mediated through the MDM2-p53 pathway.