MicroRNA-26a Is Strongly Downregulated in Melanoma and Induces Cell Death through Repression of Silencer of Death Domains (SODD)

Melanoma is an aggressive cancer that metastasizes rapidly and is refractory to conventional chemotherapies. Identifying microRNAs (miRNAs) that are responsible for this pathogenesis is therefore a promising means of developing new therapies. We identified miR-26a through microarray and quantitative reverse-transcription–PCR (qRT-PCR) experiments as an miRNA that is strongly downregulated in melanoma cell lines as compared with primary melanocytes. Treatment of cell lines with miR-26a mimic caused significant and rapid cell death compared with a negative control in most melanoma cell lines tested. In surveying targets of miR-26a, we found that protein levels of SMAD1 (mothers against decapentaplegic homolog 1) and BAG-4/SODD were strongly decreased in sensitive cells treated with miR-26a mimic as compared with the control. The luciferase reporter assays further demonstrated that miR-26a can repress gene expression through the binding site in the 3′ untranslated region (3′UTR) of SODD (silencer of death domains). Knockdown of these proteins with small interfering RNA (siRNA) showed that SODD has an important role in protecting melanoma cells from apoptosis in most cell lines sensitive to miR-26a, whereas SMAD1 may have a minor role. Furthermore, transfecting cells with a miR-26a inhibitor increased SODD expression. Our findings indicate that miR-26a replacement is a potential therapeutic strategy for metastatic melanoma, and that SODD, in particular, is a potentially useful therapeutic target

The Combination of BH3-Mimetic ABT-737 with the Alkylating Agent Temozolomide Induces Strong Synergistic Killing of Melanoma Cells Independent of p53

Metastatic melanoma has poor prognosis and is refractory to most conventional chemotherapies. The alkylating agent temozolomide (TMZ) is commonly used in treating melanoma but has a disappointing response rate. Agents that can act cooperatively with TMZ and improve its efficacy are thus highly sought after. The BH3 mimetic ABT-737, which can induce apoptosis by targeting pro-survival Bcl-2 family members, has been found to enhance the efficacy of many conventional chemotherapeutic agents in multiple cancers. We found that combining TMZ and ABT-737 induced strong synergistic apoptosis in multiple human melanoma cell lines. When the drugs were used in combination in a mouse xenograft model, they drastically reduced tumor growth at concentrations where each individual drug had no significant effect. We found that TMZ treatment elevated p53 levels, and that the pro-apoptotic protein Noxa was elevated in TMZ/ABT-737 treated cells. Experiments with shRNA demonstrated that the synergistic effect of TMZ and ABT-737 was largely dependent on Noxa. Experiments with nutlin-3, a p53 inducer, demonstrated that p53 induction was sufficient for synergistic cell death with ABT-737 in a Noxa-dependent fashion. However, p53 was not necessary for TMZ/ABT-737 synergy as demonstrated by a p53-null line, indicating that TMZ and ABT-737 together induce Noxa in a p53-independent fashion. These results demonstrate that targeting anti-apoptotic Bcl-2 members is a promising method for treating metastatic melanoma, and that clinical trials with TMZ and Bcl-2 inhibitors are warranted

Combining a BCL2 Inhibitor with the Retinoid Derivative Fenretinide Targets Melanoma Cells Including Melanoma Initiating Cells

Investigations from multiple laboratories support the existence of melanoma initiating cells (MICs) that potentially contribute to melanoma’s drug resistance. ABT-737, a small molecule BCL-2/BCL-XL/BCL-W inhibitor, is promising in cancer treatments, but not very effective against melanoma, with the antiapoptotic protein MCL-1 as the main contributor to resistance. The synthetic retinoid fenretinide N-(4-hydroxyphenyl)retinamide (4-HPR) has shown promise for treating breast cancers. Here, we tested whether the combination of ABT-737 with 4-HPR is effective in killing both the bulk of melanoma cells and MICs. The combination synergistically decreased cell viability and caused cell death in multiple melanoma cells lines (carrying either BRAF or NRAS mutations) but not in normal melanocytes. The combination increased the NOXA expression and caspase-dependent MCL-1 degradation. Knocking down NOXA protected cells from combination-induced apoptosis, implicating the role of NOXA in the drug synergy. The combination treatment also disrupted primary spheres (a functional assay for MICs) and decreased the percentage of aldehyde dehydrogenase high cells (a marker of MICs) in melanoma cell lines. Moreover, the combination inhibited the self-renewal capacity of MICs, measured by secondary sphere-forming assays. In vivo, the combination inhibited tumor growth. Thus, this combination is a promising treatment strategy for melanoma, regardless of mutation status of BRAF or NRAS

ABT-737 synergizes with Bortezomib to kill melanoma cells

Summary The BH3 mimetic ABT-737 is a potent inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-XL, and Bcl-w. The Bcl-2 family modulates sensitivity to anticancer drugs in many cancers, including melanomas. In this study, we examined whether ABT-737 is effective in killing melanoma cells either alone or in combination with a proteasome inhibitor already in clinical use (Bortezomib) in vitro and in vivo, and further evaluated the mechanisms of action. Results showed that ABT-737 alone induced modest cytotoxicity in melanoma cells, but only at higher doses. Knock-down of the anti-apoptotic proteins Bcl-2, Bcl-XL, or Mcl-1 with siRNAs demonstrated that Mcl-1 is the critical mediator of melanoma's resistance to ABT-737 treatment. However, ABT-737 displayed strong synergistic lethality when combined with Bortezomib. Immunoblot analyses demonstrated that Bortezomib increased expression of Noxa, a pro-apoptotic Bcl-2 member that antagonizes Mcl-1. Additionally, siRNA-mediated inhibition of Noxa expression protected melanoma cells from cytotoxicity induced by the combination treatment. These results demonstrate that Bortezomib synergizes with ABT-737 by neutralizing Mcl-1's function via increased levels of Noxa. In a xenograft mouse model, although drug doses were limited due to toxicity, ABT-737 or Bortezomib slowed melanoma tumor growth compared to the control, and the drug combination significantly decreased growth compared to either drug alone. These data imply that less toxic drugs fulfilling a function similar to Bortezomib to neutralize Mcl-1 are promising candidates for combination with ABT-737 for treating melanomas

Modular organization of FDH: Exploring the basis of hydrolase catalysis

An abundant enzyme of liver cytosol, 10-formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase-homologous domain and a folate-binding hydrolase domain, which are connected by an ~100-residue linker. The amino-terminal hydrolase domain of FDH (Nt-FDH) is a homolog of formyl transferase enzymes that utilize 10-formyl-THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-formyl-THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl-tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10-formyl-THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis