The Microtubule Network and Cell Death Are Regulated by an miR-34a/Stathmin 1/ßIII-Tubulin Axis.

MicroRNA-34a (miR-34a) is a master regulator of signaling networks that maintains normal physiology and disease and is currently in development as a miRNA-based therapy for cancer. Prior studies have reported low miR-34a expression in osteosarcoma; however, the molecular mechanisms underlying miR-34a activity in osteosarcoma are not well-defined. Therefore, this study evaluated the role of miR-34a in regulating signal transduction pathways that influence cell death in osteosarcoma. Levels of miR-34a were attenuated in human osteosarcoma cells and xenografts of the Pediatric Preclinical Testing Consortium (PPTC). Bioinformatics predictions identified stathmin 1 (STMN1) as a potential miR-34a target. Biotin pull-down assay and luciferase reporter analysis confirmed miR-34a target interactions within the STMN1 mRNA 3'-untranslated region. Overexpression of miR-34a in osteosarcoma cells suppressed STMN1 expression and reduced cell growth in vitro Restoration of miR-34a led to microtubule destabilization and increased ßIII-tubulin expression, with corresponding G1-G2 phase cell-cycle arrest and apoptosis. Knockdown of the Sp1 transcription factor, by siRNA silencing, also upregulated ßIII-tubulin expression in osteosarcoma cells, suggesting that miR-34a indirectly affects Sp1. Validating the coordinating role of miR-34a in microtubule destabilization, when miR-34a was combined with either microtubule inhibitors or chemotherapy, STMN1 phosphorylation was suppressed and there was greater cytotoxicity in osteosarcoma cells. These results demonstrate that miR-34a directly represses STMN1 gene and protein expression and upregulates ßIII-tubulin, leading to disruption of the microtubule network and cell death.Implications: The miR-34a/STMN1/ßIII-tubulin axis maintains the microtubule cytoskeleton in osteosarcoma, and combining miR-34a with microtubule inhibitors can be investigated as a novel therapeutic strategy. Mol Cancer Res; 15(7); 953-64. ©2017 AACR.

Integrating mechanisms of response and resistance against the tubulin binding agent Eribulin in preclinical models of osteosarcoma.

Osteosarcoma is the most frequently occurring bone cancer in children and adolescents. Unfortunately, treatment failures are common. Eribulin is a synthetic microtubule inhibitor that has demonstrated activity in preclinical osteosarcoma models. The effects of eribulin were evaluated in two human osteosarcoma cell lines as well as in eribulin-sensitive and -resistant osteosarcoma xenograft tumors of the Pediatric Preclinical Testing Program (PPTP) by characterizing cell viability, microtubule destabilization, mitotic arrest and mechanism of cell death. Eribulin demonstrated cytotoxic activity in vitro, through promotion of microtubule dynamic instability, arrest of cells in the G2/M phase, mitotic catastrophe and cell death. The microtubule-destabilizing protein stathmin-1 (STMN1) was coimmunoprecipitated with the cyclin-dependent kinase inhibitor p27 indicating that these cytoplasmic complexes can protect cells from the microtubule destabilizing effect of eribulin. Increased tumoral expression of P-glycoprotein (P-gp) and TUBB3 were also associated with lower drug sensitivity. In summary, eribulin successfully blocked cells in G2/M phase but interfered with mitochondria activity to inhibit proteins involved in apoptosis. Understanding the complex and inter-related mechanisms involved in the overall drug response to eribulin may help in the design of therapeutic strategies that enhance drug activity and improve benefits of eribulin in pediatric patients with osteosarcoma.

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways.

Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS.

MicroRNAs and Potential Targets in Osteosarcoma: Review.

Osteosarcoma is the most common bone cancer in children and young adults. Surgery and multi-agent chemotherapy are the standard treatment regimens for this disease. New therapies are being investigated to improve overall survival in patients. Molecular targets that actively modulate cell processes, such as cell-cycle control, cell proliferation, metabolism, and apoptosis, have been studied, but it remains a challenge to develop novel, effective-targeted therapies to treat this heterogeneous and complex disease. MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating cell processes including growth, development, and disease. miRNAs function as oncogenes or tumor suppressors to regulate gene and protein expression. Several studies have demonstrated the involvement of miRNAs in the pathogenesis of osteosarcoma with the potential for development in disease diagnostics and therapeutics. In this review, we discuss the current knowledge on the role of miRNAs and their target genes and evaluate their potential use as therapeutic agents in osteosarcoma. We also summarize the efficacy of inhibition of oncogenic miRNAs or expression of tumor suppressor miRNAs in preclinical models of osteosarcoma. Recent progress on systemic delivery as well as current applications for miRNAs as therapeutic agents has seen the advancement of miR-34a in clinical trials for adult patients with non-resectable primary liver cancer or metastatic cancer with liver involvement. We suggest a global approach to the understanding of the pathogenesis of osteosarcoma may identify candidate miRNAs as promising biomarkers for this rare disease.