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MicroRNAs (miRNA), a class of natural RNA-interfering agents, have recently been identified as attractive targets for therapeutic intervention. The rationale for developing miRNA therapeutics is based on the premise that aberrantly expressed miRNAs play key roles in the development of human disease, and that correcting these miRNA deficiencies by either antagonizing or restoring miRNA function may provide a therapeutic benefit. Although miRNA antagonists are conceptually similar to other inhibitory therapies, restoring the function of a miRNA by miRNA replacement is a less well characterized approach. Here, we discuss the specific properties of miRNA replacement and review recent examples that explored the therapeutic delivery of miRNA mimics in animal models of cancer. Cancer Res; 70(18); 7027–30. ©2010 AACR.

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) binds to death receptors 4/5 and selectively induces caspase-dependent apoptosis. The RNA interference screening approach has led to the discovery and characterization of several TRAIL pathway components in human cells. Here, libraries of synthetic small interfering RNA (siRNA) and microRNAs (miRNA) were used to probe the TRAIL pathway. In addition to known genes, siRNAs targeting CDK4, PTGS1, ALG2, CLCN3, IRAK4, and MAP3K8 altered TRAIL-induced caspase-3 activation responses. Introduction of the miRNAs let-7c, mir-10a, mir-144, mir-150, mir-155, and mir-193 also affected the activation of the caspase cascade. Putative targets of these endogenous miRNAs included genes encoding death receptors, caspases, and other apoptosis-related genes. Among the novel genes revealed in the screen, CDK4 was selected for further characterization. CDK4 was the only member of the cyclin-dependent kinase gene family that bore a unique function in apoptotic signal transduction. [Cancer Res 2007;67(22):10782–8]

Tumor suppressor microRNAs (miRNA) provide a new opportunity to treat cancer. This approach, “miRNA replacement therapy,” is based on the concept that the reintroduction of miRNAs depleted in cancer cells reactivates cellular pathways that drive a therapeutic response. Here, we describe the development of a therapeutic formulation using chemically synthesized miR-34a and a lipid-based delivery vehicle that blocks tumor growth in mouse models of non–small-cell lung cancer. This formulation is effective when administered locally or systemically. The antioncogenic effects are accompanied by an accumulation of miR-34a in the tumor tissue and downregulation of direct miR-34a targets. Intravenous delivery of formulated miR-34a does not induce an elevation of cytokines or liver and kidney enzymes in serum, suggesting that the formulation is well tolerated and does not induce an immune response. The data provide proof of concept for the systemic delivery of a synthetic tumor suppressor mimic, obviating obstacles associated with viral-based miRNA delivery and facilitating a rapid route for miRNA replacement therapy into the clinic. Cancer Res; 70(14); 5923–30. ©2010 AACR.

MicroRNAs play important roles in animal development, cell differentiation, and metabolism and have been implicated in human cancer. The let-7 microRNA controls the timing of cell cycle exit and terminal differentiation in Caenorhabditis elegans and is poorly expressed or deleted in human lung tumors. Here, we show that let-7 is highly expressed in normal lung tissue, and that inhibiting let-7 function leads to increased cell division in A549 lung cancer cells. Overexpression of let-7 in cancer cell lines alters cell cycle progression and reduces cell division, providing evidence that let-7 functions as a tumor suppressor in lung cells. let-7 was previously shown to regulate the expression of the RAS lung cancer oncogenes, and our work now shows that multiple genes involved in cell cycle and cell division functions are also directly or indirectly repressed by let-7. This work reveals the let-7 microRNA to be a master regulator of cell proliferation pathways. [Cancer Res 2007;67(16):7713–22]

Background: Cancer stem cells represent the cell population responsible for tumor initiation and progression. Resistance to cell death induced by cytotoxic agents is a characteristic shared by most identified cancer stem cells. In ovarian cancer, the CD44+/MyD88+ epithelial ovarian cancer (EOC) stem cells do not only represent the chemoresistant population, they are also able to repair the tumor and serve as tumor vascular progenitors. Targeting these cells is therefore necessary to improve treatment outcome. However, the previous demonstration that these cells are extremely chemoresistant, suggests that other forms of targeted therapy should be explored. We previously showed that the novel isoflavone derivative, NV-128, can induce caspase-independent cell death in ovarian cancer cells by inducing loss of mitochondrial membrane potential. We hypothesize that targeting mitochondrial bioenergetics may be a potent stimulus to induce cell death in the EOC stem cells. We demonstrate in this study that NV-128 is able to depress mitochondrial function leading to the activation of two independent pathways and cell death in these chemo-resistant cell population. Methods: A panel of CD44+/MyD88+ EOC stem cells was treated with NV-128 (1–10μg/ml). Inhibitory studies were done using the specific MEK inhibitor, U0126, or the ROS scavenger, MnTBAP. Mitochondrial integrity and function were assessed using Mitotracker Red, Mitotracker Green, MitoSox, and ApoSENSOR ADP/ATP kit. Cytoplasmic and mitochondrial fractions were prepared using the ApoAlertTM Cell Fractionation kit. Protein levels were determined using Western Blot. Results: NV-128 (10 μg/ml) significantly depressed mitochondrial function within 2h in all EOC stem cells tested. Treated cells had lower levels of ATP and Cox-IV, and higher levels of mitochondrial superoxide. Low cellular ATP led to mTOR inhibition through AMPKα1, while increased mitochondrial superoxide activated the mitochondrial MEK/ERK pathway and promoted Bax-dependent loss of mitochondrial membrane potential. Resulting cell death was characterized by Endo-G-induced but caspase-independent DNA fragmentation, as well as formation of autophagic vacuoles. Conclusion: We demonstrate that targeting the mitochondria in the EOC stem cells promotes a state of “cellular starvation” capable of activating two independent pathways: 1) AMPKα1 pathway leading to mTOR inhibition; and 2) mitochondrial MEK/ERK pathway leading to mitochondrial depolarization. The simultaneous activation of these pathways is a potent stimulus to induce cell death in cells. The demonstration that a compound can specifically target the mitochondria to induce cell death in this otherwise chemo-resistant cell population opens a new venue for treating ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-221. doi:10.1158/1538-7445.AM2011-LB-221