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By performing nine genome-wide microRNA (miRNA) screens, we recently uncovered a new class of miRNAs, which target multiple cyclins and cyclin-dependent kinases (CDKs). Systemic delivery of selected cell cycle-targeting miRNAs to mouse xenograft models resulted in potent anti-tumorigenic effects without affecting animals' health. Here, we provide an in-depth description of our miRNA screening methodology, analyses of selected cell cycle-targeting miRNAs, and discuss why miRNA therapy might be a viable therapeutic option for cancer patients.

The roles of cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs), as core components of the machinery that drives cell cycle progression are well established. Increasing evidence indicates that mammalian cyclins and CDKs also carry out important functions in other cellular processes, such as transcription, DNA damage repair, control of cell death, differentiation, the immune response and metabolism. Some of these non-canonical functions are performed by cyclins or CDKs, independently of their respective cell cycle partners, suggesting that there was a substantial divergence in the functions of these proteins during evolution.

MYC is a pleiotropic transcription factor that controls a number of fundamental cellular processes required for the proliferation and survival of normal and malignant cells, including the cell cycle. MYC interacts with several central cell cycle regulators that control the balance between cell cycle progression and temporary or permanent cell cycle arrest (cellular senescence). Among these are the cyclin E/A/cyclin-dependent kinase 2 (CDK2) complexes, the CDK inhibitor p27KIP1 (p27) and the E3 ubiquitin ligase component S-phase kinase-associated protein 2 (SKP2), which control each other by forming a triangular network. MYC is engaged in bidirectional crosstalk with each of these players; while MYC regulates their expression and/or activity, these factors in turn modulate MYC through protein interactions and post-translational modifications including phosphorylation and ubiquitylation, impacting on MYC's transcriptional output on genes involved in cell cycle progression and senescence. Here we elaborate on these network interactions with MYC and their impact on transcription, cell cycle, replication and stress signaling, and on the role of other players interconnected to this network, such as CDK1, the retinoblastoma protein (pRB), protein phosphatase 2A (PP2A), the F-box proteins FBXW7 and FBXO28, the RAS oncoprotein and the ubiquitin/proteasome system. Finally, we describe how the MYC/CDK2/p27/SKP2 axis impacts on tumor development and discuss possible ways to interfere therapeutically with this system to improve cancer treatment. [ABSTRACT FROM AUTHOR]

The MYC and RAS oncogenes are frequently activated in cancer and. together, are sufficient to transform rodent cells. The basis for this cooperativity remains unclear. We found that although Ras interfered with Myc-induced apoptosis, Myc repressed Ras-induced senescence, together abrogating two main barriers of tumorigenesis. Inhibition of cellular senescence required phosphorylation of Myc at Ser-62 by cyclin E/cyclin-dependent kinase (Cdk) 2. Cdk2 interacted with Myc at promoters, where it affected Myc-dependent regulation of genes, including Bmi-1, p16, p21, and hTERT, which encode proteins known to control senescence. Repression of senescence by Myc was abrogated by the Cdk inhibitor p27Kip1, which is induced by antiproliferative signals like IFN1 or by pharmacological inhibitors of Cdk2 but not by inhibitors of other Cdks. In contrast, a phospho-mimicking Myc-S62D mutant was resistant to these manipulations. Inhibition of cyclin E/Cdk2 reversed the senescence-associated gene expression pattern imposed by Myc/cyclin EICdk2. This indicates a role of Cdk2 as a transcriptional cofactor and activator of the antisenescence function of Myc and provides mechanistic insight into the Myc-p27Kip1 antagonism. Finally, our findings highlight that pharmacological inhibition of Cdk2 activity is a potential therapeutical principle for cancer therapy, in particular for tumors with activated Myc or Ras. [ABSTRACT FROM AUTHOR]

Activated oncogenes induce compensatory tumour-suppressive responses, such as cellular senescence or apoptosis, but the signals determining the main outcome remain to be fully understood. Here, we uncover a role for Cdk2 (cyclin-dependent kinase 2) in suppressing Myc-induced senescence. Short-term activation of Myc promoted cell-cycle progression in either wild-type or Cdk2 knockout mouse embryo fibroblasts (MEFs). In the knockout MEFs, however, the initial hyper-proliferative response was followed by cellular senescence. Loss of Cdk2 also caused sensitization to Myc-induced senescence in pancreatic β-cells or splenic B-cells in vivo, correlating with delayed lymphoma onset in the latter. Cdk2−/− MEFs also senesced upon ectopic Wnt signalling or, without an oncogene, upon oxygen-induced culture shock. Myc also causes senescence in cells lacking the DNA repair protein Wrn. However, unlike loss of Wrn, loss of Cdk2 did not enhance Myc-induced replication stress, implying that these proteins suppress senescence through different routes. In MEFs, Myc-induced senescence was genetically dependent on the ARF–p53–p21Cip1 and p16INK4a–pRb pathways, p21Cip1 and p16INK4a being selectively induced in Cdk2−/− cells. Thus, although redundant for cell-cycle progression and development, Cdk2 has a unique role in suppressing oncogene- and/or stress-induced senescence. Pharmacological inhibition of Cdk2 induced Myc-dependent senescence in various cell types, including a p53-null human cancer cell line. Our data warrant re-assessment of Cdk2 as a therapeutic target in Myc- or Wnt-driven tumours. [ABSTRACT FROM AUTHOR]

Summary Cyclins and cyclin-dependent kinases (CDKs) are hyperactivated in numerous human tumors. To identify means of interfering with cyclins/CDKs, we performed nine genome-wide screens for human microRNAs (miRNAs) directly regulating cell-cycle proteins. We uncovered a distinct class of miRNAs that target nearly all cyclins/CDKs, which are very effective in inhibiting cancer cell proliferation. By profiling the response of over 120 human cancer cell lines, we derived an expression-based algorithm that can predict the response of tumors to cell-cycle-targeting miRNAs. Using systemic administration of nanoparticle-formulated miRNAs, we inhibited tumor progression in seven mouse xenograft models, including three treatment-refractory patient-derived tumors, without affecting normal tissues. Our results highlight the utility of using cell-cycle-targeting miRNAs for treatment of refractory cancer types. [ABSTRACT FROM AUTHOR]

Summary: D-cyclins represent components of cell cycle machinery. To test the efficacy of targeting D-cyclins in cancer treatment, we engineered mouse strains that allow acute and global ablation of individual D-cyclins in a living animal. Ubiquitous shutdown of cyclin D1 or inhibition of cyclin D-associated kinase activity in mice bearing ErbB2-driven mammary carcinomas triggered tumor cell senescence, without compromising the animals’ health. Ablation of cyclin D3 in mice bearing Notch1-driven T cell acute lymphoblastic leukemias (T-ALL) triggered tumor cell apoptosis. Such selective killing of leukemic cells can also be achieved by inhibiting cyclin D associated kinase activity in mouse and human T-ALL models. Inhibition of cyclin D-kinase activity represents a highly-selective anticancer strategy that specifically targets cancer cells without significantly affecting normal tissues. [ABSTRACT FROM AUTHOR]

Summary: Cyclin D-dependent kinases (CDK4 and CDK6) are positive regulators of cell cycle entry and they are overactive in the majority of human cancers. However, it is currently not completely understood by which cellular mechanisms CDK4/6 promote tumorigenesis, largely due to the limited number of identified substrates. Here we performed a systematic screen for substrates of cyclin D1-CDK4 and cyclin D3-CDK6. We identified the Forkhead Box M1 (FOXM1) transcription factor as a common critical phosphorylation target. CDK4/6 stabilize and activate FOXM1, thereby maintain expression of G1/S phase genes, suppress the levels of reactive oxygen species (ROS), and protect cancer cells from senescence. Melanoma cells, unlike melanocytes, are highly reliant on CDK4/6-mediated senescence suppression, which makes them particularly susceptible to CDK4/6 inhibition. [ABSTRACT FROM AUTHOR]