Clinical applications of microRNAs

MicroRNAs represent a class of small RNAs derived from polymerase II controlled transcriptional regions. The primary transcript forms one or several bulging double stranded hairpins which are processed by Drosha and Dicer into hetero-duplexes. The targeting microRNA strand of the duplex is incorporated into the RNA Induced Silencing Complex from where it silences up to hundreds of mRNA transcript by inducing mRNA degradation or blocking protein translation. Apart from involvement in a variety of biological processes, microRNAs were early recognized for their potential in disease diagnostics and therapeutics. Due to their stability, microRNAs could be used as biomarkers. Currently, there are microRNA panels helping physicians determining the origins of cancer in disseminated tumors. The development of microRNA therapeutics has proved more challenging mainly due to delivery issues. However, one drug is already in clinical trials and several more await entering clinical phases. This review summarizes what has been recognized pre-clinically and clinically on diagnostic microRNAs. In addition, it highlights individual microRNA drugs in running platforms driven by four leading microRNA-therapeutic companies

D-Cyclins Repress Apoptosis in Hematopoietic Cells by Controlling Death Receptor Fas and Its Ligand FasL

D-type cyclins (D1, D2, and D3) are components of the mammalian core cell-cycle machinery and function to drive cell proliferation. Here, we report that D-cyclins perform a rate-limiting antiapoptotic function in vivo. We found that acute shutdown of all three D-cyclins in bone marrow of adult mice resulted in massive apoptosis of all hematopoietic cell types. We demonstrate that adult hematopoietic stem cells are particularly dependent on D-cyclins for survival and that they are especially sensitive to cyclin D loss. Surprisingly, we found that the antiapoptotic function of D-cyclins also operates in quiescent hematopoietic stem and progenitor cells. Our analyses revealed that D-cyclins repress the expression of the death receptor Fas and its ligand, FasL. Acute ablation of D-cyclins upregulated these proapoptotic genes and led to Fas- and caspase 8-dependent apoptosis. These results reveal an unexpected function of cell-cycle proteins in controlling apoptosis in normal cell homeostasis

Myc Stimulates Cell Cycle Progression Through the Activation of Cdk1 and Phosphorylation of p27

Cell cycle stimulation is a major transforming mechanism of Myc oncoprotein. This is achieved through at least three concomitant mechanisms: upregulation of cyclins and Cdks, downregulation of the Cdk inhibitors p15 and p21 and the degradation of p27. The Myc-p27 antagonism has been shown to be relevant in human cancer. To be degraded, p27 must be phosphorylated at Thr-187 to be recognized by Skp2, a component of the ubiquitination complex. We previously described that Myc induces Skp2 expression. Here we show that not only Cdk2 but Cdk1 phosphorylates p27 at the Thr-187. Moreover, Myc induced p27 degradation in murine fibroblasts through Cdk1 activation, which was achieved by Myc-dependent cyclin A and B induction. In the absence of Cdk2, p27 phosphorylation at Thr-187 was mainly carried out by cyclin A2-Cdk1 and cyclin B1-Cdk1. We also show that Cdk1 inhibition was enough for the synthetic lethal interaction with Myc. This result is relevant because Cdk1 is the only Cdk strictly required for cell cycle and the reported synthetic lethal interaction between Cdk1 and Myc.The work was supported by grant SAF2017-88026-R from MINECO, Spanish Government, to JL and MDD (partially funded by FEDER program from European Union). L.G.G. was recipient of FPI fellowship from Spanish Government. We are grateful Sandra Zunzunegui for technical assistance and John Sedivy and M. Dolores Delgado for helpful discussion

MYC Modulation around the CDK2/p27/SKP2 Axis

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

Modulating the activity of the c-Myc oncoprotein

The Myc oncoprotein regulates numerous cellular processes and is frequently deregulated in cancer due to genetic lesions. However, in addition to its tumor promoting activity, Myc and other oncoproteins induce intrinsic safe-guard mechanisms against tumorigenesis like apoptosis and cellular senescence, which have to be overcome by additional genetic lesions for cellular transformation. In this work, we identify ways of reactivating these anti-tumorigenic pathways in cells with deregulated Myc. First, we uncover an unexpected capacity of transforming growth factor-β (TGF-β) to force hematopoietic cells with deregulated Myc into cellular senescence despite continuous Myc expression. This involved upregulation of the Myc antagonist Mad1, leading to repressed transcription of Myc target genes. We further reveal a novel role of Myc in Myc/Ras dependent transformation. While Ras induced cellular senescence and suppressed Myc-activated apoptosis, we found that Myc repressed Ras-induced senescence. This required phosphorylation of Myc at Ser-62 by cyclin dependent kinase 2 (Cdk2). Further, pharmacological inhibitors of Cdk2 forced Myc+Ras expressing cells into senescence. In addition, although redundant for cell cycle progression, Cdk2 was shown to have a unique role in suppressing Myc-induced senescence, and depletion of Cdk2 in a mouse Eµ-myc lymphoma model led to regression of tumor development. Taken together, this highlights Cdk2-targeting in Myc and Ras-driven tumors. Finally, we uncover a novel interplay between Myc and the protein deacetylase SIRT1. While Myc induced SIRT1 expression and activity, SIRT1 fed back to Myc by stabilizing the Myc protein. Further, SIRT1 repressed Myc-induced apoptosis and senescence, pointing out SIRT1 as a promising target in neoplasia driven by Myc. In summary, this thesis demonstrates potential new strategies for therapeutic intervention of tumors with deregulated Myc by targeting its essential cofactors and collaboration partners

Identification of cell cycle-targeting microRNAs through genome-wide screens

ABSTRACT 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

a systematic substrate screen links CDK4/6 to senescence suppression through FOXM1

Cyclin D-CDKs (CDK4, CDK6) are critical regulators of cell cycle entry, and their aberrant expression and activation is observed 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 applied a proteome-wide substrate screen and demonstrate that individual cyclin D-CDK complexes possess major differences in substrate specificity. We identified the Forkhead Box M1 (FOXM1) transcription factor as a functionally critical phosphorylation target. While modification of its N-terminal sequence is linked to stabilization of the protein by preventing CDH1/APC-mediated degradation, phosphorylation of the transactivation domain leads to direct transcriptional activation, resulting in suppression of cellular senescence. Moreover, FOXM1 is required for CDK4-induced expression of proliferation-associated genes, including cyclin E. Accordingly, acute inhibition of CDK4/6 activity in cancer cells disables FOXM1, results in downregulation of cell cycle and DNA repair genes, and enforces a ROS-dependent senescence program. Thus, pharmacological inhibition of CDK4/6 catalytic activity might be particularly effective in tumors that highly depend on FOXM1

MYC is downregulated by a mitochondrial checkpoint mechanism

The MYC proto-oncogene serves as a rheostat coupling mitogenic signaling with the activation of genes regulating growth, metabolism and mitochondrial biogenesis. Here we describe a novel link between mitochondria and MYC levels. Perturbation of mitochondrial function using a number of conventional and novel inhibitors resulted in the decreased expression of MYC mRNA. This decrease in MYC mRNA occurred concomitantly with an increase in the levels of tumor-suppressive miRNAs such as members of the let-7 family and miR-34a-5p. Knockdown of let-7 family or miR-34a-5p could partially restore MYC levels following mitochondria damage. We also identified let-7-dependent downregulation of the MYC mRNA chaperone, CRD-BP (coding region determinant-binding protein) as an additional control following mitochondria damage. Our data demonstrates the existence of a homeostasis mechanism whereby mitochondrial function controls MYC expression.Funding Agencies|Cancerfonden; Vetenskapsradet; Radiumhemmets forskningsfonder; Knut och Alice Wallenbergs Stiftelse; Barncancerfonden</p