Small Molecule Inhibition of miR-544 Biogenesis Disrupts Adaptive Responses to Hypoxia by Modulating ATM-mTOR Signaling.

Hypoxia induces a complex circuit of gene expression that drives tumor progression and increases drug resistance. Defining these changes allows for an understanding of how hypoxia alters tumor biology and informs design of lead therapeutics. We probed the role of microRNA-544 (miR-544), which silences mammalian target of rapamycin (mTOR), in a hypoxic breast cancer model by using a small molecule (1) that selectively impedes the microRNA's biogenesis. Application of 1 to hypoxic tumor cells selectively inhibited production of the mature microRNA, sensitized cells to 5-fluorouracil, and derepressed mRNAs affected by miR-544 in cellulo and in vivo, including boosting mTOR expression. Thus, small molecule inhibition of miR-544 reverses a tumor cell's physiological response to hypoxia. Importantly, 1 sensitized tumor cells to hypoxia-associated apoptosis at a 25-fold lower concentration than a 2'-O-methyl RNA antagomir and was as selective. Further, the apoptotic effect of 1 was suppressed by treatment of cell with rapamycin, a well-known inhibitor of the mTOR signaling pathway, illustrating the selectivity of the compound. Thus, RNA-directed chemical probes, which could also serve as lead therapeutics, enable interrogation of complex cellular networks in cells and animals.

[1]  M. Disney,et al.  Molecular recognition of 6'-N-5-hexynoate kanamycin A and RNA 1x1 internal loops containing CA mismatches. , 2011, Biochemistry.

[2]  Juan J. Marugan,et al.  Induction and Reversal of Myotonic Dystrophy Type 1 Pre-mRNA Splicing Defects by Small Molecules , 2013, Nature Communications.

[3]  D. Phinney,et al.  MicroRNAs in the Imprinted DLK1-DIO3 Region Repress the Epithelial-to-Mesenchymal Transition by Targeting the TWIST1 Protein Signaling Network* , 2012, The Journal of Biological Chemistry.

[4]  L. Huang,et al.  HIF-1α Mediates Tumor Hypoxia to Confer a Perpetual Mesenchymal Phenotype for Malignant Progression , 2011, Science Signaling.

[5]  Stuart L. Schreiber,et al.  A mammalian protein targeted by G1-arresting rapamycin–receptor complex , 1994, Nature.

[6]  Adrian L. Harris,et al.  Hypoxia — a key regulatory factor in tumour growth , 2002, Nature Reviews Cancer.

[7]  M. Disney,et al.  Bottom‐up Design of Small Molecules that Stimulate Exon 10 Skipping in Mutant MAPT Pre‐mRNA , 2014, Chembiochem : a European journal of chemical biology.

[8]  Eric T. Wang,et al.  Design of a bioactive small molecule that targets the myotonic dystrophy type 1 RNA via an RNA motif-ligand database and chemical similarity searching. , 2012, Journal of the American Chemical Society.

[9]  David E. Housman,et al.  Molecular basis of myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member , 1992, Cell.

[10]  P. Houghton,et al.  mTORC1 signaling under hypoxic conditions is controlled by ATM-dependent phosphorylation of HIF-1α. , 2010, Molecular cell.

[11]  B. Martín-Castillo,et al.  Metformin and the ATM DNA damage response (DDR): Accelerating the onset of stress-induced senescence to boost protection against cancer , 2011, Aging.

[12]  M. Roizen,et al.  Hallmarks of Cancer: The Next Generation , 2012 .

[13]  S. Zeuzem,et al.  Inhibition of RNAse A family enzymes prevents degradation and loss of silencing activity of siRNAs in serum. , 2006, Biochemical pharmacology.

[14]  Steven M. Gallo,et al.  Sequence-based design of bioactive small molecules that target precursor microRNAs , 2014, Nature chemical biology.

[15]  D. Isenberg,et al.  Analysis of antibodies to RNA in patients with systemic lupus erythematosus and other autoimmune rheumatic diseases , 1991, Clinical and experimental immunology.

[16]  T. Paull,et al.  The ATM protein kinase and cellular redox signaling: beyond the DNA damage response. , 2012, TIBS -Trends in Biochemical Sciences. Regular ed.

[17]  J. Au,et al.  Delivery of siRNA Therapeutics: Barriers and Carriers , 2010, The AAPS Journal.

[18]  C. Michiels,et al.  Tumour hypoxia affects the responsiveness of cancer cells to chemotherapy and promotes cancer progression. , 2008, Anti-cancer agents in medicinal chemistry.

[19]  W. Mark Saltzman,et al.  Therapeutic siRNA: Principles, Challenges, and Strategies , 2012, The Yale journal of biology and medicine.

[20]  A. Judge,et al.  Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA , 2005, Nature Biotechnology.

[21]  M. Johnson,et al.  Syntheses of some highly substituted pyridines, 2,7-naphthyridines and 1H-pyrimido[4,5,6-i,j][2,7]naphthyridines , 1968 .

[22]  T. Cramer,et al.  Hypoxia-mediated drug resistance: novel insights on the functional interaction of HIFs and cell death pathways. , 2011, Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy.

[23]  A. Judge,et al.  siRNA and innate immunity. , 2009, Oligonucleotides.

[24]  S. Zeuzem,et al.  RNAse A‐like enzymes in serum inhibit the anti‐neoplastic activity of siRNA targeting polo‐like kinase 1 , 2007, International journal of cancer.

[25]  G. Semenza.  Hypoxia-inducible factors: mediators of cancer progression and targets for cancer therapy. , 2012, Trends in pharmacological sciences.

[26]  G. Semenza,et al.  Hypoxia-induced resistance to anticancer drugs is associated with decreased senescence and requires hypoxia-inducible factor-1 activity , 2008, Molecular Cancer Therapeutics.

[27]  Jessica L. Childs-Disney,et al.  Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive. , 2012, ACS chemical biology.