Competitive endogenous (ce)RNAs cross-regulate each other through sequestration of shared microRNAs and form complex regulatory networks based on their microRNA signature. However, the molecular requirements for ceRNA cross-regulation and the extent of ceRNA networks remain unknown. Here, we present a mathematical mass-action model to determine the optimal conditions for ceRNA activity in silico. This model was validated using phosphatase and tensin homolog (PTEN) and its ceRNA VAMP (vesicle-associated membrane protein)-associated protein A (VAPA) as paradigmatic examples. A computational assessment of the complexity of ceRNA networks revealed that transcription factor and ceRNA networks are intimately intertwined. Notably, we found that ceRNA networks are responsive to transcription factor up-regulation or their aberrant expression in cancer. Thus, given optimal molecular conditions, alterations of one ceRNA can have striking effects on integrated ceRNA and transcriptional networks.

Many microRNAs (miRNAs), posttranscriptional regulators of numerous cellular processes and developmental events, are downregulated in tumors. However, their role in tumorigenesis remains largely unknown. In this work, we examined the role of the muscle-specific miRNAs miR-1 and miR-206 in human rhabdomyosarcoma (RMS), a soft tissue sarcoma thought to arise from skeletal muscle progenitors. We have shown that miR-1 was barely detectable in primary RMS of both the embryonal and alveolar subtypes and that both miR-1 and miR-206 failed to be induced in RMS cell lines upon serum deprivation. Moreover, reexpression of miR-206 in RMS cells promoted myogenic differentiation and blocked tumor growth in xenografted mice by switching the global mRNA expression profile to one that resembled mature muscle. Finally, we showed that the product of the MET proto-oncogene, the Met tyrosine-kinase receptor, which is overexpressed in RMS and has been implicated in RMS pathogenesis, was downregulated in murine satellite cells by miR-206 at the onset of normal myogenesis. Thus, failure of posttranscriptional modulation may underlie Met overexpression in RMS and other types of cancer. We propose that tissue-specific miRNAs such as miR-1 and miR-206, given their ability to modulate hundreds of transcripts and to act as nontoxic differentiating agents, may override the genomic heterogeneity of solid tumors and ultimately hold greater therapeutic potential than single gene-directed drugs.

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. In here we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.

SUMMARY Mutations in the metabolic enzymes isocitrate dehydrogenase-1 (IDH1) and IDH2 that produce the oncometabolite D-2-hydroxyglutarate (2-HG) occur frequently in human acute myeloid leukemia (AML). 2-HG modulates numerous biological pathways implicated in malignant transformation, but the contribution of mutant IDH proteins to maintenance and progression of AML in vivo is currently unknown. To answer this crucial question we have generated transgenic mice that express IDH2R140Q in an on/ off- and tissue-specific manner using a tetracycline-inducible system. We found that IDH2R140Q can cooperate with overexpression of HoxA9 and Meis1a and with mutations in FMS-like tyrosine kinase 3 (FLT3) to drive acute leukemia in vivo. Critically, we show that genetic deinduction of mutant IDH2 in leukemic cells in vivo has profound effects on their growth and/or maintenance. Our data demonstrate the proto-oncogenic role of mutant IDH2 and support its relevance as a therapeutic target for the treatment of human AML.