cyclin - dependent kinase non - small cell lung cancer pumilio rna - binding family member 1/2 e3 ubitiquitin ligase
oncosuppressor mirnas have emerged as powerful post - transcriptional inhibitors of genetic programs controlling cancer cell proliferation , survival , invasion , metastasis , and stemness . moreover , the in vivo inhibition of tumor growth achieved in mouse cancer models by re - expression of well - characterized tumor suppressor mirnas , such as mir-34 and let-7 , suggests strong therapeutic potential .
experimentally validated bioinformatics analyses show that cell cycle components are highly enriched among targets of the major tumor suppressor mirnas .
several g1- and s - phase cyclins ( d1 , d3 , and e2 ) and cyclin - dependent kinases ( cdk4 and cdk6 ) represent key targets of let-7 , mir-15/16 , and mir-34 families .
conversely , several oncomirs target the expression of cdk inhibitors ; for example members of the mir-1792 and mir-106b25 oncogenic clusters target the p21 transcript .
the cdk inhibitor p27 is lost or inactivated in cancer cells by multiple mechanisms , including decreased synthesis , increased proteolysis , and mislocalization
. the p27 and p57 transcripts are critical targets of the closely related mir-221 and mir-222 oncomirs , which are overexpressed in multiple solid tumors including non - small cell lung cancer ( nsclc ) .
downregulation of mir-340 has been reported in multiple tumors such as breast , colon , neuroblastoma , and osteosarcoma , in which mr-340 expression positively correlates with better prognosis .
experimentally validated mir-340 targets include disparate cellular components such as the tyrosine kinase met in breast cancer , the transcription factors sox2 in neuroblastoma and mitf in melanoma , and the cytoskeletal regulator rock1 in osteosarcoma .
mir-340 expression inversely correlates with clinical staging in nsclc patients , whereas exogenous mir-340 inhibits proliferation and survival in nsclc - derived cells .
mir-340induced growth arrest correlates with p27 accumulation in both lung adenocarcinoma and glioblastoma cells . in a549 cells
mir-340 controls p27 at both translational and post - translational levels by directly targeting 3 negative regulators of p27 ( pum1 , pum2 , and skp2 ) ( fig .
mir-340 induces p27 at the translational level by targeting the rna - binding proteins ( pum1 and pum2 ) required for mir-221/222mediated inhibition of the p27 transcript .
the blue lines indicate both validated ( solid lines : pum1 , pum2 , and skp2 ) and preliminarily characterized ( dashed lines : cyclins d1 and d2 ) mir-340 target transcripts in a549 cells
mir-340 induces p27 at the translational level by targeting the rna - binding proteins ( pum1 and pum2 ) required for mir-221/222mediated inhibition of the p27 transcript .
the blue lines indicate both validated ( solid lines : pum1 , pum2 , and skp2 ) and preliminarily characterized ( dashed lines : cyclins d1 and d2 ) mir-340 target transcripts in a549 cells .
human pum1 and pum2 genes encode 2 evolutionary conserved rna - binding proteins related to the pumilio gene products in drosophila and fem-3 in c. elegans . the functional role of the pum1/2 binding sites has been characterized for only a few human transcripts .
binding of pum1 to the p27 3-utr induces a conformational switch that positively controls mir-221/222 accessibility .
growth factor - induced phosphorylation of pum1 ser714 increases its rna - binding activity , suggesting a role of pum1 post - translational modification in the control of cell cycle re - entry .
our results show that mirna - mediated downregulation of pum1 and pum2 antagonizes the mir-221/222mediated inhibition of p27 .
remarkably , transcriptome - wide analyses of pum1- and pum2-bound mrnas show significant enrichment for multiple cell cycle regulators in addition to p27 .
therefore , the mir-340pum1/2 axis might control cell cycle progression by targeting multiple transcripts in addition to cdkn1b , which encodes p27 .
for example , pum1/2 have also been implicated in the mirna - mediated control of the cell cycle regulator e2f3 in bladder cancer cells .
pum1 belongs to a growing list of rna - binding proteins including hur , dnd1 , crd - bp , and ptb that are implicated in the modulation of mirna targeting in mammalian cells .
interestingly , the mir-340 target site in the mitf 3-utr is controlled by the crd - bp rna - binding protein , which interferes with mir-340 binding thus protecting the mitf transcript from mir-340mediated degradation .
intriguingly , in addition to pum1/2 , mir-340 also targets 2 distinct rna - binding proteins , pbp1/hnrnp1 and hnrnpa2 , in colorectal cancer , suggesting a complex interplay between mir-340 and rna - binding proteins in cancer .
p27 levels largely depend on protein stability , which is reduced by scf - mediated ubiquitylation . through investigation of the mechanism of p27 stabilization in mir-340-overexpressing cells we have identified s - phase kinase - associated protein 2 , e3 ubitiquitin ligase ( skp2 ) , the substrate - recognizing component of the scf complex , as a target of mir-340 . to our knowledge , this is the first evidence of mirna - mediated regulation of the human skp2 oncoprotein . in summary , in nsclc cells mir-340 induces p27 accumulation by affecting both synthesis ( through pum1/2 ) and degradation ( through skp2 ) of the cdk inhibitor .
single nucleotide polymorphisms ( snps ) or 3-utr shortening events are known to affect the mirna binding sites of transcripts coding for oncoproteins , such as kras .
the identification of a skp2 mrna species harboring a short 3-utr lacking the mir-340 target site suggests that , depending on the splicing pattern , some tumors could express a skp2 transcript isoform that is resistant to mir-340mediated repression .
interestingly , we have identified cell lines in which p27 is unaffected by mir-340 . since mir-340 retains its antiproliferative activity in these cell lines , we investigated other putative mir-340 targets . among various oncogenically relevant target transcripts , our preliminary experiments identified both cyclin d1 and cyclin d2 , whose expression shows a significant inverse correlation with that of the mir-340 host gene ( rnf130 ) .
therefore , mir-340 could influence g1/s transition by affecting the accumulation of cyclins d1/d2 and the activity of cyclin d / cdk4/6 complexes , together with the induction of p27 ( via pum1/2 and skp2 ) and inhibition of the cyclin e / cdk2 complex .
in addition , having observed that mir-340 is responsive to serum induction we postulate that mir-340 might participate in the control of cell cycle progression in response to extracellular mitogenic signals .
in addition to further studies aimed at the transcriptome - wide identification of target mrnas and oncogenic networks modulated by mir-340 , future investigations will address the applications of mir-340 .
importantly , systemic delivery of pre - mir-340 has recently been shown to inhibit the growth of xenografts of human colorectal cancer cells in mice . therefore , multiple lines of evidence point to mir-340 as a novel , highly promising bullet for mirna - based anticancer therapeutics .
this work was supported by funding from airc ( associazione italiana per la ricerca sul cancro ) grant-10489 and aicr ( association for international cancer research , uk ) grant-08182 to pasquale verde .
 oncosuppressor mirnas inhibit cancer cell proliferation by targeting key components of the cell cycle machinery . in our recent report 
 we showed that mir-340 is a novel tumor suppressor in non - small cell lung cancer . 
 mir-340 inhibits neoplastic cell proliferation and induces p27kip1 by targeting multiple translational and post - translational regulators of this cyclin - dependent kinase inhibitor . 
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