Molecular Mechanism Of The MiR-302/367 Cluster And Numb In Prostatic Tumorigenesis And Progression

Objective:(1)To explore the functional role and mechanism of the miR-302/367 cluster in prostate tumorigenesis and castration resistance.(2)To elucidate the role and molecular mechanism of Numb in PCa and the functional contribution of Numb^-/low PCa cells in castration resistance.Methods:(1)The expression of the miR-302/367 cluster in twenty-nine human prostatic cancer specimens and twenty-two normal tissues as well as in ten Pten-deleted murine prostate tumor tissues and seven normal murine prostates was examined by quantitative polymerase chain reaction.The biological effects of overexpression and knockdown of the miR-302/367 in human prostate cancer cell lines were investigated in vitro and in vivo.Downstream signaling molecules of miR-302/367 were examined by biochemical approaches.(2)The expression of Numb was assessed from multiple Oncomine datasets as well as PCa tissues of both human and mouse origins.The biological effects of overexpression and knockdown of Numb in human PCa cell lines were investigated in vitro and in vivo.In addition,we developed a reliable approach to distinguish PCa cell populations with high or low endogenous Numb protein amount through a Numb promoter based lentiviral reporter system.The difference of Numb-/low and Numbhigh PCa cells in response to androgen deprivation therapy(ADT)was thereafter tested.Possible downstream factors of Numb were analyzed by luciferase reporter assays,immunoblotting and quantitative real-time PCR.Results:(1)The expression of the miR-302/367 cluster was upregulated in prostate tumors compared to normal/benign tissues.Besides,the level of the miR-302/367 cluster in androgen independent prostate cancer cell lines was significantly higher than androgen sensitive cell lines.Overexpression of the miR-302/367 cluster significantly promoted the sphere-formation,cell proliferation,migration,androgen independence and xenograft tumor growth of prostate cancer cells.On the other hand,knock down of the miR-302/367 cluster markedly inhibited xenograft prostatic tumor formation incidence.Mechanistically,we found that LATS2,a key component of the tumor suppressive Hippo signaling pathway,acted as a direct target of the miR-302/367 cluster in prostate cancer cells.Down-regulation of LATS2 by the miR-302/367 cluster led to reduced phosphorylation and enhanced nuclear translocation of the YAP oncoprotein.Restoration of LATS2 expression abrogated the tumor promoting effects induced by forced expression of the miR-302/367 cluster.(2)We show here that Numb was down-regulated and negatively correlated with PCa advancement.Functionally,Numb exerted an inhibitory role in xenograft prostate tumor growth and CRPC development via suppression of Notch and Hedgehog signaling.Through a Numb promoter based lentiviral reporter system,we were able to separate Numb^-/low PCa cells from Numb^high cells.Numb^-/low PCa cells were smaller and quiescent,preferentially express Notch and Hedgehog downstream and stem-cell-associated genes,and were associated with greater resistance to ADT.Inhibition of Notch and Hedgehog signaling pathways significantly increased the apoptosis of Numb^-/low cells in response to ADT.Conclusion:(1)The miR-302/367/LATS2/YAP pathway is essential for prostate tumor-propagating cells and acts to promote castration resistance.(2)Numb^-/low classifies a castration resistant PCa cell subpopulation associated with unregulated Notch and Hedgehog signaling.