Abstract
ObjectiveWe aimed to investigate the roles and underlying mechanisms of YAP in the proliferation of neuroblastoma cells.MethodsThe expression level of YAP was evaluated by Western blotting and immunocytochemistry. Cell viability, cell proliferation and growth were detected by CCK‐8, PH3 and Ki67 immunostaining, and the real‐time cell analyser system. The nuclear and cytoplasmic proteins of p27Kip1 were dissociated by the nuclear‐cytosol extraction kit and were detected by Western blotting and immunocytochemistry. mRNA levels of Akt, CDK5 and CRM1 were determined by qRT‐PCR.ResultsYAP was enriched in SH‐SY5Y cells (a human neuroblastoma cell line). Knock‐down of YAP in SH‐SY5Y cells or SK‐N‐SH cell line (another human neuroblastoma cell line) significantly decreased cell viability, inhibited cell proliferation and growth. Mechanistically, knock‐down of YAP increased the nuclear location of p27Kip1, whereas serum‐induced YAP activation decreased the nuclear location of p27Kip1 and was required for cell proliferation. Meanwhile, overexpression of YAP in these serum‐starved SH‐SY5Y cells decreased the nuclear location of p27Kip1, promoted cell proliferation and overexpression of p27Kip1 in YAP‐activated cells inhibited cell proliferation. Furthermore, knock‐down of YAP reduced Akt mRNA and protein levels. Overexpression of Akt in YAP‐downregulated cells decreased the nuclear location of p27Kip1 and accelerated the proliferation of SH‐SY5Y cells.ConclusionsOur studies suggest that YAP promotes the proliferation of neuroblastoma cells through negatively controlling the nuclear location of p27Kip1 mediated by Akt.
Highlights
B, YAP knock-down significantly decreased the level of p-p27kip1-Ser[10], indicating more p27kip[1] molecules will accumulate in the nucleus. These results suggest that knock-down of YAP increases the nuclear location of p27Kip[1], which may inhibit the proliferation of SH-SY5Y cells
D, YAP overexpression significantly increased the level of p-p27kip1-Ser[10], indicating that less p27kip[1] molecules will stay in the nucleus. These results strongly suggest that activation of YAP decreases the nuclear location of p27Kip[1] in SH-SY5Y cells, which may promote cell proliferation
We examined whether the decrease of nuclear location of p27 Kip[1] by YAP activation was required for serum-induced cell proliferation
Summary
The Hippo signalling pathway is a critical regulator of stem cell self-renewal, tissue regeneration and organ size.[1,2,3,4,5] Dysfunction of this classical pathway will lead to several diseases such as tumours including lung cancer, liver cancer, breast cancer, colorectal cancer and gliomas.[6,7] As the principle effector of the Hippo pathway and a key transcriptional co-factor, YAP plays important roles in organ size control through regulating cell differentiation and proliferation.[8,9,10] Uncontrolled cell proliferation caused by excessive YAP activation will give rise to brain tumours that are usually fatal, such as neuroblastoma and medulloblastoma.[11,12,13]. Cyclindependent kinases inhibitors (CDKIs), such as p27Kip[1] and p21Cip[1], by binding to CDK, inhibit the kinase activity of most CDKs, thereby inhibit cell proliferation and exert anti-tumour activity.[17] There are evidences showing that p27kip[1] is related to neuroblastoma as well as p16, p21 and p53.18-21 Decreased transcript levels of p27Kip[1] increases human susceptibility to neuroblastoma,[22] positive expression of p27Kip[1] increases survival in patients with neuroblastoma[18] and accumulation of p27Kip[1] inhibits the growth of human neuroblastoma cells.[23] Several studies have shown that p27Kip[1] can be an important regulatory target of YAP, affecting cell proliferation; the conclusions seem to be contradictory to each other. Our findings suggest that nuclear p27Kip[1] entrapment by targeting YAP-Akt signalling may be a potential therapeutic strategy for neuroblastoma
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