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EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase.

ABSTRACT: Ciliogenesis is generally inhibited in dividing cells, however, it has been unclear which signaling cascades regulate the phenomenon. Here, we report that epidermal growth factor receptor (EGFR) kinase suppresses ciliogenesis by directly phosphorylating the deubiquitinase USP8 on Tyr-717 and Tyr-810 in RPE1 cells. These phosphorylations elevate the deubiquitinase activity, which then stabilizes the trichoplein-Aurora A pathway, an inhibitory mechanism of ciliogenesis. EGFR knockdown and serum starvation result in ciliogenesis through downregulation of the USP8-trichoplein-Aurora A signal. Moreover, primary cilia abrogation, which is induced upon IFT20 or Cep164 depletion, ameliorates the cell cycle arrest of EGFR knockdown cells. The present data reveal that the EGFR-USP8-trichoplein-Aurora A axis is a critical signaling cascade that restricts ciliogenesis in dividing cells, and functions to facilitate cell proliferation. We further show that usp8 knockout zebrafish develops ciliopathy-related phenotypes including cystic kidney, suggesting that USP8 is a regulator of ciliogenesis in vertebrates.

SUBMITTER: Kasahara K

PROVIDER: S-EPMC5823934 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The identification of therapeutic approaches to improve response to platinum-based therapies is an urgent need for ovarian carcinoma. Deubiquitinases are a large family of ubiquitin proteases implicated in a variety of cellular functions and may contribute to tumor aggressive features through regulation of processes such as proliferation and cell death. Among the subfamily of ubiquitin-specific peptidases, USP8 appears to be involved in modulation of cancer cell survival by still poorly understood mechanisms. Thus, we used ovarian carcinoma cells of different histotypes, including cisplatin-resistant variants with increased survival features to evaluate the efficacy of molecular targeting of USP8 as a strategy to overcome drug resistance/modulate cisplatin response. We performed biochemical analysis of USP8 activity in pairs of cisplatin-sensitive and -resistant cells and found increased USP8 activity in resistant cells. Silencing of USP8 resulted in decreased activation of receptor tyrosine kinases and increased sensitivity to cisplatin in IGROV-1/Pt1 resistant cells as shown by colony forming assay. Increased cisplatin sensitivity was associated with enhanced cisplatin-induced caspase 3/7 activation and apoptosis, a phenotype also observed in cisplatin sensitive cells. Increased apoptosis was linked to FLIPL decrease and cisplatin induction of caspase 3 in IGROV-1/Pt1 cells, cisplatin-induced claspin and survivin down-regulation in IGROV-1 cells, thereby showing a decrease of anti-apoptotic proteins. Immunohistochemical staining on 65 clinical specimens from advanced stage ovarian carcinoma indicated that 40% of tumors were USP8 positive suggesting that USP8 is an independent prognostic factor for adverse outcome when considering progression free survival as a clinical end-point. Taken together, our results support that USP8 may be of diagnostic value and may provide a therapeutic target to improve the efficacy of platinum-based therapy in ovarian carcinoma.

Project description:IntroductionSpinal N-methyl-D-aspartate receptor (NMDAR) is vital in chronic pain, while NMDAR antagonists have severe side effects. NMDAR has been reported to be controlled by G protein coupled receptors (GPCRs), which might present new therapeutic targets to attenuate chronic pain. Dopamine receptors which belong to GPCRs have been reported could modulate the NMDA-mediated currents, while their exact effects on NMDAR in chronic bone cancer pain have not been elucidated.ObjectivesThis study was aim to explore the effects and mechanisms of dopamine D1 receptor (D1DR) and D2 receptor (D2DR) on NMDAR in chronic bone cancer pain.MethodsA model for bone cancer pain was established using intra-tibia bone cavity tumor cell implantation (TCI) of Walker 256 in rats. The nociception was assessed by Von Frey assay. A range of techniques including the fluorescent imaging plate reader, western blotting, and immunofluorescence were used to detect cell signaling pathways. Primary cultures of spinal neurons were used for in vitro evaluation.ResultsBoth D1DR and D2DR antagonists decreased NMDA-induced upregulation of Ca2+ oscillations in primary culture spinal neurons. Additionally, D1DR/D2DR antagonists inhibited spinal Calcitonin Gene-Related Peptide (CGRP) and c-Fos expression and alleviated bone cancer pain induced by TCI which could both be reversed by NMDA. And D1DR/D2DR antagonists decreased p-NR1, p-NR2B, and Gαq protein, p-Src expression. Both Gαq protein and Src inhibitors attenuated TCI-induced bone cancer pain, which also be reversed by NMDA. The Gαq protein inhibitor decreased p-Src expression. In addition, D1DR/D2DR antagonists, Src, and Gαq inhibitors inhibited spinal mitogen-activated protein kinase (MAPK) expression in TCI rats, which could be reversed by NMDA.ConclusionsSpinal D1DR/D2DR inhibition eliminated NMDAR-mediated spinal neuron activation through Src kinase in a Gαq-protein-dependent manner to attenuate TCI-induced bone cancer pain, which might present a new therapeutic strategy for bone cancer pain.

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EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase.

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