Cancer Letters

Cancer Letters

Volume 376, Issue 1, 28 June 2016, Pages 83-94
Cancer Letters

Original Articles
Gankyrin activates mTORC1 signaling by accelerating TSC2 degradation in colorectal cancer

https://doi.org/10.1016/j.canlet.2016.03.013Get rights and content

Highlights

  • Gankyrin contributes to the initiation and development of colorectal carcinogenesis.

  • Aberrant activation of mTORC1 through Gankyrin overexpression drives malignant growth.

  • Gankyrin activates mTORC1 signaling by promoting TSC2 degradation.

  • On-target inhibition of Gankyrin restores TSC2 protein and inhibits CRC growth.

Abstract

Gankyrin is overexpressed in some malignancies. However its roles in colorectal carcinogenesis and underlying mechanisms remain largely unexplored. Here we report that gankyrin promotes the initiation and development of colorectal carcinogenesis by activating mTORC1 signaling through TSC/Rheb dependent mechanism. We further show that Gankyrin overexpression accelerated TSC2 degradation, while knockdown in a panel of colorectal cancer (CRC) cell lines, cell line derived xenografts and CRC patient derived xenograft (PDX) tumors delayed TSC2 degradation, restored the TSC2 protein level, and inhibited mTORC1 signaling and CRC growth. Our findings reveal a unique mechanism by which gankyrin promotes colorectal carcinogenesis and show that gankyrin is a potential therapeutic target to improve the clinical management of CRC.

Introduction

Gankyrin (also known as PSMD10) was initially characterized as the p28 protein, a component of the 26S proteasome complex (shown by others, but see also Fig. S1). Gankyrin was recently identified as an oncoprotein when its expression level is high [1], [2], [3], [4]. Although the protein levels of Gankyrin are high in hepatocellular carcinomas [2], [3], the exact mechanism of the oncogenic effect of Gankyrin in the initiation and development of human cancers remains elusive.

Colorectal cancer (CRC) remains one of the most lethal forms of cancer worldwide, causing almost 0.7 million deaths in 2012 [5]. It is urgently necessary to explore the molecular signature of CRC pathogenesis, potential biomarkers and new targets for drug development. The mammalian Target of Rapamycin (mTOR) is a serine–threonine protein kinase and serves as a central regulating axis that coordinates cell growth and proliferation [6]. We previously reported the inhibition of mTOR signaling results in a considerable decrease of CRC tumor cell growth in vitro and in vivo, as well as significant inhibition of the initiation and progression of CRC [7]. mTOR is positioned downstream of the tuberous sclerosis complex 1 (TSC1) and TSC2 tumor suppressors [8]. TSC1 and TSC2 form a dimer and function as a GTPase-activating protein (GAP) to inactivate the small GTPase “Ras homolog enriched in brain” (Rheb), which directly binds to mTORC1 and activates kinase activity [9], [10]. Many oncogenes, such as Ras and Rab1A, are known to lie upstream of mTOR and elicit part of their oncogenic effect through the mTOR signaling pathway [11], [12] .

In this study, we show for the first time that gankyrin is an essential regulator of CRC initiation and development by activating the mTOR signaling pathway through a PI3K/AKT independent, TSC dependent mechanism. The data strongly suggest that TSC and mTOR play pivotal roles in mediating the oncogenic effect of gankyrin, and highlight the potential of gankyrin/TSC/mTOR as a therapeutic target in CRC.

Section snippets

Experimental animals

Housing and all procedures using BALB/c nude mice were approved by the Institutional Animal Care and Use Committee and in accordance with the Animals (Scientific Procedures) Act, 1986 (UK) (amended 2013). All sections of this report adhere to the ARRIVE Guidelines for reporting animal research. All mice were housed in individually ventilated cages (5 per cage) under specific pathogen free (SPF) condition. Housing was temperature-controlled, with a 12-h/12-h light/dark cycle. Animal weights were

Gankyrin is highly expressed in precancerous and colorectal cancer tissues and predicts poor survival

We investigated the expression of gankyrin in a large panel of colorectal precancerous and cancerous clinical samples and human CRC cell lines to assess its involvement in colorectal carcinogenesis. Expression of gankyrin was elevated in adenoma with low-grade intraepithelial neoplasia (LIN) and was significantly higher in adenoma with high-grade intraepithelial neoplasia (HIN), compared to healthy colon epithelial tissue. The gankyrin staining positive rate and Hscore in HIN (positive

Discussion

Recent studies linked gankyrin with several cell signaling pathways in different cancer types [23], including IL-1β/IRAK-1, STAT3/Akt, β-catenin and PI3K/AKT/HIF1a in hepatocarcinogenesis [24], [25], [26], [27], IL-6/STAT3 in cholangiocarcinoma [28], PTEN/PI3K/AKT signaling in endometrial carcinoma [29] and the RhoA/ROCK pathway in lung cancer [30]. Since PI3K/AKT locates upstream of mTOR, it is reasonable to speculate that gankyrin regulates mTOR signaling in a PI3K/AKT dependent manner.

Funding

This work was supported by grants from the National Natural Science Foundation of China (81270035 to Y.Z. and 81572796 to F.L.) and the Shanghai Pujiang Program (15PJ1404900 to Y.Z.).

Authors' contributions

X.Y.Q., X.X.W., F.L, X.W.W. and L.E.M. conducted the experiments, collected data and prepared figures; Y.J.Z. conceived the project; Y.J.Z. and B.J. directed the project, designed the experiments, interpreted data and wrote the manuscript.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

We thank Dr. Géraldine Guérin-Peyrou (Polyplus Transfection, France) for technical advice on in vivo siRNA transfection, and Drs. Chenwei Li and Tingan Yan for assistance with Patient-Derived Xenograft experiments. Special thanks to Dr. Steven Zheng for his rigorous evaluation of the manuscript and his many suggestions to improve it.

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