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ADAMTS9 is a functional tumor suppressor through inhibiting AKT/mTOR pathway and associated with poor survival in gastric cancer

Oncogene volume 32, pages 3319–3328 (2013)Cite this article

Abstract

Using genome-wide promoter methylation analysis, we identified a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9) is methylated in cancer. We aim to clarify its epigenetic inactivation, biological function and clinical implication in gastric cancer. ADAMTS9 was silenced in 6 out of 8 gastric cancer cell lines. The loss of ADAMTS9 expression was regulated by promoter hypermethylation and could be restored by demethylation agent. Ectopic expression of ADAMTS9 in gastric cancer cell lines (AGS, BGC823) inhibited cell growth curve in both the cell lines (P<0.0001), suppressed colony formation (P<0.01) and induced apoptosis (P<0.001 in AGS, P<0.01 in BGC823). Moreover, conditioned culture medium from ADAMTS9-transfected cell lines significantly disrupted the human umbilical vein endothelial cell tube formation capacity on Matrigel (P<0.01 in AGS, P<0.001 in BGC823). The in vivo growth of ADAMTS9 cells in nude mice was also markedly diminished after stable expression of ADAMTS9 (P<0.001). On the other hand, ADAMTS9 knockdown promoted cell proliferation (P<0.001). We further revealed that ADAMTS9 inhibited tumor growth by blocking activation of Akt and its downstream target the mammalian target of rapamycin (mTOR). ADAMTS9 also reduced phosphorylation of mTOR downstream targets p70 ribosomal S6 kinase, eIF4E-binding protein and downregulated hypoxia-inducible factor-1α. Therefore, this is the first demonstration that ADAMTS9 is a critical tumor suppressor of gastric cancer progression at least in part through suppression of oncogenic AKT/mTOR signaling. Moreover, promoter methylation of ADAMTS9 was detected in 29.2% (21/72) of primary gastric tumors. Multivariate analysis showed that patients with ADAMTS9 methylation had a poorer overall survival (relative risk (RR)=2.788; 95% confidence interval, 1.474–5.274; P=0.002). Kaplan–Meier survival curves showed that ADAMTS9 methylation was significantly associated with shortened survival in gastric cancer patients (P=0.001, log-rank test). In conclusion, ADAMTS9 acts as a functional tumor suppressor in gastric cancer through inhibiting oncogenic AKT/mTOR signaling pathway. Methylation of ADAMTS9 is an independent prognostic factor of gastric cancer.

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Abbreviations

7-AAD:

7-amino-actinomycin

5-Aza:

5-Aza-2′-deoxycytidine

ADAMTS9:

a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9

BCL-2:

B-cell CLL/lymphoma 2

BGS:

bisulfite genomic sequencing

CDK4:

cyclin-dependent kinase 4

eIF4E:

eukaryotic translation initiation factor 4E

4E-BP1:

eIF4E-binding protein

EGF:

epidermal growth factor

GLUT-1:

glucose transporter 1

HIF1α:

hypoxia-inducible factor 1α

HK2:

hexokinase 2

HUVEC:

human umbilical vein endothelial cell

MSP:

methylation specific PCR

mTOR:

the mammalian target of rapamycin

PARP:

nuclear enzyme poly (ADP-ribose) polymerase

PGK1:

phospho-glycerate kinase 1

PI3K:

phosphoinositide 3-kinase

PCNA:

proliferating cell nuclear antigen

VEGFA:

vascular endothelial growth factor A.

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Acknowledgements

This project was supported by National Basic Research Program of China (973 Program, 2010CB529305); Shenzhen Basic Research Program (JC20110520111A); Research Grants Council RGC CERG CUHK (473008); Group Research Scheme CUHK (3110043); CUHK Focused Investment Gant (1903026), and RFCID (10090942, 11100022).

Author contributions: WD, SW, QZ, XL, KFC and JC performed the experiments; QT, participated in initial conception, technical and material support; ZC provided technical and material support; WD and SW analyzed the data and drafted the paper; JF and EKON provided material support; JJYS supervised and commented on the study; JY designed, supervised study and wrote the paper.

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Author notes

  1. W Du and S Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Digestive Disease and Department of Medicine and Therapeutics, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong

    W Du, S Wang, X Li, J J Y Sung & J Yu

  2. Division of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Renji Hospital, Shanghai Institution of Digestive Disease; State Key Laboratory of Oncogene and Related Genes,

    W Du & J Fang

  3. Key Laboratory of Gastroenterology & Hepatology, Ministry of Health, Shanghai, China

    W Du & J Fang

  4. Department of Clinical Oncology, The Chinese University of Hong Kong, Shatin, Hong Kong

    Q Zhou & Q Tao

  5. Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong

    J Chu

  6. Department of Biological Sciences and Biotechnology, School of Medicine, State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing, China

    Z Chang

  7. Department of Surgery, The Chinese University of Hong Kong, Shatin, Hong Kong

    E K O Ng

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Correspondence to J Fang or J Yu.

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Du, W., Wang, S., Zhou, Q. et al. ADAMTS9 is a functional tumor suppressor through inhibiting AKT/mTOR pathway and associated with poor survival in gastric cancer. Oncogene 32, 3319–3328 (2013). https://doi.org/10.1038/onc.2012.359

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