Cannabinoid receptor 2 as a novel target for promotion of renal cell carcinoma prognosis and progression

Abstract

Purpose

Renal cell carcinoma (RCC) is the most common malignancy of urogenital system, and patients with RCC may face a poor prognosis. However, limited curable therapeutic options are currently available. The aim of this study is to investigate the role of Cannabinoid receptor 2 (CB2) in RCC progression.

Methods

Immunohistochemistry was to investigate the expression pattern of CB2 in 418 RCC tissues and explore its prognostic function in RCC patients. Furthermore, the role of used CB2 si-RNA knockdown and inhibited by AM630, a CB2 inverse agonist, on cell proliferation, migration, and cell cycle of RCC cell lines in vitro was also investigated.

Results

We observed that CB2 was up-regulated in RCC tissues, and presented as an independent prognostic factor for overall survival of RCC patients and higher CB2 expression tends to have poor clinical outcomes in survival analyses. Moreover, we also observed that CB2, incorporated with pN stage, pathological grade, and recurrence or distant metastasis after surgery, could obviously enhance their prognostic accuracy in a predictive nomogram analysis. In addition, knockdown or inhibition by AM630 for the expression of CB2 in vitro could significantly decreased cell proliferation and migration, and obviously induced cell cycle arrest in G2/M of RCC cells.

Conclusions

CB2 expression is functionally related to cellular proliferation, migration, and cell cycle of RCC cells. Our data suggest that CB2 might be a potential therapeutic target for RCC.

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Abbreviations

RCC:

Renal cell carcinoma

CB2:

Cannabinoid receptor 2

ccRCC:

Clear-cell renal cell carcinoma

OS:

Overall survival

TMA:

Tissue microarrays

FBS:

Fetal bovine serum

DMSO:

Dimethyl sulfoxide

SRB:

Sulforhodamine B

C-index:

Harrell’s concordance index

AIC:

Akaike information criteria

IHC:

Immunohistochemistry

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Acknowledgements

This work was supported by National Natural Science Foundation of China (nos. 81472378, 81272841, and 91129725), Shanghai Committee of Science and Technology (13ZR1425100). All these study sponsors have no roles in the study design, in the collection, analysis, and interpretation of data.

Author information

Affiliations

Authors

Contributions

Jianfeng Wang, Yunze Xu, and Liangsong Zhu conceived and designed the experiments. Jianfeng Wang the manuscript and performed the experiments. Jianfeng Wang and Yunze Xu performed the immunohistochemistry assay. Jianfeng Wang and Liangsong Zhu performed the RNA interference (RNAi) transfection. Jianfeng Wang and Yun Zou performed the proliferative ability analysis. Jianfeng Wang, Wen Kong, Baijun Dong, and Jiwei Huang performed cell migration assay. Jianfeng Wang, Yonghui Chen and Wei Xue performed cell cycle analysis and statistical analysis. Yiran Huang and Jin Zhang oversight of all aspects of the study. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yiran Huang or Jin Zhang.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethics statement

This investigation was approved by the Ethics and Research Committees of Renji Hospital, Shanghai Jiao Tong University School of Medicine, and was conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guidelines. Tissue samples were obtained with written consent from all the patients.

Electronic supplementary material

Below is the link to the electronic supplementary material.

S1 Figure. Expression of CB2 is elevated in RCC tissues.

CB2 mRNA expression analyzed by real-time RT-PCR in RCC tissues. CB2 mRNA expression levels were normalized to GAPDH mRNA expression. CB2 mRNA was determined to be higher in the RCC tissues compared to adjacent normal tissues. (TIFF 39 kb)

S2 Figure. Inhibition of CB2 suppresses the migration and invasion ability of RCC cells.

(A and B) AM630 reduced the number of invasion cells in 786-O cell line at 10 μM and 20 μM concentrations (Bar: 1 mm); (C and D) 786-O cells were seeded on six-well plates. A single scratch was made after the cells grew about 90% confluence. After treatment of AM630 for 6 h, the cells were photographed (20 ×). The lines indicate that the area occupied by the initial scraping was quantified. (TIFF 21727 kb)

S3 Figure. Knockdown of CB2 significantly reduced the levels of cyclin B1 and cdc-25c expression in RCC cells.

Western blotting analysis revealed that knockdown of CB2 significantly reduced the expression levels of cyclin B1 and cdc-25c in 786-O and CAKI-1 cells. (TIFF 1643 kb)

S4 Figure. Inhibition of CB2 has little effect on the proliferation of HK-2.

HK-2 cells were treated at the indicated concentrations, and the cell viability was analyzed by SRB assay. AM630 shows little effect on cell proliferation ability of HK-2. (TIFF 20 kb)

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Cite this article

Wang, J., Xu, Y., Zhu, L. et al. Cannabinoid receptor 2 as a novel target for promotion of renal cell carcinoma prognosis and progression. J Cancer Res Clin Oncol 144, 39–52 (2018). https://doi.org/10.1007/s00432-017-2527-y

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Keywords

  • Renal cell carcinoma
  • Cannabinoid receptor 2
  • AM630