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

  • Jianfeng Wang
  • Yunze Xu
  • Liangsong Zhu
  • Yun Zou
  • Wen Kong
  • Baijun Dong
  • Jiwei Huang
  • Yonghui Chen
  • Wei Xue
  • Yiran HuangEmail author
  • Jin ZhangEmail author
Original Article – Cancer Research



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.


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.


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.


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.


Renal cell carcinoma Cannabinoid receptor 2 AM630 



Renal cell carcinoma


Cannabinoid receptor 2


Clear-cell renal cell carcinoma


Overall survival


Tissue microarrays


Fetal bovine serum


Dimethyl sulfoxide


Sulforhodamine B


Harrell’s concordance index


Akaike information criteria





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.

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.

Compliance with ethical standards

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.

Supplementary material

432_2017_2527_MOESM1_ESM.tif (39 kb)
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)
432_2017_2527_MOESM2_ESM.tif (21.2 mb)
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)
432_2017_2527_MOESM3_ESM.tif (1.6 mb)
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)
432_2017_2527_MOESM4_ESM.tif (21 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)


  1. Brown I, Cascio MG, Rotondo D, Pertwee RG, Heys SD, Wahle KW (2013) Cannabinoids and omega-3/6 endocannabinoids as cell death and anticancer modulators. Prog Lipid Res 52(1):80–109CrossRefPubMedGoogle Scholar
  2. Caffarel MM, Andradas C, Mira E, Perez-Gomez E, Cerutti C, Moreno-Bueno G et al (2010) Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition. Mol Cancer 9:196CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chan PC, Sills RC, Braun AG, Haseman JK, Bucher JR (1996) Toxicity and carcinogenicity of delta 9-tetrahydrocannabinol in Fischer rats and B6C3F1 mice. Fundam Appl Toxicol 30(1):109–117CrossRefPubMedGoogle Scholar
  4. Chow WH, Dong LM, Devesa SS (2010) Epidemiology and risk factors for kidney cancer. Nat Rev Urol 7(5):245–257CrossRefPubMedPubMedCentralGoogle Scholar
  5. De Jesus ML, Hostalot C, Garibi JM, Salles J, Meana JJ, Callado LF (2010) Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas. Neurochem Int 56(6–7):829–833CrossRefPubMedGoogle Scholar
  6. Elbaz M, Ahirwar D, Ravi J, Nasser MW, Ganju RK (2016) Novel role of cannabinoid receptor 2 in inhibiting EGF/EGFR and IGF-I/IGF-IR pathways in breast cancer. Oncotarget 8(18):29668–29678PubMedCentralGoogle Scholar
  7. Elmore JM, Kadesky KT, Koeneman KS, Sagalowsky AI (2003) Reassessment of the 1997 TNM classification system for renal cell carcinoma. Cancer 98(11):2329–2334CrossRefPubMedGoogle Scholar
  8. Engeli S (2012) Central and peripheral cannabinoid receptors as therapeutic targets in the control of food intake and body weight. Handb Exp Pharmacol 209:357–381CrossRefGoogle Scholar
  9. Felder CC, Glass M (1998) Cannabinoid receptors and their endogenous agonists. Annu Rev Pharmacol Toxicol 38:179–200CrossRefPubMedGoogle Scholar
  10. Ficarra V, Novara G, Galfano A, Brunelli M, Cavalleri S, Martignoni G et al (2009) The ‘Stage, Size, Grade and Necrosis’ score is more accurate than the University of California Los Angeles Integrated Staging System for predicting cancer-specific survival in patients with clear cell renal cell carcinoma. BJU Int 103(2):165–170CrossRefPubMedGoogle Scholar
  11. Gupta K, Miller JD, Li JZ, Russell MW, Charbonneau C (2008) Epidemiologic and socioeconomic burden of metastatic renal cell carcinoma (mRCC): a literature review. Cancer Treat Rev 34(3):193–205CrossRefPubMedGoogle Scholar
  12. Guzman M, Sanchez C, Galve-Roperh I (2002) Cannabinoids and cell fate. Pharmacol Ther 95(2):175–184CrossRefPubMedGoogle Scholar
  13. Hang J, Hu H, Huang J, Han T, Zhuo M, Zhou Y et al (2016) Sp1 and COX2 expression is positively correlated with a poor prognosis in pancreatic ductal adenocarcinoma. Oncotarget 7(19):28207–28217CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hong SK, Jeong CW, Park JH, Kim HS, Kwak C, Choe G et al (2011) Application of simplified Fuhrman grading system in clear-cell renal cell carcinoma. BJU Int 107(3):409–415CrossRefPubMedGoogle Scholar
  15. Hu G, Ren G, Shi Y (2011) The putative cannabinoid receptor GPR55 promotes cancer cell proliferation. Oncogene 30(2):139–141CrossRefPubMedGoogle Scholar
  16. Jia N, Zhang S, Shao P, Bagia C, Janjic JM, Ding Y et al (2014) Cannabinoid CB2 receptor as a new phototherapy target for the inhibition of tumor growth. Mol Pharm 11(6):1919–1929CrossRefPubMedGoogle Scholar
  17. Klein Nulent TJ, Van Diest PJ, van der Groep P, Leusink FK, Kruitwagen CL, Koole R et al (2013) Cannabinoid receptor-2 immunoreactivity is associated with survival in squamous cell carcinoma of the head and neck. Br J Oral Maxillofac Surg 51(7):604–609CrossRefPubMedGoogle Scholar
  18. Larrinaga G, Sanz B, Perez I, Blanco L, Candenas ML, Pinto FM et al (2010) Cannabinoid CB(1) receptor is downregulated in clear cell renal cell carcinoma. J Histochem Cytochem 58(12):1129–1134CrossRefPubMedPubMedCentralGoogle Scholar
  19. Larrinaga G, Sanz B, Blanco L, Perez I, Candenas ML, Pinto FM et al (2013) Cannabinoid CB1 receptor is expressed in chromophobe renal cell carcinoma and renal oncocytoma. Clin Biochem 46(7–8):638–641CrossRefPubMedGoogle Scholar
  20. Ligresti A, Bisogno T, Matias I, De Petrocellis L, Cascio MG, Cosenza V et al (2003) Possible endocannabinoid control of colorectal cancer growth. Gastroenterology 125(3):677–687CrossRefPubMedGoogle Scholar
  21. Ligresti A, De Petrocellis L, Di Marzo V (2016) From phytocannabinoids to cannabinoid receptors and endocannabinoids: pleiotropic physiological and pathological roles through complex pharmacology. Physiol Rev 96(4):1593–1659CrossRefPubMedGoogle Scholar
  22. Liu CS, Chau SA, Ruthirakuhan M, Lanctot KL, Herrmann N (2015) Cannabinoids for the treatment of agitation and aggression in Alzheimer’s Disease. CNS Drugs 29(8):615–623CrossRefPubMedGoogle Scholar
  23. Ljungberg B, Campbell SC, Choi HY, Jacqmin D, Lee JE, Weikert S et al (2011) The epidemiology of renal cell carcinoma. Eur Urol 60(4):615–621CrossRefPubMedGoogle Scholar
  24. Ljungberg B, Bensalah K, Canfield S, Dabestani S, Hofmann F, Hora M et al (2015) EAU guidelines on renal cell carcinoma: 2014 update. Eur Urol 67(5):913–924CrossRefPubMedGoogle Scholar
  25. Malik Z, Baik D, Schey R (2015) The role of cannabinoids in regulation of nausea and vomiting, and visceral pain. Curr Gastroenterol Rep 17(2):429CrossRefPubMedGoogle Scholar
  26. Martínez-Martínez E, Gómez I, Martín P, Sánchez A, Román L, Tejerina E et al (2015) Cannabinoids receptor type 2, CB2, expression correlates with human colon cancer progression and predicts patient survival. Oncoscience 2(2):131–141CrossRefPubMedPubMedCentralGoogle Scholar
  27. Martínez-Martínez E, Martín-Ruiz A, Martín P, Calvo V, Provencio M, García JM (2016) CB2 cannabinoid receptor activation promotes colon cancer progression via AKT/GSK3β signaling pathway. Oncotarget 7(42):68781–68791CrossRefPubMedPubMedCentralGoogle Scholar
  28. McKallip RJ, Nagarkatti M, Nagarkatti PS (2005) Delta-9-tetrahydrocannabinol enhances breast cancer growth and metastasis by suppression of the antitumor immune response. J Immunol 174(6):3281–3289CrossRefPubMedGoogle Scholar
  29. Mills B, Yepes A, Nugent K (2015) Synthetic cannabinoids. Am J Med Sci 350(1):59–62CrossRefPubMedGoogle Scholar
  30. Palazzo E, Luongo L, Novellis V, Rossi F, Maione S (2010) The role of cannabinoid receptors in the descending modulation of pain. Pharmaceuticals (Basel) 3(8):2661–2673CrossRefGoogle Scholar
  31. Patard JJ, Kim HL, Lam JS, Dorey FJ, Pantuck AJ, Zisman A et al (2004) Use of the University of California Los Angeles integrated staging system to predict survival in renal cell carcinoma: an international multicenter study. J Clin Oncol 22(16):3316–3322CrossRefPubMedGoogle Scholar
  32. Perez-Gomez E, Andradas C, Blasco-Benito S, Caffarel MM, Garcia-Taboada E, Villa-Morales M et al (2015) Role of cannabinoid receptor CB2 in HER2 pro-oncogenic signaling in breast cancer. J Natl Cancer Inst 107(6):077CrossRefGoogle Scholar
  33. Reimers M, Carey VJ (2006) Bioconductor: an open source framework for bioinformatics and computational biology. Methods Enzymol 411:119–134CrossRefPubMedGoogle Scholar
  34. Ross RA, Brockie HC, Stevenson LA, Murphy VL, Templeton F, Makriyannis A et al (1999) Agonist-inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656, and AM630. Br J Pharmacol 126(3):665–672CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sarfaraz S, Afaq F, Adhami VM, Mukhtar H (2005) Cannabinoid receptor as a novel target for the treatment of prostate cancer. Cancer Res 65(5):1635–1641CrossRefPubMedGoogle Scholar
  36. Siegel RL, Miller KD, Jemal A (2016) Cancer statistics, 2016. CA Cancer J Clin 66(1):7–30CrossRefPubMedGoogle Scholar
  37. Smaletz O (2015) Current management and future directions in the treatment of advanced renal cell carcinoma-a latin american perspective: 10 years in review. Int Braz J Urol 41(5):835–843CrossRefPubMedPubMedCentralGoogle Scholar
  38. Velasco G, Hernandez-Tiedra S, Davila D, Lorente M (2016) The use of cannabinoids as anticancer agents. Prog Neuropsychopharmacol Biol Psychiatry 64:259–266CrossRefPubMedGoogle Scholar
  39. Vemuri VK, Makriyannis A (2015) Medicinal chemistry of cannabinoids. Clin Pharmacol Ther 97(6):553–558CrossRefPubMedPubMedCentralGoogle Scholar
  40. Waalkes S, Kramer M, Herrmann TR, Schrader AJ, Kuczyk MA, Merseburger AS (2010) Present state of target therapy for disseminated renal cell carcinoma. Immunotherapy 2(3):393–398CrossRefPubMedGoogle Scholar
  41. Wang L, Ma L, Wang X, Li B, Guo S, Qiao Q (2015) Therapeutic effects and associated adverse events of first-line treatments of advanced renal cell carcinoma (RCC): a meta-analysis. Int Urol Nephrol 47(4):617–624CrossRefPubMedGoogle Scholar
  42. Xu X, Liu Y, Huang S, Liu G, Xie C, Zhou J et al (2006) Overexpression of cannabinoid receptors CB1 and CB2 correlates with improved prognosis of patients with hepatocellular carcinoma. Cancer Genet Cytogenet 171(1):31–38CrossRefPubMedGoogle Scholar
  43. Zurier RB, Burstein SH (2016) Cannabinoids, inflammation, and fibrosis. Faseb J 30(11):3682–3689CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of Urology, Renji Hospital, School of MedicineShanghai Jiaotong UniversityShanghaiChina

Personalised recommendations