P. W. Underwood1, D. Zhang1, A. Delitto1, M. H. Gerber1, D. Delitto1, S. J. Hughes1, A. R. Judge1, J. G. Trevino1 1University Of Florida,Gainesville, FL, USA
Introduction:
Nicotine promotes pancreatic cancer (PC) progression and chemoresistance. Interleukin 8 (IL-8) contributes to metastasis and chemoresistance in PC cells and promotes cachexia. The relationship between nicotine and the tumor microenvironment is poorly understood. We hypothesize that nicotine induces IL-8 secretion through effects on the tumor microenvironment in PC.
Methods:
Patient-derived tumor associated stroma (TAS) and primary pancreatic ductal adenocarcinoma cancer cells (PCC) were treated with varying doses of nicotine or control (PBS). Cell lysates and mRNA were collected at different time points and ELISA and quantitative PCR was performed to assess IL-8 levels. PCR was used to assess for nicotinic acetylcholine receptors (nAChR) and IL-8 receptors on cells. Western blot was performed to assess for Src tyrosine kinase, Akt, and ERK activation. Cells were treated with ERK inhibitor UO126 to assess expression of IL-8 levels. To confirm the effects of IL-8 on cachexia, mice were then injected with 50 µg/kg of recombinant IL-8 for a week and assessed for body weight, tibialis anterior and gastrocnemius weight, and epididymal fat weight. Patient-derived xenografts (PDX) were implanted in NSG mice and treated with nicotine or control (PBS). Tumors were harvested and IL-8 expression was assessed.
Results:
nAChR receptors (α3, α5, α7, α9, β2) are present on TAS cells. Nicotine induces increased expression of these receptors on TAS cells. IL-8 levels were significantly higher in TAS cells treated with nicotine than control (18.8 ng/ml ± 4.3 vs. 7.6 ng/ml ± 1.3, p < 0.001). Increased exposure of nicotine induced higher levels of IL-8 expression. IL-8 levels were significantly higher in nicotine treated TAS compared to PCC (4.7 ng/ml ± 0.4 vs. 0.4 ng/ml ± 0.22, p <0.001). Nicotine induces activation of ERK MAPK but not Src tyrosine kinase or Akt. Pharmacologic inhibition of ERK significantly reduced IL-8 levels in nicotine treated TAS cells. Relative IL-8 receptor expression was 10 fold greater on PCC cells compared to TAS before treatment with nicotine (p < 0.001). IL-8 receptor expression increased to > 20 fold higher on PCC after nicotine exposure (p < 0.001). Treatment of mice with IL-8 resulted in a significant decrease in body weight (11%), tibialis anterior weight (17%), gastrocnemius weight (15%), and epididymal fat weight (34%). In nicotine treated PDX mice, relative IL-8 expression was increased four-fold in tumor tissue when compared to control (p = 0.009).
Conclusion:
Nicotine significantly increases the expression of IL-8. TAS cells, rather than PCC, seem to be the primary source of IL-8. The pathway appears to be ERK dependent and may contribute to a cachectic phenotype in PC patients. Further investigations into molecular targets that contribute to cancer cachexia are warranted.