DPP4 Promotes High Fat Diet Induced Cancer Progression Via CCL2 Activation

Obesity has been increasingly recognized as a high risk factor for several common types of cancer.Compared with lean patients,obese patients are more likely to be diagnosed with a larger,higher grade tumor,an increased incidence of tumor metastases,and elevated risk of distant recurrence.Recent studies have shown that obesity was usually accompanied by an elevated level of serum Dipeptidyl Peptidase 4(DPP4)which is a novel adipokine potentially linking obesity to the metabolic syndrome.DPP4 is a multifunctional cell surface protein and is widely expressed in most cell types.It is also present in serum and other body fluids in a truncated form which preferentially cleaves N-terminal dipeptides from polypeptides with proline or alanine in the penultimate position,and in doing so,regulates the activities of a number of cytokines and chemokines.Although DPP4 has been documented as an important diagnostic or prognostic biomarker in several clinical settings,its role in the pathogenesis of cancer,especially in the context of obesity,is largely unknown.Here we aim to investigate whether the increased serum DPP4 activity contributes to obesity-associated tumor progression and explore the therapeutic potential of DPP4 inhibitor in cancer treatment.We chronically treated wild type or DPP4-deficient rats with diethylnitrosamine(DEN)to produce a multistage hepatocarcinogenesis model.The rats were also placed either on high fat diet(HFD;45 kcal% fat)to induce obesity or on low fat diet(LFD;10 kcal% fat)representing the lean one.HCC development was monitored at 15 weeks and 20 weeks after DEN treatment.We found that high-fat diet dramatically promoted hepatocarcinogenesis and induced extensive lung metastasis.At the 20 th week of the experiment,the survival rate of HFD-treated rats was 20% compared to nearly 85% in lean rats.However,DPP4-defecient rats displayed a significant amelioration in DEN-induced liver cirrhosis and tumorigenesis.More importantly,DPP4 deficiency prevented lung metastasis induced by high-fat diet and increased the survival rate to 80% at 20 th week of experiment.We then administered the DPP4 inhibitor vildagliptin in drinking water(150mg/L)to investigate its preventive effect on obesity-accelerated liver cancer development and metastasis.As expected,DPP4 inhibitor treatment led to a significant reduction in tumor formation and lung metastasis.Animal survival was also significantly improved after vildagliptin treatment.The therapeutic effect of vildagliptin was further confirmed in a xenografted tumor model in nude mice fed with high fat-diet.To directly test the hypothesis that DPP4 has a role in obesity-associated tumor metastasis,we used two well-studied mouse models.Experimental metastasis was established by injecting B16F10 melanoma cells into the spleen of mice fed with HFD and liver metastasis was monitored.Mice treated with DPP4 inhibitor showed much lower number of metastatic tumors and longer survival rate compared with saline control.We then determined the effects of DPP4 activity on spontaneous metastasis using LLC cells by injection of the cells subcutaneously.The metastatic tumor number was significantly lower in vildagliptin-treated mice than in control mice,supporting the notion that DPP4 contributes to tumor metastasis.As angiogenesis constitutes an important point in the control of cancer metastasis,we evaluated whether DPP4 inhibition reduced the angiogenic process in HFD-accelerated tumor progression.Immunohistochemistry analyses revealed that the capacity of angiogenesis is strongly enhanced in HFD-maintained wild-type rats but not in DPP4-deficient rats,as shown by the expression of endothelial marker CD31.Similarly,vildagliptin treatment resulted in a reduced number of CD31-positive cells inside tumors.These results suggest that DPP4 played a critical role in HFD-induced tumor angiogenesis and metastasis.Next,we explored the mechanism by which DPP4 promoted tumor neovascularization.The substrates of DPP4 are proline(or alanine)-containing peptides and include growth factors,chemokines and neuropeptides.Given the key roles of chemokines in tumor angiogenesis,we performed protein array analysis(Ray Biotech Mouse Chemokine Array Q1)to screen chemokines attributable to HFD-induced angiogenesis.We found that the serum levels of CCL2,a known substrate of DPP4,were significantly reduced in DPP4-deficient or vildagliptin-treated animals on high-fat diet.It was well documented that CCL2 had potent facilitating effect on angiogenesis and macrophages recruitment.In accordance,the number of infiltrating CD68-positive macrophages was evidently decreased in tumors from obese animals without DPP4 or treated with vildagliptin.To confirm the vasoactive effects of CCL2 in vitro,we isolated primary endothelial cell and treated them directly with CCL2 or with sera from control and HFD-fed animals.Interestingly,we found that both CCL2 and serum from HFD-fed animals were able to enhance neovascularization.In contrast,addition of CCL2 blocking antibody prevented the vasoactive effects of serum from obese animals,suggesting that CCL2 might,at least in part,mediate the angiogenesis-promoting effects of DPP4.Finally,clinical study indicates that the DPP4 activities are significantly correlated with the body mass index(BMI)index and serum levels of CCL2 concentrations.Taken together,our results demonstrate a tumor promoting effect of obesity-associated DPP4 and support the use of DPP4 or CCL2 inhibitors for preventing tumor angiogenesis and metastasis.