The Study On The Role Of TAMs CGI-58 In Promoting Colorectal Cancer Progression

colorectal cancer(CRC) is the third most common gastrointestinal malignancy. Like many other solid tumors, colorectal cancers are infiltrated with a variety of immune cells, including macrophages. Recently reported,Macrophages infiltrated in tumor tissues are commonly termed as tumor-associated macrophages(TAMs)。TAMs plays an important role in the malignant progression in inflammatory microenvironment. Recent studies suggest that metabolic reprogramming in stromal cells plays a pivotal role in regulating tumor biology. comparative gene identification-58(CGI-58), also termed as AB-hydrolase containing 5(ABHD5), is a co-activator of adipose triglyceride lipase(ATGL) which is a key enzyme involvement in the lipolysis from triglyceride to diglyceride and free fatty acid. human colorectal cancer tissue-derived macrophages expressed notably higher CGI-58 than that from the corresponding adjacent normal tissues in mRNA levels by Realtime PCR and in protein levels by immunohistochemistry. We use our lab macrophages- colon cancer cell co-culture system, specific knockdown macrophages CGI-58 and over-expression of macrophage CGI-58.In vivo and in vitro experiments show that the effects of macrophages CGI-58 in colon cancer cell proliferation, apoptosis and invasion, It reveals that Macrophages CGI-58 affect the malignant progression of colon cancer. At the same time, we further explore the possible mechanism by Western blot and Real-time PCR, ATMs CGI-58 deficiency-induced augment IL-1βtransciption inhibit the phosphorylation of FOXO1 activates FOXO1 transcription. As we know FOXOI can regulate apoptosis.Our study tumor-associated macrophages(TAMs) lipid metabolism as the starting point, the first analytical tumor associated macrophages(TAMs) CGI-58 in chronic inflammation and malignant progression of colon cancer, the results have important guiding significance for looking effective cancer control strategies from the perspective of the tumor microenvironment strategies.Objective:Revealing the role of cancer-associated macrophages participating in the progression of colorectal cancerMethod:1.expression level of CGI-58 in ATMsFor researching the association between Lipid metabolic reprogramming and cancer-associated macrophages, we utilize immunohistochemical method to exam the expression of CGI-58 in homo and mus colon cancer tissues and their para cancinoma tissues, the result of immunohistochemical have been scored on the basis of Immunoreactive Score. After been cultured in conditional media coming from CT26 and MC38 cells, Western blot and RT-PCR have been used to detect the expression level of CGI-58 in RAW2647. RAW2647 cultured in normal medium were used as control.2.CGI-58 in ATMs promot colorectal cancer progressionFor revealing the role of CGI-58 in ATMs participating in colorectal cancerprogression, we use lentivirus-mediated method to ablate and overexpress CGI-58 in RAW264.7. Western blot was used to identify the deletion and overexpression of CGI-58 in cellsIn vitro, conditional medium from control, shCGI-58, CGI-58 were used to treat CT26. CCK8 assay, Annexin V FITC assay.colone formation assay were used to check the implication of CGI-58 in colorectal cancer progressionIn vivo, BABL/C mice were seeded with CT26 and treated with conditional medium from control, shCGI-58 and control, CGI-58 sparately. Size of tumor was detected3.Preliminary discussion on the mechanism of macrophages CGI-58 promoting colorectal cancer progressionFour groups(normal group, CGI-58 overexpressed group, shCGI-58 group and inhibitor treated group) were used to detect the regulation of CGI-58 to FOXO1.The result were detected by Western blot and RT-PCRConclusion:1.In immunohistochemical experiment, expression level of CGI-58 is higher in ATMs compared to that in para-cancinoma macrophages. After treating with conditional medium from CT26 and MC38, CGI-58 in macrophages is up-regulated. So, we concluded that CGI-58 is up-regulated in TAMs, and it is due to the possible lipolysis caused by CGI-58 in lipid-rich TAMs.2. Compared with control group, CT26 treated with conditional medium from shCGI-58 macrophages had a decreased cell proliferation, increased cell cycle and apoptosis, and lower clone formation rate. In Subcutaneous tumor models, Treated with conditional medium from shCGI-58 macrophages, Subcutaneous tumor and metastasis is larger, survival rate of the mice is lower. We concluded that Ablation of CGI-58 in cancer-associated macrophages can inhibite colorectal cancerprogression3. After ablating CGI-58 in macrophages, IL-1β is up-regulated, pFOXO1 is down-regulated, and identical results were obtained when CGI-58 is overexpressed. So we concluded that ablating CGI-58 in macrophages can up-regulate IL-1β through inhibiting FOXO1 activity. Providing that CGI-58 expression was increased in tumor associated macrophages, we presumed that the CGI-58/FOXO1 pathway might potentiate the polarization of M2 macrophages and exert pro-tumor activity.