Galectin-1 Mediates NFs Transformation Into CAFs And Promotes Apatinib Resistance In Gastric Cancer

Background:Gastric cancer (GC) is the fourth most commonly diagnosed cancers, and the second leading cause of cancer-related deaths worldwide. Neoangiogenesis is an important process in tumor growth and metastasis. Vascular endothelial growth factor (VEGF) signaling is an important regulator of tumor angiogenesis, and anti-VEGF therapy is widely used in cancer therapy. Apatinib potently suppresses the kinase activity of VEGFR-2. A phase Ⅲ study of apatinib used as second-line therapy for advanced gastric cancer has shown encouraging progression-free survival and overall survival improvement. Currently, there are no validated biomarkers that can be used reliably to determine which patients with GC will respond to apatinib therapy. Cancer-associated fibroblasts (CAFs) contribute to tumor angiogenesis through increased secretion of growth factors and cytokines. Galectin-1 is expressed both in cancer cells and CAFs. Galectin-1 promotes tumor angiogenesis involving in multiple mechanisms. However, there is no evidence about the role of CAFs with galectin-1 overexpression in apatinib sensitivity in gastric cancer.Part ⅠBackground:Accumulating evidence suggests that crosstalk between cancer cells and cancer-associated fibroblasts (CAFs) play a pivotal role in tumor invasion and metastasis. Cancer cells induce normal fibroblasts (NFs) transformation into CAFs via secreted factors such as TGF-β, and subsequently activated CAFs promote tumor progression and chemotherapy resistance. Galectin-1 is expressed both in cancer cells and CAFs. In this section, we investigated the galectin-1/TGF-β axis in the interaction between cancer cells and CAFs.Methods:In this study, CAFs were isolated from freshly-resected gastric cancer tissues, and NFs were isolated from paried normal gastric tissues at least 5cm distant from cancer tissues. CAFs and NFs were identified using immunofluorescence and western blotting. Different expression of galectin-1 between CAFs and NFs was examined by quantitative real-time PCR and western blotting. Galectin-1 expression was upregulated in NFs via transfected with galectin-1 overexpression lentiviral vectors, and knockdown in CAFs via transfection with small interfering RNA (siRNA). Induction of transformation from NFs into CAFs by cancer cells via adding conditioned media (CMs) to the culture media of NFs. Galectin-1 and TGF-β expression in gastric cancer were detected by immunohistochemical staining. Kaplan-Meier curves described the survival of patients with gastric cancer, and clinical data was statistical analyzed by Log-rank test. Multivariate analysis was performed to analyze the gastric cancer prognosis and the clinical parameters with Cox regression hazards regression model.Results:Galectin-1 was overexpressed in CAFs compared with NFs, and promoted the transformation from NFs into CAFs. TGF-β secreted from cancer cells induced galectin-1 and the CAFs marker a-SMA expression in NFs. TGF-β-induced α-SMA expression in NFs was mediated by galectin-1. Galectin-1 expression in stroma was positively associated with TGF-β expression in cancer cells of gastric cancer tissues.Galectin-1 expression was associated with histological degree, tumor T stage, lymph node metastasis, TNM staging and poor prognosis. Multivariate analysis showed that histo logical degree, TNM staging, and galectin-1 expression is an independent risk factor for gastric cancer prognosis.Conclusion:Galectin-1 mediates TGF-β-driven NFs transformation into CAFs. Galectin-1 overexpression is associated with shorter overall survival time in patients with gatric cancer, and is an independent prognostic risk factor for gastric cancer.Part ⅡBackground:We have showed that galectin-1 was overexpression in CAFs, and negatively correlated with overall survival of patients with gastric cancer. This part of the study we explore the mechanisms by which galectin-1 contributes to tumor progression.Methods:The conditioned media (CMs) model was applied for determinating the role of fibroblasts expressing different levels of galectin-1 in cancer cells in vitro. Flow cytometry and transwell assay were used for cancer cell apoptosis, migration and invasion in vitro experiments. The relationship between Gall expression in fibroblasts and tumor growth was detected in nude mice subcutaneous transplanted tumors experiments.Results:CMs derived from CAFs with galectin-1 high expression significantly promoted cancer cell anti-apoptosis, migration and invasion compared with control, NFs group and CAFs with galectin-1 knockdown group (P<0.05). Western blotting evaluation demonstrated that anti-apoptotic protein Bcl-2 was upregulated, and pro-apoptotic protein Bax was downregulated in cancer cells treated with CMs from CAFs with galectin-1 overexpression compared with other groups. The results also exhibited that MMP9 degrading extracellular matrix was upregulated in cancer cells treated with CMs from CAFs with Gall overexpression. Meanwhile, the tumor volume was bigger in groups of NFs transfected with galectin-1 overexpression vector and CAFs, compared with the group of NFs (P<0.05). There was no difference in tumor volume between goups of NFs transfected with galectin-1 overexpression vector and CAFs.Conclusion:CAFs with galectin-1 overexpression promote tumor progression possibily through enhancing anti-apoptosis, migration and invasion of cancer cells. The effects of CAFs on cancer cell apoptosis dependent on regulating apoptotic-related gene expression, and on invasion dependents on regulating MMP9 expression.Part ⅢBackground:Apatinib potently suppresses the kinase activity of VEGFR-2, and significantly improved overall survival of advanced gastric cancer. But there are no validated biomarkers that can be used reliably to determine which patients with GC will respond to apatinib therapy. Galectin-1 contributes to tumor angiogenesis involving in multiple mechanisms. Therefore, the impact of galectin-1 expression in CAFs on apatinib sensitivity in gastric cancer needs to determine.Methods:CCK-8 assay and HUVEC tube formation assay were applied for evaluating the impact of galectin-1 on apatinib sensitivity in vitro. Models of nude mice subcutaneous tumors were used for evaluating the anti-tumor activity of apatinib in vivo.Results:Galectin-1 overexpression promoted bFGF expression in CAFs, and therefore suppressed the inhibitory role of apatinib in HUVEC proliferation and tube formation in vitro. The ability of anti-tumor growth and anti-angiogenesis of apatinib was abolished by galectin-1 upregulation in fibroblasts.Conclusion:Galectin-1 overexpression in fibroblasts induced apatinib resistance in gastric cancer.