ABSTRACT
OBJECTIVE: Cancer stromal fibroblasts are important members of the cancer microenvironment. In this study, we determined the effect of sunitinib, a small molecule tyrosine kinase inhibitor, on the primary human colonic fibroblasts. METHODS: Cell cycle analysis and cell proliferation assays were performed to evaluate the inhibitory effect of sunitinib in vitro. Western-blot analysis was performed to evaluate variations in the levels of phosphorylated platelet-derived growth factor receptor ß (PDGFR-ß), Akt, and ERK proteins. Co-injection of SW620 cells and colonic fibroblasts in nude mice was employed to test anti-growth efficacy in vivo. RESULTS: Sunitinib was found to effectively inhibit the growth of primary colonic fibroblasts. Low-dose sunitinib blocked the PDGF-BB-induced cell proliferation and PDGFR-ß signaling. Co-injection of SW620 cells and colonic fibroblasts in nude mice generated greater tumor volumes than single injection of SW620 cells. Sunitinib treatment inhibited the SW620 cell+colonic fibroblast tumor growth more effectively than treatment of 5-fluorouracil. CONCLUSIONS: Sunitinib mesylate inhibited the proliferation of primary human colonic fibroblasts through target-inhibited PDGFR signaling in vitro and in vivo.
Subject(s)
Colon/drug effects , Colon/pathology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Indoles/pharmacology , Pyrroles/pharmacology , Animals , Antineoplastic Agents/pharmacology , Becaplermin , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Colon/metabolism , Colonic Neoplasms/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Fluorouracil/pharmacology , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-sis/antagonists & inhibitors , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Signal Transduction/drug effects , Stromal Cells/drug effects , Stromal Cells/metabolism , Stromal Cells/pathology , Sunitinib , Tumor Cells, Cultured , Tumor Microenvironment/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The cooperation and communication between different cell signaling transduction pathways are considered critical in the development of various types of cancer as well as drug resistance. There is evidence of crosstalk between the G protein-coupled receptor 30 (GPR30), the newly discovered estrogen receptor (ER), and the ErbB family. Heregulin (HRG)-ß1, the ligand for ErbB3 and ErbB4, upregulates GPR30 expression in MCF-7, T-47D and BT-474 breast cancer cell lines that express ERα. In the present study, recombinant human HRG-ß1 was used to investigate the upregulation of GPR30 expression by HRGs in MCF-7 breast cancer cells which were ERα-positive. In MCF-7 cells, the ErbB2 inhibitor, AG825, the MAPK inhibitor, PD98059, and the MEK1/2 inhibitor, U0126, blocked the HRG-ß1-induced GPR30 expression. 17-ß-estradiol (E2) boosted the HRG-ß1-induced proliferation, migration and invasion of MCF-7 cells. Similar to E2, the specific GPR30 agonist, G-1, promoted HRG-ß1-induced migration and invasion, but inhibited growth. Using the specific GPR30 antagonist, G-15, or the small interfering RNA for GPR30, the functions of GPR30 after treatment with HRG-ß1 were further investegated. The results from our study indicate that the interruption between GPR30 signaling and the ErbB family system may serve as a promising therapeutic strategy for breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Cell Movement/genetics , Neuregulin-1/pharmacology , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic , Humans , MAP Kinase Signaling System , Receptor, ErbB-2/metabolism , Receptors, Estrogen/genetics , Receptors, G-Protein-Coupled/genetics , Up-Regulation/geneticsABSTRACT
Estrogen receptor (ER)-negative breast cancer cells are probably more aggressive with larger metastatic potential than ER-positive cells. Loss of ER in recurrent breast cancer is associated with poor response to endocrine therapy. G protein-coupled receptor 30 (GPR30) is expressed in half of ER-negative breast cancers. Tumor cell-derived heregulin-ß1 (HRG-ß1) is also found mainly in ER-negative cancer. In SkBr3 breast cancer cells that lack ER but express GPR30, HRG-ß1 upregulates mRNA and protein levels of GPR30 by promoting ErbB2-ErbB3 heterodimerization and activating the downstream MAPK-ERK signaling pathway. Moreover, GPR30 boosts HRG-ß1-induced migration and invasion of SkBr3 cells after combinative treatment with E2, 4-hydroxy-tamoxifen or the specific GPR30 agonist G-1, which are blocked by the specific GPR30 antagonist G-15 or the transfection with the small interfering RNA for GPR30. The ErbB2 inhibitor AG825 and the MEK1/2 inhibitor U0126 also partly inhibit the enhanced migration and invasion. Therefore, HRG-ß1-induced migration and invasion partly depend on the upregulation of GPR30 expression through activation of the ErbB2-ERK pathway in SkBr3 cells. The results of this study indicate that the crosstalk between GPR30 and HRGs signaling is important for endocrine therapy resistance and may provide a new therapeutic way to treat breast cancer.
Subject(s)
Breast Neoplasms/pathology , Cell Movement , Neuregulin-1/metabolism , Receptors, Estrogen/biosynthesis , Receptors, G-Protein-Coupled/biosynthesis , Breast Neoplasms/metabolism , Cell Line, Tumor , Female , Humans , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase Kinases/metabolism , Neoplasm Invasiveness , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/metabolism , Up-RegulationABSTRACT
OBJECTIVE: Cancer-associated fibroblasts (CAFs) are one of the hallmarks of the cancer microenvironment. Recent evidence has indicated that CAFs are more competent in enhancing cancer cell growth and migration than normal fibroblasts. However, the unique protein expression of CAFs has not been fully elucidated. This study aims to investigate the characterizations of colon CAFs by comparing the differential protein expression between CAFs and normal fibroblasts. METHODS: Primary fibroblasts were isolated from surgical specimen of human colon cancer and matched normal colonic tissue. Purity of the cell population was verified through immunostain analysis. Total cell lysates and conditioned media from each group of cells were extracted, and protein expression analysis was conducted using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip platform. RESULTS: Most primary cells showed typical fibroblast-like features after two weeks. Increased proportion of α-smooth muscle actin-positive myofibroblasts was detected within the CAFs in four of the six pairs of primary cells. Fibroblast activation protein was weakly expressed in most cells without differences. Using SELDI-TOF-MS ProteinChip platform, four protein peaks mass over charge ratio (m/z) 1142, 3011, 4035, and 4945 were detected in the total cell lysates, and two protein peaks m/z 1368 and 1389 were detected in the conditioned media. The potential candidate proteins found in the Swiss-Prot database include morphogenetic neuropeptides, FMRFamide-related peptides, insulin-like growth factor II, thymosin ß-4-like protein 3, and tight junction-associated protein 1. CONCLUSIONS: Using the SELDI-ProteinChip platform, differential protein expressions were identified in colon CAFs compared with normal colonic stromal fibroblasts. The complex proteomic alternations in colon CAFs may play important roles related to the colon cancer microenvironment.
