During selleck kinase inhibitor tumor progression, however, the loss of TGFb growth inhibitory effects is frequently due to defects in c myc and p15 regulation by TGFb. Mean while, other TGFb responses prevail, unrelated to growth inhibition and favoring tumor progression and metastasis. Indeed, TGFb induces degradation of the ECM, inhibits cell adhesion and stimulates cell migration and invasion, thereby promoting tumor metastasis. Moreover, during cancer progression, tumor cells secrete increasing quantities of TGFb, which in turn alter the stroma environment, leading to stimulation of tumor angiogenesis and causing local and systemic immunosup pression, thus further contributing to tumor progression and metastasis. Together these studies highlight an important role for TGFb in advanced breast cancer.
However, the function for p21 downstream of TGFb has not been described in breast cancer. In this study, we found that high p21 expression corre lates with poor survival in breast cancer patients. The expression of p21 is required to promote tumor cell migration and invasion in vitro and local invasion in vivo. Furthermore, p21 expression is tightly regulated by TGFb/Smad3 signaling in a panel of human basal like tri ple negative breast cancer cell lines. We found p21 to physically interact with Smad3 and the histone acetyl transferase p/CAF in response to TGFb and identified p21 and p/CAF as key regulators of TGFb mediated breast cancer cell migration and invasion. We also showed that p21 and p/CAF regulate TGFb transcrip tional activity on multiple tumor promoting target genes by controlling Smad3 acetylation and Smad3 occupancy on its DNA binding elements.
Immunohistochemical analysis of tissue arrays from breast cancer patients revealed a significant correlation between active TGFb/ Smad3 signaling and high expression levels of both p21 and p/CAF in lymph node positive invasive ductal carci nomas. Together, our findings identified p21 and p/CAF as critical regulators of cell migration and invasion down stream of TGFb/Smad3 pathway in advanced breast cancer. Methods Cell culture and transfection Human breast carcinoma MDA MB231, SCP2 and SCP25 cells and HEK293 cells were grown in DMEM supplemented with 10% fetal bovine serum and 2 mM L glutamine at 37 C in 5% CO2. SUM149PT, SUM159PT and SUM229PE were grown in F 12 HAMS nutrient mixture supplemented with 5% FBS, 5 ��g/ml insulin, 1 ��g/ml hydrocortisone at 37 C in 5% CO2.
SUM1315MO2 were grown in F 12 HAMS nutrient mixture supplemented with 5% FBS, 5 ��g/ml insulin, 10 ng/ml epidermal growth factor at 37 C in 5% CO2. Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs, 6�� myc Smad2, myc Smad3, p/CAF and Flag tagged human p21 cDNAs using Lipofectamine 2000 reagent, according to the manufacturers Batimastat protocol.