TGF-β1-induced cell migration in pancreatic carcinoma cells is RAC1 and NOX4-dependent and requires RAC1 and NOX4-dependent activation of p38�MAPK

Abstract

Transforming growth factor (TGF)-β promotes epithelial-mesenchymal transition and cell invasion of cancer cells in part through the small GTPase RAC1. Since RAC1 can signal through reactive oxygen species (ROS), we probed the role of the ROS-producing NADPH oxidase (NOX) and p38 mitogen-activated protein kinase (MAPK) in mediating TGF-β1/RAC1-driven random cell migration (chemokinesis). Although the NOX isoforms NOX2, 4, 5, 6, and RAC1 were readily detectable by RT-PCR in pancreatic ductal adenocarcinoma (PDAC)-derived Panc1 and Colo357 cells, only NOX4 and RAC1 were expressed at higher levels comparable to those in peripheral blood monocytes. TGF-β1 treatment resulted in upregulation of NOX4 (and NOX2) and rapid intracellular production of ROS. To analyze whether RAC1 functions through NOX and ROS to promote cell motility, we performed real-time cell migration assays with xCELLigence® technology in the presence of the ROS scavenger N-acetyl-L-cysteine (NAC) and various NOX inhibitors. NAC, the NOX4 inhibitor diphenylene iodonium or small interfering RNA (siRNA) to NOX4, and the NOX2 inhibitor apocynin all suppressed TGF-β1-induced chemokinesis of Panc1 and Colo357 cells as did various inhibitors of RAC1 used as control. In addition, we showed that blocking NOX4 or RAC1 function abrogated phosphorylation of p38 MAPK signaling by TGF-β1 and that inhibition of p38 MAPK reduced TGF-β1-induced random cell migration, while ectopic expression of a kinase-active version of the p38 activating kinase MKK6 was able to partially rescue the decline in migration after RAC1 inhibition. Our data suggest that TGF-β1-induced chemokinesis in PDAC cells is mediated through a RAC1/NOX4/ROS/p38 MAPK cascade.

Vascular Endothelial Growth Factor-A Exerts Diverse Cellular Effects via Small G Proteins, Rho and Rap

“…Other signaling molecules, including p38 mitogen-activated protein (MAP) kinase, focal adhesion kinase, and Src, are activated by VEGF-A and contribute to angiogenesis in endothelial cells [ 17 , 18 , 19 ]. The regulation and function of these signaling pathways are directly or indirectly associated with small G proteins [ 20 , 21 , 22 , 23 ].…”

The Role of P TEN Protein Phosphatase in Nasopharyngeal Carcinoma: The Ability of TGF-β1 Inducing Migration and Invasion was Inhibited by PTEN Protein Phosphatase through Down-regulating p-P38

“…TGF-β1 plays a key role in the control of metastatic capacity of many malignancies [30,36].To demostrate the function of TGF-β1 on NPC cells,5-8F and HONE1 cells were cultured in 10% FBS medium with 0ng/ml,1ng/ml,5ng/ml,10 ng/ml of TGF-β1,respectively.After 48h,Scratch,Treanswell and Boyden asasay was used to detect the change in ability of migration and invasion( Figure.2A,B, C,D,E,F).As for 5-8F and HONE1 cells,metastatic capacity was enhanced in a concentration-dependent behavior.But,the diference between the experimental groups and the control group(0ng/ml TGF-β1 group) was statistical significance when the concentration of TGF-β1 was≥5ng/μl.And Western Blot was used to prove the change of EMT transformation,we found that the expression of E-cadherin was reduced,whereas,N-cadherin and VIMENTIN was increased( Figure.3 A,B).The results showed,compared to 0ng/ml TGF-β1,5ng/ml or 10ng/ml TGF-β1 promoted migration,invasion and EMT in NPC cells,and we gave that appropriate stimulus concentration of TGF-β1 was 5ng/μl.…”

“…Especially in the terms of TGF-β1-induced EMT in malignant tumor cells [21 -23].The positive rates of TGF-β1 protein in NPC tissues obviously exceed that in nasopharyngeal inflammation tisses,and may correlate with NPC distant metastases [24][25].In fact,recent researches show TGF-β1 could participate in EMT of nasopharyngeal carcinoma cells [26][27][28][29].TGF-β1-induced migration requires RAC1-dependent activation of P38 in pancreatic carcinoma [30].Interesting,PTEN can inhibit Rac1 activity [10,31].…”

NOX4, a new genetic target for anti‐cancer therapy in digestive system cancer

“…Interestingly, NOX4 may function as an antiapoptotic gene via another novel signaling pathway mediated by the state of JAK2 phosphorylation, which can be regulated by the production of ROS . During pancreatic cancer invasion, NOX4 induces the EMT mechanism and upregulates E‐cadherin to augment cell migration through the TGF‐β pathway, which has been correlated with the RAC/P38MAPK pathway.…”