Targeting p21-activated kinase 1 inhibits growth and metastasis via Raf1/MEK1/ERK signaling in esophageal squamous cell carcinoma cells

Abstract

Background p21-activated kinase 1 (PAK1) plays a fundamental role in promoting the development and progression of several cancers and is a potential therapeutic target. However, the biological function and underlying mechanism of PAK1 in esophageal squamous cell carcinoma (ESCC) remain unclear. Methods The expression of PAK1 was detected in both ESCC cell lines and clinical samples. Cell growth was measured by MTT, focus formation and soft agar assays. Cell migration and invasion were detected by wound healing and transwell assays. Animal models of subcutaneous tumourigenicity and tail vein metastasis were performed to determine the inhibitory effect of pharmacological inhibitor IPA-3 on tumor growth and metastasis of ESCC cells. Results We found that PAK1 was frequently overexpressed in ESCC. Ectopic expression of PAK1 promoted cellular growth, colony formation and anchorage-independent growth. Overexpressing PAK1 also enhanced migration, invasion and the expression of MMP-2 and MMP-9 in ESCC cells. In contrast, silencing PAK1 by lentiviral knockdown or a specific inhibitor IPA-3 resulted in a contrary effect. Subsequent investigations revealed that Raf1/MEK1/ERK signaling pathway was involved in PAK1-mediated effect. Enhanced expression of Raf1 attenuated the inhibitory functions of PAK1 shRNA. Whereas blocking of Raf1 by shRNA or specific inhibition of MEK1 by U0126 antagonized the oncogenetic effect of PAK1 on ESCC cells. More importantly, Pharmacological inhibition of PAK1 by IPA-3 significantly suppressed tumor growth and lung metastasis of ESCC cells in vivo. Conclusions These data support that PAK1 is an ideal target for the development of potential therapeutic drugs for ESCC patients even with metastasis. Electronic supplementary material The online version of this article (10.1186/s12964-019-0343-5) contains supplementary material, which is available to authorized users.

Maternal Embryonic Leucine Zipper Kinase Promotes Tumor Growth and Metastasis via Stimulating FOXM1 Signaling in Esophageal Squamous Cell Carcinoma

“…Human ESCC cell lines TE-1, EC109, KYSE70, KYSE30, KYSE450, KYSE150, and EC9706 and one immortalized normal esophageal epithelial cell line Het-1A were obtained and cultured as our previously described (23). All cells were maintained in a humidified atmosphere (5% CO 2 ) at 37 • C and were recently tested for STR profiling and mycoplasma contamination.…”

“…qRT-PCR was performed as our previously described by using an Applied Biosystems 7900HT sequence detection system (Applied Biosystems) and SYBR Premix Ex Taq II (TaKaRa, Dalian, China) (23). PCR was conducted in a 20-µL volume reaction system containing 20 ng cDNA, 0.4 µmol/L paired primers and 10 µL SYBR Premix Ex Taq II according to the manufacture's manual.…”

“…Cells were then cultured in serum-free medium and recorded at 0 h, 24 h, and 48 h postscratching by using an inverted microscope. The percentage of wound closure was calculated by the following formula: wound closure (%) = (original gap distance-gap distance at the indicated time)/original gap distance × 100% as our previously described (23,29).…”

“…Plasmids including pCMV-Flag-His-puro-MELK (MELK), pCMV-Flag-His-puro-FOXM1 (FOXM1) and the empty vector pCMV-Flag-His-puro (Vector) were purchased from Transheep (Shanghai, China). For MELK overexpression, cells were transfected with 2 µg plasmids (MELK or Vector) for 48 h by using the Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, Inc.) following the manufacturer's manual (23). Cells were then selected in the presence of 1.5 µg/mL puromycin (Sigma-Aldrich).…”

“…The indicated sequences were described in Table S1. Lentiviruses production and subsequently tranfection were performed as our previously described (23). The cells were then cultured with puromycin for 3 weeks to establish stable cells.…”