ARAP1 negatively regulates stress fibers formation and metastasis in lung adenocarcinoma via controlling Rho signaling

Abstract

Small GTPases regulate multiple important cellular behaviors and their activities are strictly controlled by a mass of regulators. The dysfunction or abnormal expression of small GTPases or their regulators was frequently observed in various cancers. Here, we analyzed the expression and prognostic correlation of several GTPases and related regulators based on the TCGA database and found that Ankyrin Repeat and PH Domain 1 (ARAP1), a GTPase activating protein (GAP), is reduced in lung adenocarcinoma tissues compared to normal tissues and displays a positive correlation with overall survival (OS) and progression-free survival (PFS) of patients with lung adenocarcinoma. qPCR and western blot verified that ARAP1 is frequently downregulated in lung adenocarcinoma tumor tissues and cancer cells, and its downregulation might be mediated by epigenetic modification. Moreover, metastatic assays showed that overexpression of ARAP1 significantly inhibits metastasis of lung adenocarcinoma in vitro and in vivo. We further demonstrated that Rho signaling inhibition, mediated by RhoGAP activity of ARAP1, majorly contributes to suppressing migration and invasion of lung adenocarcinoma cancer cells via inhibiting stress fibers formation. In summary, this study indicates that ARAP1 may serve as a potential prognostic predictor and a metastatic suppressor in lung adenocarcinoma via its RhoGAP activity.

Keywords: ARAP1; GTPase; Lung adenocarcinoma; Metastasis; Rho; Stress fiber.

Fig. 1

Ankyrin Repeat and PH Domain 1 (ARAP1) is frequently down-regulated with a worse prognosis in lung adenocarcinoma (LUAD) patients. TCGA data analysis showed that ARAP1 expression is negatively correlated with poor OS (A) and PFS (B) of LUAD patients. Western blot (C) or qRT-PCR (E, F) examined the expression of ARAP1 in tumor and paired adjacent non-cancerous tissues of LUAD patients. T: tumor; A: paired adjacent non-cancerous. (D) The density of bands in C was quantified using Image J and relative expression to loading control was calculated. Western blot (G) and qRT-PCR (H) detected ARAP1 protein and mRNA level in different LUAD cancer cells and a normal lung epithelial tissue. ARAP1 mRNA level was normalized to the expression of GAPDH (mean ± SD, n = 3). ***P < 0.001 compared to N1 by Student t test

Fig. 2

ARAP1 expression in LUAD is regulated by epigenetic modification. A, B A549 cells were transfected with indicated siRNA for 72 h, and the cells were collected and used for qRT-PCR to detect the expression of ARAP1 mRNA. C A549 cells were treated with Aza (5 μM for 72 h), SAHA (2.5 μM for 24 h), GSK126 (5 μM for 72 h) alone or in combination. The cells were then collected and used for qRT-PCR to examine the expression of ARAP1 mRNA. The value in each group was displayed as fold change relative to siNC or DMSO (mean ± SD, n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 compared to siNC by Student t test

Fig. 3

ARAP1 inhibits metastasis and EMT of LUAD cancer cells. Wound healing (A, B) and transwell (C, D) assays detected the effects of ARAP1 OE on migration and invasion of A549 cells (mean ± SD, n = 3). Scale bar: 200 μm. E Western blot analyzed EMT markers expression in LUAD cells after ARAP1 OE. F The effect of ARAP1 OE on metastasis of A549 cells in vivo. Red arrows indicate metastatic nodules on the lung surface and the tumor number is counted. The relative area of tumor to lung of each H&E image was calculated. Scale bar: 500 μm (n = 6). *P < 0.05 and ***P < 0.001 compared to control by Student t test

Fig. 4

ARAP1 inhibits stress fibers formation. A Immunostaining analyzed stress fibers after ARAP1 OE. F-actin was labeled with phalloidin-FITC (green), Flag and Flag-ARAP1 transfected cells were labeled with anti-Flag antibody (red), and DNA was labeled with DAPI (blue). Scale bar: 10 μm. B Western blot examined the effects of ARAP1 OE on the expression of actin rearrangement related proteins and their phosphorylation including FAK and cofilin

Fig. 5

ARAP1 regulates stress fibers formation depending on its RhoGAP activity. A The schematic diagram of ARAP1 protein. B Immunostaining analyzed the effects of ARAP1 and its different mutants OE on stress fibers in A549 cells. DNA was labeled with DAPI. Scale bar: 10 μm. C Western blot examined the effects of ARAP1 and indicated mutants OE on the expression of actin rearrangement related proteins. The level of p-FAK and p-cofilin were quantified using Scion Image software and normalized to the density of the GAPDH bands. D Western blot detected the effects of ARAP1 OE on expression of Rho GTPases. E IP and western blot detected the effects of ARAP1 OE on Rho activity

Fig. 6

ARAP1-mediated inhibition of LUAD cancer cell metastasis is mainly dependent on its RhoGAP activity. Wound healing (A) and transwell (B, C) assays detected the effects of ARAP1 and indicated mutants OE on migration and invasion of A549 cells (mean ± SD, n = 3). Scale bar: 200 μm. D Western blot examined EMT markers expression after ARAP1 and indicated mutants OE. E The model of ARAP1 regulates metastasis in LUAD. ns: no significant; #P < 0.05; *P < 0.05 and ***P < 0.001 compared to control by Student t test