Low Expression of Rasal2 Promotes Non-small Cell Lung Cancer Metastasis through Ras/ERK Pathway

Abstract

The RAS protein activator like 2 (Rasal2) has been reported to be a tumor suppressor in variety of cancers; while an oncogenic protein in ovarian cancer and triple negative breast cancer (TNBC). However, the exact role of Rasal2 in non-small cell lung cancer (NSCLC) is lacking. This study aimed to investigate the role of Rasal2 in NSCLC and the underlying mechanisms. Rasal2 expression level was measured in NSCLC tissue and cells by using quantitative (q)-PCR and immunoblotting analysis. The clinical implication of Rasal2 in NSCLC patients was also analyzed. The function role of Rasal2 in NSCLC cells were measured by small interfering RNA (si-RNA), immunostaining, transwell assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Low Rasal2 expression level was observed in human NSCLC tissue and cell lines and significantly related to tumor thickness, ulceration and TNM staging in NSCLC patients. Rasal2 knockdown significantly increased NSCLC cell invasion and migration. Mechanistically, we showed that Rasal2 knockdown significantly increased the phosphorylation level of extracellular signal-regulated kinase (ERK)/Raf1/mitogen-activated protein extracellular kinase (MEK) thus activated Ras/ERK signal pathway. Thus, our data showed that Rasal2 is downregulated in NSCLC cells and act as an epithelial–mesenchymal transition (EMT) and metastasis suppressor through the Ras/ERK pathway. Rasal2 may be a prognostic biomarker for NSCLC in the future.

INTRODUCTION

Lung cancer is most common and the leading cause of cancer death around the world.¹⁾ According to the microscopic features, non-small cell lung cancer (NSCLC) accounts for approximately 85% of overall reported cases.²⁾ The main cause of high mortality in NSCLC is metastatic spread.³⁾ Therefore, identification of key regulators and prognostic markers in NSCLC metastasis and investigation of its underlying mechanism become the prerequisites for improving NSCLC therapeutics.

RAS protein activator like 2 (Rasal2), as a RasGap, can inhibit the Ras signal pathway through catalyzing the hydrolysis of Ras-guanosine 5′-triphosphate (GTP) to Ras-guanosine 5′-diphosphate (GDP).⁴⁾ Rasal2 was first reported as a tumor suppressor and down-regulated in luminal B breast cancer, which correlated with metastasis and poor outcomes.⁵⁾ In addition, Rasal2 knockdown enhances lung cancer invasion and migration through epithelial–mesenchymal transition (EMT) in nude mouse.⁶⁾ However, some reports showed that Rasal2 was an oncogenic protein and upregulated in triple-negative breast cancer (TNBC), where it promoted cell invasion and metastasis.⁷⁾ Moreover, Rasal2 can interact with ECT2 to regulate RHO activity in astrocytoma cells.⁸⁾ Recently, report suggested that Rasal2 S351 phosphorylation promoted breast tumorigenesis and correlated with a poor prognosis in breast cancer patients.⁹⁾ Therefore, the function role of Rasal2 in cancer seems complicated.

This study aimed to investigate the clinical features of Rasal2 expression in NSCLC and to explore the biological effects of Rasal2 in NSCLC cell metastasis.

RESULTS

Rasal2 Was Down-Regulated in Human NSCLC and Correlated with Poor Clinical Outcomes

We first investigated Rasal2 mRNA expression in 74 NSCLC tissues and paired normal tissues adjacent to NSCLC tissues by using quantitative (q)-PCR assays. The results showed that Rasal2 mRNA in human NSCLC tissues was lower than paired tissues adjacent to cancer (Fig. 1A). In addition, immunoblotting results showed that Rasal2 protein level was decreased in NSCLC tumor tissues (Fig. 1B). We then measured the relationship between Rasal2 expression level and clinic-pathological parameters in NSCLC patients. The result showed that Rasal2 expression was significantly correlated with tumor thickness (χ² test, p = 0.027), ulceration (χ² test, p = 0.035) and TNM staging (χ² test, p = 0.044) but not with age or gender (p > 0.05; Table 1). Moreover, the prognostic significance of Rasal2 by using Kaplan–Meier method showed that patients with low Rasal2 expression had shorter survival than patients with high Rasal2 expression (log-rank, p = 0.016; Fig. 1C).

Fig. 1. Rasal2 Was Down-Regulated in Human NSCLC and Correlated with Poor Clinical Outcomes

(A) RT-PCR analysis of Rasal2 expression level in 74 pairs of NSCLC tissues and adjacent non-tumor tissues. (B) Rasal2 protein expression was measured in NSCLC and adjacent non-tumor tissues. (C) Kaplan–Meier survival curve in NSCLC patients as defined by low and high expression of Rasal2. ** p < 0.01.

Table 1. Relationship between Rasal2 Expression and Clinic-Pathological Parameters of NSCLC Patients

Parameters	Case (n = 74)	Rasal2 expression		p
		Low (n = 42)	High (n = 32)
Age
≤65	24	14	10	0.82
>65	50	28	22
Gender
Male	47	27	20	0.47
Female	27	15	12
Tumor thickness
≤3 mm	32	6	26	0.027*
>3 mm	42	36	6
Ulceration
No	29	7	22	0.035*
Yes	45	35	10
TNM staging
I	33	4	29	0.044*
II/III	41	38	3

Results were represented as the means ± standard deviation (S.D.). * p < 0.05 considered as statistically significant.

Expression of Rasal2 in NSCLC Cell Lines

We then measured the Rasal2 mRNA expression in human NSCLC cell lines A549, H1299, H2009 and benign cell line BEAS-2B by q-PCR assays. Results showed that Rasal2 mRNA level was highly expressed in benign cell line BEAS-2B when compared to NSCLC cell lines A549, H1299 and H2009 (Fig. 2A). Consistently, immunoblotting assays revealed that Rasal2 protein level was lower in NSCLC cells (Fig. 2B), indicating that Rasal2 was also down-regulated in NSCLC cells.

Fig. 2. Relative Rasal2 Expression Level in NSCLC Cell Lines

(A) Rasal2 mRNA expression level in human NSCLC cell lines A549, H1299, H2009 and benign cell line BEAS-2B. (B) Rasal2 protein expression level in human NSCLC cell lines A549, H1299, H2009 and benign cell line BEAS-2B. All data were measured in triplicate as mean ± S.D. * p < 0.05, ** p < 0.01.

Rasal2 Inhibits NSCLC Cell Metastasis

As Rasal2 relatively high expressed in NSCLC cell line H2009, we therefore knockdown Rasal2 in H2009 cells by using small interfering RNA (si-RNA). The mRNA and protein level of Rasal2 was significantly decreased after cell transfected with Rasal2 si-RNA (Figs. 3A, B). We then use these cells to measure the function role of Rasal2 in NSCLC cell invasion and migration. As showed in Fig. 3C, both the cell invasion and migration were greatly increased after Rasal2 knockdown in H2009 cells. We also overexpressed Rasal2 by transfection of Myc-Rasal2 to A549 cells (relatively low Rasal2 expression) and found that Rasal2 overexpression dramatically inhibited A549 cell migration and invasion (Figs. 3D, E). These results suggested that Rasal2 might inhibit H2009 cells metastasis.

Fig. 3. Rasal2 Inhibits NSCLC Cell Metastasis

(A, B) H2009 cells were transfected with si-Rasal2-1, si-Rasal2-2 or si-NC. After 48 h, q-PCR and Western blot were performed. (C) Cell generated in (A) were used of transwell migration or invasion assays respectively. (D) A549 cells were transfected with Myc-Rasal2 or Myc-pcDNA3.1 vector. After 24h, Western blot were performed. (E) Cell generated in (D) were used of transwell migration or invasion assays respectively. All data were measured in triplicate as mean ± S.D. * p < 0.05, ** p < 0.01.

Rasal2 Inhibits NSCLC Cell Metastasis via EMT

To further explore whether EMT was involved in this Rasal2-regulated cancer cell metastasis, we examine the expression level of metastasis related proteins in H2009 cells by immunoblotting assay (Fig. 4A). We found that Rasal2 knockdown greatly increased the expression level of vimentin, N-cadherin and snail (mesenchymal markers) while reduced the expression level of E-cadherin (epithelial marker). Consistently, immunofluorescence showed that Rasal2 knockdown reduced E-cadherin protein level while increased vimentin protein level, suggesting that Rasal2 regulates NSCLC cell invasion and migration through EMT (Fig. 4B).

Fig. 4. Rasal2 Inhibits NSCLC Cells Metastasis via EMT

(A, B) H2009 cells were transfected with si-Rasal2-1, si-Rasal2-2 and si-NC for 48 h. Western blot analysis or immunostaining assays were performed with indicated antibodies. Scale bars, 20 µm. All data were measured in triplicate as mean ± S.D. * p < 0.05.

Rasal2 Inhibits Ras/Extracellular Signal-Regulated Kinase (ERK) Signal Pathway

Rasal2 is one of the RasGAPs, so we next investigated whether Rasal2 knock down could activated Ras/ERK pathway by detecting Raf-1, mitogen-activated protein extracellular kinase (MEK) and ERK phosphorylation status. As showed in Fig. 5A, Rasal2 knockdown significantly increased Raf-1, MEK1/2 and ERK phosphorylation in H2009 cells as compared with control cells. However, mitogen-activated protein kinase (MAPK) pathways like c-Jun N-terminal kinase (JNK) and p38 did not affect much after Rasal2 knockdown in H2009 cells. To determine whether Rasal2 knockdown affected activity, we performed Ras activation assay. The results showed that, Ras GTPase activity was higher after Rasal2 knockdown (Fig. 5B). Moreover, overexpression of Rasal2 in A549 cells dramatically reduced Raf-1, MEK and ERK phosphorylation (Fig. 5C), indicating that Rasal2 could inhibits Ras/ERK pathway in NSCLC cells.

Fig. 5. Rasal2 Inhibits Ras/ERK Signal Pathway

(A) H2009 cells were transfected with si-Rasal2-1, si-Rasal2-2 and si-NC for 48 h and Western blot analysis were performed with indicated antibodies. (B) H2009 cells were transfected with si-Rasal2-1, si-Rasal2-2 and si-NC for 48 h and were used of Ras activation assay. (C) A549 cells were transfected with Myc-Rasal2 or Myc-pcDNA3.1 vector. After 24h, Western blot were performed as indicated. All data were measured in triplicate as mean ± S.D. * p < 0.05, ** p < 0.01.

Ras/ERK Pathway Is Involved in Rasal2-Relugated EMT Inhibition

It is well known that EMT can be regulated by Ras/ERK signal pathway,¹⁰⁾ we thus examined whether Rasal2 regulate EMT inhibition through ERK pathway. We transfected H2009 cells with si-Rasal2-1 or si-Rasal2-2 and incubated in the present or absent of PD98059, an ERK pathway inhibitor.¹¹⁾ As expected, Rasal2 knockdown significantly increased the phosphorylation level of ERK. Moreover, PD98059 treatment greatly decreased Vimentin expression while increased E-cadherin expression level as compared with untreated group in Rasal2 knockdown H2009 cells (Fig. 6A). In addition, we found that the cell invasion and migration were promoted in Rasal2 knockdown cells, while this effect was rescued after PD98059 added in. Together, these results suggested that Rasal2 regulated EMT through Ras/ERK pathway (Figs. 6B, C).

Fig. 6. ERK Pathway Is Involved in Rasal2-Relugated EMT Inhibition

H2009 cells transfected with si-Rasal2 or si-NC for 48 h and then incubated with/without PD98059 (10 µg/mL) for 6h. (A) Immunoblotting was performed with indicated antibodies and (B, C) migration or invasion assays were performed respectively. All data were measured in triplicate as mean ± S.D. * p < 0.05, ** p < 0.01.

DISCUSSION

Ras signal pathway is one of the most frequently activated pathways in cancer and it is regulated by upstream or downstream regulators.¹²⁾ Rasal2 is a Ras-GAP protein that can negatively regulate the Ras signal pathway. Therefore, Rasal2 function as a tumor suppressor and often down-regulated in a variety of cancers.^5,13) However, Rasal2 was reported as an oncogene and correlated with poor outcomes in TNBC patients.⁷⁾ Rasal2 also promoted colon carcinogenesis.¹⁴⁾ Therefore, the effect of Rasal2 in cancer seems context dependent. In this study, we found that Rasal2 expression was lower in NSCLC tissues and NSCLC cell lines. The lower level of Rasal2 was showed significantly associated with tumor thickness, ulceration and TNM staging and correlated with poor survival in NSCLC patients. Thus, we suggested that Rasal2 might be a prognostic marker in NSCLC patients.

The function role of Rasal2 in cell metastasis is also complicated. Rasal2 significantly inhibit the cell invasion and migration in luminal B breast cancer, ovarian cancer.^5,6) However, Rasal2 could promote TNBC cell migration and invasion through increasing RAC1 activity by antagonizing its GAP protein ARHGAP24.⁷⁾ Rasal2 also promoted metastasis through activation of hippo pathway in colorectal cancer.¹⁴⁾ However, whether Rasal2 could promote or inhibit cell EMT in NSCLC cells is unclear. Our data showed that Rasal2 knockdown greatly promote NSCLC cell invasion and migration through EMT process as evidence by the up-regulation of mesenchymal markers and down-regulation the epithelial markers.

Previous studies showed that Rasal2 could regulate EMT and cell migration through Ras/ERK pathway in luminal B breast cancer and ovarian cancer.^5,6) In line with previously report, our study found that Rasal2 knockdown greatly activated Ras/ERK pathway and promoted cell migration in NSCLC cells. Moreover, blocking ERK pathway by ERK inhibitor PD98059 could reverse Rasal2-knockdown-induced promotion of mesenchymal markers and cell migration ability. These results suggested Rasal2 regulated EMT and cell metastasis through Ras/ERK pathway in NSCLC cells.

In conclusion, we demonstrated that Rasal2 served as an EMT and metastasis suppressor and down-regulated in NSCLC cells. Rasal2 knockdown significantly increased NSCLC cell migration and invasion. Mechanistically, Rasal2 knockdown could activate Ras/ERK signal pathway, thus greatly increase NSCLC cell epithelial–mesenchymal transition and cell metastasis. Rasal2 may be a prognostic biomarker and a potential therapeutic target for NSCLC in the future.

MATERIALS AND METHODS

Tissue Specimens

The study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University and was performed in compliance with government policies and the Helsinki Declaration. Human malignant NSCLC tissues and normal tissues adjacent to tumor tissues surgically excised from 74 patients at the Department of Respiratory in the First Affiliated Hospital of Harbin Medical University from 2014 to 2019. Complete pathological data of the patients were collected as shown in Table 1.

Materials and Cell Lines

PD98059 (ERK pathway inhibitor) was obtained from Sigma (St. Louis, U.S.A.). Antibodies for Rasal2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Abcam (Cambridge, U.S.A.). Antibodies for E-cadherin, vimentin, N-cadherin, snail, Raf-1, p-Raf1, MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 were obtained from Cell Signaling Technology (Danvers, U.S.A.).

All cell lines were obtained from cell bank of Chinese Academy of Sciences, Shanghai (Shanghai, China) and cultured in RPMI-1640 with 10% fetal bovine serum (FBS). Cells were cultured in 37 °C with 5% CO₂ in humidified incubator.

Western Blot

Total protein were extracted from tissues and cells by using radio immunoprecipitation assay (RIPA) buffer (Beyotime, China). Thirty microgram protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose (NC) membranes. After blocking with 5% bovine serum albumin (BSA) for 1 h, the indicated primary antibodies were added into NC membranes and incubated overnight. After that, the membrane was washed with TBS + Tween20 (TBST) for 3 times and cultured with the secondary antibodies for 1h. The blot was detected by using enhanced chemiluminescence (ECL) resolution (Beyotime) and quantified by ImageJ.

Rasal2 Knockdown

We knockdown Rasal2 by si-RNA targeting the Rasal2-siRNA1 (5′-CCC TCG TGT TCT TGC TGA TAT-3′) and the Rasal2-siRNA2 (5′-GCC TTC CAC CTC TTC ATA GTA-3′) and si-NC (sc-37007, Santa Cruz, U.S.A.) as a control. Lipofectamine 2000 (Invitrogen, U.S.A.) was used as a transfection reagent. Briefly, cells were seeded in 6-well plates overnight and transfected with Rasal2 si-RNA or si-NC separately. After 48 h, the transfected cells were harvested for immunoblotting and subsequent assays.

RT-PCR Assay

RT-qPCR rection was performed by SYBR^® Fast qPCR Mix (TaKaRa, China) according to the manufacturer’s instructions in the StepOnePlus™ RT-PCR instrument (Thermo Fisher Scientific, U.S.A.). The primer for RT-PCR were follows: Rasal2 F: 5′-AAA AGA GTC ACG TTC CCA TGA AT-3′ R: 5′-CCA AGG ATG CTA CTA TGA AGT GG-3′; GAPDH F: 5′-AAG AAG GTG GTG AAG CAG G-3′, R: 5′-GAA GGT GGA AGA GTG GGA GT-3′; PCR results were compared using the relative Delta–Delta Ct method.

Cell Migration and Invasion Assay

Cultrex^® 96 Well Cell Migration/Invasion Assay (Trevigen, U.S.A.) was used to measure cell migration or invasion according to the instructions.¹⁵⁾ Briefly, cells were starved and seed to the top chamber and added medium to the bottom chamber. Incubate this chamber at 37 °C for 24 h. Then wash the top and bottom chamber carefully, added cell dissociation solution/calcein-AM to the bottom chamber. Cell migration or invasion was determined by using a microplate reader at 520 nm.

Ras Activation Assay

Ras activation assay was performed by using Ras GTPase enzyme-linked immunosorbent assay (ELISA) Kit (ab134640, Abcam, Cambridge, U.K.) according to the manufacturer’s instructions.

Immunofluorescence Microscopy

After fixing and permeabilizing, cells were washed and blocked with 3% BSA for 1h. Then the cells were incubated with indicated primary antibodies overnight. After incubation with secondary antibodies (Thermo Fisher Scientific), cells were stained by 4′-6-diamidino-2-phenylindole (DAPI) (Vectashield, U.S.A.). Images were acquired and analyzed with an LSM510 confocal microscope (Carl Zeiss, Germany).

Statistical Analyses

The data were presented as mean ± standard deviation (S.D.) and all of the experiments were repeated at least three times. The difference among treatment groups were performed by Student’s t-test (between 2 groups), one-way ANOVA (3 or more groups). The χ² test was used to analyze the relationship between Rasal2 expression and pathological characteristics. The prognostic value of Rasal2 in NSCLC was analyzed using the Kaplan–Meier curve and Cox regression analysis. * p < 0.05 considered as statistically significant.

Author Contributions

SY and DF conceived and designed the experiments; DF, YY and SQ performed the experiments and contributed to molecular analysis; DF and YY analyzed the data; SY wrote the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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