Axl mediates tumor invasion and chemosensitivity through PI3K/Akt signaling pathway and is transcriptionally regulated by slug in breast carcinoma

Authors


Abstract

The invasion and chemoresistance are crucial causes of morbidity and relapse for cancer patients. Axl is implicated in the modulation of cell invasion, cancer metastasis, and chemosensitivity in human breast carcinoma cell lines. Both breast cancer cell lines and tissues displayed increased expression of Axl, and it over expressed in highly metastatic breast cancer. The altered expression level of Axl was corresponding to the changed invasive phenotype and chemosensitivity of MDA-MB-231 cells both in vitro and in vivo. Further data indicated that experimental inhibition of Axl by RNAi assay inhibited phosphatidylinositol 3-kinase (PI3K)/Akt/GSK3β signaling pathway, resulted in the decrease of Slug expression, and further suppressed cell invasion properties and chemosensitivity. What is more, after the detection and statistics in human breast cancer specimens, we found the Axl expression was closely correlated with histological grade, lymph node metastasis, and clinical stage (P < 0.01). Taken together, these findings indicate that Axl exerts the role of tumor metastasis and chemosensitivity through activation of the PI3K/Akt/GSK3β signaling pathway, which is transcriptionally regulated by Slug. Our findings support the possibility that Axl is a novel regulator. It means by targeting Axl or its related signaling pathways, we can reduce the invasion and chemosensitivity of breast tumor. © 2014 IUBMB Life, 66(7):507–518, 2014

Introduction

Breast cancer is the most common malignant disease to women, and the probability of women being attacked by this disease is 1 in 10 approximately. Metastasis of tumor cells and the development of resistance to antitumor therapies are the major causes of death in cancer patients [1]. Traditional treatment modalities of it such as screening, surgery, adjuvant radiation, and systemic therapy have limited to their benefits especially in advanced disease, so that the majority of the patients who underwent these treatments have to succumb to the related complications [2]. Nowadays the frequent use of targeting drug has solved these problems. For instance, it is reported that the receptor tyrosine kinase (RTK) targeted drugs such as trastuzumab, cetuximab, imatinib, and gefitinib inhibitors for the treatment of certain cancers have achieved good therapeutic effect [3, 4]. Meanwhile, the continuous generation of new targeted drug has great effects on the treatment of cancer.

The Axl (also named UFO and Ark), first identified as a transforming gene in chronic myeloid leukemia, is the founding member of the TAM family of receptor tyrosine kinases, which include Tyro3 (or SKY), AXL, and MER [5, 6]. It is a transmembrane receptor that contains an intracellular(C-terminal) kinase domain including three autophosphorylation sites (Tyr779, Tyr821, and Tyr866) and a unique extracellular (N-terminal) domain composed of two N-terminal immunoglobulin-like domains and two fibronectin type III repeats similar to the structure of neural cell adhesion molecules [7, 8]. The primary ligand for Axl, Growth arrest-specific 6 (Gas6), is a vitamin K-dependent protein that binds Axl with high affinity [5, 9]. In our previous study [10], we found 3.05-fold increase of Axl in MCF-7/ADR (adriamycin resistance) cells as compared with MCF-7 cells and suggested Axl represents a promising gene for overcoming MDR (multiple drug resistance) in breast cancer therapy. What is more, Axl was also reported to be associated with invasion or chemosensitivity in some types of human cancers including human non-small cell, T-cell acute lymphoblastic leukemia, gastric, uterine endometrial, certain types of breast cancer, and ovarian cancers [11-16]. It strengthened many essential biological functions for cancer formation and progression including invasion, migration, survival, angiogenesis, resistance to chemotherapeutic and targeting drugs, and cell transformation and proliferation [14, 17]. Adequate evidences have supported that the downstream pathways activated by the Gas6/Axl signaling include the phosphatidylinositide 3-kinase/Akt pathway [18], mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway [19], and NF-κB pathway [20]. However, the detailed mechanism of Axl promoting tumor metastasis and its clinical implications in breast carcinoma have not been studied extensively.

The altered invasion ability and drug resistance of tumor cells are reminiscent of the events at EMT (epithelial-to-mesenchymal transition), during which, the epithelial makers E-cadherin is down-regulated, whereas the mesenchymal markers N-cadherin and fibronectin are up-regulated [21, 22]. Several transcription factors have been implicated in the transcriptional repression of E-cadherin, including the Snail family of zinc-finger transcription factors (Snail, Slug, and Smuc), the two-handed zinc-finger members of the dEF family (ZEB1/dEF1 and ZEB-2/SIP1), and the basic helix-loop-helix factor Twist [23, 24]. Asiedu et al. [25] indicated that Axl can induce epithelial-to-mesenchymal transition. But whether Slug is involved in Axl signal transduction has not been confirmed. According to our experiments, we proved that Slug expression is essential for the invasion-promoting activity and chemosensitivity of Axl.

In the previous studies, we concluded that Axl had a critical role in the PI3K/Akt-PAK1 signal pathway and knockdown of it inhibits the metastasis properties of hepatocellular carcinoma [26]. We also found that differential expressions of Axl are correlated with invasion and multidrug resistance in cancer cells [10]. Given that Axl may have an important role in tumor metastasis and drug resistance, we have prompted in this study to explore its detailed mechanism in the regulation of tumor invasion and chemosensitivity via PI3K/Akt pathway. Based on our results, we proposed that Axl, via activation of PI3K/Akt/GSK3β signaling, enhanced the expression of EMT transcriptional regulator Slug, further changed cancer cell invasion and chemosensitivity potential. Our study supports the possibility that Axl is a novel regulator of tumor metastasis and chemosensitivity in breast cancer and a promising target for breast cancer therapy.

Materials and Methods

Cell Culture and Tissues

Human breast carcinoma cell lines MCF-7 and MDA-MB-231, purchased from Nanjing KeyGEN company (Nanjing, China), with high and low metastatic potential, respectively, were cultured in 90% Roswell Park Memorial Institute RPMI 1640 (Gibco) and 10% fetal bovine serum (FBS) (Gibco). Cells were incubated in a humidified atmosphere at 37 °C with 5% CO2.

After obtaining informed consent, breast cancer and transitional tissues (3 cm from the tumor edge) were collected from the same 101 patients who underwent surgical resections from July 2010 to May 2012 at the Second Affiliated Hospital of Dalian Medical University. Prior consents from the patients and approval from the Ethics Committees of the second affiliated hospital of Dalian medical University were obtained and all the procedures have been performed in compliance with the Helsinki Declaration. All specimens had confirmed pathological diagnosis and were staged according to the 2013 breast carcinomas staging system of the International Union against Cancer (UICC). Samples were taken from mastectomy or wide local excision surgical specimens under the supervision of a pathologist. Fresh tissue samples were snap frozen in liquid nitrogen within 3 Min of harvesting and stored at −80 °C.

Whole Protein Extract and Western Blot Analysis

After digested with trypsin at room temperature for 2 Min, cells were centrifuged at room temperature at 1,000 × g for 10 Min, then rinsed twice with phosphate buffered saline (PBS) at 1,000 × g for 5 Min, and lysed with a protease inhibitor cocktail (whole protein extraction kit KGP2100, KeyGEN). Cells were suspended on a swing bed at 4 °C for 15 Min, and centrifuged at 4 °C at 14,000 × g for 15 Min. The supernatant of harvest is whole protein we needed.

Tissues (100 mg) per sample were cut into about 3 mm × 3 mm pieces with surgical scissors, added in commercially available lysis buffer containing a cocktail of protease and phosphatase inhibitors (whole protein extraction kit KGP2100, KeyGEN), the suspensions were further subjected to a high pressure homogenization by passing 15 times through a homogenizer on the ice. Then the homogenized samples were centrifuged at 10,000 rpm for 5 Min at 4 °C, and the supernatant (vitreous humor) was collected. Protein concentration of the whole cells and tissues was measured with a bicinchoninic acid protein assay kit (KGPBCA, KeyGEN), and the protein was used for western blot.

Extracted proteins were electrophoresed under reducing conditions in 10% sodium dodecyl sulfate-polyacrylamide gels, and then blotted onto a polyvinylidene difluoride membrane. After blocking with 5% skimmed milk in PBS containing 0.1% Tween 20 (PBST) for 2 H, the membrane was incubated with antibody (anti-Axl, 1/200 diluted, Santa Cruz Biotech; anti-Slug, 1/500 diluted, Abcam; anti-Snail, 1/200 diluted, Abgent; anti-PI3K, 1/200 diluted, Thermo; anti-p-Akt T308, 1/200 diluted, thermo; anti-p-Akt S473, 1/200 diluted Abgent; anti-Akt 1/500 diluted, Abgent; anti-p-GSK3β,1/200 diluted Abgent; anti-GSK3β1/500 diluted Abgent) overnight in 5% powdered skim milk buffer, washed thrice with PBS with 0.1% Tween 20, and then incubated with peroxidase-conjugated anti-rabbit IgG (1/5,000 diluted; Santa Cruz Biotech). GAPDH antibody (1/200 diluted; Santa Cruz Biotech) was used as a loading control. All bands were detected using ECL western blotting kit (Amersham Biosciences, UK), according to the manufacturer's instruction.

RNA Isolation and Real-Time Polymerase Chain Reaction Analysis

For real-time polymerase chain reaction (PCR) analysis, total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Valencia, CA), then 3 µg of each RNA was used for cDNA synthesis in a final volume of 20 L with QuantiTect Reverse Transcription Kit (QIAGEN, Valencia, CA) as described by the manufacturer. Quantitative real-time PCR was conducted with QuantiTect SYBR Green PCR Kit (QIAGEN, Valencia, CA) on ABI Prism 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA). RNA was converted to cDNA with the use of oligo d (T) 12–18 primers to preserve the relative mRNA profile and to produce a template suitable for PCR. The PCR primers Axl (F: 5′-GGTGGCTGTGAAGACGATGA-3′, R: 5′-CTCAGATACT CCATGCCA-3′) were used to confirm cDNA integrity and normalization of cDNA yields. Two microliters of cDNA conversion mixture was used for PCR amplification in a final volume of 25 L with Quantitect Probe PCR kit (QIAGEN) according to the manufacturer's instruction. Amplification was programmed to hold at 50 °C for 2 Min, hold at 95 °C for 15 Min, and complete 45 cycles of 94 °C for 15 Sec and 60 °C for 30 Sec. The level of mRNA expression for each gene was expressed as the value of the quantity for each gene relative to that for GAPDH. Each assay was performed in triplicate.

RNAi Assay

MDA-MB-231 cells were incubated in appropriate antibiotic-free medium with 10% fetal bovine serum (Gibco), transferred to a six-well tissue culture and incubated at 37 °C, in a CO2 incubator to obtain 60–80% confluence. MDA-MB-231 cells were stably co-transfected with a plasmid vector containing the puromycin-resistance marker and the specific shRNA, respectively, which was prepared according to the protocol. Scrambled shRNA was used as the negative control. The shRNA sequences were listed in Table 1. The transfection efficiency calculated by the percentage of fluorescent cells was about 80%, and cell viability was 90% by trypan blue dye exclusion assay. Four weeks later, we used puromycin to screen the cells stably expression shRNA. Several colonies were picked and expanded for further study. The knockdowns had no effects on the cell morphology.

Table 1. Summary of target sequences for RNA interference
TargetTarget sequence
AXL-shRNA15′-GGAACTGCATGCTGAATGA-3′
AXL-shRNA25′-CAGTACCAGTGTTTGGTGT-3′
AXL-shRNA35′-GGTTCTAGGTTTCAAAGAT-3′
Akt-shRNA5′-GAATTGTAGTCCAACTTCA-3′
Slug-shRNA5′-CAGCTGTAAATACTGTGACAA-3′

Immunohistochemical Staining Analysis

Immunohistochemical (IHC) staining was conducted using formalin-fixed paraffin-embedded sections of tissues by the avidin–biotin–peroxidase complex (ABC) method. Four micron sections of formalin-fixed paraffin-embedded tissues were cut with a microtome and dried overnight at 37 °C on a silicanized slide (Dako, Carpinteria, CA). Samples were deparaffinized in xylene at room temperature for 80 Min and washed with a graded ethanol/water mixture and then with distilled water. The samples were soaked in a citrate buffer and then microwaved at 100 °C for 10 Min. The following steps were used. Before addition of the primary antibodies, endogenous peroxidase activity was blocked by incubation in methanol containing 1% H2O2 for 20 Min, followed by 60 Min incubation with normal donkey serum to reduce background staining. The primary antibodies, goat antihuman, Axl antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), were incubated at 4 °C for 8 H, followed by incubation with the biotinylated secondary antibodies (donkey anti-goat IgG; Santa Cruz Biotechnology) for 30 Min and ABC complex for 30 Min. The primary and secondary antibodies were used at 1:80 and 1:100 dilutions, respectively. The peroxidase binding sites were demonstrated by the diaminobenzidine method. A phosphate-buffered solution instead of the primary antibody was used in the protocols for negative controls. The level of expression level is measured by Image-Pro Plus software.

In Vitro Extracellular Matrix Invasion Assays

The cell invasion in vitro was demonstrated using 24-well transwell units (Corning, NY) with 8 µm pore size polycarbonate filter coated with ECMatrix gel (Chemicon) to form a continuous thin layer. Cells (3 × 105) were harvested in serum-free medium containing 0.1% BSA and added to the upper chamber. The lower chamber contained 500 µL DMEM. Cells were incubated for 24 H at 37 °C, 5% CO2 incubator. At the end of incubation, the cells on the upper surface of the filter were completely removed by wiping with a cotton swab. Then the filters were fixed in methanol and were stained with Wright-Giemsa. Cells that had invaded the Matrigel and reached the lower surface of the filter were counted under a light microscope at a magnification of 400×. Triplicate samples were acquired and the data were expressed as the average cell number of five fields.

In Vitro Drug Sensitivity Assay

Drug sensitivity was measured using an MTT assay. Cells (1 × 104) were plated in 96-well plates (Costar, Charlotte, NC), and incubated with 5-fluorouracilx (5-FU, Sigma) for 48 H, respectively. Then cells were treated with 100 µL MTT (5 mg/mL, Sigma). After 4 H incubation at 37 °C in 5% CO2, 100 µL DMSO (Gibco) was pipetted to solubilize the formazan product for 30 Min at room temperature. Spectrometric absorbance was measured at 490 nm using microplate reader. Each group contained three wells and was repeated three times. The concentrations required for 50% growth inhibition (IC50 values) were determined by the drug dose that caused 50% cell viability.

In Vivo Antitumor Activity

To investigate whether Axl is related to drug sensitivity of tumor cell in vivo, the antitumor activity of 5-FU was examined in nude mice bearing tumor cell xenografts. Five-week-old female athymic nude mice were obtained from Animal Facility of Dalian Medical University, and were provided with sterilized food and water. Approximately 1 × 107 cells were injected subcutaneously into the right flank of each nude mouse, respectively. Once bearing palpable tumors (about 3 weeks after tumor cell inoculation), tumor-bearing mice were randomly divided into control and treatment groups (n = 6 animals per group). The treatment groups received 30 mg/kg 5-FU i.p. (intraperitoneal) three times per week for 3 weeks, and the control groups received physiological saline alone. Mice were sacrificed and their tumors were isolated and weighed. The tumor inhibition rate (IR) was calculated according to the follow equation: IR (%) = ((Wc − Wt)/Wc) × 100%, wherein Wc and Wt represent the mean tumor weight of the control group and treatment group, respectively. Experiments were approved by the Committee on the Ethics of Animal Experiments of the Dalian Medical University, China (Permit Number: 12-896).

Inhibition of the PI3K/Akt Signaling

LY294002 (Sigma) were used to suppress the activity of the PI3K/Akt signaling in MDA-MB-231 cells. Briefly, MDA-MB-231 cells (1 × 104 cells per well) were incubated with dimethyl sulfoxide (DMSO) or the PI3K inhibitor LY294002 (20 µmol/L) dissolved in DMSO, and collected after 24 H. In addition, Akt expression was also silenced by RNAi, and GSK3β inhibitor SB415286 was also applied to the research of PI3K/Akt signaling. Changes in protein expression were measured by western blot analysis. The tumorigenicity and chemosensitivity were also analyzed when PI3K/Akt signaling was blocked in xenograft tumor model. Sixty female athymic nude mice (5-week-old) were divided into six groups and 1 × 107 MDA-MB-231 cells (with DMSO, LY294002, control shRNA, AKt shRNA, SB415286(+), and SB415286(−), respectively) were injected subcutaneously into the mammary fat pads area of each nude mouse, respectively. Once bearing palpable tumors (about 3 weeks after tumor cell inoculation), mice were sacrificed and their tumors were isolated and weighed.

Activation of the PI3K/Akt Signaling

Insulin-like growth factor-1 (IGF-1) (Sigma) was used to promote the activity of the PI3K/Akt signaling in MDA-MB-231 cells. In addition, Wortmannin is an special activator of GSK3β. Briefly, MDA-MB-231-Axl shRNA cells (1 × 104 cells per well) were incubated with IGF-1 (20 µmol/L) or Wortmannin (20 µmol/L), and collected after 24 H. Changes in protein expression were measured by western blot analysis. The invasion ability and chemosensitivity were also analyzed when PI3K/Akt signaling was activated in the MDA-MB-231-Axl shRNA cells.

Statistical Analysis

The data were expressed as mean ± SD, Student's t-test and Chi-square test were used to determine the significance of differences in multiple comparisons. All data were analyzed with SPSS statistics software (Version 13.0, Chicago, IL) and a P value <0.05 was considered statistically significant.

Results

Differential Expression Profile of Axl in MDA-MB-231 and MCF-7 Cell Lines

We used real-time PCR and western blot analysis to evaluate the expression level of Axl in mRNA (Fig. 1A) and protein expression (Fig. 1B). It was found that MDA-MB-231 cells have a higher Axl expression compared with the MCF-7 cells on both mRNA and protein levels (P < 0.05).

Figure 1.

Different expression levels of Axl in MDA-MB-231 and MCF-7 cells. (A) The mRNA levels of Axl gene analyzed by real-time RT-PCR. The relative amount of gene mRNA level was normalized to GAPDH level. (B) Relative signal intensity of Axl expression in MDA-MB-231 was significantly higher than that in MCF-7 cells (*P < 0.05). Asterisk indicates significant difference from the groups without an asterisk (P < 0.05).

Knockdown of Axl Alters Cell Invasion Ability and Chemoresistance of MDA-MB-231 Cells In Vitro

Because of the higher expressions of Axl mRNA and protein in MDA-MB-231 cells (Figs. 1A and 1B), Axl was silenced by shRNA so as to elucidate its direct effect on the invasive ability and chemosensitivity of MDA-MB-231 cells. As shown in Figs. 2A and 2B, the expression level of Axl was significantly reduced in MDA-MB-231 transfectants compared to the control (Figs. 2A and 2B).

Figure 2.

Silence of Axl inhibits the invasive and chemoresistance of MDA-MB-231 cells in vitro. (A) Silencing of Axl in MDA-MB-231 cells was analyzed by RNAi approach. Axl transcripts were decreased apparently in MDA-MB-231 cells by shRNA treatment. (B) After shRNA transfection, distinct reduction of Axl was observed at protein levels by western blot analysis. GAPDH was also examined and served as controls for sample loading. Relative signal intensities of Axl protein levels were normalized against those of GAPDH by LabWorks (TMver4.6, UVP, BioImaging Systems) analysis, respectively. (C) The average number of cells that invaded through the filter was counted by in vitro ECMatrix gel analysis. MDA-MB-231-Axl shRNA cells were significantly less invasive (P < 0.05) than the MDA-MB-231 cells and MDA-MB-231-control shRNA cells. (D) Cell chemosensitivity was assessed by MTT assay. The reported values were the IC50 (mean ± SD) of three independent experiments. IC50 represents the drug concentration producing 50% decrease of cell growth. Data are the average ± SD of triplicate determinants (*P < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

We performed ECMatrix gel analysis to further evaluate the invasion capability of cells with Axl knockdown on tumor cells in vitro. It revealed that the invasion capability of MDA-MB-231 cells transfected with control shRNA was not affected, while the invasion capability obviously decreased in Axl shRNA cells (Fig. 2C).

Cell chemosensitivity was assessed by MTT assay .The reported values were the IC50 (mean ± SD) of three independent experiments. IC50 represents the drug concentration producing 50% decrease of cell growth. The IC50 value of 5-FU was significantly less in the MDA-MB-231-Axl shRNA groups that in the MDA-MB-231-control shRNA groups, suggesting that cell proliferation was inhibited when MDA-MB-231 cells were treated with Axl shRNA. (Fig. 2D) (P < 0.05).

Knockdown of Axl Mediates Cell Invasion Ability and Chemoresistance of MDA-MB-231 Cells In Vivo

Nude mice bearing MDA-MB-231, MDA-MB-231-control shRNA, and MDA-MB-231-Axl shRNA xenografts were used to analyze the differences of tumor weight with or without 5-FU administrated. As shown in Fig. 3A, the mean tumor weight in nude mice bearing MDA-MB-231-Axl shRNA was significantly reduced compared with the control (Fig. 3A). The IR of 5-FU was 10.7%, 10.82%, and 44.4% in these shRNA treated groups.

Figure 3.

Knockdown of Axl inhibits the tumorigenicity of MDA-MB-231 cells in vivo. (A) A decrease of mean tumor weight in mice group with MDA-MB-231-Axl shRNA tumors was observed, as compared to the control group. (B) Reduced regulation of Axl was also shown by IHC staining in xenograft tumors derived from Axl shRNA cells (×400). The data are means ± SD of three independent assays (*P < 0.05).

Furthermore, the tumor tissues isolated from mice were used to perform IHC studies. It also showed that Axl was highly suppressed in tumors tissues derived from Axl knockdown cells group (Fig. 3B).

Modulation of Various EMT Markers by AXL in Breast Cancer Cells

In the course of exploring the role of Axl in the induction of the EMT process in breast cancer cells, we used MDA-MB-231, MDA-MB-231-control shRNA, and MDA-MB-231-Axl shRNA cells to study the EMT marker protein expression. It showed that knockdown of endogenous Axl resulted in the increased expression of epithelial maker-E-cadherin and the reduced expression of various mesenchymal makers, namely N-cadherin, Snail, and Slug (Fig. 4A).

Figure 4.

Axl gene knockdown regulates various EMT makers and inhibits the activity of PI3K/Akt/GSK3β signal pathway. (A) MDA-MB-231 cells were transfected with Axl-specific shRNA or control shRNA. After 48 H, cell lysates were prepared and subjected to western blot analysis for E-cadherin, N-cadherin, Snail, Slug, and GAPDH served as internal controls. (B) With western blot analysis, the main signal molecules of PI3K/Akt/GSK3β signal pathway were found to be down-regulated at protein phosphorylation level (p-Akt, p-GSK3β) in MDA-MB-231 cells with Axl shRNA, as compared with those in control shRNA cells(*P < 0.05).

Axl Induces Slug Expression Through the PI3K/Akt/GSK3β Pathway

Resent study has suggested that activation of PI3K/Akt/GSK3β signaling induces the EMT process [27]. GSK3β is a multifunctional serine/threonine(ser/thr) kinase that has a fundamental role in a wide variety of functions, including cell division, proliferation, differentiation, and adhesion [28, 29].

In our study, following the silence of Axl expression level, we found the expression and activity of the PI3K/Akt pathway were inhibited. What is worth mentioning, p-GSK3β and Slug were also revealed significant changes at protein level between MDA-MB-231-Axl shRNA cells and untreated cells (Fig. 4B).

PI3K/Akt/GSK3β Signaling Pathway Modulates the Invasive Ability and Chemosensitivity of MDA-MB-231 Cells Both In Vitro and In Vivo

To further determine the potential involvement of PI3K/AKT signaling, we applied LY294002, Akt shRNA, and SB415286, selective antagonist of the PI3K, Akt, and GSK3β, respectively. Our studies showed that treatment with 20 µM LY294002 significantly reduced protein level of p-Akt, p-GSK3β, and Slug particular in MDA-MB-231-Axl shRNA cells (Fig. 5A). What is more, transwell experiment revealed that the invasion ability of it also be restrained (Fig. 5D).

Figure 5.

PI3K/Akt inhibition modulates the invasive ability and chemosensitivity of MDA-MB-231 cells both in vitro and in vivo. (A) Cells were treated with 20 µM LY294002 and further subjected to Akt, GSK3β, and Slug analysis, respectively, in MDA-MB-231 cells and MDA-MB-231 Axl shRNA cells. The phosphorylation levels of Akt and GSK3β, Slug were found to be down-regulated in cells treated with LY294002. (B) MDA-MB-231 cells were treated with 20 µM of the specific GSK3β inhibitor SB415286 for 48 H. The phosphorylation protein levels of GSK3β, Snail, and Slug were up-regulated in MDA-MB-231 cells by western blot analysis. (C) After MDA-MB-231 cells been treated with 20 µM Akt shRNA (T308A/S473A) for 48 H, we found that the protein levels of p-Akt, GSK3β, and Slug have a tendency to descend. (D) MDA-MB-231 cells were treated, respectively, according to the processes described as A–C. Then the invasion property was appraised by counting the mean number of cells that invaded through the filter described as ECMatrix gel analysis. (E) Chemoresistance of MDA-MB-231 cells was evaluated by the value of IC50 in vitro. (F) The average tumor weight in different mice groups was weighted after injecting LY294002, Akt shRNA, and SB415286 in mice with or without 5-FU treatment (P < 0.05). (G and I) After inhibition of PI3K/Akt pathway associated genes in MDA-MB-231 cells, we knocked down Axl by RNAi assay. Then the invasion and chemosensitivity were evaluated by ECMatrix gel analysis and the value of IC50, respectively. (H and J) We knocked down Axl in MDA-MB-231 cells and subsequently activated PI3K/Akt pathway associated genes. Then the invasion and chemosensitivity were evaluated by ECMatrix gel analysis and the value of IC50, respectively.*,#,△ Indicate a significant difference compared with the control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Then we used the same experiment to survey whether the invasion and metastasis process were regulated by Akt and GSK3β signal transduction. The result just as we expected, downstream signal molecules and invasion property exhibit a significant reduction when we knocked down Akt (Fig. 5C). Whereas in SB415286-treated cells we found p-GSK3β, Snail, and Slug have a tendency to rise in protein expression (Fig. 5B). Meanwhile, the invasiveness obtained corresponding ascension (Fig. 5D).

The inhibition of PI3K or Akt pathway made the MDA-MB-231 cells susceptible to chemotherapy (Fig. 5E). The similar results were also observed in vivo analysis that reduced tumor weight was measured in mice group MDA-MB-231 tumors with LY294002 or Akt treatment (Fig. 5F). While SB415286 treatment increased chemoresistance of MDA-MB-231 cells both in vitro and in vivo (Figs. 5E and 5F). These data implicated a role of PI3K/Akt signaling in modulating the invasive properties and chemosensitivity of MDA-MB-231 cells.

After inhibition of PI3K/Akt pathway associated genes, we knocked down Axl by RNAi assay. Interestingly, we detected the Axl knockdown affected the invasion and chemosensitivity when PI3K/Akt/p-GSK3β pathway was overinhibited (Figs. 5G and 5I).

To further verify that the function of Axl was PI3K/Akt signaling pathway dependent, we knocked down Axl in MDA-MB-231 cells and subsequently activated the PI3K/Akt pathway associated genes. Finally, we found the activation of PI3K/Akt pathway rescued the cells from suppressed invasion and chemoresistance caused by Axl knockdown (Figs. 5H and 5J).

Slug Expression Is Essential for the Chemosensitivity and Invasion-Promoting Activity of Gas6/Axl Signaling

Gas6 as the primary ligand of Axl has become an accepted conclusion, Mishra et al. have confirmed the ability of Gas6 to activate the endogenous Axl [9]. To determine whether Slug expression is essential for the invasion promoting activity and chemosensitivity of Gas6/Axl signaling, MDA-MB-231-control and MDA-MB-231-Slug knockdown cells were serum starved in 1% fetal bovine serum DMEM for 48 H and then incubated with 200 ng/mL of Gas6 for 1 H. First of all, we verified that the Slug protein expression is lower in MDA-MB-231-Slug shRNA cells (Fig. 6A). Then, by transwell experiments, we found Gas6 enhanced the invasiveness of control cells but not cells with the knockdown of Slug (Fig. 6B). In the groups without Gas6 treatment, the invasion ability of control cells is higher than that in the Slug shRNA cells. Similarly, it showed Gas6 intensified the chemosensitivity of control cells but not cells with the silence of Slug (Fig. 6C). Above all, it manifested Slug expression is imperative for the chemosensitivity and invasion activity of the Gas6/Axl signaling in breast cancer.

Figure 6.

Slug expression is imperative for the chemosensitivity and invasion-promoting activity of Axl. (A) MDA-MB-231 cells were transfected with Slug shRNA or control shRNA for 30 H, and then protein level of Slug was assessed by western blot analysis. (B) MDA-MB-231-control shRNA and MDA-MB-231-Slug shRNA cells were treated with or without Gas6, respectively. The invasion property was appraised by counting the average number of cells that invaded through the filter described as ECMatrix gel analysis. (C) The chemosensitivity was assessed by the value of IC50 in vitro after treatment described as in Fig. 6B (*P < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Clinical Implications of Axl Protein Expression in Breast Carcinoma

The Axl expression status was investigated in 101 paraffin-embedded primaries breast carcinoma with the corresponding transitional tissue samples by immunohistochemistry staining, and Axl protein was stained at the membrane of tumor cells. It is shown that breast cancer tissues had a higher expression level of Axl compared with transitional tissues (P < 0.05). In cancer tissue, high and low Axl protein expressions were observed in 69 of 101 (68.3%) and in 32 of 101 (31.7%) cases, respectively. The data as shown in Table 2 are numbers of cases, and the expression of Axl is classified as high if >30% of tumor cells are stained and as low if <30% of cancer cells are stained. Finally, no significant evidence indicated Axl has relationship with age, and distant metastasis in breast cancer patients (P > 0.05). Interestingly, we observed that the protein expression of Axl was closely correlated with histological grade, lymph node metastasis, and clinical stage in patients with breast cancer (P < 0.05) (Table 2).

Table 2. Correlation between the clinicopathologic characteristics and expression of Axl protein in breast carcinoma
CharacteristicsnAxl (%)P value
Axl (high)Axl (low)
Group
Cancer tissues10172 (71.3%)29 (28.7%)0.009
Transitional tissues10154 (53.5%)47 (46.5%) 
Age (years)
≥506839 (57.4%)29 (42.6%)0.790
<503318 (54.5%)15 (45.5%) 
Histological grade
G17632 (42.1%)44 (57.9%)0.003
G2, G32519 (76.0%)6 (24.0%) 
Lymph node metastasis
Absent4822 (45.8%)26 (54.2%)0.004
Present5339 (73.6%)14 (26.4%) 
Distant metastasis
Yes2014 (70.0%)6 (30.0%)0.144
No8143 (51.9%)38 (48.1%) 
Clinical stage
I–II4520 (44.4%)25 (55.6%)0.003
III–IV5641 (73.2%)15 (26.8%) 

Discussion

For most solid malignancies, metastasis remains the major driver of mortality and relapse. Cancer metastasis is the process by which cancer cells spread from a primary tumor to other non-adjacent organs and tissues, forming viable secondary deposits of cancer and representing the end stage of a complex multistep cellular process terms as the invasion metastasis cascade [1, 30]. Our growing body of knowledge regarding this process provides the basis for the development of molecularly targeted therapeutics aimed at the tumor cell or its interaction with the host microenvironment. Here, our final goal is to identify genes, which could contribute to the differences in the invasion ability and chemosensitivity, and research the mechanism of how this interested gene changes invasion properties and chemosensitivity.

We previously showed that Axl has a crucial role in promoting invasion, lymphatic metastasis, and multidrug resistance [10, 26]. In this study, we identified down-regulating of Axl not only could effectively regulate the invasiveness, drug resistance of breast tumor cells in vitro, and animal models in vivo, but also related with clinicopathologic characteristics of human breast cancer. These results clearly demonstrate that Axl confers increasing invasion and decreasing chemosensitivity in breast cancer cell lines. In addition, we showed that Axl induces a mesenchymal phenotype and this in turn translates into increased cell invasiveness and tumor metastasis.

The molecular mechanism of Axl gene in physiological process and disease development is of great interest to academics and activists [11-13, 15, 16]. It has been reported that Axl is associate with several signal transduction pathways, in which the PI3K/Akt pathway is one of the core intracellular signaling pathways [8, 18]. In this study, we intensively elucidated the possible signaling transduction mechanism of Axl on the tumor invasion and chemosensitivity in human breast cell lines MDA-MB-231 and MCF-7 with high, low metastatic potential, and affirmed that knockdown of Axl suppresses the PI3K/Akt pathway expression, which been inhibited by LY294002 or Akt shRNA can reduce the invasion phenotype and chemoresistance of breast cancer cells. Furthermore, we found the activation of PI3K/Akt pathway rescued the cells from suppressed invasion and chemoresistance caused by Axl knockdown, and Axl knockdown affected invasion and chemosensitivity when PI3K/Akt pathway was overinhibited. All in all, these results demonstrated invasion ability and chemosensitivity of Axl was, at least in part, PI3K/Akt signaling pathway dependent.

Stabilization of Slug is reported to be regulated through a modulation of PI3K/Akt/GSK3β signaling [31, 32]. The zinc fingers of Slug as the major epithelial–mesenchymal transition (EMT)-inducing transcriptional factor [33, 34] were also contributed to cancer progression inducing by Axl. We observed that silence of Axl restrained the expression of Snail and Slug. In addition, inhibition of the PI3K and Akt pathway also blocked the level of Slug, and further reversed the invasive properties and chemoresistance of MDA-MB-231 cells. While the level of Slug increased in MDA-MB-231 cells with GSK3β inhibitor treatment. Besides, the SB415286 as the inhibitor of GSK3β made the MDA-MB-231 cells more invasive and less susceptible to chemotherapy. These results indicated the role of the regulation of Slug expression in Axl-mediated EMT and showed that Slug serves as a downstream factor of Axl via PI3K/Akt/GSK3β pathway participate in promoting invasive property and chemoresistance.

To observe the relationship between Axl and clinical features, we used immunohistochemistry and pathology evaluates protein expression of Axl in breast cancer specimens of 101 cases. Some academics had suggested that Axl was localized in the membrane of the human breast cancer cells, and the number of cells expressing Axl was found to be higher in cancerous tissue than in the normal breast [35], which been sustained by our results that breast cancer tissues had a higher expression level of Axl compared with transitional tissues. In addition, we analyzed the correlation of Axl expression with clinicopathologic features in breast cancer. The results indicated that significantly increased protein expression of Axl closely associated with histological grade (P < 0.01), lymph node metastasis (P < 0.01), and clinical stage (P < 0.01) in patients with breast carcinoma, which supported that Axl as a growth factor might play an important role in promoting tumor invasion and metastasis [36]. Thus, we should pay high attention to inhibiting it by targeted drugs for alleviating the development of patient's condition.

Gjerdrum et al. showed that EMT program activation leads to Axl up-regulation, which is essential for invasiveness and spontaneous metastasis in breast epithelial cells [37]. Vuoriluoto et al. confirmed that expression of Slug significantly induced Axl levels and EMT changes (cell migration, morphology) in MCF10A cells [38]. Interestingly, here we showed that Axl-activated tumor invasion and chemoresistance were depended on the upregulation of EMT transcription factor Slug. This, together with others findings, suggests that Axl could participate in a positive feedback loop contributing to EMT, which sustains the malignant mesenchymal phenotype of breast tumor cells. However, the specific mechanism remains to be further studied.

In this study, our results indicated that Axl played an important role in association with breast cancer cells invasion and chemoresistance via modulating the PI3K/Akt/GSK3β signaling pathway, and it is transcriptionally regulated by Slug. We also found that elevated expression of Axl was not only shown in breast cancer tissue compared with corresponding noncancerous tissues but also closely associated with histological grade, lymph node metastasis, and clinical stage in patients with breast cancer. These findings were of potential clinical importance for understanding the integration of migration and chemoresistance-related signaling and provided a basis for designing future therapeutic strategy to block breast metastasis and drug resistance in patients.

Acknowledgements

This work was supported by a grant from The National Natural Science Foundation of China director fund (81250025). No conflict of interest exists in the submission of this manuscript, and manuscript is approved by all authors for publication.

Ancillary