AXL and GAS6 co-expression in lung adenocarcinoma as a prognostic classifier
- Authors:
- Published online on: April 21, 2017 https://doi.org/10.3892/or.2017.5594
- Pages: 3261-3269
Abstract
Introduction
Lung cancer is the leading cause of cancer-associated deaths worldwide, contributing to approximately 1.4 million deaths each year despite major advances in diagnostics and treatment in the last decade (1). Approximately 80% of lung cancers are classified histologically as non-small cell lung cancers (NSCLCs), of which the most common type is adenocarcinoma (AD), accounting for approximately half of all NSCLCs (2). Therapeutic strategies for lung AD patients currently focus on inhibiting target molecules or oncogenic pathways such as receptor tyrosine kinases (3–5). Unfortunately, despite initial marked responses to tyrosine kinase inhibitors (TKIs), most AD patients with oncogenic driver mutations eventually acquire resistance. Therefore, identification of predictive and prognostic biomarkers and precision medicine using the biomarkers could have a clinically significant impact on treatment strategies for lung AD patients.
Signaling by AXL, a receptor tyrosine kinase, induces the downstream activation of phosphoinositide 3-kinase (PI3K)/AKT, signal transducer and activator of transcription 3 (STAT3), mitogen-activated protein kinase (MAPK), and nuclear factor κB (6–8). Growth arrest-specific 6 (GAS6) is a ligand of AXL and a member of the vitamin K-dependent protein family. AXL overexpression is associated with cell survival, proliferation, invasion, migration, cell-to-cell adhesion, metastasis, and anti-apoptosis in different types of tumors (9,10). In human cancers, increased expression of AXL has been observed in glioma and cancer cells of the breast, stomach and lung, and is associated with invasion and metastasis (11–14). Furthermore, recent studies revealed that AXL overexpression led to resistance to EGFR-TKI in NSCLC cells undergoing epithelial-to-mesenchymal transition (EMT), making AXL a potential therapeutic target in patients with acquired resistance to EGFR-TKIs (15,16).
In this study, we examined the correlation of AXL and GAS6 expression with clinicopathologic parameters and prognoses in patients with complete lung AD resection. We ultimately found that the combination of AXL and GAS6 expression was useful in distinguishing those with a worse prognosis, particularly among stage I AD patients.
Materials and methods
Patients and tumor samples
We carried out a retrospective study of 113 Japanese patients who had been diagnosed with lung AD and had undergone complete surgical resection at Nippon Medical School Hospital between 2001 and 2009. The patients were enrolled consecutively into the cohort upon undergoing surgery. During a 5-year follow-up, overall survival was measured from the date of lung cancer surgery until the date of death, and disease-free survival (DFS) was measured from the date of surgery until relapse. All tumor samples were freshly collected during surgery, quickly snap-frozen and stored at −80°C. TNM staging, including T factor, N factor and tumor differentiation grade (G), was assessed by the latest TNM staging system and by following the 7th edition of the American Joint Committee on Cancer Staging Manual (17–19). Specimens from lung AD patients were used only for immunohistochemistry (IHC) analysis. The study protocol was approved by an ethics committee review board at Nippon Medical School Hospital. Written informed consent was obtained from all patients and the specimen of the patients was inspected according to the Declaration of Helsinki 2008.
Cell culture
Ten lung AD cell lines were used in the present study: PC-9, HCC827, NCI-H1975, A549, RERF-LC-KJ, RERF-LC-MC, NCI-H441, PC-14, LC-2/ad and ABC-1. Cell lines were grown in RPMI-1640 medium (Gibco, Carlsbad, CA, USA), except ABC-1 and RERF-LCMS, which were grown in minimum essential medium Eagle (Sigma-Aldrich, St. Louis, MO, USA). All media were supplemented with 10% fetal bovine serum. The cell line, PC-14, was obtained from Immuno-Biological Laboratories (Gunma, Japan); HCC827, NCI-H441, and NCI-H1975 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA); A549, LC-2/ad, PC-9, and RERF-LCKJ were obtained from the RIKEN BRC Cell Bank (Ibaraki, Japan); and ABC-1 and RERF-LCMS were obtained from the Japanese Collection of Research Bioresources Collection (JCRB) Cell Bank (Osaka, Japan). Of the three cell lines with activating EGFR mutations, PC-9 and NCI-HCC827 contained EGFR deletions (delE746-A750) at exon 19, and NCI-H1975 showed double mutations: L858R at exon 21 and T790M at exon 20. The other cells all had wild-type EGFR.
Detection of EGFR mutations
The PNA-LNA PCR clamp method was used to identify EGFR mutations in tissue or cytology specimens by LSI Medience Corp. (Tokyo, Japan), as previously described (20).
Immunohistochemistry
Immunohistochemistry (IHC) was consecutively performed on formalin-fixed, paraffin-embedded sections. After deparaffinization, sections were quenched for endogenous activity with 0.3% hydrogen peroxide plus absolute methanol for 20 min. Thereafter, antigen retrieval was carried out in a 10 mmol/l citrate buffer solution (pH 6.0; LSI Medience Corp.) for 10 min using an autoclave. After blocking with 2% normal swine serum (Vector Laboratories Inc., Burlingame, CA, USA), sections were washed and incubated with goat anti-AXL polyclonal antibody (#AF154, Rot: DMG0514051) and goat anti-GAS6 polyclonal antibody (#AB885, Rot: DNH0113121; R&D Systems Inc., Minneapolis, MN, USA), or anti-vimentin antibody (#3932, Rot: 3; Cell Signaling Technology Inc., Danvers, MA, USA) overnight at 4°C. After washing, slides were incubated for 30 min with biotinylated anti-goat antibody for AXL and GAS6, or anti-rabbit antibody for vimentin (1:200 dilution; Vector Laboratories), and treated with an avidin-biotin complex kit (Funakoshi Co., Ltd., Tokyo, Japan). Finally, slides were exposed to 3, 3′-diaminobenzidine tetrahydrochloride (Muto Pure Chemicals Co., Ltd., Tokyo, Japan), followed by counterstaining with Mayer's hematoxylin. Negative control slides were prepared by omitting the primary antibody in the above steps.
Evaluation of immunohistochemical expression of AXL, GAS6, and vimentin
IHC scoring was performed using a Histoscore (H-score) as previously described (21), where the staining intensity was graded as follows: 0 (none), 1 (weak), 2 (moderate), and 3 (strong). The percentage of immunoreactive positive tumor cells for AXL and GAS6 were graded as follows: 0, <10% positive cells; 1, 10–25% positive cells; 2, 25–50% positive cells; 3, 50–75% positive cells; and 4, ≥75% positive cells. The percentage of vimentin-positive tumor cells was graded differently as follows: 0, <5% positive cells; 1, 5–30% positive cells; 2, 30–70% positive cells; and 3, ≥70% positive cells (22). The final H-score was obtained by multiplying the intensity grade by the percentage grade. All slides were reviewed and scored independently by two investigators (C.-H. Kim and F. Zou) who were blinded to clinical information pertaining to patients. A tumor was defined as positive for IHC staining if the AXL H-score ≥1.0, GAS6 H-score, ≥3.0 and vimentin H-score ≥1.0; in all other cases, a tumor was defined as negative (Fig. 1).
Western blotting
Western blotting was performed as previously described (23,24). Protein samples (10 µg) were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated in solutions of primary antibodies: anti-AXL (#AF154, Rot: DMG0514051), anti-GAS6 (#AB885, Rot: DNH0113121), anti-vimentin (#3932, Rot: 3) and β-actin (#A5316, Rot: 123M4876). Anti-β-actin was obtained from Sigma-Aldrich. Anti-goat antibody for AXL and GAS6, anti-rabbit antibody for vimentin or anti-mouse antibody for β-actin were used as secondary antibodies (1:5000 dilution; Vector Laboratories). Proteins were detected with Clarity™ ECL Western Blotting Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and visualized using a chemiluminescence system (GE Healthcare Japan Corp., Tokyo, Japan).
Statistical analysis
Correlations between IHC staining and clinicopathological factors were determined using the chi-square test or the Fisher's exact test. Kaplan-Meier survival curves were drawn for overall survival and DFS and compared by log-rank test. The 5-year survival rate was analyzed by the Wilcoxon rank test. Univariate and multivariate analyses were performed using the Cox proportional hazard model. All tests were two-sided, and a P-value of <0.05 was considered statistically significant. Statistical analyses were performed using IBM SPSS Statistics version 21 (IBM SPSS, Inc., Armonk, NY, USA).
Results
AXL and GAS6 expression in lung AD
Among 113 patients with IHC staining for AXL and GAS6 proteins, 43 (38.1%) were positive for AXL (AXL+), 38 (33.6%) were positive for GAS6 (GAS6+), and 20 (17.7%) were positive for both AXL and GAS6 (AXL+/GAS6+; Fig. 1; Table I). Associations between patient clinicopathological parameters and AXL+ or GAS6+ status are shown in Table II. There were no significant associations between AXL+ or GAS6+ and parameters such as age, sex, smoking, T factor, N factor, tumor grade, post-operative recurrence, and EGFR status. The 5-year survival rates for patients who were AXL+ or GAS6+ were significantly lower than those for AXL− or GAS6− patients (51% vs. 75%; P=0.028; 53% vs. 72%; P=0.040).
 | Table II.Univariate and multivariate Cox proportional hazards models of factors associated with death in stages I–III and stage I patients. |
Association between AXL or GAS6 and vimentin expression
The overexpression of AXL and GAS6 is associated with an EMT phenotype (15,16). Therefore, we evaluated vimentin expression as a mesenchymal marker. Of 113 patients, 84 were assessed by IHC assessment for vimentin. Eighteen cases (21.4%) were vimentin positive (vimentin+; Fig. 1; Table I). A vimentin+ status significantly correlated with AXL+, GAS6+, and AXL6+/GAS6+ status (P=0.044, P=0.023 and P=0.004, respectively; Fig. 2A-C). The frequency of vimentin+ increased with the extent of AXL+ or GAS6+, which was highest for the AXL+/GAS6+ group (44.4%), followed by the single-positive group (AXL+/GAS6− plus AXL−/GAS6+; 33.3%) and then the AXL−/GAS6− group (22.2%; P=0.006, linear-by-linear association; Fig. 2D). With regard to EGFR status, none of the 18 patients who were vimentin+ had a mutant EGFR. On the other hand, 16 (24.2%) of 66 patients who were vimentin− showed EGFR mutations (P=0.018; Fig. 2E). Similarly, two (10%) of 20 patients with AXL+/GAS6+ had a mutant EGFR, while 20 (21.5%) of 93 patients in the other groups (AXL+/GAS6− plus AXL−/GAS6+ plus AXL−/GAS6−) showed the presence of EGFR mutations, although the difference was not statistically significant (P=0.354; Fig. 2F). This suggested that high expression levels of AXL and GAS6 were associated with vimentin overexpression and a wild-type EGFR status.
Association between AXL and/or GAS6 expression and clinical outcome
We next evaluated correlations between AXL, GAS6, vimentin expression, and patient prognosis. Overall survival was not significantly different between vimentin+ and vimentin− for stages I–III lung AD (P=0.167; data not shown). In contrast, AXL+ or GAS6+ status was significantly associated with poor overall survival compared to AXL− or GAS6− among patients with stages I–III lung AD (P=0.027 and P=0.042, respectively; Fig. 3A and B). Furthermore, patients showing AXL+/GAS6+ were also significantly associated with poor overall survival compared to AXL−/GAS6− or other groups (AXL+/GAS6− plus AXL−/GAS6+ plus AXL−/GAS6−; P=0.004 and P=0.008, respectively; Fig. 3C and D); however, there was no significant association between AXL+/GAS6+ and AXL+/GAS6− plus AXL−/GAS6+ (P=0.094; Fig. 3C). These results suggested that AXL and GAS6 co-expression (AXL+/GAS6+) was associated with a poor prognosis for lung AD. Next, we investigated whether a patient's prognosis was affected by AXL+ or GAS6+ expression among patients stratified according to stage and EGFR status. For stage I cases, overall survival and DFS rates for AXL+/GAS6+ cases were significantly shorter than those for other cases (P=0.007 and P=0.006, respectively; Fig. 3E and F). In stage I patients with wild-type EGFR, the overall survival and DFS rates for AXL+/GAS6+ patients were also significantly shorter than those for the other patients (P=0.0001 and P=0.0004, respectively; Fig. 3G and H). In contrast, AXL+/GAS6+ as a negative prognostic factor was not observed in stage I patients with mutant EGFR (data not shown). Thus, AXL and GAS6 expression in combination significantly correlated with a poor outcome for stage I lung AD and an EGFR wild-type status.
The impact of AXL/GAS6 expression on lung AD patient survival
We finally evaluated whether the prognostic ability of AXL+/GAS6+ was affected by underlying clinicopathological covariates using univariate and multivariate Cox regression analyses. Among stages I–III patients, N factor [hazard ratio (HR)=2.94, P=0.002], p-stage (HR=2.61, P=0.007) and AXL/GAS6 classification (HR=2.56, P=0.011) were significant predictors of survival in univariate analysis (Table II). Multivariate analysis, adjusted for N factor, p-stage, and AXL/GAS6 classification, showed that only AXL+/GAS6+ (HR=2.45, P=0.018) was a statistically significant predictor of survival (Table II). In stage I cases, univariate analysis showed that the EGFR mutation status (HR=6.22, P=0.001) and AXL/GAS6 classification (HR=3.89, P=0.012) were significantly associated with death. Finally, the EGFR mutation status (HR=9.30, P=0.0001) and AXL/GAS6 classification (HR=5.90, P=0.0024) were found to be independent predictors of death in multivariate analysis (Table II). Thus, co-expression of AXL and GAS6 significantly correlated with death for stages I–III and I lung AD patients.
AXL, GAS6, and vimentin expression in lung AD cell lines
We also evaluated protein expression levels of AXL, GAS6, and vimentin in 10 lung AD cell lines (Fig. 4). Unfortunately, GAS6 proteins were not detected in cells, probably because this is a secreted protein. Among seven AD cell lines with wild-type EGFR, five cell lines (A549, RERF-LC-KJ, RERF-LC-MS, PC-14, and LC-2/ad) strongly expressed AXL and vimentin protein. Of three mutant EGFR cell lines, PC-9 and H1975 showed strong AXL and vimentin expression, respectively.
Discussion
In this study, we found that the positive expression of AXL and GAS6 in combination could be used as a marker of a poor prognosis in lung AD patients. AXL protein expression has, in the past, correlated with lymph node metastasis and clinical stage (13,25), while high expression levels of AXL and GAS6 have been associated with poor survival in lung AD patients with stage I–III (25). Consistent with these findings, we observed that AXL and GAS6 expression levels significantly correlated with poor survival in lung AD cases. Furthermore, we showed the negative impact that the high expression of both AXL and GAS6 had on the survival of patients with stage I lung AD. Of note, AXL+ expression significantly correlated with vimentin+ expression, as reported in an earlier in vitro study (26). The co-expression of AXL and GAS6 was mostly associated with vimentin positive expression in this study. Thus, rather than the individual expression of either protein, the expression of both in combination may be more closely associated with the biological features of vimentin, suggesting that AXL+/GAS6+ tumor cells may represent abundant vimentin. Vimentin may actually induce AXL expression (27). However, vimentin expression was not a prognostic factor in this study. Besides vimentin, AXL expression could be also regulated by other factors, including TGF-β1 (26). Therefore, AXL and GAS6 co-expression, but not vimentin expression, may be critical for patient survival, as well as in the carcinogenesis of lung AD. Furthermore, high vimentin expression correlated with an EGFR wild-type status. AXL and vimentin-positive expression were also found for most AD cell lines showing wild-type EGFR. Therefore, AXL and GAS6 may play a critical role in tumor progression and patient survival in lung AD patients with wild-type EGFR. As for the significance of AXL/GAS6/vimentin for the EGFR mutant-type, small numbers of stage I–III AD patients as well as AD cells with the EGFR mutation have been analyzed. Further studies are planned to perform using large-scale samples with an EGFR mutation, including stage IV, to evaluate the correlation between AXL/GAS6/vimentin expression and EGFR status as a prognostic factor.
Recently, AXL upregulation and activation by GAS6 has been implicated in the EMT of breast cancer and hepatocellular carcinoma (28,29). Likewise, the expression of both AXL and GAS6 is deemed to be closely related to full-blown EMT in a subset of lung AD. AXL-related EMT resulting in drug resistance has been reported in patients with prior EGFR-TKI therapy, as well as in in vitro studies using NSCLC cell lines (15,16,30). Aberrant AXL signaling and the development of the EMT phenotype were also associated with ALK inhibitor resistance in ALK-driven neuroblastoma cells (31). Our clinical data support the concept that EMT under AXL or GAS6 high expression apparently exists in patients with prior surgical resection for lung AD, which consequently leads to de novo resistance to EGFR-TKI (15,30).
Unfortunately, approximately 20–30% of early stage NSCLC patients undergo a relapse, even after complete surgical treatment (32). Sensitive biomarkers can help identify patients with early-stage or locally advanced NSCLC who have a high risk of relapse and a poor prognosis. High expression levels of excision repair cross-complementation group 1 (ERCC1), ribonucleotide reductase subunit M2 (RRM2), and thymidylate synthase (TS) were suggested as negative prognostic factors for patients with resected NSCLC (33,34). In addition, cyclooxygenase-2 and amplification of the actin-4 (ACTN4) gene were considered markers for a poor prognosis in stage I disease (35,36). However, a conceivable prognostic biomarker for patients with stage I AD has not yet been established. In the present study, we demonstrated that the co-expression of AXL and GAS6 had a greater effect on survival in patients with stage I lung AD, especially in those with wild-type EGFR. AXL has been recognized as a potential therapeutic target for overcoming EGFR-TKI resistance (30). The BATTLE study using AXL inhibitor and EGFR-TKI demonstrated synergistic effects in some patients with wild-type EGFR (15). Our findings suggest that the combination of AXL and GAS6 was significantly associated with poor overall survival and DFS in the AD subgroup of stage I disease with wild-type EGFR. Therefore, AXL and GAS6 may be promising predictive biomarkers of a drug response and crucial therapeutic targets in lung AD with wild-type EGFR. The prognostic significance of the co-expression of AXL and GAS6 needs to be further validated in large-scale studies of AD samples. Further investigation is also needed to determine whether the overexpression of AXL and/or GAS6 modulate different internal signaling pathways, depending on the EGFR status and EMT signature.
Our study demonstrated that the co-expression of AXL and GAS6 in a tumor was a significant independent predictor of a poor outcome in patients with stage I lung AD, as well as stages I–III lung AD. An AXL and GAS6 expression status may be useful for the identification of lung AD patients at high risk of post-operative death and who will benefit from adjuvant chemotherapy.
Acknowledgements
This study was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant no. 25461172 to A.G.), and the Clinical Rebiopy Bank Project for Comprehensive Cancer Therapy Development in Nippon Medical School.
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