Therapeutic Activity of Anti-AXL Antibody against
Triple-Negative Breast Cancer Patient-Derived
Xenografts and Metastasis
Wilhem Leconet
1, Myriam Chentouf
1, Stanislas du Manoir
1, Clement Chevalier
2,
Audrey Sirvent
2, Imade A
€t-Arsa
1, Muriel Busson
1, Marta Jarlier
3, Nina Radosevic-Robin
4,
Charles Theillet
1, Dany Chalbos
1, Jean-Max Pasquet
5, Andr
e Pelegrin
1, Christel Larbouret
1,
and Bruno Robert
1Abstract
Purpose: AXL receptor tyrosine kinase has been described as a relevant molecular marker and a key player in invasiveness, especially in triple-negative breast cancer (TNBC).
Experimental Design: We evaluate the antitumor efficacy of the anti-AXL monoclonal antibody 20G7-D9 in several TNBC cell xenografts or patient-derived xenograft (PDX) models and deci-pher the underlying mechanisms. In a dataset of 254 basal-like breast cancer samples, genes correlated with AXL expression are enriched in EMT, migration, and invasion signaling pathways.
Results: Treatment with 20G7-D9 inhibited tumor growth and bone metastasis formation in AXL-positive TNBC cell xenografts or PDX, but not in AXL-negative PDX, highlighting AXL role in
cancer growth and invasion. In vitro stimulation of AXL-positive cancer cells by its ligand GAS6 induced the expression of several EMT-associated genes (SNAIL, SLUG, and VIM) through an intra-cellular signaling implicating the transcription factor FRA-1, important in cell invasion and plasticity, and increased their migration/invasion capacity. 20G7-D9 induced AXL degradation and inhibited all AXL/GAS6–dependent cell signaling implicated in EMT and in cell migration/invasion.
Conclusions: The anti-AXL antibody 20G7-D9 represents a promising therapeutic strategy in TNBC with mesenchymal fea-tures by inhibiting AXL-dependent EMT, tumor growth, and metastasis formation.Clin Cancer Res; 23(11); 2806–16. 2016 AACR.
Introduction
Breast cancer is the most common female malignancy in western countries. Triple-negative breast cancers (TNBC) account for 15% to 20% of all breast cancer cases and are defined by the absence of estrogen receptors (ER), progesterone receptors (PR), and HER2 expression. TNBC prognosis is poor and chemotherapy remains the sole therapeutic option (1). No targeted therapy is available for TNBC due to the lack of identified molecular targets
in most of these cancers. Moreover, TNBC is associated with a significant higher probability and faster occurrence of relapse compared with other breast cancer subgroups. Epithelial-to-mes-enchymal transition (EMT) is believed to be a main contributor to the invasion process (2, 3). During EMT, cells lose some of their epithelial characteristics, typically E-cadherin downregulation, and acquire mesenchymal phenotypic marks, characterized by upregulation of vimentin, N-cadherin, andfibronectin. Transcrip-tional regulators activated in the EMT program include TWIST1, ZEB1, and SNAIL transcription factors (SNAI1/SNAI2). These regulators are expressed in many tumors and have an important role in breast cancer metastasis formation (4).
Several studies reported that AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, has a major role in the control of tumor cell migration and invasion (5–8). AXL was first identified as a transforming gene in cells from patients with chronic myelogenous leukemia (9). Following binding to its ligand, growth arrest–specific 6 (GAS6) leads to TAM receptors dimerization and, consequently, to the activation of various intracellular pathways that regulate many biological processes, such as cell invasion and proliferation, angiogenesis, and drug resistance (10).
High AXL expression has been reported in many cancers and is associated with tumor progression and shorter overall survival (10, 11). In breast cancer, Gjerdrum and colleagues found that AXL expression is increased in metastatic lesions compared with matched primary tumors suggesting a crucial role in breast cancer cell invasiveness and metastatic potential (12). However, it is unclear whether AXL directly induces EMT by regulating EMT 1IRCM-INSERM-U1194, Universite de Montpellier, ICM, Montpellier, France.
2CNRS UMR5237, University of Montpellier, CRBM, Montpellier, France.3Unite
de Biometrie, ICM, Montpellier, France.4Department of Biopathology, The Jean
Perrin Comprehensive Cancer Center and ERTICa Research Group, University of Auvergne, Clermont-Ferrand, France. 5INSERM-U876, Hematopo€ese Leucemique et Cible Therapeutique, Universite Victor Segalen, Laboratoire d'hematologie CHU de Bordeaux, Bordeaux, Cedex, France.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
Current address for W. Leconet: Department of Urology, Weill Cornell Medical College, 525 East 68th Street, New York, New York.
C. Larbouret and B. Robert contributed equally to this article.
Corresponding Author: Bruno Robert, IRCM-INSERM-U1194, 208, rue des Apothicaires, Montpellier 34298, France. Phone: 467-612-418; Fax: 467-613-783; E-mail: bruno.robert@inserm.fr
doi: 10.1158/1078-0432.CCR-16-1316
2016 American Association for Cancer Research.
effectors or whether it is a target of these EMT regulators. Indeed, AXL downregulation in breast cancer using siRNAs inhibits AKT phosphorylation and decreases cell invasion, motility, and metas-tasis. On the other hand, ectopic expression of AXL in human mammary epithelial cells leads to a downregulation of E-cadherin expression and increased expression of mesenchymal markers (13). Along similar lines, transfection of SLUG and SNAIL genes in MCF10A breast cancer cells induces the acquisition of mesenchy-mal markers and loss of epithelial characteristics together with increased AXL expression (12).
In this study, we examined the potential therapeutic effect of a recently developed anti-AXL mAb (20G7-D9; ref. 14) in TNBC cell xenografts and in basal-like patient-derived xenografts (PDX). We observed a dual role of our antibody consisting of inhibiting tumor growth and AXL/GAS6-dependent migration/invasion of cancer cells. In fact, we demonstrated, in vitro, that stimulation of several TNBC cell lines with recombinant human GAS6 (rhGAS6) induced the expression of EMT transcription factors, mesenchymal markers, and phosphorylation of FRA-1, a key transcription factor in cancer cell invasion, migration, and plasticity (15). Treatment with 20G7-D9 resulted in the degradation of AXL receptor, avoid-ing any AXL/GAS6-dependent signalavoid-ing to occur and, in vivo, reducing spreading of MDA-MB-231-Luc to bones after intracar-diac injection. In conclusion, in AXL-positive TNBC tumors, AXL and GAS6, have a critical role in the maintenance of the EMT phenotype. Therefore, AXL should be considered as a therapeutic target of choice for tumor growth and metastasis inhibition.
Materials and Methods
Cell lines and reagents
The 231, 435, 436, MDA-MB-453, MDA-MB-468, BT549, BT20, HCC38, Hs578T, HBL-100 breast cancer cell lines were obtained from ATCC. Cells were cultured for at most 3 months and then cultures were renewed by thawing a new vial from our master cell bank. Authentication by typical morphology observation and mycoplasma testing using the MycoAlert Mycoplasma Detection Kit (Lonza) were routinely
performed. All cell lines were cultured following the ATCC recommendations. Control (sh-Luc) and anti-AXL shRNAs were previously described (14). Luciferase-transfected MDA-MB-231 cells (MDA-MB-231-Luc) were previously described (16). RhGAS6 and the goat anti-human GAS6 antibody were purchased from R&D Systems. The anti-GAPDH antibody was from Milli-pore. All other antibodies used in Western blotting were from Cell Signaling Technology. The anti-FRA1 siRNA corresponds to the 124–144 coding region relative to the first nucleotide of the start codon (15). The specific anti-human AXL murine IgG1 antibody 20G7-D9 that induces AXL internalization and degradation was previously described (14).
Western blotting
Western blotting was performed using protein lysates from cell lines or PDXs, as previously described (17). Western blot analysis was performed using the G:BOX iChemi imaging system (Syngene). Protein expression was quantitated with the ImageJ software and the Student t test was used to evaluate differences.
Boyden chamber migration and invasion assays
Cancer cells were serum-starved in the presence or absence of 50 mg/mL 20G7-D9 overnight. This incubation time was chosen as we previously showed a massive degradation of AXL at this time point (14). After trypsin digestion, cells were counted and migration assays were performed by seeding 3 104cells resuspended in serum-free DMEM in 8-mm pore size BD-Falcon 24 Fluoroblock Transwell inserts. For GAS6-dependent migration, 200 ng/mL rhGAS6 was added to the bottom chamber containing the migra-tion medium (DMEM/5% serum). Invasion assays were carried out using Matrigel Matrix Growth Factor Reduced (BD Biosciences) and 5 104cells. After 4 hours of migration and 24 hours of invasion, cells in the bottom chamber were stained with 4mg/mL calcein and then counted under an invertedfluorescence microscope. In vivo studies
All in vivo experiments were performed in compliance with the French regulations and ethical guidelines for experimental animal studies in an accredited establishment (agreement no. C34-172-27). MDA-MB-231 or MDA-MB-436 cells (5 106) were injected subcutaneously in 6-week-old female athymic mice (Harlan). When tumors reached a minimum volume of 100 mm3, tumor-bearing mice were randomized in different treatment groups (n ¼ 6) and treated with 200 mg of 20G7-D9 or saline by intraperitoneal injection twice a week for four consecutive weeks. Tumors were measured using a caliper and volume was calculated using the formula V ¼ (tumor length tumor width tumor depth)/2, until the tumor volume reached 2,000 mm3. For metastasis studies, 5 105 MDA-MB-231-Luc cells were injected intracardially in anesthetized BALB/c nude mice. Intraperitoneal treatments with 200 mg 20G7-D9/injection or saline were done twice a week for four consecutive weeks. Bone metastases were quantified by in vivo bioluminescence imaging at day 25, as described previously (16). For PDX models, approximately 60 mm3of B3804 and
B3467 tumors were transplanted in the interscapular fat pads of Swiss nude female mice (Charles River Laboratories) and treated with 200 mg 20G7-D9 twice weekly or saline, when
Translational Relevance
The receptor tyrosine kinase AXL is overexpressed in several cancers and particularly in breast cancer, the most common female malignancy in western countries. Triple-negative breast cancer (TNBC), which accounts for 15% to 20% of all breast cancer cases, has a poor prognosis and chemotherapy is the only available therapeutic option due to the lack of identified molecular targets. In this preclinical study, we show that in vivo AXL immunotargeting with the specific therapeutic antibody 20G7-D9 significantly decreases the growth of TNBC cell xenografts and AXL-positive patient-derived breast tumor xenografts (PDX). Moreover, 20G7-D9 inhibits breast cancer cell migration in vitro and bone metastasis development in vivo. Mechanistically, the 20G7-D9 antibody acts by downregulat-ing AXL expression at cell membranes and consequently inhibiting cell migration, invasion, and epithelial-to-mesen-chymal transition (EMT) induced by the AXL ligand GAS6. AXL might therefore be a target for immunotherapy with the antibody 20G7-D9 in TNBC.
tumor volume reached a minimum volume of 100 mm3. Tumor volumes were measured as described above.
PDX and basal-like breast cancer transcriptome analysis Expression profiling of PDXs was performed using Affymetrix U133plus2. Raw data were normalized using robust multi-array average (RMA). Data were aggregated around HUGO gene symbols with max argument (18). The CIT classification devel-oped by our group was applied using the R script referred in the publication (19). The dataset of 1,809 breast cancer samples was downloaded from http://kmplot.com/analysis/index.php? p¼download. The CIT classification R script was run on the 1,809 breast cancer samples. The AXL expression level was
dis-played for each molecular subtypes (Supplementary Fig. S2). The 254 samples identified as Basal-like were extracted. The 100 genes most correlated with AXL expression in the 254 basal-like tumors were obtained using the gene neighbor command of the Gene-Pattern suite (Broad Institute, Boston) with standard settings (including Pearson correlation). Annotations were made with the GSEA software (Broad Institute, Boston),first with hallmark gene sets (Fig. 3) and then with the MSigDB collections. Statistical analyses
A linear mixed regression model was used to determine the relationship between tumor growth and number of days after implantation. The fixed part of the model included variables
Figure 1.
AXL surface expression in TNBC cell lines and growth inhibition of MDA-MB-436 and MDA-MB-231 cell xenografts by the anti-AXL 20G7-D9 mAb. A, Cell membrane detection of AXL (black peak) in TNBC cell lines byflow cytometry analysis. B, Athymic mice with established MDA-MB-436 and MDA-MB-231 cell xenografts were treated or not with 200mg of 20G7-D9 (D9) twice per week (n ¼ 6). Results are presented as the mean tumor volume (SEM) in each group.,P < 0.01.
corresponding to the number of days after implantation and the different treatment groups. Interaction terms were built into the model. Random intercepts and random slopes were included to take into account the time effect. The model coefficients were estimated by maximum likelihood and considered significant at the 0.05 level. Statistical analysis was carried out using the STATA 10.0 software.
Results
The anti-AXL 20G7-D9 mAb inhibits growth of AXL-positive TNBC cell xenografts
Before testing the effect of the 20G7-D9 anti-AXL mAbs in TNBC xenografts, we assessed AXL surface expression by flow
cytometry (Fig. 1A) and TAM receptors expression by Western blotting (Supplementary Fig. S1) in 10 TNBC cell lines and found that AXL was significantly expressed in 7. These results are con-sistent with previous studies reporting that AXL is often upregu-lated in basal B and occasionally in basal A TNBC (20, 21). Moreover, the cell lines in which AXL was most overexpressed (MDA-MB-231, BT549, HBL-100, BT20, and HS578T) are con-sidered as highly invasive (22). Interestingly, we analyzed AXL mRNA expression in a wide range of breast cancer tumors and we did not observe a significant upregulation of this receptor in basal-like breast tumors in comparison with the other breast cancer subtypes (Supplementary Fig. S2).
AXL GAS6 SNAIL VIM MMP1 SLUG TUBULIN FRA-1 MMP9 TWIST B2164B3160B3977B2412B3467B3029B1995B3804B4122B1915 0 PDTX id. CTRL D9 0.0 5.0×1006 1.0×1007 1.5×1007
**
Bioluminescence intensity (p/s) Data 1High level AXL Low level AXL 0 50 100 150 200 250 Av era g e p a ss ag e du rat io n ( d ays )
***
B2164B316 0 B3977B2412B3467B30 29 B19 95 B3804B41 22 B1915 0 100 200 300 400 500 50 1,500 1,600 1,700 PDTX id. AX L m R NA Exp res s io n (a rb it ra ry u n it ) B3804 10 15 20 25 30 35 0 500 1,000 1,500 2,000 CTRL D9**
Time post graft (days)Time post graft (days)
Tu m o r v o lum e ( m m 3) B3467 20 30 40 50 60 0 200 400 600 800 1,000 Tu m o r v o lum e ( m m 3) CTRL D9
A
B
C
D
E
F
G
CTRL
20G7
-D9
Figure 2.Effect of the anti-AXL 20G7-D9 monoclonal antibody in mice with basal-like TNBC PDX. A, AXL mRNA expression (arbitrary units) in 10 basal-like TNBC PDXs based on Affymetrix microarray data. B, Western blot analysis of AXL, GAS6, and several EMT factors [VIM (Vimentin), MMP9, MMP1, FRA-1, TWIST, SNAIL, SLUG]. TUBULIN served as loading control. C, Average passage duration for thefirst three passages in PDXs with high and low AXL protein level. D, Mice engrafted with the AXL-positive PDX B3804 were treated or not with 200mg of 20G7-D9 twice/week. Results are presented as the mean tumor volume (SEM) in each group (n ¼ 6). E, Mice harboring the AXL-negative PDX B3467 were treated or not with 200mg of 20G7-D9 twice/week. Results are presented as the mean tumor volume (SEM) in each group (n ¼ 10). F, Bone metastasis development inhibition by the anti-AXL mAb 20G7-D9. The day after intracardiac injection of 5 105MDA-MB-231-luc cells, mice (n ¼ 7/group) were treated or not with 200mg of 20G7-D9 or saline twice per week for 4 weeks. The
bioluminescence intensity was measured weekly after intraperitoneal injection of 100 mg/kg luciferin. Results are presented as the bioluminescence signal (p/s) of treated and untreated mice at day 25. G, Representative bioluminescence images of treated (D9) and untreated mice. This experiment has been reproduced twice. ,P < 0.01;,P < 0.001.
Treatment of mice harboring established MDA-MB-436 (basal B, moderate AXL expression) and MDA-MB-231 (basal B, high AXL expression) xenografts with 20G7-D9 for 4 weeks led to a statistically significant growth inhibition in both models com-pared with saline-treated animals (P ¼ 0.015 and 0.047, respec-tively). Specifically, the mean tumor volume in 20G7-D9–treated mice was reduced by 62.5% at day 53 in the MDA-MB-436 model, and by 57% at day 68 in the MDA-MB-231 model (Fig. 1B). The anti-AXL 20G7-D9 mAb inhibits PDX growth
With the aim of using a more relevant preclinical model, we also included 10 TNBC PDXs generated in our institute (18). Microarray analysis showed that AXL mRNA was expressed in six PDXs (>50 arbitrary units). For instance, the PDX derived from the B3804 tumor showed a very high expression level of AXL (Fig. 2A). The other four showed minimal or no AXL expression. AXL protein expression, assessed by Western blotting, was well corre-lated with the mRNA values (Fig. 2B). As AXL (protein and mRNA) was not detected in the B3467 tumor, this PDX was used as negative control for further experiments. Moreover, the four PDXs with the highest AXL protein content (Fig. 2B) also showed the fastest growth rate (average passage duration for the threefirst passages¼ 52.3 days) compared with the other PDXs (151.1 days, t test; P ¼ 0.0003; Fig. 2C).
We then investigated the effect of 20G7-D9 on the growth of the AXL-positive B3804 and the AXL-negative B3467 PDXs. At the end of the treatment, B3804 growth was significantly reduced (by 51%)
in 20G7-D9–treated mice compared with the untreated group (saline; P < 0.01; Fig. 2D). Conversely, growth of the AXL-negative B3467 PDX was not affected by antibody treatment (Fig. 2E). The anti-AXL 20G7-D9 mAb inhibits metastatic spread of MDA-MB-231 cells to bone
As AXL expression has been linked to EMT and metastasis formation (13), we next assessed whether 20G7-D9 could inhibit in vivo migration and invasion of cancer cells. To this aim, one day after intracardiac injection of MDA-MB-231-Luc cells, mice were treated with 20G7-D9 or saline (control) for 4 weeks (biweekly injection) and at day 25, the development of MDA-MB-231-Luc metastases in bone was quantified using a luciferin/luciferase assay. The bioluminescence intensity was significantly lower in the group of mice treated with 20G7-D9 than in untreated controls (Fig. 2F; P ¼ 0.0038). This result was confirmed by bioluminescence imaging performed at the end of the 4 weeks of treatment (Fig. 2G).
AXL expression in basal-like PDXs and basal-like breast tumors is correlated with invasive and mesenchymal gene signatures
Thefinding that treatment with 20G7-D9 reduces cancer cell invasion to bone in mice with TNBC cell xenografts was in accordance with the AXL gene being a major actor in EMT induction. Accordingly, Western blot analysis of the expression of EMT markers involved in breast cancer metastasis formation in TNBC PDXs showed that all AXL-positive PDXs expressed FRA1, GENE SET NAME
[# Genes]
HALLMARK EPITHELIAL
MESENCHYMAL TRANSITION [200] HALLMARK TGF BETA SIGNALING [54]
A
DESCRIPTION
Genes defining epithelial-mesenchymal transion as in wound healing, fibrosis, metastasis.
Genes up-regulated in response to TGFB1 [GeneID=7040] # Genes in Overlap P-value FDR q-value 9.65E–18 14 4.82E–16 1.04E–07 5 2.61E–06 -3 -2 -1 0 1 2 3 Normalized expression Low High
B
AXL SERPINE1 TGFB1 TIMP1 DOC1 LOXL1 VEGFC MARCKS EMP3 COLGA2 SGPP1 SERPINF1 CDH11GENE CorrelaonPearson
1 0.52 0.51 0.50 0.47 0.46 0.46 0.46 0.45 0.44 0.44 0.43 0.43
Epithelial Mesenchymal Transi on, TGFβ signaling Epithelial Mesenchymal Transi on, TGFβ signaling Epithelial Mesenchymal Transi on
Epithelial Mesenchymal Transi on Epithelial Mesenchymal Transi on Epithelial Mesenchymal Transi on Epithelial Mesenchymal Transi on
Epithelial Mesenchymal Transi on
Hallmark
Figure 3.
The 100 genes most correlated with AXL expression in 254 basal-like breast cancers. A, Annotation of these 100 genes using GSEA revealed a striking enrichment in genes defining the EMT and TGFb signaling pathways. B, Heatmap of the 12 genes most correlated with AXL expression in 254 basal-like breast cancers with members of the MSigDB hallmark EMT signature.
SNAIL, and TWIST1, whereas SLUG was detected only in three of them (Fig. 2B). Generally, all AXL-positive PDXs expressed mole-cules involved in EMT, with the exception of GAS6, MMP1, and VIMENTIN that were not correlated with AXL expression. More-over, microarray analysis of the 100 genes most correlated with AXL expression in 254 basal-like breast cancers (Supplementary Fig. S3) using the MSigDB hallmark gene signatures indicated a strong enrichment in genes that define the EMT and TGFB1 pathways (Fig. 3A). Particularly, 8 of the 12 genes most correlated with AXL expression, were annotated as EMT markers and includ-ed TGFB1 and VEGFC (Fig. 3B). Further annotation analysis with the MSigDB collections indicated a strong enrichment in signal transduction and cell proliferation. Membrane components, including the integrin pathway and focal adhesion processes, were also overrepresented. Significant overlaps were also found with gene signatures present in adult stem cells, early embryonic stem cells, or epithelial cell lines submitted to EMT-inducing factor TGFB1 and mesenchymal breast cancer cell lines. These enrichments reveal the influence of AXL in human tumors through integrin-mediated signaling impacting cell proliferation and EMT in accordance with our in vitro experiments (Supple-mentary Table S1). These findings in basal-like breast cancer samples and PDXs suggest that high AXL expression is associated with acquisition of mesenchymal features. The extent of this effect might depend on the cell context and tumor environment. The anti-AXL 20G7-D9 mAb inhibitsin vitro migration and invasion of TNBC cells
To further understand how 20G7-D9 inhibits metastasis for-mation, we then performed Boyden chamber assays using the AXL-positive MDA-MB-231 and HBL-100 cell lines (basal B TNBC). Preincubation with 20G7-D9 inhibited migration and invasion by more than 50% in both lines compared with untreat-ed cells (Fig. 4A and B). Conversely, addition of rhGAS6 in the bottom chamber increased significantly the migration and inva-sion of both cell lines compared with untreated cells (Fig. 4A and B). This effect was abolished by preincubation with the anti-AXL antibody. We then assessed whether 20G7-D9 could inhibit
degradation of the extracellular matrix by breast cancer cells using in situ zymography. Preincubation of MDA-MB-231 cells with 20G7-D9 significantly reduced the number of invadopodia and the areas of focal gelatin degradation compared with untreated cells (Supplementary Fig. S4A and S4B). Collectively, these data suggest that GAS6 and AXL are involved in TNBC cell migration and invasion.
GAS6 stimulation triggers expression of EMT factors
To further investigate the role of GAS6/AXL signaling in EMT and metastasis formation, we incubated MDA-MB-231, HBL-100 (two cell lines with high AXL expression), and MDA-MB-436 (moderate AXL expression) cells with rhGAS6 and then assessed AXL and FRA-1 phosphorylation and the expression of EMT effectors by Western blotting. GAS6 induced phosphorylation of AXL and FRA-1 (at S265) and increased AXL expression in all three cell lines (Fig. 5A). Moreover, expression of SNAIL, SLUG, TWIST1, and ZEB-1/2 was also increased in the three TNBC cell lines upon addition of GAS6 (Fig. 5A and B, quantification in Supplementary Fig. S5). These effects were abolished when AXL was knocked down by shRNA in MDA-MB-231 cells. Similarly, FRA1 silencing in MDA-MB-231 cells inhibited ZEB1 and SNAIL upregulation following stimulation with GAS6 (Fig. 5C). This is in agreement with thefinding that FRA-1 can induce EMT-related genes, such as SLUG and ZEB1/2 (23, 24), and suggests that some of the effects of activated AXL could result from increased expres-sion of phosphorylated FRA-1.
Finally, incubation with GAS6 also induced expression of the mesenchymal markers vimentin, MMP1 and MMP9 in HBL100 and MDA-MB-436 cells (Fig. 5B). In MDA-MB-231 cells, induc-tion was less clear-cut due to the high basal expression of these markers. Nevertheless, AXL silencing reduced the expression level of the three EMT markers (Fig. 5B). Moreover, the level of induction of these EMT markers varied according to the TNBC cell line and could be explained by the involvement of other, still unidentified factors in AXL/GAS6 signaling. Globally, these results confirm an important role of GAS6 and AXL in the expression of EMT markers involved in breast cancer invasion.
Figure 4.
20G7-D9 inhibits TNBC cell migration (A) and invasion (B)in vitro. MDA-MD-231 and HBL-100 cells were preincubated with 20G7-D9 overnight, trypsinized, and plated in the top compartment of a Boyden chamber in serum-free medium with or without 200 ng/mL rhGAS6. At the end of the experiment, cells were stained with calcein to evaluate cell migration/invasion.,P < 0.01;,P < 0.001.
The anti-AXL mAb dramatically reduces the mesenchymal phenotype of breast cancer cells
We then asked whether 20G7-D9 could influence EMT marker expression and migration of MDA-MB-231 cells. Incubation with
20G7-D9 induced downregulation of AXL (14) and consequently its expression could not be induced by GAS6 (Fig. 6A and D). Similarly, GAS6-induced AXL phosphorylation was reduced com-pared with untreated cells.
Figure 5.
GAS6 regulates expression of EMT markers in TNBC. The effect of incubation with rhGAS6 on the expression of EMT transcription factors (A) and mesenchymal factors (B) was analyzed by Western blotting. MDA-MB-231-shCONTROL, -shAXL, HBL-100, and MDA-MB-436 cells were incubated with 200 ng/mL of rhGAS6 for the indicated times. GAPDH served as loading control. C, FRA-1 silencing in MDA-MB-231 cells inhibits the GAS6-induced expression of EMT transcription factors. Cells were stimulated with 200 ng/mL of rhGAS6 for 30 minutes. Control cells express a scrambled siRNA. TUBULIN and GAPDH served as loading control. Quantification of each protein band was normalized to GAPDH or TUBULIN and then represented as fold change relative to nontreated siCTRL MDA-MB-231 cells.
Figure 6.
Effect of the anti-AXL mAb 20G7-D9 on AXL activation (A) and on the expression of EMT effectors (B) and their targets (C). MDA-MD-231 cells were incubated with 20G7-D9 or an isotype control antibody (Px) overnight. AXL phosphorylation (Tyr702) was analyzed by Western blotting after 30-minute incubation with 200 ng/mL rhGAS6. Expression of FRA-1 (and its phosphorylation on Ser265), ZEB-1, ZEB-2, TWIST, SLUG, SNAIL, and of the mesenchymal markers Vimentin, MMP1, and MMP9 was analyzed after 120-minute incubation with 200 ng/mL rhGAS6. GAPDH served as loading control. All Western blots experiments were repeated at least three times. D, Quantification of each protein band was normalized to GAPDH and then represented as fold change relative to the nontreated cell band (NT¼ 1). Statistical analyses were determined between the effect of GAS6 (200 ng/mL) and 20G7-D9 (24 hours) þ GAS6 (200 ng/mL) on MDA-MB-231 cells (,P < 0.05;,P < 0.01).
A significant inhibition of the GAS6-induced upregulation of the EMT inducers SNAIL, SLUG, TWIST, and ZEB1/2 as well as of FRA-1 was observed in MDA-MB-231 cells preincubated with 20G7-D9 (Fig. 6B and D) compared with untreated cells. GAS6-induced FRA-1 phosphorylation was also strongly inhib-ited upon AXL downregulation by 20G7-D9. Finally, the anti-AXL antibody treatment also reduced GAS6 effect on the expression of the mesenchymal markers vimentin, MMP1, and MMP9 (Fig. 6C and D). In BT20 cell line, GAS6 treatment induced a strong downregulation of E-cadherin that is abrogated by pre-treatment with 20G7-D9 anti-AXL antibody (Supplementary Fig. S6).
Discussion
Among the large number of drug approved for breast cancer, the use of targeted therapies [hormonal, tyrosine kinase inhibitors (TKI), or antibodies] highlighted the existence of an aggressive breast cancer subtype, the TNBC, characterized by frequent and rapid relapse. Although this subtype is sensitive to chemotherapy, the risk of relapse and metastasis formation is high and the development of novel therapies to suppress cancer metastasis is needed. In this study, we show that, in vitro, AXL and its ligand GAS6 enhance TNBC cell migration and invasiveness. Further-more, we also demonstrate that after activation by GAS6, AXL promotes EMT by upregulating the transcription factors FRA1, SLUG, SNAIL, ZEB1/2, and TWIST1. AXL also increases cell motility by inducing expression of MMP1 and MMP9, thus promoting cell migration and invasion. Inhibition of AXL by shRNA or with the anti-AXL mAb 20G7-D9 suppresses both cancer cell migration and invasion, even following GAS6 addition to the medium.
In vivo treatment with 20G7-D9 reduces tumor growth in TNBC xenograft models and also in AXL-positive basal-like breast cancer PDXs. A limitation of cancer cell line xenograft models is that the genetic and physiologic backgrounds of these cell lines strongly diverge from those of primary tumors (25). Moreover, tumor heterogeneity is often lost after cell culture, whereas it is an important characteristic of breast cancer and can affect the tumor response to treatment. PDX are today, with genetically engineered cancer mouse models, a more relevant model because they are based on the graft of fresh primary tumor fragments directly from the patient to immunodeficient mice. PDX maintain the genetic and histologic features of the original tumor (18, 26, 27). For instance, a good prediction of patient treatment efficacy using PDXs has been reported (28). Nevertheless, it is important to note that, even if PDXs are today recognized as a robust in vivo model for preclinical assays, the microenvironment remains of murine origin. This context might have an effect on AXL expression and role that only clinical trial, targeting the receptor with an antibody or a TKI, would be able to answer. Today, various AXL-specific TKIs (5, 29, 30) and mAbs (14, 31, 32) have been tested in preclinical models and only one AXL inhibitor, BGB324 (R428), is evaluated in clinical trials for acute myeloid leukemia (33). Phase I studies demonstrated that BGB324 could be safely administered for prolonged periods at doses that inhibit AXL activation and exhibit antileukemic activity.
In the third in vivo experiment, we demonstrated the impor-tance of AXL in cancer cell migration and invasion. Indeed, treatment with 20G7-D9 after intracardiac injection of MDA-MB-231-Luc cells significantly reduced the number of bone metastases. Therefore, in the context of AXL-expressing TNBC,
AXL seems a valuable therapeutic target because it drives both tumor growth and cancer cell migration/invasion.
In cancer, EMT involves complex cell signaling cascades that result in the alteration of cell–cell and cell–extracellular matrix interactions, leading to the acquisition by epithelial cells of a mesenchymal phenotype and ultimately to metastasis formation (34). Among the EMT cellular pathways, receptor tyrosine kinases can be inducers (for instance, c-MET), or can be activated (for instance, PDGFRa) following induction of the expression of different EMT transcription factors (35, 36). In breast cancer, AXL was known as an EMT-induced regulator of cancer metastasis, under the control of SLUG (13). Although AXL is required to maintain the invasiveness of mesenchymal-like cells, EMT starting point is the overexpression of intracellular proteins, such as SLUG or RAS (12, 37). More recently, Asiedu and colleagues described AXL as an inducer of EMT using the TKI MP470 (13). AXL inhibition led to the downregulation of EMT markers, such as SNAIL and N-cadherin. However, these studies did not assess the role of GAS6, and MP470 can target different tyrosine kinases that have been already defined as EMT inducers (for instance, c-KIT; ref. 38). Here, we show that AXL activation by GAS6 is necessary to promote strong EMT signaling through the involvement of many EMT transcription factors. Moreover, AXL inhibition by shRNA or with the specific antibody 20G7-D9 blocks GAS6/AXL-dependent EMT. Thus, AXL may play a dual role in TNBC as inducer and target of EMT signaling during metastasis formation. This GAS6-induced signaling, as well as the basic expression of EMT markers, vary in the different TNBC cell lines studied and could imply other intracellular factors that remain to be identified.
Furthermore, we show the involvement of the transcription factor FRA-1, an AP-1 family member, in GAS6/AXL signaling. Several studies suggest that FRA-1 can regulate cancer cell motility and invasion. Direct or indirect transcriptional FRA-1 targets include proteins that promote extracellular matrix degradation (MMP1, MMP9; ref. 15), EMT transcription factors (SLUG and ZEB1/2; refs. 24, 39), and proteins involved in cell adhesion or migratory signaling (CD44, VEGF, c-MET; ref. 40). In bladder cancer, FRA-1 promotes AXL transcription that mediates FRA-1 effect on cancer cell motility (41). Ourfindings that in TNBC cell lines AXL activation by GAS6 increases FRA-1 expression and FRA1 knockdown abolishes SNAIL and ZEB1 upregulation by GAS6/AXL signaling bring new insights into the cross-talk between these proteins in EMT signaling.
Although TNBC cell lines are frequently AXL-positives, we observed that AXL expression in patients was not specific to this breast cancer subtype, confirming previous studies (22). It would be thus interesting to extend the characterization of AXL, as well as the therapeutic use of our antibody, in other breast cancer sub-types, like luminal models, to know whether the receptor has the same behavior and implication in EMT signaling.
Several of the basal-like PDXs we established express elevated levels of AXL and interestingly those with the highest AXL expres-sion level grow faster in vivo. Moreover, analysis of the genes correlated with AXL upregulation in basal-like breast tumors indicated that most of these genes are involved in cancer cell invasion and EMT, TGFb signaling, mesenchymal markers, stem cells, cytoskeleton, and ECM remodeling. Of note, the transcrip-tion factor FRA-1 induces EMT through TGFb modulation (42). AXL is also overexpressed in breast cancer stem cells (13) and EMT features are often associated with stemness (43).
In conclusion, our results highlight the expression and role of AXL and its ligand GAS6 in TNBC cell invasion. Our results suggest that AXL is a modulator, and not only a target, of EMT signaling. The use of anti-AXL antibodies, which degrade the receptor, is an interesting approach to inhibit metastatic process in such breast cancers.
Disclosure of Potential Conflicts of Interest
W. Leconet, C. Larbouret, A. Pelegrin, and B. Robert are listed as coinventors on a patent on the development of a murine antibody (20G7-D9) against the human receptor tyrosine kinase AXL for cancer therapy, which is owned by the French Research Agency of Health (INSERM), University of Montpellier, and Regional Clinical Cancer Center (ICM), and licensed to Oribase-Pharma. No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: W. Leconet, D. Chalbos, A. Pelegrin, C. Larbouret, B. Robert
Development of methodology: W. Leconet, A. Sirvent, M. Busson, N. Radosevic-Robin
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W. Leconet, M. Chentouf, S. du Manoir, I. A€t-Arsa, M. Busson, N. Radosevic-Robin, C. Theillet, D. Chalbos, J.-M. Pasquet Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W. Leconet, S. du Manoir, M. Busson, M. Jarlier, B. Robert
Writing, review, and/or revision of the manuscript: W. Leconet, S. du Manoir, C. Theillet, D. Chalbos, A. Pelegrin, C. Larbouret, B. Robert
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W. Leconet, C. Chevalier, B. Robert Study supervision: W. Leconet, B. Robert
Acknowledgments
The authors wish to thank Dr. Rui Bras-Gon¸calves for his expert help with the PDXs. We thank all the staff of the IRCM animal facility, the RHEM, and the Small Animal Imaging Platform of Montpellier (IPAM) facilities for IHC and mice imaging, respectively. We thank also to Dr. Pierre Martineau, Dr. Antonio Maraver, and Dr. Claude Sardet for their input concerning the manuscript. We thank Beatrice Orsetti for supplying PDX samples for Western blot experiments.
Grant Support
This publication has been funded with support from the French National Research Agency under the program "Investissements d'avenir" Grant Agree-ment LabEx MAbImprove: ANR-10-LABX-53. This work was supported by the INSERM; Oribase Pharma. This work benefited from the support of INCa grants MOPRECLI and "Role of cancer stem cells during metastatic progression of breast cancer."
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received May 26, 2016; revised October 11, 2016; accepted November 10, 2016; published OnlineFirst December 6, 2016.
References
1. Metzger-Filho O, Tutt A, de Azambuja E, Saini KS, Viale G, Loi S, et al. Dissecting the heterogeneity of triple-negative breast cancer. J Clin Oncol 2012;30:1879–87.
2. Thiery JP, Acloque H, Huang RYJ, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009;139:871–90. 3. Taube JH, Herschkowitz JI, Komurov K, Zhou AY, Gupta S, Yang J, et al.
Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes. Proc Natl Acad Sci U S A 2010;107:15449–54.
4. Moyret-Lalle C, Ruiz E, Puisieux A. Epithelial-mesenchymal transition transcription factors and miRNAs: "Plastic surgeons" of breast cancer. World J Clin Oncol 2014;5:311–22.
5. Holland SJ, Pan A, Franci C, Hu Y, Chang B, Li W, et al. R428, a selective small molecule inhibitor of Axl kinase, blocks tumor spread and prolongs survival in models of metastatic breast cancer. Cancer Res 2010;70: 1544–54.
6. Koorstra J-BM, Karikari CA, Feldmann G, Bisht S, Rojas PL, Offerhaus GJA, et al. The Axl receptor tyrosine kinase confers an adverse prognostic influence in pancreatic cancer and represents a new therapeutic target. Cancer Biol Ther 2009;8:618–26.
7. Sawabu T, Seno H, Kawashima T, Fukuda A, Uenoyama Y, Kawada M, et al. Growth arrest-specific gene 6 and Axl signaling enhances gastric cancer cell survival via Akt pathway. Mol Carcinog 2007;46:155–64.
8. Zhang Y-X, Knyazev PG, Cheburkin YV, Sharma K, Knyazev YP, Orfi L, et al. AXL is a potential target for therapeutic intervention in breast cancer progression. Cancer Res 2008;68:1905–15.
9. O'Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B, Prokop C, et al. axl, a transforming gene isolated from primary human myeloid leuke-mia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol 1991;11:5016–31.
10. Linger RMA, Keating AK, Earp HS, Graham DK. TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer. Adv Cancer Res 2008;100:35–83.
11. Linger RMA, Keating AK, Earp HS, Graham DK. Taking aim at Mer and Axl receptor tyrosine kinases as novel therapeutic targets in solid tumors. Expert Opin Ther Targets 2010;14:1073–90.
12. Gjerdrum C, Tiron C, Høiby T, Stefansson I, Haugen H, Sandal T, et al. Axl is an essential epithelial-to-mesenchymal transition-induced regulator of
breast cancer metastasis and patient survival. Proc Natl Acad Sci U S A 2010;107:1124–9.
13. Asiedu MK, Beauchamp-Perez FD, Ingle JN, Behrens MD, Radisky DC, Knutson KL. AXL induces epithelial-to-mesenchymal transition and regulates the function of breast cancer stem cells. Oncogene 2014;33: 1316–24.
14. Leconet W, Larbouret C, Chardes T, Thomas G, Neiveyans M, Busson M, et al. Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy. Oncogene 2014;33:5405–14. 15. Belguise K, Kersual N, Galtier F, Chalbos D. FRA-1 expression level
regulates proliferation and invasiveness of breast cancer cells. Oncogene 2005;24:1434–44.
16. Bossard C, Busson M, Vindrieux D, Gaudin F, Machelon V, Brigitte M, et al. Potential role of estrogen receptor beta as a tumor suppressor of epithelial ovarian cancer. PLoS One 2012;7:e44787.
17. Gaborit N, Larbouret C, Vallaghe J, Peyrusson F, Bascoul-Mollevi C, Crapez E, et al. Time-resolvedfluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR/HER2 dimers: a new method to evaluate the efficiency of targeted therapy using monoclonal antibodies. J Biol Chem 2011;286:11337–45.
18. du Manoir S, Orsetti B, Bras-Gon¸calves R, Nguyen T-T, Lasorsa L, Boissiere F, et al. Breast tumor PDXs are genetically plastic and correspond to a subset of aggressive cancers prone to relapse. Mol Oncol 2014;8:431–43. 19. Guedj M, Marisa L, de Reynies A, Orsetti B, Schiappa R, Bibeau F, et al.
A refined molecular taxonomy of breast cancer. Oncogene 2012;31: 1196–206.
20. D'Alfonso TM, Hannah J, Chen Z, Liu Y, Zhou P, Shin SJ. Axl receptor tyrosine kinase expression in breast cancer. J Clin Pathol 2014;67: 690–6.
21. Wilson C, Ye X, Pham T, Lin E, Chan S, McNamara E, et al. AXL inhibition sensitizes mesenchymal cancer cells to antimitotic drugs. Cancer Res 2014;74:5878–90.
22. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 2006;10:515–27.
23. Chen H, Zhu G, Li Y, Padia RN, Dong Z, Pan ZK, et al. Extracellular signal-regulated kinase signaling pathway regulates breast cancer cell migration by maintaining slug expression. Cancer Res 2009;69:9228–35.
24. Shin S, Blenis J. ERK2/Fra1/ZEB pathway induces epithelial-to-mesenchy-mal transition. Cell Cycle 2010;9:2483–4.
25. Siolas D, Hannon GJ. Patient-derived tumor xenografts: transforming clinical samples into mouse models. Cancer Res 2013;73:5315–9. 26. DeRose YS, Wang G, Lin Y-C, Bernard PS, Buys SS, Ebbert MTW, et al.
Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes. Nat Med 2011;17:1514–20.
27. Zhang H, Cohen AL, Krishnakumar S, Wapnir IL, Veeriah S, Deng G, et al. Patient-derived xenografts of triple-negative breast cancer reproduce molecular features of patient tumors and respond to mTOR inhibition. Breast Cancer Res 2014;16:R36.
28. Stebbing J, Paz K, Schwartz GK, Wexler LH, Maki R, Pollock RE, et al. Patient-derived xenografts for individualized care in advanced sarcoma. Cancer 2014;120:2006–15.
29. Byers LA, Diao L, Wang J, Saintigny P, Girard L, Peyton M, et al. An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance. Clin Cancer Res 2013;19:279–90. 30. Mollard A, Warner SL, Call LT, Wade ML, Bearss JJ, Verma A, et al. Design, synthesis and biological evaluation of a series of novel Axl kinase inhibi-tors. ACS Med Chem Lett 2011;2:907–12.
31. Ye X, Li Y, Stawicki S, Couto S, Eastham-Anderson J, Kallop D, et al. An anti-Axl monoclonal antibody attenuates xenograft tumor growth and enhances the effect of multiple anticancer therapies. Oncogene 2010;29: 5254–64.
32. Liu R, Gong M, Li X, Zhou Y, Gao W, Tulpule A, et al. Induction, regulation, and biologic function of Axl receptor tyrosine kinase in Kaposi sarcoma. Blood 2010;116:297–305.
33. Loges S, Gjertsen BT, Heuser M, Ben-Batalla I, Micklem D, Jorg C, et al. A first-in-patient phase I study of BGB324, a selective Axl kinase inhibitor in
patients with refractory/relapsed AML and high-risk MDS. J Clin Oncol 34, 2016(suppl; abstr 2561)
34. Nieto MA. The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu Rev Cell Dev Biol 2011;27:347–76.
35. Mazzone M, Comoglio PM. The Met pathway: master switch and drug target in cancer progression. FASEB J 2006;20:1611–21.
36. Eckert MA, Lwin TM, Chang AT, Kim J, Danis E, Ohno-Machado L, et al. Twist1-induced invadopodia formation promotes tumor metastasis. Can-cer Cell 2011;19:372–86.
37. Vuoriluoto K, Haugen H, Kiviluoto S, Mpindi J-P, Nevo J, Gjerdrum C, et al. Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer. Oncogene 2011;30:1436–48.
38. Tang Y, Fan Y, Jiang J, Li K, Zheng M, Chen W, et al. C-kit induces epithelial-mesenchymal transition and contributes to salivary adenoid cystic cancer progression. Oncotarget 2014;5:1491–501.
39. Verde P, Casalino L, Talotta F, Yaniv M, Weitzman JB. Deciphering AP-1 function in tumorigenesis: fraternizing on target promoters. Cell Cycle 2007;6:2633–9.
40. Ramos-Nino ME, Scapoli L, Martinelli M, Land S, Mossman BT. Microarray analysis and RNA silencing link fra-1 to cd44 and c-met expression in mesothelioma. Cancer Res 2003;63:3539–45.
41. Sayan AE, Stanford R, Vickery R, Grigorenko E, Diesch J, Kulbicki K, et al. Fra-1 controls motility of bladder cancer cells via transcriptional upregula-tion of the receptor tyrosine kinase AXL. Oncogene 2012;31:1493–503. 42. Bakiri L, Macho-Maschler S, Custic I, Niemiec J, Guío-Carrion A, Hasenfuss
SC, et al. Fra-1/AP-1 induces EMT in mammary epithelial cells by mod-ulating Zeb1/2 and TGFb expression. Cell Death Differ 2015;22:336–50. 43. Mani SA, Guo W, Liao M-J, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008;133:704–15.
