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The Receptor AXL Diversifies EGFR Signaling and Limits the Response to EGFR-targeted Inhibitors in Triple-Negative Breast Cancer Cells

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The Receptor AXL Diversifies EGFR Signaling and Limits the Response to EGFR-targeted Inhibitors in Triple-Negative Breast Cancer Cells

Aaron S Meyer et al. Sci Signal.

Abstract

The relationship between drug resistance, changes in signaling, and emergence of an invasive phenotype is well appreciated, but the underlying mechanisms are not well understood. Using machine learning analysis applied to the Cancer Cell Line Encyclopedia database, we identified expression of AXL, the gene that encodes the epithelial-to-mesenchymal transition (EMT)-associated receptor tyrosine kinase (RTK) AXL, as exceptionally predictive of lack of response to ErbB family receptor-targeted inhibitors. Activation of EGFR (epidermal growth factor receptor) transactivated AXL, and this ligand-independent AXL activity diversified EGFR-induced signaling into additional downstream pathways beyond those triggered by EGFR alone. AXL-mediated signaling diversification was required for EGF (epidermal growth factor)-elicited motility responses in AXL-positive TNBC (triple-negative breast cancer) cells. Using cross-linking coimmunoprecipitation assays, we determined that AXL associated with EGFR, other ErbB receptor family members, MET (hepatocyte growth factor receptor), and PDGFR (platelet-derived growth factor receptor) but not IGF1R (insulin-like growth factor 1 receptor) or INSR (insulin receptor). From these AXL interaction data, we predicted AXL-mediated signaling synergy for additional RTKs and validated these predictions in cells. This alternative mechanism of receptor activation limits the use of ligand-blocking therapies and indicates against therapy withdrawal after acquired resistance. Further, subadditive interaction between EGFR- and AXL-targeted inhibitors across all AXL-positive TNBC cell lines may indicate that increased abundance of EGFR is principally a means to transactivation-mediated signaling.

Figures

Fig. 1
Fig. 1. Support vector classification to identify mechanisms of drug resistance
(A) Spearman correlations of expression for a subset of RTKs. Only statistically significant correlations are shown (P < 0.01). (B) Classification of cell lines as resistant or sensitive to AEW541, erlotinib, and lapatinib based on RTK expression. Classification accuracy using randomized expression data (black), a model considering the expression of the gene encoding the drug target receptor (blue), or a model considering the expression of both the gene encoding the drug target receptor and that of AXL (dotted line) are shown. (C) Fraction of cell lines that are sensitive to erlotinib after separation according to those that exhibit greater or less than median expression of EGFR or AXL. (D) AXL expression probe values for resistant and sensitive cell lines to each drug (**P < 0.01, Kruskal-Wallis test, n = 91 to 396 cell lines per grouping). FI, fluorescence intensity; n.s., not significant. (E) Dose-response curves for R428 and erlotinib in three TNBC cell lines that have abundant EGFR and AXL. Bars on the side indicate the range of viability between the highest and lowest erlotinib dose to illustrate subadditivity (P < 10−6, BT549; P < 0.05, MB436; P < 0.01, MB231 by Loewe’s synergy analysis; see Materials and Methods) between erlotinib and R428. Data are means ± SEM from three independent biological measurements.
Fig. 2
Fig. 2. EGF stimulation transactivates AXL and MET
(A) ELISA-based pan-phosphotyrosine (pan-pY) measurement of alternative receptors after EGF stimulation in MDA-MB-231 (*P < 0.05, Student’s t test). (B) AXL knockdown, measured by ELISA. (C) Total and surface amounts of alternative receptors in AXL-silenced MDA-MB-231 cells (*P < 0.05, Student’s t test). Data are means ± SEM of three biological measurements. (D) Downstream signaling assessed by kinase phosphorylation in MDA-MB-231 cells 5 min after stimulation with EGF, TGFα, or HGF in the presence or absence (siAXL) of AXL. Each phosphosite was mean-centered and variance-normalized.
Fig. 3
Fig. 3. AXL knockdown attenuates downstream signaling in MDA-MB-231
(A) Ratios of fold activation after treatment with growth factor in AXL knockdown cells relative to wild-type cells: ([siAXL GF]/[siAXL Unstim]) ÷ ([siControl GF]/[siControl Unstim]). The unstimulated bar indicates the ratio of unstimulated abundance: [siAXL Unstim]/[siControl Unstim]. Inset shows the Spearman correlation across all phosphosites between the unstimulated and stimulated ratios (*P < 0.05). (B) PCA score plot of signaling data after AXL knockdown. Line colors indicate stimulation conditions denoted in (A). (C) Loading plot of signaling data after AXL knockdown.
Fig. 4
Fig. 4. AXL amplifies the EGFR signaling response
(A) ELISA of pan-pY EGFR in wild-type (siControl) or AXL-silenced (siAXL) MDA-MB-231 after 5 min of treatment with varying EGF amounts. Data are means ± SEM. P < 0.05, Student’s t test. n = 3. (B) Phosphorylation of Akt in response to a range of EGF doses. Data are means ± SEM. P < 0.05, Student’s t test. n = 3. (C) ELISA for the abundance of pan-pY on EGFR versus the phosphorylation of Akt in MDA-MB-231 cells. Lines show a Hill regression to each set of data with SE of biological triplicate measurements. (D) Hill regression of each plot shows similar Kd values but significantly different maximal activation (F test). Error bars indicate SE of the fit.
Fig. 5
Fig. 5. AXL signaling is required for EGF-elicited protrusion
(A) Mean loadings of the reduced partial least-squares regression models. The red point corresponds to the projection of the phenotype. Error bars indicate the SE for the family of reduced models. (B) Protrusion predictions from reduced partial least-squares regression models for wild-type (by cross-validation) and AXL knockdown (by prediction) cells. Error bars indicate the SE of prediction across the family of reduced models. (C) EGF-elicited protrusion response of MDA-MB-231 cells upon AXL knockdown (***P < 0.001, Mann-Whitney test; n = 13 to 25 from three independent experiments). (D) EGF-elicited protrusion responses with or without 0.3 µM R428 (***P < 0.001, Mann-Whitney test; n = 17 to 35 from three independent experiments). MDA-MB-231 and MDA-MB-157 cells express AXL, whereas MCF7 and T47D cells do not.
Fig. 6
Fig. 6. AXL colocalizes with ErbB receptors and MET
(A) Illustration of the multivariate cross-linking–mediated coimmunoprecipitation procedure adopted. After ethylene glycolbis(succinimidylsuccinate) (EGS) cross-linking, cells are lysed and incubated with identifiable beads targeting non-AXL RTKs, then with an antibody for AXL to quantify the amount of receptor coimmunoprecipitation. (B) Quantification of AXL in complex with the indicated receptor in MDA-MB-231 and MCF7 cells. To account for possible antibody crosstalk, samples were always compared to those with AXL modulated by either siRNA-mediated knockdown (siAXL) or exogenous expression (AXL) in MDA-MB-231 or MCF7 cells, respectively. Data are means ± SE FI from six technical replicates across biological duplicates; P < 0.05, Student’s t test. (C) Summary of the relative amount of the indicated receptor found in AXL-linked complexes assessed by direct ELISA (TOTAL) or cross-receptor measurement (XLINK) from each cell line in cross-linked lysates. Data are representative of two separate experiments, each with technical triplicates. (D) Relative likelihood of each parameter being 0 (no complex occurring with AXL) for each receptor across all five cell lines.
Fig. 7
Fig. 7. Cross-linking predicts HRG-AXL crosstalk in MDA-MB-453
(A) Fold activation of downstream signaling after HRG stimulation in cells with or without exogenous AXL expression. (B) For the effect of clustered receptors in MDA-MB-231, the signaling measurement of IGF-stimulated cells was z score–normalized to the mean measurement of EGF-, TGFα-, HGF-, HRG-, and HBEGF-stimulated cells. For synergy in MDA-MB-453 cells (which do not normally express AXL), the ratio of fold activation after HRG treatment in cells overexpressing AXL to that in cells that do not express AXL is shown. P = 0.027, Spearman correlation.

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