Targeting HER2-AXL heterodimerization to overcome resistance to HER2 blockade in breast cancer

Abstract

Anti-HER2 therapies have markedly improved prognosis of HER2-positive breast cancer. However, different mechanisms play a role in treatment resistance. Here, we identified AXL overexpression as an essential mechanism of trastuzumab resistance. AXL orchestrates epithelial-to-mesenchymal transition and heterodimerizes with HER2, leading to activation of PI3K/AKT and MAPK pathways in a ligand-independent manner. Genetic depletion and pharmacological inhibition of AXL restored trastuzumab response in vitro and in vivo. AXL inhibitor plus trastuzumab achieved complete regression in trastuzumab-resistant patient-derived xenograft models. Moreover, AXL expression in HER2-positive primary tumors was able to predict prognosis. Data from the PAMELA trial showed a change in AXL expression during neoadjuvant dual HER2 blockade, supporting its role in resistance. Therefore, our study highlights the importance of targeting AXL in combination with anti-HER2 drugs across HER2-amplified breast cancer patients with high AXL expression. Furthermore, it unveils the potential value of AXL as a druggable prognostic biomarker in HER2-positive breast cancer.

Fig. 1.. Trastuzumab-resistant HER2⁺ BC cell line characterization.

(A) Cell proliferation analysis by WST-1 in trastuzumab-sensitive and trastuzumab-resistant cell lines with and without trastuzumab treatment at 15 μg/ml for 7 days. (B) IHC staining of Ki67 in the indicated cell lines. (C) Comparison of basal cell proliferation by WST-1 in trastuzumab-sensitive and trastuzumab-resistant cell lines over 144 hours. (D) IHC staining ER, PR, and HER2 in the indicated cell lines. MDA-MB-231 was used as a negative control for HER2, ER, and PR staining. Flow cytometry analysis of HER2 expression in cell membrane (E), Western blot analysis for total HER2 expression (F) and RT-qPCR analysis of ERBB2 mRNA expression (G) in trastuzumab-sensitive and trastuzumab-resistant cell lines. Western blot analysis of AXL and phospho-AXL (H) and RT-qPCR analysis of AXL mRNA expression (I) and in trastuzumab-sensitive and trastuzumab-resistant cell lines. Scale bars, 100 μm; magnifications, ×20 (B) and ×10 (D). GAPDH was used as an endogenous control in (F) to (I). Flow cytometry negative control was stained with isotype primary antibody in (E). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test in (A), (C), (E), (G), and (I).

Fig. 2.. AXL has a role in trastuzumab response in HER2⁺ BC cell lines.

(A) Western blot analysis of AXL and phospho-AXL protein expression after 72 hours of siRNA-mediated AXL knockdown. (B) Cell proliferation analysis by WST-1 in sensitive cell lines and siRNA-mediated AXL knockdown acquired resistant cell lines treated with trastuzumab for 7 days. (C) Western blot analysis of AXL and phospho-AXL protein expression after 72 hours of TP-0903 treatment. (D) Cell proliferation analysis by WST-1 in sensitive cell lines and TP-0903–treated resistant cell lines with trastuzumab for 7 days. (E) Western blot analysis of AXL and phospho-AXL protein expression after 72 hours of AXL overexpression. (F) Cell proliferation analysis by WST-1 in resistant cell lines and AXL-overexpressed sensitive cell lines with trastuzumab treatment for 7 days. GAPDH was used as an endogenous control in (A), (C), and (E).

Fig. 3.. AXL expression is associated with EMT-like phenotype.

RT-qPCR analysis of mRNA expression (A) and Western blot analysis of protein expression (B) of EMT markers: VIM, FN1, CTNNB1, CDH2, and CDH1 in sensitive versus trastuzumab-resistant cell lines. Transwell migration and invasion assay in SKBR3 versus SKBR3R (C), siRNA-mediated AXL knockdown (D), and pharmacological AXL inhibition by TP-0903 (G) in the SKBR3R cell line. Western blot analysis of EMT markers in siRNA-mediated AXL knockdown (E) and pharmacological AXL inhibition by TP-0903 (F) in SKBR3R and in AXL-overexpressed SKBR3 (H). (I) Transwell migration and invasion assay in AXL-overexpressed SKBR3. GAPDH was used as an endogenous control in (A), (B), (E), (F), and (H). Images show representative migrating/invading cells, and bars show percentage of the mean (±SD, n = 8) of migrating/invading cells. Scale bar, 100 μm; magnification, ×10 (C, D, G, and I). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test in (A), (C), (D), (G), and (I).

Fig. 4.. AXL-HER2 heterodimer regulates PI3K/AKT and MAPK/ERK pathways.

(A) Co-immunoprecipitation (IP) of AXL, HER2, and IgG as a negative control (NC) followed by immunoblotting of AXL and HER2 in acquired trastuzumab-resistant cell lines. (B) PLA of AXL and HER2 (red) in acquired resistant cell lines by flow cytometry. Negative control: primary antibodies were omitted (gray). (C) PLA of AXL and HER2 in trastuzumab-sensitive and acquired resistant cell lines by confocal microscopy. Red spots indicate AXL-HER2 interaction. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (D) Western blot analysis of AXL-HER2 downstream proteins in trastuzumab-sensitive versus trastuzumab-resistant cell lines at basal level. Western blot analysis of AXL-HER2 downstream proteins with and without trastuzumab for 72 hours in siRNA-mediated AXL knockdown (E) after pharmacological AXL inhibition by TP-0903 (F) in acquired trastuzumab-resistant cell lines and in AXL-overexpressed sensitive cell lines (G). GAPDH was used as an endogenous control in (D) to (G). Scale bar, 25 μm; magnification, ×40 (C).

Fig. 5.. AXL inhibition overcomes trastuzumab resistance in PDX-derived HER2⁺ BC cell lines.

(A) Schematic representation of in vitro trastuzumab–resistant PDX cell lines (PDX118-TR1 and TR2) and in vivo trastuzumab–resistant PDX model (PDX118-TR4) generation. (B) Cell proliferation assay of PDX-derived cell lines treated with trastuzumab for 6 days. Cell numbers were estimated with the crystal violet staining assay. (C) RT-qPCR analysis of ERBB2, AXL, and VIM mRNA expression of PDX-derived cell lines. (D) Western blot analysis of HER2 downstream pathways with and without trastuzumab and TP-0903 treatment for 72 hours in acquired trastuzumab-resistant PDX-derived cell lines. (E) Representative images of organoids treated with trastuzumab, TP-0903, and combination. (F) Cell proliferation analysis of PDX-derived cell lines treated with trastuzumab combined with pharmacological AXL inhibition by TP-0903. Cell number (G) and apoptosis assay (H) of PDX118, TR1, and TR2 organoids treated with trastuzumab (100 μg/ml), TP-0903 (1 μM in PDX118 and TR1, 0.15 μM in TR2), or combination. Viable and apoptotic cells were quantified by flow cytometry using propidium iodide and annexin V staining. GAPDH was used as an endogenous control in (C) and (D). Scale bar, 100 μm; magnification, ×4 (E). *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed Student’s t test in (B), (C), (G), and (H).

Fig. 6.. AXL inhibition overcomes trastuzumab resistance in HER2⁺ BC PDX models.

(A) Generation of an in vivo trastuzumab–resistant model (PDX118-TR4). Parental PDX118 was implanted in an immunocompromised mouse, and when tumors reached ~200 mm³, animals were treated intraperitoneally with trastuzumab (left). Next, resistant tumors were reimplanted in immunocompromised mice and treated with trastuzumab (right). Red arrows represent days of trastuzumab treatment. (B) RT-qPCR analysis of ERBB2, AXL, and VIM mRNA expression of in vivo PDX–resistant model PDX118-TR4. GAPDH was used as an endogenous control. (C) Cell number analysis of trastuzumab-resistant patient-derived organoids treated with trastuzumab at 100 μg/ml, TP-0903 at 150 nM, and a combination of these two drugs. Viable cells were quantified by flow cytometry using EpCAM as a marker. (D) Representative images of organoids treated with trastuzumab, TP-0903, and combination of these two drugs. Tumor growth of PDX118-TR4 in vivo model injected in NOD-SCID mice treated with vehicle, trastuzumab alone (10 mg/kg), TP-0903 alone (50 mg/kg), or a combination of these two drugs represented in time-course line (E), endpoint dot plot (G), and percentage change from baseline of each individual (H). (F) Tumor growth of PDX118-TR4 in vivo model in NOD-SCID mice treated with trastuzumab and TP-0903 combination. Black line represents treatment time period. (I) OS Kaplan-Meier curve of PDX mice divided by treatment. (J) Animals’ weight in grams during treatment and follow-up period. Treatment was performed between days 89 and 110 after injection. N = 30 mammary fat pad tumors; data represent means ± SEM in (E) to (G). Scale bar, 100 μm; magnification, ×4 (D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed Student’s t test in (B) and (C). ****P < 0.0001 by two-way ANOVA with Bonferroni correction posttest in (E). **P < 0.01 and ***P < 0.001 by Mann-Whitney test in (G).

Fig. 7.. AXL expression correlates with a strong negative prognostic factor in HER2⁺ BC patients.

(A) RT-qPCR analysis of AXL mRNA expression in HER2⁺ BC patients’ FFPE samples. (B) ROC curve analysis for relapse prediction based on AXL mRNA expression in HER2⁺ BC patients. DFS (C) and OS (D) Kaplan-Meier curves of HER2⁺ BC patients stratified by median AXL expression levels. (E) RT-qPCR analysis of VIM mRNA expression in HER2⁺ BC patients’ FFPE samples. DFS (F) and OS (G) Kaplan-Meier curves of HER2⁺ BC patients stratified by median VIM expression levels. (H) Correlation matrix of AXL, VIM, ERRB2, and GAS6 mRNA expression in HER2⁺ BC patients. N = 50. Red lines represent median and interquartile range. GAPDH was used as an endogenous control in (A) and (E). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test with Welch’s correction in (A), (E), and (H).

Fig. 8.. AXL mRNA biological changes after dual HER2 blockade.

AXL mRNA expression changes between baseline and day 14 of treatment in all 139 tumor samples from PAMELA clinical trial (A) and across histological subtypes (B) and PAM50 subtypes (C). (D) AXL mRNA expression changes between baseline, day 14, and surgery in 97 residual tumors at surgery. (E) Correlation between AXL, VIM, and ERBB2 in 96 tumor samples from the PAMELA clinical trial. Each line represents a tumor sample. Increases are represented in orange, and decreases in blue. P values were determined by two-tailed paired t tests.