Paradoxical role of the proto-oncogene Axl and Mer receptor tyrosine kinases in colon cancer

Abstract

The receptor tyrosine kinases Axl and Mer, belonging to the Tyro3, Axl and Mer (TAM) receptor family, are expressed in a number of tumor cells and have well-characterized oncogenic roles. The therapeutic targeting of these kinases is considered an anticancer strategy, and various inhibitors are currently under development. At the same time, Axl and Mer are expressed in dendritic cells and macrophages and have an essential function in limiting inflammation. Inflammation is an enabling characteristic of multiple cancer hallmarks. These contrasting oncogenic and anti-inflammatory functions of Axl and Mer posit a potential paradox in terms of anticancer therapy. Here we demonstrate that azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced inflammation-associated cancer is exacerbated in mice lacking Axl and Mer. Ablation of Axl and Mer signaling is associated with increased production of proinflammatory cytokines and failure to clear apoptotic neutrophils in the intestinal lamina propria, thereby favoring a tumor-promoting environment. Interestingly, loss of these genes in the hematopoietic compartment is not associated with increased colitis. Axl and Mer are expressed in radioresistant intestinal macrophages, and the loss of these genes is associated with an increased inflammatory signature in this compartment. Our results raise the possibility of potential adverse effects of systemic anticancer therapies with Axl and Mer inhibitors, and underscore the importance of understanding their tissue and cell type-specific functions in cancer.

Fig. 1.

Genetic ablation of Axl and Mer leads to increased susceptibility to AOM/DSS-induced colon cancer. (A) Schematic representation of AOM/DSS-induced colon cancer treatment. In brief, WT and Axl^−/−Mer^−/− mice were injected with the DNA-methylating agent AOM and were subsequently treated with the indicated cycles of DSS. (B) Representative colonoscopy images after 90 d of treatment in WT and Axl^−/−Mer^−/− mice. (C) Tumor number and tumor score in WT and Axl^−/−Mer^−/− mice after 90 d of treatment as determined by colonoscopy. (D) Representative luminal views of WT and Axl^−/−Mer^−/− colons treated as indicated in A. Tumors in the rectum and distal colon of Axl^−/−Mer^−/− can be seen. (E) Representative image of colon sections stained with H&E, showing adenomas in Axl^−/−Mer^−/− mice. (Scale bar: 500 μm.) Data are presented as representative individual samples or as the mean of 14–16 independent samples per group. **P < 0.01.

Fig. 2.

Genetic ablation of Axl and Mer leads to increased susceptibility to DSS-induced colitis. (A and B) Body weight (A) and representative colonoscopy images and colonoscopy scores (B) in WT and Axl^−/−Mer^−/− mice in the steady-state condition (untreated) and after DSS treatment (DSS-treated). (C) Representative images and colon length in WT and Axl^−/−Mer^−/− mice after DSS treatment. (D) Representative images of H&E-stained colon sections showing normal colon morphology in untreated mice and increased colitis in Axl^−/−Mer^−/− mice compared with WT after DSS treatment. Histological scoring of colitis is reported. (Scale bars: 500 μm.) Data are representative images or mean ± SEM of at least six independent samples per group. *P < 0.05; **P < 0.01; ****P < 0.0001.

Fig. 3.

Increased number of apoptotic neutrophils and production of proinflammatory cytokines in the lamina propria of Axl^−/−Mer^−/− mice. (A) Percentage of neutrophils (CD45⁺CD3⁻Ly6G⁺) in the lamina propria of WT and Axl^−/−Mer^−/− before (untreated) and after 7 d of DSS treatment (DSS) as detected by FACS analysis. Representative FACS plots and independent data are shown. (B) Representative immunofluorescence staining for TUNEL in colon sections from WT and Axl^−/−Mer^−/− mice after 7 d of DSS treatment. Nuclei are counterstained with DAPI. (Scale bar: 75 μm.) (C) TUNEL staining in CD11b^lowLy6G⁺ cells in the lamina propria leukocytes of WT and Axl^−/−Mer^−/− mice as detected by FACS analysis. (D) Rate of phagocytosis of apoptotic neutrophils, labeled with CellTracker and incubated with WT or Axl^−/−Mer^−/− BM macrophages for 60 min. FACS plots show WT or Axl^−/−Mer^−/− F4/80⁺ BM macrophages that have engulfed CellTracker-labeled apoptotic neutrophils. The phagocytic index normalized to WT macrophages is shown on the right. (E) Expression of IFN-γ and TNF-α in the lamina propria leukocytes from WT and Axl^−/−Mer^−/− mice after 7 d of DSS treatment as detected by qPCR. Data are presented as representative images or as mean ± SEM of at least five samples per group. n.s., nonsignificant. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Loss of Axl and Mer in the hematopoietic compartment does not confer susceptibility to colitis. (A) Schematic representation of BM chimeras and chronic DSS treatment. (B) FACS analysis of chimerism of blood leukocytes in BM chimeras as indicated in A. (C) Colonoscopy score determined at the end of each DSS cycle in the indicated BM chimeras. (D) TUNEL staining in CD11b^lowF4/80⁻Ly6G⁺ cells in the lamina propria leukocytes of WT > WT and Axl^−/−Mer^−/− >WT BM chimera mice, as detected by FACS, after 7 d of DSS treatment. Representative histograms (Left) and bar graphs (Right) indicating mean fluorescence intensity (MFI) of TUNEL expression are shown. (E) Representative FACS plots (Left) and bar graphs (Right) showing the frequency of IL17-α⁺, IFN-γ⁺, and IL17-α⁺IFN-γ⁺ CD4⁺ T cells in the lamina propria of BM chimeras at the end of the chronic DSS treatment. Data are presented as representative images or as mean ± SEM of at least four independent samples per group. n.s., nonsignificant.

Fig. 5.

Axl and Mer are expressed in radioresistant intestinal macrophages, limiting the inflammatory response to DSS-induced colitis. (A) FACS analysis of chimerism of lamina propria leukocytes in the indicated BM chimeras, showing the persistence of a radioresistant CD45.1⁺ population. (B) Percentage of F4/80⁺CD11b⁺ cells in the radioresistant CD45.1⁺ host population, as shown by FACS analysis. (C) Representative histogram showing Mer expression in host CD45.1⁺ (blue), donor CD45.2⁺ WT (green), and donor CD45.2⁺ Axl^−/−Mer^−/− (red) F4/80⁺CD11b⁺ lamina propria leukocytes and the MFI of Mer in the indicated F4/80⁺CD11b⁺ populations isolated at 2 d after the completion of DSS treatment (day 14). MFI of Mer expression in each population is represented on the right. (D) Expression of Axl and Mer in indicated cell-sorted macrophages from the lamina propria of WT (open bar) and Axl^−/−Mer^−/− (black bar) mice before (day 0), during the course of DSS treatment (days 3 and 7), and 2 d after the completion of DSS treatment (day 14) as detected by qPCR. Lamina propria macrophages isolated from Axl^−/−Mer^−/− mice were used as internal negative controls. (E) Expression of the indicated genes in sorted macrophages from the lamina propria of WT and Axl^−/−Mer^−/− mice during DSS treatment, as detected by qPCR. Shown are mRNA levels for Nos2, IL-17, TGF-β, and IL-10 in the F4/80^highCD11b⁺ population after 7 d of DSS treatment, along with mRNA levels for IL-6, TNF-α, IL-12, and RELM-α on F480⁺CD11b⁺ population at 2 d after the completion of DSS treatment (day 14). Data are presented as representative images or as mean ± SEM of at least four independent samples per group. *P < 0.05; **P < 0.01.