Interleukin-17 receptor a signaling in transformed enterocytes promotes early colorectal tumorigenesis

Abstract

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine linked to rapid malignant progression of colorectal cancer (CRC) and therapy resistance. IL-17A exerts its pro-tumorigenic activity through its type A receptor (IL-17RA). However, IL-17RA is expressed in many cell types, including hematopoietic, fibroblastoid, and epithelial cells, in the tumor microenvironment, and how IL-17RA engagement promotes colonic tumorigenesis is unknown. Here we show that IL-17RA signals directly within transformed colonic epithelial cells (enterocytes) to promote early tumor development. IL-17RA engagement activates ERK, p38 MAPK, and NF-κB signaling and promotes the proliferation of tumorigenic enterocytes that just lost expression of the APC tumor suppressor. Although IL-17RA signaling also controls the production of IL-6, this mechanism makes only a partial contribution to colonic tumorigenesis. Combined treatment with chemotherapy, which induces IL-17A expression, and an IL-17A neutralizing antibody enhanced the therapeutic responsiveness of established colon tumors. These findings establish IL-17A and IL-17RA as therapeutic targets in colorectal cancer.

Figure 1. IL-17RA exerts its protumorigenic activity within radio-resistant cells

(A–D) 5-months old CPC-APC mice heterozygous (+/−) or null (−/−) for the Il17ra gene were sacrificed for cryosectioning and immunostaining (A, B), ELISA (C) or immunoblot analysis (E) of colonic tumors. Data in B were determined by counting tumor epithelial cells positive for the indicated markers in 5 high magnification fields (HMF), AU: artificial unit. (D) CAF isolated from colonic tumors were stimulated with 50 ng/ml recombinant IL-17A for 4 hrs and analyzed by Q-RT-PCR analysis for IL-6 mRNA expression (n=5). (F) CPC-APC mice heterozygous (HE) or null (KO) for Il17ra were lethally irradiated at 6 weeks of age and transplanted with bone marrow of the indicated Il17ra genotypes. At 5 months of age the mice were sacrificed and tumor number and size (diameter) were measured. Tumor load is the sum of all tumor diameters. N=4. Data represent averages ± S.E.M. * p < 0.05. Scale bar = 100 μm. See also Figures S1–S3.

Figure 2. IL-17RA signals within APC-deficient enterocytes to promote tumor development and activate ERK, p38 MAPK and NF-κB signaling

(A) Agarose gel image of PCR-mediated genotyping of the floxed and wildtype (WT) Il17ra alleles (upper panel), and the Cre-mediated deletion product (KO, lower panel). The Il17ra^F allele came from the newly generated Il17ra^F/+ strain, whereas the null allele (−) was from a whole body Il17ra ablation (Ye et al., 2001). (B, C) Colonic IECs isolated by EpCAM-biotin staining and magnetic bead enrichment were stimulated with 50 ng/ml IL-17A for indicated periods before immunoblot (B) and Q-RT-PCR analysis (C, n=4). (D) Tumor number, size and load in CPC-APC mice (Cdx2^Cre Apc^F/+) harboring a specific deletion of IL-17RA in APC-deleted cells (F/−). Il17ra^F/+ CPC-APC mice were used as controls. N=6. (E) Representative images of CPC-APC tumor-bearing colons that either express (F/+) or do not express (F/−) IL-17RA in APC-deleted enterocytes. (F, G) APC-deleted intestinal organoids were stimulated with 50 ng/ml IL-17A for the indicated times. Whole-cell-lysates (F), cytosol and nuclear extract (G) were collected, gel separated and immunoblotted for the indicated proteins. Data are averages ± S.E.M. * p < 0.05. See also Figure S4.

Figure 3. IL-6 contributes to the development of colonic tumors but not to IL-17A expression

CPC-APC mice heterozygous (+/−) or null (−/−) for the Il6 gene were sacrificed at 5 months of age and analyzed for tumor parameters (A, n=5) or IL-17A mRNA by Q-RT-PCR of mesenteric lymph node (MLN), normal colon (N) and tumor (T) tissues (B, n=9). Data represent averages ± S.E.M. * p < 0.05. See also Figure S5.

Figure 4. IL-17RA signaling is required for the growth of aberrant crypt foci

Cdx2^Cre-ERT2Apc^F/F mice were injected with tamoxifen for intestinal tumor induction. (A) Q-RT-PCR analysis of IL-17A mRNA in colon tissues following tamoxifen injection. N=8. (B) Immunostaining of MUC2 in colon cryosections 1 week after tamoxifen injection. Tamoxifen-injected Cdx2^Cre-ERT2 negative mice were used as controls. Arrows indicate areas of MUC2 loss. Representative images of 3 slides of each specimen are shown. (C) Cdx2^Cre-ERT2 Apc^F/F mice heterozygous (+/−) or null (−/−) for Il17ra were sacrificed for colon tumor count 4 weeks after tamoxifen injection. n=5. (D) Cdx2^Cre-ERT2 Apc^F/F mice were given tamoxifen via i.p. injection. Starting one day after the last tamoxifen dose, mice received i.p. injections of 500 ug of isotype control or anti-IL-17A antibody on a weekly basis. Colon tumors were counted 4 weeks after last tamoxifen injection. N=7. (E, F) Immunostaining of colon cryosections from Cdx2^Cre-ERT2 Apc^F/F mice heterozygous (+/−) or null (−/−) for Il17ra 2 weeks after tamoxifen injection. (F) Quantification of Ki-67 (n=4) and phospho-NF-κB p65 (P-p65; n=7) positive transformed cells in HMF of tamoxifen-induced ACF lesions. Data shown in arbitrary units (AU) represent averages ± S.E.M. * p < 0.05. Scale bar = 100 μm. See also Figure S6.

Figure 5. IL-17A neutralizing antibody reduces colonic tumor load

(A–C) 5-month-old CPC-APC mice were injected with isotype control or anti-IL-17A antibodies for 2 weeks at 500 ug/injection/week. (A) Immunostaining of cryosections of colonic tumors. (B) Quantitation of P-p65 (n=10) and Ki-67 (n=6) positive cells in HMF from (A). (C, D) 2-month-old CPC-APC mice were injected weekly with isotype control or anti-IL-17A antibodies and sacrificed at 5 months of age for bright field imaging (C) and analysis of tumor parameters (D, n=6). Data are averages ± S.E.M. * p < 0.05. Scale bar = 100 μm.

Figure 6. Combined treatment with anti-IL-17A and 5-FU results in a stronger therapeutic effect

(A) CPC-APC mice were treated with 5-FU (50 mg/kg) for two consecutive days each week for 6 weeks. PBS was used as a control. Colonic tumors were analyzed by Q-RT-PCR for IL-17A and IL-17F mRNAs. (B, C) 3.5-month-old CPC-APC mice were treated with isotype control (Con), anti-IL-17A (Ab), 5-FU + isotype control (5-FU) or 5-FU + anti-IL-17A (Ab + 5-FU) for 6 weeks and sacrificed for analysis of tumor parameters (B, n=8) and Q-RT-PCR analysis of IL-6 mRNA (C, n=21). (D, E) 5-month-old CPC-APC mice were treated with isotype control (Con), anti-IL-17A (Ab), 5-FU + isotype control (5-FU) or 5-FU + anti-IL-17A (Ab + 5-FU) for 2 weeks and sacrificed. Colonic tumors were embedded in paraffin blocks and analyzed for Ki-67 (D) and cleaved caspase 3 (E). 8 HMF images were used for quantification in D and E. Data are averages ± S.E.M. * p < 0.05. * p < 0.05. Scale bar = 100 μm.