Cell-type specific MyD88 signaling is required for intestinal tumor initiation and progression to malignancy

Abstract

The signal adapter MyD88, an essential component of Toll-like receptor (TLR) signaling, is important for gut-microbiome interactions. However, its contribution to cancer and its cell-type specific functions are controversially discussed. Therefore, we generated new tissue-specific mouse models and analyzed the clinical importance in human colorectal cancer. A gene-trap was inserted into the murine Myd88 gene (Myd88^LSL), yielding MyD88-deficient background with Cre-mediated re-expression in myeloid (MYEL) or intestinal epithelial cells (IECs). These lines were bred with the Apc^1638N model that develops invasive adenocarcinoma and analyzed at 12 months. Further, two patient collectives of colorectal cancer (n = 61, and n = 633) were analyzed for expression of Myd88 and TLRs. MyD88 expression was significantly increased in carcinomas, and increased intratumoral levels of MyD88 and TLR pathway components were associated with significantly shorter disease-free (P = .011), and overall survival (P < .0001). In accordance, fully MyD88-deficient mice showed highly significantly decreased tumor incidence, tumor numbers, increased survival, and, importantly, fully lacked malignant lesions. Thus, MyD88 is essential for tumorigenesis and especially progression to malignancy. Tissue-specific re-expression of MyD88 highly significantly increased tumor initiation by differing mechanisms. In intestinal epithelia, MyD88 enhanced epithelial turnover, whereas in myeloid cells, it led to increased production of tumor- and stemness-enhancing cytokines, significantly associated with altered expression of adaptive immune genes. However, neither re-expression of MyD88 in IECs or myeloid cells was sufficient for malignant progression to carcinoma. Thus, MyD88 crucially contributes to colorectal cancer initiation and progression with non-redundant and cell-type specific functions, constituting an attractive therapeutic target.

Keywords: APC; Toll-like receptors; Tumor immunology; colorectal cancer; mouse model.

Figure 1.

Myd88/TLR signaling is frequently overexpressed in colorectal cancer and associated with prognosis. A, TLR signaling components are upregulated in human (left) and murine (right) intestinal tumors compared to normal mucosa, as verified by qRT-PCR for Myd88, TLR2, TLR4 and TLR9. Human colorectal cancer (n = 51 patients) shows significant upregulation of Myd88 and TLR2 transcripts. Right panel: colon cancer model Apc^1638N (n = 15 mice per group) shows a highly significant intratumoral upregulation of TLR2 and TLR4. *P < .05; ***P < .01; ns: not significant. B, Alterations in the TLR pathway are highly significantly associated with poor overall survival (log-rank test: P = .0009), as well as with poor disease-free survival (log-rank test: P = .0013). (C-H) Genetic “switch on” mouse models demonstrate that intestinal carcinogenesis depends on MyD88 expression in both epithelial and myeloid cells. C, Tissue-specific re-expression of Myd88 in intestinal epithelial cells (IEC) was achieved in MyD88^IEC mice, or in bone marrow derived macrophages in the MyD88^MYEL strain. Expression was analyzed on mRNA level by qRT-PCR (n = 4 mice/group; top panel). No expression was detected in control tissue (brain). Bottom panel: representative example for successful and tissue-specific “switch on” of MyD88 expression on protein level (immunoblot). Loading control: total ERK1/2. D, Macroscopic analysis of representative tissue samples from mice at 12 months of age: wildtype control is tumor-free, Apc^1638N/+-model shows several tumors in proximal duodenum (arrows), Apc^1638N/+ MyD88^LSL mice have strongly reduced tumor formation (arrow). E, Median tumor numbers per animal. Compared to parental line (Apc^1638N/+), tumors per animal are significantly reduced in MyD88-deficient mice (Apc^1638N/+ MyD88^LSL; P = .00018), as well as in mice with re-expression in IECs (Apc^1638N/+ MyD88^IEC; P = .0123), or in myeloid cells (Apc^1638N/+ MyD88^LSL; P = .0245). Re-expression of MyD88 in IECs, as well as in myeloid cells is sufficient for a significant, but partial restoration of the tumor phenotype, (Apc^1638N/+ MyD88^IEC: P = .0256; Apc^1638N/+ MyD88^MYEL: P = .0037). F, Kaplan-Meier survival analysis show significantly enhanced tumor-specific survival for mice with global MyD88-deficiency, or re-expression of MyD88 in intestinal epithelia, as compared to the parental Apc^1638N/+ strain. G, No differences in tumor size were observed. Macroscopically visible lesions were measured along the largest diameter.

Figure 2.

MyD88 is required for progression to malignancy and turnover of the intestinal epithelium. A, Typical histology of lesions from the different mouse strains, HE staining of tissue sections. Carcinoma in situ (Apc^1638N/+), low to intermediate benign dysplasia (Apc^1638N/+ MyD88^LSL, Apc^1638N/+ MyD88^IEC, and Apc^1638N/+ MyD88^MYEL). Size bars: 100 µm. B, Tumor grading as performed by an experienced pathologist. Lesions were staged as adenomas, low to intermediate intraepithelial neoplasia, as well as invasive carcinoma. C, Extent of the transit amplifying compartment (TA) in jejunum, marked by Ki67-staining, depends on epithelial MyD88-expression. Nuclear counterstaining by DAPI, size bars: 40 µm. Quantification of TA according to published protocols showed a highly significant decrease in MyD88-deficient mice, rescued by re-expression in IECs (n≥5 per group). D, Immunoblot analysis of small intestinal tissue lysates from all four animal models revealed a-dependency of phospho-ERK1/2 levels from MyD88 expression in IECs (tissue from n = 6 mice analysed per group, shown here are two representative mice per genotype). The density of immunoblot signals was measured and is shown in relation to total ERK1/2 protein level (n = 6 mice). The relative amount of phospho-ERK1/2 was significantly reduced in Apc^1638N/+ MyD88^LSL (P = .0058), this decrease in pERK was rescued upon re-expression of MyD88 in IECs (not significantly differing from MyD88-proficient mice), but not by re-expression in myeloid cells in the line Apc^1638N/+ MyD88^MYEL (P = .0487), compared to the parental Apc^1638N/+ strain. E, MyD88 deficiency does not alter aberrant activation of the canonical Wnt-pathway in lesions (L) compared to normal tissue (N), verified by detection of β-catenin in tumor lysates by immunoblot analysis, and by expression of the surrogate markers osteopontin (Spp1), CyclinD1 (Ccnd1) and dual specificity phosphatase 4 (Dusp4).

Figure 3.

MyD88 signaling in macrophages shapes expression of cytokines and intratumoral T-cell marker transcripts. A, Expression of pro-inflammatory cytokines IL1β and IL6 is increased in tumors (L) compared to normal tissue in parental mice, crucially depending on Myd88 expression in myeloid cells, and only to a minor extent in IECs (n≥6 per group, error bars: s.d.), whereas TNFα is increased in tumors independent of MyD88, as verified by qRT-PCR. B, The tumor-promoting cytokine IL6 is derived from myeloid cells after stimulation with microbial-derived TLR ligands, but not from primary intestinal epithelial cells. Bone marrow derived macrophages were stimulated in vitro with Pam3Cys (TLR2 ligand), LPS (TLR4 ligand), or CpG DNA (TLR9 ligand), and IL6 secretion was tested by ELISA (n = 4), demonstrating exclusive dependency on MyD88 expression in the myeloid compartment. In contrast, isolated primary intestinal epithelial cells showed basically no detectable IL6 secretion. C, Intratumoral immune-regulatory transcripts of T-cell populations and T-cell produced cytokines were quantified by qRT-PCR. Of note, general T-cell transcripts (T-cell receptor subunit CD3e) were significantly reduced in parental tumors compared to normal tissue, similar to TH1-type cell transcripts (Tbx21). This intratumoral decrease in expression depends on MyD88-signalling in macrophages. Gata3 (Gata3, T_H2 signature marker) was increased in tumors, essentially independent of MyD88. In contrast, Foxp3 (FoxP3, T_reg signature marker) was significantly reduced in tumors upon global MyD88-deficiency. None of the other markers, including TH17-population markers, was found to depend on MyD88 expression (Gzmb (Granzyme B, effector CTLs and NK-cells), Il22 (Interleukin 22), Rorgt (RorgT, T_H17 signature marker), Il23 (IL-23, ligand for IL23r), Il23r (IL23-receptor, T_H17 signature marker).

Figure 4.

MyD88 signaling critically contributes to epithelial-mesenchymal transition (EMT) and stemness. A, MyD88 signaling is required for increased intratumoral expression of the EMT-inducing transcription factor Slug (Snai2), and the stem cell markers Lgr5 and Sox9. Re-expression of MyD88 in myeloid cells rescues the increase of Slug, whereas Lgr5 and SoX9 are partially rescued by expression in epithelial cells as well, as judged by qRT-PCR (n = 10 per group). B, Immunofluorescence staining on tumor cryosections (n = 5 per group). Slug is markedly expressed in the nuclei of tumor cells at the invasive front in the parental Apc-model, but is completely absent in MyD88-deficient mice. Partial and full rescue of Slug-expression can be seen upon re-expression in IECs, and in myeloid cells, respectively. Anti-Slug staining in red (fluorophore Cy3), nuclear counterstaining with DAPI in blue (size bars: 40 µm). C, Quantification of long-term label retaining cells in tumor tissue sections. Mice (n = 4 from each group) were subjected to BrdU injections for 10 days, sacrificed after 55 days of chase period. The number of BrdU-positive cells per low-power field (ROI) was significantly reduced in MyD88 deficient mice compared to parental Apc mice, and was essentially not rescued by tissue-specific re-expression. Right side: representative tumor tissue cryosections, stained with anti-BrdU antibody (red, positive cells marked with arrows), nuclear counterstaining with DAPI in blue (size bars: 50 µm). *P < .05; ns: not significant.