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A cell-intrinsic role for TLR2–MYD88 in intestinal and breast epithelia and oncogenesis

Nature Cell Biology volume 16, pages 1238–1248 (2014)Cite this article

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Abstract

It has been postulated that there is a link between inflammation and cancer. Here we describe a role for cell-intrinsic toll-like receptor-2 (TLR2; which is involved in inflammatory response) signalling in normal intestinal and mammary epithelial cells and oncogenesis. The downstream effectors of TLR2 are expressed by normal intestinal and mammary epithelia, including the stem/progenitor cells. Deletion of MYD88 or TLR2 in the intestinal epithelium markedly reduces DSS-induced colitis regeneration and spontaneous tumour development in mice. Limiting dilution transplantations of breast epithelial cells devoid of TLR2 or MYD88 revealed a significant decrease in mammary repopulating unit frequency compared with the control. Inhibition of TLR2, its co-receptor CD14, or its downstream targets MYD88 and IRAK1 inhibits growth of human breast cancers in vitro and in vivo. These results suggest that inhibitors of the TLR2 pathway merit investigation as possible therapeutic and chemoprevention agents.

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Figure 1: TLR2 and MYD88 are functional in murine intestinal epithelial cells.
Figure 2: TLR2 and MYD88 protect Apcmin/+ mice from adenomas.
Figure 3: TLR2 is functionally expressed in normal murine mammary epithelial cells.
Figure 4: Limiting dilutions of mammary epithelial cells.
Figure 5: TLR2 expression on human ERneg breast cancer influences in vitro colony formation.
Figure 6: TLR2 expression on colon cancer influences in vitro colony formation.
Figure 7: Analysis of TLR2 mutations.

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Acknowledgements

This study was supported by the National Institutes of Health (NCI), the Breast Cancer Research Foundation, the Ludwig Institute, The California Institute for Regenerative Medicine and the Department of Defense (DOD). F.A.S. was supported by NWO-Rubicon grant, a fellowship from the Dutch Cancer Society and by a seed grant of the organization My Blue Dots. We thank T.N. Schumacher and M.A. Child for scientific input, S. Sim for her assistance with single-cell PCR assays, P. Lovelace for her assistance with flow cytometry and K. Montgomery for IHC. Some research was performed on a FACS Aria that was purchased using NIH S10 Shared Instrumentation Grant (1S10RR02933801) funds.

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Author notes

  1. Debashis Sahoo & Tomer Kalisky

    Present address: Present addresses: Department of Pediatrics, University of California at San Diego, La Jolla, California 92093, USA (D.S.); Faculty of Engineering, Bar-Ilan University, Ramat Gan 52900, Israel (T.K.),

  2. Ferenc A. Scheeren and Angera H. Kuo: These authors contributed equally to this work.

Authors and Affiliations

  1. Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 265 Campus Drive Stanford, California 94305, USA,

    Ferenc A. Scheeren, Angera H. Kuo, Linda J. van Weele, Shang Cai, Shaheen S. Sikandar, Maider Zabala, Dalong Qian, Jessica S. Lam, Darius Johnston, Jens P. Volkmer, Debashis Sahoo, Michael E. Rothenberg & Michael F. Clarke

  2. The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands

    Ferenc A. Scheeren & Iris Glykofridis

  3. Department of Pathology, Stanford University, Stanford, California 94305, USA

    Matt van de Rijn

  4. Department of Surgery, Stanford University, Stanford, California 94305, USA

    Frederick M. Dirbas

  5. Department of Medical Oncology, Beckman Research Institute and City of Hope Comprehensive Cancer Center, Duarte, California 91010, USA

    George Somlo

  6. Department of Bioengineering and Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA

    Tomer Kalisky & Stephen R. Quake

  7. Department of medicine, Division of Oncology, Stanford University, Stanford, California 94305, USA

    Michael F. Clarke

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Contributions

F.A.S. and A.H.K. performed, designed and analysed research and wrote the paper; L.J.v.W., S.C., M.Z., S.S.S., I.G., D.J. and M.E.R. performed, designed and analysed research; D.Q. and J.S.L. performed research; J.P.V., T.K., S.R.Q., D.S. and M.v.d.R. designed and analysed research; F.M.D. and G.S. provided critical reagents; M.F.C. designed research and wrote paper.

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Correspondence to Michael F. Clarke.

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Integrated supplementary information

Supplementary Figure 1 TLR2 and MyD88 expression in murine mammary epithelial subpopulations.

(a) Representative FACS dot plot and histogram of CD24, CD49f and CD61. Data are representative of 6 mice. (b) Quantitative rt-PCR on MEC populations as indicated in a. Basal markers: Krt5, Krt14, Krt17; Luminal marker: Krt19; N = 3 mice; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.005; ∗∗∗∗P < 0.001. Error bars represent s.e.m. One-tail unpaired t-test analysis was used. (c) Ma-CFCs were sorted based on expression of CD24highCD49flow/neg luminal phenotype in combination with CD14. 200 cells luminal cells sorted for CD14pos or CD14neg were plated out on matrigel in triplicates. After 7-12 days colonies were counted and passaged as a single cell suspension again (N = 4 mice). 4 independent experiments were performed and the average is shown, P = 0.7, 0.9 (NS: non-significant). Values represent mean ± s.d. Student’s unpaired t-test for independent samples was used.

Supplementary Figure 2 Phenotypic characterization of mammary epithelial cells.

(a) Flow cytometric analysis of mammary epithelial cells from 6 weeks old mice of wild-type, Tlr2−/− and Cd14−/− mice. Cells were gated on life and lineage negative cells. (b) Immunohistochemistry on mammary glands from 6 weeks old mice of wild-type, Tlr2−/− and Cd14−/− mice. Myoepithelial cells (Cytokertain-14 (KRT14)) and luminal cells (E-CADHERIN) are present and properly organized in both wild-type as well as the knock out mammary glands. Scale bar is 50 μm. (c) Carmine Alum staining on whole mount of lactation of wild-type, Tlr2−/− and Cd14−/− mice. Scale bar is 1 mm. (d) Flow cytomety analysis of mammary epithelial cells from 6 to 8 weeks old mice. Cells were gated on live and lineage negative and stained for CD24, CD49f and CD14, TLR4, IL-1R1 or IL-18R1. In red is the isotype control for each specific sub-population, in blue is the CD14, TLR4, IL-1R1 or IL-18R1 staining. (e) Flow cytometry analysis of Myd88−/− mammary epithelial cells, from 6 weeks old mice of Krt14-CrenegMyd88f/f and Krt14-CreposMyd88f/f mice. Cells were gated on life and lineage negative cells. (f) Immunohistochemistry on mammary glands from 6 weeks old mice of Krt14-CrenegMyd88f/f and Krt14-CreposMyd88f/f mice. Myoepithelial cells (Cytokertain-14 (KRT14)) and luminal cells (E-CADHERIN) are present and properly organized in the MyD88 knockout mammary gland. Scale bar is 50 μm. All analyses were done with at least 3 mice.

Supplementary Figure 3 Limiting dilutions of mammary epithelial cells.

To determine the MRU frequency of each donor genotype we injected sorted cells, as indicated from donor mice of 10 to 14 weeks old mice in cleared mammary fat pads of three weeks old recipients. Recipient mice were syngeneic C57BL/6J mice. Numbers of successful out growths and numbers of total injections are shown for each dilution and genotype. MRU frequency and confidence was determined by ELDA graph and analysis. (a) Raw numbers for limiting dilutions of Tlr2−/− and Cd14−/− linneg MECs for Fig. 4a. (b,c) Raw numbers for limiting dilutions of Tlr4−/− linneg MECs, including ELDA analysis. Data for WT (N = 25 samples), Tlr4−/− (N = 30 samples) pooled from 3 independent experiments. P = 0.0085. (d) Raw numbers for limiting dilutions of Il-1r1−/− (N = 74 samples) and Il-18r1−/− linneg (N = 39 samples) MECs. (e,f) Limiting dilutions of Ink4a-Arf+/+Tlr2+/+(WT), Ink4a-Arf+/+Tlr2−/− (Tlr2KO), Ink4a-Arf−/−Tlr2+/+ (Ink4a-Arf KO) and Ink4a-Arf−/− Tlr2−/− (Ink4a-Arf KO Tlr2 KO) linneg MECs, including ELDA graph and analysis. Data for WT (N = 41 samples), Tlr2−/− KO (N = 75 samples), P < 0.0001, Ink4a-Arf KO (N = 66 samples), P = N.S. and Ink4a-Arf KO Tlr2 KO (N = 45 samples), P = N.S. are pooled from 4 independent experiments.

Supplementary Figure 4 Single cell gene expression of MRU.

Single cell expression showing expression of wild-type (black) and Tlr2−/− (red) MRUs for Fig. 3e. Cells were double sorted and subjected to multiplexed single cell rt-PCR. Normalized Ct values as visualized by comparing the distribution of Ct values in histograms. (a) Gene that was significantly up-regulated in Tlr2−/− cells as compared to wild-type cells. (b) Genes that were significantly down-regulated in Tlr2−/− as compared to wild-type cells. Each analysis was done on 2 different mice.

Supplementary Figure 5 Antibody and/or shRNA mediated blockade of TLR2, CD14, MYD88 and IRAK1.

(a) Representative examples colony formation on matrigel of primary ERneg breast cancer cells (N = 4). Scale bar is 20 μm. Cells were treated with control antibody or with neutralizing TLR2 and CD14 antibodies. (b,c) Two breast cell lines were transduced with the lentivirus to knockdown TLR2 and after puromycin selection cells were plated out and stained with crystal violet blue after 9 days in culture. Data are representative of 2 experiments. Scale bar is 1 cm. (d) MDA-MD-231 cells were transduced with control or indicated shRNA and knock-down efficiency was determined by qPCR. All shRNA mediated knock-down constructs resulted in a 4 to 12x reduction of its target mRNA. Data are representative of 2 experiments. (e) Knock-down of MYD88 and IRAK1 in the breast cell line MCF7. After transduction cells were sorted for Cherry and plated out on matrigel in triplicate to determine colony forming capacity. The number of colonies on matrigel is shown and a representative photo of the colonies is shown. N = 6 samples pooled from 2 independent experiments. ∗∗∗P < 0.001. Scale bar is 20 μm. Values represent mean ± s.d. Student’s unpaired t-test for independent samples was used. (f) MYD88 and IRAK1 knock down resulted in decreased expression of the NF-κB target gene IL-1B indicating that the knock down resulted in decreased NF-κB activity. Data are representative of 2 experiments.

Supplementary Figure 6 TLR2 immunohistochemistry on normal and tumour colon tissue.

(a) 10X magnification of immunohistochemistry for TLR2. Boxed is normal colon tissue, the rest are colon tumours. Scale bar is 200 μm. (b) 40X magnification of immunohistochemistry for TLR2 of colon tumours. Scale bar is 50 μm.

Supplementary Figure 7 Genome editing the Tlr2 locus using CAS9.

(a) Schematic of the targeted region of human TLR2 locus and targeting strategy. sgRNA cuts around + 944. The homologues recombination (HR) cassette has a left arm (including the E283* mutation) and a right arm. In between there is a CMV Cherry pA cassette (yellow box) in the anti sense orientation. Primer pairs (Pp1 & Pp2) are shown to detect homologues recombination. (b) Region of the sgRNA complementary to the protospacer. (c) Sequenced region show correct integration of HR construct as determined by sequencing the genomic junctions.

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Scheeren, F., Kuo, A., van Weele, L. et al. A cell-intrinsic role for TLR2–MYD88 in intestinal and breast epithelia and oncogenesis. Nat Cell Biol 16, 1238–1248 (2014). https://doi.org/10.1038/ncb3058

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