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Formyl peptide receptors (FPR1, FPR2 and FPR3) are innate immune sensors of pathogen and commensal bacteria and have a role in colonic mucosa homeostasis. We identified FPR1 as a tumour suppressor in gastric cancer cells due to its ability to sustain an inflammation resolution response with antiangiogenic potential. Here, we investigate whether FPR1 exerts similar functions in colorectal carcinoma (CRC) cells. Since it has been shown that the commensal bacterium Lactobacillus rhamnosus GG (LGG) can promote intestinal epithelial homeostasis through FPR1, we explored the possibility that it could induce proresolving and antiangiogenic effects in CRC cells. We demonstrated that pharmacologic inhibition or genetic deletion of FPR1 in CRC cells caused a reduction of proresolving mediators and a consequent upregulation of angiogenic factors. The activation of FPR1 mediates opposite effects. Proresolving, antiangiogenic and homeostatic functions were also observed upon treatment of CRC cells with supernatant of LGG culture, but not of other lactic acid or nonprobiotic bacteria (i.e. Bifidobacterium bifidum or Escherichia coli). These activities of LGG are dependent on FPR1 expression and on the subsequent MAPK signalling activation. Thus, the innate immune receptor FPR1 could be a regulator of the balance between microbiota, inflammation and cancer in CRC models.

// Maria Rosaria Ambrosio 1, * , Vittoria D’Esposito 1, * , Valerio Costa 2 , Domenico Liguoro 1 , Francesca Collina 3 , Monica Cantile 3 , Nella Prevete 1 , Carmela Passaro 1 , Giusy Mosca 1 , Michelino De Laurentiis 4 , Maurizio Di Bonito 3 , Gerardo Botti 3 , Renato Franco 5 , Francesco Beguinot 1 , Alfredo Ciccodicola 2, 6 and Pietro Formisano 1 1 Department of Translational Medicine, University of Naples “Federico II” & URT “Genomic of Diabetes” of Institute of Experimental Endocrinology and Oncology “G. Salvatore”, National Council of Research (CNR), Naples 80131, Italy 2 Institute of Genetic and Biophysics “A. Buzzati-Traverso”, CNR, Naples 80131, Italy 3 Pathology Unit, National Cancer Institute “G. Pascale Foundation”, Naples 80131, Italy 4 Department of Breast Surgery and Cancer Prevention, National Cancer Institute “G. Pascale Foundation”, Naples 80131, Italy 5 Pathology Unit, Universita degli Studi della Campania “Luigi Vanvitelli”, Naples 80138, Italy 6 Department of Science and Technology, University of Naples “Parthenope”, Naples 80131, Italy * These authors have equally contributed to this work Correspondence to: Pietro Formisano, email: fpietro@unina.it Keywords: breast cancer; tamoxifen; glucose; adipose tissue; connective tissue growth factor Received: April 21, 2017 Accepted: July 25, 2017 Published: November 20, 2017 ABSTRACT Type 2 diabetes and obesity are negative prognostic factors in patients with breast cancer (BC). We found that sensitivity to tamoxifen was reduced by 2-fold by 25 mM glucose (High Glucose; HG) compared to 5.5 mM glucose (Low Glucose; LG) in MCF7 BC cells. Shifting from HG to LG ameliorated MCF7 cell responsiveness to tamoxifen. RNA-Sequencing of MCF7 BC cells revealed that cell cycle-related genes were mainly affected by glucose. Connective Tissue Growth Factor (CTGF) was identified as a glucose-induced modulator of cell sensitivity to tamoxifen. Co-culturing MCF7 cells with human adipocytes exposed to HG, enhanced CTGF mRNA levels and reduced tamoxifen responsiveness of BC cells. Inhibition of adipocyte-released IL8 reverted these effects. Interestingly, CTGF immuno-detection in bioptic specimens from women with estrogen receptor positive (ER + ) BC correlated with hormone therapy resistance, distant metastases, reduced overall and disease-free survival. Thus, glucose affects tamoxifen responsiveness directly modulating CTGF in BC cells, and indirectly promoting IL8 release by adipocytes.

Additional file 3: Supplementary Figure 2. Functional activity of intrinsic PD-1 circuit in TC cells. A. Expression levels of PD-1 in 8505c and TPC1 cells or in 8505c and TPC-1 transiently transfected with pFLAG or pFLAG PD-1, assessed by western blot. A representative experiment is shown. B. Cell cycle distribution of 8505c and TPC-1 cells transiently transfected with pFLAG or pFLAG PD-1, measured by Propidium Iodide (PI) staining by means of Flow Cytometry. The percent of the cells distributed in G0/G1, S, G2/M was indicated in each panel. Representative experiments are shown. C. Percent of apoptotic cells assessed by TUNEL reaction in 8505c and TPC-1 cells transiently transfected with pFLAG or pFLAG PD-1 and treated or not with soluble PD-L1 (sPD-L1 - 1 μg/ml). Data are presented as mean ± SD of 5 independent experiments. D. Cytofluorimetric evaluation of PD-1 expression in 8505c cells treated with siPD-1 (solid lines) or scrambled siCTR (dotted line) (100 nM). A representative experiment is shown. E. Cell cycle distribution of 8505c and TPC-1 cells treated with Nivolumab (Nivo - 10 μg/ml) or control IgG4 (10 μg/ml), measured by Propidium Iodide (PI) staining by means of Flow Cytometry. The percent of the cells distributed in G0/G1, S, G2/M was indicated in each panel. Representative experiments are shown. F. Percent of apoptotic cells assessed by TUNEL reaction in 8505c and TPC-1 cells treated with siPD-1 (100 nM) or Nivolumab (Nivo - 10 μg/ml) or the relative controls. Data are presented as mean ± SD of 5 independent experiments. G. DNA synthesis of 8505c cells transiently transfected with pCMV3, pCMV3 PD-L1 or pCMV3 PD-L2 or treated with anti-PD-L1, anti-PD-L2 blocking antibodies or IgG1 isotype control (10 μg/ml) assessed by BrdU incorporation. Data are presented as mean ± SD of 5 independent experiments. * P

Additional file 2: Supplementary Figure 1. Immune checkpoint expression in thyroid cancer (TC) cells. Protein expression levels assessed by western blot (A) and mRNA expression indicated as ΔCt for PD-1, PD-L1 and PD-L2 (B) in H-6040 normal thyroid epithelial cells, PTC-derived cell lines (BcPAP and TPC-1), and ATC-derived cell lines (8505c, CAL62, SW1736, FRO, BHT101, HTH7, OCUT1). A representative western blot experiment is shown. PCR data are presented as mean ± SD of 5 independent experiments.

Additional file 5: Supplementary Figure 4. Effects of intrinsic PD-1 on SHP2 localization and functions. A. Expression levels of PD-1, phospho-PD-1, SHP2 and phospho-SHP2 in 8505c and TPC-1 cells transiently transfected with pFLAG PD-1 or the empty vector pFLAG, assessed by western blot. A representative experiment is shown. B. Immunofluorescence microscopy of 8505c cells, transiently transfected with pFLAG PD-1 or the empty vector, with antibody specific for SHP2. Arrows indicate the surface signal of SHP2. Bars, 5 μm. A representative experiment is shown. C. Immunofluorescence microscopy of 8505c cells transiently transfected with pCMV6 PD-1-GFP and stained with antibody specific for SHP2, and the merged signal. Arrows indicate the surface signal of SHP2. Bars, 5 μm. A representative experiment is shown. D. Total protein extracts from TPC-1 cells transiently transfected with pFLAG-PD-1 or the empty vector pFLAG were subjected to a pull-down assay using the indicated recombinant proteins or to immunoprecipitation using the indicated antibodies. Proteins were immunoblotted with antibody against SHP2 or GRB2. A representative experiment is shown. E. Total cell protein extracts from 8505c cells transiently transfected with combination of pCEFL H-Ras AU5, pFLAG PD-1 or empty vector (pFLAG + pCEFL) were subjected to immunoprecipitation with anti-phospho tyrosine followed by western blotting with pan (RAS) antibody. A representative experiment is shown, together with the mean densitometric analysis ± SD of 5 independent assays. * P

Additional file 4: Supplementary Figure 3. Signalling pathways downstream PD-1overexpression. A. Expression levels of PD-1 in some clones or mass populations obtained from 8505c cells stably transfection with PD-1, assessed by western blot. A representative experiment is shown. B. Expression levels of phosphorylated forms of BRAF, MEK1/2 and MAPK (p44/p42) in 8505c cells stably transfected with PD-1 or the empty vector, assessed by western blot. A representative experiment is shown. C. Activation of AKT, SRC, S6, S6K, 4EBP1 in 8505c and TPC-1 cells, transiently transfected or not with PD-1 or the relative empty vector, assessed by western blot for their phosphorylated forms. A representative experiment is shown.

Additional file 6: Supplementary Figure 5. Immunohistochemical evaluation of 8505c xenografts. A. Proliferation index (Ki-67) assessed by immunohistochemistry of 8505c pCMV3 and pCMV3 PD-1 cl13 xenografts harvested 28 days post-inoculation. Representative images are shown. B. Proliferation index (Ki-67) assessed by immunohistochemistry of 8505c xenografts harvested 35 days post-inoculation in mice treated with Nivolumab or control IgG4. Representative images are shown.

Additional file 1: Supplementary Table 1. Mouse immune cell density (expressed as percentage of CD45+ leukocytes) in 8505c xenografts.