Global proteomic identifies multiple cancer-related signaling pathways altered by a gut pathobiont associated with colorectal cancer

Abstract

In this work, we investigated the oncogenic role of Streptococcus gallolyticus subsp. gallolyticus (SGG), a gut bacterium associated with colorectal cancer (CRC). We showed that SGG UCN34 accelerates colon tumor development in a chemically induced CRC murine model. Full proteome and phosphoproteome analysis of murine colons chronically colonized by SGG UCN34 revealed that 164 proteins and 725 phosphorylation sites were differentially regulated. Ingenuity Pathway Analysis (IPA) indicates a pro-tumoral shift specifically induced by SGG UCN34, as ~ 90% of proteins and phosphoproteins identified were associated with digestive cancer. Comprehensive analysis of the altered phosphoproteins using ROMA software revealed up-regulation of several cancer hallmark pathways such as MAPK, mTOR and integrin/ILK/actin, affecting epithelial and stromal colonic cells. Importantly, an independent analysis of protein arrays of human colon tumors colonized with SGG showed up-regulation of PI3K/Akt/mTOR and MAPK pathways, providing clinical relevance to our findings. To test SGG's capacity to induce pre-cancerous transformation of the murine colonic epithelium, we grew ex vivo organoids which revealed unusual structures with compact morphology. Taken together, our results demonstrate the oncogenic role of SGG UCN34 in a murine model of CRC associated with activation of multiple cancer-related signaling pathways.

Figure 1

SGG UCN34 induces acceleration of tumorigenesis in AOM-induced CRC model. (a) Experimental design used for the development of colonic tumors in an AOM-induced CRC model. (b) CFU (colony forming units) per g of stools. Fecal materials were collected at different time points (1, 5, 8 and 11 weeks), homogenized, and serial dilutions plated onto Enterococcus Selective Agar plates to enumerate SGG UCN34 and SGM bacteria; ns, not significant; Mann–Whitney test. (c) Sum of tumors per mouse. Macroscopic tumors were evaluated by an experimented observer. ns not significant; *p < 0.05; **p < 0.01; Mann–Whitney test. (d) Representative pictures of mouse colon after dissection for SGM and SGG UCN34 groups (T tumor, LA lymphoid aggregate). (e) Representative histological (H&E) sections of colon tumors found in AOM-induced CRC mouse model for each experimental group: NT (low-grade dysplasia), SGM (low-grade dysplasia) and SGG UCN34 (low-grade adenoma).

Figure 2

Proteome and phosphoproteome analyses on colonic tissue colonized by SGG UCN34 or SGM over 12 weeks reveals a pro-tumoral shift specific to SGG UCN34. (a) Sample preparation for proteome and phosphoproteome LC–MS analysis. All samples were collected at the end of the AOM-induced CRC model. Tissue samples were from macroscopically tumor-free colon sections for SGM and SGG UCN34 groups and from colon tumors found in this experiment only for the SGG UCN34 group. Proteins were extracted by urea lysis and trypsin digestion. For phosphoproteome MS analysis a phosphopeptide enrichment step was added. All samples were processed by label free LC–MS/MS analysis. (b) Principal component analysis (PCA) of proteome samples in 3D representation. (c) PCA of phosphoproteome samples in 3D representation. Proteome and phosphoproteome analyses were done using myProMS web server. (d) Top disease and bio functions (IPA) of proteome and phosphoproteome changes between SGG UCN34 and SGM. The diseases and functions analysis identified biological functions and/or diseases that were most significant from the data set. Data sets used in this analysis were proteins or phosphoproteins differentially expressed between SGG UCN34 and SGM detected in macroscopically tumor-free colonic tissue. Molecules indicate the number of proteins or phosphoproteins associated with indicated diseases and disorders. A right-tailed Fisher’s Exact Test was used to calculate a p value determining the probability that each biological function and/or disease assigned to that data set is due to chance alone.

Figure 3

ROMA analysis reveals multiple cancer-related pathways altered by SGG UCN34 in comparison to SGM. (a) Heat map view of up and downregulated pathways between three groups: SGM tumor-free, SGG UCN34 tumor-free and SGG UCN34 tumors. (b) Schematic diagram of SGG UCN34-induced activation of MAPK cascades, mTOR signaling and integrin/ILK/actin cytoskeleton pathways. ROMA pathway analysis together with IPA indicates activators of MAPK cascades by UCN34. Apart from the MAPK cascades we can point out activation of the mTOR signaling pathway as well as integrin/ILK/actin cytoskeleton signaling. ROMA: representation and quantification of module activities. Created with Biorender.com.

Figure 4

SGG induces up-regulation of PI3K/Akt/mTOR, MAPK and AMPK pathways in human colon tumors. Proteins were extracted from human colon biopsies enriched for SGG (n = 32) vs negative ones (n = 29) and subjected to RPPA. The table displays up-regulated by SGG pathways for which at least 3 RPPA targeted antibody were attributed. RPPA: reverse phase protein array. Created with BioRender.com.

Figure 5

SGG UCN34 induces pre-cancerous transformation of murine colon tissue. (a) Immunofluorescence analysis of Pdpn (green) and CD34 (red) in sections of colonic tumor-free regions of mice colonized with SGM (top) or SGG UCN34 (bottom) (DAPI/nuclei in blue and E-cadherin in grey). Scale bar 50 µm. Images were acquired with SP8 microscope (Leica) and a ×40 objective. Graph presents the percentage of colocalization of Pdpn and CD34 signals per field of view (FOV). The quantification was done by Imaris (version8) software on SGM (n = 8) and SGG UCN34 (n = 7) colonized mice, using at least 6 images per mouse and showing 1.1% vs 3.1% of Pdpn + CD34 + signal respectively (*t-test p = 0.01). (b) Experimental design of ex vivo organoid formation originating from macroscopically tumor-free sections of colon tissue colonized by SGG UCN34 or SGM over 12 weeks in the AOM-induced CRC model. (c) The number of organoids with normal or cystic morphology defined as colonospheres or colonoids with a central empty lumen and polarized epithelium (actin staining). (d) The number of organoids with compact morphology defined as colonospheres or colonoids with a lumen full of cells and depolarized, restructured actin filaments. (e). Microscopic pictures showing DAPI/nuclei labeling (blue), Phalloidin/actin (green) and Ki67/proliferation marker (red). Images were acquired with Opterra Multipoint Scanning Confocal microscope (Bruker) at ×63 objective and analyzed by Fiji software. Scale bar 50 µm.

Figure 6

Working model of the oncogenic potential of SGG UCN34. Chronic colonization of host colon with SGG UCN34 induces the activation of MAPK cascades, PI3K/AKT/mTOR signaling and integrin/ILK/actin cytoskeleton pathways. SGG UCN34 alters multiple signaling pathways found downstream of EGF and FGF receptors present on colonic epithelial cells, but also downstream of PDGFA receptor present in stromal cells. SGG UCN34 induces the expansion of Pdpn⁺CD34⁺ cell in the stromal compartment, which regulates epithelial cells differentiation/proliferation. All these events contribute to pre-cancerous transformation of colon epithelium and acceleration of tumor development. Created with BioRender.com.