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CROSS-TALK BETWEEN THE GUT MICROBIOME, CEACAM'S, AND TGF-β SIGNALLING IN GASTROINTESTINAL CANCERS
Purpose: Evaluate whether a fiber supplement added to the usual diet of AN volunteers will increase the microbial production of butyrate and suppress fecal bile acids, thus reducing CRC risk.
Methods: Forty-eight healthy AN adult volunteers were enrolled a 4-week randomized, double-blind, placebo-controlled trial in which they received either a resistant starch supplement, RS, a non-chemically modified food starch extracted from high amylose corn, or a digestible starch supplement, DS, an isocaloric amylopectin supplement, occurring naturally as a branched glucose polymer. Quantitation of fecal SCFA and bile acids was performed by on stool samples collected pre- and post-supplementation by gas chromatography-flame-ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) (Figure 1). The primary endpoint was to evaluate whether RS could suppress epithelial proliferation measured by immunohistological staining of proliferative cells with Ki67 in well orientated crypts. The magnitude of the Ki67 suppression was also used to define clinically significant responses of <20%, termed “responders” (Figure 2). Statistical analyses were performed with paired t-tests on all SCFA and bile acids.
Results: Butyrate levels showed increases in both groups, significant in the RS group. Conversely, there were reductions in LCA and DCA in both groups, significant for DCA in the RS group.
Conclusions: RS significantly increased fecal anticarcinogenic butyrate and suppressed inflammatory and potentially carcinogenic secondary bile acids which might explain the suppression of cancer biomarkers in the colonic mucosa. Interestingly, DS had similar but less pronounced effects. It is noteworthy that previous dietary analysis indicated the AN diet was also low in carbohydrate (Ocvirk et al. AJCN 2016) and so it is possible that their diet could be improved by the increased consumption of all forms of complex carbohydrate.
Acknowledgements: NIH grant R01 CA204403



Methods: KLF4 expression was examined by Real time-PCR, Western blot and Immunohistochemical staining in CRC tissue and cells. The clinical significance of KLF4 in two independent cohorts of CRC patients was assessed by Kaplan-Meier analysis and the Multivariate Cox proportional hazards model. The biological function of KLF4 in tumor growth and metastasis was detected both in vivo and in vitro. Glucose consumption, lactate production and extracellular acidification rate (ECAR) were analyzed by the specific fluorescent probe. Serial deletion, site-directed mutagenesis luciferase report assays and chromatin immunoprecipitation (ChIP) were used to determine transcriptional regulation of TKT, PGM1 and ACO2 promoters by KLF4.
Results: The key enzymes of glucose metabolism were differentially expressed in CRC and adjacent non-tumorous tissues, in which the ectopic expression of KLF4 is closely related to multiple glucose metabolic pathways. Downregulation of KLF4 was significantly correlated with more aggressive CRC and indicated a poorer prognosis in CRC patients. KLF4 downregulation was an independent and significant risk factor for recurrence and poor survival. Knockdown of KLF4 promoted CRC proliferation and metastasis both in vitro and in vivo, whereas upregulation of KLF4 hampered CRC aggressiveness. Mechanically, dysregulation of KLF4 facilitated CRC glucose metabolism by transcriptionally inhibiting TKT, whereas transactivating PGM1 and ACO2. Deregulation of TKT, PGM1 and ACO2 blocked KLF4 downregulation-mediated CRC progression, while ectopic expression of TKT, PGM1 and ACO2 rescued KLF4-suppressed proliferation and metastasis. Furthermore, the cAMP-response element binding protein (CREB) that is inhibited by lactate receptor GPR81, binds directly to the KLF4 promoter and activates its expression. Inhibitor of GPR81 attenuated KLF4 deregulation-mediated tumor growth and metastasis.
Conclusions: Lactate-induced downregulation of KLF4 promotes CRC proliferation and metastasis by regulating the expression of three key enzymes for glucose metabolism TKT, PGM1 and ACO2. The lactate receptor GPR81 inhibitor deregulates KLF4 deficiency-promoted CRC proliferation and metastasis, implicating KLF4 as a potential tumor-suppressive prognostic biomarker and a novel therapeutic target for CRC.

Figure 1. KLF4 downregulation correlates with glucose metabolism and promotes CRC progression. (A-C)TCGA dataset shows that the key enzymes of glucose metabolism were differentially expressed in CRC and adjacent non-tumorous tissues. (D)The expression profile of KLF gene family in 473 CRC tissues and 41 normal colorectal epithelial tissues. (E)Gene set enrichment analysis shows that the dysregulation of KLF4 is closely related to multiple glucose metabolic pathways. (F-G)The downregulation of KLF4 indicates a poor prognosis for CRC in TCGA datasets. (I-J)Immunohistochemistry staining analysis of KLF4 expression in adjacent non-tumorous tissues and CRC tissues. (K)Correlation between KLF4 expression and prognosis in CRC patients. (L-O)Cell counting kit-8, clone formation, transwell analysis of CRC cell proliferation, migration and invasion. (M) In vivo tumorigenesis assay. Representative bioluminescent images, tumor growth curves, and the overall survival in different groups are shown.

Figure 2. KLF4, transcriptionally inhibiting TKT whereas transactivating PGM1 and ACO2 promotes glucose metabolism in CRC cells. (A)Transcriptome screening in KLF4 knockdown CRC cells vs control. (B)The sequence of KLF4 DNA-binding sites predicted by JASPAR, ENCODE, MotifMap, TRANSFAC, TRUST and CHEA. (C-D)Venn diagram and volcano plot for KLF4 target genes. (E-G)Glucose consumption, lactate production, and ECAR analysis for glucose metabolism. (H-I)KLF4 represses TKT while activating PGM1 and ACO2 promoter activities. (J-O)Deletion and selective mutation analysis and ChIP assays identify the KLF4-responsive regions and their direct binding sites to the TKT, PGM1 and ACO2 promoters. (P) KLF4 expression was inhibited by lactate stimulation. (Q)GPR81 and PKA inhibitors inhibited lactate-induced downregulation of KLF4 expression. (R-S)Methylation analysis indicates hypermethylation of the KLF4 promoter. Protein interaction analysis implies KLF4 may interact with HDAC2, TP53 and LIN28A.
Methods: Germ-free wildtype mice were colonized with a simplified bacterial consortium, the bile acid converting microbiota (BACOMI). BACOMI is able to produce DCA due to the 7αDH activity of Clostridium scindens. Control mice were colonized with BACOMI without C. scindens, thus lacking 7αDH activity and DCA. Some mice were treated with AOM/DSS to induce colonic tumors. Cecal bile acid composition was analyzed using LC-MS/MS, bacterial abundances were measured in colonic content via qPCR and colonic tissue was analyzed using immunofluorescence-staining and qPCR. Primary T -cells were isolated from mesenteric and iliac lymph nodes and further analyzed by flow cytometry.
Results: Successful colonization of BACOMI revealed distinct bile acid profiles of BACOMI vs. control mice as well as higher abundances of C. scindens in mice treated with AOM/DSS compared to mice without AOM/DSS treatment. In the AOM/DSS experiment, presence of DCA led to significantly higher colonic tumor numbers (+7αDH = 20,5 tumors ±7,5 SD vs. -7αDH = 7,9 tumors ±2,0 SD), and shorter colon lengths compared to mice devoid of DCA. The difference was most pronounced for small- and medium-sized tumors. In mice colonized with BACOMI without AOM/DSS treatment, the presence of DCA lead to significantly higher numbers of Ki67+ cells in colonic crypts and more pronounced hypoxia in the colonic epithelium. Colonic expression of genes associated with CRC (e.g., Axin2, Ccnd1, Myc) and bile acid metabolism (e.g., Nr1h4, Nr0b2, Slc51b) were significantly upregulated in mice having DCA compared to control mice lacking DCA. Different subtypes of Foxp3+ regulatory T -cells were significantly more abundant in mesenteric and iliac lymph nodes of mice where DCA was present compared to mice lacking DCA.
Conclusions: Microbially-derived DCA promotes colonic tumorigenesis in vivo in an experimental gnotobiotic model for CRC. Even in the absence of chemically-induced tumorigenesis, DCA promotes markers associated with CRC, suggesting a causal role of DCA in colonic tumorigenesis.
Methods: DNA from fecal samples collected from wild-type mice with disruption of TGF-β signaling (Smad4+/-Sptbn1+/-) were analyzed through shotgun metagenomics sequencing to identify differences in gut microbiota composition. We examined CEACAM expression, changes in the T cell populations, liver inflammation, and cancer in tissues from wild-type mice and liver-specific βII-spectrin knockout (LSKO) mice fed either a normal chow diet or DEN and Western diet (WD) induced NASH-HCC model.
Results: Doubly heterozygous Smad4+/-Sptbn1+/- mice (C57BL/6J) spontaneously develop GI cancers. These mice exhibit increased bacterial species associated with CRC in humans (C. septicum) and decreased commensal gut microbes associated with a healthy microbiome (B. vulgatus, P. distasonis). Single-cell RNA sequencing analyses revealed that Ceacam1 expression decreases in early CRC and in hepatocytes of WD fed mice (NASH). This result was corroborated using immunohistochemical analyses showing a decreased CEACAM1 expression in NASH livers of WD fed wild-type mice. Strikingly, CEACAM1 levels are increased and restored to normal in tissues from liver-specific βII-spectrin knockout (LSKO) mice. In addition, diet-induced changes in the T cell populations, liver inflammation, and cancer are blocked in LSKO mice.
Conclusion: Our results indicate that two critical pathways, TGF-β signaling, and immune mediator CEACAM1 can suppress inflammation and early GI cancers that arise with microbiome alterations.
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