CROSS-TALK BETWEEN THE GUT MICROBIOME, CEACAM'S, AND TGF-β SIGNALLING IN GASTROINTESTINAL CANCERS

Background: Diets high in animal fat and low in plant fiber increase colorectal cancer (CRC) risk, while diets high in fiber, but low in animal fat, are protective. Bacteria present in the colon produce beneficial and harmful metabolites from our diets. Fiber is fermented by these bacteria, producing short-chain fatty acids (SCFA). One of them, butyric acid, is the major energy source for colonic cells and has strong anti-inflammatory/anti-carcinogenic properties. Excess consumption of animal fats is known to increase levels of carcinogenic secondary bile acids in the colon, namely lithocholic acid (LCA) and deoxycholic acid (DCA). Alaska Native (AN) people, at highest risk globally for CRC, were interested in whether increasing dietary fiber intake could reduce CRC risk, as their diets are high in meat and fat, but deficient in fiber.
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

Background: Obesity, particularly abdominal obesity, is a major risk factor of cancer. However, it remains unknown how visceral fat may influence cancer risk independently of traditional measures of overall and abdominal obesity, due to the lack of large-scale data on visceral fat. Methods: Leveraging the body (abdominal) magnetic resonance imaging (MRI) measurements among 23,148 participants who attended the first imaging test since 2014 in the UK Biobank (UKB) cohort, we prospectively examined visceral adipose tissue volume (VAT) in relation to the risk of overall, obesity-related, and gastrointestinal (GI) cancer and major individual cancers in men and women, using multivariable Cox proportional hazards regression (see the table 1 footnote for the covariate list). We also trained and validated a prediction equation for VAT based on demographic factors and traditional anthropometric measures (see the predictor list and brief summary of process in the table footnote). We compared the association of the measured and predicted VAT with cancer risk among individuals with the MRI data, and then assessed the predicted VAT in relation to cancer risk among 471,089 participants in the overall UKB cohort who attended the initial assessment visit (2006-2010). To examine the added value of VAT beyond traditional measures of overall and abdominal obesity, we conducted a joint association analysis for predicted VAT with body mass index (BMI) and waist circumference (WC) in relation to total cancer risk in the overall UKB cohort. Results: In the MRI subcohort and overall UKB cohort, respectively, we documented 719 and 47,940 cancer cases during a median follow-up of 4 and 12 years. In the MRI subcohort, each unit (90^th to 10^th difference) increase in the measured VAT was associated with 33% and 74% higher risk of total cancer in men (multivariable HR=1.33, 95% CI: 1.05-1.67) and women (1.74, 1.11-2.72), respectively. The final equation for predicted VAT showed a similarly high R² in the training (0.76) and validation (0.74) sets; no difference was found between the measured and predicted VAT in the validation set (p-value>0.05), with Spearman correlation (r=0.87). In the MRI subcohort, the predicted VAT showed similar HRs to those of the measured VAT. After applying the prediction equation to the overall UKB cohort, we found that the predicted VAT was associated with higher risk of total cancer and many individual cancers, particularly GI, kidney, liver, and endometrial cancers. Furthermore, within each of the BMI and WC groups, predicted VAT was associated with a higher risk of total cancer. Conclusions: VAT was associated with an increased risk of total cancer and several individual cancers. VAT also provided additional information regarding total cancer risks within each of the BMI or WC groups.

Background and aims: Colorectal cancer (CRC) cells experience fluctuating metabolic vulnerability to facilitate tumor progression and metastasis. However, the molecular mechanism underlying CRC metabolism reprogramming remains unclear. Kruppel-like factor (KLF) family proteins play crucial roles in carcinogenesis and progression. Here we report a novel function of KLF4 in promoting CRC aggressiveness by regulating glucose metabolism.
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.

Background: The secondary bile acid deoxycholic acid (DCA) is produced by intestinal bacteria with 7α-dehydroxylating (7αDH) activity. Higher concentrations of DCA are positively associated with colorectal cancer (CRC) risk, but underlying molecular mechanisms demonstrating a causal role of DCA are still not fully revealed. In this study, we investigate the tumor-promoting role of DCA in vivo in a gnotobiotic CRC mouse model.
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.

Background: Gut microbiota imbalances associated with inflammation and altered metabolism are prominent risk factors for GI cancers, such as colorectal cancer (CRC) and hepatocellular carcinoma (HCC). CEACAM proteins are known immune and epithelial cell regulators, and CEACAM1, 3, 5, and 6 bind to pathogenic microbes. Mutations of the CEACAM-encoding genes produce significant alterations in the gut microbiome through altered regulation of host-immune response, and changes correlate with scores of TGF-β pathway activity in GI cancers. Nearly 40% of human GI cancers have genetic alterations in the TGF-β pathway members, and cancers occur in mouse models with disruption of TGF-β signaling only in the presence of an altered microbiome. Yet, in-depth analyses of how cancer pathways such as TGF-β signaling are involved remain to be elucidated. We have recently found that in NASH-HCC murine model, hepatocyte-specific βII-spectrin knockout (LSKO) blocked diet-induced NASH and HCC (Sci Transl Med. 2021;13(624): eabk2267). We, therefore, investigated crosstalk between pathogen-binding CEACAM1, the gut microbiome, and the multifunctional intrinsically-disordered protein βII-spectrin (SPTBN1), a Smad3 adaptor, in driving GI cancers.
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.