Aaron C. Shang

Slc10a2-null mice uncover colon cancer-promoting actions of endogenous fecal bile acids.

Although epidemiological evidence in humans and bile acid feeding studies in rodents implicate bile acids as tumor promoters, the role of endogenous bile acids in colon carcinogenesis remains unclear. In this study, we exploited mice deficient in the ileal apical sodium-dependent bile acid transporter (ASBT, encoded by SLC10A2) in whom fecal bile acid excretion is augmented more than 10-fold. Wild-type and Asbt-deficient (Slc10a2 (-/-) ) male mice were treated with azoxymethane (AOM) alone to examine the development of aberrant crypt foci, the earliest histological marker of colon neoplasia and a combination of AOM and dextran sulfate sodium to induce colon tumor formation. Asbt-deficient mice exhibited a 54% increase in aberrant crypt foci, and 70 and 59% increases in colon tumor number and size, respectively. Compared to littermate controls, Asbt-deficient mice had a striking, 2-fold increase in the number of colon adenocarcinomas. Consistent with previous studies demonstrating a role for muscarinic and epidermal growth factor receptor signaling in bile acid-induced colon neoplasia, increasing bile acid malabsorption was associated with M3 muscarinic and epidermal growth factor receptor expression, and activation of extracellular signal-related kinase, a key post-receptor signaling molecule.

Differential expression of M3 muscarinic receptors in progressive colon neoplasia and metastasis

M3 muscarinic receptor (M3R) activation promotes colon cancer cell proliferation, migration, and invasion in vitro. Although over-expression of CHRM3, the gene encoding M3R, is reported in primary colon cancers, expression of M3R itself has not been studied in colon neoplasia. We compared M3R expression in normal colon to colon adenomas, and primary and metastatic colon cancers. Compared to adjacent normal colon, CHRM3 expression was increased up to 128-fold in 10 of 18 consecutive surgical cancer specimens (56%) and associated with metastatic spread (P < 0.05). To analyze M3R protein expression we interrogated 29 consecutive paraffin-embedded colon adenocarcinomas and adjacent normal colon using a specific anti-M3R antibody and immunoperoxidase staining. This revealed weak M3R expression in normal colonocytes, primarily on basolateral surfaces. In contrast, in 25 of 29 cancer tissues (86%) we observed both cytoplasmic and plasma membrane over-expression of M3R; compared to normal epithelium, mean M3R staining intensity was increased more than two-fold in colon cancer (P < 0.001). M3R staining was also increased in 22 colon adenomas compared to adjacent normal colon (P < 0.001). In contrast, M3R staining intensity was not increased in lymph node or liver metastases. These findings suggest M3R expression plays an important role in early progression and invasion of colon neoplasia but is less important once tumors have spread.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Diminished gallbladder filling, increased fecal bile acids, and promotion of colon epithelial cell proliferation and neoplasia in fibroblast growth factor 15-deficient mice

Fibroblast growth factor-19 (human FGF19; murine FGF15) suppresses bile acid synthesis. In FGF19 deficiency, diarrhea resulting from bile acid spillage into the colon mimics irritable bowel syndrome. To seek other consequences of FGF19/15 deficiency, we used Fgf15-/- and wild-type (WT) mice to assess gallbladder filling, the bile acid pool, fecal bile acid levels, and colon neoplasia. We fasted mice for six hours before assessing gallbladder size by magnetic resonance imaging (MRI). We measured bile acid levels in different compartments by enzymatic assay, and induced colon neoplasia with azoxymethane (AOM)/dextran sodium sulfate (DSS) and quantified epithelial Ki67 immunostaining and colon tumors 20 weeks later. In vivo MRI confirmed the gross finding of tubular gallbladders in FGF15-deficient compared to WT mice, but fasting gallbladder volumes overlapped. After gavage with a bile acid analogue, ex vivo MRI revealed diminished gallbladder filling in FGF15-deficient mice (P = 0.0399). In FGF15-deficient mice, the total bile acid pool was expanded 45% (P <0.05) and fecal bile acid levels were increased 2.26-fold (P <0.001). After AOM/DSS treatment, colons from FGF15-deficient mice had more epithelial cell Ki67 staining and tumors (7.33 ± 1.32 vs. 4.57 ± 0.72 tumors/mouse; P = 0.003 compared to WT mice); carcinomas were more common in FGF15-deficient mice (P = 0.01). These findings confirm FGF15, the murine homolog of FGF19, plays a key role in modulating gallbladder filling and bile acid homeostasis. In a well-characterized animal model of colon cancer, increased fecal bile acid levels in FGF15-deficient mice promoted epithelial proliferation and advanced neoplasia.

Two sides to colon cancer: mice mimic human anatomical region disparity in colon cancer development and progression

Aim: Strong evidence reveals important differences between cancers in the proximal vs. distal colon. Animal models of metastatic colon cancer are available but with varying degrees of reproducibility and several important limitations. We explored whether there were regional differences in the location of murine colon cancers and assessed the utility of murine models to explore the biological basis for such differences. Methods: We re-analyzed data from our previous studies to assess the regional distribution of murine colon cancer. In survival surgery experiments, we injected HT-29 human colon cancer cells into the wall of the cecum or distal colon of Nu(NCr)-Foxn1nu or NOD.Cg-PrkdcscidIl2rgTim1Wji/SzJ mice and compared the development of primary tumors and metastases. Results: Within 7–17 weeks after intramural cecal injection of HT-29 cells, eight mice failed to develop solid primary tumors or metastases. In contrast, within four weeks after cell injection into the distal colon, 13 mice developed metastases - 12 mice developed subcutaneous metastases; of these, four developed liver metastases and one developed both liver and lung metastases. One mouse developed liver metastases only. Histological examination confirmed these lesions were adenocarcinomas. Conclusion: Our findings reveal the preferential growth of murine colon neoplasia and invasive human orthotopic xenografts in the distal mouse colon. The new approach of injecting cells into the distal colon wall results in a pattern of colon cancer development that closely mimics the progression of metastatic colon cancer in humans. This novel model of colon neoplasia has great potential for exploring anatomical differences in colon cancer and testing novel therapeutics.