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. 2016 Dec 1;7(1):181.
doi: 10.1186/s13287-016-0439-4.

Bile acid: a potential inducer of colon cancer stem cells

Lulu Farhana  1   2 , Pratima Nangia-Makker  1   3   2 , Evan Arbit  1 , Kathren Shango  1 , Sarah Sarkar  1 , Hamidah Mahmud  1 , Timothy Hadden  1   2 , Yingjie Yu  1   2 , Adhip P N Majumdar  4   5   6
Affiliations

Bile acid: a potential inducer of colon cancer stem cells

Lulu Farhana et al. Stem Cell Res Ther. .

Abstract

Background: Although the unconjugated secondary bile acids, specifically deoxycholic acid (DCA) and lithocholic acid (LCA), are considered to be risk factors for colorectal cancer, the precise mechanism(s) by which they regulate carcinogenesis is poorly understood. We hypothesize that the cytotoxic bile acids may promote stemness in colonic epithelial cells leading to generation of cancer stem cells (CSCs) that play a role in the development and progression of colon cancer.

Methods: Normal human colonic epithelial cells (HCoEpiC) were used to study bile acid DCA/LCA-mediated induction of CSCs. The expression of CSC markers was measured by real-time qPCR. Flow cytometry was used to isolate CSCs. T-cell factor/lymphoid-enhancing factor (TCF/LEF) luciferase assay was employed to examine the transcriptional activity of β-catenin. Downregulation of muscarinic 3 receptor (M3R) was achieved through transfection of corresponding siRNA.

Results: We found DCA/LCA to induce CSCs in normal human colonic epithelial cells, as evidenced by the increased proportion of CSCs, elevated levels of several CSC markers, as well as a number of epithelial-mesenchymal transition markers together with increased colonosphere formation, drug exclusion, ABCB1 and ABCG2 expression, and induction of M3R, p-EGFR, matrix metallopeptidases, and c-Myc. Inhibition of M3R signaling greatly suppressed DCA/LCA induction of the CSC marker ALDHA1 and also c-Myc mRNA expression as well as transcriptional activation of TCF/LEF.

Conclusions: Our results suggest that bile acids, specifically DCA and LCA, induce cancer stemness in colonic epithelial cells by modulating M3R and Wnt/β-catenin signaling and thus could be considered promoters of colon cancer.

Keywords: ABCCB1; ABCG2; Cancer stem cells; Colonic epithelial cell; Colonospheres; Deoxycholic acid; Lithocholic acid; Wnt/β-catenin signaling; matrix metallopeptidases.

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Figures

Fig. 1
Fig. 1
DCA and LCA induce increases in CSC markers. Quantitative real-time PCR showed significantly increased mRNA expression of CD44 (a), CD166 (b), and ALDHA1 (c) in HCoEpiC following incubation with 100 μM DCA or LCA for 72 h. Flow cytometric analysis of HCoEpiC showing an increased proportion of CD44-positive and CD166-positive cells following 12-day incubation in the absence (control) or presence of 100 μM DCA or LCA (d). DCA/LCA mediated the increase in spheroid formation by the CD44+CD166– CSC phenotype of HCoEpiC (e); photomicrographs showing spheroids formed in response to DCA or LCA at the end of the 12-day incubation period. For spheroid formation, CD44+CD166– cells were sorted by flow cytometry and 200–300 cells were seeded with B27 containing DMEM/F12 medium in 96-well low-attachment plates; 24 h after seeding, 100 μM DCA or LCA was added to the incubation medium, and cells were incubated for an additional 12 days. 0 Day, cell size at the time of plating. The sizes of spheres were photographed and measured on a 100 μm scale at a magnification of 400× using an Olympus microscope. Data represent the mean ± standard deviation of 15 sphere determinations. **P < 0.01, ***P < 0.001, compared with control. Data were analyzed by ANOVA, Tukey HSD test for multiple comparisons (e). DCA deoxycholic acid, LCA lithocholic acid
Fig. 2
Fig. 2
Exposure of DCA/LCA in HCoEpiC increased the expression of pluripotency genes. Levels of mRNA encoding the pluripotency genes KLF-4, Nanog, OCT4, and SOX2 was significantly higher in cells incubated with DCA or LCA than control cells (a). Likewise, expression of EMT regulators N-Cadherin, Slug, Twist, Vimentin, Zeb1, and Zeb2 was also increased in response to 100 μM DCA or LCA (b). Results expressed as mean ± standard deviation of three separate experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. DCA (deoxycholic acid), LCA (lithocholic acid)
Fig. 3
Fig. 3
DCA/LCA induction of drug exclusion in HCoEpiC. Ability of HCoEpiC cells to exclude Hoechst dye (H33342) was greatly increased following 30-day incubation with DCA or LCA (a). Likewise, the expression of ABCB1 and ABCG2 was also increased in HCoEpiC cells following 18-day exposure to 50 μM LCA (b, c). Controls contained the appropriate vehicle. Data represent the mean ± standard deviation of three separate determinations. **P < 0.01 and ***P <0.001 Statistical significance determined by t test. DCA (deoxycholic acid), LCA (lithocholic acid)
Fig. 4
Fig. 4
DCA/LCA induction of CSC phenotypic characters in HCoEpiC is mediated by M3R and knockdown of M3R decreased ALDHA1, c-Myc, and TCF/LCF activity in cells treated with DCA. Induction of M3R in HCoEpiC following incubation with 100 μM DCA or LCA after 72 h (a). Downregulation of M3R in cells following transfection with either of two siRNAs (si-M3RT2 and si-M3RT3) for M3R (b). ALDHA1 and c-Myc expression is reduced in M3R-downregulated cells (c). Suppression of DCA-induced stimulation of ALDH1, CD166, and c-Myc expression in M3R-downregulated cells (d–f). Data represent the mean ± standard deviation of three separate determinations. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the control. DCA (deoxycholic acid), LCA (lithocholic acid)
Fig. 5
Fig. 5
Bile acids induce Wnt-β-catenin signaling pathways and increase the expression of the target gene c-Myc in HCoEpiC. Real-time qPCR showing an increased expression of β-catenin mRNA in HCoEpiC cells following 72-h incubation in the presence of 100 μM DCA or LCA (a). Induction of transcriptional activity of TCF/LEF in HCoEpiC in response to 100 μM DCA or LCA treatments for 72 h (b). Photomicrographs showing increased nuclear localization of β-catenin in HCoEpiC following 72 h incubation with 100 μM DCA or LCA; controls were incubated with an equivalent volume of the vehicle (c): left panel , β-catenin immunostained cells; right panel, merged photograph of β-catenin and nucleus stained with DAPI; arrow, nuclear localization of β-catenin in cells. Increased expression of c-Myc in HCoEpiC following 72-h exposure to 100 μM DCA or LCA (d). DCA/LCA-mediated induction of transcriptional activity of TCF/LEF is greatly suppressed in M3R-downregulated HCoEpiC (e). Cells were photographed on a 100 μm scale at a magnification of 400×. Results expressed as mean ± standard deviation of three separate experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. DCA (deoxycholic acid), LCA (lithocholic acid), TCF/LEF (T-cell factor/lymphoid-enhancing factor). Diamonds represent significant reduction in M3R down-regulated cells in response to DCA compared to those without siM3R transfected cells
Fig. 6
Fig. 6
DCA/LCA increased the expression and activation of EGFR in HCoEpiC. Real-time qPCR showing increased expression of EGFR mRNA in cells in response to DCA and LCA (a). Western blot analysis indicates increased tyrosine phosphorylation (Y992) of EGFR in response to DCA (b). Real-time qPCR showing changes in the expression of the MMPs in cells in response to DCA or LCA (c). Results represent the mean of three separate determinations ± standard deviation. ***P <0.001, compared with control. DCA (deoxycholic acid), EGFR (epidermal growth factor receptor), LCA (lithocholic acid), MMP (matrix metallopeptidase)
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