Michael Bordonaro

Butyrate and Wnt signaling: a possible solution to the puzzle of dietary fiber and colon cancer risk?

Studies on the protective role of dietary fiber and its breakdown product butyrate against colorectal cancer (CRC) have yielded inconsistent findings. We have reported that butyrate treatment of CRC cells in vitro modulates canonical Wnt signaling, a pathway which is constitutively activated in the majority of CRCs. Analyses of ten human CRC cell lines exposed to butyrate have established that the levels of apoptosis in these cells are dependent upon the fold induction of canonical Wnt transcriptional activity. It is likely that the observed variability in the levels of induced Wnt activity and apoptosis in CRC cells in vitro reflects the existence of different CRC subtypes in vivo. The existence of CRC subtypes, individual- and population-specific variation in butyrate producing colonic microflora, and the time at which the colorectal lesions (early vs. late stage) are exposed to fiber/butyrate are all factors that may influence the protective role of fiber against CRC. We discuss the evidence by which these factors influence the effects of fiber on colonic tumorigenesis and outline experimental approaches for testing these hypotheses.

Mechanisms linking dietary fiber, gut microbiota and colon cancer prevention.

Many epidemiological and experimental studies have suggested that dietary fiber plays an important role in colon cancer prevention. These findings may relate to the ability of fiber to reduce the contact time of carcinogens within the intestinal lumen and to promote healthy gut microbiota, which modifies the hosts metabolism in various ways. Elucidation of the mechanisms by which dietary fiber-dependent changes in gut microbiota enhance bile acid deconjugation, produce short chain fatty acids, and modulate inflammatory bioactive substances can lead to a better understanding of the beneficial role of dietary fiber. This article reviews the current knowledge concerning the mechanisms via which dietary fiber protects against colon cancer.

Linear relationship between Wnt activity levels and apoptosis in colorectal carcinoma cells exposed to butyrate

We have reported that butyrate, a fatty acid produced by dietary fiber that induces cell cycle arrest, differentiation and/or apoptosis in colorectal carcinoma (CRC) cells in vitro, modulates Wnt activity in 2 CRC cell lines (Bordonaro et al., Int. J. Cancer, 2002; 97:42–51). Our study determines how changes in the levels of Wnt activity induced by butyrate relate to the effects of butyrate on apoptosis, cell cycle arrest and differentiation of CRC cells. In 10 human CRC cell lines a direct relationship was shown between apoptosis and butyrate‐induced increase in Wnt activity, as well as between suppressed clonal growth and increased Wnt activity. No correlation existed between butyrate‐induced increase in Wnt activity and differentiation. The direct relationship between apoptosis and Wnt activity was supported by analyses of DLD‐1 and HCT‐116 cells expressing a dominant negative form of Tcf4, and therefore, with repressed Wnt activity, as well as by measuring the ratio of apoptotic to live cells in flow cytometry‐sorted cell fractions with high and low Wnt activity. Novel flow cytometric methodology was utilized to show that butyrate differentially increases the number of cells with Wnt activity in different CRC cell lines. Thus, CRC cell lines in which butyrate upregulated Wnt activity to relatively high levels were most susceptible to the apoptotic effects of butyrate, whereas cell lines in which butyrate modestly modulated Wnt activity were less affected.

Cell type‐ and promoter‐dependent modulation of the Wnt signaling pathway by sodium butyrate

The Wnt signaling pathway modulates the transcription of genes linked to proliferation, differentiation and tumor progression. β‐Catenin‐Tcf (BCT)‐dependent Wnt signaling is influenced by the short‐chain fatty acid sodium butyrate, which induces growth arrest and/or maturation of colonic carcinoma cells. We have compared the effects of sodium butyrate on BCT‐dependent signaling in 2 colon carcinoma cell lines that differ in their physiologic response to butyrate, with SW620 cells responding to butyrate by undergoing terminal differentiation and apoptosis, and HCT‐116 cells undergoing reversible growth arrest, but no significant apoptotic cell death. Furthermore, these colon carcinoma cell lines differ in their mechanism of Wnt pathway activation, with adenomatous polyposis coli (APC) mutant SW620 cells having high levels of BCT complexes and APC wild‐type HCT‐116 cells having mutant β‐catenin, low levels of BCT complexes and correspondingly higher levels of free Tcf. We have demonstrated that in SW620 cells, butyrate downregulates BCT‐dependent expression of the Tcf‐TK, matrilysin and cyclin D1 promoters, whereas in HCT‐116 cells, butyrate upregulates expression of these promoters. Cotransfection with expression vectors that interfere with the Wnt pathway suggests that butyrate enhances BCT complex‐DNA binding. Butyrate reduces the expression of Tcf4 in HCT‐116 cells, consistent with the induction by butyrate of Tcf‐repressible promoters in these cells. These findings indicate that sodium butyrate modulates the Wnt pathway in SW620 and HCT‐116 cells in a different manner and that these differences have consequences for promoter activity that may influence the physiologic response to butyrate.

A Switch from Canonical to Noncanonical Wnt Signaling Mediates Drug Resistance in Colon Cancer Cells

Butyrate, a fermentation product of fiber in the colon, acts as a histone deacetylase inhibitor (HDACi) and induces apoptosis in colon cancer (CC) cells in vitro. We have reported that the apoptotic effects of butyrate are dependent upon the hyperactivation of the Wnt/beta-catenin pathway. However, prolonged exposure of CC cells to increasing concentrations of butyrate results in the acquisition of resistance to the Wnt/beta-catenin- and apoptosis-inducing effects of this agent, as well as cross-resistance to structurally different HDACis. Here we report that one mechanism whereby HDACi resistance arises is through the increase of beta-catenin-independent (noncanonical) Wnt signaling. Compared to HDACi-sensitive HCT-116 CC cells, HDACi-resistant HCT-R cells exhibit higher levels of AKT/PKB cell survival signaling, which is in part induced by WNT5A and its receptor ROR2. The induction of AKT signaling by HDACis is also detected in other CC cell lines, albeit to a lesser extent than in the drug-resistant HCT-R cells. The observations suggested that the apoptotic effect of butyrate and other HDACis in CC cells can be augmented by inhibitors of pAKT. In agreement with the hypothesis, the combination of MK2206, a pAKT inhibitor, and a HDACi (butyrate or LBH589) induced higher apoptosis in CC cells compared to each agent alone. The exposure to both agents also re-sensitized the HCT-R cells to apoptosis. Finally, the concept of simultaneously inducing canonical Wnt activity and suppressing AKT signaling was translated into a combination of diet-derived agents. Diet-derived pAKT inhibitors (caffeic acid phethyl ester, sulforaphane, dilallyl trisulfide) suppressed the butyrate-induced levels of pAKT, and increased the apoptotic effects of butyrate in both drug-sensitive and drug-resistant CC cells. Our findings can be translated into (a) CC therapy employing combinations of synthetic HDACis and inhibitors of pAKT, as well as (b) CC prevention based upon diets that result in sufficient amounts of butyrate and pAKT inhibitors.

CBP Activity Mediates Effects of the Histone Deacetylase Inhibitor Butyrate on WNT Activity and Apoptosis in Colon Cancer Cells.

Mutations in the WNT/beta-catenin pathway are responsible for initiating the majority of colorectal cancers (CRCs). We have previously shown that hyperactivation of this signaling by histone deacetylase inhibitors (HDACis) such as butyrate, a fermentation product of dietary fiber, promotes CRC cell apoptosis. The extent of association between beta-catenin and the transcriptional coactivator CREB-binding protein (CBP) influences WNT/catenin signaling and, therefore, colonic cell physiology. CBP functions as a histone acetylase (HAT); therefore, we hypothesized that the modulation of WNT/catenin activity by CBP modifies the ability of the HDACi butyrate to hyperinduce WNT signaling and apoptosis in CRC cells. Our findings indicate that CBP affects the hyperinduction of WNT activity by butyrate. ICG-001, which specifically blocks association between CBP and beta-catenin, abrogates the butyrate-triggered increase in the number of CRC cells with high levels of WNT/catenin signaling. Combination treatment of CRC cells with ICG-001 and butyrate results in cell type-specific effects on apoptosis. Further, both butyrate and ICG-001 repress CRC cell proliferation, with additive effects in suppressing cell growth. Our study strongly suggests that ICG-001-like agents would be effective against butyrate/HDACi-resistant CRC cells. Therefore, ICG-001-like agents may represent an important therapeutic option for CRCs that exhibit low-fold hyperactivation of WNT activity and apoptosis in the presence of HDACis. The findings generated from this study may lead to approaches that utilize modulation of CBP activity to facilitate CRC therapeutic or chemopreventive strategies.

The Notch Ligand Delta-Like 1 Integrates Inputs from TGFBeta/Activin and Wnt Pathways

Unlike the well-characterized nuclear function of the Notch intracellular domain, it has been difficult to identify a nuclear role for the ligands of Notch. Here we provide evidence for the nuclear function of the Notch ligand Delta-like 1 in colon cancer (CC) cells exposed to butyrate. We demonstrate that the intracellular domain of Delta-like 1 (Dll1icd) augments the activity of Wnt signaling-dependent reporters and that of the promoter of the connective tissue growth factor (CTGF) gene. Data suggest that Dll1icd upregulates CTGF promoter activity through both direct and indirect mechanisms. The direct mechanism is supported by co-immunoprecipitation of endogenous Smad2/3 proteins and Dll1 and by chromatin immunoprecipitation analyses that revealed the occupancy of Dll1icd on CTGF promoter sequences containing a Smad binding element. The indirect upregulation of CTGF expression by Dll1 is likely due to the ability of Dll1icd to increase Wnt signaling, a pathway that targets CTGF. CTGF expression is induced in butyrate-treated CC cells and results from clonal growth assays support a role for CTGF in the cell growth-suppressive role of butyrate. In conclusion, integration of the Notch, Wnt, and TGFbeta/Activin signaling pathways is in part mediated by the interactions of Dll1 with Smad2/3 and Tcf4.

Cellular Mechanisms of Risk and Transformation

ABSTRACT Our early work using the first array and imaging methods for the quantitative analysis of the expression of 4000 cDNA sequences suggested that modulation of mitochondrial gene expression was a factor in determining whether colonic epithelial cells displayed a differentiated or transformed phenotype. We have since dissected a pathway in which mitochondrial function is a key element in determining the probability of cells undergoing cell‐cycle arrest, lineage‐specific differentiation, and cell death. Moreover, this pathway is linked to signaling through β‐catenin‐Tcf, but in a manner that is independent of effects of the APC gene on β‐catenin‐Tcf activity. Utilization of unique mouse genetic models of intestinal tumorigenesis has confirmed that mitochondrial function is an important element in generation of apoptotic cells in the colon in vivo and has demonstrated that modulation of cell death may be involved in intestinal tumor progression rather than initiation. Normal spatial and temporal patterns of cell proliferation, differentiation, and apoptosis in the colonic mucosa are determined by developmentally programmed genetic signals and external signals generated by homo‐ and heterotypic cell interactions, humoral agents, and lumenal contents. Mitochondrial function may play a pivotal role in integrating these signals and in determining probability of cells entering different maturation pathways. How this is accomplished is under investigation using high‐density cDNA microarrays.

Role of Wnt signaling in the development of type 2 diabetes.

Type 2 diabetes is characterized by insulin resistance, insulin deficiency, and hyperglycemia. Susceptibility to type 2 diabetes has been linked to Wnt signaling, which plays an important role in intestinal tumorigenesis. Carriers of variants of the transcription factor 7-like 2 gene, an important component of the Wnt pathway, are at enhanced risk for developing type 2 diabetes. The modulation of proglucagon expression by Wnt activity may partially explain the link between Wnt signaling and diabetes, and one of the transcriptional and processing products of the proglucagon gene, the glucagon-like peptide-1 (GLP-1), exhibits a wide variety of antidiabetogenic activities. GLP-1 stimulates Wnt signaling in pancreatic beta cells, enhancing cell proliferation; thus, positive feedback between GLP-1 and Wnt signaling may result in increased proliferation, and suppressed apoptosis, of pancreatic cells. Since beta-cell protection is a potential treatment for type 2 diabetes, stimulation of Wnt activity may represent a valid therapeutic approach.

Tcf3 and cell cycle factors contribute to butyrate resistance in colorectal cancer cells

Butyrate, a fermentation product of dietary fiber, inhibits clonal growth in colorectal cancer (CRC) cells dependent upon the fold induction of Wnt activity. We have developed a CRC cell line (HCT-R) that, unlike its parental cell line, HCT-116, does not respond to butyrate exposure with hyperactivation of Wnt signaling and suppressed clonal growth. PCR array analyses revealed Wnt pathway-related genes, the expression of which differs between butyrate-sensitive HCT-116 CRC cells and their butyrate-resistant HCT-R cell counterparts. We identified overexpression of Tcf3 as being partially responsible for the butyrate-resistant phenotype, as this DNA-binding protein suppresses the hyperinduction of Wnt activity by butyrate. Consequently, Tcf3 knockdown in HCT-R cells restores their sensitivity to the effects of butyrate on Wnt activity and clonal cell growth. Interestingly, the effects of overexpressed Tcf3 differ between HCT-116 and HCT-R cells; thus, in HCT-116 cells Tcf3 suppresses proliferation without rendering the cells resistant to butyrate. In HCT-R cells, however, the overexpression of Tcf3 inhibits Wnt activity, and the cells are still able to proliferate due to the higher expression levels of cell cycle factors, particularly those driving the G(1) to S transition. Knowledge of the molecular mechanisms determining the variable sensitivity of CRC cells to butyrate may assist in developing approaches that prevent or reverse butyrate resistance.