J Cancer. 2013 Jul 15;4(6):481-90. doi: 10.7150/jca.6583.

CBP Activity Mediates Effects Of The Histone Deacetylase Inhibitor Butyrate On WNT Activity And Apoptosis In Colon Cancer Cells.

Darina L Lazarova1, Christopher Chiaro1, Terrence Wong1, Eric Drago1, Anthony Rainey1,2, Shannon O’Malley1,3, and Michael Bordonaro1*

1Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA18509, USA

2 Marywood University, 2300 Adams Avenue, Scranton, PA18509

3 The Pennsylvania State University, 201 Old Main, University Park, PA16802

 

*Corresponding author.

The Commonwealth Medical College, 525 Pine Street, Scranton, PA18509.

Tel: 570-504-9646; Fax: 570-504-9636; Email: mbordonaro@tcmedc.org

 

Abstract

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 acetylases (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.

KEYWORDS: CBP, ICG-001, WNT, butyrate, colorectal cancer, fiber.

PMID: 23901348

 

Additional Information

In 2011, the protective effect of dietary fiber against colorectal cancer (CRC) was upgraded from “probable” to “convincing” (1).  One of the fermentation products of fiber in the colonic lumen is the short-chain fatty acid butyrate, which functions as a potent histone deacetylase inhibitor (HDACi); most CRC cells respond to physiological concentrations of butyrate with apoptosis and cell growth arrest.  At the same time, mutations in WNT signaling initiate the vast majority of CRCs, and we had previously discovered that WNT signaling is upregulated by physiologically relevant concentrations of butyrate.  We have shown that the ability of butyrate to induce CRC cell apoptosis is directly and causally related to the ability of this agent to hyperactivate WNT signaling (2).  This observation is consistent with the “just right hypothesis” of colonic tumorigenesis, which states that either low or high levels of WNT activity can result in apoptosis, while moderate levels of WNT signaling result in proliferation (3).  These findings provide a possible mechanism for the preventive role of fiber against CRC, and for the potential therapeutic action of butyrate and other HDACis against CRC.

Thus, it is important to understand the mechanisms by which butyrate influences WNT signaling and to identify molecular targets for therapeutic intervention. Findings from the Kahn laboratory have demonstrated the important of the histone acetylases (HATs) CBP and p300 in the control of WNT-mediated gene expression in colonic cells (4).  Given the importance of WNT signaling for the development of CRC, and given the crucial role played by WNT hyperactivation in mediating the effects of butyrate (e.g., apoptosis of CRC cells), it is crucial to more precisely understand how CBP and p300 influence WNT activity, and how these HAT factors influence the effects of butyrate on CRC cell signaling and apoptosis. The Kahn group has identified and characterized an agent, ICG-001, that represses CBP-WNT activity, while allowing for continued p300-WNT activity (4).  Using ICG-001 and other approaches to modulate the levels and activity of CBP and p300, we evaluated the roles played by these HAT factors in the ability of butyrate to induce WNT signaling and apoptosis in CRC cells.

In our manuscript (5), we evaluated the role of CBP in the effects of butyrate on WNT signaling and on CRC cell physiology. We demonstrated that CBP is essential for WNT hyperactivation by butyrate, and that the effects of ICG-001 on butyrate-induced CRC cell apoptosis differ in a cell-type specific manner.  The apoptosis findings are particularly important for evaluating the potential of combinatorial anti-CRC therapy utilizing ICG-001-like agents with HDACis such as butyrate.  On the one hand, as individual agents, both ICG-001 and butyrate induce CRC cell apoptosis and therefore the combination of these agents might be expected to effectively induce programmed cell death in cancer cells.  However, since ICG-001 represses butyrate-induced WNT hyperactivation, and since we have previously shown that this hyperactivation contributes to the ability of butyrate to upregulate apoptosis (2 and refs. therein), it is possible that ICG-001 may actually inhibit apoptosis induced by butyrate and other HDACis.

Our findings indicated that ICG-001 and butyrate cooperatively enhance caspase activation in certain CRC cell lines, but act antagonistically with respect to caspase activation in other CRC cells.  Further, these differential effects of butyrate and ICG-001 on apoptosis are likely influenced by variable levels of p21 (a cell cycle inhibitor) and survivin (an inhibitor of apoptosis) in different CRC cell lines.

These findings demonstrate the importance of (a) understanding the mechanisms whereby ICG-001 and butyrate act upon cell signaling pathways influencing apoptosis, and (b) identifying those neoplasms that would be sensitive to the cooperative action of these agents vs. those that are not, and would exhibit repressed apoptosis with combinatorial treatment.  Our hypothesis on the relative roles played by CBP and p300 in CRC cell physiology and the effects of butyrate is shown in Fig. 1.

Our findings could contribute to anti-CRC therapeutic approaches based upon the modulation of CBP-WNT signaling in conjunction with treatment with agents such as butyrate.

Figure CBPFig. 1.  Role of CBP-WNT activity in CRC cell physiology.  CBP-mediated WNT signaling likely promotes colonic cell proliferation, while p300-mediated WNT signaling promotes differentiation.  ICG-001, which can promote apoptosis in certain CRC cell lines, inhibits CBP-mediated, but not p300-mediated, WNT signaling.  Treatment with butyrate hyperactivates WNT signaling and induces apoptosis; we believe that it is the CBP-mediated component of WNT signaling that is primarily responsible for enhancing apoptosis upon hyperactivation of WNT signaling.  Thus, lower basal levels of CBP-mediated WNT signaling are pro-proliferative, but the very high levels of that signaling induced by butyrate promote apoptosis.  The effects of cotreatment of CRC cells with ICG-001 and butyrate are complex, and are cell type-dependent.  In some CRC cell lines, ICG-001 interferes with the apoptotic induction stimulated by butyrate, while in other CRC cell lines, this interference does not occur.  It is likely that variation in the levels of p21 and survivin in different CRC cell lines influences the manner in which ICG-001/butyrate cotreatment affects apoptosis.  Figure 1 is based on our findings in ref. 5, as well as the findings of ref. 4.

 

References

1.  World Cancer Research Fund/American Institute for Cancer Research. Continuous Update Project Interim Report Summary. Food, Nutrition, Physical Activity, and the Prevention of Colorectal Cancer. 2011

2. Lazarova DL, Bordonaro M, Carbone R, Sartorelli AC. Linear relationship between WNT activity levels and apoptosis in colorectal carcinoma cells exposed to butyrate. Internat J Cancer 2004; 110:523-31.

3. Albuquerque C, Breukel C, van der Luijt R, Fidalgo P, Lage P, Slors  FGM, Leitao CN, Fodde R, Smits R. The just-right signaling model: APC somatic mutations are selected based on a special level of activation of the beta-catenin signaling cascade. Hum Mol Genet 2002; 11:1549-60.

4.  Emami KH, Nguyen C, Ma H, Kim DH, Jeong KW, Eguchi M, Moon RT, Teo JL, Oh SW, Kim HY, Moon SH, Ha JR, Kahn M. A small molecule inhibitor of b-catenin/CREB-binding protein transcription. Proc Natl Acad Sci USA 2004; 101:12682-7.

5. Lazarova DL, Chiaro C, Wong T, Drago E, Rainey A, O’Malley S, Bordonaro M.  CBP activity mediates effects of the histone deacetylase inhibitor butyrate on WNT activity and apoptosis in colon cancer cells. J Cancer 2013; 4: 481-90.

 

Acknowledgments

This work was supported by NIH NCI grant 1R15CA149589-01.

 

Corresponding author:

Michael Bordonaro, PhD

Associate Professor of Molecular Biology

Department of Basic Sciences

The Commonwealth Medical College

525 Pine Street

Scranton, PA 18509

Email: mbordonaro@tcmedc.org

Tel: 570-504-9646

Fax: 570-504-9636

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