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      Determination of the Role of CBP- and p300-Mediated Wnt Signaling on Colonic Cells

      , PhD 1 , , , PhD 1
      (Reviewer), (Reviewer)
      JMIR Research Protocols
      JMIR Publications Inc.
      Wnt, butyrate, beta-catenin, CBP, p300, histone acetylation, colorectal cancer

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          The Wnt signaling pathway, mediated through active beta-catenin, is responsible for initiating the majority of cases of human colorectal cancer (CRC), and we have previously shown that hyperactivation of this pathway by histone deacetylase inhibitors (HDACis), such as butyrate, can induce the death of CRC cells. An important cellular switch that mediates the effects of Wnt-signaling activation is variation in the association between beta-catenin and the transcriptional coactivators cAMP response element binding (CREB) binding protein (CBP) and p300. Association of CBP with beta-catenin is thought to activate a set of genes linked to cell proliferation, while the p300-mediated Wnt genetic program is believed to promote cell differentiation. Small molecule agents have been discovered that modulate CBP/p300 Wnt transcriptional programs by altering the association of CBP and p300 to beta-catenin. ICG-001 and ICG-427 inhibit CBP- and p300-mediated Wnt activity, respectively, while IQ-1 prevents the shift from CBP-mediated to a p300-mediated Wnt activity.


          Aim 1 of this proposal is designed to determine the role of CBP- and p300-mediated Wnt signaling in the response of CRC cells to HDACis. Aim 2 is to determine the role of CBP and p300 in the maintenance of high- and low-Wnt fractions in CRC cell line. Aim 3 will compare the effects of CBP- and p300-mediated Wnt activity on CRC initiation and progression.


          In Aim 1, cells will be cotreated with HDACis and ICG-001, ICG-427, or IQ-1 and the levels of Wnt activity, apoptosis, proliferation, differentiation, and CBP- or p300-beta-catenin binding measured. Aim 2 of this proposal may mirror similar heterogeneity observed in human tumors and which may be of clinical significance. Aim 3 will use CRC cell line model systems of initiation and progression: the normal colon cell lines CCD-841CoN, the adenoma line LT97, the primary colon carcinoma cell line SW480, and the lymph node metastasis cell line SW620. Cells will be treated with HDACis and the small molecule agents, and assayed as described above.


          We will also attempt to use changes in CBP- and p300-mediated Wnt signaling to shift colonic cells between cell type, modifying CBP- and p300-mediated gene expression in the LT97 adenoma line to shift the adenoma phenotype to more characteristic of the CCD-841CoN normal cells, or the SW480 carcinoma cells. We will use microarray analyses to determine the patterns of gene expression responsible for these CBP- or p300-mediated changes in colonic neoplastic phenotype.


          The findings generated from this study will lead to future, more in-depth projects to further dissect the action of CBP/p300 Wnt–mediated transcriptional programs in colonic neoplasia, with an emphasis on methods to modulate these genetic programs for chemopreventive effect.

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          Most cited references67

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          Opinion: migrating cancer stem cells - an integrated concept of malignant tumour progression.

          The dissemination of tumour cells is the prerequisite of metastases and is correlated with a loss of epithelial differentiation and the acquisition of a migratory phenotype, a hallmark of malignant tumour progression. A stepwise, irreversible accumulation of genetic alterations is considered to be the responsible driving force. But strikingly, metastases of most carcinomas recapitulate the organization of their primary tumours. Although current models explain distinct and important aspects of carcinogenesis, each alone can not explain the sum of the cellular changes apparent in human cancer progression. We suggest an extended, integrated model that is consistent with all aspects of human tumour progression - the 'migrating cancer stem (MCS)-cell' concept.
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            A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected].

            Inherited and somatic mutations in the adenomatous polyposis coli occur in most colon cancers, leading to activation of beta-catenin-responsive genes. To identify small molecule antagonists of this pathway, we challenged transformed colorectal cells with a secondary structure-templated chemical library, looking for compounds that inhibit a beta-catenin-responsive reporter. We identified ICG-001, a small molecule that down-regulates beta-catenin/T cell factor signaling by specifically binding to cyclic AMP response element-binding protein. ICG-001 selectively induces apoptosis in transformed cells but not in normal colon cells, reduces in vitro growth of colon carcinoma cells, and is efficacious in the Min mouse and nude mouse xenograft models of colon cancer.
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              • Article: not found

              Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer.

              Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers. It occurs through mutations mainly of APC and less often of CTNNB1 (encoding beta-catenin) or AXIN2 (encoding axin-2, also known as conductin). These mutations allow ligand-independent WNT signaling that culminates in abnormal accumulation of free beta-catenin in the nucleus. We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzled-related proteins (SFRPs) in colorectal cancer. SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development. Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.

                Author and article information

                JMIR Res Protoc
                JMIR Res Protoc
                JMIR Research Protocols
                JMIR Publications Inc. (Toronto, Canada )
                Apr-Jun 2016
                13 May 2016
                : 5
                : 2
                : e66
                [1] 1The Commonwealth Medical College Scranton, PAUnited States
                Author notes
                Corresponding Author: Michael Bordonaro mbordonaro@ 123456tcmedc.org
                Author information
                ©Michael Bordonaro, Darina Lazarova Lazarova. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 13.05.2016.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on http://www.researchprotocols.org, as well as this copyright and license information must be included.

                : 1 January 2016
                : 20 January 2016
                : 21 January 2016
                Original Paper
                Original Paper

                wnt,butyrate,beta-catenin,cbp,p300,histone acetylation,colorectal cancer


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