Stephen W. Byers

Serine phosphorylation-regulated ubiquitination and degradation of beta-catenin.

Several lines of evidence suggest that accumulation of cytoplasmic β-catenin transduces an oncogenic signal. We show that β-catenin is ubiquitinated and degraded by the proteosome and that β-catenin stability is regulated by a diacylglycerol-independent protein kinase C-like kinase activity, which is required for β-catenin ubiquitination. We also define a six-amino acid sequence found in both β-catenin and the NF-κB regulatory protein IκBα, which, upon phosphorylation, targets both proteins for ubiquitination. Mutation of a single serine within the ubiquitination targeting sequence prevents ubiquitination of β-catenin. Mutations within the ubiquitination targeting sequence of β-catenin may be oncogenic.

Liver stem cells and hepatocellular carcinoma.

Although the existence of cancer stem cells (CSCs) was first proposed over 40 years ago, only in the past decade have these cells been identified in hematological malignancies, and more recently in solid tumors that include liver, breast, prostate, brain, and colon. Constant proliferation of stem cells is a vital component in liver tissues. In these renewing tissues, mutations will most likely result in expansion of the altered stem cells, perpetuating and increasing the chances of additional mutations and tumor progression. However, many details about hepatocellular cancer stem cells that are important for early detection remain poorly understood, including the precise cell(s) of origin, molecular genetics, and the mechanisms responsible for the highly aggressive clinical picture of hepatocellular carcinoma (HCC). Exploration of the difference between CSCs from normal stem cells is crucial not only for the understanding of tumor biology but also for the development of specific therapies that effectively target these cells in patients. These ideas have drawn attention to control of stem cell proliferation by the transforming growth factor beta (TGF‐β), Notch, Wnt, and Hedgehog pathways. Recent evidence also suggests a key role for the TGF‐β signaling pathway in both hepatocellular cancer suppression and endoderm formation, suggesting a dual role for this pathway in tumor suppression as well as progression of differentiation from a stem or progenitor stage. This review provides a rationale for detecting and analyzing tumor stem cells as one of the most effective ways to treat cancers such as HCC. (HEPATOLOGY 2009;49:318–329.)

Cross-regulation of β-catenin–LEF/TCF and retinoid signaling pathways

Abstract Vitamin A derivatives (retinoids) are potent regulators of embryogenesis, cell proliferation, epithelial cell differentiation and carcinogenesis [1]. In breast cancer cells, the effects of retinoids are associated with changes in the cadherin–β-catenin adhesion and signaling system [2,3]. β-catenin is a component of the Wnt signaling pathway, which regulates several developmental pathways [4]. Increases in cytoplasmic β-catenin and β-catenin signaling are also associated with numerous cancers, and are particularly important in colon cancer [5]. The oncogenic and developmental effects of β-catenin are mediated by its interaction with and activation of members of the LEF/TCF family of transcription factors [6–8]. Here, we shown that retinoic acid (RA) decreases the activity of the β-catenin–LEF/TCF signaling pathway. This activity of RA was independent of the adenomatous polyposis coli (APC) tumor suppressor and ubiquitination-dependent degradation of cytoplasmic β-catenin. Consistent with this finding, β-catenin interacted directly with the RA receptor (RAR) in a retinoid-dependent manner, but not with the retinoid X receptor (RXR), and RAR competed with TCF for β-catenin binding. The activity of RA on RAR-responsive promoters was also potentiated by β-catenin. The data suggest that direct regulation of β-catenin–LEF/TCF signaling is one mechanism whereby RA influences development, cell differentiation and cancer.

β-catenin binds to the activation function 2 region of the androgen receptor and modulates the effects of the N-terminal domain and TIF2 on ligand-dependent transcription

ABSTRACT β-Catenin is a multifunctional molecule that is activated by signaling through WNT receptors. β-Catenin can also enhance the transcriptional activity of some steroid hormone receptors such as the androgen receptor and retinoic acid receptor α. Androgens can affect nuclear translocation of β-catenin and influence its subcellular distribution. Using mammalian two-hybrid binding assays, analysis of reporter gene transcription, and coimmunoprecipitation, we now show that β-catenin binds to the androgen receptor ligand-binding domain (LBD) and modulates the transcriptional effects of TIF2 and the androgen receptor N-terminal domain (NTD). In functional assays, β-catenin bound to androgen receptor only in the presence of ligand agonists, not antagonists. β-Catenin binding to the androgen receptor LBD was independent of and cooperative with the androgen receptor NTD and the p160 coactivator TIF2, both of which bind to the activation function 2 (AF-2) region of the androgen receptor. Different mutations of androgen receptor helix 3 amino acids disrupted binding of androgen receptor NTD and β-catenin. β-Catenin, androgen receptor NTD, and TIF2 binding to the androgen receptor LBD were affected similarly by a subset of helix 12 mutations, but disruption of two sites on helix 12 affected only binding of β-catenin and not of TIF2 or the androgen receptor NTD. Mutational disruption of each of five LXXLL peptide motifs in the β-catenin armadillo repeats did not disrupt either binding to androgen receptor or transcriptional coactivation. ICAT, an inhibitor of T-cell factor 4 (TCF-4), and E-cadherin binding to β-catenin also blocked binding of the androgen receptor LBD. We also demonstrated cross talk between the WNT and androgen receptor signaling pathways because excess androgen receptor could interfere with WNT signaling and excess TCF-4 inhibited the interaction of β-catenin and androgen receptor. Taken together, the data show that β-catenin can bind to the androgen receptor LBD and modulate the effects of the androgen receptor NTD and TIF2 on transcription.

Tumor suppressor BRCA1 epigenetically controls oncogenic microRNA-155

BRCA1, a well-known tumor suppressor with multiple interacting partners, is predicted to have diverse biological functions. However, so far its only well-established role is in the repair of damaged DNA and cell cycle regulation. In this regard, the etiopathological study of low-penetrant variants of BRCA1 provides an opportunity to uncover its other physiologically important functions. Using this rationale, we studied the R1699Q variant of BRCA1, a potentially moderate-risk variant, and found that it does not impair DNA damage repair but abrogates the repression of microRNA-155 (miR-155), a bona fide oncomir. Mechanistically, we found that BRCA1 epigenetically represses miR-155 expression via its association with HDAC2, which deacetylates histones H2A and H3 on the miR-155 promoter. We show that overexpression of miR-155 accelerates but the knockdown of miR-155 attenuates the growth of tumor cell lines in vivo. Our findings demonstrate a new mode of tumor suppression by BRCA1 and suggest that miR-155 is a potential therapeutic target for BRCA1-deficient tumors.

Cyclin D1 Determines Mitochondrial Function In Vivo

ABSTRACT The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.

The role of TGF-β and Wnt signaling in gastrointestinal stem cells and cancer

The past three decades have seen an unremitting quest to identify and understand gastrointestinal stem cells, their plasticity in differentiating across cell types, as well as their role in normal, regenerative, and cancer cells. A fascinating hallmark of stem cells is their ability to undergo assymetric cell division, which entails replication of the DNA followed by division of the nucleus and partitioning of the cytoplasm to yield two different daughter cells: a stem cell as well as a committed progenitor cell, the latter proliferating into differentiated progeny. We are only just beginning to understand how normally quiescent, tissue-specific stem cells interpret a vast array of signals to develop into the gastrointestinal system. These signaling pathways include the transforming growth factor-β (TGF-β) superfamily, Wnt, FGFs, Hedgehog, Hox proteins that originate from surrounding mesodermal/stromal tissue as well as endodermal/epithelial tissue. TGF-β and wnt proteins are key morphogens that ultimately influence cell division and cell fate, so that gut endodermal stem cells enter the cell cycle, and undergo cell division that ultimately leads to differentiated cells such as functional hepatocytes, gastric parietal cells, or gut epithelial cells. Disruptions and errors in this process usually lead to tissue-specific gastrointestinal cancers such as hepatocellular cancers, gastric adenocarcinomas, and colonic adenocarcinomas. An increasingly complex and coherent view of stem/progenitor cell signaling networks, which coordinate cell growth, proliferation, stress management, and survival, is helping to define the fragile areas where malignancies are likely to develop and shows promise for the development of better cancer therapies.

Pathway-specific differences between tumor cell lines and normal and tumor tissue cells.

BackgroundCell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue.MethodsThis study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM).ResultsCellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs), and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways.ConclusionSignificantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative phosphorylation. Signaling pathways involved in adhesion and communication of cultured cancer cells were downregulated. The three way pathways comparison presented in this study brings light into the differences in the use of cellular pathways by tumor cells and cancer cell lines.

Cyclin D1 genetic heterozygosity regulates colonic epithelial cell differentiation and tumor number in ApcMin mice

ABSTRACT Constitutive β-catenin/Tcf activity, the primary transforming events in colorectal carcinoma, occurs through induction of the Wnt pathway or APC gene mutations that cause familial adenomatous polyposis. Mice carrying Apc mutations in their germ line (ApcMin ) develop intestinal adenomas. Here, the crossing of ApcMin with cyclin D1−/− mice reduced the intestinal tumor number in animals genetically heterozygous or nullizygous for cyclin D1. Decreased tumor number in the duodenum, intestines, and colons of ApcMin /cyclin D1+/− mice correlated with reduced cellular proliferation and increased differentiation. Cyclin D1 deficiency reduced DNA synthesis and induced differentiation of colonic epithelial cells harboring mutant APC but not wild-type APC cells in vivo. In previous studies, the complete loss of cyclin D1 through homozygous genetic deletion conveyed breast tumor resistance. The protection of mice, genetically predisposed to intestinal tumorigenesis, through cyclin D1 heterozygosity suggests that modalities that reduce cyclin D1 abundance could provide chemoprotection.

Trans-repression of β-Catenin Activity by Nuclear Receptors

The signaling/oncogenic activity of β-catenin can be repressed by the activation of nuclear receptors such as the vitamin A, vitamin D, and androgen receptors. Although these receptors directly interact with β-catenin and can sequester it away from its transcription factor partner T-cell factor, it is not known if this is the mechanism of trans-repression. Using several different promoter constructs and nuclear receptors and mammalian two-hybrid and mutation analyses we now show that interaction with the co-activator, p300, underlies the trans-repression of β-catenin signaling by nuclear receptors and their ligands.