Kira T. Pate

NOTCH Signaling Is Required for Formation and Self-Renewal of Tumor-Initiating Cells and for Repression of Secretory Cell Differentiation in Colon Cancer

NOTCH signaling is critical for specifying the intestinal epithelial cell lineage and for initiating colorectal adenomas and colorectal cancers (CRC). Based on evidence that NOTCH is important for the maintenance and self-renewal of cancer-initiating cells in other malignancies, we studied the role of NOTCH signaling in colon cancer-initiating cells (CCIC). Tumors formed by CCICs maintain many properties of the primary CRCs from which they were derived, such as glandular organization, cell polarity, gap junctions, and expression of characteristic CRC molecular markers. Furthermore, CCICs have the property of self-renewal. In this study, we show that NOTCH signaling is 10- to 30-fold higher in CCIC compared with widely used colon cancer cell lines. Using small-molecule inhibition and short hairpin RNA knockdown, we show that NOTCH prevents CCIC apoptosis through repression of cell cycle kinase inhibitor p27 and transcription factor ATOH1. NOTCH is also critical to intrinsic maintenance of CCIC self-renewal and the repression of secretory cell lineage differentiation genes such as MUC2. Our findings describe a novel human cell system to study NOTCH signaling in CRC tumor initiation and suggest that inhibition of NOTCH signaling may improve CRC chemoprevention and chemotherapy.

Wnt signaling directs a metabolic program of glycolysis and angiogenesis in colon cancer

Much of the mechanism by which Wnt signaling drives proliferation during oncogenesis is attributed to its regulation of the cell cycle. Here, we show how Wnt/β‐catenin signaling directs another hallmark of tumorigenesis, namely Warburg metabolism. Using biochemical assays and fluorescence lifetime imaging microscopy (FLIM) to probe metabolism in vitro and in living tumors, we observe that interference with Wnt signaling in colon cancer cells reduces glycolytic metabolism and results in small, poorly perfused tumors. We identify pyruvate dehydrogenase kinase 1 (PDK1) as an important direct target within a larger gene program for metabolism. PDK1 inhibits pyruvate flux to mitochondrial respiration and a rescue of its expression in Wnt‐inhibited cancer cells rescues glycolysis as well as vessel growth in the tumor microenvironment. Thus, we identify an important mechanism by which Wnt‐driven Warburg metabolism directs the use of glucose for cancer cell proliferation and links it to vessel delivery of oxygen and nutrients.

Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH

There is a lack of fast and high resolution methods to measure metabolic activity of single cells in their native environment. Here we develop a straightforward, non-invasive and sensitive method to measure metabolic phenotype of single cells in a live tissue. By using NADH as optical biomarker and the phasor approach to Fluorescence Lifetime microscopy (FLIM) we identify cellular metabolic fingerprints related to different rates of oxidative phosphorylation and glycolysis. For the first time we measure a three dimensional metabolic gradient in the small intestine (SI) epithelia that appears tightly associated with epithelial cell proliferation, differentiation and the Wnt gradient. The highest free/bound NADH ratios are measured at the base of the crypt within the highly proliferative stem cells, indicating high levels of glycolysis. For the first time mouse small intestinal stem cells in intact live crypts are identified within the tissue by their metabolic fingerprint.

Groucho binds two conserved regions of LEF-1 for HDAC-dependent repression

BackgroundDrosophila Groucho and its human Transducin-like-Enhancer of Split orthologs (TLEs) function as transcription co-repressors within the context of Wnt signaling, a pathway with strong links to cancer. The current model for how Groucho/TLEs modify Wnt signaling is by direct competition with β-catenin for LEF/TCF binding. The molecular events involved in this competitive interaction are not defined and the actions of Groucho/TLEs within the context of Wnt-linked cancer are unknown.MethodsWe used in vitro protein interaction assays with the LEF/TCF family member LEF-1, and in vivo assays with Wnt reporter plasmids to define Groucho/TLE interaction and repressor function.ResultsMapping studies reveal that Groucho/TLE binds two regions in LEF-1. The primary site of recognition is a 20 amino acid region in the Context Dependent Regulatory domain. An auxiliary site is in the High Mobility Group DNA binding domain. Mutation of an eight amino acid sequence within the primary region (RFSHHMIP) results in a loss of Groucho action in a transient reporter assay. Drosophila Groucho, human TLE-1, and a truncated human TLE isoform Amino-enhancer-of-split (AES), work equivalently to repress LEF-1•β-catenin transcription in transient reporter assays, and these actions are sensitive to the HDAC inhibitor Trichostatin A. A survey of Groucho/TLE action in a panel of six colon cancer cell lines with elevated β-catenin shows that Groucho is not able to repress transcription in a subset of these cell lines.ConclusionOur data shows that Groucho/TLE repression requires two sites of interaction in LEF-1 and that a central, conserved amino acid sequence within the primary region (F S/T/P/xx y I/L/V) is critical. Our data also reveals that AES opposes LEF-1 transcription activation and that both Groucho and AES repression require histone deacetylase activity suggesting multiple steps in Groucho competition with β-catenin. The variable ability of Groucho/TLE to oppose Wnt signaling in colon cancer cells suggests there may be defects in one or more of these steps.

Molecular functions of the TLE tetramerization domain in Wnt target gene repression

Wnt signaling activates target genes by promoting association of the co‐activator β‐catenin with TCF/LEF transcription factors. In the absence of β‐catenin, target genes are silenced by TCF‐mediated recruitment of TLE/Groucho proteins, but the molecular basis for TLE/TCF‐dependent repression is unclear. We describe the unusual three‐dimensional structure of the N‐terminal Q domain of TLE1 that mediates tetramerization and binds to TCFs. We find that differences in repression potential of TCF/LEFs correlates with their affinities for TLE‐Q, rather than direct competition between β‐catenin and TLE for TCFs as part of an activation–repression switch. Structure‐based mutation of the TLE tetramer interface shows that dimers cannot mediate repression, even though they bind to TCFs with the same affinity as tetramers. Furthermore, the TLE Q tetramer, not the dimer, binds to chromatin, specifically to K20 methylated histone H4 tails, suggesting that the TCF/TLE tetramer complex promotes structural transitions of chromatin to mediate repression.

A role for YY1 in repression of dominant negative LEF-1 expression in colon cancer

Lymphoid enhancer factor 1 (LEF-1) mediates Wnt signaling via recruitment of β-catenin to target genes. The LEF1 gene is aberrantly transcribed in colon cancers because promoter 1 (P1) is a Wnt target gene and is activated by TCF–β-catenin complexes. A second promoter in intron 2 (P2) produces dominant negative LEF-1 isoforms (dnLEF-1), but P2 is silent because it is repressed by an upstream distal repressor element. In this study we identify Yin Yang 1 (YY1) transcription factor as the P2-specific factor necessary for repression. Site-directed mutagenesis and EMSA were used to identify a YY1-binding site at +25 in P2, and chromatin immunoprecipitation assays detected YY1 binding to endogenous LEF1 P2. Mutation of this site relieves P2 repression in transient transfections, and knockdown of endogenous YY1 relieves repression of integrated P2 reporter constructs and decreases the H3K9me3 epigenetic marks. YY1 is responsible for repressor specificity because introduction of a single YY1-binding site into the P1 promoter makes it sensitive to the distal repressor. We also show that induced expression of dnLEF-1 in colon cancer cells slows their rate of proliferation. We propose that YY1 plays an important role in preventing dnLEF-1 expression and growth inhibition in colon cancer.

Abstract 138: A role for Wnt signaling in regulation of Warburg metabolism in colon cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA For most aerobic organisms, oxidative phosphorylation (OXPHOS) is the predominant metabolic pathway for production of cellular energy. Compared to anaerobic metabolism, OXPHOS is much more efficient at producing ATP. However, despite an abundance of environmental oxygen and the induction of angiogenesis by a colon cancer tumor, the predominant metabolic pathway utilized is glycolysis. This paradox is also known as Warburg metabolism. In mouse xenograft tumors grown from SW620 colon cancer cells, partial inhibition of beta-catenin dependent Wnt signaling shifts metabolism of the tumor, where glycolysis markers decrease. We also determined that transcription of the pyruvate dehydrogenase kinase-1 gene (PDK1), is regulated by Wnt signaling. PDK1 protein normally inactivates mitochondrial pyruvate dehydrogenase (PDH) through phosphorylation in order to increase the conversion of pyruvate to lactate in the cytosol. We concluded that Wnt signaling directs Warburg metabolism in colon cancer via regulation of a key regulator of glycolysis. When staining for phosphorylated PDH in the xenograft tumors as a measure of active PDK, we discovered a unique spotted pattern of discrete regions of increased phospho-PDH at regular intervals throughout the xenograft tumor. The pattern was accentuated when Wnt signaling was reduced and it was completely abolished with PDK1 expression. This regular spotted pattern was also seen in beta-catenin expression, suggesting that Wnt signaling may be responsible for establishing the spotted pattern. We hypothesized that this patterning could be modeled mathematically as a Turing pattern. Beta-catenin dependent Wnt signaling and its associated inhibitors have been previously characterized to form Turing patterns, or reaction-diffusion systems, in multiple developmental biology systems. Our collaborative group has developed a system of reaction-diffusion equations that describes the formation of these spots in relation to varying concentrations of Wnt signaling, Wnt inhibitors, and nutrients. Included in the model are equations for glycolytic cells, oxidative cells, Wnt signaling activity, Wnt inhibitors, PDK activity, lactate, HIF, and a general nutrient term. Wnt activity and Wnt inhibitor equations are based on the Gierer-Meinhardt activator-inhibitor model. The tumor cells switch metabolic regimes based on PDK activity level (high activity implies a tendency towards glycolysis, and low activity tends toward oxidative phosphorylation). Using novel imaging techniques and mathematical modeling, we have demonstrated that beta-catenin dependent Wnt signaling regulates expression of PDK1 to drive glycolysis in xenograft tumors. This increased glycolysis exists in a regular Turing pattern throughout the tumor. Our mathematical models will allow us to predict changes to tumor metabolism and behavior in response to modulation of Wnt signaling or external stimuli. Citation Format: George T. Chen, Mary Lee, Kira Pate, Kehui Wang, Robert A. Edwards, John S. Lowengrub, Marian L. Waterman. A role for Wnt signaling in regulation of Warburg metabolism in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 138. doi:10.1158/1538-7445.AM2014-138