Kehui Wang

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.

Epigenetic Repression of DNA Mismatch Repair by Inflammation and Hypoxia in Inflammatory Bowel Disease–Associated Colorectal Cancer

Sporadic human mismatch repair (MMR)-deficient colorectal cancers account for approximately 12.5% of all cases of colorectal cancer. MMR-deficient colorectal cancers are classically characterized by right-sided location, multifocality, mucinous histology, and lymphocytic infiltration. However, tumors in germ-line MMR-deficient mouse models lack these histopathologic features. Mice lacking the heterotrimeric G protein alpha subunit Gialpha2 develop chronic colitis and multifocal, right-sided cancers with mucinous histopathology, similar to human MMR-deficient colorectal cancer. Young Gialpha2-/- colonic epithelium has normal MMR expression but selectively loses MLH1 and consequently PMS2 expression following inflammation. Gialpha2-/- cancers have microsatellite instability. Mlh1 is epigenetically silenced not by promoter hypermethylation but by decreased histone acetylation. Chronically inflamed Gialpha2-/- colonic mucosa contains patchy hypoxia, with increased crypt expression of the hypoxia markers DEC-1 and BNIP3. Chromatin immunoprecipitation identified increased binding of the transcriptional repressor DEC-1 to the proximal Mlh1 promoter in hypoxic YAMC cells and colitic Gialpha2-/- crypts. Treating Gialpha2-/- mice with the histone deacetylase inhibitor suberoylanilide hydroxamic acid significantly decreased colitis activity and rescued MLH1 expression in crypt epithelial cells, which was associated with increased acetyl histone H3 levels and decreased DEC-1 binding at the proximal Mlh1 promoter, consistent with a histone deacetylase-dependent mechanism. These data link chronic hypoxic inflammation, epigenetic MMR protein down-regulation, development of MMR-deficient colorectal cancer, and the firstmouse model of somatically acquired MMR-deficient colorectal cancer.

Angiogenic sprouting is regulated by endothelial cell expression of Slug.

ABSTRACT The Snail family of zinc-finger transcription factors are evolutionarily conserved proteins that control processes requiring cell movement. Specifically, they regulate epithelial-to-mesenchymal transitions (EMT) where an epithelial cell severs intercellular junctions, degrades basement membrane and becomes a migratory, mesenchymal-like cell. Interestingly, Slug expression has been observed in angiogenic endothelial cells (EC) in vivo, suggesting that angiogenic sprouting may share common attributes with EMT. Here, we demonstrate that sprouting EC in vitro express both Slug and Snail, and that siRNA-mediated knockdown of either inhibits sprouting and migration in multiple in vitro angiogenesis assays. We find that expression of MT1-MMP, but not of VE-Cadherin, is regulated by Slug and that loss of sprouting as a consequence of reduced Slug expression can be reversed by lentiviral-mediated re-expression of MT1-MMP. Activity of MMP2 and MMP9 are also affected by Slug expression, likely through MT1-MMP. Importantly, we find enhanced expression of Slug in EC in human colorectal cancer samples compared with normal colon tissue, suggesting a role for Slug in pathological angiogenesis. In summary, these data implicate Slug as an important regulator of sprouting angiogenesis, particularly in pathological settings.

Mathematical modeling links Wnt signaling to emergent patterns of metabolism in colon cancer

Cell‐intrinsic metabolic reprogramming is a hallmark of cancer that provides anabolic support to cell proliferation. How reprogramming influences tumor heterogeneity or drug sensitivities is not well understood. Here, we report a self‐organizing spatial pattern of glycolysis in xenograft colon tumors where pyruvate dehydrogenase kinase (PDK1), a negative regulator of oxidative phosphorylation, is highly active in clusters of cells arranged in a spotted array. To understand this pattern, we developed a reaction–diffusion model that incorporates Wnt signaling, a pathway known to upregulate PDK1 and Warburg metabolism. Partial interference with Wnt alters the size and intensity of the spotted pattern in tumors and in the model. The model predicts that Wnt inhibition should trigger an increase in proteins that enhance the range of Wnt ligand diffusion. Not only was this prediction validated in xenograft tumors but similar patterns also emerge in radiochemotherapy‐treated colorectal cancer. The model also predicts that inhibitors that target glycolysis or Wnt signaling in combination should synergize and be more effective than each treatment individually. We validated this prediction in 3D colon tumor spheroids.

An alternative flow cytometry strategy for peripheral blood dendritic cell enumeration in the setting of repetitive GM-CSF dosing

BackgroundEnumeration of circulating peripheral blood dendritic cells (DCs) is complicated by the absence of a unique cell surface marker expressed on all DC subsets and by the use of various biological adjuvants to modulate the DC compartment, including granulocyte macrophage colony stimulating factor (GM-CSF). Common methods employ a cocktail of antibodies, typically including anti-CD14, to define a lineage negative, MHC class II positive, putative DC population. Reported flow cytometry protocols include highly variable gating strategies and DC identification criteria. Increasing appreciation of DC pleiomorphism, GM-CSF biology, and recognition of CD14 expression in some DC subsets led us to consider an alternative lineage cocktail to improve identification of the circulating DC pool.MethodsStandard whole blood staining with appropriate fluorochrome conjugated antibodies to MHC class II and either standard CD14 containing, or an alternate CD66acde containing, lineage cocktail was performed on samples obtained from normal donors and breast cancer patients before and after administration of dose-dense, cytotoxic chemotherapy with daily GM-CSF hematopoetic growth factor support. Putative DCs were enumerated by standard flow cytometry. Data set differences were evaluated using two tailed Mann-Whitney or Wilcoxon signed rank tests. Cellular morphology was examined in cell-sorted populations from post GM-CSF samples.ResultsUse of either antibody cocktail defined comparably sized lineage negative, MHC class II positive populations in normal donors and at baseline in cancer patients. However, selection of lineage negative subsets with increasing MHC class II expression levels yielded larger putative DC populations identified with the alternate cocktail. Both cocktails yielded highly reproducible data. Use of the alternate cocktail: 1) yielded a putative DC population, post GM-CSF that was more homogenous and consistent with DCs, 2) resulted in less data variation across gating strategies, and 3) resulted in more uniform and concordant longitudinal data, consistent with established GM-CSF biological activity.ConclusionAn alternative lineage negative cocktail substituting anti-CD66 antibody for anti-CD14 is a viable option for enumerating the circulating DC population, potentially more accurately defining the circulating DC pool by including CD14 positive immature DCs, and thus, may give more reliable data, particularly in the setting of sustained GM-CSF administration.

Role for Epithelial Dysregulation in Early-onset Colitis-associated Colon Cancer in Gi2-alpha−/− Mice

Background: Inflammatory bowel disease (IBD) is a risk factor for developing colorectal cancer but the mechanisms are poorly characterized. Mice lacking the G‐protein alpha subunit Gi2‐alpha spontaneously develop colitis and colon cancer with high penetrance. Compared to canonical Wnt/APC signaling‐based animal models of colon cancer, the tumors in Gi2‐alpha−/− mice more closely recapitulate the features of IBD‐associated cancers seen in humans. They are predominantly right‐sided, multifocal, mucinous, and arise from areas of flat dysplasia. Methods: In evaluating the potential contribution of epithelial Gi2‐alpha signaling to this phenotype, we found that Gi2‐alpha−/− colonic epithelium is hyperproliferative even before the onset of colitis, and resistant to the induction of apoptosis. We generated colon cancer cell lines overexpressing dominant‐negative Gi2‐alpha. Results: Like other cells lacking Gi2‐alpha, these cells release less arachidonic acid, an important antiinflammatory and epithelial growth regulator. They are also hyperproliferative and resistant to camptothecin‐induced apoptosis and caspase‐3 activation. Conclusions: The colitis‐associated cancers in Gi2‐alpha−/− mice appear very similar to those seen in human IBD patients, and Gi2‐alpha is a direct negative regulator of colonic epithelial cell growth.

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