Research Interests: Biology, Biological Sciences, DNA, Cell line, Environmental Sciences, and 15 moreHumans, Animals, Alternative splicing, Expression, Genes, Introns, Domain, HeLa cells, Enhancer, Alternative Splicing, Amino Acid Sequence, Base Sequence, DNA binding proteins, Gene Expression Regulation, and binding sites
Research Interests: Fluorescence Microscopy, Biology, Cell Biology, Metastasis, Cancer Research, and 15 moreTranscription Factors, Medicine, Extracellular Matrix, Angiogenesis, Western blotting, Integrative Biology, In Vitro, Fibrin, Humans, Anoxia, Endothelial Cells, In Vivo, Neoplasms, Vasculogenic Mimicry, and In Vitro Techniques
The constitutive tyrosine kinase activity of Bcr-Abl leads to aberrant expression of multiple genes by several mechanisms, including dysregulation of transcription. Recently though, increasing attention has been focused on the effect of... more
The constitutive tyrosine kinase activity of Bcr-Abl leads to aberrant expression of multiple genes by several mechanisms, including dysregulation of transcription. Recently though, increasing attention has been focused on the effect of Bcr-Abl in dysregulating translation. Our group has previously documented the effects of Bcr-Abl on key regulators of cap-dependent translation and the role that this mechanism plays in transformation (Ly et al, Cancer Research, 2003; Prabhu et al., Oncogene, in press). Here we describe a novel form of translational control by Bcr-Abl. Specifically, we show how Bcr-Abl regulates cap-independent translation of Lymphoid Enhancer Factor-1 (LEF-1) via a bona fide internal ribosome entry site (IRES) in the 5′ untranslated region (UTR) of LEF1. Lymphoid Enhancer Factor-1 (LEF-1), a transcription factor that mediates Wnt signals via interaction with β-catenin, is often expressed in cancers derived from aberrant Wnt signaling. Lately, it has been reported that LEF1 transcripts are elevated in CML. We examined LEF-1 expression in primary CML cells and cell lines (K562 and Ba/F3-Bcr-Abl) and show that LEF-1 protein is detected in all patient-derived cells. Treatment of these cells with the Bcr-Abl imatinib mesylate (imatinib) inhibits LEF-1 expression in imatinib-sensitive cancers, but not in cancers that exhibit clinical resistance even though such cancers express imatinib-sensitive Bcr-Abl. For those cancers that are sensitive, inhibition of Bcr-Abl has a partial effect on LEF1 mRNA levels, and a significant effect on LEF-1 protein levels. LEF-1 protein is produced via two IRESs in it’s 5′ UTR. IRES-driven translation of LEF-1 was highly sensitive to Bcr-Abl as treatment with imatinib reduced IRES activity 5 fold. Transfection of CML cells with dicistronic mRNAs suggests that Bcr-Abl stimulates LEF-1 protein production through steps in the nucleus and cytoplasm. We propose that, in addition to its strong effects on cap-dependent translation in CML, Bcr-Abl is an important regulator of alternative translation pathways. mRNAs that are translated via IRES-dependent mechanisms are particularly relevant to cancer since they encode proteins that regulate cell proliferation and survival. Together, these observations underscore the important role which dysregulated translation plays in transformation, and suggest novel approaches with which to counteract the transforming properties of Bcr-Abl.
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Introduction: Tumor growth is dramatically affected by the microenvironment, including supporting cells such as the stroma and vasculature, mechanical factors, such as interstitial flow and extracellular matrix and aspects of the tumor... more
Introduction: Tumor growth is dramatically affected by the microenvironment, including supporting cells such as the stroma and vasculature, mechanical factors, such as interstitial flow and extracellular matrix and aspects of the tumor mass itself, including shape. However, current tumor growth models, including xenograft models, 2D and/or simplistic 3D cultures, are unable to address these interactions in a high throughput human-derived system. We have developed a novel microfluidic platform that combines human derived perfused microvessels, stroma, and interstitial flow with 3D culture. This platform was used to quantitatively compare the role of these microenvironmental factors on tumor growth. Methods: A microfluidic device was fabricated consisting of two supply channels on either side of a central tissue compartment. The inner stromal compartment consists of normal human fibroblasts (NHLFs) and GFP-labeled human colorectal adenocarcinoma tumor cells, SW620, seeded in a fibrin matrix. To simulate a vasculogenic-like process, human cord blood endothelial colony forming cells endothelial cells (ECFC-ECs) were distributed throughout the stromal channel with the fibroblasts and tumor cells. Tumor growth rate and area was compared across day, interstitial flow rates and tumor shape (fractal dimension, perimeter to area) with ANOVA. Results: Cell viability within the device was maintained under interstitial flow conditions for a period of 21 days. Within one week of culture, microvessel formation and significant tumor growth into spheroids (n=636) were observed. On average, tumor growth rate was 26% ±62% per day with the highest growth rates observed on the first days. By day 7, many tumor masses had died off, with 2-3 large, fast growing tumors remaining per chamber. Highest tumor growth rates and areas were observed in tumor masses with a characteristic morphology of high perimeter to area and lower cohesion. Interstitial flow rates ranging from essentially static to supraphysiologic were generated. Differences in tumor growth rates were not statistically significant across chambers with different mean flow rates. To demonstrate intraluminal flow within the vascular network, fluorescently labeled dextran (40, 70, and 150 kDa) was introduced into the microfluidic lines. Dextran was retained in the vessel network, and showed tumor cells residing in the intraluminal space of the formed vasculature. By day 14, the network eroded, as the tumor masses overgrew and encompassed more than 60% of the chamber volume. Discussion: We have developed a novel 3D microfluidic system of the tumor microenvironment that features perfused capillaries and controlled interstitial flow. Tumor growth was affected by tumor characteristic shape in this model though interstitial flow appeared to play a lesser role. Vascular development was observed and its interaction with tumor growth will be analyzed in future work. Citation Format: Luis F. Alonzo, Claire J. Robertson, Monica L. Moya, Marian L. Waterman, Christopher C. Hughes, Steven C. George. Recapitulating the microenvironment in vitro for comparative study of factors affecting tumor growth and vascularization. [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 3930. doi:10.1158/1538-7445.AM2014-3930
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In experiments designed to study the mechanism by which peptide hormones binding to their plasma membrane receptors stimulate the expression of specific genes, the transcription of two neuroendocrine genes, prolactin and growth hormone,... more
In experiments designed to study the mechanism by which peptide hormones binding to their plasma membrane receptors stimulate the expression of specific genes, the transcription of two neuroendocrine genes, prolactin and growth hormone, was analyzed in a rat pituitary cell line. The results showed that cyclic adenosine monophosphate (cyclic AMP) stimulates the transcription of discrete subsets of eukaryotic genes by at least two independent molecular mechanisms. Cyclic AMP stimulated growth hormone gene transcription and phosphorylation of a 19,000-dalton nuclear protein; this appears to reflect direct nuclear actions of the cyclic AMP-dependent protein kinase. In contrast, the stimulation by cyclic AMP of prolactin gene transcription appears to reflect activation of a discrete calcium-dependent event.
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Research Interests: Biology, Membrane Proteins, Cell Biology, Medicine, Signal Transduction, and 15 moreHumans, Mice, G protein-coupled receptors, Secretory Pathway, Wnt Signaling, Animals, Beta-Catenin, T cells, Differentiation, LRP, Secretion, Biochemistry and cell biology, Gene expression profiling, G Protein Coupled Receptors, and Paediatrics and reproductive medicine
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Research Interests: Crystallography, Biology, Medicine, Signal Transduction, Biological Sciences, and 15 moreCercopithecus aethiops, Cell line, Humans, Mutation, Mice, Methylation, Wnt Signaling, Animals, Structural equation models, Chromatin, Histones, Protein Binding, Gene Expression Regulation, Tetramer, and Medical and Health Sciences
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Research Interests: Molecular Biology, Biology, Transcription Factors, Medicine, T cell receptor, and 12 moreBiological Sciences, Humans, T lymphocytes, Enhancer, S100 protein family, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, High mobility group proteins, Psychology and Cognitive Sciences, Thymus Gland, and Medical and Health Sciences
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Genetic-driven deregulation of the Wnt pathway is crucial but not sufficient for colorectal cancer (CRC) tumourigenesis. Here, we show that environmental glutamine restriction further augments Wnt signaling in APC mutant intestinal... more
Genetic-driven deregulation of the Wnt pathway is crucial but not sufficient for colorectal cancer (CRC) tumourigenesis. Here, we show that environmental glutamine restriction further augments Wnt signaling in APC mutant intestinal organoids to promote stemness and leads to adenocarcinoma formation in vivo via decreasing intracellular alpha-ketoglutarate (aKG) levels. aKG supplementation is sufficient to rescue low-glutamine induced stemness and Wnt hyperactivation. Mechanistically, we found that aKG promotes hypomethylation of DNA and histone H3K4me3, leading to an upregulation of differentiation-associated genes and downregulation of Wnt target genes, respectively. Using CRC patient-derived organoids and several in vivo CRC tumour models, we show that aKG supplementation suppresses Wnt signaling and promotes cellular differentiation, thereby significantly restricting tumour growth and extending survival. Together, our results reveal how metabolic microenvironment impacts Wnt signaling and identify aKG as a potent antineoplastic metabolite for potential differentiation therapy for CRC patients.
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TCF‐1 and TCF‐4 are the major transducers of Wnt signaling in the intestine and the oncogenic drivers of colon cancer. However, knock‐out of the TCF1 gene leads to intestinal tumors while knockout of TCF‐4 suppresses growth and... more
TCF‐1 and TCF‐4 are the major transducers of Wnt signaling in the intestine and the oncogenic drivers of colon cancer. However, knock‐out of the TCF1 gene leads to intestinal tumors while knockout of TCF‐4 suppresses growth and maintenance of crypt stem cells. These phenotypes suggest that TCF‐4 is Wnt‐promoting and TCF‐1 is a tumor suppressor. We show that a truncated dominant negative isoform of TCF‐1 (dnTCF‐1) predominates in the normal colon and is equally distributed between nuclear and cytoplasmic compartments in normal crypts. However, in primary human colon tumors, TCF‐1 is often cytoplasmic while TCF‐4 remains nuclear. This shift to the cytoplasm is regulated by a CaMKII/TAK1/NLK kinase cascade that triggers secretion of a Wnt ligand to selectively export TCF‐1 from the nucleus. In colon cancer, TCF‐1 expression is altered so that dnTCF‐1 is downregulated while to full‐length, Wnt‐promoting isoforms are upregulated. Our study shows that control of nuclear export is an impor...
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Research Interests: Genetics, Biology, Medicine, Colorectal cancer, Signal Transduction, and 15 moreHumans, Alternative splicing, Transcription Factor, Cell nucleus, Cancer Metastasis, Colon cancer, Enhancer, Alternative Splicing, Protein isoforms, Colon Carcinoma, Protein Binding, DNA binding proteins, Nuclear Localization, Colorectal Neoplasms, and Activation Function
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Standard-of-care treatment for metastatic colorectal cancer combines chemotherapy with bevacizumab, an angiogenesis inhibitor that depletes nutrients and triggers hypoxia. Unfortunately, this strategy extends patient survival only a few... more
Standard-of-care treatment for metastatic colorectal cancer combines chemotherapy with bevacizumab, an angiogenesis inhibitor that depletes nutrients and triggers hypoxia. Unfortunately, this strategy extends patient survival only a few months because tumors acquire adaptive resistance, enabling resumption of angiogenesis. Adaptive resistance arises from reprogrammed metabolism, but the regulatory networks that govern reprogramming are not defined. Our studies have shown that overactive Wnt signaling, which is the most common cause of colon cancer, programs cancer metabolism by promoting glycolysis and angiogenesis. Interestingly, metabolic genes targeted by Wnt are also regulated by the hypoxia transcription factor HIF1alpha suggesting there may be crosstalk between the two pathways. Several studies suggest that HIF1alpha can influence Wnt signaling, but the reported effects are variable ranging from positive to negative. We find that HIF1alpha and LEF/TCF/beta-catenin complexes co-regulate metabolic targets including LEF1 and TCF1 (TCF7) expression. Using luciferase reporters driven by LEF/TCF promoters, we observe significantly increased LEF1/TCF1 but not TCF3(TCF7L1)/TCF4(TCF7L2) promoter activity in hypoxic colon cancer cells. We identified putative hypoxia responsive elements (HREs) in the human LEF1 promoter and deletion of these HREs reduced responsiveness to hypoxia. Furthermore, when colon cancer cells were treated with inhibitors that either inactivate or reduce HIF1alpha protein, there was a significant decrease in Wnt reporter activity. These results suggest that hypoxia and the Wnt pathway crosstalk wherein hypoxia co-regulates metabolism genes and increases Wnt signaling capacity via LEF/TCF expression. Current studies are underway to investigate the mechanisms of crosstalk and joint regulation of metabolic gene programs during adaptive resistance. Citation Format: Yung Lyou, Amber Habowski, Stephanie Sprowl-Tanio, Kira Pate, George Chen, Marian L. Waterman. Hypoxia via HIF1alpha can regulate Wnt signaling in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5494. doi:10.1158/1538-7445.AM2017-5494
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While there have been significant advances in using 3D culture systems to recapitulate physiological aspects of the tumor microenvironment, few systems are able to capture the full range of authentic, complex in vivo processes such as... more
While there have been significant advances in using 3D culture systems to recapitulate physiological aspects of the tumor microenvironment, few systems are able to capture the full range of authentic, complex in vivo processes such as neovascularization and metastasis. Furthermore, these systems generally utilize established cell lines which compromise the clinical relevance. The goal of this study was to create a 3D in vitro model of patient-specific vascularized tumor tissue that could be used to evaluate the sensitivity to F-fluoruracil (5-FU). Colon cancer cells isolated directly from patient biopsies and endothelial colony forming cell-derived endothelial cells (EC) from cord blood were combined to form multicellular spheroids, and then embedded in a fibrin gel containing normal stromal cells (primary human lung fibroblasts). After 7 days in culture, these “prevascularized tumors” (PVTs) exhibited robust sprouting angiogenesis into the surrounding matrix, and also formed a distinct and contiguous vessel network within the spheroid itself. Network formation and branching of the sprouting vessels was comparable to those generated by co-culture spheroids of EC and cells from the colon cancer cell line SW620. These spheroids in particular demonstrated intravasation of single cells within the lumens of the vessel networks. This phenomenon was enhanced in low oxygen, and abrogated by silencing the transcription factor Slug. Thus, this phenomena is consistent with endothelial-mesenchymal transition. The PVTs have furthermore been used to distinguish the effects of EC on SW620 viability in response to 5-FU. Spheroids composed of EC and SW620 cells demonstrated decreased sensitivity to low dosage 5-FU treatment compared to spheroids composed of SW620 cells only, implicating potential EC-induced drug resistance. Our results demonstrate the utility of a 3D model system of the tumor microenvironment that may be used to study intravasation, microvessel formation, and sensitivity to anti-cancer drugs. Citation Format: Seema M. Ehsan, Masahiro Inoue, Marian L. Waterman, Christopher CW Hughes, Steven C. George. Drug sensitivity of vascularized tumor tissue in vitro. [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 14. doi:10.1158/1538-7445.AM2014-14
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Standard-of-care treatment for KRAS mutant metastatic colorectal cancer (mCRC) combines a cytotoxic chemotherapy backbone (FOLFOX) with an antiangiogenesis biologic agent (i.e., bevacizumab). Despite significant efforts to improve... more
Standard-of-care treatment for KRAS mutant metastatic colorectal cancer (mCRC) combines a cytotoxic chemotherapy backbone (FOLFOX) with an antiangiogenesis biologic agent (i.e., bevacizumab). Despite significant efforts to improve treatment, the prognosis for this type of cancer remains poor with an average 5-year overall survival of ~11%. Oncogenic KRAS mutations lead to the overactivation of RAS/ERK/MAPK pathwa,y driving many hallmarks of cancer such as cell proliferation, growth, and altered metabolism. In particular, several studies have shown that oncogenic activation of KRAS leads to normoxic stabilization of HIF1A protein, which can then reprogram cellular metabolism towards aerobic glycolysis. Our studies have discovered that overactive Wnt signaling can also reprogram cancer cell metabolism towards aerobic glycolysis. Of note, KRAS (~40%) and Wnt signaling (~90%) are very frequently coactivated in colorectal cancer. Interestingly, an important, key metabolic gene targeted by Wnt signaling, Pyruvate Dehydrogenase Kinase I (PDK1), is also directly regulated by HIF1A, suggesting there may be crosstalk between the HIF1A and Wnt pathways. Indeed, several published studies support the idea that HIF1A can influence Wnt signaling, and our data show this may occur through regulation of the Wnt mediating transcription factors LEF1 and TCF1 (TCF7 gene). Using luciferase reporters driven by LEF/TCF promoters, we observed significantly increased LEF1/TCF7 promoter activity in hypoxic colon cancer cells but not for other TCF family members (TCF7L1/TCF7L2). We identified putative hypoxia responsive elements (HREs) in the human LEF1 promoter, and deletion of these HREs reduced responsiveness to hypoxia. Furthermore, we have found that targeting glycolysis and Wnt can be synergistic in inhibiting colon cancer cell colony growth. We find that HIF1A and LEF/TCF/beta-catenin complexes coregulate gene expression of metabolic targets as well as Wnt signaling components (e.g., LEF1 and TCF7). These data suggest that cross-regulation between the HIF1A and Wnt signaling pathway is important and may be a potential Achilles heel for novel therapeutics in targeting KRAS mutant colon cancer. Current studies are under way to further investigate the mechanisms of crosstalk and joint regulation of metabolic reprogramming in KRAS mutant colon cancer cells. Citation Format: Yung Lyou, George Chen, Marian Waterman. HIF1A can regulate Wnt signaling in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 385.
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Recognition of a nonclassical Wnt-response element by the transcription factor TCF results in β-catenin acting as a transcriptional repressor of certain Wnt target genes.