Modulation of LEF/TCFs and Wnt Signaling in Colon Cancer

Oncogenic Wnt signaling is implicated as the major driving force in colon cancer. Actions of misregulated Wnt signaling rely on the upregulation of Lymphoid Enhancer Factor/ T-cell Factor (LEF/TCF) transcription factor-dependent Wnt target genes. In colon cancer, Wnt-activating, full-length LEF/TCFs are expressed, while their dominant negative, Wnt-suppressing counterparts (dnLEF/TCFs) are not. Therefore, full-length isoforms act unopposed, leading to overactive, inappropriate expression of target genes. We use re-expression of dnLEF/TCFs as a tool to modulate Wnt signaling in colon cancer and discover what cancer phenotypes and gene programs are contributing to the oncogenic drive to cell transformation. Using expression of dnLEF-1 and dnTCF-1, this thesis illustrates how Wnt/β-catenin signaling directs Warburg metabolism. We identify Pyruvate dehydrogenase kinase 1 (PDK1) - a kinase that directs the conversion of pyruvate to lactate, and SLC16A1/MCT-1 - a transporter of small metabolites such as lactate, as important direct targets within a larger gene program for metabolism. Given recent preclinical development of small molecules that target Wnt signaling and metabolism, and given interest in developing new combination therapies for cancer treatment, we tested how Wnt inhibition affects the ability of 3-bromopyruvate to kill cancer cells in vitro. We report that this toxic molecule kills colon cancer cells, but that Wnt signaling inhibition lowers its efficacy. We conclude that both PDK1 and MCT-1 are part of a core Wnt gene program for glycolysis in colon cancer and that modulation of this program could play an important role in shaping sensitivity to drugs that target cancer metabolism. Wnt ligands are secreted morphogens that control multiple developmental processes during embryogenesis and adult homeostasis. A diverse set of receptors and signals have been linked to individual Wnts, but the lack of tools for comparative analysis has limited the ability to determine which of these signals are general for the entire Wnt family, and which define subsets of differently acting ligands. We have created a versatile Gateway expression library of clones for all 19 human Wnts. An analysis comparing epitope-tagged and untagged versions of each ligand shows that despite their similar expression at the mRNA level, Wnts exhibit considerable variation in stability, processing and secretion. This comprehensive toolkit provides critical tools and new insights into human Wnt gene expression and function

Modulation of LEF/TCFs and Wnt Signaling in Colon Cancer

Oncogenic Wnt signaling is implicated as the major driving force in colon cancer. Actions of misregulated Wnt signaling rely on the upregulation of Lymphoid Enhancer Factor/ T-cell Factor (LEF/TCF) transcription factor-dependent Wnt target genes. In colon cancer, Wnt-activating, full-length LEF/TCFs are expressed, while their dominant negative, Wnt-suppressing counterparts (dnLEF/TCFs) are not. Therefore, full-length isoforms act unopposed, leading to overactive, inappropriate expression of target genes. We use re-expression of dnLEF/TCFs as a tool to modulate Wnt signaling in colon cancer and discover what cancer phenotypes and gene programs are contributing to the oncogenic drive to cell transformation. Using expression of dnLEF-1 and dnTCF-1, this thesis illustrates how Wnt/β-catenin signaling directs Warburg metabolism. We identify Pyruvate dehydrogenase kinase 1 (PDK1) - a kinase that directs the conversion of pyruvate to lactate, and SLC16A1/MCT-1 - a transporter of small metabolites such as lactate, as important direct targets within a larger gene program for metabolism. Given recent preclinical development of small molecules that target Wnt signaling and metabolism, and given interest in developing new combination therapies for cancer treatment, we tested how Wnt inhibition affects the ability of 3-bromopyruvate to kill cancer cells in vitro. We report that this toxic molecule kills colon cancer cells, but that Wnt signaling inhibition lowers its efficacy. We conclude that both PDK1 and MCT-1 are part of a core Wnt gene program for glycolysis in colon cancer and that modulation of this program could play an important role in shaping sensitivity to drugs that target cancer metabolism. Wnt ligands are secreted morphogens that control multiple developmental processes during embryogenesis and adult homeostasis. A diverse set of receptors and signals have been linked to individual Wnts, but the lack of tools for comparative analysis has limited the ability to determine which of these signals are general for the entire Wnt family, and which define subsets of differently acting ligands. We have created a versatile Gateway expression library of clones for all 19 human Wnts. An analysis comparing epitope-tagged and untagged versions of each ligand shows that despite their similar expression at the mRNA level, Wnts exhibit considerable variation in stability, processing and secretion. This comprehensive toolkit provides critical tools and new insights into human Wnt gene expression and function

Lactate/pyruvate transporter MCT-1 is a direct Wnt target that confers sensitivity to 3-bromopyruvate in colon cancer.

BackgroundThere is increasing evidence that oncogenic Wnt signaling directs metabolic reprogramming of cancer cells to favor aerobic glycolysis or Warburg metabolism. In colon cancer, this reprogramming is due to direct regulation of pyruvate dehydrogenase kinase 1 (PDK1) gene transcription. Additional metabolism genes are sensitive to Wnt signaling and exhibit correlative expression with PDK1. Whether these genes are also regulated at the transcriptional level, and therefore a part of a core metabolic gene program targeted by oncogenic WNT signaling, is not known.ResultsHere, we identify monocarboxylate transporter 1 (MCT-1; encoded by SLC16A1) as a direct target gene supporting Wnt-driven Warburg metabolism. We identify and validate Wnt response elements (WREs) in the proximal SLC16A1 promoter and show that they mediate sensitivity to Wnt inhibition via dominant-negative LEF-1 (dnLEF-1) expression and the small molecule Wnt inhibitor XAV939. We also show that WREs function in an independent and additive manner with c-Myc, the only other known oncogenic regulator of SLC16A1 transcription. MCT-1 can export lactate, the byproduct of Warburg metabolism, and it is the essential transporter of pyruvate as well as a glycolysis-targeting cancer drug, 3-bromopyruvate (3-BP). Using sulforhodamine B (SRB) assays to follow cell proliferation, we tested a panel of colon cancer cell lines for sensitivity to 3-BP. We observe that all cell lines are highly sensitive and that reduction of Wnt signaling by XAV939 treatment does not synergize with 3-BP, but instead is protective and promotes rapid recovery.ConclusionsWe conclude that MCT-1 is part of a core Wnt signaling gene program for glycolysis in colon cancer and that modulation of this program could play an important role in shaping sensitivity to drugs that target cancer metabolism

Additional file 4: Figure S4. of Lactate/pyruvate transporter MCT-1 is a direct Wnt target that confers sensitivity to 3-bromopyruvate in colon cancer

MCT-1 and β-catenin staining in human colon tumor samples. Immunohistochemical staining of four human colon tumor samples (β-catenin and MCT-1) shows heterogeneous patterns for both β-catenin and MCT-1. β-catenin and MCT-1 were stained in adjacent tumor slices. Images shown at ×20 magnification. (PDF 31 kb

Additional file 1: Figure S1. of Lactate/pyruvate transporter MCT-1 is a direct Wnt target that confers sensitivity to 3-bromopyruvate in colon cancer

Blocking Wnt with XAV939 reduces MCT-1 levels. qRT-PCR analysis was performed on RNA collected from SW480 (A), SW620 (B) HCT116 (C) and DLD-1 (D) cells treated with XAV939 (10 μM) for 24 h. Graphs shown represent the average of three trials (+/− SEM). Whole cell lysates from each cell line (A-D) were harvested 72 h (for MCT-1) and 24 h (for β-catenin) after XAV939 treatment (10 μM) and were probed with the antibodies shown. (*p value < 0.05; **p value < 0.01; ***p value < 0.001). (PDF 678 kb

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

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

Rational identification of a colorectal cancer targeting peptide through phage display

Colorectal cancer is frequently diagnosed at an advanced stage due to the absence of early clinical indicators. Hence, the identification of new targeting molecules is crucial for an early detection and development of targeted therapies. This study aimed to identify and characterize novel peptides specific for the colorectal cancer cell line RKO using a phage-displayed peptide library. After four rounds of selection plus a negative step with normal colorectal cells, CCD-841-CoN, there was an obvious phage enrichment that specifically bound to RKO cells. Cell-based enzyme-linked immunosorbent assay (ELISA) was performed to assess the most specific peptides leading to the selection of the peptide sequence CPKSNNGVC. Through fluorescence microscopy and cytometry, the synthetic peptide RKOpep was shown to specifically bind to RKO cells, as well as to other human colorectal cancer cells including Caco-2, HCT 116 and HCT-15, but not to the normal non-cancer cells. Moreover, it was shown that RKOpep specifically targeted human colorectal cancer cell tissues. A bioinformatics analysis suggested that the RKOpep targets the monocarboxylate transporter 1, which has been implicated in colorectal cancer progression and prognosis, proven through gene knockdown approaches and shown by immunocytochemistry co-localization studies. The peptide herein identified can be a potential candidate for targeted therapies for colorectal cancer.Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte and the Project FCOMP-01–0124-FEDER-021053 (PTDC/SAU-BMA/121028/2010). Débora Ferreira is recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Franklin L. Nobrega, Sara Granja and Ligia R Rodrigues acknowledge FCT for the grants SFRH/BD/86462/2012, SFRH/BPD/117858/2016 and SFRH/BSAB/142991/2018, respectively. Catarina Barbosa-Matos also acknowledge her research grant UMINHO/BI/395/2018info:eu-repo/semantics/publishedVersio