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

Abstract

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.

Keywords: Wnt; angiogenesis; colon cancer; fluorescence lifetime imaging; metabolism.

Figure 1. Blocking Wnt alters the expression of metabolism-linked genes

Panther Gene Ontology analysis of microarray data from DLD-1 cells overexpressing either dnLEF-1 (23 h), dnTCF-1Emut (8 h), or dnTCF-1EWT (8 h).

1. Schematic of dnLEF/TCF isoforms expressed (beige box = context-dependent regulatory domain; red box = HMG DNA binding domain; yellow box = nuclear localization signal; green box = alternatively spliced C-terminal tails; yellow star = mutation in E-tail).

2. Ontology analysis of dnLEF/TCF-downregulated genes reveals a large category of genes linked to metabolism. PANTHER binomial statistical analysis determined statistically significant overrepresentation of regulated genes within each category compared to representation in the human genome (*P-value < 0.01).

3. List of metabolism-linked genes downregulated by all three dnLEF/TCF isoforms. List of transporter genes regulated by at least 2 dnLEF/TCF isoforms. For all genes on the list changes are P < 0.05, and range of fold changes is −1.3 to −6.0.

Figure 2. Blocking Wnt alters the metabolic program of colon cancer cells

A Lactate levels are reduced with lentiviral transduction of dnLEF-1 or dnTCF-1Emut in SW480 cells growing under standard culture conditions for 10 days. Representative graph of three replicates is shown with error bars representing the SEM between three internal replicates. B Fold change of lactate levels produced in 3D cultures. Images are of representative wells for each condition. Measurements performed on media collected from SW480 cells grown in soft agar after 22 days. Representative graph of three replicates is shown with error bars representing the SEM between three internal replicates. C Fold change in lactate levels of SW480 cells treated with Wnt inhibitor XAV939 (10 μM) for a minimum of 4 days. Data represent the average of six independent trials (± SEM). D ATP levels in SW480 cells collected 7 days post-transduction. Data represent the average of three independent trials (± SEM). E–G Fold changes in glucose consumption in SW480 cells expressing dnLEF/TCFs (E), DLD-1 cells [dnLEF-1(2)] treated with doxycycline to induce dnLEF-1 expression (F), and SW480 cells treated with XAV939 (10 μM) (G). Data represent the average of four trials (± SEM). H Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in SW480 cells transduced with MOCK, dnLEF-1, or dnTCF-1Emut virus. Data represent the average of three independent trials (± SD). I Data from (H) represented as an OCR/ECAR ratio. J OCR/ECAR ratio of SW480 cells treated with XAV939 (10 μM). Data represent the average of three independent trials (± SD). Data information: *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001.

Figure 3. Fluorescence lifetime analysis of NADH reveals shifts in glycolysis upon expression of dnLEF/TCF

A Phasor plot of a FLIM analysis showing DLD-1 cells before and after a 1-min treatment with 4 mM potassium cyanide (KCN). The phasor position of pure free NADH is also shown. B, C Top panel of images in (B) shows the autofluorescent intensity at 740 nm. Bottom panels in (B) correspond to the free/bound NADH coloring of the same field of cells according to the color map shown on the phasor plot in (A). Changes indicate an increase in free/bound NADH after KCN treatment. This is also shown in (C), a scatterplot analysis of average phasor positions of individual cells before and after KCN treatment. D Phasor position of DLD-1/dnLEF-1(1) cells, and a color map to represent the gradient of relative levels of free and bound NADH. E–H The top row of images show two-photon fluorescence intensity images excited at 740 nm for two clones of DLD-1 dnLEF-1 stable cells [dnLEF-1(1) and dnLEF-1(2)] and SW480 cells. The bottom row shows free/bound NADH color mapping as indicated by the color map in (D). The bottom panels show scatterplots where each point represents the average phasor position from one cell. Induction of dnLEF-1 or dnTCF-1Emut expression, or treatment with the Wnt inhibitor XAV939, results in a phasor shift toward bound NADH. All images and measurements were taken 5 days after seeding under confluent conditions. Representative data from single trials are shown from among at least three replicate experiments for each cell line. Each treatment resulted in a population on the scatterplot distinct from mock cells with P < 0.0001.

Figure 4. Blocking Wnt directly reduces PDK1 levels via regulation of transcription

A Whole-cell lysates from DLD-1 dnLEF-1(1) cells were collected 48, 72, and 96 h after 0.01 μg/ml doxycycline treatment and were probed with the antibodies shown. SW480 cells were harvested 48 h post-transduction. B, C 9RT-qPCR analysis was performed on RNA collected from DLD-1 dnLEF-1(2) cells harvested 24 h (B) or 120 h (C) after the addition of doxycycline. Graphs shown represent the average of three trials (± SEM). D RT-qPCR analysis was performed on 4-thiouridine-labeled RNA isolated from a 30-min pulse in the presence/absence of dnTCF-1Emut, induced by 2-h doxycycline treatment in DLD-1 cells. Known Wnt target genes Axin2 and Uba52 were used as positive and negative controls, respectively. A representative graph is shown of two replicates, with error bars representing the SD among three internal replicates. E RT-qPCR analysis of chromatin immunoprecipitated from DLD-1 cells with or without induction of FLAG-dnTCF-1Emut, using anti-FLAG antibody. PCR primers designed to detect the indicated PDK1 genomic regions show that dnTCF-1Emut associates with distal regions that flank the PDK1 locus. A representative graph is shown of two replicates, with error bars representing the SD among three internal replicates. F Luciferase reporter activity in SW480 cells shows that Peak 1 and Peak 2 regions confer elevated transcription activity to the heterologous thymidine kinase (TK) promoter. Expression of transduced dnLEF-1, or treatment with the Wnt inhibitor XAV939 (10 μM), eliminates the regulatory activity of these fragments. Graph shown represents the average of three independent replicates (± SD). Data information: *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001). Source data are available online for this figure.

Figure 5. PDK1 overexpression rescues the altered metabolic phenotype induced by blocking Wnt

1. Western blot analysis of lysates collected from DLD-1 dnLEF-1(2) cells treated with or without 0.01 μg/ml doxycycline for 24 h and with or without PDK1 lentivirus for 72 h. FLIM imaging was performed at confluency (96 h doxycycline and 7 days post-transduction). Intensity images are shown on the left. FLIM results, shown through the free/bound NADH color mapping (right), as well as the scatterplot, show that doxycycline induction of dnLEF-1 shifted the phasor toward bound NADH, while PDK1 rescue shifted it back to its original position (P < 0.0001 comparing −Dox to +Dox and comparing +Dox to +Dox+PDK1).

2. FLIM analysis of SW480 cells treated with 50 mM DCA for 48 h shows a phasor shift toward bound NADH (P < 0.0001 comparing +DCA to mock).

3. OCR/ECAR ratio of DCA (10 mM)-treated SW480 cells shows that blocking PDK1 activity doubles the oxygen consumption rate (mitochondrial activity) relative to the extracellular acidification rate (glycolysis-produced lactate). Data shown represent the average of three independent trials (± SD; *P-value < 0.05).

4. Sulforhodamine B cell proliferation assay of SW480 cells treated with or without 2.5 μM irinotecan, 20 mM DCA, and dnLEF-1 lentivirus shows an increased sensitivity to irinotecan when treated with DCA or dnLEF-1. A representative graph of two trials is shown. Error bars represent the standard deviation between eight internal replicates.

Source data are available online for this figure.

Figure 6. Blocking Wnt reduces in vivo tumor growth

1. Expression of dnLEF-1 or dnTCF-1Emut in SW480 cells results in smaller xenograft tumors. Images of tumors are shown with quantification of the tumor mass and volume at the time of harvest. Data include measurements of eight tumors for each condition. Error bars represent the SEM among eight replicates (***P-value < 0.001).

2. Ki67 staining of paraffin-embedded sections from xenograft tumors shows fewer Ki67-positive cells with dnLEF-1/dnTCF-Emut expression. Data shown represent the average of counts from at least eight fields. Error bars represent the SEM among at least eight replicates (*P-value < 0.05).

3. Western blot was performed on protein lysates prepared from freshly extracted xenograft tumors. Western blot analysis of endogenous PDK1 and its target, pyruvate dehydrogenase (pSer293-PDH), shows decreases when dnLEF-1 or dnTCF-1Emut tumors are expressed. cMYC expression shows variable levels of expression.

4. Phasor plot representation for the color mapping of in vivo tumor FLIM analysis, fluorescence intensity (top image panels), FLIM color mapping (bottom panels), and scatterplot analysis are as described in Fig5 except that these analyses were performed on living, surgically exposed, yet still actively perfused, xenograft tumors. Both the free/bound NADH color mapping (bottom row) and scatterplot (average phasor position of individual cells within each tumor) show a shift in the phasor position toward bound NADH with dnLEF-1 expression and a return to free NADH with PDK1 overexpression (P < 0.0001 comparing dnLEF-1 to mock and comparing dnLEF-1+PDK1 to dnLEF-1). A minimum of three fields of view per tumor were analyzed. Data shown are from one mouse representative of eight replicates (additional tumor analysis in Supplementary Fig S7).

Source data are available online for this figure.

Figure 7. Blocking Wnt reduces tumor vessel density, while reintroduction of PDK1 restores it

A Tumor vessel density within the subcapsular region is greatly diminished with dnLEF/TCF, but is restored with PDK1 expression. Images show FITC dextran-labeled vasculature in the xenograft tumors (˜ 50–200 μm deep). B Quantification of vessel density derived from the CD31 staining of paraffin-embedded sections of each tumor. For quantification, the number of vessels per field of view (40× objective) was averaged from every cross-section (at least 20 for each condition) within the subcapsular region for each tumor. Error bars represent the SEM among at least 20 replicate fields. C Western blot analysis was performed on protein lysates prepared from freshly extracted xenograft tumors. D, E RT-qPCR analysis of (D) HIF-1α and (E) VEGF expression in xenograft tumor RNA. HIF-1α mRNA levels do not change among the various tumors (normalized to GAPDH), but VEGF expression levels are downregulated in the dnLEF-1 and dnTCF-1Emut tumors. However, PDK1 rescue expression does not change the levels of either HIF-1α or VEGF mRNA. RT-qPCR data shown are representative of four replicate tumor sets. Error bars represent the standard error between three internal replicates. F Lactate levels in dnLEF/TCF-expressing tumors are reduced compared to MOCK levels and are partially restored with PDK1 rescue expression. Data show the average fold lactate levels (compared to mock) in five tumors for each condition, with error bars representing the SEM (*P-value < 0.05; ***P-value < 0.001). Source data are available online for this figure.

Figure 8. Wnt promotes aerobic glycolysis through PDK1

1. Model of Wnt regulation of cancer metabolism and angiogenesis through PDK1.

2. Immunohistochemical staining of normal human small intestine (PDK1) and normal human colon (phospho-PDH and HIF1α) shows correlations between high levels of Wnt signaling, PDK1 activity, and HIF1α levels. Bottom row shows higher-power images of boxed portions in the top row.