The Warburg effect as a therapeutic target for bladder cancers and intratumoral heterogeneity in associated molecular targets

Abstract

Bladder cancer is the 10th most common cancer worldwide. For muscle-invasive bladder cancer (MIBC), treatment includes radical cystectomy, radiotherapy, and chemotherapy; however, the outcome is generally poor. For non-muscle-invasive bladder cancer (NMIBC), tumor recurrence is common. There is an urgent need for more effective and less harmful therapeutic approaches. Here, bladder cancer cell metabolic reprogramming to rely on aerobic glycolysis (the Warburg effect) and expression of associated molecular therapeutic targets by bladder cancer cells of different stages and grades, and in freshly resected clinical tissue, is investigated. Importantly, analyses indicate that the Warburg effect is a feature of both NMIBCs and MIBCs. In two in vitro inducible epithelial-mesenchymal transition (EMT) bladder cancer models, EMT stimulation correlated with increased lactate production, the end product of aerobic glycolysis. Protein levels of lactate dehydrogenase A (LDH-A), which promotes pyruvate enzymatic reduction to lactate, were higher in most bladder cancer cell lines (compared with LDH-B, which catalyzes the reverse reaction), but the levels did not closely correlate with aerobic glycolysis rates. Although LDH-A is expressed in normal urothelial cells, LDH-A knockdown by RNAi selectively induced urothelial cancer cell apoptotic death, whereas normal cells were unaffected-identifying LDH-A as a cancer-selective therapeutic target for bladder cancers. LDH-A and other potential therapeutic targets (MCT4 and GLUT1) were expressed in patient clinical specimens; however, positive staining varied in different areas of sections and with distance from a blood vessel. This intratumoral heterogeneity has important therapeutic implications and indicates the possibility of tumor cell metabolic coupling.

Keywords: Warburg effect; epithelial-mesenchymal transition; intratumoral heterogeneity; lactate dehydrogenase A; non-muscle-invasive and muscle-invasive bladder cancers.

FIGURE 1

Aerobic glycolysis of bladder cancer cell lines. A, Rate of cellular lactate release into the culture media of the indicated bladder cancer cell lines (nmoles of lactate released into the cell culture media per 10⁶ cells per hour). Values presented represent the mean ± SD from a minimum of three independent experiments. Green dotted line represents rate of lactate release by proliferating normal human urothelial cells (NHUC), and the red dotted line represents rate of lactate release by confluent NHUC. B, Rate of cellular glucose consumption from the culture media of the indicated bladder cancer cell lines (nmoles of glucose consumed per 10⁶ cells per hour). Values presented represent the mean ± SD from a minimum of three independent experiments. C, Ratio of the rate of lactate release to the rate of glucose consumption. The ratios are calculated from the mean rates shown in (A) and (B), respectively. Dotted line indicates two molecules of lactate released per glucose consumed. D, Box and whisker plot showing the median, mean (x), and minimum and maximum rates of cellular lactate release by cell lines clustered by grade (left) or stage (right). E, Box and whisker plot showing the median, mean (x), and minimum and maximum rates of cellular lactate release by cell lines clustered by p53 status. F, Box and whisker plot showing the median, mean (x), and minimum and maximum rates of cellular lactate release by cell lines clustered by Ras mutational status

FIGURE 2

Oxygen consumption rates of bladder cancer cell lines are independent of aerobic glycolysis and cellular rates of lactate production. A, Rate of cellular oxygen consumption of the indicated human bladder cancer cell lines (nmoles of oxygen consumed per 10⁶ cells per minute). Values presented represent the mean ± SD from three independent experiments. Cell lines are ordered from left to right on the basis of cellular lactate release (low to high rate of lactate release). B, Ratio of the rate of cellular oxygen consumption to the rate of cellular lactate release for the indicated human bladder cancer cell lines. The ratios are calculated from the mean rates shown in Figure 2A (O₂ consumed) and Figure 1A (lactate released). Scatter plots of cellular oxygen consumption rate against the rate of cellular lactate release (C), or glucose consumption rate (D) with the mean rates for each individual cell line indicated by a black circle. Box and whisker plots showing the median, mean (x), and minimum and maximum rates of cell line oxygen consumption following cell line clustering by grade (E), p53 status (F), or Ras status (G)

FIGURE 3

Protein expression of LDH‐A, LDH‐B, and phosphorylated pyruvate dehydrogenase (PDH) and correlation analysis with cell line rates of extracellular lactate release. A, Immunoblots showing relative protein expression levels of LDH‐A, LDH‐B, phosphorylated PDH (S293 PDH‐E1a), and total PDH in the indicated bladder cell lines. Actin was used as a loading control with equivalent exposures (normalized to RT112) for all cell line panels. Correlation analysis of cell line mRNA and protein levels for LDH‐A (B) and LDH‐B (C). Mean value for each cell line is represented by a black circle. D, Scatter plot of cell line LDH‐A/LDH‐B mRNA ratio relative to LDH‐A/LDH‐B protein ratio for each cell line. Mean value for each cell line is represented by a black circle; R ² as indicated. E, F, Scatter plots of cell line LDH‐A/LDH‐B protein ratios and PDHP/PDH protein ratios relative to the mean rate of cellular lactate release (Figure 1A), with each individual cell line represented by a black or green circle. For the LDH‐A/LDH‐B protein ratio scatter plot, cell lines above the horizontal red dotted line express more LDH‐A protein relative to LDH‐B. PDHP/PDH total protein ratio is expressed relative to the LUCC8 cell line which showed the highest proportion of phosphorylated PDH relative to total PDH levels (LUCC8 PDHP/PDH ratio arbitrarily set at 1). Cell lines within the two horizontal red dotted lines in (F) represent cell lines with ≥50% of total PDH being phosphorylated at S293 assuming 100% phosphorylation of LUCC8 total PDH. Green circles in both scatter plots represent cell lines which have an LDH‐A/LDH‐B ratio ≥1 and a PDHP/PDH ratio ≥0.5

FIGURE 4

LDH‐A knockdown induces apoptotic cell death of bladder cancer cell lines but not of normal human urothelial cells. A, Immunoblots showing selective depletion of LDH‐A protein by LDH‐A siRNA in the indicated cell lines and effects of LDH‐B siRNA at 72 h following siRNA transfection. LDH‐A/LDH‐B blots for normal human urothelial cells (NHUC) and RT112 bladder cancer cells are overexposed relative to the 97‐7 and UMUC3 blots in order to obtain similar LDH‐A/B protein levels in control‐transfected cells (no siRNA) for all three lines to facilitate comparison of extent of LDH‐A knockdown between the cell lines. Actin was used as a loading control. As a positive control for cell death induction in NHUC, these cells were additionally treated with SIRT1 siRNA. B, Representative phase contrast images of 97‐7, RT112, and UMUC3 human bladder cancer cells and of normal human urothelial cells (NHUCs) 72 h post transfection with the indicated siRNAs. C, Quantification of apoptotic cell death induced by LDH‐A and LDH‐B siRNAs 72 h post transfection as determined by the proportion of annexin V–positive cells; values are expressed as a fold change in the proportion of annexin V–positive cells relative to background levels in control cells

FIGURE 5

Enhanced rates of lactate release following epithelial mesenchymal transition (EMT) induction in an in vitro FGFR1‐inducible bladder cancer model of EMT. A, Immunoblots showing the induction of FGFR1 signaling in FGFR1‐expressing 94‐10 human bladder cancer cells by FGF2, or FGF2 plus heparin, as indicated by increased phosphorylated ERK and increased EMT as indicated by decreased E‐cadherin expression. Effect of FGFR1‐induced EMT on LDH‐A, LDH‐B, and LDH‐A phosphorylation levels are also shown. B, Phase contrast images showing the induction of a mesenchymal‐like morphology (indicated by black arrows) following treatment of FGFR1‐expressing 94‐10 human bladder cancer cells with FGF2 or FGF2 plus heparin, consistent with EMT. C, Effect of EMT induction on the rate of cellular lactate release (nmoles lactate released/10⁶ cells/hour). **P < .001, Student's t‐test

FIGURE 6

Lactate release by freshly resected bladder cancer tissue and intratumoral heterogeneous expression of key targets associated with the Warburg effect. A, Mean rate of lactate release into fresh growth medium per milligram of tumor tissue per hour. Stage and grading of tumor tissue as indicated. Freshly resected TURBTs were weighed prior to overnight (16 h) incubation in 500 µL of fresh growth media at 37°C in a 5% CO₂ incubator and determination of levels of lactate released into the medium during the incubation period (see "Methods” section). Four‐ (or three‐) digit numbers indicate anonymized clinical specimen identifiers for different patient samples. Mean ± SD from three independent lactate determinations. B, Box plot of the mean rates of cellular lactate release by clinical specimens clustered by stage and grade, *P < .05, **P < .01, Student's t‐test. C, Tissue sections of the indicated clinical specimens (analyzed in [A] for lactate release) that have been immunostained for the indicated metabolic targets. Representative IHC images are shown; location of blood vessel (BV) indicated for 2165 specimens

FIGURE 7

mRNA expression of putative targets associated with the Warburg effect in Ta, T1, and muscle‐invasive bladder cancer (MIBC) clinical specimens. mRNA expression analysis (log2) of the indicated targets associated with the Warburg effect (LDH‐A, GLUT1, MCT4) and intratumoral metabolic coupling (lactate import transporter MCT1) in 263 urothelial tumor specimens (125 Ta G2, 106 T1 G3, and 32 MIBC clinical specimens). LDH‐A/LDH‐B indicates ratio of LDH‐A expression relative to LDH‐B. Box and whisker plots showing the median, mean (x), and minimum and maximum expression following clustering by stage. *P < .05, **P < .01; Student's t‐test