Dichloroacetate reverses the hypoxic adaptation to bevacizumab and enhances its antitumor effects in mouse xenografts

Abstract

Inhibition of vascular endothelial growth factor increases response rates to chemotherapy and progression-free survival in glioblastoma. However, resistance invariably occurs, prompting the urgent need for identification of synergizing agents. One possible strategy is to understand tumor adaptation to microenvironmental changes induced by antiangiogenic drugs and test agents that exploit this process. We used an in vivo glioblastoma-derived xenograft model of tumor escape in presence of continuous treatment with bevacizumab. U87-MG or U118-MG cells were subcutaneously implanted into either BALB/c SCID or athymic nude mice. Bevacizumab was given by intraperitoneal injection every 3 days (2.5 mg/kg/dose) and/or dichloroacetate (DCA) was administered by oral gavage twice daily (50 mg/kg/dose) when tumor volumes reached 0.3 cm(3) and continued until tumors reached approximately 1.5-2.0 cm(3). Microarray analysis of resistant U87 tumors revealed coordinated changes at the level of metabolic genes, in particular, a widening gap between glycolysis and mitochondrial respiration. There was a highly significant difference between U87-MG-implanted athymic nude mice 1 week after drug treatment. By 2 weeks of treatment, bevacizumab and DCA together dramatically blocked tumor growth compared to either drug alone. Similar results were seen in athymic nude mice implanted with U118-MG cells. We demonstrate for the first time that reversal of the bevacizumab-induced shift in metabolism using DCA is detrimental to neoplastic growth in vivo. As DCA is viewed as a promising agent targeting tumor metabolism, our data establish the timely proof of concept that combining it with antiangiogenic therapy represents a potent antineoplastic strategy.

Figure 1

Analysis of Bevacizumab treated U87-MG tumors. (A) Tumor growth of U87-MG in vivo, treated with Bevacizumab (BVC) or vehicle control (CTRL), started from day 12 to the end of the experiment. Mean ± SE, N=5. **P<0.01. (B) Immunohistochemical staining of tumor sections from CTRL or BVC treated tumors show increased hypoxia using HIF-1 and CA9; and a marker of vessel morphology using CD34. Main images are 10x magnification, and inset images 20x magnification. Sections were stained and scored for (C) Vessel Q (CD31). (D) CA9 levels. (E) Necrosis. Mean ± SE. *P<0.05; ***P<0.001.

Figure 2

Gene expression changes in control versus Bevacizumab-treated groups. X-axis represents control and Bevacizumab-treated tumors. Array data were preprocessed using GCRMA, normalized using quantile normalization and log base 2. Expression is shown standardized per gene (Z values; color key at the top). Y-axis: Gene Symbols shown on the right. Blue bands on side indicate mitochondrial gene complexes (from light blue =I to dark blue =V). Green band indicate Krebs cycle genes. Red band indicates HIF targets.

Figure 3

Downregulation of metabolic mitochondrial genes in Bevacizumab–resistant tumors. Quantitative PCR analysis was performed to investigate the effects of BVC treatment or hypoxia on U87 cells. For normoxia versus hypoxia comparisons, cells were incubated at 1% oxygen for 72 hr. The following mitochondrial genes were tested: (A) Mitochondrial ribosomal genes. (B). Krebs cycle enzymes. (C) Electron transport complex I and V components. Gene levels are expressed as fold changes by comparison in vivo to PBS controls and in vitro to normoxic controls. Mean ± SE. *P<0.05; **P<0.01; ***P<0.001.

Figure 4

DCA amplifies the effects of BVC on tumor growth in vivo. (A) Tumor growth of U87-MG in vivo, treated with either CTRL, BVC, DCA or BVC/DCA, starting from day 7 until the end of the experiment. Mean ± SE, N=4–6. *P<0.05; (B) Tumor growth of U118 in vivo, treated with either CTRL, BVC, DCA or BVC/DCA. The U118 tumors were slow growing and so day 0 is treatment start time. Mean ± SE, N=4–8. *P<0.05; **P<0.01; (black stars is comparison to CTRL, grey stars to BVC alone); (C) Growth of U87-MG spheroids treated with either vehicle control (CTRL) or 10mM DCA. Spheroids were allowed to grow to 0.2 mm³ before being treated with DCA every 3 days. NB: Error bars are small and within the symbols. (D) Pictures (at 5x magnification) show representative images of the CTRL and DCA treated spheroids.

Figure 5

Effect of Bevacizumab and DCA on U87-MG tumor histology. (A) Immunohistochemical staining for CA9, CD31, Ki-67 (MIB-1) and necrosis in FFPE sections of U87-MG tumor xenografts treated with BVC, DCA, a combination of BVC and DCA or vehicle control (CTRL). Quantification of (B) Percentage of the viable tissue positive for CA9; (C) Vessel score, VQ, (CD31); (D) proliferation index, PQ, (Ki-67); (E) Necrosis. All images are shown at 20x magnification. Mean ± SE. *P<0.05; **P<0.01; ***P<0.001.

Figure 6

Effect of combination therapy of Bevacizumab and DCA on key metabolic genes. Quantitative PCR analysis was performed to investigate the effects of CTRL, BVC, DCA or BVC/DCA treatment on U87-MG and U118 tumors by measuring fold changes in the RNA (A and B) PDK1 (C and D) CA9. Mean ± SE, N=4–8. *P<0.05; **P<0.01; ***P<0.001.