Background. Hypoxia is recognised to induce a multigene programme mainly via HIF1a and also HIF2a transcription factors. Bioinformatics analysis of multiple gene array data sets in breast cancer showed a core hypoxia response programme of approximately 90 genes associated with poor outcome independently of other factors. This core response was significantly over-expressed in triple receptor negative cancers. Additionally, microRNAs associated with hypoxia were shown to give additional worse prognosis associations (mir-210). mir-210 targeted the mitochondrial iron chaperone responsible for regulation of key enzymes in the Krebs cycle and showed an adaptive response to hypoxia involving switching off the mitochondrial metabolism. Aims and methods. To assess in human breast cancer the hypoxia transcriptome we conducted gene microarray studies before and after 2 weeks of bevacizumab 15mg/kg single dose before neoadjuvant chemotherapy. This was correlated with imaging by DCE-MRI Ktrans analysis. Results. The study showed that high Ktrans was an excellent predictor for the biological affect of VEGF inhibition and extensive gene induction occurred, including many components of the HIF pathway, but also glycogen metabolism and lipid metabolism. We investigated these further in xenograft models to see which of the adaption pathways may be most important for survival under hypoxic conditions. We showed that induction of CA9, a key enzyme regulating extracellular pH, was critical for survival under anti-angiogenic therapy and blocking CA9 could synergise and also produce radiosensitivity. Reactivating mitochondria under hypoxic conditions induced by angiogenesis also showed additional anti-cancer benefits and is the basis now for a new phase I study in our department. Additionally, surprisingly, induction of glycogen and lipid storage occurred and this was essential for survival on reoxygenation and for protection against free radical damage, which greatly increased when either pathway was inhibited. We investigated, by bioinformatic approaches, the expression of 133 key enzymes in metabolism, showed that they were strongly associated with different subtypes of breast cancer, which may help in selection of patients for future intervention studies. To additionally define the hypoxia transcription, we conducted RNA sequencing of MCF7 cells in normoxia and mild hypoxia. This revealed marked induction of many long non-coding RNAs, suppression of all transfer RNAs and induction of novel antisense RNAs. Conclusions. Overall, therefore, although anti-angiogenic therapy alone is now withdrawn from clinical utility in breast cancer, the massive induction of hypoxic microenvironment and synergy with many other therapeutics, suggests that as new approach using induced essentiality should be reassessed in breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr BS02-1.

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Background: Recently the FDA has withdrawn the indication for bevacizumab in metastatic breast cancer after several clinical studies failed to demonstrate an overall survival benefit. These studies however did report an increase in response rates to chemotherapy and improvement in progression free survival, suggesting a pattern of response to the drug followed by the development of resistance. We have little knowledge of the molecular mechanisms driving the development of resistance to bevacizumab. To better understand these mechanisms, we have conducted a window of opportunity study using a single cycle of bevacizumab with detailed pharmacodynamic assessments using gene expression arrays and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Methods: After ethical approval, 47 newly diagnosed locally advanced breast cancer patients were prospectively enrolled in this trial. Patients received single dose bevacizumab (15mg/ kg) 2 weeks prior to neoadjuvant chemotherapy and underwent core biopsies for gene expression and immunohistochemistry analysis and DCE-MRI scans before and 2 weeks after bevacizumab. 35 patients who had invasive ductal carcinoma together with good quality MRI scans and core biopsies before and after bevacizumab were included in this analysis. Pharmacokinetic (PK) modelling techniques were used to quantify PK parameters (Ktrans, kep, ve) from the DCE-MRI data. Gene expression profiling was performed using the Affymetrix Human Exon 1.0 ST arrays. Results: The majority of patients (28/35) showed a significant reduction in vessel permeability and blood flow of at least 30% following bevacizumab, with a mean decrease in the forward transfer constant (P < 0.0001) and the reverse rate constant kep (P < 0.0001). From gene expression and immunohistochemistry analyses, we identified several key metabolism-related genes that are significantly up-regulated after bevacizumab treatment, including pyruvate dehydrogenase kinase isozyme 1 (PDK1) (fig.1) and carbonic anhydrase 9 (CA9). In addition, we found a number of interesting genes that are down-regulated after bevacizumab treatment, including sulfatase-1 (SULF1), and cyclin E1 (CCNE1). Discussion: This study highlights that the combination of DCE-MRI and gene expression arrays can lead to an improved understanding of the molecular mechanisms governing response and resistance to anti-angiogenic therapy. Heterogeneity of response to bevacizumab was demonstrated, with some tumours showing increases or no change in Ktrans and others marked reductions, which may be of value in early stratification for therapy maintenance. Furthermore, the gene expression analysis showed activation of pathways, which could contribute to the development of resistance. For example, we observed significant up regulation of genes involved in regulating the switch from mitochondrial metabolism to glycolysis, such as PDK1. This suggests that using bevacizumab with the other targeted agents such as Dichloroacetate, a PDK1 inhibitor might be helpful in overcoming the development of resistance and ultimately lead to improved patient survival. Our preclinical studies strongly support this possibility. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-06-01.