Ioanna Ledaki

Carbonic Anhydrase IX Promotes Tumor Growth and Necrosis In Vivo and Inhibition Enhances Anti-VEGF Therapy

Purpose: Bevacizumab, an anti-VEGFA antibody, inhibits the developing vasculature of tumors, but resistance is common. Antiangiogenic therapy induces hypoxia and we observed increased expression of hypoxia-regulated genes, including carbonic anhydrase IX (CAIX), in response to bevacizumab treatment in xenografts. CAIX expression correlates with poor prognosis in most tumor types and with worse outcome in bevacizumab-treated patients with metastatic colorectal cancer, malignant astrocytoma, and recurrent malignant glioma. Experimental Design: We knocked down CAIX expression by short hairpin RNA in a colon cancer (HT29) and a glioblastoma (U87) cell line which have high hypoxic induction of CAIX and overexpressed CAIX in HCT116 cells which has low CAIX. We investigated the effect on growth rate in three-dimensional (3D) culture and in vivo, and examined the effect of CAIX knockdown in combination with bevacizumab. Results: CAIX expression was associated with increased growth rate in spheroids and in vivo. Surprisingly, CAIX expression was associated with increased necrosis and apoptosis in vivo and in vitro. We found that acidity inhibits CAIX activity over the pH range found in tumors (pK = 6.84), and this may be the mechanism whereby excess acid self-limits the build-up of extracellular acid. Expression of another hypoxia inducible CA isoform, CAXII, was upregulated in 3D but not two-dimensional culture in response to CAIX knockdown. CAIX knockdown enhanced the effect of bevacizumab treatment, reducing tumor growth rate in vivo. Conclusion: This work provides evidence that inhibition of the hypoxic adaptation to antiangiogenic therapy enhances bevacizumab treatment and highlights the value of developing small molecules or antibodies which inhibit CAIX for combination therapy. Clin Cancer Res; 18(11); 3100–11. ©2012 AACR.

Role of Hypoxia‐Inducible Factors in Epigenetic Regulation via Histone Demethylases

Eukaryotic chromatin is subject to multiple posttranslational histone modifications such as acetylation, methylation, phosphorylation, and ubiquitination. These various covalent modifications have been proposed to constitute a “histone code,” playing important roles in the establishment of global chromatin environments, transcription, DNA repair, and DNA replication. Among these modifications, histone methylation specifies regulatory marks that delineate transcriptionally active and inactive chromatin. These histone methyl marks were considered irreversible; however, recent identification of site‐specific histone demethylases demonstrates that histone methylation is dynamically regulated, which may allow cells to rapidly change chromatin conformation to adapt to environmental stresses or intrinsic stimuli. Of major interest is the observation that these histone demethylase enzymes, which are in the Jumonji gene family, require oxygen to function and, in some cases, are induced by hypoxia in an HIFα‐dependent manner. This provides a new mechanism for regulation of the response to hypoxia.

The BET inhibitor JQ1 selectively impairs tumour response to hypoxia and downregulates CA9 and angiogenesis in triple negative breast cancer.

The availability of bromodomain and extra-terminal inhibitors (BETi) has enabled translational epigenetic studies in cancer. BET proteins regulate transcription by selectively recognizing acetylated lysine residues on chromatin. BETi compete with this process leading to both downregulation and upregulation of gene expression. Hypoxia enables progression of triple negative breast cancer (TNBC), the most aggressive form of breast cancer, partly by driving metabolic adaptation, angiogenesis and metastasis through upregulation of hypoxia-regulated genes (for example, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A). Responses to hypoxia can be mediated epigenetically, thus we investigated whether BETi JQ1 could impair the TNBC response induced by hypoxia and exert anti-tumour effects. JQ1 significantly modulated 44% of hypoxia-induced genes, of which two-thirds were downregulated including CA9 and VEGF-A. JQ1 prevented HIF binding to the hypoxia response element in CA9 promoter, but did not alter HIF expression or activity, suggesting some HIF targets are BET-dependent. JQ1 reduced TNBC growth in vitro and in vivo and inhibited xenograft vascularization. These findings identify that BETi dually targets angiogenesis and the hypoxic response, an effective combination at reducing tumour growth in preclinical studies.

Disrupting hypoxia-induced bicarbonate transport acidifies tumor cells and suppresses tumor growth

Tumor hypoxia is associated clinically with therapeutic resistance and poor patient outcomes. One feature of tumor hypoxia is activated expression of carbonic anhydrase IX (CA9), a regulator of pH and tumor growth. In this study, we investigated the hypothesis that impeding the reuptake of bicarbonate produced extracellularly by CA9 could exacerbate the intracellular acidity produced by hypoxic conditions, perhaps compromising cell growth and viability as a result. In 8 of 10 cancer cell lines, we found that hypoxia induced the expression of at least one bicarbonate transporter. The most robust and frequent inductions were of the sodium-driven bicarbonate transporters SLC4A4 and SLC4A9, which rely upon both HIF1α and HIF2α activity for their expression. In cancer cell spheroids, SLC4A4 or SLC4A9 disruption by either genetic or pharmaceutical approaches acidified intracellular pH and reduced cell growth. Furthermore, treatment of spheroids with S0859, a small-molecule inhibitor of sodium-driven bicarbonate transporters, increased apoptosis in the cell lines tested. Finally, RNAi-mediated attenuation of SLC4A9 increased apoptosis in MDA-MB-231 breast cancer spheroids and dramatically reduced growth of MDA-MB-231 breast tumors or U87 gliomas in murine xenografts. Our findings suggest that disrupting pH homeostasis by blocking bicarbonate import might broadly relieve the common resistance of hypoxic tumors to anticancer therapy. Cancer Res; 76(13); 3744-55. ©2016 AACR.

Abstract 4053: Hypoxic trans-activation of sodium dependent bicarbonate co-transporters regulates pH in 3D spheroids and promotes growth.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Carbonic anhydrase IX (CAIX) is strongly induced by hypoxia. CAIX extracellularly hydrates CO2 to HCO3− and H+, and we previously showed CAIX regulates pH in 3D spheroids. CAIX expression correlates with poor prognosis in most tumour types. We recently showed CAIX regulates tumour growth, necrosis and that knockdown enhances Bevacizumab treatment, highlighting the role of pH regulation in the hypoxic tumour milieu. We hypothesised that transport of HCO3−, which had been produced by CAIX extracellularly could facilitate quenching of intracellular H+. We identified increased expression of SLC4A4 (which encodes a bicarbonate transporter), in response to Bevacizumab, a treatment which increases the hypoxic fraction of cells, in vivo. Method: We investigated a panel of cells lines (4 colon cancers, 2 glioblastomas, 1 breast cancer and 1 head and neck cancer) for changes in RNA expression of bicarbonate transporters in response to hypoxia (0.1% O2, 72 hours), and the role of HIF1α and HIF2α in key identified changes. We used a specific Na+ dependent bicarbonate transport inhibitor (SO859) to examine the effect of inhibition on pH regulation and growth in spheroids. Further to this we used shRNA knockdown of SLC4A4 and SLC4A9 in cell lines with increased hypoxic expression to further investigate their role. Results: Hypoxia increased the RNA expression of one or more bicarbonate transporter in 6/8 cancer cell lines. The largest increases in expression change were for SLC4A4 in Ls174T and SCC25 (79.4 fold, p<0.01 n=3: 2.4 fold, p<0.05, n=3) and SLC4A9 in U87 and Ls174T (10.5 fold, p<0.01 n=3; 19 fold, p<0.01 n=3). SLC4A4 expression was regulated by HIF1α in Ls174T and SCC25 (p<0.05, n=3). HIF1α bound directly to the HRE in the promoter of SLC4A4 in SCC25. SLC4A9 expression increase was regulated by HIF2α (80%) and HIF1α (29%) in Ls174T. SO859 treatment acidified the intracellular pH of cells in U87 spheroids. The most significant differences in pH were seen at the spheroid core (p<0.001, n=20) and periphery (p<0.001, n=20). U87 spheroids have HIF1α stabilisation and CAIX expression throughout, as detected by immunohistochemistry. Longer SO859 treatment of 24 hours resulted in a reduced effect on pH, suggesting a compensation mechanism. SO859 treatment reduced the spheroid growth rate of 4/4 cell lines tested, including MDA-MB-468 a cell line that showed no hypoxic increase in bicarbonate transport expression. In U87 spheroids, CAIX knockdown further reduced the reduction in growth rate seen with SO859. SLC4A4 and SLC4A9 knockdown reduced growth rate in Ls174T spheroids and SLC4A9 knockdown reduced spheroid growth rate in U87. Conclusion: This work highlights the value of developing small molecules or antibodies, which inhibit bicarbonate transport to clinically deregulate pH regulation in the hypoxic microenvironment of tumours. Citation Format: Alan McIntyre, Alzbeta Hulikova, Ioanna Ledaki, Peter Seden, Helen Turley, Dylan Jones, Angela Russell, Pawel Swietach, Adrian L. Harris. Hypoxic trans-activation of sodium dependent bicarbonate co-transporters regulates pH in 3D spheroids and promotes growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4053. doi:10.1158/1538-7445.AM2013-4053

Abstract BS02-1: Hypoxia metabolism in breast cancer – How to overcome resistance to anti-angiogenic therapy

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.