Targeting aerobic glycolysis in breast and ovarian cancer

Cancer cells, unlike normal tissue, frequently rely on glycolysis for the production of energy and the metabolic intermediates required for their growth regardless of cellular oxygenation levels. This metabolic reconfiguration, termed the Warburg effect, provides a potential strategy to preferentially target tumours from a therapeutic perspective. The present study sought to investigate the glycolytic phenotype of breast and ovarian cancer, and assess the possibility of exploiting several glycolytic targets therapeutically. Initially the growth dependency of breast and ovarian cancer cells on the availability of glucose was established. An array of 10 compounds reported to inhibit key enzymes of the glycolytic pathway were investigated and compared against an extended panel of breast and ovarian cancer cell line models. All inhibitors investigated, targeted against multiple points of the pathway, were shown to block the glycolytic pathway as demonstrated by glucose accumulation in the culture media combined with decreased lactate secretion, and attenuated breast and ovarian cancer cell proliferation in a concentration dependent manner. Furthermore their mechanism of action was investigated by flow cytometric analysis and their antiproliferative effect was associated with induction of apoptosis and G0/G1 cell cycle arrest. The glycolytic inhibitors were further assessed in combination strategies with established chemotherapeutic and targeted agents and several synergistic interactions, characterised by low combination index values, were revealed. Among them, 3PO (a novel PFKFB3 inhibitor) enhanced the effect of cisplatin in both platinum sensitive and platinum resistant ovarian cancer cells suggesting a strategy for treatment of platinum resistant disease. Furthermore robust synergy was identified between IOM-1190 (a novel GLUT1 inhibitor) and metformin, an antidiabetic inhibitor of oxidative phosphorylation, resulting in strong inhibition of breast cancer cell growth. This combination is proposed for the treatment of highly aggressive triple negative breast tumours. An additional objective of this research was to investigate the effect of the oxygen level on sensitivity to glycolysis inhibition. Breast cancer cells were found to be more sensitive to glycolysis inhibition in high oxygen conditions. This enhanced resistance at low oxygen levels was associated with upregulation of the targeted glycolytic enzymes as demonstrated at both the mRNA (by gene expression microarray profiling, Illumina BeadArrays) and protein level (by Western blotting). Manipulation of LDHA (Lactate Dehydrogenase A) by siRNA knockdown provided further evidence that downregulation of this target was sufficient to significantly suppress breast cancer cell proliferation. Finally, the expression of selected glycolytic targets was examined in a clinical tissue microarray set of a large cohort of ovarian tumours using quantitative immunofluorescence technology, AQUA. The role of the glycolytic phenotype in ovarian cancer was suggested and interesting associations between the glycolytic profile and clear cell and endometrioid ovarian cancers revealed. Increased PKM2 (Pyruvate kinase isozyme M2) and LDHA expression were demonstrated in clear cell tumours and also low expression of these enzymes was significantly correlated with improved survival of endometrioid ovarian cancer patients. Taken together the findings of this study support the glycolytic pathway as a legitimate target for further investigation in breast and ovarian cancer treatment

HER2 regulates HIF-2α and drives an increased hypoxic response in breast cancer

Abstract Background Tumour hypoxia is a driver of breast cancer progression associated with worse prognosis and more aggressive disease. The cellular response to hypoxia is mediated by the hypoxia-inducible transcription factors HIF-1 and HIF-2, whose transcriptional activity is canonically regulated through their oxygen-labile HIF-α subunits. These are constitutively degraded in the presence of oxygen; however, HIF-1α can be stabilised, even at high oxygen concentrations, through the activation of HER receptor signalling. Despite this, there is still limited understanding on how HER receptor signalling interacts with HIF activity to contribute to breast cancer progression in the context of tumour hypoxia. Methods 2D and 3D cell line models were used alongside microarray gene expression analysis and meta-analysis of publicly available gene expression datasets to assess the impact of HER2 overexpression on HIF-1α/HIF-2α regulation and to compare the global transcriptomic response to acute and chronic hypoxia in an isogenic cell line model of HER2 overexpression. Results HER2 overexpression in MCF7 cells leads to an increase in HIF-2α but not HIF-1α expression in normoxia and an increased upregulation of HIF-2α in hypoxia. Global gene expression analysis showed that HER2 overexpression in these cells promotes an exaggerated transcriptional response to both short-term and long-term hypoxia, with increased expression of numerous hypoxia response genes. HIF-2α expression is frequently higher in HER2-overexpressing tumours and is associated with worse disease-specific survival in HER2-positive breast cancer patients. HER2-overexpressing cell lines demonstrate an increased sensitivity to targeted HIF-2α inhibition through either siRNA or the use of a small molecule inhibitor of HIF-2α translation. Conclusions This study suggests an important interplay between HER2 expression and HIF-2α in breast cancer and highlights the potential for HER2 to drive the expression of numerous hypoxia response genes in normoxia and hypoxia. Overall, these findings show the importance of understanding the regulation of HIF activity in a variety of breast cancer subtypes and points to the potential of targeting HIF-2α as a therapy for HER2-positive breast cancer

Adipocytes disrupt the translational programme of acute lymphoblastic leukaemia to favour tumour survival and persistence

The specific niche adaptations that facilitate primary disease and Acute Lymphoblastic Leukaemia (ALL) survival after induction chemotherapy remain unclear. Here, we show that Bone Marrow (BM) adipocytes dynamically evolve during ALL pathogenesis and therapy, transitioning from cellular depletion in the primary leukaemia niche to a fully reconstituted state upon remission induction. Functionally, adipocyte niches elicit a fate switch in ALL cells towards slow-proliferation and cellular quiescence, highlighting the critical contribution of the adipocyte dynamic to disease establishment and chemotherapy resistance. Mechanistically, adipocyte niche interaction targets posttranscriptional networks and suppresses protein biosynthesis in ALL cells. Treatment with general control nonderepressible 2 inhibitor (GCN2ib) alleviates adipocyte-mediated translational repression and rescues ALL cell quiescence thereby significantly reducing the cytoprotective effect of adipocytes against chemotherapy and other extrinsic stressors. These data establish how adipocyte driven restrictions of the ALL proteome benefit ALL tumours, preventing their elimination, and suggest ways to manipulate adipocyte-mediated ALL resistance

Expression of glycolytic enzymes in ovarian cancers and evaluation of the glycolytic pathway as a strategy for ovarian cancer treatment

Table S2. Spearman correlation of the expression of four glycolytic enzymes in a cohort of 380 ovarian cancers. Spearman rho correlation values (top value) along with the respective adjusted P value (bottom value) of statistically significant correlations thresholded at FDR P < 0.01 are summarised. (DOCX 21 kb

High-Yield Production of a Rich-in-Hydroxytyrosol Extract from Olive (Olea europaea) Leaves

The aim of the present study was to explore the high-yield production of hydroxytyrosol, a phenolic compound with very high antioxidant capacity. Olea europaea leaves were chosen as feedstock as they contain significant amounts of oleuropein, which can be hydrolyzed to hydroxytyrosol. The chosen techniques are widely used in the industry and can be easily scaled up. Olive leaves underwent drying and mechanical pretreatment and extractives were transported to a solvent by solid&ndash;liquid extraction using water&ndash;ethanol mixtures. The use of approximately 60&ndash;80% ethanol showed an almost 2-fold increase in extracted phenolics compared to pure water, to approximately 45 g/kg of dry leaves. Extracted oleuropein was hydrolyzed with hydrochloric acid and the hydrolysate was extracted with ethyl acetate after pH adjustment. This step led to a hydroxytorosol content increase from less than 4% to approximately 60% w/w of dry extract, or 10&ndash;15 g of hydroxytyrosol recovery per kg of dry leaves

Inhibition of pH regulation as a therapeutic strategy in hypoxic human breast cancer cells

Hypoxic cancer cells exhibit resistance to many therapies. This study compared the therapeutic effect of targeting the pH regulatory proteins (CAIX, NHE1 and V-ATPase) that permit cancer cells to adapt to hypoxic conditions, using both 2D and 3D culture models. Drugs targeting CAIX, NHE1 and V-ATPase exhibited anti-proliferative effects in MCF-7, MDA-MB-231 and HBL-100 breast cancer cell lines in 2D. Protein and gene expression analysis in 2D showed that CAIX was the most hypoxia-inducible protein of the 3 targets. However, the expression of CAIX differed between the 3 cell lines. This difference in CAIX expression in hypoxia was consistent with a varying activity of FIH-1 between the cell lines. 3D expression analysis demonstrated that both CAIX and NHE1 were up-regulated in the hypoxic areas of multicellular tumor spheroids. However, the induction of CAIX expression in hypoxia was again cell line dependent. 3D invasion assays conducted with spheroids showed that CAIX inhibition significantly reduced the invasion of cells. Finally, the capability of both NHE1 and CAIX inhibitors to combine effectively with irradiation was exhibited in clonogenic assays. Proteomic-mass-spectrometric analysis indicated that CAIX inhibition might be combining with irradiation through stimulating apoptotic cell death. Of the three proteins, CAIX represents the target with the most promise for the treatment of breast cancer