Many cancer cells rely on aerobic glycolysis for energy production and targeting of this pathway is a potential strategy to inhibit cancer cell growth. In this study, inhibition of five glycolysis pathway molecules (GLUT1, HKII, PFKFB3, PDHK1 and LDH) using 9 inhibitors (Phloretin, Quercetin, STF31, WZB117, 3PO, 3-bromopyruvate, Dichloroacetate, Oxamic acid, NHI-1) was investigated in panels of breast and ovarian cancer cell line models. All compounds tested blocked glycolysis as indicated by increased extracellular glucose and decreased lactate production and also increased apoptosis. Sensitivity to several inhibitors correlated with the proliferation rate of the cell lines. Seven compounds had IC50 values that were associated with each other consistent with a shared mechanism of action. A synergistic interaction was revealed between STF31 and Oxamic acid when combined with the antidiabetic drug metformin. Sensitivity to glycolysis inhibition was also examined under a range of O2 levels (21% O2, 7% O2, 2% O2 and 0.5% O2) and greater resistance to the inhibitors was found at low oxygen conditions (7% O2, 2% O2 and 0.5% O2) relative to 21% O2 conditions. These results indicate growth of breast and ovarian cancer cell lines is dependent on all the targets examined in the glycolytic pathway with increased sensitivity to the inhibitors under normoxic conditions.
BackgroundNovel therapeutic approaches are required to treat ovarian cancer and dependency on glycolysis may provide new targets for treatment. This study sought to investigate the variation of expression of molecular components (GLUT1, HKII, PKM2, LDHA) of the glycolytic pathway in ovarian cancers and the effectiveness of targeting this pathway in ovarian cancer cell lines with inhibitors.MethodsExpression of GLUT1, HKII, PKM2, LDHA were analysed by quantitative immunofluorescence in a tissue microarray (TMA) analysis of 380 ovarian cancers and associations with clinicopathological features were sought. The effect of glycolysis pathway inhibitors on the growth of a panel of ovarian cancer cell lines was assessed by use of the SRB proliferation assay. Combination studies were undertaken combining these inhibitors with cytotoxic agents.ResultsMean expression levels of GLUT1 and HKII were higher in high grade serous ovarian cancer (HGSOC), the most frequently occurring subtype, than in non-HGSOC. GLUT1 expression was also significantly higher in advanced stage (III/IV) ovarian cancer than early stage (I/II) disease. Growth dependency of ovarian cancer cells on glucose was demonstrated in a panel of ovarian cancer cell lines. Inhibitors of the glycolytic pathway (STF31, IOM-1190, 3PO and oxamic acid) attenuated cell proliferation in platinum-sensitive and platinum-resistant HGSOC cell line models in a concentration dependent manner. In combination with either cisplatin or paclitaxel, 3PO (a novel PFKFB3 inhibitor) enhanced the cytotoxic effect in both platinum sensitive and platinum resistant ovarian cancer cells. Furthermore, synergy was identified between STF31 (a novel GLUT1 inhibitor) or oxamic acid (an LDH inhibitor) when combined with metformin, an inhibitor of oxidative phosphorylation, resulting in marked inhibition of ovarian cancer cell growth.ConclusionsThe findings of this study provide further support for targeting the glycolytic pathway in ovarian cancer and several useful combinations were identified.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4521-4) contains supplementary material, which is available to authorized users.
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.
BackgroundTumour 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.Methods2D 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.ResultsHER2 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (10.1186/s13058-019-1097-0) contains supplementary material, which is available to authorized users.
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.
Background: The abnormal regulation of H+ ions, leading to a reversed pH gradient in tumor cells in comparison to normal cells, is considered to be one of the hallmarks of cancer. This feature, however, has yet to be exploited as a therapeutic target. The aim of this study was to assess whether targeting proteins (CAIX, NHE1 and V-ATPase) that permit hypoxic cancer cell adaptation to acidosis in the tumor microenvironment can produce an effective therapeutic response in breast cancer, using 2D and 3D models. Method: Western blotting and gene expression analysis were performed on MCF-7, MDA-MB-231 and HBL-100 cancer cells to assess target protein expression in differing O2 conditions in 2D, while IHC was used to measure protein levels in 3D using multicellular tumor spheroids. Sulforhodamine B assays were executed to analyze the effects of inhibitors targeting CAIX, NHE1 and V-ATPase on breast cancer cell proliferation in 2D. 3D invasion assays were performed with MDA-MB-231 spheroids and explant tissue derived from human patients to see if CAIX inhibition had any effect on cancer cell invasion. An MDA-MB-231 xenograft model was used to investigate the effects of CAIX inhibition in vivo. Clonogenic assays were performed with MDA-MB-231 spheroids to evaluate whether any of the drugs combined effectively with irradiation. Results: 2D and 3D expression analysis showed that CAIX levels were extremely responsive to changes in O2 conditions in each of the cell lines, with HBL100 cells exhibiting the largest changes in both mRNA (42-fold increase) and protein (78-fold increase) levels at low (0.5%) O2 concentrations. NHE1 and V-ATPase mRNA/protein levels were, however, much more consistently expressed across the cell lines in different O2 conditions. Drugs targeting CAIX, NHE1 and V-ATPase had anti-proliferative effects on the breast cancer cells in 2D. Normoxic cancer cells were the most sensitive to drug treatment, acute hypoxic cancer cells showed increased resistance to the anti-proliferative effects of these drugs, while chronic hypoxic cells had IC50 values more similar to the normoxic cells. The results for the CAIX inhibitor were unexpected, as we had predicted that the increased levels of CAIX in the acute hypoxic cells would make them more sensitive to treatment. CAIX inhibition did, however, significantly reduce the invasion of cancer cells from both MDA-MB-231 spheroids (p≤0.01) and explant tissue (p≤0.001). Targeting pH regulation was also shown to have an effect in vivo on MDA-MB-231 xenografts, with CAIX inhibition significantly reducing the growth (p≤0.05) and proliferation (p≤0.05) of tumors within mice. Finally, clonogenic assays showed that drugs targeting both CAIX and NHE1 led to a significant reduction in colony number when combined with radiation (p≤0.05), compared to either drug individually or radiation treatment alone. Conclusions: This study shows that drugs targeting pH regulation molecules have potential in the treatment of breast cancer. This is highlighted by their ability to affect the proliferation and invasion of breast cancer cells, along with their ability to be combined with radiation. Of the 3 pH regulatory molecules, CAIX represents the target with the most promise. Citation Format: Meehan J, Ward C, Jarman E, Xintaropoulou C, Martinez-Perez C, Turnbull A, Supuran C, Dixon M, Kunkler I, Langdon SP. Targeting the pH regulatory mechanisms of breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-05.
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