James Meehan

Targeting tumour hypoxia to prevent cancer metastasis: from biology, biosensing and technology to drug development : the METOXIA consortium

Abstract The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation–deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009–2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [18F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O2), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Evaluation of carbonic anhydrase IX as a therapeutic target for inhibition of breast cancer invasion and metastasis using a series of in vitro breast cancer models

Triple negative, resistant or metastatic disease are major factors in breast cancer mortality, warranting novel approaches. Carbonic anhydrase IX (CAIX) is implicated in survival, migration and invasion of breast cancer cells and inhibition provides an innovative therapeutic strategy. The efficacy of 5 novel ureido-substituted sulfamate CAIX inhibitors were assessed in increasingly complex breast cancer models, including cell lines in normoxia and hypoxia, 3D spheroids and an ex-vivo explant model utilizing fresh biopsy tissue from different breast cancer subtypes. CAIX expression was evaluated in a tissue microarray (TMA) of 92 paired lymph node and primary breast cancers and 2 inhibitors were appraised in vivo using MDA-MB-231 xenografts. FC11409B, FC9398A, FC9403, FC9396A and S4 decreased cell proliferation and migration and inhibited 3D spheroid invasion. S4, FC9398A and FC9403A inhibited or prevented invasion into collagen. FC9403A significantly reversed established invasion whilst FC9398A and DTP348 reduced xenograft growth. TMA analysis showed increased CAIX expression in triple negative cancers. These data establish CAIX inhibition as a relevant therapeutic goal in breast cancer, targeting the migratory, invasive, and metastatic potential of this disease. The use of biopsy tissue suggests efficacy against breast cancer subtypes, and should provide a useful tool in drug testing against invasive cancers.

Antitumour activity of the novel flavonoid oncamex in preclinical breast cancer models

Background:The natural polyphenol myricetin induces cell cycle arrest and apoptosis in preclinical cancer models. We hypothesised that myricetin-derived flavonoids with enhanced redox properties, improved cell uptake and mitochondrial targeting might have increased potential as antitumour agents.Methods:We studied the effect of a second-generation flavonoid analogue Oncamex in a panel of seven breast cancer cell lines, applying western blotting, gene expression analysis, fluorescence microscopy and immunohistochemistry of xenograft tissue to investigate its mechanism of action.Results:Proliferation assays showed that Oncamex treatment for 8 h reduced cell viability and induced cytotoxicity and apoptosis, concomitant with increased caspase activation. Microarray analysis showed that Oncamex was associated with changes in the expression of genes controlling cell cycle and apoptosis. Fluorescence microscopy showed the compound’s mitochondrial targeting and reactive oxygen species-modulating properties, inducing superoxide production at concentrations associated with antiproliferative effects. A preliminary in vivo study in mice implanted with the MDA-MB-231 breast cancer xenograft showed that Oncamex inhibited tumour growth, reducing tissue viability and Ki-67 proliferation, with no signs of untoward effects on the animals.Conclusions:Oncamex is a novel flavonoid capable of specific mitochondrial delivery and redox modulation. It has shown antitumour activity in preclinical models of breast cancer, supporting the potential of this prototypic candidate for its continued development as an anticancer agent.

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.

Carbonic Anhydrase IX (CAIX), Cancer, and Radiation Responsiveness

Carbonic anhydrase IX has been under intensive investigation as a therapeutic target in cancer. Studies demonstrate that this enzyme has a key role in pH regulation in cancer cells, allowing these cells to adapt to the adverse conditions of the tumour microenviroment. Novel CAIX inhibitors have shown efficacy in both in vitro and in vivo pre-clinical cancer models, adversely affecting cell viability, tumour formation, migration, invasion, and metastatic growth when used alone. In co-treatments, CAIX inhibitors may enhance the effects of anti-angiogenic drugs or chemotherapy agents. Research suggests that these inhibitors may also increase the response of tumours to radiotherapy. Although many of the anti-tumour effects of CAIX inhibition may be dependent on its role in pH regulation, recent work has shown that CAIX interacts with several of the signalling pathways involved in the cellular response to radiation, suggesting that pH-independent mechanisms may also be an important basis of its role in tumour progression. Here, we discuss these pH-independent interactions in the context of the ability of CAIX to modulate the responsiveness of cancer to radiation.

Implantable biosensors and their contribution to the future of precision medicine

Precision medicine can be defined as the prevention, investigation and treatment of diseases taking individual variability into account. There are multiple ways in which the field of precision medicine may be advanced; however, recent innovations in the fields of electronics and microfabrication techniques have led to an increased interest in the use of implantable biosensors in precision medicine. Implantable biosensors are an important class of biosensors because of their ability to provide continuous data on the levels of a target analyte; this enables trends and changes in analyte levels over time to be monitored without any need for intervention from either the patient or clinician. As such, implantable biosensors have great potential in the diagnosis, monitoring, management and treatment of a variety of disease conditions. In this review, we describe precision medicine and the role implantable biosensors may have in this field, along with challenges in their clinical implementation due to the host immune responses they elicit within the body.

Biocompatibility of common implantable sensor materials in a tumor xenograft model: Biocompatibility of common implantable sensor material

Abstract Real‐time monitoring of tumor microenvironment parameters using an implanted biosensor could provide valuable information on the dynamic nature of a tumors biology and its response to treatment. However, following implantation biosensors may lose functionality due to biofouling caused by the foreign body response (FBR). This study developed a novel tumor xenograft model to evaluate the potential of six biomaterials (silicon dioxide, silicon nitride, Parylene‐C, Nafion, biocompatible EPOTEK epoxy resin, and platinum) to trigger a FBR when implanted into a solid tumor. Biomaterials were chosen based on their use in the construction of a novel biosensor, designed to measure spatial and temporal changes in intra‐tumoral O2, and pH. None of the biomaterials had any detrimental effect on tumor growth or body weight of the murine host. Immunohistochemistry showed no significant changes in tumor necrosis, hypoxic cell number, proliferation, apoptosis, immune cell infiltration, or collagen deposition. The absence of biofouling supports the use of these materials in biosensors; future investigations in preclinical cancer models are required, with a view to eventual applications in humans. To our knowledge this is the first documented investigation of the effects of modern biomaterials, used in the production of implantable sensors, on tumor tissue after implantation.

Personalisation of Radiotherapy for Breast Cancer

The role of radiotherapy is well established in the multidisciplinary management of breast cancer. However, its use could be customised further with the intent of enhancing tumour cell radiosensitisation and reducing normal cell toxicity. The importance of tumour heterogeneity and the microenvironment in the response to radiotherapy is under intense scrutiny, and the value of molecular profiling is being increasingly recognised. Genome-wide association studies are likely to play an important role in elucidating the molecular pathogenesis of radiotoxicity in the emerging area of radiogenomics. Biomarkers of tumour radiosensitivity should help indicate potentially responsive and unresponsive cancers. Further understanding of the tumour microenvironment and better preclinical models will help identify targets to enhance radiosensitivity or reverse radioresistance.

Abstract P4-08-06: Modulation of hypoxia-inducible factors and the HIF transcriptional response to hypoxia by ERBB2 overexpression in the MCF7 breast cancer cell line

Objective: To explore the role of HIF2α in growth factor receptor-driven HIF modulation and investigate the relationship between growth factor- and hypoxia-driven HIF activation. HIF-mediated transcriptional activity is known to drive genes involved in various processes which are associated with cancer pathology such as glycolysis, angiogenesis and metastasis. Therefore, understanding the implications of hypoxia-independent HIF regulation for both HIF1α and HIF2α, may give new insight into the mechanisms by which HIF drives cancer pathology in vivo and a greater understanding of when HIF inhibitory agents may be effective therapies. Methods: We used an ERBB2 overexpressing MCF7 cell line (MCF7-HER2) to investigate the effect of ERBB2 on the HIF-axis. Western blotting was used to assess protein level in these cell lines. HIF protein expression was compared with and without ERBB stimulation by ERBB3 ligand neuregulin 1β. Illumina BeadChip analysis was used to compare mRNA levels between these cell lines in normoxia (20% oxygen), acute hypoxia (0.5% oxygen for 24 hours) and chronic hypoxia (0.5% oxygen for 10 weeks). Differentially expressed genes were identified using rank products analysis with a cut-off P-value of 0.01. This allowed an in-depth comparison of hypoxia responses at the level of transcription between the cell lines to ascertain the effect of ERBB2 overexpression on hypoxia driven transcriptional changes. Results: Immunoblotting shows that HIF1α protein level is comparable between MCF7 and MCF7-HER2 cell lines, and is inducible in normoxia by stimulation with neuregulin 1β. Conversely, HIF2α protein is unaffected, but is constitutively expressed in MCF7-HER2 only. This suggests that both HIF isoforms can be up-regulated in normoxia but by different mechanisms. Microarray data suggests that the constitutively higher HIF2α levels in the MCF7-HER2 cell line may be due, at least in part, to the increased transcription of the HIF2A gene which is higher in normoxia and in response to hypoxia when compared to wild-type MCF7. Overexpression of ERBB2 in MCF7-HER2 cells appears to prime cells for their response to hypoxia, as 14% (N= 591) of the genes which are induced in acute hypoxia are also expressed at significantly higher levels in normoxic MCF7-HER2 cells. However, only 1% are more highly expressed in wild-type MCF7 cells. For chronic hypoxic genes, 18% (N= 514) were more highly expressed in normoxic MCF7-HER2 cells and just 8% in wild-type MCF7 cells. These up-regulated genes include both HIF1 and HIF2 target genes which may have important consequences for glycolysis (ALDOC, PFKFB), tumour cell survival (E4BP4, STC2) and proliferation (FOS, KDM5B). Conclusions: We have demonstrated that both HIF1α and HIF2α can be regulated independently of hypoxia, however these appear to be controlled through distinct mechanisms. Whilst the implications of HIF1 in breast cancer pathology have been appreciated for some time, relatively little is known about the impact of HIF2. Here we show that ERBB2 overexpression can not only increase HIF2α protein levels in normoxia, but may also prime cells for hypoxia by allowing the constitutively higher expression of HIF1 and HIF2 target genes. Citation Format: Jarman EJ, Turnbull AK, Martinez-Perez C, Meehan J, Xintralopoulou C, Ward C, Langdon SP. Modulation of hypoxia-inducible factors and the HIF transcriptional response to hypoxia by ERBB2 overexpression in the MCF7 breast cancer cell line. [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 P4-08-06.

Abstract P5-04-05: Targeting the pH regulatory mechanisms of breast cancer cells

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.