Dan Cojocari

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.

AMPK regulates metabolism and survival in response to ionizing radiation

BACKGROUND AND PURPOSE AMPK is a metabolic sensor and an upstream inhibitor of mTOR activity. AMPK is phosphorylated by ionizing radiation (IR) in an ATM dependent manner, but the cellular consequences of this phosphorylation event have remained unclear. The objective of this study was to assess whether AMPK plays a functional role in regulating cellular responses to IR. METHODS The importance of AMPK expression for radiation responses was investigated using both MEFs (mouse embryo fibroblasts) double knockout for AMPK α1/α2 subunits and human colorectal carcinoma cells (HCT 116) with AMPK α1/α2 shRNA mediated knockdown. RESULTS We demonstrate here that IR results in phosphorylation of both AMPK and its substrate, ACC. IR moderately stimulated mTOR activity, and this was substantially exacerbated in the absence of AMPK. AMPK was required for IR induced expression of the mTOR inhibitor REDD1, indicating that AMPK restrains mTOR activity through multiple mechanisms. Likewise, cellular metabolism was deregulated following irradiation in the absence of AMPK, as evidenced by a substantial increase in oxygen consumption rates and lactate production. AMPK deficient cells showed impairment of the G1/S cell cycle checkpoint, and were unable to support long-term proliferation during starvation following radiation. Lastly, we show that AMPK proficiency is important for clonogenic survival after radiation during starvation. CONCLUSIONS These data reveal novel functional roles for AMPK in regulating mTOR signaling, cell cycle, survival and metabolic responses to IR.

A three-dimensional engineered tumour for spatial snapshot analysis of cell metabolism and phenotype in hypoxic gradients

The profound metabolic reprogramming that occurs in cancer cells has been investigated primarily in two-dimensional cell cultures, which fail to recapitulate spatial aspects of cell-to-cell interactions as well as tissue gradients present in three-dimensional (3D) tumours. Here, we describe an engineered model to assemble 3D tumours by rolling a scaffold-tumour composite strip. By unrolling the strip, the model can be rapidly disassembled for snap-shot analysis, allowing spatial mapping of cell metabolism in concert with cell phenotype. We also show that the establishment of oxygen gradients within samples are shaped by oxygen-dependent signalling pathways, as well as the consequential variations in cell growth, response to hypoxic gradients extending from normoxia to severe hypoxia, and therapy responsiveness, are consistent with tumours in vivo. Moreover, by using liquid chromatography tandem mass spectrometry, we mapped cellular metabolism and identified spatially defined metabolic signatures of cancer cells to reveal both known and novel metabolic responses to hypoxia.

The autophagy associated gene, ULK1, promotes tolerance to chronic and acute hypoxia

BACKGROUND AND PURPOSE Tumor hypoxia is associated with therapy resistance and malignancy. Previously we demonstrated that activation of autophagy and the unfolded protein response (UPR) promote hypoxia tolerance. Here we explored the importance of ULK1 in hypoxia tolerance, autophagy induction and its prognostic value for recurrence after treatment. MATERIAL AND METHODS Hypoxic regulation of ULK1 mRNA and protein was assessed in vitro and in primary human head and neck squamous cell carcinoma (HNSCC) xenografts. Its importance in autophagy induction, mitochondrial homeostasis and tolerance to chronic and acute hypoxia was evaluated in ULK1 knockdown cells. The prognostic value of ULK1 mRNA expression was assessed in 82 HNSCC patients. RESULTS ULK1 enrichment was observed in hypoxic tumor regions. High enrichment was associated with a high hypoxic fraction. In line with these findings, high ULK1 expression in HNSCC patients appeared associated with poor local control. Exposure of cells to hypoxia induced ULK1 mRNA in a UPR and HIF1α dependent manner. ULK1 knockdown decreased autophagy activation, increased mitochondrial mass and ROS exposure and sensitized cells to acute and chronic hypoxia. CONCLUSIONS We demonstrate that ULK1 is a hypoxia regulated gene and is associated with hypoxia tolerance and a worse clinical outcome.

New small molecule inhibitors of UPR activation demonstrate that PERK, but not IRE1α signaling is essential for promoting adaptation and survival to hypoxia

BACKGROUND AND PURPOSE The unfolded protein response (UPR) is activated in response to hypoxia-induced stress in the endoplasmic reticulum (ER) and consists of three distinct signaling arms. Here we explore the potential of targeting two of these arms with new potent small-molecule inhibitors designed against IRE1α and PERK. METHODS We utilized shRNAs and small-molecule inhibitors of IRE1α (4μ8c) and PERK (GSK-compound 39). XBP1 splicing and DNAJB9 mRNA was measured by qPCR and was used to monitor IRE1α activity. PERK activity was monitored by immunoblotting eIF2α phosphorylation and qPCR of DDIT3 mRNA. Hypoxia tolerance was measured using proliferation and clonogenic cell survival assays of cells exposed to mild or severe hypoxia in the presence of the inhibitors. RESULTS Using knockdown experiments we show that PERK is essential for survival of KP4 cells while knockdown of IRE1α dramatically decreases the proliferation and survival of HCT116 during hypoxia. Further, we show that in response to both hypoxia and other ER stress-inducing agents both 4μ8c and the PERK inhibitor are selective and potent inhibitors of IRE1α and PERK activation, respectively. However, despite potent inhibition of IRE1α activation, 4μ8c had no effect on cell proliferation or clonogenic survival of cells exposed to hypoxia. This was in contrast to the inactivation of PERK signaling with the PERK inhibitor, which reduced tolerance to hypoxia and other ER stress inducing agents. CONCLUSIONS Our results demonstrate that IRE1α but not its splicing activity is important for hypoxic cell survival. The PERK signaling arm is uniquely important for promoting adaptation and survival during hypoxia-induced ER stress and should be the focus of future therapeutic efforts.

PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote tumour survival during hypoxia

Tumours exist in a hypoxic microenvironment and must limit excessive oxygen consumption. Hypoxia-inducible factor (HIF) controls mitochondrial oxygen consumption, but how/if tumours regulate non-mitochondrial oxygen consumption (NMOC) is unknown. Protein-tyrosine phosphatase-1B (PTP1B) is required for Her2/Neu-driven breast cancer (BC) in mice, although the underlying mechanism and human relevance remain unclear. We found that PTP1B-deficient HER2+ xenografts have increased hypoxia, necrosis and impaired growth. In vitro, PTP1B deficiency sensitizes HER2+ BC lines to hypoxia by increasing NMOC by α-KG-dependent dioxygenases (α-KGDDs). The moyamoya disease gene product RNF213, an E3 ligase, is negatively regulated by PTP1B in HER2+ BC cells. RNF213 knockdown reverses the effects of PTP1B deficiency on α-KGDDs, NMOC and hypoxia-induced death of HER2+ BC cells, and partially restores tumorigenicity. We conclude that PTP1B acts via RNF213 to suppress α-KGDD activity and NMOC. This PTP1B/RNF213/α-KGDD pathway is critical for survival of HER2+ BC, and possibly other malignancies, in the hypoxic tumour microenvironment.

Development of TRACER: tissue roll for analysis of cellular environment and response

The tumour microenvironment is heterogeneous and consists of multiple cell types, variable extracellular matrix (ECM) composition, and contains cell-defined gradients of small molecules, oxygen, nutrients and waste. Emerging in vitro cell culture systems that attempt to replicate these features often fail to incorporate design strategies to facilitate efficient data collection and stratification. The tissue roll for analysis of cellular environment and response (TRACER) is a novel strategy to assemble layered, three-dimensional tumours with cell-defined, graded heterogeneous microenvironments that also facilitates cellular separation and stratification of data from different cell populations from specific microenvironments. Here we describe the materials selection and development of TRACER. We find that cellulose fibre scaffolding is an ideal support to generate tissue constructs having homogenous cell seeding and consistent properties. We explore ECM remodeling and long-term cell growth in the scaffold, and characterize the tumour microenvironment in assembled TRACERs using multiple established analysis methods. Finally, we confirm that TRACERs replicate small molecule gradients of glucose and lactate, and explore cell phenotype associated with these gradients using confocal microscopy, flow cytometry, and quantitative PCR analysis. We envision this technology will provide a platform to create complex, yet controlled tumour microenvironments that can be easily disassembled for snapshot analysis of cell phenotype and response to therapy in relation to microenvironment properties.

A three-dimensional engineered heterogeneous tumor model for assessing cellular environment and response

This protocol describes how to build and implement a three-dimensional (3D) cell culture system, TRACER (tissue roll for analysis of cellular environment and response), that enables analysis of cellular behavior and phenotype in hypoxic gradients. TRACER consists of infiltrating cells encapsulated in a hydrogel extracellular matrix (ECM) within a thin strip of porous cellulose scaffolding that is then rolled around an oxygen-impermeable mandrel for assembly of thick and layered 3D tissue constructs that develop cell-defined oxygen gradients. TRACER differs from other stacked-paper cell culture models because it is assembled from a single-piece scaffold, which facilitates rapid disassembly for analysis of different cell populations and metabolites. The protocol describes how to fabricate TRACER components, cell seeding in the scaffold, and scaffold assembly and disassembly. Furthermore, it provides methods to quantify live, dead, or proliferating cells, as well as gradients of oxygen using the nitroimidazole derivative EF5, in a layer-by-layer analysis with confocal microscopy or by flow cytometry of cells isolated from the TRACER scaffold. Additional methods to isolate live cells from TRACER layers for dose–response analysis with a clonogenic assay, as well as steps to extract RNA or fast-changing metabolites from TRACER layers, are also presented. Finally, we provide alternative steps to establish TRACER co-cultures for assessment of tumor cell invasion and metastasis, in this case in the absence of a hypoxic gradient. Although analysis time varies according to the assay chosen, scaffold fabrication and seeding typically take 2 h, and TRACER assembly takes 20 min on the day following scaffold seeding. The TRACER platform is designed for use by researchers and students who have basic tissue culture experience.This protocol describes TRACER, a 3D cell culture system that enables the assembly of heterogeneous model tumors or tissues that easily disassemble for rapid analysis of different cell populations from particular microenvironments.

Abstract LB-302: PTP1B regulates the Moyamoya disease-associated E3 ligase, RNF213 and cellular dioxygenase activity to allow breast tumor survival in hypoxia

Deletion of Ptpn1 , which encodes Protein-Tyrosine Phosphatase-1B (PTP1B), delays the onset of Her2/Neu -driven breast cancers in mice, but the underlying mechanism(s) remains controversial. Moreover, the role of PTP1B in HER2 + human breast cancer is unresolved. We found that, unexpectedly, PTP1B protects HER2 + breast cancer (BC) cell lines and tumors from hypoxia-induced death. Although there was no consistent effect of PTPN1 depletion or PTP1B inhibition on growth factor signaling or proliferation of HER2 + BC cells in vitro , PTP1B-deficient HER2 + xenografts showed increased hypoxia, necrosis and impaired growth. PTPN1 -knockdown (1B-KD) also sensitized HER2 + BC lines to hypoxia-induced death in vitro . Studies using catalytically inactive mutants or an allosteric PTP1B inhibitor demonstrated that the ability of PTP1B to promote survival in hypoxia requires catalytic activity. Metabolic analysis revealed increased non-mitochondrial oxygen consumption, accompanied by decreased α-ketoglutarate (α-KG) levels, in 1B-KD cells, suggestive of enhanced activity of one or more α-KG-dependent dioxygenases. Consistent with this notion, addition of the pan-oxygenase inhibitors IOXI or DMOG protected 1B-KD HER2 + BC cells from hypoxia-induced death. Studies with “substrate-trapping” mutants identified the product of the Moyamoya disease-associated gene RNF213 , as a PTP1B substrate in HER2 + BC cells. Remarkably, RNF213 -knockdown ( RNF213 -KD) rescued the effects of PTP1B-deficiency on non-mitochondrial oxygen consumption and hypoxia-induced death of HER2 + BC cells. RNF213 -KD also partially restored growth of tumors evoked by 1B-KD HCC1954 cells. RNF213 is a 591kDa E3-ligase with RING finger and AAA+ ATPase domains, not previously implicated in PTP1B action. Preliminary proteomic characterization revealed that BT474 1B-KD cells have RNF213-dependent alterations in the ubiquitylome. Future work will determine how these changes affect α-KG-dependent dioxygenase(s) activity. Our results reveal a new function for PTP1B, acting via RNF213, to control one or more α-KG-dependent dioxygenases in HER2 + BC cells. This novel PTP1B/RNF213 hypoxia-regulatory pathway is critical for the survival of breast cancer and possibly other malignant cells in the tumor microenvironment. Note: This abstract was not presented at the meeting. Citation Format: Robert S. Banh, Caterina Iorio, Richard Marcotte, Yang Xu, Dan Cojocari, Anas Abdel Rahman, Judy Pawling, Ankit Sinha, Toshiaki Hitomi, Toshiyuki Habu, Akio Koizumi, Sarah Wilkins, Thomas Kislinger, Christopher J. Schofield, James W. Dennis, Bradly G. Wouters, Benjamin G. Neel. PTP1B regulates the Moyamoya disease-associated E3 ligase, RNF213 and cellular dioxygenase activity to allow breast tumor survival in hypoxia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-302. doi:10.1158/1538-7445.AM2015-LB-302

Abstract A203: An in vitro tumor model to characterize tumor cell metabolism in heterogeneous complex microenvironments.

Tumor cells often exhibit altered metabolic behaviors that are influenced by the heterogeneity of the tumor microenvironment. This heterogeneity arises from the presence of multiple cell types, variable ECM compositions, and variable protein expression due to the presence of oxygen and nutrient gradients. Conventional methods of identifying tumor therapies fail to account for these metabolic variations in the context of a heterogeneous microenvironment. To address this shortfall, we have developed a layered, three-dimensional, heterogeneous tumor model that allows spatiotemporal metabolite collection on a timescale relevant for metabolomics profiling. SKOV-3 ovarian cancer cells were mixed with collagen and infiltrated into a scaffolding material to create a thin layer. Multiple layers were then stacked to make a thick section of tumor tissue which is then combined with a customized oxygen impermeable bioreactor. EF5 immunofluorescence was used to confirm the presence of an oxygen gradient and to quantify the spatial PO2 values, which mimic those found in solid tumors. Cell viability and nutrient, and drug distribution profiles were also characterized and profiles were found to resemble those found in spheroid culture and in vivo. Cellular response to hypoxia was confirmed through qPCR analysis of mRNA of hypoxia-inducible genes such as CA9 and CHOP, and with HIF1α staining. Our technology allows for the creation of complex, yet controlled tumor microenvironments while facilitating “snap shot” data acquisition, and will allow researchers to decouple the influence of heterogeneity on metabolic response in cancerous solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A203. Citation Format: Darren Rodenhizer, Dan Cojocari, Bradley G. Wouters, Alison P. McGuigan. An in vitro tumor model to characterize tumor cell metabolism in heterogeneous complex microenvironments. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A203.