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Hypoxia and the Hallmarks of Cancer: Angiogenesis and Metastasis

Hanahan and Weinberg’s “Hallmarks of Cancer” are at the root of the multi-step progression of cancer, and they are all influenced by hypoxia in the tumor microenvironment. In this mini-review series, HypOxygen has been taking a closer look at the way HypOxystation users worldwide are delineating the effects of hypoxia on the Hallmarks of Cancer: so far, we’ve showcased Avoiding Immune Destruction and Tumor Promoting Inflammation and Genome Instability and Mutation and Enabling Replicative Immortality.  

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Hypoxia and the Hallmarks of Cancer: Genome Instability and Immortality

HypOxygen continues to look at the way the iconic “Hallmarks of Cancer”, as first described by Douglas Hanahan and Robert Weinberg, are influenced by hypoxia in the tumor microenvironment. Oxygen around and within the tumor cells is central to metabolism, immunology, epigenetics and therapy resistance of all the cancers; in the lab, oxygen levels during tumor cell culture exert effects on metabolism, maintenance, cell yield, and cell survival. 

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Hypoxia and the Hallmarks of Cancer: Inflammation and Immunity

The iconic “Hallmarks of Cancer” first described by Douglas Hanahan and Robert Weinberg in 2000 continue to develop explosively as efforts to illuminate the “acquired capabilities of cancer” evolve. Hypoxia in the fast-growing, poorly perfused tumor setting is one of the main factors selecting for survival of cancer cells and driving mutagenesis and metastasis. Oxygen in the tumor microenvironment and within the cells is central to metabolism, immunology, epigenetics and therapy resistance of all the cancers.  

Read more: Hypoxia and the Hallmarks of Cancer: Inflammation and Immunity

Don Whitley Visits The Francis Crick Institute

DWSCrickDr Don Whitley, chairman and founder of DWS, recently visited The Francis Crick Institute in London to see the recently installed Whiltley H45 Hypoxystation at the site. 

The Crick has moved into a brand new state-of-the-art building in the centre of London. Situated next to Kings Cross and St Pancreas stations, The Crick brings together 1500 scientists and staff working collaboratively in the biggest biomedical research facility under one roof in Europe. The work at The Crick covers many disciplines and applications in biomedical research, all with the aim of improving understanding of human health and disease. 

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Therapeutic Targeting of Hypoxia and HIF's in Cancer

“Tumor hypoxia and HIF’s affect most of the cancer hallmarks … and contribute to chemo- and radiotherapy resistance.” In their review from 2016, Wigerup, Pahlman and Bexell of Lund University in Sweden discuss how hypoxia inducible factors HIF’s regulate the hypoxic microenvironment in cancer, and the therapeutic strategies that are being developed to improve patients’ prognosis. Dr. Sven Pahlman’s lab has been using the H35 HypOxystation for more than 5 years now, to research SCLC and neuroblastoma, and their data is contributing to the understanding of the role of oxygen levels in the progression of cancer.

Hypoxia and HIF-1α and 2α expression in cancer usually signify a worse prognosis, but most hypoxia-induced transcriptional, translational, and epigenetic changes are cell-type specific. Many effects engendered by hypoxia are mediated directly or indirectly via HIF pathways, and most are causative of the iconic “Hallmarks of Cancer” that Hanahan and Weinberg introduced in 2000 and expanded in 2011. Hypoxia induces increased autophagy, apoptosis, and aberrant cell proliferation; neoangiogenesis mediated by VEGF and PDGF-β; proliferation of cancer stem cells; metabolic reprogramming to satisfy energy and synthetic requirements in proliferating cells; modulation of inflammation and immune responses; genomic instability through increased mutagenesis and diminished DNA repair; and metastasis as hypoxia induces epithelial-to-mesenchymal transition and degradation of the extracellular matrix. Assaying the relationship between hypoxia and the Hallmarks of Cancer benefits significantly from the physiological atmosphere mimicked in the HypOxystation, a closed-culture hypoxia workstation controlling gasses, temperature and humidity.

Read more: Therapeutic Targeting of Hypoxia and HIF's in Cancer

Jumping Off the Redox Cliff

Increased production of ROS, which is both a symptom and a driver of cancer Hallmarks, can push cancer cells over the cliff of oxygen homeostasis. Compounds adding oxidative pressure can thus be utilized for selective tumor therapy. In their paper “Triggering apoptosis in cancer cells with an analogue of cribrostatin 6 that elevates intracellular ROS” , HypOxystation users Asby et al. describe their approach to chemically modifying a natural compound from marine sponges, cribrostatin 6, to enhance its cytotoxic potential. They synthesized a modified molecule 8-phenylcribrostatin 6 (8PC6) that was both more potent and more selective for breast cancer cells.  Co-author Ali Tavassoli from the University of Southampton says, "We study HIF-1, so working in a hypoxic environment is critical. Besides culturing our cells in the H35, we also harvest proteins and collect RNA inside the chamber. The H35 is very easy to use; the touch screen controls are straightforward and intuitive. We have used the workstation to incubate cells in hypoxia for ~5 days, and the atmosphere remains stable over time."

Annexin V/7-AAD staining indicated that 8PC6 induces apoptosis in cancer cells. Treatment of MCF7 cancer cells with ROS-sensing dyes and siRNA to knock down ROS-protective TIGAR demonstrated that 8PC6 increases intracellular reactive oxygen species, upsetting the delicate redox balance in highly susceptible cancer cells and leading to cell death. Hypothesizing that reduction of the cribrostatin analogue yields a semi-quinone that reacts with molecular oxygen to generate superoxide, Asby’s group decided to withdraw oxygen from the equation by incubating the MCF7 cells in the HypOxystation at 1% O2. Pre-incubation and subsequent incubation with increasing doses of 8PC6 in normoxia versus hypoxia showed that, indeed, the IC50 was increased up to 46-fold in hypoxia due to lack of oxygen. The HypOxystation’s closed workstation format and rigorous control of oxygen, CO2, temperature and humidity facilitates authentic cell behavior as in vivo conditions are replicated. Thus, hypoxia in the workstation equated to significant reduction in the intracellular availability of oxygen for the generation of ROS. For research being conducted on highly hypoxic tumors, the workstation atmosphere represents a close approximation of the actual conditions cells encounter. 

Read more: Jumping Off the Redox Cliff