Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response

Abstract

Hypoxia and free radicals, such as reactive oxygen and nitrogen species, can alter the function and/or activity of the transcription factor hypoxia-inducible factor 1 (HIF1). Interplay between free radicals, hypoxia and HIF1 activity is complex and can influence the earliest stages of tumour development. The hypoxic environment of tumours is heterogeneous, both spatially and temporally, and can change in response to cytotoxic therapy. Free radicals created by hypoxia, hypoxia-reoxygenation cycling and immune cell infiltration after cytotoxic therapy strongly influence HIF1 activity. HIF1 can then promote endothelial and tumour cell survival. As discussed here, a constant theme emerges: inhibition of HIF1 activity will have therapeutic benefit.

Figure 1. Features of hypoxia-inducible factor (HIF1) regulation

HIF1 transcriptional activity regulates numerous genes that control a variety of cellular functions, including anaerobic metabolism, angiogenesis stimulation and mechanisms for resistance to therapy. HIF1 transcriptional activity requires formation of a heterodimer consisting of HIF1α and HIF1β. The heterodimer binds to hypoxia response elements (HREs) in promoter regions of its target genes, where it activates transcription. Whereas HIF1β is constitutively expressed, HIF1α protein levels are subject to a number of points of regulation. HIF1α consists of the following regulatory domains: bHLH (basic-helix-loop-helix), PAS (Per-ARNT-Sim), NTAD (N-terminal transactivation domain), CTAD (C-terminal transactivation domain) and ODD (oxygen-dependent degradation domain). The rate of synthesis of HIF1α is controlled by activation of the phosphatidylinositol 3-kinase (PI3K)-Akt and Ras pathways by a variety of stimuli. HIF1α protein is rapidly targeted by the von Hippel-Lindau protein (VHL) complex for proteasomal degradation under normoxic conditions, following an oxygen-dependent prolyl hydroxylation of proline residues in the ODD. Activity of the prolyl hydroxylases (PHDs) is influenced by protein kinase C (PKC), PTEN and reactive oxygen species (ROS). Nitric oxide (NO) has variable effects on the stability of HIF1α, depending on the oxygenation state of the cell. Once HIF1α forms a heterodimer with its partner, HIF1β, the transcriptional activity is further regulated by cofactors, such as CREB binding protein (CBP) or factor-inhibiting HIF (FIH). ARD, acetyl transferase.

Figure 2. Seven points of regulation of tumour oxygenation

Seven features are often present in tumours, contributing in a multifactorial and interrelated fashion to hypoxia. a | Intravascular partial pressure of O₂ (pO₂) drops as oxygen is unloaded from haemoglobin in red blood cells (RBCs) as they traverse distally from feeding arterioles; this is termed a longitudinal oxygen gradient. The distance that oxygen can diffuse radially from a vessel is dependent on how much oxygen is present in the vessel. The extent and severity of hypoxia surrounding blood vessels will become more severe as intravascular pO₂ drops. b | At the extreme, red blood cells lose virtually all oxygen and there is no oxygen available to diffuse out into the tumour. c | Shunt flow diverts blood around the tumour, stealing nutrients from the tumour bed. d | Low vascular density creates hypoxia in extravascular tumour tissue because of limitations on the diffusion distance of oxygen once it leaves the blood vessel. e | Blood vessels that are haphazardly oriented will be less efficient in supplying adequate oxygen to all regions of tissue. f | Oxygen consumption rate is the most dynamic feature of oxygen transport in tumours. Small changes in demand for oxygen create large changes in the extent and severity of hypoxia, because higher demand not only limits the diffusion distance of oxygen, it also more severely depletes vessels of oxygen, thereby exacerbating longitudinal oxygen gradients. g | Intravascular hypoxia reduces red blood cell deformability, increasing blood viscosity, which in turn reduces flow rate. Relative oxygen concentrations are colour-coded. The direction of blood flow is from the red vessel(s) to the blue vessel(s) as indicated by the grey arrows. These diagrams provide a general pictorial depiction of these basic features. In reality, within any tumour there are microregional variations in oxygenation, with oxygen concentration decreasing radially from microvessels (FIG. 3).

Figure 3. Composite model for the effect of cycling tumour hypoxia on radial diffusion of oxygen

In this figure, the effect of four different types of change in red cell flux (the primary determinant of oxygen delivery) on the location and severity of hypoxia are depicted within a tumour microregion. a | The baseline case depicts balanced red cell fluxes in all microvessels. The hypoxic zone, defined in this discussion as regions where the partial pressure of O₂ (pO₂) <10 mmHg, is centrally located. b | If the red cell flux drops by an equal proportion in all microvessels, the size and severity of the hypoxic zone will increase, but it will still be centrally located. Spontaneous fluctuations in red blood cell flux within small networks such as this have been shown to change the oxygen gradient, creating a switch from a to b, on a timescale of 1–3 cycles per hour. c | Vasodilation of a downstream vessel steals red blood cells from the bottom segment, because flow resistance is much less in the dilated vessel. This change will shift the hypoxic zone downward. This may occur during the process of ongoing angiogenesis. d | Vascular remodelling (here indicated by intussusception) changes the flow resistance in the split downstream vessel, diverting flow upstream. This will reduce the hypoxic zone and shift the gradient upward. If the pO₂ of the blood entering the microregion is higher overall, then the effects of these shifts will be less apparent on the tumour cells in terms of severity of hypoxia. If the pO₂ of the blood entering the microregion is lower, then the severity will be greater. Vascular remodelling and/or variations in shunt flow can also alter the extent and severity of hypoxia from day to day, by changing the oxygen delivery to the tumour microregion.

Figure 4. Models for role of hypoxia in initiation and acceleration of angiogenesis

The vascular crisis model was hypothesized by Holash et al. to explain how hypoxia leads to angiogenesis initiation. It is characterized by an early overexpression of angiopoietin 2 (ANGPT2) in the absence of vascular endothelial growth factor (VEGF), which leads to vascular regression and hypoxia. Once hypoxia occurs, VEGF is upregulated, promoting initiation of angiogenesis. The acceleration model was hypothesized by Cao et al.. In this model, hypoxia is not responsible for initiation of angiogenesis; rather it is driven by non-hypoxia-mediated mechanisms, such as VEGF upregulation by oncogenes. Once angiogenesis is initiated, proliferation of tumour cells occurs, creating hypoxia. Hypoxia-inducible factor 1 (HIF1) is then upregulated to accelerate angiogenesis. Both models incorporate the concept of cooption before angiogenesis and both emphasize the importance of hypoxia in robust and disregulated angiogenesis. The two models converge on a cycle of unchecked angiogenesis, keyed by instability in perfusion, reactive oxygen species, hypoxia and persistent HIF1 activity. Further work is needed to establish how accurate these models are in both primary and metastatic tumour models.

Figure 5. Mechanisms for hypoxia-inducible factor 1 (HIF1) upregulation and consequences after radiation therapy

a | Untreated tumours often have hypoxic subregions. In hypoxic regions, stress granules form containing HIF1-mediated transcripts, sequestering the transcripts from being translated into protein. b | After radiation treatment, better oxygenated cells die and there is an increase in perfusion, leading to reoxygenation of the previously hypoxic cells. Stress granules disaggregate, releasing HIF1-regulated mRNAs, which can then go on to be translated into protein. Reoxygenation also causes hypoxia–reoxygenation injury, causing byproduction of reactive oxygen species (ROS), which stabilize HIF1α, even in the presence of improved oxygenation. c | Macrophages are attracted to the dying tumour cells, become activated and release nitric oxide (NO), which also stabilizes HIF1α. d | The increase in HIF1 activity increases vascular endothelial growth factor (VEGF) levels, promoting endothelial cell survival, angiogenesis and tumour cell survival and proliferation. This is highly simplified. Reoxygenation does not completely eliminate hypoxia and there are oxygen gradients within tumours as opposed to categorical differences between aerobic and hypoxic cells.