Vitamin C & Cancer – HIF – The Oxygen Sensor

The 2019 Nobel Prize in Physiology or Medicine was awarded jointly to William G. Kaelin Jr., Sir Peter J. Ratcliffe and Gregg L. Semenza for their discoveries of how cells sense and adapt to oxygen availability by using specific molecules that regulate the activity of genes (both mitochondrial and nuclear).  This ensures the survival of cells under different levels of oxygen tension [1]. The molecules that help cells adapt to oxygen fluctuations belong to a complex called the Hypoxia-Inducible Factors (HIF).  

The hypoxia pathway was first recognized in 1991 [2] when scientists connected the production of red blood cells to low oxygen levels, or hypoxia. The HIF complex was formally identified in 1995 by Semenza et al [3].  HIFs can orchestrate the production of red blood cells in response to systemic hypoxia by inducing epigenetic changes in gene expressions that increases erythropoietin production in liver and kidney [4] Why is cellular response to oxygen so important that the discovery of HIF led to the award of the Nobel Prize in 2019?

Hypoxia, Oxygen & HIF

Hypoxia is a condition when inadequate oxygen supply restricts functions in tissues and cells.  To counter the effects of low oxygen, our bodies have evolved extremely effective response systems mediated by the hypoxia-inducible transcription factors (HIFs) [5].  

Ever since the Great Oxygenation Event 2.45 billion years ago, when the earth’s atmosphere was changed from a reducing one to an oxidizing one, oxygen became the irreplaceable final electron acceptor for all species that utilize aerobic metabolism. Having an electronegativity value of 3.44, oxygen is an ideal element for the bonding of electrons during energy metabolism [6]. 

Oxygen partial pressure (pO2) measures the balance between oxygen delivery and consumption in cells.  In mammals, oxygen (O2) is transported by red blood cells and delivered to organs and tissues depending on their different levels of metabolic requirements.  Therefore, under physiological conditions, organs and tissues all have their own unique levels of ‘normoxia’, or normal levels of oxygen. Whereas tissue oxygenation is highly dysregulated in pathological settings including cancer, diabetes, coronary heart disease and stroke, where hypoxia, or a decrease in pO2 is observed [7]. 

Ambient oxygen levels in earth’s atmosphere is currently around 21%. Yet human tissues and organs require varying levels of oxygen, from 19.7% in trachea, to 3.8% in muscles.  The brain only requires 4.4% oxygen! [7]. Even though oxygen is critical for energy production in mitochondria, different oxygen gradients play important roles in that low oxygen or physiological hypoxia (under 2% oxygen) is actually required for providing extracellular stimulus in embryogenesis [8], wound healing [9] and the maintenance of stem cell pluripotency [10].  

Low oxygen levels can initiate the HIF response pathway, leading to epigenetic changes in gene expression.  The difference between physiological and non-physiological hypoxia is key to understanding the fine line maintained by HIF in health and disease. 

Cancer, Wound Healing & Angiogenesis: The HIF Connection

In 1966, H.F. Dvorak coined the famous phrase describing tumors as “wounds that do not heal”. The implication being the processes associated with wound healing are similar to the conditions involved in the development of tumor stroma [11].  Those conditions are chronic inflammation and active angiogenesis. 

Angiogenesis is the physiological process where new blood vessels are formed from pre-existing vessels. Blood vessels deliver oxygen and nutrients to every part of the body. The growth of additional blood vessels also nourish cancer cell proliferation. Insufficient vessel growth can lead to stroke, myocardial infarction, ulcerative disorders and neurodegeneration. Whereas abnormal vessel increase fuels cancer [12], inflammatory disorders and pulmonary hypertension [13].

The balance between activators and inhibitors regulate angiogenesis.  Tumor cells can stimulate activators of angiogenesis and also down-regulate inhibitors of angiogenesis. Therefore, the balance between activators and inhibitors of angiogenesis determines whether it follows a physiological (wound healing) or pathological (tumor) progression [14]. 

The primary stimulus of angiogenesis is the activation of HIF-1.  Angiogenesis is regulated by a balance between stimulatory and inhibitory growth factors and stressful conditions such as alterations in oxygen levels [15].  Decreased oxygen concentration initiates the HIF pathway that will activate the expression of a wide array of genes to enhance glucose uptake, increased red blood cell production, and the formation of new blood vessels in angiogenesis [16].

Angiogenesis is indispensable in the establishment of a mature vascular system that can delivery proper levels of oxygen and nutrients to all cells in both normal tissues and hypoxic areas.  Angiogenesis is therefore essential for normal growth as well as tumorigenesis. Tumor cells must be able to develop an infrastructure that can support increased metabolic demands. Understanding the role of hypoxia and the HIF pathway in tumor development is critical to the management and treatment of cancer [17]. 

Hypoxia-Inducible Factors (HIF)

Hypoxia-inducible factors (HIFs) are a family of transcription factors that are activated by hypoxia, or a decrease in cellular oxygen levels. What type of conditions can activate hypoxia-inducible factors?

If you live in a low lying area and travel to high altitudes, you may experience environmental hypoxia.  Atmospheric hypoxia occurs at high altitudes when total atmospheric pressure decreases as altitude increases, causing  a lower partial pressure of oxygen, even though oxygen remains at 20.9% of the total gas mixture. This condition is called hypobaric hypoxia [18], which is different from hypoxic hypoxia, where the actual percentage of oxygen is decreased in the air or organs and tissues in the body. 

When the body transitions from a low altitude to a high altitude environment, profound physiological changes, including haematological, respiratory and cardiovascular adaptations induced by hypoxia-inducible factors occur,  assisting the acclimation to lower oxygen availability at high altitudes [19]. How does HIF accomplish these processes?

HIF – Quintessential Oxygen Sensor & Gene Regulator

Hypoxia-inducible factor (HIF) is a DNA-binding protein that can promote or repress the transcription of a large number of genes involved in the maintenance of biological homeostasis. Genes targeted by HIF assist cells to adapt and survive in stressful microenvironments.  HIF is mostly non-functional in oxygenated cells but is activated under specific conditions, including low-oxygen stress or hypoxia. Since the central role of HIF is in the sensing of oxygen, it is therefore critical for the survival of organisms. 

In disease states such as cancer, ischemic disorders of the heart and the brain, hypoxia is often the result of inadequate supply of oxygen due to defective or inadequate vasculature [20]. During tumor progression, expansive growth of cells create large distances between tumor cells and blood vessels that carry oxygen and nutrients, resulting in a hypoxic microenvironment that would activate HIF [21].  The activation and stabilization of HIF initiates adaptive survival processes that maintain metabolic equilibrium, pH homeostasis that encourages further tumor growth, promoting metastasis [22, 23]. 

HIF is a heterodimeric complex, which means it is composed of two polypeptide chains with different compositions.  The HIF complex consists of an oxygen-destructible α-subunit and an oxygen-indestructible β-subunit, (HIF-1a, HIF-1b respectively).  Under hypoxic conditions, three isoforms of the α-subunit (HIF-1a, HIF-2a, HIF-3a), and two isoforms of the β-subunit (HIF-1b, HIF-2b) are believed to be involved in the in vivo responses to hypoxia [24].  

Under normoxia with sufficient oxygen, enzymes proceed to degrade and inactivate the α-subunits.  In hypoxic conditions when oxygen levels are low, these enzyme-mediated processes are inhibited, resulting in the stabilization and translocation of the α-subunits [25].  Stabilized HIF α-subunits interact with HIF β-subunits to produce epigenetic changes by binding to specific DNA sequences [26]. 

HIF is known to increase the expression of more than 60 well-defined gene products, including the one that encodes vascular endothelial growth factor A (VEGF-A), responsible for the promotion of angiogenesis [27].  Other gene expressions induced by HIF are involved in metabolism, vasodilation, erythropoiesis, pH homeostasis, oxygen sensing and autophagy, to name only a few. HIF activation can also lead to the repression of a broad range of gene products [28]. 

Is oxygen the only regulator of HIF? 

If you think about how life began with the cyanobacteria over 3.5 billion years ago [40], the creation and evolution of life forms required not only oxygen, but light. Cyanobacteria filled the earth’s atmosphere with oxygen using the powerful energy from light photons to split water into oxygen, protons and electrons [41].  

Light, Circadian Rhythm & Clock Genes – The HIF Connection

The cyanobacteria use light, carbon dioxide and water to produce energy and oxygen.  The backbone of photosynthesis is built upon reduction and oxidation reactions where electrons are constantly being exchanged.  The flow and exchange of electrons in both photosynthesis or chemical redox couples in respiration is the foundation of life on earth. During the exchange of electrons, reactive oxygen species (ROS) are created naturally and sometimes in abundance [29].  It is therefore not surprising that both light and ROS can regulate HIF. 

Intense light has always been the main regulator of human circadian rhythm and associated clock genes [34]. Yet both clock genes and HIF proteins belong to the same PAS domain superfamily of signal sensors for oxygen, light or metabolism [30, 31].  This evolutionary conserved relationship between light and oxygen sensing pathways suggests that light-induced circadian rhythm proteins, or clock genes, are intimately related to oxygen sensing proteins like HIF [37].  

HIF1a mRNA have been observed to cycle naturally in a circadian manner in murine cardiac tissues [32].  Changes in oxygen levels have been shown to be able to effectively RESET circadian clocks through HIF1a activation [33].    

In August 2019, a groundbreaking paper released by Yoshimasa Oyama et al. revealed that intense light can protect the heart from myocardial ischemia by using HIF1A transcriptional reprogramming of the endothelium in a PER2 clock gene-dependent manner [35].

Oyama et al. showed that under both normoxia and hypoxia, the clock gene PER2 activated by intense light, is an essential co-factor for HIF-1a in gene transcription processes that control the switch from energy-efficient lipid metabolism to oxygen efficient glucose metabolism during myocardial ischemia. The switch in metabolism is what allows the myocardium to function under stress [35].  This also means that the HIF pathway is deeply tied to mitochondria. 

Mitochondria, Oxidative Stress & HIF – The ROS Connection

Mitochondria consumes as much as 90% of cellular oxygen in order to maintain electron transport chain requirements that sustain ATP energy production by oxidative phosphorylation (OXPHOS) [42].  The reduction in oxygen availability under hypoxia forces cells to adapt their metabolic program in order to maintain biological processes that normally rely on ATP energy supplied by oxidative phosphorylation (OXPHOS) [43].  

Science has known for a long time that HIF signalling supports anaerobic ATP production in glycolysis, while down-regulating oxidative phosphorylation (OXPHOS) in mitochondria.  This metabolic switch effectively relieves the reliance on oxygen-dependent energy production during hypoxia [36]. However, the relationship between HIF and mitochondria extends far beyond metabolic adaptations during low oxygen stress.

During hypoxia, electron transport chains in mitochondria generate a large amount of superoxide, resulting in excessive oxidative stress [38].  HIF1a has been found to be recruited to mitochondria in direct response to oxidative stress, whether under hypoxia or excessive accumulation of reactive oxygen species (ROS).  When HIF1a is translocated to mitochondria, it is able to reduce ROS levels, reverse mitochondrial damage and protect mitochondria against oxidative stress induced apoptosis. HIF1a was demonstrated to directly regulate mitochondria using mechanisms that are independent of its gene transcription activity in the nucleus [39].

Translocated to mitochondria, HIF1a was able to lower production of reactive oxygen species (ROS) without having to increase activity of antioxidant enzymes like SOD or catalase.  In fact, mRNA of SOD2 and catalase were found to be markedly reduced in HIf1a expressing mitochondria compared to controls [39].

Yet HIF1a have been observed to cause mitochondria fission (fragmentation) that leads to the death of pancreatic beta cells, resulting in the loss of function in Type 1 and Type 2 diabetes [44].  How can the same protein cause such different effects in mitochondria?  

Clearly the activation and stabilization of HIF can result in opposing results of protection or damage. HIF can be activated and stabilized not only under hypoxic conditions, but under normoxia.  The primordial elements of oxygen and its counterpart reactive oxygen species; iron; and Vitamin C (ascorbic acid) play major roles in the degradation, activation and stabilization of HIF under normoxia and hypoxia, affecting the development and progression of a wide array of diseases including cancer. —— To Be Continued ——-



[1] The Nobel Prize in Physiology or Medicine 2019 – Press release

[2] Hypoxia-inducible nuclear factors bind to an enhancer element located 3′ to the human erythropoietin gene.

[3] Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.

[4] Novel Masters of Erythropoiesis: Hypoxia Inducible Factors and Recent Advances in Anemia of Renal Disease – FullText – Blood Purification 2016, Vol. 42, No. 2 – Karger Publishers 

[5] Hypoxia-inducible factors – Wikipedia 

[6] Electronegativities of the elements (data page) – Wikipedia

[7] Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia

[8] The Role of Hypoxia in Development of the Mammalian Embryo: Developmental Cell

[9] The Role of Hypoxia-Inducible Factor in Wound Healing

[10] Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions

[11] Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. – PubMed – NCBI

[12] Angiogenesis in Cancer  

[13] Angiogenesis: an organizing principle for drug discovery? – PubMed – NCBI

[14] Normal Wound Healing and Tumor Angiogenesis as a Game of Competitive Inhibition 

[15] Signal Transduction by Vascular Endothelial Growth Factor Receptors

[16] The Role of Hypoxia-Inducible Factor in Wound Healing

[17] Molecular and Functional Imaging Insights Into the Role of Hypoxia in Cancer Aggression

[18] The physiological effects of hypobaric hypoxia versus normobaric hypoxia: a systematic review of crossover trials

[19] The human side of hypoxia‐inducible factor – Smith – 2008 – British Journal of Haematology – Wiley Online Library

[20] Role of hypoxia-inducible factor in cell survival during myocardial ischemia-reperfusion. – PubMed – NCBI

[21] Hypoxia and cancer. – PubMed – NCBI

[22] Hypoxia-Inducible Carbonic Anhydrase IX and XII Promote Tumor Cell Growth by Counteracting Acidosis through the Regulation of the Intracellular pH | Cancer Research

[23] Lysyl oxidase is essential for hypoxia-induced metastasis. – PubMed – NCBI 

[24] Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-α-prolyl-4-hydroxylases

[25] Activation of hypoxia-induced transcription in normoxia. – PubMed – NCBI

[26] The hypoxia-inducible factor and tumor progression along the angiogenic pathway. – PubMed – NCBI

[27] Targeting HIF-1 for cancer therapy. – PubMed – NCBI

[28 Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1 | Blood | American Society of Hematology

[29] Reactive oxygen species, oxidative signaling and the regulation of photosynthesis – ScienceDirect

[30] PAS domains: internal sensors of oxygen, redox potential, and light. – PubMed – NCBI

[31] The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. – PubMed – NCBI  

[32] Adora2b-elicited Per2 stabilization promotes a HIF-dependent metabolic switch crucial for myocardial adaptation to ischemia. – PubMed – NCBI 

[33] Rhythmic Oxygen Levels Reset Circadian Clocks through HIF1α. – PubMed – NCBI

[34] Humans Entrain to Sunlight – Impact of Social Jet Lag on Disease and Implications for Critical Illness. – PubMed – NCBI 

[35] Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium 

[36] HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. – PubMed – NCBI 

[37] Reciprocal Regulation between the Circadian Clock and Hypoxia Signaling at the Genome Level in Mammals. – PubMed – NCBI

[38] Redox signaling during hypoxia in mammalian cells. – PubMed – NCBI

[39] HIF-1α protects against oxidative stress by directly targeting mitochondria – ScienceDirect  

[40] Early Archean origin of Photosystem II – Cardona – 2019 – Geobiology – Wiley Online Library 

[41] The mechanism of photosynthetic water splitting. – PubMed – NCBI

[42] Measurement of steady-state values of respiration rate and oxidation levels of respiratory pigments at low oxygen tensions. A new technique. – PubMed – NCBI

 [43] Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. – PubMed – NCBI

[44] Increased mitochondrial fission is critical for hypoxia-induced pancreatic beta cell death. 

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