Heme Oxygenase: Disease Relevance

HO-1 in Cancer

Heme oxygenase 1 (HO-1) has generally been considered as being a cytoprotective mediator, crucially involved in cancer progression by augmenting cell proliferation, metastasis, and angiogenesis as well as by conferring resistance to phytodynamic therapy and radio- and chemotherapy ²⁰². Compared to healthy tissues, the expression of HO-1 is up-regulated in different tumor types including lymphosarcoma, adenocarcinoma, hepatoma, glioblastoma, melanoma, prostate cancer, Kaposi sarcoma, squamous carcinoma, pancreatic cancer, renal cell carcinoma as well as in brain tumors and hematological malignancies (for a review see ^{202, 363-365}). Anti-cancer approaches can further increase the expression of HO-1 and thus attenuate the efficiency of the therapy ^{25, 366, 367}. However, pharmacological inhibitors or knockdown of HO-1 have been found to sensitize tumors to anti-cancer therapies ³⁶⁸. It is interesting to note that HO-1 over-expression in tumors confers resistance to anoikis, a certain subtype of cell death induced by the detachment of cells from the extracellular matrix and responsible for the metastatic spread of tumor cells ³⁶⁹. A critical factor in metastasis is the epithelial-to-mesenchymal transition (EMT) which is not only able to activate the Nrf-2/HO-1 pathway ³⁷⁰ but also to suppress migration and invasion of cancer cells by reversing EMT after HO-1 inhibition³⁷¹.

 

Clinically, patients with up-regulated HO-1 expression often show a reduced survival rate and poor outcome²². An HO-1 up-regulation was reported to correlate with enhanced metastasis and poor outcome in patients with non-small cell lung cancer (NSCLC) ³⁷², bladder cancer ³⁷³, and gall bladder cancer ³⁷⁴. Moreover, enhanced expression of HO-1 has been associated with an unfavorable prognosis in patients with astrocytoma ³⁷⁵, glioma ³⁷⁵, neuroblastoma ³⁷⁶, and cholangiocarcinoma ³⁷⁷. In patients with chronic myeloid leukemia (CML), HO-1 expression was enhanced in comparison to controls and culmulated markedly in relapsing disease ³⁷⁸. HO-1 is also up-regulated in further hematological malignancies such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and multiple myeloma where it functions as an important survival factor (for a review see Li Volti et al., 2017) ³⁶³. From this finding one can hypothesize that HO-1 might be considered as being a potential indicator for disease progression.

 

The definite role of HO-1 in carcinogenesis is controversial and multifaceted. In contrast to its well documented pro-tumorigenic actions, HO-1 has also been found to exhibit anti-tumorigenic effects, e.g., in breast ²⁴, prostate ²³, and pancreatic cancer ²⁵ as well as in NSCLC ²⁶. In the latter, HO-1 functions as a tumor supressor which blocks the proliferation, migration, growth and angiogenesis of cancer cells by simultaneously up-regulating tumor-suppressive miRNA and down-regulating oncogenic miRNA ²⁶. A tumor-suppressive activity of HO-1 can also be observed in lung mucoepidermoid carcinoma (MEC) where HO-1 activation was found as being associated with a significant down-regulation of matrix-degrading MMP-9 and MMP-13 and the concomitant impairment of the metastatic potential ³⁷⁹.

 

As described before, HO-1 can be found in several extra- and intracellular compartments such as the smooth ER ⁶⁷, cytosol ^{5, 6}, mitochondria ^{7, 8}, plasma membrane caveolae ⁹, chloroplasts ^10-13, extracellular space ¹⁴, and the nucleus ¹⁵. Nuclear HO-1 is catalytically inactive and lacks the C-terminal TMS ²³⁵, but it might function as a transcriptional regulator ^{15, 191} and a modulator of cytoprotective mechanisms ²²⁸, respectively. Several studies revealed a nuclear localization of HO-1 in different tumors such as lung ²³⁵, prostate ³⁸⁰, and head and neck cancer ³⁸¹ where it correlates with poor prognosis and disease progression. Apart from its expression in cancer cells, the positive HO-1 immunoreactivity is also detectable in stromal compartments, particularly in tumor-associated macrophages of cancer tissues ^382-384, indicating the impact of HO-1 in cancer progression by modifying the tumor microenvironment ³⁸⁵. Controversially, anti-tumorigenic actions of nuclear HO-1 have also been observed in prostate cancer where HO-1 serves as a repressor of its transcriptional activity by binding to the promoter region of prostate-specific antigen (PSA) ³⁸⁶. A further study in prostate cancer unveiled that the down-stream effector CO inhibits tumor growth and increases sensitivity to chemotherapy by augmenting metabolic exhaustion ²³². These findings clearly highlight the paradoxical role of HO-1 in carcinogenesis.

 

 HMOX1 Promoter Polymorphisms

The group of Michael Bauer recently identified the (GT)_n microsatellite polymorphism as a novel regulatory mechanism in HMOX1 translation as it affects alternative splicing within the 5’-untranslated region ²⁸⁸. Microsatellite dinucleotide (GT)_n length polymorphisms in the untranslated regions of the human HMOX1 gene are known to impede the transcriptional regulation and thus to down-regulate the expression of HO-1 in persons carrying the long allele of this polymorphism ²⁸⁹. Previous studies have linked the HMOX1 (GT)_n length polymorphism to cancer risk ³⁸⁷. Long (GT)_n repeats within the HMOX1 promoter have been linked to a higher risk of various human malignancies such as breast cancer ³⁸⁸, esophageal squamous cell carcinoma ³⁸⁹, gastric adenocarcinoma ³⁹⁰, lung adenocarcinoma ³⁹¹, malignant mesothelioma ³⁸⁷, and laryngeal squamous cell carcinoma ²⁵². On the contrary, shorter (GT)_n repeats within the HMOX1 promoter have been attributed to a higher risk of pancreatic cancer ³⁹² and melanoma ³⁹³, respectively.

 

Apart from its role in tumorigenesis, HMOX1 promoter polymorphisms have been found as being positively associated with numerous human diseases including atherosclerosis and cardiovascular disease ³⁹⁴, chronic kidney disease ³⁹⁵, coronary artery disease ³⁹⁵, necrotizing acute pancreatitis ³⁹⁶, and pre-eclampsia ³⁹⁷. In particular, the long (GT)_n allele has been reported as being associated with susceptibility to chronic obstructive pulmonary disease (COPD) ³⁹⁸, acute respiratory distress syndrome ³⁹⁹, vascular restenosis ⁴⁰⁰, rheumatoid arthritis ⁴⁰¹, and outcome of renal transplantation ⁴⁰². On the other hand, shorter (GT)_n HMOX1 promoter polymorphisms are significantly associated with hyperbilirubinemia risk in Indian newborns ⁴⁰³. Also small nucleotide polymorphisms (SNPs) in the HMOX1 gene have been identified, such as rs2071746 T/A which was found as being associated with protection from atherosclerotic stroke ⁴⁰⁴, and with increased risk of Parkinson’s disease (PD) ¹⁴³, respectively. However, consideration should be given to negative studies demonstrating no correlation between HMOX1 polymorphisms and the outcome of certain diseases, e.g., in urothelial carcinoma ⁴⁰⁵, sporadic colorectal cancer ⁴⁰⁶, coronary artery disease ⁴⁰⁷, and alcoholic liver disease ⁴⁰⁸. In line with these data, a meta-analysis evaluating the association of an HMOX1 (GT)_n repeat polymorphism and cancer susceptibility did not reveal any correlation between the (GT)_n repeat length and cancer risk at both, the allelic and genotypic level ⁴⁰⁹. These data clearly emphasize the occurrence of multi-factorial determinants of the final disease outcome.

 

HO-1 in Diabetes and Neurodegeneration

HO-1 has been implicated in the pathogenesis of diabetes mellitus. HO-1 expression is diminished in peripheral blood mononuclear cells (PBMCs) ⁴¹⁰ and the serum ²⁵⁰ from patients with type 2 diabetes mellitus (T2DM). Accordingly, serum levels of HO-1 are reduced in early pregnancy and associated with a higher risk of developing gestational diabetes mellitus ^{250, 411}. HMOX1 gene expression is markedly reduced in leukoctes from T2DM patients with and without diabetic microangiopathy compared to control individuals, negatively correlating with oxidative stress, glycosylated hemoglobin, and diabetes duration ⁴¹². Evidence is emerging to highlight the role of HO-1 in experimental diabetes as well. Importantly, up-regulation of HO-1 has been shown to increase insulin release from pancreatic beta-cells and to decrease hyperglycemia in different diabetic models ⁴¹³. Moreover, Lundquist et al. detected an increased insulin secretion in pancreatic beta-cells from diabetic mice upon exposure to the HO product CO ⁴¹⁴. As type 1 diabetes mellitus (T1DM) is accompanied by a massive production of inflammatory mediators coupled to elevated apoptotic cell death, HO-1 over-expression has been characterized to exert beneficial effects in the pathogenesis of T1DM ⁴¹⁵. Data raised by the group of Olga Pol recently demonstrated that the induction of HO-1 attenuates diabetic neuropathy and in turn improves anti-nociceptive effects of morphine in diabetic mice ⁴¹⁶. HO-1 induction was further correlated to lower blood pressure, enhanced serum adiponectin and expansion of insulin-sensitive adipocytes in obese mice ⁴¹⁷. These findings offer the opportunity of treating detrimental metabolic consequences of obesity.

 

Emerging evidence indicates a neuroprotective role of HO-1 in the pathogenesis of various neurodegenerative diseases, including Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington’s disease, and Parkinson’s disease (PD), ⁴¹⁸. Expression of HO-1 is restricted to a few scattered neuroglia and neurons in the normal brain, and can be induced by a variety of stressors in neuronal and non-neuronal cells ²⁶⁰. An up-regulated expression of HO-1 has been found in neurons and astrocytes of the hippocampus and cerebral cortex from AD patients ⁴¹⁹ as well as in astrocytes from PD patients ⁴²⁰. Moreover, enhanced serum/plasma levels of HO-1 were noted in AD patients ²⁴⁰ and PD patients ²⁴¹, respectively. Over-expression of HO-1 is also present in glial cells in the vicinity of cerebral infarcts, in contusions and hemorrhages, within multiple sclerosis (MS) plaques, and in alternative inflammatory and neurodegenerative disorders ⁴²¹. In PD, AD, and other neurodegenerative disorders, constitutive over-expression and thus hyperactivation of HO-1 culminates in pathological iron accumulation that promotes mitochondrial associated non-transferrin iron sequestration and macroautophagy ⁴²². It is noteworthy that HO-1 mediates dopaminergic cell injury upon exposure to polychlorinated biphenyls, and this might be a critical aspect in the pathophysiology of PD ⁴²³.

 

Extracellular HO-1

HO-1 can be found in various extracellular compartments including body fluids. HO-1 was identified not only in human milk ¹⁹⁶ but also the serum and plasma as well as the cerebrospinal fluid of healthy and diseased human individuals (summarized by Vanella et al., 2016) ¹⁷. Extracellularly located HO-1 might function as a potential biomarker in disease ^{18, 19} or as an extracellular receptor ligand ²⁰. Compared to controls, enhanced serum/plasma levels of HO-1 were determined in patients with pre-eclampsia ¹⁴, Alzheimer’s disease (AD) ²⁴⁰, Parkinson’s disease (PD) ²⁴¹, in patients resuscitated from out-of-hospital cardiac arrest ²⁴², acute kidney injury ²⁴³, interstitial pneumonia ²⁴⁴, silicosis ^{245, 246}, in patients with acute respiratory distress syndrome (ARDS) ^{247, 248}, and adult-onset Still’s disease (AOSD) ²⁴⁹. Decreased serum/plasma levels are observed in patients with type 2 diabetes mellitus (T2DM) ²⁵⁰, gestational diabetes mellitus ²⁵¹, and laryngeal squamous cell carcinoma ²⁵².

 

HO-1 in Vascular Diseases and Diseases of the Pulmonary Circuit

HO-1 is implicated in vascular diseases such as atherosclerosis ^{351, 424}. HO-1 is expressed in atherosclerotic lesions of LDL receptor-deficient mice ⁴²⁵ as well as apolipoprotein E-deficient mice and humans ⁴²⁶, where it is supposed to serve as an intrinsic protective factor against atherosclerotic lesion formation. In HMOX1^-/- mice, intimal hyperplasia was found in animals with aortic injury compared to controls ⁴²⁴. The protective nature of HO-1 could be confirmed in apolipoprotein E-deficient mice where over-expression of HMOX1 was noted to attenuate the development of atherosclerosis ⁴²⁷. Similarly, HMOX1 gene transfer improved vascular responsiveness and inhibited vascular smooth muscle cell proliferation in a pig model of arterial injury ⁴²⁴. HO-1 also plays a protective role in clinical cardiovascular diseases as evidenced not only by in vitro and animal experimentation but also by clinical trials in humans ⁴²⁸. Protection against cardiopathology is attributable to the anti-fibrinolytic and vasodilative activity of CO ^{217, 429-431}, anti-oxidant activity of bilirubin ^432-434, and iron sequestration by ferritin ⁴³⁵. In patients with coronary heart disease, the levels of HO-1 expression were found to vary in relation to the quantity of coronary lesions with highest levels found in acute myocardial infarction, followed by patients with unstable angina pectoris while lowest levels could be detected in stable angina pectoris ^{436, 437}. It should be pointed out that, in patients with angiographically-defined coronary artery disease, HO-1 expression reached maximum levels in patients with a greater disease burden ⁴³⁷. This is in line with observations demonstrating the presence of HO-1 exclusively in endothelial cells (ECs) from patients with advanced atherosclerotic lesions, and this expression positively correlated with VEGF protein levels in ECs ⁴³⁸.

 

As outlined before, HO-1 expression has been shown to exert neuroprotective effects not only in human neurodegenerative disorders but also during distinct pathological changes to the brain such as subarachnoidal hemorrhage (SAH), and ischemia and traumatic brain injury (TBI). The group of Richard Meyermann noted an increased accumulation of HO-1-positive microglia/macrophages at hemorrhagic lesions as early as 6 h after traumatic brain injury which was still detectable after 6 months ⁴³⁹. Conversely, after focal cerebral infarctions HO-1-positive microglia/macrophages accumulated exclusively within focal hemorrhages and were not detectable in non-hemorrhagic regions. A weak HO-1 expression was instead determined in astrocytes in the perifocal penumbra. From these findings the authors hypothesized that continuing expression of HO-1 in human glial cells after traumatic brain injury and cerebral infarction supports the recovery of neuronal tissue after these insults. A protective role of an up-regulated HO-1 expression has also been proposed in the development of intracranial aneurysms, but these data must be treated with caution due to the low number of patients enrolled ⁴⁴⁰. In this context, evaluation of correlative human data revealed that SAH patients have significantly higher HO-1 activity in cerebrospinal fluid compared to patients with unruptured cerebral aneurysms ⁴⁴¹. In a murine SAH model, the expression of HO-1 in microglia has been found as being required for alleviating neuronal cell death, vasospasm, cognitive dysfunction, and clearance of cerebral blood burden ⁴⁴¹. Since CO inhalation after SAH rescued the absence of microglial HO-1 and reduced neuronal injury and cognitive dysfunction, CO may be considered as being a potential therapeutic modality in patients with ruptured cerebral aneurysms ⁴⁴¹.

 

HO-1 plays an important role in the pathogenesis of diseases of the pulmonary circuit. As demonstrated by Solari and co-workers, lung tissues from patients with congenital diaphragmatic hernia and persistent pulmonary hypertension (PPH) harbored a reduced expression of HO-1 and eNOS which might add to modified vascular tone causing PPH ⁴⁴². Interestingly, HMOX1^-/- mice showed signs of pulmonary hypertension under hypoxic conditions such as right ventricle dilatation, and right ventricular infarcts with organized mural thrombi ⁴³¹. In transgenic mice, HO-1 over-expression effectively inhibited pulmonary inflammation and prevented pulmonary hypertension induced by hypoxia implying a crucial protective function of HO-1 products as inhibitors of hypoxia-induced vasoconstrictive and pro-inflammatory pathways ⁴⁴³. HO-1 gene transfer has also been reported to diminish hypoxia/reoxygenation-induced stasis in mice with sickle cell disease (SCD), an archetypal example of hemolysis, by inducing anti-inflammatory responses ⁴⁴⁴.

 

HO-1 in other Diseases

HO-1 plays a critical role in later stages of myocarditis as HO-1 was reported to mediate apoptosis of heart muscle cells via activation of caspase-3 ⁴⁴⁵. HO-1 also contributes to muscle atrophy, since HO-1 over-expression has been shown to cause muscle damage in vitro and in vivo while HO-1 deficiency significantly reduced muscle atrophy ⁴⁴⁶.

 

To date, only two cases of HMOX1 deficiency in humans have become apparent ^{447, 448}. Both cases show a similar phenotype featuring generalized inflammation, asplenia, anemia, nephropathy, and tissue iron deposition. In addition, growth retardation and vascular injury were noted in both patients, indicating the impact of HO-1 in vascular physiology. The low penetrance of this gene deficiency in humans might be elucidated by the observation that deletion of the Hmox1 gene in mouse embryos is lethal in most cases, most likely due to defects in the placental vasculature ⁴⁴⁹. Hmox1-deficient mice share a common phenotype with the human HMOX1 deficiency, characterized by thorough inflammation, iron overload, splenomegaly, hepatomegaly, hepatic fibrosis, growth retardation, and premature death ⁴⁴⁹. Additionally, Hmox1^-/- mice are further characterized by leukocytosis and predominance of activated CD4⁺ T cells, production of pro-inflammatory cytokines as well as protein and lipid peroxidation ^{450, 451}.