Abstract
The hallmarks of cancer are described as the distinctive and complementary capacities that cells must acquire during the multistep development of becoming a cancer cell that allow them to survive, proliferate and disseminate. The renin-angiotensin system (RAS) was first discovered and extensively studied in the physiological regulation of systemic arterial pressure. RAS signalling increases cell proliferation in malignancy by directly affecting tumour and stromal cells and by indirectly modulating the growth of vascular cells during angiogenesis. We aim to describe and give a general view of how the RAS is involved in several hallmarks of cancer and how this could open a window to several interesting treatments.
Introduction
In 2000, Hanahan and Weinberg defined the hallmarks of cancer as the distinctive and complementary capacities that cells must acquire during the multistep development of becoming a cancer cell that allow them to survive, proliferate and disseminate. In that classic article, six hallmarks were established: sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. 1 Eleven years after establishing the hallmarks of cancer paradigm, four new hallmarks were added, including: reprogramming of energy metabolism, evading immune destruction, genome instability and inflammation, which promote multiple hallmarks, resulting in 10 total hallmarks. Acquiring multiple hallmarks depends, in large part, on a series of alterations in the genomes of neoplastic cells; certain mutant genotypes confer selective advantage on subclones of cells, enabling their overgrowth and eventual dominance in a local tissue environment. 2 Several biochemical networks are involved in these processes, and many of them are obvious targets of study because of their roles in the cell cycle and in maintaining the integrity of the genome. But others, such as the renin-angiotensin system (RAS), which is known for controlling the homeostasis of the organism and not for any apparent involvement in cancer, are now being added to the complex networks of carcinogenesis. 3
The RAS was first discovered and extensively studied in the physiological regulation of systemic arterial pressure and, therefore, in the pathogenesis of hypertension.
4
As a gatekeeper system, RAS is involved in more than one function.
RAS: The classical and novel view
The classical view of the RAS is presented as a hormonal circulating system that is centred on the active peptide Angiotensin II (AngII). Angiotensinogen (AGT) is synthesised and released by the liver into the blood flow in response to decreased blood pressure or plasma sodium. The juxtaglomerular cells of the kidneys release aspartyl protease renin, which cleaves AGT at its N-terminus, generating the inactive decapeptide Angiotensin I (AngI) and leaving the remaining 98% of the molecule as a much larger fragment called des (angiotensin I) Angiotensinogen (des(AngI)AGT). AngI is further hydrolysed by angiotensin-converting enzyme (ACE), which is expressed in the lungs, to give the active octapeptide AngII that increases plasma aldosterone, promotes vasoconstriction, retains water and sodium, and increases thirst and salt appetite primarily through the angiotensin II receptor type 1 (AT1R). Together, these activities increase blood pressure and maintain fluid and electrolyte homeostasis; hence, deregulation of the RAS has been associated with hypertension and cardiovascular disease. 6
The novel view of the RAS is more complex and interesting, involving a balance between multiple processing pathways for angiotensin peptide generation and the belief that bioactive angiotensin peptides can be generated not only in the systemic circulation but also as local hormones in several tissues and organs. AngII is not the only active component of the RAS. New peptides, with similar or different functional properties, were identified as products of AngII: the heptapeptide angiotensin III (2–8) (AngIII), hexapeptide angiotensin IV (3–8) (AngIV) and heptapeptide (1–7). 6
The intracellular effects of the angiotensin peptides are mainly mediated by two receptors, the AT1R and the Angiotensin II receptor type 2 (AT2R), but also by the MAS receptor, the insulin-regulated aminopeptidase receptor, the Angiotensin II receptor type 4 and ACE, which are recent additions to the RAS network. 6 Ang-(1–7) acts mainly through the G protein-coupled receptor Mas. Ang-(1–7)/Mas axis and AngII/ATR2 are antagonists of the ACE/Ang II/ATR1 receptor axis, especially under pathological conditions. 7
RAS and cancer
Proliferative signalling
RAS signalling increases cell proliferation in malignancy by directly affecting tumour and stromal cells and by indirectly modulating the growth of vascular cells during angiogenesis. 8
The most studied components of the RAS, in the process of cell proliferation, are AngII and its receptors, which induce hyperplasia or hypertrophy in cultured vascular smooth muscle cells derived from the aorta. Interestingly, AngII has antagonist functions. When signalling through the AT1R, AngII facilitates cellular proliferation and angiogenesis, whereas AngII signalling through AT2R has anti-proliferative properties. 9 In most tissues, the proportion of AT1R and AT2R changes dramatically during the postnatal period. In foetal tissues, AT2R expression is dominant, and in adult animals, it is the AT1R that is more abundant. The observation of transient expression of the AT2R during development, together with an AT2R-induced stimulation of phosphotyrosine phosphatase activity, suggests that this receptor subtype is involved in the control of cell proliferation and differentiation. 10
The expression of the AT2R is subject to significant changes during development and differentiation. In addition, an increased expression of AT2R has been described in pathophysiological conditions, such as vascular neointima proliferation or left ventricular hypertrophy due to aortic banding. 11
The mitotic effect of AngII is not limited to the circulatory system. This peptide is also involved in the control of proliferation of breast, anterior pituitary, adrenocortical and endometrial cells. AngII is a potentially important growth promoter because, in a variety of cell types, it activates phosphatidylinositol via AT1R, which utilises two main second messengers, triphosphoinositol (IP3) and diacylglycerol, and causes a rise in cytosolic Ca2+, a process linked to mitogenesis in several cell types. 12
Another intracellular mechanism by which angiotensin peptides can control cell growth is the modulation of protein tyrosine kinases (PTKs). Tyrosine kinases are coupled to the receptors of several growth factors and are involved in the transduction of growth-inducing signals. AngII, acting via AT1R, may modulate cell growth and tyrosine kinase activity in various normal and neoplastic tissues, such as vascular muscle cells, normal and tumoural pituitary gland and a hormone-independent prostate cancer. There is also evidence that AngII-induced biological effects may depend on the concentration and presence of gonadal steroids. Breast cancer cell lines and primary breast cancer cells exhibit increased proliferation in response to AngII stimulation that is partly mediated through MEK and PI3K signaling. 13
Ang(1–7) is generally thought to inhibit cellular proliferation; however, it has antagonist effects in some cell types, including fibroblasts, epidermal stem cells, keratinocytes and haematopoietic progenitors in which this peptide stimulates cell proliferation. Therefore, whether Ang(1–7) is pro- or anti-proliferative for a particular tumour/host cell may be an important consideration for understanding the role of RAS in the carcinogenesis process and the applicability of RAS blockade as a cancer treatment. 13
Evading growth suppressors
In addition to the hallmark capability of inducing and sustaining growth-stimulatory signals, cancer cells must also avoid the effect of genes that act as tumour suppressors by limiting cell growth and proliferation. 1
Arrieta and colleagues studied the presence of AT1R and AT2R in high-grade astrocytomas and their correlation with mitotic and proliferation indexes, as well as with vascular density. They found that there is co-expression of the AT1R and the AT2R in malignant tumours, suggesting a possible relationship between both receptors. The stimulation of AT2R can inhibit AT1R activation pathways, leading to growth inhibition. The presence of both receptors in the same tumour suggests that selective blockage of one receptor could increase the effect of the other. It is possible that AT1R inhibition produces disequilibrium in the AT1R/AT2R stimulation, which in turn favours AT2R stimulation, thus opening the possibility of the pathway leading mainly to apoptosis. 14
Recently, several downstream proteins have been reported to have unique roles in AT2R induction, including AT2 receptor-interacting protein (ATIP) and Src homology 2 domain-containing protein-tyrosine phosphatase 1 (SHP-1). ATIP is also reported to potentially act as a membrane-associated Golgi protein that dictates delivery of the AT2R to the cell surface. Therefore, ATIP seems to act as a potential novel early component of the growth and/or differentiation-regulating signalling cascade mediated by the AT2 receptor. 15
Resisting cell death
The activation of the AT1R in malignant cells can enhance pro-survival signalling though at least two distinct pathways: the first via activation of NF-KB and the subsequent production of the anti-apoptotic molecules BCL and survivin and the second through the PI3K-Akt pathway and subsequent suppression of caspase. 8
Signaling through the AT2R is linked to the promotion of apoptosis. In prostate cancer cells,
Inducing angiogenesis
Angiogenesis is defined as the development of new blood capillaries from pre-existing vessels; it is involved in a number of physiological processes such as embryonic development and wound healing and also in the progression of disorders, including diabetic retinopathy and tumour progression. Tumour growth depends on angiogenesis to first provide oxygen and nutrients to proliferating cells and to then provide a physical route for metastasis transport. 17
They also reduced neovascularisation in a chicken egg model of angiogenesis, using a recombinant adenovirus carrying the human angiotensinogen gene under the control of the cytomegalovirus promoter (AdAGT).
Most recently, this group demonstrated that when using AdAGT in double transgenic mice with hepatocarcinoma, the expression of human angiotensinogen enhanced survival and reduced tumour growth and angiogenesis. Together, these results provide convincing evidence for the AngII-independent, anti-angiogenic effects of angiotensinogen. 5
It is likely that dysregulation of the normal physiological actions of the RAS in inflammation and angiogenesis might function to synergistically promote growth during malignancy. 8
Some reports using an AT1R antagonist and AT1R gene-deficient (
In 2007, Imai and colleagues determined that AT1R-induced vascular endothelial growth factor a (VEGFa) signalling, in both host and tumour tissues, is one of the key regulators of tumour growth and tumour-associated angiogenesis. 23
The role of AT2R in cancer inflammation and angiogenesis is less clear. A recent study demonstrated that the inhibition of AT2R delays tumour growth by impairing VEGF expression and that tumours inserted into
Ang(1–7) has been involved in angiogenesis with paradoxical effects in a tissue-dependent manner; i.e. in human prostate cancer xenografts it has been shown to reduce proliferation and angiogenesis by decreasing the angiogenic factors placental growth factor (PlGF) and VEGF; 25 also in rodents models Ang(1–7) has been demonstrated to promote angiogenesis by stimulating the expression of cardiac VEGF-D and matrix metalloproteinase-9 (MMP-9), thus facilitating cardiac repair and ventricular function. 26 This opens the possibility of considering the use of Ang(1–7) as a cancer treatment or even following myocardial infarction. Certainly, further studies are required to elucidate its exact mechanism of action in both situations. 26
Reprogramming of energy metabolism
The chronic and usually uncontrolled cell proliferation that represents the essence of neoplastic disease involves not only deregulated control of cell proliferation but also the resultant modifications of energy metabolism, like aerobic glycolysis, fatty-acid synthesis and glutaminolysis to fuel cell growth and division.2,27
In cancer cells, the rate of glycolysis is abnormally high, but aerobic glycolysis is preferably used rather than mitochondrial oxidative phosphorylation, a phenomenon known as the ‘Warburg effect’. 27 The study of the RAS involved in the metabolism of glucose has been focused on the metabolic syndrome; therefore direct evidence of RAS involvement in the Warburg effect is lacking. On the other hand, high-glucose levels in several tissues can increase the expression of (pro)renin receptor, AGT, ACE, and AT1R and stimulates renin. 4 These results suggest that the RAS could be either directly or indirectly implicated in the Warburg effect.
Direct RAS activation by AngII in the tumour microenvironment may lead to the generation of reactive oxygen species (ROS) and subsequent pro-inflammatory and pro-angiogenic signalling, which depends on cell-type-specific NADPH oxidases that activate downstream signalling cascades, including the activation of the MAPK and PI3K pathways and other redox-sensitive transcriptional factors such as hypoxia-inducible factor 1α (HIF1α). 8
A hypoxic tumour environment, coupled with excessive accumulation of ROS, leads to oxidative stress, followed by increased protein modifications, cellular damage and death or increased growth factor signalling. 28
AngII induces the expression of p47phox and the production of the O2-radical through the activation of the AT1R in prostate cancer cells. AngII increased 8-hydroxy-2′-deoxyguanosine, a marker of DNA damage that is induced by oxidative stress, coupled with the increased expression of the inflammatory marker inducible nitric oxide synthase, all of which were diminished with candesartan treatment. 28
Inflammation
The link between cancer-related inflammation and RAS signalling may converge on the response to tumour hypoxia and oxidative stress mechanisms. The RAS is a key mediator of inflammation, with the AT1 receptors governing the transcription of pro-inflammatory mediators both in resident tissue and in infiltrating cells, such as macrophages. 29
Several inflammatory proteins are induced by the AT1 receptor; these include interleukin-1 beta (IL-1b), tumour necrosis factor-alpha (TNF-α), plasminogen activator inhibitor type 1 (PAI-1), adrenomedullin transforming growth factor-beta (TGF)-β, and signal transducer and activation of transcription (STAT) family members and HIF1α. The expression of AT1 could be a consequence of stress and cell damage in the stromal cells of the neoplastic cells. 29
AngII exerts pleiotropic actions on the vasculature, such as vasoconstriction, migration, proliferation and hypertrophy, increased extracellular matrix formation, and activation of NADPH oxidases. Through these actions, AngII promotes vascular inflammation as well as endothelial dysfunction and structural remodelling. 30 An increased expression of two essential factors for vascular inflammation – lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) and NF-KB – incites Ang II-stimulated inflammation. Ang (1–7) can inhibit Ang II-activated inflammation by reducing LOX-1 expression. 31
Cell migration, invasion and metastasis
Mutations, polymorphism and changes in the expression of RAS components have been associated with the increased capacity of transformed cells to migrate, invade and metastasise. 8 For example, in gastric cancer, ACE and AT1R expression in the tumour and the ACE I/D polymorphism (rs1799752) influence metastatic behaviour and, in combination with AT1R expression, positively correlate with nodal spread. 32
AT1R expression is also strongly associated with ovarian tumour invasiveness and with the histological classification: AT1R is expressed in a high proportion of invasive ovarian adenocarcinomas and borderline malignant tumours, but it is expressed in few benign cystadenomas. 33
Recently, Krishnan and colleagues studied the capacity of Ang(1–7) to reduce prostate cancer metastasis in mice. They found in pre-treated mice with Ang(1–7) a prevention in metastatic tumour formation while the untreated mice developed tumours in metastatic sites. 34
In a retrospective study, Keizman et al. evaluated the concomitant use of angiotensin system inhibitors with sunitinib treatment in metastatic renal cell carcinoma. The patients who received angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers, before or within one month of sunitinib treatment, presented a significant (hazard ratio (HR) 0.537,
In preclinical models, angiotensin II type-1 receptor blockers (ARBs) and ACEIs have shown efficacy in reducing metastases, whereas AT1R expression frequently correlates with the degree of tumour invasiveness. 8
Finally, AT1R activation of PI3K signalling has been shown to induce the migration and invasion of choriocarcinoma cells, 36 whereas the invasion of gastric cancer cell lines was inhibited in some, but not all, of the cell lines treated with enalapril (an ACEI) or olmesartan (an ARB). 37
RAS blockers and cancer
Four groups of RAS blockers have been developed: direct renin inhibitor (DRI), ACEIs, ARBs and aldosterone antagonist (AA). 38 The use of this group of drugs has two potential benefits. First, the use of RAS blockers could improve the prognosis of the patients, and second, the finding of a new therapeutic use for a proven drug has the advantage of decreased development costs and decreased time to market compared to traditional discovery efforts because of the availability of previously collected pharmacokinetic, toxicology and safety data. 39 RAS is expressed in many tissues and has diverse functions; when considering the use of its inhibitors as part of a coadjutant treatment in cancer, the potential benefits of their use should be balanced against their adverse effects such as angioedema, hypotensive symptoms, cough, syncope, diarrhoea, renal insufficiency and hyperkalaemia. 40
Epidemiologic studies have been controversial regarding the use of antihypertensive drugs as a risk or protective factor for cancer. One of the strongest pieces of data comes from the Rotterdam Study, a population-based, prospective cohort study with 7983 participants. The effect of medication, ACE I/D genotypes, and their interaction with cancer risk and progression was studied using Cox proportional hazard models. The authors observed a dose-dependent, protective association between RAS inhibitor use and cancer risk in individuals with the ACE DD genotype (HR, 0.28; 95% confidence interval (CI), 0.10–0.79), while short-term, high-dose users were at risk for colorectal cancer progression in the II/ID stratum (HR, 3.83; 95% CI, 1.67–8.79).
41
On the other hand, in a meta-analysis reported by Sipahi et al. with 61,590 patients enrolled in five trials with an ARB randomly given in at least one group, a modest risk to develop cancer was found in the ARB-receiving group (7.2% vs 6.0%, relative risk (RR) 1.08, 95% CI 1.01–1.15;
A retrospective study analysing 287 patients with advanced non-small-cell lung cancer undergoing first-line platinum-based chemotherapy to examine the use of long-term medication with ACEI and ARB found that patients receiving ACEIs/ARBs had a remarkable estimated survival advantage of 3.1 months (HR 0.56,
Furthermore, several reports in the literature have demonstrated that blocking the AT1 receptors using specific receptor antagonists (ARBs) effectively reduces tumour growth and metastasis in preclinical models. 21
Epidemiological studies provide further evidence that the RAS may influence tumour progression. 13 A retrospective cohort study based on 5207 patients found that the incidence of fatal cancers was reduced in patients treated with ACEIs for four months. 46 But other studies have failed to confirm these data, 47 which could be affected by variables intrinsic to the populations or the use of different inclusion criteria. In an attempt to avoid these differences, other epidemiological studies have combined the genotype with the use of RAS inhibitors, for example the Rotterdam Study. The mean follow-up was 9.6 years, during which 730 incidents of cancers occurred. They found that carriers of the high-activity genotype DD had an increased risk of breast cancer compared with low-activity II/ID genotype, but DD carriers who were exposed to long-term and high-dose medication were at a lower risk for other types of cancer. 41 All these results suggest that pre-clinical and clinical studies suggested that the blockade of the renin-angiotensin pathway with angiotensin system inhibitors might inhibit tumour growth in several cancer types. 35
Conclusions
RAS as a gatekeeper system is involved in more than one function.
Drugs that target the RAS, in particular ACEIs and AT1R antagonists, are commonly used in the treatment of hypertension, so they are widely used, have few side effects and have relatively low costs; thus, they could be good coadjuvants in the treatment of cancer. One major challenge is the complex nature of RAS signalling, which seems to be context dependent, making the response to RAS therapeutics, either individually or in combination with other drugs, difficult to predict. 8
The future of RAS blockers in cancer treatment could take two directions. As a first approach, protocols using these drugs as chemo-prophylactic agents might be considered to reduce cancer incidence. In this case, the limited efficacy of long-term use to compensatory increases in renin of ACEIs and ARBs could be a limitation, where strategies such as the combination of RAS blockers with a renin inhibitor should be contemplated. Despite the multiple limitations, chemoprevention is thought to be a realistic approach for reducing the incidence of cancer, and RAS blockers constitute a great potential approach.
The other direction could be the use of RAS blockers as coadjuvant drugs in cancer treatment, in this case the adverse effects may be a major challenge as previously discussed. Also, the regulation of the RAS system in the tissue of interest could be a challenge and should be addressed in future studies. In both directions the study of the genotype/phenotype relationship is mandatory, giving us the advantage of avoiding the use of this medications in individuals that will only suffer from the adverse effects instead of the benefit of their use; but mainly it will help cancer patients to receive a specific treatment for this disease or a specific drug to reduce their risk to develop cancer.
The time when RAS was considered only in the context of hypertension has passed. Since the RAS has met the hallmarks of cancer, RAS should be added as a new player involved in the molecular process of carcinogenesis and in consequence in the treatment and prophylaxis of these complex diseases.
Footnotes
Conflict of interest
None declared.
Funding
This research was supported by grants from the División de Investigación of the Instituto Nacional de Cancerologia of Mexico (INCan). Talia Wegman-Ostrosky is supported by a PhD fellowship from CONACyT (224956). Additional support was provided by the PhD Graduate Program of Doctorado en Genética Humana of the Universidad de Guadalajara.