High-impact MR mutations based on MutationAssessor in curated TCGA and non-TCGA dataset collection (
In the last decades, advances that take place in the next-generation sequencing and bioinformatics research have helped reveal tissue- and cancer-specific gene expression patterns and mutation landscapes. Indeed, such data are now easily accessible via online genome browsers and different types and levels of public data compendia. Appropriate use of these tools eventually can lead to better patient stratification for diagnosis, prognosis, and therapy of cancers. Mineralocorticoid receptor (MR), encoded by NR3C2 gene, has long been implicated in the development and progression of multiple cancers. Nevertheless, MR has remained relatively understudied at the genomic and transcriptomic levels. In this review, we present the current, literature-based state of knowledge on the role of MR primarily in epithelial cancers. At the same time, we summarize the gene expression, mutation, and copy number variation data on MR obtained from The Cancer Genome Atlas (TCGA). We also show that MR expression could be a promising prognostic marker in different cancers using online tools for survival data analysis. Accordingly, this review strongly demonstrates the emerging potential of studying MR using available tools from the genomics/transcriptomics field for improving cancer diagnosis and prognostication.
- mineralocorticoid receptor
- epithelial cancers
- The Cancer Genome Atlas
MR/NR3C2 belongs to the steroid receptor family and it adopts important roles in human physiology and pathology. Although MR has long been studied in renal and cardiovascular contexts, identification of MR in multiple epithelial cancers and presence of cross talk between steroid receptors in cancer-related processes make MR a promising candidate for cancer diagnosis and prognosis. Nevertheless, a focused yet comprehensive literature review about MR’s expression in cancers and established role in cancer-associated hallmarks is lacking. Our literature search reveals that MR is expressed in cancerous as well as adjacent and/or normal tissue although the expression of MR can become deregulated during cancer development. Moreover, we provide an account of changes in ligand-dependent or -independent MR signaling in association with cell proliferation, apoptosis, and senescence of cancer cells. We also identify future directions that can help target novel aspects of MR signaling for mechanistic studies as well as cancer therapeutics. In addition, we point out an emergent need for analyzing the range of genomic alterations and variability in MR expression and its potential association with prognosis across epithelial solid tumors using the existing genomic and transcriptomic resources. We exemplify the extent of variability in MR expression within and among patients based on the patient data found in The Cancer Genome Atlas (TCGA) . Moreover, we demonstrate the profound potential of MR expression as a biomarker for cancer prognostication, i.e., estimation of the likelihood of developing future risks for cancer over a time period using TCGA datasets .
2. A concise literature review on MR in cancer biology
Herein an overview of the scientific literature on MR is provided using examples mainly from cancers of epithelial origin including the lung, colon, liver, kidney, pancreas, prostate, breast, and adrenal gland, revealing the understudied aspects of MR in the context of cancer biology.
2.1 Lung cancer
The presence of MR protein in lung cancer tissues has opened new avenues for MR research. Suzuki et al.  demonstrated by immunohistochemistry (IHC) that primary lung cancer tissues expressed MR protein along with HSD11B2 enzyme, required for MR receptor specificity through conversion of cortisol to cortisone. However, MR and HSD11B2 proteins, although present and significantly correlated with each other in lung adenocarcinomas, were non-existent in squamous cell, small cell, or large-cell carcinomas . Next, Jeong and colleagues  studied gene expression signatures of all 48 nuclear receptors (NRs) including MR in non-small-cell lung cancers (NSCLCs) and corresponding normal lung tissues and found that short heterodimer partner (SHP) and progesterone receptor (PGR) predicted survival in patients with early-stage lung tumors. In the same study, the prognostic role of NRs was also investigated in corresponding normal tissues; and higher expressions of MR and nerve growth factor-induced gene B3, NGFIB3, were identified as predictors of good prognosis for survival and disease recurrence . Furthermore, increasing aldosterone levels in the presence of VEGF inhibitors also proved to be a better indicator of prognosis in NSCLC patients . However, future epidemiological as well as mechanistic studies are needed to address the cross talk between antiangiogenic drugs and MR signaling in lung cancer.
2.2 Colorectal cancer
In colon cancer, an observed decrease in the expression of MR in cancerous tissue in comparison to the adjacent normal mucosa has attributed MR a tumor suppressive role . Tiberio et al.  further investigated how MR expression correlated with patient survival in colorectal carcinomas. In this study, the expressions of MR and tumor microvessel density marker protein CD34 were evaluated in tumor and normal colorectal mucosa by IHC, and an inverse correlation of expression was detected between them in colorectal cancers. Kaplan–Meier survival analysis has led to a conclusion that MR could be a tumor suppressor whose decreased expression is correlating well with poor patient survival based on a relatively small number of patients . Recently, spironolactone, an MR antagonist, has also arisen as a tumor suppressor in colon carcinoma yet independent of MR and through RXRγ receptor signaling . Moreover, recent studies showed that HSD11B2 inhibition, and hence potential dysregulation of MR signaling, could modify gut microbiota, known to be an important factor in colon carcinogenesis . A better understanding of MR cross talk with other nuclear receptors and interaction with gut microbiota from patients treated with MR antagonists is needed in the future.
2.3 Breast cancer
MR expression in normal and diseased breast tissues was initially identified in the 1990s [10, 11]. MR and HSD11B2 proteins were shown to co-localize predominantly in the duct epithelia and to exhibit higher expressions in invasive ductal carcinoma than invasive lobular carcinoma . More recently a study by Conde et al.  found that MR expression was peculiar to the cytoplasm of benign and cancerous breast lesions, whereas GR/NR3C1 expression was nuclear in benign breast lesions but showed cytoplasmic as well as nuclear distribution in cancer tissues. These findings might suggest a potential deregulation of GR signaling in malignant tissues, while the effects of MR on tumor development could be less ligand-dependent and/or ligand-insensitive in breast cancer. However, this remains to be assessed.
Induction of growth of lobulo-alveolar structures in mouse mammary gland by MR ligand aldosterone also pointed to the importance of MR signaling in breast biology [13, 14, 15]. Furthermore, the presence of progesterone, a potential MR ligand, in the breast tissue highlighted the significance of MR in breast cancers . In addition, in vitro culturing of breast cancer cell lines provided further opportunities for understanding the impact of MR signaling in tumor growth. For example, in the breast cancer cell line PMC42 with detectable MR and HSD11B2 expression levels, aldosterone, alone, did not have an effect on cell proliferation yet when given together with the anti-mineralocorticoid spironolactone resulted in a significant decrease in cell numbers . In another study, aldosterone and cortisol exerted progesterone-like effects such as induction of focal adhesion and reduced cell growth in the progesterone receptor-transfected MDA-MB-231 breast cancer cell line . Recently, genomic and non-genomic actions of aldosterone through G-coupled estrogen receptor (GPER) and MR signaling pathways were demonstrated . Rapid aldosterone exposure activated EGFR and ERK1/2 transduction pathway through MR and GPER in the HER2+ breast cancer cell line SkBr3 and breast tumor-derived endothelial cells . Furthermore, direct interactions among GPER and MR as well as GPER and EGFR were shown, while a long-term exposure to aldosterone increased cell growth which could be inhibited by the silencing of MR expression . These findings indicated possible contributions of GPER activity in the MR-dependent aldosterone signaling and EGFR activation in the regulation of cancer cell growth. However, the relationship between aldosterone and other modulators of GPER, e.g., estrogen, remains to be investigated in breast cancer. MR receptors can exhibit affinity to aldosterone and cortisol as well as other potential ligands likely to be found in the milieu of breast cancer, and thus studying MR, GR, GPER, and/or other receptor crosstalk could be important for better evaluating mammary gland physiology and pathology.
2.4 Liver cancer
As in the lung and colon cancers, MR expression was shown to be downregulated in a large cohort of liver cancer patient tissues . Furthermore, in the same study, overexpression of MR suppressed cancer progression by inhibition of proliferation and induction of cell cycle arrest eventually leading to apoptosis. Aldosterone’s effect on tumor growth was also tested, and its antiproliferative and apoptotic effects were reversed by spironolactone. All of these significantly implicated an evidence for decreased MR signaling in liver cancer pathogenesis . Additionally, this study showed that MR suppressed the Warburg effect  by which cancer cells gain growth advantage over normal cells  leading to novel insights about MR signaling through cancer research.
Apart from cancer, fibrosis is another significant pathology of the liver. Fibrosis, the leading factor of liver carcinogenesis, occurs due to the accumulation of fibrogenic cells and extracellular matrix (ECM) proteins in excess [22, 23]. ECM is central to sustain cellular homeostasis and integrity, and deregulation of ECM is considered as a hallmark of cancer . The role of aldosterone on ECM synthesis and potentially on liver fibrosis was previously shown in rats, although independent of MR itself . On the other hand, spironolactone was shown to act as a potent provocateur of liver regeneration following partial hepatectomy . As a result, these studies demonstrate the current need for better understanding the role of ligand-dependent and ligand-independent signaling of MR in different facets of liver pathologies including fibrosis, regeneration, and carcinoma.
2.5 Renal cancer
MR and aldosterone signaling have been extensively studied in kidney physiology and pathology for decades [27, 28] and to some degree, in renal carcinomas. Initially, the protein expressions of MR and HSD11B2 were characterized in a large cohort of renal cell neoplasms of different cellular origins using IHC. In this study, co-expression HSD11B2 and MR was shown in normal distal nephron, in chromophobe renal cell carcinoma (chRCC) and oncocytoma of distal nephron origin . In renal cell carcinoma, aldosterone led to upregulation of KRAS oncogene (KRAS4A splice variant) resulting in increased survival and cell proliferation . Yet another study revealed that aldosterone exerted its migratory/metastatic actions through G-protein-coupled estrogen receptor (GPER) in a murine renal cortical adenocarcinoma cell line and also in mice in vivo . These findings implicated important oncogenic pathways and their crosstalk with MR and aldosterone signaling in renal cancers.
Hypertension is a well-established risk factor in kidney cancers potentially due to the genotoxic nature of aldosterone [32, 33]. Indeed, supraphysiological levels of aldosterone treatment induced DNA breaks and chromosomal aberrations in epithelial porcine kidney cells, whereas MR blockade by antagonists prevented formation of such aberrations . Genotoxic effects of aldosterone were investigated further in the DOCA-salt-treated rat model used for inducing MR-dependent hypertension. DOCA-salt treatment caused inflammation, oxidative stress, DNA damage, and increased kidney cell proliferation . Another study highlighted aldosterone-dependent induction of oxidative stress and DNA damage as well as activation of MR-dependent NFKB signaling pathway in kidney tubule cells . Queisser et al.  further addressed the downstream signaling pathways triggered by aldosterone-induced oxidative stress both in vitro (porcine kidney cells with proximal tubular properties) and in vivo (rat kidneys). In these models, aldosterone treatment resulted in MR-dependent activation of ERK1/2 and its target, STAT3; and hence aldosterone exposure led to higher proliferation rates while diminishing apoptosis . Accordingly, the role of aldosterone-induced MR signaling in deregulation of DNA damage response needs to be studied also in other epithelial cancers in more detail.
Senescence, evasion of which is another hallmark of cancer , was studied in the context of aldosterone signaling in different renal models. For example, p16INK4a, a cyclin-dependent kinase inhibitor and a cellular senescence marker [39, 40], was induced in the kidneys and hearts of DOCA-salt-treated rats . These effects could be reversed by antihypertensives and spironolactone, suggesting a potential role of MR signaling in the regulation of the senescent phenotype . In another study, senescence was investigated in aldosterone-infused rats and cultured human proximal tubular cells. In both models, aldosterone-induced senescence-like characteristics were marked by senescence-associated beta-galactosidase staining, p21/Cdkn1a and p53 overexpression, and SIRT1 under-expression. MR blockade either using eplerenone (in vivo) or through gene silencing (in vitro) sufficiently reversed the aldosterone-induced senescence-like characteristics . In line with this study, Kitada et al.  also showed the presence of aldosterone-induced senescence, characterized by increased p21 expression and beta-galactosidase staining, in human proximal tubular cells. Furthermore, a prolonged exposure to aldosterone triggered p21-mediated cytokine release, e.g., TNF alpha, which in turn led to apoptosis . All of these have implicated MR signaling through interaction with aldosterone in the induction of senescence, an inherent autoregulatory mechanism of proliferating cells with established tumor suppressive activity. The role of MR/aldosterone-induced senescence in epithelial cancers however needs to be further studied since this can provide an effective route for therapeutic invention.
2.6 Pancreas cancer
Recently, MR has been ascribed a tumor suppressive role also in pancreatic ductal adenocarcinoma (PDAC) . In PDAC patients, dysregulated expression of macrophage migration inhibitory factor (MIF) was associated with disease aggressiveness, and MIF-driven upregulation of miR-301b was shown to suppress MR expression . In turn, MR expression resulted in the inhibition of epithelial to mesenchymal transition (EMT) and increased chemotherapeutic drug (gemcitabine) sensitivity. Consistently, survival data analysis further associated downregulation of MR expression with poor survival in PDAC patients . However, PDAC remains one of the cancers receiving less attention in the MR field; future studies can address the role of genomic and non-genomic effects of MR signaling in PDAC.
2.7 Prostate cancer
Detection of 11 beta-hydroxysteroid dehydrogenase enzyme  and a functional MR in the androgen-dependent prostate cancer cell line LNCaP cells dates back to the early 1990s . More recently, Dovio et al.  assessed GR and MR expression together with HSD11B-1 and HSD11B-2 enzyme activity upon inflammatory stimulus (IL1B stimulation) or basal conditions in the androgen-dependent and androgen-independent prostate cancer cell lines. Diverse expression patterns of MR, GR, and HSD11B enzyme activities were detected among cell lines, while downstream effects of IL1B exposure were inhibited by cortisol or dexamethasone in a cell line-dependent manner .
Another lead for the role of mineralocorticoids in prostate cancer has come from abiraterone acetate (AA), an androgen synthesis inhibitor, used for metastatic castration-resistant prostate cancer (mCRPC), which results in secondary mineralocorticoid excess . Androgen-induced conformational changes in androgen receptor (AR) could be inhibited in the presence of mineralocorticoids (corticosterone or deoxycorticosterone). However, administration of corticosterone alone resulted in repression of AR transcriptional activity and cellular growth at concentrations present in the serum of AA-administered patients . Pia et al.  focusing on identifying ways of eliminating adrenocorticotropic hormone (ACTH)-dependent AA-induced mineralocorticoid excess showed that a low effective dose of glucocorticoid together with MR antagonist and salt deprivation could be an ideal treatment. Indeed, the use of prednisone, a synthetic glucocorticoid, could overcome the effects of secondary mineralocorticoid excess in mCRPC patients treated with abiraterone . Effect of eplerenone-abiraterone co-administration on secondary mineralocorticoid excess syndrome and progression-free survival (PFS) was evaluated and compared to prednisone-abiraterone co-administered in patients. No significant difference was obtained by means of mineralocorticoid excess syndrome characteristics and PFS between these two experimental groups; and this has raised AA-eplerenone as an alternative therapy for overcoming prednisone-induced side effects . Enzalutamide is another antiandrogen drug used for treating metastatic prostate cancer patients; however, resistance gained against enzalutamide therapy remains a challenge. GR signaling induces antiandrogen resistance by hijacking AR function [53, 54]; hence, the therapeutic effects of enzalutamide-corticosteroid co-administration were addressed in prostate cancer cells . Dexamethasone decreased the therapeutic effects of enzalutamide as well as increasing resistance. However, prednisolone and aldosterone diminished resistance to enzalutamide. Consistently, silencing of MR resulted in enhanced resistance to enzalutamide and AR activity . Moreover, the effects of diverse antihypertensive medication on prostate cancer survival following radical prostatectomy were tested in the Finnish population, and overall, the antihypertensive treatment was associated with increased death risk . These findings clearly establish the importance of AR and MR cross talk and complex ligand interactions in prostate cancer, which could be further studied in other cancers and cancer subtypes where AR signaling can play a role.
2.8 Adrenocortical cancer
Adrenal incidentalomas include adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytoma [57, 58]. Aldosterone-producing adenomas (APA, i.e., benign tumors of the adrenal glands) account for 35% of the diseases of the primary aldosteronism spectrum [59, 60]. Somatic mutations occurring in KCNJ5, CACNA1D, ATP1A1, ATP2B3, and CTNNB1 genes give rise to sporadic APA . In addition to the abovementioned mutations, regulatory RNAs such as miRNAs have also been shown to be important in modulating aldosterone levels and tumorigenesis [62, 63, 64]. On the other hand, adrenocortical cancers (ACC), some of which are hormone-producing, occur relatively rarely, and patients with ACC exhibit poor prognosis with a median survival of 5.5 years . In the adrenocortical cell line H295R, aldosterone in vitro is shown to upregulate T-type calcium channel expression and currents, an effect reversed by spironolactone . H295R cells have also been shown to express components of aldosterone signaling pathway including ENaC subunits, NEDD4L, SGK1, MR, and HSD11B2 [66, 67]. Although several factors, such as age, resection margin and proliferation scores, uterine steroid profiles, CpG island hypermethylation status, as well as levels of selected biomarkers, have been tested for their contribution in ACC prognosis, the importance of MR expression status is yet to be evaluated in ACC patients [65, 68, 69]. Future studies should investigate the role of activation/inactivation of MR-aldosterone signaling in diagnosis/prognosis of ACC for which significant amounts of genomic and transcriptomic data have recently become available (please see the next section for details).
3. Cancer genome and transcriptome analyses for MR using online tools
The advent of whole-genome sequencing and development and availability of genome browsers, such as UCSC genome browser [70, 71] and Ensembl [72, 73], in the early 2000s have enabled researchers to identify genes/genomic intervals that are important in human physiology and pathology. This is mainly done by association of genome fragments with informative tracks that range from expression values to the presence of copy number and single nucleotide variations (CNVs and SNVs, respectively). Cataloging and annotation of the human genome with regard to genomic and transcriptomic variation have also revolutionized cancer research [74, 75]. The NIH-driven giant effort named The Cancer Genome Atlas (TCGA) has published its first results in 2008 on the genomic and transcriptomic landscape of gliomas . Over the years, TCGA has expanded to house thousands of cancer genomes/transcriptomes/proteomes from tens of different cancers allowing researchers from all over the world to have unlimited access to cancer-related datasets . Many different web-based tools nowadays use TCGA and Catalog of Somatic Mutations in Cancer (COSMIC)  as primary resources and build on them to extract data from user-provided queries and/or to perform gene-specific or genome-wide secondary analyses, such as
The expression of MR along with those of other nuclear receptors has been studied in a recent TCGA PanCancer transcriptomics study of bladder, breast, colon, head and neck, liver, and prostate cancers; and MR expression is shown to be downregulated in all . To demonstrate the potential of TCGA in revealing the importance of MR in cancer research, we have used
Overall, the observed rate of somatic mutations of MR has been shown to be significantly lower with respect to the expected rate suggesting MR does not tolerate well mutations with functional constraints . On the other hand, functional mutations of MR have been identified in different contexts including renal pseudohypoaldosteronism as well as hypertension [81, 82]. In addition, relatively different residues in the DNA-binding domain of MR seem to be affected between type I pseudohypoaldosteronism and cancers . Herein we have compiled the MR mutation landscape for cancers found in TCGA provisional datasets (
Moreover, using TCGA provisional datasets, we analyzed whether MR accumulated different rates of genetic alterations in different cancers (Figure 2B). The mean proportion of genetic alterations per cancer was 0.024 (0.017–0.031, 95% confidence interval, CI). The results in percentages showed that around 7% of uterine carcinosarcoma patients exhibited genomic alterations (mutation, amplification; 7.02% in 57 cases), while the second and third ranking cancers were those of esophageal carcinoma (5.38% in 186 cases) and stomach adenocarcinomas (5.23% in 478 cases) (Figure 2B). As mentioned above the ranking of these cancers in terms of percent genomic alterations can be dynamic depending on which data collection has been used. For example, when using TCGA PanCancer sample collection, which reports a more complete mutation/CNV annotation information, the first ranking cancer with the highest percentage of genetic alterations has become melanoma (8.71% out of 448 skin cutaneous melanoma) followed by uterine carcinomas (7.75% out of 529 uterine corpus endometrial carcinoma and 7.02% out of 57 uterine carcinosarcomas). Future studies may focus on uterine carcinomas and melanoma to address the mechanisms and effects of these observed alterations.
4. Investigating the role of MR in cancer patient stratification and prognosis using genomics resources
Survival analysis is often used for studying the association of an event of interest, e.g., death and disease recurrence, with another clinical or biological variable . Analysis of TCGA-associated survival data (overall survival (OS) and/or relapse-free survival (RFS)) is available through several online webservers including GEPIA , OncoLnc , Kaplan–Meier Plotter , and KM-Express . These tools help evaluate survival of cancer patients whose genomic/transcriptomic and clinical data are stored in TCGA, by using Cox coefficient statistics, hazard ratio (HR) and/or logrank tests, and Kaplan–Meier plots. GEPIA, which has previously been used in prognostic identification of several biomarkers in ACC [93, 94], performs survival statistics in addition to providing other functionalities such as co-expression analysis and diagnostic marker prediction. We analyzed data from all cancers available in GEPIA and showed that an NR3C2/MR expression higher than the median level predicted a significantly better prognosis (OS) and low HR in adrenocortical carcinoma (ACC), kidney renal clear cell carcinoma (KIRC), breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), brain lower-grade glioma (LGG), and liver hepatocellular carcinoma (LIHC) (logrank p-value <0.1; Figure 3).
Similar analyses can be performed using other webservers, such as KM plotter (
A comprehensive look at the history of MR in cancer research strongly implicates the dysregulation of MR signaling in the development and progression of epithelial cancers. However, the interactions with its natural ligand aldosterone and/or with other potential ligands, such as tissue-specific progesterone as well as the growing evidence on the presence of receptor cross talk, complicate the “tumor suppressive” role often attributed to MR. MR’s relatively well-established effects in renal tissue senescence, oxidative stress and DNA damage, as well as its emerging potential in the regulation of Warburg effect and fibrosis/regeneration in liver tissue represent novel avenues to pursue especially in the context of cancer therapy. In addition, the genome-wide availability of CNV, SNV, and mRNA expression profiles from cancer patients enables comparisons within and between different tumors providing an enhanced level of accessibility to researchers in the field of cancer biology. Indeed, online examination of the interaction between different data sources such as expression and patient survival data is now effectively possible for MR and can be extended to other genes participating in MR signaling. Generation of large-scale genome-wide data along with the development of tools that help analyze and integrate such data is likely to further enhance our understanding of MR in the development and progression of different cancers.
This publication is based upon work from the EU COST Action ADMIRE BM1301 in Aldosterone and Mineralocorticoid Receptor Physiology and Pathophysiology (