A tumour suppressive relationship between mineralocorticoid and retinoic acid receptors activates a transcriptional program consistent with a reverse Warburg effect in breast cancer.

BACKGROUND: The role of nuclear receptors in both the aetiology and treatment of breast cancer is exemplified by the use of the oestrogen receptor (ER) as a prognostic marker and treatment target. Treatments targeting the oestrogen signalling pathway are initially highly effective for most patients. However, for the breast cancers that fail to respond, or become resistant, to current endocrine treatments, the long-term outlook is poor. ER is a member of the nuclear receptor superfamily, comprising 48 members in the human, many of which are expressed in the breast and could be used as alternative targets in cases where current treatments are ineffective. METHODS: We used sparse canonical correlation analysis to interrogate potential novel nuclear receptor expression relationships in normal breast and breast cancer. These were further explored using whole transcriptome profiling in breast cancer cells after combinations of ligand treatments. RESULTS: Using this approach, we discovered a tumour suppressive relationship between the mineralocorticoid receptor (MR) and retinoic acid receptors (RAR), in particular RARß. Expression profiling of MR expressing breast cancer cells revealed that mineralocorticoid and retinoid co-treatment activated an expression program consistent with a reverse Warburg effect and growth inhibition, which was not observed with either ligand alone. Moreover, high expression of both MR and RARB was associated with improved breast cancer-specific survival. CONCLUSION: Our study reveals a previously unknown relationship between MR and RAR in the breast, which is dependent on menopausal state and altered in malignancy. This finding identifies potential new targets for the treatment of breast cancers that are refractory to existing therapeutic options.

Clinical, genetic, and structural basis of apparent mineralocorticoid excess due to 11ß-hydroxysteroid dehydrogenase type 2 deficiency.

Mutations in 11ß-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) cause an extraordinarily rare autosomal recessive disorder, apparent mineralocorticoid excess (AME). AME is a form of low renin hypertension that is potentially fatal if untreated. Mutations in the HSD11B2 gene result either in severe AME or a milder phenotype (type 2 AME). To date, ∼40 causative mutations have been identified. As part of the International Consortium for Rare Steroid Disorders, we have diagnosed and followed the largest single worldwide cohort of 36 AME patients. Here, we present the genotype and clinical phenotype of these patients, prominently from consanguineous marriages in the Middle East, who display profound hypertension and hypokalemic alkalosis. To correlate mutations with phenotypic severity, we constructed a computational model of the HSD11B2 protein. Having used a similar strategy for the in silico evaluation of 150 mutations of CYP21A2, the disease-causing gene in congenital adrenal hyperplasia, we now provide a full structural explanation for the clinical severity of AME resulting from each known HSD11B2 missense mutation. We find that mutations that allow the formation of an inactive dimer, alter substrate/coenzyme binding, or impair structural stability of HSD11B2 yield severe AME. In contrast, mutations that cause an indirect disruption of substrate binding or mildly alter intramolecular interactions result in type 2 AME. A simple in silico evaluation of novel missense mutations could help predict the often-diverse phenotypes of an extremely rare monogenic disorder.

Primary Aldosteronism: The Next Five Years.

The management of primary aldosteronism is widely varied within various published guidelines, with very little in the way of data supporting the choice of one variation over others. Current estimates of prevalence are probably accurate for aldosterone producing adenoma, but fall very short of that for bilateral adrenal hyperplasia. Discovery at the level of basic science has proven illuminating over the past 6 years in terms of unilateral disease and both somatic and germline mutations, with much less focus on the much more common bilateral disease; Attempts at harmonization have begun - for example, criteria for complete/partial/absent cure after adrenalectomy for unilateral disease; again focus on bilateral disease is muted. A number of possibilities are suggested as agenda for active consideration and change, across a wide range of areas - referral patterns, screening, confirmation and lateralization, what will be needed is discussion and agreement, to fill the lacunae within the current guidelines. Those involved will want to change to make such an agenda possible.

Aldosterone and Mineralocorticoid Receptors-Physiology and Pathophysiology.

Aldosterone is a uniquely terrestrial hormone, first appearing in lungfish, which have both gills and lungs. Mineralocorticoid receptors (MRs), on the other hand, evolved much earlier, and are found in cartilaginous and bony fish, presumptive ligand cortisol. MRs have equivalent high affinity for aldosterone, progesterone, and cortisol; in epithelia, despite much higher cortisol circulating levels, aldosterone selectively activates MRs by co-expression of the enzyme 11ß-hydroxysteroid dehydrogenase, Type 11. In tissues in which the enzyme is not expressed, MRs are overwhelmingly occupied but not activated by cortisol, which normally thus acts as an MR antagonist; in tissue damage, however, cortisol mimics aldosterone and acts as an MR agonist. The risk profile for primary aldosteronism (PA) is much higher than that in age-, sex-, and blood pressure-matched essential hypertensives. High levels of aldosterone per se are not the problem: in chronic sodium deficiency, as seen in the monsoon season in the highlands of New Guinea, plasma aldosterone levels are extraordinarily high, but cause neither hypertension nor cardiovascular damage. Such damage occurs when aldosterone levels are out of the normal feedback control, and are inappropriately elevated for the salt status of the individual (or experimental animal). The question thus remains of how excess salt can synergize with elevated aldosterone levels to produce deleterious cardiovascular effects. One possible mechanism is through the agency of the elusive ouabain-like factors (OLFs). Such factors are secreted from the adrenal in response to ACTH (adrenalocortical tropic hormone), to angiotensin via AT2R, and-the polar opposite of aldosterone-to sodium loading. They act on blood vessels to cause vasoconstriction and thus elevate blood pressure to dump excess sodium through pressure natriuresis. Their levels are chronically elevated in PA in response to the continually elevated sodium status, and they thus act to constrict coronary and systemic arteries. In the context of the elevated blood volume and total body sodium in a PA patient, this raises blood pressure and acts as the proximate cause of cardiovascular damage. If this is the case, it would appear to offer new insights into therapy for PA. One would be the use of digibindin, or its more recent successors as antagonists of OLFs acting on Na/K ATPase at the vessel wall. A second would be to routinely combine a low dose MR antagonist, an ENaC inhibitor, and sodium restriction as first-line therapy for bilateral aldosterone overproduction. Finally, for unilateral cases post-surgery, there is good reason to include low-dose MRs in drug therapy if required, given the ability of cortisol in damaged blood vessels to mimic aldosterone vasoconstrictor action.

Primary aldosteronism and salt.

For many years, primary aldosteronism was thought (and taught) to be a relatively rare (< 1 %) and benign form of high blood pressure: now we know that neither is the case. Currently, the prevalence is considered to be 5-10 % of hypertensives, on the basis of more or less stringent cutoffs for the aldosterone/renin ratio and plasma aldosterone concentration: increasingly, evidence is mounting that the true prevalence of (relatively) autonomous aldosterone secretion may be ∼ 30 % of hypertensives. There is, in addition, a consensus that the risk profile for patients with primary aldosteronism is substantially higher than in age-, sex-, and blood pressure-matched essential hypertensives; the cardiovascular/renal damage in primary aldosteronism is thus not a primary effect of raised blood pressure. The nexus between salt and primary aldosteronism is clear, as equivalently raised or even higher levels of plasma aldosterone in chronic sodium deficiency are homeostatic and do not cause cardiovascular damage, thus ruling out deleterious effects of aldosterone acting alone. In primary aldosteronism the normal homeostatic feedback loops between sodium status and aldosterone levels are disturbed, so that cardiovascular/renal damage reflects inappropriate aldosterone levels for sodium status. One possible actor in such a scenario is endogenous ouabain (or similar compounds), which is elevated in the sodium-loaded state and a vasoconstrictor, and thus potentially be able both to raise blood pressure and to cause cardiovascular/renal damage. A second consideration is that of the epidemiologic data linking a chronically high salt intake to a raised blood pressure. If autonomous aldosterone secretion is in fact present in ∼ 30 % of hypertensives, this may be the group sensitive to the pressor effects of high salt, with the remainder much less affected. Finally, at a practical level given even the currently accepted prevalence of primary aldosteronism, a radical reconsideration of first-line antihypertensive therapy is proposed.

Distinct nuclear receptor expression in stroma adjacent to breast tumors.

The interaction between breast tumor epithelial and stromal cells is vital for initial and recurrent tumor growth. While breast cancer-associated stromal cells provide a favorable environment for proliferation and metastasis, the molecular mechanisms contributing to this process are not fully understood. Nuclear receptors (NRs) are intracellular transcription factors that directly regulate gene expression. Little is known about the status of NRs in cancer-associated stroma. Nuclear Receptor Low-Density Taqman Arrays were used to compare the gene expression profiles of all 48 NR family members in a collection of primary cultured cancer-associated fibroblasts (CAFs) obtained from estrogen receptor (ER)α positive breast cancers (n = 9) and normal breast adipose fibroblasts (NAFs) (n = 7). Thirty-three of 48 NRs were expressed in both the groups, while 11 NRs were not detected in either. Three NRs (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1); estrogen-related receptor beta (ERR-ß); and RAR-related orphan receptor beta (ROR-ß)) were only detected in NAFs, while one NR (liver receptor homolog-1 (LRH-1)) was unique to CAFs. Of the NRs co-expressed, four were significantly down-regulated in CAFs compared with NAFs (RAR-related orphan receptor-α (ROR-α); Thyroid hormone receptor-ß (TR-ß); vitamin D receptor (VDR); and peroxisome proliferator-activated receptor-γ (PPAR-γ)). Quantitative immunohistochemistry for LRH-1, TR-ß, and PPAR-γ proteins in stromal fibroblasts from an independent panel of breast cancers (ER-positive (n = 15), ER-negative (n = 15), normal (n = 14)) positively correlated with mRNA expression profiles. The differentially expressed NRs identified in tumor stroma are key mediators in aromatase regulation and subsequent estrogen production. Our findings reveal a distinct pattern of NR expression that therefore fits with a sustained and increased local estrogen microenvironment in ER-positive tumors. NRs in CAFs may provide a new avenue for the development of intratumoral-targeted therapies in breast cancer.

Biphasic time course of the changes in aldosterone biosynthesis under high-salt conditions in Dahl salt-sensitive rats.

OBJECTIVE: The comorbidity of excess salt and elevated plasma aldosterone has deleterious effects in cardiovascular disease. We evaluated the mechanisms behind the paradoxical increase in aldosterone biosynthesis in relation to dietary intake of salt. METHODS AND RESULTS: Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were fed a high-salt diet, and plasma and tissue levels of aldosterone in the adrenal gland and heart were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry. In Dahl-S rats, we found that the delayed and paradoxical increase in aldosterone biosynthesis after the initial and appropriate response to high salt. The late rise in aldosterone biosynthesis was accompanied by upregulation of CYP11B2 expression in the zona glomerulosa and increased adrenal angiotensin II levels and renin-angiotensin system components. It preceded the appearance of left ventricular systolic dysfunction and renal insufficiency. Blockade of angiotensin AT(1) receptors reversed the paradoxical increase in aldosterone biosynthesis. In contrast, Dahl-R rats maintained the initial suppression of aldosterone biosynthesis. Aldosterone levels in the heart closely paralleled those in the plasma and adrenal gland and disappeared after bilateral adrenalectomy. CONCLUSIONS: Chronic salt overload in Dahl-S rats stimulates aberrant aldosterone production via activation of the local renin-angiotensin system in the adrenal gland, thereby creating the comorbidity of excess salt and elevated plasma aldosterone.

Who and How Should We Screen for Primary Aldosteronism?

There are mounting data that at least 30% of hypertensives who are appropriately screened have primary aldosteronism (PA), rather than the commonly reported figure of 5% to 10%. Second, there are similar data that undertreated patients with PA have a 3-fold higher risk profile than essential hypertensives with the same blood pressure levels. Third, clinicians managing hypertension measure success as sustainable lowering of blood pressure; untreated hypertensive patients with PA are thus in double jeopardy. Finally, and crucially, fewer than 1% of patients with hypertension are ever screened-let alone investigated-for PA. Accordingly, for "Who should we screen?" the answer is simple-all patients with hypertension. For "How they should be screened?" the answer is also simple-add spironolactone 25 mg/day for 4 weeks and measure the blood pressure response. In established hypertension, a fall of <10 mm Hg means PA is unlikely; above 12 mm Hg PA, it is probable. Newly presenting hypertension is much the same-hold off on first-order antihypertensive(s) and prescribe spironolactone 25 mg/day for 4 weeks. If blood pressure falls into the normal range, continue; if it does not, prescribe a standard antihypertensive. It is likely that the above protocols-a first start, amenable to refinement-will find additional hypertensives with unilateral PA; it is probable that the overwhelming majority will have bilateral disease. What this means is that we have a major public health issue on our hands: how can this be the case?

The genetic basis of primary aldosteronism.

Primary aldosteronism is commonly regarded as largely sporadic, but both germline and somatic mutations are increasingly recognized as underlying the condition. Three germline mutations causing familial hyperaldosteronism have been described, dubbed FH I (due to a CYP11B1/CYP11B2 chimera), FH II (localized to chromosome 7p22, exact location of mutation[s] unknown to date), and FH III (reflecting a T158A mutation in the potassium channel subunit KCNJ5). Major contributions (FH I, FH III) have been by Lifton and his associates; more recently they have also described somatic mutations (G151R, L168R) in KCNJ5 in over a third of aldosterone-producing adenomas, with results confirmed, refined, and extended in a much larger study from Europe. These findings have sparked considerable interest, and over the next 12 months a number of additional reports can be confidently expected to throw light on both normal and abnormal adrenocortical zonation and the genesis of primary aldosteronism.

Primary Aldosteronism: Where Are We Now? Where to From Here?

Primary aldosteronism, the most common secondary form of hypertension, is thought to be present in ≈5% to 10% of hypertensive adults. However, recent studies indicate that its prevalence may be at least 3-fold higher based on the identification of renin-independent (autonomous) aldosterone production that is not suppressible with dietary sodium loading in a large fraction of adults with primary hypertension. Currently, the screening rate for primary aldosteronism in adults with primary hypertension is <1%. This review summarizes current thinking about primary aldosteronism from the standpoint of 3 key questions: Where are we now? Where to from here? So how do we get there?

Aldosterone: a cardiovascular risk factor?

The hormone aldosterone has a well-recognized physiological role in epithelial fluid and electrolyte homeostasis, and more recently defined pathophysiological roles in the cardiovascular system. The term "risk factor" implies an active role in pathophysiology, with levels lower (e.g. HDL) or higher (e.g. LDL, BP) than normal contributing to an increased likelihood of morbidity and/or mortality. In this regard, primary aldosteronism represents a classic illustration of aldosterone as a cardiovascular risk factor. In this syndrome of (relatively) autonomous aldosterone secretion, the effects of elevated hormone levels are on a range of organs and tissues-the heart, blood vessels and brain, inter alia. In other cardiovascular disorders (e.g. CCF, EH) while an elevation of aldosterone levels is often regarded as a risk factor, it is more correctly a response to the severity of disease (or to treatment intervention), rather than necessarily a risk factor with a primary role in disease progression. An enduring enigma relevant to any discussion of aldosterone as a risk factor is that very high levels of aldosterone in response to chronic sodium deficiency have homeostatic (and protective of cardiovascular) functions, while the considerably lower levels commonly seen in primary aldosteronism are incontrovertibly damaging. A final section of the paper will thus propose a mechanism which might solve this enigma, based on the commonalities-and a single crucial difference-in the factors stimulating the secretion of aldosterone and endogenous ouabain from the zona glomerulosa of the adrenal gland.

Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney.

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na(+)) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na(+)/H(+) exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na(+)/H(+) exchange activity by Na(+)-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na(+)/H(+) exchange activity by >30%. Moreover, the sgk2-mediated increase in Na(+)/H(+) exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na(+) transport through NHE3 in the proximal tubule.

Medicine as a profession.

Over half a century ago, a Canadian judge defined a profession in a way that resonates still today, not only for lawyers and doctors, but for the current wide variety of professions and professionals. This article is a reflection on this definition. It briefly considers the historical context within which the knowledge base that characterises a profession evolved and what the various component parts of the judge's definition entail. A final consideration goes beyond the terms of the definition proposed--that of our ethical responsibility as professionals to stand up and be counted and, in the context of the disorder around us, to speak out.

The nongenomic actions of aldosterone.

Aldosterone has physiological effects to regulate fluid and electrolyte homeostasis across epithelia and proinflammatory effects on a variety of nonepithelial cells in the context of inappropriate salt status. These effects are mediated by mineralocorticoid receptors, members of a large family of nuclear transcription factors, by DNA-directed, RNA-mediated protein synthesis. Rapid effects of aldosterone, insensitive to actinomycin D or cycloheximide and thus clearly nongenomic, have been convincingly documented in a variety of epithelial and nonepithelial tissues. Despite strenuous attempts, isolation of a nonclassical membrane receptor for aldosterone has proven unsuccessful, and rapid nongenomic effects mediated by classical mineralocorticoid receptors are increasingly recognized in the kidney, heart, and vascular wall. The mechanism of rapid nongenomic actions of aldosterone may vary between tissues in terms of pathways; in addition, what remains to be established is the physiological role of aldosterone action via such rapid nongenomic mechanisms and how they might synergize with the longer time course genomic actions of mineralocorticoids.

Aldosterone and mineralocorticoid receptors: Clinical studies and basic biology.

With the enormous advances in our understanding of cellular and molecular biology over the past 30 years, it is perhaps not surprising that the term 'Translational Research' is commonly thought of as unidirectional, from benchtop to bedside. Just as enzymes are bidirectional, however much one reaction may be predominant, so is translation. This review sets out to chart how three sets of clinical observations, in hypertension and heart failure, have triggered a radical re-evaluation (and expansion) of our previous concepts of the basic (patho)physiology of aldosterone and mineralocorticoid receptors (MR). The first of these is an example of levering off classical translational research from benchtop to bedside, to revisit the accepted sequence of evolution of the MR, GR, AR and PR subfamily nuclear transactivating factors from a common primordial ancestor. The second example is a meta-analysis of two studies using the selective MR antagonist eplerenone as monotherapy in essential hypertension, which clearly distinguishes the effects of MR blockade on blood pressure from these on urinary electrolyte excretion; this in turn clearly calls into question current teaching of a primarily renal role for aldosterone in raising blood pressure. The final example is the demonstrated efficacy of MR blockade in clinical trials in heart failure and hypertension, when plasma aldosterone levels are in low normal range and sodium status unremarkable. This poses the question of what is activating cardiac and vascular smooth muscle MR under such circumstances, which in turn leads to a radical reconsideration of the role of glucocorticoids in non-epithelial mineralocorticoid receptors.

Mediators of mineralocorticoid receptor-induced profibrotic inflammatory responses in the heart.

Coronary, vascular and perivascular inflammation in rats following MR (mineralocorticoid receptor) activation plus salt are well-characterized precursors for the appearance of cardiac fibrosis. Endogenous corticosterone, in the presence of the 11betaHSD2 (11beta hydroxysteroid dehydrogenase type 2) inhibitor CBX (carbenoxolone) plus salt, produces similar inflammatory responses and tissue remodelling via activation of MR. MR-mediated oxidative stress has previously been suggested to account for these responses. In the present study we thus postulated that when 11betaHSD2 is inhibited, endogenous corticosterone bound to unprotected MR in the vessel wall may similarly increase early biomarkers of oxidative stress. Uninephrectomized rats received either DOC (deoxycorticosterone), CBX or CBX plus the MR antagonist EPL (eplerenone) together with 0.9% saline to drink for 4, 8 or 16 days. Uninephrectomized rats maintained on 0.9% saline for 8 days served as controls. After 4 days, both DOC and CBX increased both macrophage infiltration and mRNA expression of the p22(phox) subunit of NADPH oxidase, whereas CBX, but not DOC, increased expression of the NOX2 (gp91(phox)) subunit. eNOS [endothelial NOS (NO synthase)] mRNA expression significantly decreased from 4 days for both treatments, and iNOS (inducible NOS) mRNA levels increased after 16 days of DOC or CBX; co-administration of EPL inhibited all responses to CBX. The responses characterized over this time course occurred before measurable increases in cardiac hypertrophy or fibrosis. The findings of the present study support the hypothesis that endogenous corticosterone in the presence of CBX can activate vascular MR to produce both inflammatory and oxidative tissue responses well before the onset of fibrosis, that the two MR ligands induce differential but overlapping patterns of gene expression, and that elevation of NOX2 subunit levels does not appear necessary for full expression of MR-mediated inflammatory and fibrogenic responses.

Primary aldosteronism: cardiovascular, renal and metabolic implications.

For many years primary aldosteronism was considered a relatively benign form of hypertension. This assumption reflects the primacy accorded to elevated levels of angiotensin in terms of deleterious cardiovascular effects, and the fact that in primary aldosteronism renin and angiotensin levels are low. We now know that primary aldosteronism causes a constellation of cardiovascular, renal and metabolic sequelae which make it far from benign and that these are not merely effects of blood pressure elevation. In primary aldosteronism, tissue damage, on several indices, is higher than in age-, sex- and blood pressure-matched controls, reflecting the ability of inappropriately elevated aldosterone for salt status to produce structural and functional changes over and above those produced by high blood pressure.