Epigenetic Regulation in Hypertension: Mechanistic Insights and Environmental Influences.

BackgroundA critical area of recent cancer research is the emergence of transition states between normal and cancer that exhibit increased cell plasticity which underlies tumor cell heterogeneity. Pancreatic ductal adenocarcinoma (PDAC) can arise from the combination of a transition state termed acinar-to-ductal metaplasia (ADM) and a gain-of-function mutation in the proto-oncogene KRAS. During ADM, digestive enzyme-producing acinar cells acquire a transient ductal epithelium-like phenotype while maintaining their geographical acinar organization. One route of ADM initiation is the overexpression of the Krüppel-like factor 4 gene (KLF4) in the absence of oncogenic driver mutations. Here, we asked to what extent cells acquire and retain an epigenetic memory of the ADM transition state in the absence of oncogene mutation.Methods We profiled the DNA methylome and transcriptome of KLF4-induced ADM in transgenic mice at various timepoints during and after recovery from ADM. We validated the identified DNA methylation and transcriptomic signatures in the widely used caerulein model of inducible pancreatitis.ResultsWe identified differential DNA methylation at Kras-downstream PI3K and Rho/Rac/Cdc42 GTPase pathway genes during ADM, as well as a corresponding gene expression increase in these pathways. Importantly, differential methylation persisted after gene expression returned to normal. Caerulein exposure, which induces widespread digestive system changes in addition to ADM, showed similar changes in DNA methylation in ADM cells. Regions of differential methylation were enriched for motifs of KLF and AP-1 family transcription factors, as were those of human pancreatic intraepithelial neoplasia (PanIN) samples, demonstrating the relevance of this epigenetic transition state memory in human carcinogenesis. Finally, single-cell spatial transcriptomics revealed that these ADM transition cells were enriched for PI3K pathway and AP1 family members.ConclusionsOur comprehensive study of DNA methylation in the acinar-ductal metaplasia transition state links epigenetic memory to cancer-related cell plasticity even in the absence of oncogenic mutation.

Epigenetic mechanisms have been recognized to be important for the physiologic regulation of cognitive functions and their pathologic alterations contribute to many neurologic and neuropsychiatric diseases. We survey here an important mechanism of epigenetic regulation of brain function, the histone acetylation state modulated by histone deacetylases (HDACs), and its impact on cognitive behaviour as well as on hippocampal adult neurogenesis, which is presently recognized as an essential process for learning and memory. A potent way to modulate histone acetylation state is through HDAC inhibitors, which are currently tested as potential drugs for treatment of major diseases including cancer and neurologic/neuropsychiatric disorders. Various HDAC inhibitor molecules and their impact on brain function are examined with particular reference to neuroprotective action of some of these substances. The core of the review deals with the current status of the literature on the use of some of these inhibitors as memory enhancers and dementia-fighting agents as well as on their possible role in regulation of adult neurogenesis in the hippocampal dentate gyrus, a process linked to the ability of acquiring new memories. Possible reasons for the many controversial results arising from the literature are discussed in view of establishing a more reliable ground for the future use of HDAC inhibitors to improve memory process in brain health and disease. Keywords: Epigenetics, Histone acetylation state, Transcription regulation, Memory, Hippocampus, Dentate gyrus, Adult neurogenesis, neuropsychiatric disorders, phenotypical, HISTONE DEACETYLASE, basolateral amygdala, hippocampus-dependent, ameliorated, kinase, astrocytes

The CaSR regulates Ca 2+ -mediated release of parathyroid hormone (PTH) from parathyroid cells by sensing small changes in serum Ca 2+ concentration. The receptor is also involved in the regulation of cell Ca 2+ levels independent of PTH secretion. The renin-angiotensin-aldosterone system (RAAS) plays vital roles in the development of hypertension, which is associated with vascular and renal dysfunction, and treatment is difficult because the mechanisms are not fully understood. Studies show that adequate Ca 2+ intake (1.0 to 1.5 g/day) is critical for optimal BP regulation and randomized controlled trials have revealed significant reductions in hypertension risk and BP levels in humans. In the kidney, the CaSR is expressed along the nephron and plays an important role, with the Na + /Ca 2+ exchanger (NCX), in the regulation of tubular Ca 2+ , Na + and water reabsorption. In the present study, we analyzed the effects of mutation, sex hormones and high salt diets on the expression of the CaSR, its downstream signaling complements and proteins in the RAAS in both female and male salt-sensitive (SS) rats. Our data indicate that CaSR gene mutation and high salt diets modulate the expression of the CaSR, GRK2, PKCα, NCX1 and RAAS proteins in the kidney of SS female rats than male rats. And also showed statistically significant difference in electrolytes as shown in the data below. Thus, CaSR signaling is linked to RAAS to control hypertension.

This study aimed to examine the effects of salt loading, with or without simultaneous angiotensin receptor blocker (ARB) treatment, on the systemic and tissue renin-angiotensin system (RAS) in spontaneously hypertensive rats (SHRs). Evaluation was performed early (4 weeks) in the course of salt loading in order to examine initial mediating events of cardiovascular and renal damage produced by salt excess. Four groups of rats were studied. Group 1 received regular rat chow (normal-salt diet); group 2 received normal-salt diet and an ARB (losartan, 30 mg/kg per day); group 3 received high-salt (8%) chow; and group 4 received high-salt diet and losartan. High-salt diet increased systolic pressure to 193±1 mmHg compared to 180±2 in normal-salt diet group. Losartan reduced SBP in SHRs fed normal-salt diet but did not reduce SBP in the SHRs fed high-salt diet (192±2 mmHg). High-salt diet markedly increased urinary protein excretion from 27±4 to 64±13 mg/day and this increase was ameliorated by losartan (40±9 mg/day). In SHRs on high-salt diet, plasma angiotensin II concentration increased three to four-fold, whereas urinary angiotensinogen excretion increased 10-fold; and these changes were significantly reduced by losartan. High-salt diet accelerated glomerular injury and interstitial fibrosis in SHRs which were reduced by losartan. These results demonstrate that the activity of RAS was either not suppressed or, even augmented, after 4 weeks of salt loading despite high salt intake and increased SBP. The data suggest that an augmented intrarenal RAS during high-salt diet may contribute to the development of renal injury in this experimental model.

To assess changes in the activity of the brain renin-angiotensin system during (i) the development of salt-sensitive hypertension; and (ii) the prevention of salt-sensitive hypertension by blocking brain 'ouabain'. In protocol I, angiotensin converting enzyme (ACE) mRNA and activity and angiotensin I and II levels were assessed in the hypothalamus and pons of Dahl salt-sensitive (Dahl S) and salt-resistant (Dahl R) rats on regular (120 micromol Na+ per g) or high (1370 micromol Na+ per g) salt diet from 4-6 weeks or 4-9 weeks of age. In protocol II, ACE mRNA and activity were assessed in the hypothalamus and pons in Dahl S on regular or high salt treated with intracerebroventricular (i.c.v.) Fab fragments blocking brain 'ouabain' or gamma-globulins, and in Dahl R on high or regular salt ACE mRNA was assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay and angiotensin I and II by radioimmunoassay after high-performance liquid chromatography. In protocol III, effects of i.c.v. angiotensin I and i.c.v. bradykinin on renal sympathetic nerve activity (RSNA), heart rate and blood pressure before and after i.c.v. captopril were assessed in Dahl S and R rats on regular or high salt intake from 4-8 weeks of age. High salt diet caused a gradual, but marked increase in blood pressure in Dahl S but not Dahl R rats. Dahl S rats showed small but significant increases in ACE mRNA in the hypothalamus on regular salt diet. In Dahl S rats on high salt diet for 2 or 5 weeks ACE mRNA levels significantly increased in both hypothalamus and pons, compared with Dahl R rats on either diet or Dahl S rats on regular diet. After 5 weeks of high salt diet, ACE mRNA levels in the hypothalamus in Dahl S rats were almost three-fold higher and in the pons two-fold higher than in Dahl R rats on either diet or Dahl S on regular salt diet. High salt diet also increased ACE activity of the hypothalamus and pons in Dahl S but not Dahl R. Consistent with this increased ACE activity, central responses to angiotensin I were clearly enhanced and to bradykinin markedly diminished in Dahl S on high salt intake. Chronic blockade of brain 'ouabain' by i.c.v. Fab fragments prevented the increases in blood pressure, ACE mRNA and activity in the hypothalamus and pons by high salt intake in Dahl S rats. Angiotensin I levels in the hypothalamus and pons were similar in both groups of rats and there were no significant changes caused by high salt diet in Dahl S and R rats. On regular salt intake angiotensin II levels in the hypothalamus of Dahl S rats showed a significant decrease as compared with Dahl R rats on regular salt diet, and were similar in the pons of the two strains. High salt intake did not affect angiotensin II levels in either hypothalamus or pons in Dahl S and R rats. These results indicate that high salt intake increases blood pressure, ACE expression and activity in the hypothalamus and pons of Dahl S rats without a parallel increase in angiotensin II levels. Effects of high salt intake on ACE mRNA and activity appear to be secondary to activation of brain 'ouabain'.

Perinatal iron deficiency causes changes in offspring mesenteric artery function in adulthood, particularly in males, which can be exacerbated by chronic intake of a high salt diet. Perinatal iron deficient male offspring exhibit enhanced conversion of big endothelin-1 to active endothelin-1, coinciding with decreased nitric oxide levels. Perinatal iron deficient male offspring have reduced nitric oxide-mediated endothelial-dependent vasodilatation coincident with increased vascular superoxide levels following consumption of a high salt diet. Perinatal iron deficiency has no apparent effects on vascular function in female offspring, even when fed a high salt diet. These results help us better understand underlying vascular mechanisms contributing to increased cardiovascular risk from perinatal stressors such as iron deficiency. Pre- and immediate postnatal stressors, such as iron deficiency, can alter developmental trajectories and predispose offspring to long-term cardiovascular dysfunction. Here, we investigated the impact of perinatal iron deficiency on vascular function in the adult offspring, and whether these long-term effects were exacerbated by prolonged consumption of a high salt diet in adulthood. Female Sprague Dawley rats were fed either an iron-restricted or -replete diet prior to and throughout pregnancy. Six weeks prior to experimentation at 6months of age, adult offspring were fed either a normal or high salt diet. Mesenteric artery responses to vasodilators and vasoconstrictors were assessed ex vivo by wire myography. Male perinatal iron deficient offspring exhibited decreased reliance on nitric oxide with methacholine-induced vasodilatation (interaction P=0.03), coincident with increased superoxide levels when fed the high salt diet (P=0.01). Male perinatal iron deficient offspring exhibit enhanced big endothelin-1 conversion to active endothelin-1 (P=0.02) concomitant with decreased nitric oxide levels (P=0.005). Female offspring vascular function was unaffected by perinatal iron deficiency, albeit the high salt diet was associated with impaired vasodilation and decreased nitric oxide production (P=0.02), particularly in the perinatal iron deficient offspring. These findings implicate vascular dysfunction in the sex-specific programming of cardiovascular dysfunction in the offspring by perinatal iron deficiency.

Loss of salt-inducible kinase 1 (SIK1) triggers an increase in blood pressure (BP) upon a chronic high-salt intake in mice (Circ Res 2015;116:642-52). Here, we address possible acute mechanisms that may relate to the observed high BP in mice lacking SIK1. SIK1 knockout ( sik1 -/- ) and wild-type ( sik1 +/+ ) littermate mice were challenged for seven days with a normal- (0.3% NaCl) or high-salt (8% NaCl) diet. Systolic BP (SBP) was significantly increased in sik1 -/- mice (137.0±17.2 mmHg) after seven days of high-salt intake, as compared to sik1 +/+ mice counterparts (120.6±4.5 mmHg). The renin-angiotensin system (RAS) and the sympathetic nervous system (SNS) were assayed in order to investigate the possible causes for the increase in SBP in sik1 -/- mice fed a high-salt diet. No differences in renin (normal-salt: 463.4±17.9, high-salt: 462.9±28.9 pg/ml) and angiotensin II (normal-salt: 45.8±10.0, high-salt: 39.0±8.5 pg/ml) serum levels were observed. The activity of dopamine β-hydroxylase (DβH), the enzyme that converts dopamine (DA) to norepinephrine (NE), was significantly increased in the adrenal glands of sik1 -/- mice fed a high-salt diet (356.7±32.8 nmol/mg protein) as compared to sik1 -/- mice on a normal-salt diet (184.4±14.4 nmol/mg protein). Similarly, urinary catecholamines (DA, NE, epinephrine) and L-DOPA were significantly increased (3- to 7-fold increase) in sik1 -/- mice fed a high-salt diet as compared to sik1 -/- mice on a normal-salt intake. Altogether, this data supports the view that sik1 -/- mice fed a high-salt diet develop SNS overactivity. Next, we addressed the question if reducing SNS activity in sik1 -/- mice fed a high-salt diet would ameliorate hypertension. For that purpose, the effect of etamicastat, a peripheral reversible DβH inhibitor, was evaluated on the development of high BP upon high-salt diet. Etamicastat treatment (50 mg/kg/day), started prior to high-salt feeding, completely prevented SBP increase in sik1 -/- mice fed a high-salt diet (116.8±4.7 mmHg). It is concluded that the SNS is involved in the development of salt-induced hypertension in sik1 -/- mice and that the DβH inhibitor etamicastat is able to reduce SNS overactivity and high BP in this mouse model of hypertension.

This study investigated the role of impaired angiotensin II (Ang II) modulation in contributing to reduced vascular relaxation in isolated middle cerebral arteries (MCA) (100 to 200 microm in diameter) of normotensive Dahl salt-sensitive (SS) rats maintained on low salt (LS) diet (0.4% NaCl) for 9 to 10 weeks. MCA from SS rats on LS diet (n=6 to 9) constricted in response to reduction of perfusate and superfusate PO2 to 35 to 40 mm Hg or acetylcholine (ACh). Vasodilator responses to reduced PO2 and ACh were restored in SS.13BN consomic rats that are 98% genetically identical to SS rats, but exhibit normal regulation of their renin-angiotensin system (RAS). This restored dilation could be prevented by feeding SS.13BN rats high-salt (HS) diet (4% NaCl) for 3 days to suppress Ang II. A continuous intravenous infusion of a subpressor dose (3 ng/kg per minute) of Ang II for 3 days restored vasodilator responses to ACh and reduced PO2 in SS.13BN rats on HS diet and in SS rats on LS diet. Superoxide scavenging with tempol (100 micromol/L) restored vasodilator responses to ACh and reduced PO2 in MCA of SS rats on LS diet, but did not affect vasodilator responses in MCA of SS.13BN rats on LS diet. These data indicate that exposure to chronically low Ang II levels leads to impaired vascular relaxation in SS rats, even when the animals are on LS diet and normotensive. This impaired relaxation appears to be mediated by increased levels of oxidative stress in the arteries.

Histone deacetylase (HDAC) inhibitors are gaining interest as cancer therapeutic agents. We tested the hypothesis that natural organoselenium compounds might be metabolized to HDAC inhibitors in human prostate cancer cells. Se-Methyl-L-selenocysteine (MSC) and selenomethionine are amino acid components of selenium-enriched yeast. In a cell-free system, glutamine transaminase K (GTK) and L-amino acid oxidase convert MSC to the corresponding alpha-keto acid, beta-methylselenopyruvate (MSP), and L-amino acid oxidase converts selenomethionine to its corresponding alpha-keto acid, alpha-keto-gamma-methylselenobutyrate (KMSB). Although methionine (sulfur analogue of selenomethionine) is an excellent substrate for GTK, selenomethionine is poorly metabolized. Structurally, MSP and KMSB resemble the known HDAC inhibitor butyrate. We examined androgen-responsive LNCaP cells and androgen-independent LNCaP C4-2, PC-3, and DU145 cells and found that these human prostate cancer cells exhibit endogenous GTK activities. In the corresponding cytosolic extracts, the metabolism of MSC was accompanied by the concomitant formation of MSP. In MSP-treated and KMSB-treated prostate cancer cell lines, acetylated histone 3 levels increased within 5 hours, and returned to essentially baseline levels by 24 hours, suggesting a rapid, transient induction of histone acetylation. In an in vitro HDAC activity assay, the selenoamino acids, MSC and selenomethionine, had no effect at concentrations up to 2.5 mmol/L, whereas MSP and KMSB both inhibited HDAC activity. We conclude that, in addition to targeting redox-sensitive signaling proteins and transcription factors, alpha-keto acid metabolites of MSC and selenomethionine can alter HDAC activity and histone acetylation status. These findings provide a potential new paradigm by which naturally occurring organoselenium might prevent the progression of human prostate cancer.

Enhanced angiotensin-converting enzyme activity and systemic reactivity to angiotensin II in normotensive rats exposed to a high-sodium diet

We set out to confirm the long-held, but untested, assumption that dietary salt affects proximal reabsorption through reciprocal effects on the renin-angiotensin system in a way that facilitates salt homeostasis. Wistar rats were fed standard or high-salt diets for 7 days and then subjected to renal micropuncture for determination of single-nephron GFR (SNGFR) and proximal reabsorption. The tubuloglomerular feedback (TGF) system was used as a tool to manipulate SNGFR in order to distinguish primary changes in net proximal reabsorption (Jprox) from changes due to glomerulotubular balance. The influence of Ang II over Jprox was determined by the sensitivity of Jprox to the AT1 receptor antagonist, losartan. Plasma, whole kidneys, and fluid from midproximal tubules were assayed for Ang II content by radioimmunoassay. In rats on the standard diet, losartan reduced Jprox by 25% and reduced the maximum range of the TGF response by 50%. The high-salt diet suppressed plasma and whole-kidney Ang II levels. But the high-salt diet failed to reduce the impact of losartan on Jprox or the TGF response and actually caused tubular fluid Ang II content to increase. The persistent effect of Ang II on Jprox prevented a major rise in late proximal flow rate in response to the high-salt diet. These observations challenge the traditional model and indicate that the role of proximal tubular Ang II in salt-replete rats is to stabilize nephron function rather than to contribute to salt homeostasis.

Context and ObjectivesIn obese mice and patients, mineralocorticoid receptor (MR) activation has deleterious consequences on metabolic and cardiovascular parameters. In mice, adipose tissue MR triggers mitochondrial and vascular dysfunction. Adipocyte MR activation induces metabolic and vascular dysfunction in a context of obesity, however, obese adipose tissue is also infiltrated with macrophages, which are capable of expressing MR. Thus, we hypothesised that macrophage MR could also be responsible for metabolic and cardiovascular complications in high‐fat high‐salt diet‐fed mice.MethodsWe fed myeloid cell‐specific MR knock‐out male and female mice (My‐MRKO) with a 6‐week high fat – high salt diet (60% kcal from fat, 1.2% salt in drinking water) and studied weight gain, whole body and specific organs composition by nuclear magnetic resonance and insulin sensitivity to explore the metabolic phenotype. Blood pressure measurements by plethysmography and vascular reactivity studies by wire myography were also performed to characterise the cardiovascular phenotype.ResultsMy‐MRKO male mice under high fat – high salt diet displayed reduced weight (37.8 g [interquartile range 34.1–40.8] vs 42.5 g [39.5–45.2]; P = 0.0128), preserved muscle mass (61.5 % [55.8–63.3] vs 56.8 % [54.1–58.5]; P = 0.0337) and a strong tendency to decreased fat mass (26.1 % [23.7–33.9] vs 31.7 % [27.1–35.4]; P = 0.0728). Moreover, liver fat content at sacrifice was decreased (5.8 % [2.1–9.1] vs 9.3 % [5.9–13.4]; P = 0.0491). Those effects were not observed in female mice. Fasting glycaemia was decreased in male My‐MRKO mice (9.2 mmol/L [6.4–12.8] vs 13.2 mmol/L [10.4–16.1]; P = 0.0005) and insulin resistance was decreased in both gender (area under curve 9915 [9398–10043] vs 11550 [9795–12848]; P = 0.0458 in males, 8291 [7812–8884] vs 9544 [8918–9735]; P = 0.0177 in females), as assessed by an insulin tolerance test. A clear effect was observed on blood pressure only in female mice (blood pressure 96 mmHg [94–97] vs 106 mmHg [104–109]; P = 0.0004), whereas there was no modification in mesenteric arteries vascular reactivity in both gender.ConclusionsThus, our findings show that myeloid cell‐MR regulates systemic metabolic parameters, highlighting a new role in obesity and its associated complications for MR expressed in myeloid cells.Support or Funding InformationSorbonne Université, Inserm, CARMMA‐RHU

Diabetes mellitus (DM) is a major risk factor for heart failure, independent of coronary artery disease or hypertension (HT). Therefore, our study was designed to examine the mechanisms of DM-induced left ventricular (LV) diastolic dysfunction. In this study, we made five different 10-week treatment groups of Dahl salt-sensitive rats as follows: Control; a low-salt (0.5% NaCl) diet, HT; a high-salt (5% NaCl) diet, DM; a low-salt diet with streptozotocin (STZ) injection (30 mg kg(-1) i.p.), HT+DM; a high-salt diet with STZ injection, and the Olmesartan group; a high-salt diet with STZ treated with an angiotensin receptor blocker, olmesartan (1 mg kg(-1) day(-1)). Cardiac diastolic dysfunction with a preserved systolic function was noted in the HT group, and was most prominently noted in the HT+DM group, characterized by enhanced cardiac fibrosis, whereas the extent of HT and myocardial hypertrophy was comparable between the two groups. Myocardial expressions of collagen III, transforming growth factor-beta2, angiotensin-converting enzyme (ACE), angiotensin II type-1 receptor and myocardial oxidative stress (evaluated by 4-hydroxy-2-nonenal-modified protein) were mostly enhanced in the HT+DM group. Importantly, there was a positive correlation between the extent of diastolic dysfunction and that of myocardial ACE expression. All these cardiac abnormalities induced by DM and HT were ameliorated in the olmesartan group. These results indicate that DM accelerates diastolic dysfunction in hypertensive heart disease through activation of the renin-angiotensin system, with subsequent inflammatory and oxidative stresses and myocardial fibrosis, suggesting that an inhibition of the system is effective for the treatment of diastolic dysfunction in this combined disorder.

Multiple myeloma (MM) is a neoplastic proliferation of plasma cells and remains an incurable disease because of the development of drug resistance. Histone deacytylase (HDAC) inhibitors are a new class of chemotherapeutic reagents that cause growth arrest and apoptosis of neoplastic cells. Depsipeptide, a new member of the HDAC inhibitors, was found to be safe in humans and has been shown to induce apoptosis in various cancers. In order to evaluate the effects of depsipeptide, a MM cell line, U266 [interleukin (IL)-6 dependent], was analysed for viability and apoptosis. The combined effect of depsipeptide with melphalan and changes in BCL-2 family proteins (BCL-2, BCL-XL, BAX and MCL-1) were also investigated. In addition, the RPMI 8226 cell line (IL-6 independent), and primary patient myeloma cells were also analysed for apoptosis after depsipeptide treatment. Depsipeptide induced apoptosis in both U266 and RPMI 8226 cell lines in a time- and dose-dependent fashion, and in primary patient myeloma cells. We also demonstrated that depsipeptide had an additive effect with melphalan (10 micromol/l). BCL-2, BCL-XL and MCL-1 showed decreased expression in depsipeptide-treated samples. Based on recent clinical trials demonstrating minimal clinical toxicity, our study supports the future clinical utilization of depsipeptide in the management of MM.

BackgroundThe status of estrogen receptor-α (ERα) is critical to the clinical prognosis and therapeutic approach in breast cancer. ERα-negative breast cancer is clinically aggressive and has a poor prognosis because of the lack of hormone target-directed therapies. Previous studies have shown that epigenetic regulation plays a major role in ERα silencing in human breast cancer cells. Dietary green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), is believed to be an anticancer agent in part through its regulation of epigenetic processes.ResultsIn our current studies, we found that EGCG can reactivate ERα expression in ERα-negative MDA-MB-231 breast cancer cells. Combination studies using EGCG with the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), revealed a synergistic effect of reactivation of ERα expression in ERα-negative breast cancer cells. Reactivation of ERα expression by EGCG and TSA treatment was found to sensitize ERα-dependent cellular responses to activator 17β-estradiol (E2) and antagonist tamoxifen in ERα-negative breast cancer cells. We also found that EGCG can lead to remodeling of the chromatin structure of the ERα promoter by altering histone acetylation and methylation status thereby resulting in ERα reactivation. A decreased binding of the transcription repressor complex, Rb/p130-E2F4/5-HDAC1-SUV39H1-DNMT1, in the regulatory region of the ERα promoter also contributes to ERα transcriptional activation through treatment with EGCG and/or TSA.ConclusionsCollectively, these studies show that green tea EGCG can restore ERα expression by regulating epigenetic mechanisms, and this effect is enhanced when combined with an HDAC inhibitor. This study will facilitate more effective uses of combination approaches in breast cancer therapy and will help to explore more effective chemotherapeutic strategies toward hormone-resistant breast cancer.