Pathogenicity evaluation of variants of uncertain significance at exon-intron junction by splicing assay in patients with Mowat-Wilson syndrome.

High-throughput sequencing has identified vast numbers of variants in genetic disorders. However, the significance of variants at the exon-intron junction remains controversial. Even though most cases of Mowat-Wilson syndrome (MOWS) are caused by heterozygous loss-of-function variants in ZEB2, the pathogenicity of variants at exon-intron junction is often indeterminable. We identified four intronic variants in 5/173 patients with clinical suspicion for MOWS, and evaluated their pathogenicity by in vitro analyses. The minigene analysis showed that c.73+2T>G caused most of the transcripts skipping exon 2, while c.916+6T>G led to partial skipping of exon 7. No splicing abnormalities were detected in both c.917-21T>C and c.3067+6A>T. The minigene analysis reproduced the splicing observed in the blood cells of the patient with c.73+2T>G. The degree of the exon skipping was concordant with the severity of MOWS; while the patient with c.73+2T>G was typical MOWS, the patient with c.916+6T>G showed milder phenotype which has been seldom reported. Our results demonstrate that mRNA splicing assays using the minigenes are valuable for determining the clinical significance of intronic variants in patients with not only MOWS but also other genetic diseases with splicing aberrations and may explain atypical or milder cases, such as the current patient.

Resveratrol exhibits diverse anti-cancer activities through epigenetic regulation of E-cadherin and p21 in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) has an aggressive phenotype and poor outcome, however no specific targeted therapy has been established for TNBC lacking germline BRCA1/2 pathogenic variants. To develop a novel therapeutic strategy, we explored the potential of resveratrol (RSV) for TNBC treatment. METHODS: We investigated the effects of RSV on malignant phenotypes of TNBC cells as well as on apoptosis induced by ABT263, a specific inhibitor of BCL-2 and BCL-xL, using morphological observation, migration assay, ß-galactosidase staining, and Hoechst staining. To elucidate the underlying mechanisms of RSV-mediated effects, expression levels and histone acetylation levels of cadherin 1 (CDH1, E-cadherin) and cyclin dependent kinase inhibitor 1A (CDKN1A, p21) were determined by RT-qPCR, western blotting, and chromatin immunoprecipitation. Furthermore, knockdown analysis was conducted to evaluate the involvement of E-cadherin and/or p21 in RSV potentiation on cytotoxic activity of ABT263. RESULTS: RSV treatment induced epithelial-like cellular morphology and suppressed the migration capacity in MDA-MB-231 and BT-549-Luc TNBC cells. ß-galactosidase-positive cells were increased after RSV treatment, indicating the induction of cellular senescence, in MDA-MB-231 cells but not in BT-549-Luc cells. RSV increased the expression and histone acetylation of CDH1 and CDKN1A in both cells. Interestingly, pre-treatment with RSV enhanced the induction of apoptosis in the ABT263-treated MDA-MB-231 and BT-549-Luc cells, and knockdown of CDKN1A decreased ABT263-induced apoptosis in RSV-treated MDA-MB-231 cells. CONCLUSIONS: RSV represses the metastatic capacity and enhances the cytotoxic activity of ABT263 in TNBC cells. Our results suggested that RSV can potentially be used as a repressor of metastasis or a sensitizer to ABT263 for TNBC treatment via up-regulation of CDH1 and CDKN1A through epigenetic mechanisms.

A case of non-immune hydrops fetalis with maternal mirror syndrome diagnosed by trio-based exome sequencing: An autopsy case report and literature review.

Non-immune hydrops fetalis (NIHF) indicates the risk for stillbirth. Although the causes vary and most NIHFs have no identifiable cause, recent advances in exome sequencing have increased diagnostic rates. We report a case of NIHF that developed into a giant cystic hygroma complicated by maternal mirror syndrome. Trio-based exome sequencing showed a de novo heterozygous missense variant in the RIT1 (NM_006912: c.246 T > G [p.F82L]). The RIT1 variants are known causative variants of Noonan syndrome (NS; OMIM #163950). The location of the RIT1 variants in the previously reported NS cases with NIHF or/and maternal mirror syndrome was mainly in the switch II region, including the present case. While a further accumulation of cases is needed, exome sequencing, which can identify the variant type in detail, might help predict the phenotype and severity of NIHF.

Verification of dose distribution in high dose-rate brachytherapy for cervical cancer using a normoxic N-vinylpyrrolidone polymer gel dosimeter.

The polymer gel dosimeter has been proposed for use as a 3D dosimeter for complex dose distribution measurement of high dose-rate (HDR) brachytherapy. However, various shapes of catheter/applicator for sealed radioactive source transport used in clinical cases must be placed in the gel sample. The absorbed dose readout for the magnetic resonance (MR)-based polymer gel dosimeters requires calibration data for the dose-transverse relaxation rate (R2) response. In this study, we evaluated in detail the dose uncertainty and dose resolution of three calibration methods, the multi-sample and distance methods using the Ir-192 source and the linear accelerator (linac) method using 6MV X-rays. The use of Ir-192 sources increases dose uncertainty with steep dose gradients. We clarified that the uniformly irradiated gel sample improved the signal-to-noise ratio (SNR) due to the large slice thickness of MR images and could acquire an accurate calibration curve using the linac method. The curved tandem and ovoid applicator used for intracavitary irradiation of HDR brachytherapy for cervical cancer were reproduced with a glass tube to verify the dose distribution. The results of comparison with the treatment planning system (TPS) calculation by gamma analysis on the 3%/2 mm criterion were in good agreement with a gamma pass rate of 90%. In addition, the prescription dose could be evaluated accurately. We conclude that it is easy to place catheter/applicator in the polymer gel dosimeters, making them a useful tool for verifying the 3D dose distribution of HDR brachytherapy with accurate calibration methods.

Conversion efficiency improvement of terahertz wave generation laterally emitted by a ridge-type periodically poled lithium niobate.

We demonstrate terahertz (THz) wave generation by wavelength conversion in a ridge-type/bulk periodically poled lithium niobate (RT-/bulk-PPLN) under almost the same experimental conditions. When using the RT-PPLN, the ridge structure works as a slab waveguide for the incident pump beam (wavelength: ∼1 µm), and the generated THz wave (∼200 µm) was emitted uniformly from the entire side surface of the crystal. The RT-PPLN has a much higher conversion efficiency from the pumping beam to the THz wave than the bulk-PPLN, and the ratio improved several ten times compared with those of previous studies.

Impact of Percutaneous Transluminal Renal Angioplasty on Autonomic Nervous System and Natriuresis in Hypertensive Patients With Renal Artery Stenosis.

Background We investigated the early postoperative effect of percutaneous transluminal renal angioplasty on ambulatory blood pressure (BP) and the circadian characteristics of natriuresis and autonomic nerve activity. Methods and Results A total of 64 patients with hypertension with hemodynamically significant renal artery stenosis (mean age, 60.0±21.0 years; 31.3% fibromuscular dysplasia) who underwent angioplasty were included, and circadian characteristics of natriuresis as well as heart rate variability indices, including 24-hour BP, low-frequency and high-frequency (HF) components, and the percentage of differences between adjacent normal R-R intervals >50 ms were evaluated using an oscillometric device, TM-2425, both at baseline and 3 days after angioplasty. In both the fibromuscular dysplasia and atherosclerotic stenosis groups, 24-hour systolic BP (fibromuscular dysplasia, -19±14; atherosclerotic renal artery stenosis, -11±9 mm Hg), percentage of differences between adjacent normal R-R intervals >50 ms, HF, brain natriuretic peptide, and nighttime urinary sodium excretion decreased (all P<0.01), and heart rate increased (both P<0.05) after angioplasty. In both groups, revascularization increased the night/day ratios of percentage of differences between adjacent normal R-R intervals >50 ms (both P<0.01) and HF, and decreased those of low frequency/HF (all P<0.05) and nighttime urinary sodium excretion (fibromuscular dysplasia, 1.17±0.15 to 0.78±0.09; atherosclerotic renal artery stenosis, 1.37±0.10 to 0.99±0.06, both P<0.01). Multiple logistic regression analysis indicated that a 1-SD increase in baseline low frequency/HF was associated with at least a 15% decrease in 24-hour systolic BP after angioplasty (odds ratio, 2.30 [95% CI, 1.03-5.67]; P<0.05). Conclusions Successful revascularization results in a significant BP decrease in the early postoperative period. Intrarenal perfusion might be a key modulator of the circadian patterns of autonomic nerve activity and natriuresis, and pretreatment heart rate variability evaluation seems to be important for treatment success.

Recurrent pneumonia in three patients with MECP2 duplication syndrome with aspiration as the possible cause.

INTRODUCTION: Methyl-CpG binding protein 2 gene (MECP2) is located on the X chromosome (Xq28) and is important for nervous and immune system functioning. Patients with MECP2 duplication syndrome (MDS) have recurrent respiratory infections (RRIs). Although RRIs often occur with MDS because some patients with MDS also have hypoimmunoglobulinemia and duplication of the interleukin-1-receptor-associated kinase-1 gene (IRAK1), which is also located on Xq28, the phenotype of IRAK1 duplication in patients with MDS remains unclear. METHODS: The clinical course of three patients with MDS who underwent laryngotracheal separation (LTS) at two institutions was summarized. RESULTS: Three patients with MDS were identified to have recurrent pneumonia characteristic of aspiration pneumonia, sometimes requiring artificial ventilation therapy; they had no other bacterial infections. After LTS, they rarely had pneumonia. In MDS, MECP2 expression increased two-fold naturally, while IRAK-1 expression showed no difference compared with a healthy subject. CONCLUSIONS: Since RRIs in MDS are thought to be caused by aspiration and not susceptibility to infection previously estimated to be major complication, the evaluation of aspiration is recommended for RRIs for better management of MDS.

One DNA Methylation Regulates CHIP Gene Expression of Human Breast Cancer and Predicts Recurrence.

BACKGROUND/AIM: Carboxyl terminus of Hsc70-interacting protein (CHIP) is a ubiquitin ligase that induces ubiquitination and degradation of its target proteins including oncoproteins. We reported that its down-regulation is associated with tumor progression and metastasis of breast cancer. However, the mechanism through which CHIP gene affects cancer cells is unclear. MATERIALS AND METHODS: We extracted RNA from 45 primary breast cancer samples and compared CHIP mRNA expression profiles, promoter DNA methylation status, and clinicopathological information. RESULTS: CHIP mRNA expression was significantly correlated with the tumor progression status. In several samples, a pinpoint CpG methylation in the CHIP gene promoter region was significantly correlated with CHIP mRNA expression. When this specific CpG was methylated in estrogen receptor (ER)-positive cases, a significant difference in 5-year recurrence was not found compared with ER-negative cases. CONCLUSION: CpG methylation contributes to the long-term prognosis of ER-positive breast cancer.

Molecular targeted drugs resistance impairs double-strand break repair and sensitizes ER-positive breast cancer to PARP inhibitors.

BACKGROUND: There are various treatments for estrogen-positive breast cancer, mainly hormone therapy and molecular-targeted drugs. Acquiring resistance to these drugs is a major clinical problem. Additionally, little is known about the effect of drug resistance on the DNA repair mechanism. Poly ADP ribose polymerase (PARP) inhibitors currently used for treating HER2-negative metastatic breast cancer with BRCA mutations have been shown to be effective in BRCA-deficient cells with impaired homologous recombination repair. Here, we investigated the effect of drug resistance acquisition on the DNA repair mechanism and the effect of PARP inhibitors on ER (estrogen receptor) -positive breast cancer. METHODS: We investigated changes in the expression of DNA repair mechanism-related factors and repair ability of double-strand breaks (DSB) in various drug-resistant cell lines established in our laboratory. Additionally, PARP inhibitor susceptibility was investigated using olaparib. RESULTS: DSB repairs in MCF-7 and hormone therapy-resistant model cells were normal, and these cells demonstrated low sensitivity to olaparib. The resistant cell lines against CDK4/6 inhibitors, fulvestrant and mTOR/PI3K inhibitors showed decreased DSB repair ability and high olaparib sensitivity. They showed low sensitivity to CDK4/6 inhibitors, a close link between acquiring resistance to CDK4/6 inhibitors and hypersensitivity to olaparib. CONCLUSIONS: Our study suggests some cases of acquiring drug resistance impairs DSB repair ability and sensitizes ER-positive breast cancer to PARP inhibitors.

Three-dimensional Winston-Lutz test using reusable polyvinyl alcohol-iodide (PVA-I) radiochromic gel dosimeter.

Medical linear-accelerator-based stereotactic radiosurgery (SRS) using a stereotactic apparatus or image-guided radiotherapy system for intracranial lesions is performed widely in clinical practice. In general, Winston-Lutz (WL) tests using films or electric portal imaging devices (EPIDs) have been performed as pre-treatment and routine quality assurance (QA) for the abovementioned treatment. Two-dimensional displacements between the radiation isocentre and mechanical isocentre are analysed from the test; therefore, it is difficult to identify the three-dimensional (3D) isocentre position intuitively. In this study, we developed an innovative 3D WL test for SRS-QA using a novel radiochromic gel dosimeter based on a polyvinyl alcohol-iodide (PVA-I) complex that can be reused after annealing. A WL gel phantom that was consisted of the PVA-I gel dosimeter poured into a tall acrylic container and an embedded small tungsten sphere was used as a position detector. A flatbed scanner was used to analyse the isocentre position. The measured 3D isocentre accuracy from the gel-based WL test was within 0.1 mm compared with that obtained from the EPID-based WL test. Furthermore, excellent reusability of the WL gel phantom was observed in long-term SRS isocentre verification, in which clinical SRS cases involving repeated irradiation and annealing were analysed. These results demonstrate the high accuracy and reliable evaluation of the isocentre position using an innovative test. In addition, the clinical-based routine SRS-QA using the PVA-I gel dosimeter demonstrates a highly convenience while affording an easy and fast analysis process.

R3HDM1 haploinsufficiency is associated with mild intellectual disability.

R3HDM1 (R3H domain containing 1) is an uncharacterized RNA-binding protein that is highly expressed in the human cerebral cortex. We report the first case of a 12-year-old Japanese male with haploinsufficiency of R3HDM1. He presented with mild intellectual disability (ID) and developmental delay. He had a pericentric inversion of 46,XY,inv(2)(p16.1q21.3)dn with breakpoints in intron 19 of R3HDM1 (2q21.3) and the intergenic region (2p16.1). The R3HDM1 levels in his lymphoblastoid cells were reduced to approximately half that of the healthy controls. However, the expression of MIR128-1, in intron 18 of R3HDM1, was not affected via the pericentric inversion. Knockdown of R3HDM1 in mouse embryonic hippocampal neurons suppressed dendritic growth and branching. Notably, the Database of Genomic Variants reported the case of a healthy control with a 488-kb deletion that included both R3HDM1 and MIR128-1. miR-128 has been reported to inhibit dendritic growth and branching in mouse brain neurons, which directly opposes the novel functions of R3HDM1. These findings suggest that deleting both R3HDM1 and MIR128-1 alleviates the symptoms of the disease caused by loss-of-function mutations in R3HDM1 only. Thus, haploinsufficiency of R3HDM1 in the patient may be the cause of the mild ID due to the genetic imbalance between R3HDM1 and MIR128-1.

Associations Between Arterial Stiffness Indices and Chronic Kidney Disease Categories in Essential Hypertensive Patients.

BACKGROUND: This study investigated the association between arterial stiffness indices and asymptomatic chronic kidney disease (CKD) risk categories in hypertensive patients. METHODS: Arterial stiffness indices, including 24-hour brachial and aortic systolic blood pressure (SBP) and pulse wave velocity (PWV), were measured by an oscillometric Mobil-O-Graph device, brachial-ankle PWV (baPWV) by a volume-plethysmographic method, and renal resistive index (RI) by ultrasonography, in 184 essential hypertensive patients (66.0 ± 17.1 years, 47.3% male). CKD was categorized into 3 stages based on the Kidney Disease Improving Global Outcomes (KDIGO) criteria, using a combination of estimated glomerular filtration and albuminuria. RESULTS: The 24-hour aortic PWV (aPWV), baPWV, and RI increased with worsening severity of CKD risk category (all P < 0.01 for trend). Multivariate logistic regression analysis found that a 1 SD increase of nighttime aortic SBP (odds ratio [OR] 1.52), PWV (OR 4.80), or RI (OR 1.75) was an independent predictor of high or very-high CKD stage (all P < 0.05). After adjustment for potential confounders, day-to-night change in brachial SBP as well as in aPWV differed among groups (P < 0.05, respectively). In a multivariate regression model, day-to-night changes in aortic SBP and PWV, and RI were independently associated with day-to-night brachial SBP change. CONCLUSIONS: In hypertension, circadian hemodynamics in high CKD stage are characterized by higher nighttime values of aortic SBP and PWV and disturbed intrarenal hemodynamics. Further, the blunted nocturnal BP reduction in these patients might be mediated via disturbed intrarenal hemodynamics and circadian hemodynamic variation in aortic SBP and arterial stiffness.

Insight into the Role of Angiopoietins in Ageing-Associated Diseases.

Angiopoietin (Ang) and its receptor, TIE signaling, contribute to the development and maturation of embryonic vasculature as well as vascular remodeling and permeability in adult tissues. Targeting both this signaling pathway and the major pathway with vascular endothelial growth factor (VEGF) is expected to permit clinical applications, especially in antiangiogenic therapies against tumors. Several drugs targeting the Ang-TIE signaling pathway in cancer patients are under clinical development. Similar to how cancer increases with age, unsuitable angiogenesis or endothelial dysfunction is often seen in other ageing-associated diseases (AADs) such as atherosclerosis, Alzheimer's disease, type 2 diabetes, chronic kidney disease and cardiovascular diseases. Thus, the Ang-TIE pathway is a possible molecular target for AAD therapy. In this review, we focus on the potential role of the Ang-TIE signaling pathway in AADs, especially non-cancer-related AADs. We also suggest translational insights and future clinical applications of this pathway in those AADs.

Clinical efficacy of haematopoietic stem cell transplantation for adult adrenoleukodystrophy.

Accumulated experience supports the efficacy of allogenic haematopoietic stem cell transplantation in arresting the progression of childhood-onset cerebral form of adrenoleukodystrophy in early stages. For adulthood-onset cerebral form of adrenoleukodystrophy, however, there have been only a few reports on haematopoietic stem cell transplantation and the clinical efficacy and safety of that for adulthood-onset cerebral form of adrenoleukodystrophy remain to be established. To evaluate the clinical efficacy and safety of haematopoietic stem cell transplantation, we conducted haematopoietic stem cell transplantation on 12 patients with adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy in a single-institution-based prospective study. Through careful prospective follow-up of 45 male adrenoleukodystrophy patients, we aimed to enrol patients with adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy at early stages. Indications for haematopoietic stem cell transplantation included cerebral form of adrenoleukodystrophy or cerebello-brainstem form of adrenoleukodystrophy with Loes scores up to 13, the presence of progressively enlarging white matter lesions and/or lesions with gadolinium enhancement on brain MRI. Clinical outcomes of haematopoietic stem cell transplantation were evaluated by the survival rate as well as by serial evaluation of clinical rating scale scores and neurological and MRI findings. Clinical courses of eight patients who did not undergo haematopoietic stem cell transplantation were also evaluated for comparison of the survival rate. All the patients who underwent haematopoietic stem cell transplantation survived to date with a median follow-up period of 28.6 months (4.2-125.3 months) without fatality. Neurological findings attributable to cerebral/cerebellar/brainstem lesions became stable or partially improved in all the patients. Gadolinium-enhanced brain lesions disappeared or became obscure within 3.5 months and the white matter lesions of MRI became stable or small. The median Loes scores before haematopoietic stem cell transplantation and at the last follow-up visit were 6.0 and 5.25, respectively. Of the eight patients who did not undergo haematopoietic stem cell transplantation, six patients died 69.1 months (median period; range 16.0-104.1 months) after the onset of the cerebral/cerebellar/brainstem lesions, confirming that the survival probability was significantly higher in patients with haematopoietic stem cell transplantation compared with that in patients without haematopoietic stem cell transplantation (P = 0.0089). The present study showed that haematopoietic stem cell transplantation was conducted safely and arrested the inflammatory demyelination in all the patients with adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy when haematopoietic stem cell transplantation was conducted in the early stages. Further studies are warranted to optimize the procedures of haematopoietic stem cell transplantation for adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy.

Zero-spindle spectral drill: real-time spectral measurement in a fixed Fabry-Pérot cavity.

An optical configuration for Fabry-Pérot cavity scanning using a geometric phase shifter, known as the "spectral drill," is improved to acquire a spectrum in real-time. Previously, the resonance condition of the spectral drill is swept by the mechanical rotation of a phase plate comprising a geometric phase shifter, and the acquisition time is limited. In this work, using a q-plate and a camera instead of phase plate rotation and a photo detector, we remove all the spinning mechanics and increase the acquisition rate by a factor 720. This technique will be applied to locking laser frequency.

CD4+ Resident Memory T Cells Mediate Long-Term Local Skin Immune Memory of Contact Hypersensitivity in BALB/c Mice.

In allergic contact dermatitis (ACD) and contact hypersensitivity (CHS), the healed skin shows greater swelling than the naïve skin in the same individual upon re-exposure to the same hapten. This "local skin memory" (LSM) in healed skin was maintained for a prolonged period of time and mediated by skin CD8+-resident memory T (TRM) cells in C57BL/6 mice. However, the number of CD4+ T cells is elevated in ACD-healed human skin, and the contribution of CD4+ TRM cells to the formation of LSM currently remains unclear. We herein demonstrated that immediately after CHS subsided, the healed skin in BALB/c mice showed an accumulation of hapten-specific CD4+ and CD8+ TRM cells, with a predominance of CD4+ TRM cells. The presence of CD4+ or CD8+ TRM cells in the healed skin was sufficient for the induction of a flare-up reaction upon a re-challenge. The CD4+ and CD8+ TRM cells both produced interferon-γ and tumor necrosis factor early after the re-challenge. Moreover, while CD8+ TRM cells gradually decreased over time and were eventually lost from the healed skin at 40-51 weeks after the resolution of CHS, the CD4+ TRM cell numbers remained elevated during this period. The present results indicate that the long-term maintenance of LSM is mediated by CD4+ TRM cells, and thus CD4+ TRM cells are an important target for the treatment of recurrent human ACD.

Verification of dose distribution in high-dose-rate brachytherapy using a nanoclay-based radio-fluorogenic gel dosimeter.

Dose distributions have become more complex with the introduction of image-guided brachytherapy in high-dose-rate (HDR) brachytherapy treatments. Therefore, to correctly execute HDR, conducting a quality assurance programme for the remote after-loading system and verifying the dose distribution in the patient treatment plan are necessary. The characteristics of the dose distribution of HDR brachytherapy are that the dose is high near the source and rapidly drops when the distance from the source increases. Therefore, a measurement tool corresponding to the characteristic is required. In this study, using an Iridium-192 (Ir-192) source, we evaluated the basic characteristics of a nanoclay-based radio-fluorogenic gel (NC-RFG) dosimeter that is a fluorescent gel dosimeter using dihydrorhodamine 123 hydrochloride as a fluorescent probe. The two-dimensional dose distribution measurements were performed at multiple source positions to simulate a clinical plan. Fluorescence images of the irradiated NC-RFG were obtained at a high resolution (0.04 mm pixel-1) using a gel scanner with excitation at 465 nm. Good linearity was confirmed up to a dose range of 100 Gy without dose rate dependence. The dose distribution measurement at the five-point source position showed good agreement with the treatment planning system calculation. The pass ratio by gamma analysis was 92.1% with a 2%/1 mm criterion. The NC-RFG dosimeter demonstrates to have the potential of being a useful tool for quality assurance of the dose distribution delivered by HDR brachytherapy. Moreover, compared with conventional gel dosimeters such as polymer gel and Fricke gel dosimeters it solves the problems of diffusion, dose rate dependence and inhibition of oxygen-induced reactions. Furthermore, it facilitates dose data to be read in a short time after irradiation, which is useful for clinical use.

Heregulin controls ERα and HER2 signaling in mammospheres of ERα-positive breast cancer cells and interferes with the efficacy of molecular targeted therapy.

Estrogen receptor (ER)α and the human epidermal growth factor receptor (HER) family are inversely expressed in ERα-positive cancer in association with resistance to hormonal therapy, but the mechanism underlying their relationship remains unknown. We analyzed the effect of HER family ligands on the expression of ER and the HER family in ERα-positive MCF-7 and T47D breast cancer cell lines in 3D spheroid culture. Here, we demonstrated for the first time that heregulin-1ß (HRG), a HER3 and HER4 ligand, most effectively regulated ER/HER family expression by decreasing ERα mRNA expression and increasing HER family mRNA expression. HRG treatment attenuated fulvestrant-mediated growth inhibition, and promoted the migration of MCF-7 cells. Moreover, HRG increased the CD44+/CD24- cell fraction and side population cells, both of which are recognized as prospective breast cancer stem cell markers. HRG activated both phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) and mitogen-activated protein kinase (MAPK) pathways. Inhibitors of these pathways reduced the growth of MCF-7 cells, but the addition of HRG has different effects on these pathways. HRG blocked the inhibitory effect of mTOR inhibitors, such as rapamycin and everolimus, on cell growth but not that of a PI3K inhibitor. Furthermore, HRG slightly decreased the inhibitory effect of an AKT inhibitor on cell growth. In contrast, HRG enhanced the MEK inhibitor-induced inhibition of cell growth. These findings suggest that HRG-stimulated signaling pathways allow ERα-positive breast cancer cells to escape from growth inhibition caused by everolimus, via MAPK signaling and/or other signaling pathways. Everolimus improves progression-free survival in combination with exemestane as second-line therapy for metastatic hormone receptor-positive breast cancer. Our study suggests that HRG is a novel target for ERα-positive breast cancer therapy.