HDAC inhibitors target HDAC5, upregulate microRNA-125a-5p, and induce apoptosis in breast cancer cells.

Histone deacetylase inhibitors (HDACi) are novel clinical anticancer drugs that inhibit HDAC gene expression and induce cell apoptosis in human cancers. Nevertheless, the detailed mechanism or the downstream HDAC targets by which HDACi mediates apoptosis in human breast cancer cells remains unclear. Here, we show that HDACi reduce tumorigenesis and induce intrinsic apoptosis of human breast cancer cells through the microRNA miR-125a-5p in vivo and in vitro. Intrinsic apoptosis was activated by the caspase 9/3 signaling pathway. In addition, HDACi mediated the expression of miR-125a-5p by activating RUNX3/p300/HDAC5 complex. Subsequently, miR-125a-5p silenced HDAC5 post-transcriptionally in the cells treated with HDACi. Thus, a regulatory loop may exist in human breast cancer cells involving miR-125a-5p and HDAC5 that is controlled by RUNX3 signaling. Silencing of miR-125a-5p and RUNX3 inhibited cancer progression and activated apoptosis, but silencing of HDAC5 had a converse effect. In conclusion, we demonstrate a possible new mechanism by which HDACi influence tumorigenesis and apoptosis via downregulation of miR-125a-5p expression. This study provides clinical implications in cancer chemotherapy using HDACi.

miRNA-199a-5p regulates VEGFA in endometrial mesenchymal stem cells and contributes to the pathogenesis of endometriosis.

It is believed that endometrial miRNAs contribute to the aetiology of endometriosis in stem cells; however, the mechanisms remain unclear. Here we collected serum samples from patients with or without endometriosis and characterized the miRNA expression profiles of these two groups. MicroRNA-199a-5p (miR-199a-5p) was dramatically down-regulated in patients with endometriosis compared with control patients. In addition, we found that the tumour suppressor gene, SMAD4, could elevate miR-199a-5p expression in ectopic endometrial mesenchymal stem cells. Up-regulation of miR-199a-5p suppressed cell proliferation, motility and angiogenesis of these ectopic stem cells by targeting the 3' untranslated region of VEGFA. Furthermore, we established an animal model of endometriosis and found that miR-199a-5p could decrease the size of endometriotic lesions in vivo. Taken together, this newly identified miR-199a-5p module provides a new avenue to the understanding of the processes of endometriosis development, especially proliferation, motility and angiogenesis, and may facilitate the development of potential therapeutics against endometriosis.

Benzyl butyl phthalate induces migration, invasion, and angiogenesis of Huh7 hepatocellular carcinoma cells through nongenomic AhR/G-protein signaling.

BACKGROUND: The widespread use of phthalates as plasticizers has raised public health concerns regarding their adverse effects, including an association with cancer. Although animal investigations have suggested an association between phthalate exposure and hepatocellular carcinoma, the mechanisms are unknown. METHODS: The hepatocellular carcinoma cell line Huh7 was treated with benzyl butyl phthalate (BBP), and then analyzed by total internal reflection fluorescence microscopy, confocal microscopy and double immunogold transmission electron microscopy. Following BBP treatment, mRNA levels were measured by RT-PCR, protein levels were measured using western blot, and vascular endothelial growth factor levels were measured by an enzyme-linked immunosorbent assay. Cell migration and invasion assays were evaluated by transwell, and angiogenesis were performed by a tube formation assay. Nude mice were used to investigate metastasis and angiogenesis in vivo. RESULTS: BBP affected hepatocellular carcinoma progression through the aryl hydrocarbon receptor (AhR) and that benzyl butyl phthalate (BBP) stimulated AhR at the cell surface, which then interacted with G proteins and triggered a downstream signaling cascade. BBP activated AhR through a nongenomic action involving G-protein signaling rather than the classical genomic AhR action. BBP treatment promoted cell migration and invasion in vitro and metastasis in vivo via the AhR/Gß/PI3K/Akt/NF-κB pathway. In addition, BBP induced both in vitro and in vivo angiogenesis through the AhR/ERK/VEGF pathway. CONCLUSIONS: These findings suggest a novel nongenomic AhR mechanism involving G-protein signaling induced by phthalates, which contributes to tumor progression of hepatocellular carcinoma.

Phthalates induce proliferation and invasiveness of estrogen receptor-negative breast cancer through the AhR/HDAC6/c-Myc signaling pathway.

The environmentally present group of chemical phthalates, or phthalate esters, has been recognized as a rising threat to public health, including cancer. While most studies have addressed the estrogenic effects of phthalates in malignancies of the breast and the prostate, little is known about their role in the etiology of hormone-independent cancer. Here we show that treatments with the phthalates n-butyl benzyl phthalate (BBP) and dibutyl phthalate (DBP) at 1 µM induced proliferation (BBP, 3.2-fold; DBP, 3.2-fold), migration (BBP, 2.6-fold; DBP, 2.6-fold), invasion (BBP, 2.7-fold; DBP, 3.1-fold), and tumor formation (EC(50): BBP, 0.12 µM; DBP, 0.22 µM) in estrogen receptor (ER)-negative breast cancer cells (MDA-MB-231). We further demonstrate that phthalates stimulated the cell surface aryl hydrocarbon receptor (AhR) and triggered the downstream cyclic AMP (cAMP)-PKA-CREB1 signaling cascade. The pathway led to increased expression of HDAC6, which facilitated nuclear assembly of the ß-catenin-LEF1/TCF4 transcriptional complex and transactivation of the c-Myc oncogene. This nongenomic pathway emanated from the phthalate-induced AhR promoted tumorigenesis of ER-negative breast cancer. Collectively, our findings revealed a novel oncogenic mechanism of phthalates in breast cancer independent from their estrogenic activities.

Modulation of tumorigenesis and oestrogen receptor-alpha expression by cell culture conditions in a stem cell-derived breast epithelial cell line.

BACKGROUND INFORMATION: The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ER alpha (oestrogen receptor alpha) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T-antigen transfection and X-ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. RESULTS: The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone-deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and alpha 6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44+/high/CD24-/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial-to-mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX-2 (cyclo-oxygenase-2) through a CD44-mediated mechanism. CONCLUSION: An oestrogen-responsive cell line with ER alpha and CD44+/CD24-/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ER alpha-positive breast cancers, (2) CD44+/CD24-/low breast tumour stem cells and (3) the metastatic ability of breast cancer.

Increasing CD44+/CD24(-) tumor stem cells, and upregulation of COX-2 and HDAC6, as major functions of HER2 in breast tumorigenesis.

BACKGROUND: Cancer cells are believed to arise primarily from stem cells. CD44+/CD24(-) have been identified as markers for human breast cancer stem cells. Although, HER2 is a well known breast cancer oncogene, the mechanisms of action of this gene are not completely understood. Previously, we have derived immortal (M13SV1), weakly tumorigenic (M13SV1R2) and highly tumorigenic (M13SV1R2N1) cell lines from a breast epithelial cell type with stem cell phenotypes after successive SV40 large T-antigen transfection, X-ray irradiation and ectopic expression of HER2/C-erbB2/neu. Recently, we found that M13SV1R2 cells became non-tumorigenic after growing in a growth factor/hormone-deprived medium (R2d cells). RESULTS: In this study, we developed M13SV1R2N1 under the same growth factor/hormone-deprived condition (R2N1d cells). This provides an opportunity to analyze HER2 effect on gene expression associated with tumorigenesis by comparative study of R2d and R2N1d cells with homogeneous genetic background except HER2 expression. The results reveal distinct characters of R2N1d cells that can be ascribed to HER2: 1) development of fast-growing tumors; 2) high frequency of CD44+/CD24(-) cells (~50% for R2N1d vs. ~10% for R2d); 3) enhanced expression of COX-2, HDAC6 mediated, respectively, by MAPK and PI3K/Akt pathways, and many genes associated with inflammation, metastasis, and angiogenesis. Furthermore, HER2 expression can be down regulated in non-adhering R2N1d cells. These cells showed longer latent period and lower rate of tumor development compared with adhering cells. CONCLUSIONS: HER2 may induce breast cancer by increasing the frequency of tumor stem cells and upregulating the expression of COX-2 and HDAC6 that play pivotal roles in tumor progression.

In vitro effects of arecoline on sperm motility and cyclooxygenase-2 expression.

Semen samples were obtained from 30 volunteers who had never consumed betel quid. Swim-up spermatozoa from the 30 seminal samples of non-betel quid chewers and also non-smokers, usually not exposed to passive smoking, were treated in vitro with arecoline at different concentrations to evaluate the action of these drugs on sperm motility. Highly motile sperms were collected and divided into 5 equal fractions. Four fractions were supplemented with various concentrations of arecoline and one as control. The study was carried out at time 0 and +1, +2, +3 and +4 hr of incubation. Sperm cells were also extracted and blotted with COX-2 antibody after arecoline treatment after 4 hr incubation. The sperm motility parameters, i.e., motility, average path velocity, curvilinear velocity, straight-line velocity and linearity, were significantly decreased after arecoline treatment. In vitro, arecoline induces the COX-2 expression of sperm cells in a dose-dependent manner. This is the first report to demonstrate that arecoline may mediate COX-2 expression in human sperms, resulting in inflammation response. This situation may act on the structure responsible for the flagellar motion and cause the reduction of sperm motility.

Benzyl butyl phthalate promotes breast cancer stem cell expansion via SPHK1/S1P/S1PR3 signaling.

Understanding the regulatory mechanisms unique to breast cancer stem cells (BCSCs) is required to control breast cancer metastasis. We found that phthalates promote BCSCs in human breast cancer cell cultures and xenograft tumors. A toxic phthalate, benzyl butyl phthalate (BBP), activated aryl hydrocarbon receptor in breast cancer cells to stimulate sphingosine kinase 1 (SPHK1)/sphingosine 1-phosphate (S1P)/sphingosine-1-phosphate receptor 3 (S1PR3) signaling and enhance formation of metastasis-initiating BCSCs. BBP induced histone modifications in S1PR3 in side population (SP) cells, but not in non-SP cells. SPHK1 or S1PR3 knockdown in breast cancer cells effectively reduced tumor growth and lung metastasis in vivo. Our findings suggest S1PR3 is a determinant of phthalate-driven breast cancer metastasis and a possible therapeutic target for regulating BCSC populations. Furthermore, the association between breast carcinogenesis and environmental pollutants has important implications for public health.

Atrial natriuretic peptide inhibits ovarian functions in female mice.

OBJECTIVE: To investigate the effects of atrial natriuretic peptide (ANP) on ovarian regulation in mice through intraperitoneal administration. STUDY DESIGN: Forty-two ICR strain female mice were divided into seven groups, including one control. Each mouse received ovarian hyperstimulation. ANP was prepared with saline at concentrations of 0 (as control), 2, 4, 8, 10, 20, and 30 ng/g of body weight. The solution was intraperitoneally applied to each referred group (n = 6) 1 h before the hMG and hCG injections. The mice were sacrificed next morning and blood samples were collected for analysis. RESULTS: A significant reduction in both ovarian weight and the number of oocytes was found as the ANP dosage increased. ANP treatment decreased both serum estradiol and progesterone levels. The pituitary FSH and LH contents elevated significantly by ANP at a dosage larger than 4 ng/g of body weight; however, both serum FSH and LH levels remained unchanged. Histologically, there were remarkable ovarian inhibitory features, i.e. reduction in size and number of follicles, arrest of follicular growth and diminution of ovulation. CONCLUSION: Intraperitoneal administration of ANP inhibits the follicular growth, ovulation and steroidogenesis in female mice. These effects are attributed to the direct action of ANP on ovaries.

Curcumin Suppresses Phthalate-Induced Metastasis and the Proportion of Cancer Stem Cell (CSC)-like Cells via the Inhibition of AhR/ERK/SK1 Signaling in Hepatocellular Carcinoma.

Recent evidence indicating that phthalates promote cancer development, including cell proliferation, migration, and invasion, has raised public health concerns. Here, we show that bis(2-ethylhexyl) phthalate promotes the migration, invasion, and epithelial-mesenchymal transition of hepatocellular carcinoma cells. In addition, bis(2-ethylhexyl) phthalate increased the proportion of cancer stem cell (CSC)-like cells and stemness maintenance in vitro as well as tumor growth and metastasis in vivo. The various activities of curcumin, including anticancer, anti-inflammation, antioxidation, and immunomodulation, have been investigated extensively. Curcumin suppressed phthalate-induced cell migration, invasion, and epithelial-mesenchymal transition, decreased the proportion of CSC-like cells in hepatocellular carcinoma cell lines in vitro, and inhibited tumor growth and metastasis in vivo. We also reveal that curcumin suppressed phthalate-induced migration, invasion, and CSC-like cell maintenance through inhibition of the aryl hydrocarbon receptor/ERK/SK1/S1P3 signaling pathway. Our results suggest that curcumin may be a potential antidote for phthalate-induced cancer progression.

Dynamic Trk and G Protein Signalings Regulate Dopaminergic Neurodifferentiation in Human Trophoblast Stem Cells.

Understanding the mechanisms in the generation of neural stem cells from pluripotent stem cells is a fundamental step towards successful management of neurodegenerative diseases in translational medicine. Albeit all-trans retinoic acid (RA) has been associated with axon outgrowth and nerve regeneration, the maintenance of differentiated neurons, the association with degenerative disease like Parkinson's disease, and its regulatory molecular mechanism from pluripotent stem cells to neural stem cells remain fragmented. We have previously reported that RA is capable of differentiation of human trophoblast stem cells to dopamine (DA) committed progenitor cells. Intracranial implantation of such neural progenitor cells into the 6-OHDA-lesioned substantia nigra pars compacta successfully regenerates dopaminergic neurons and integrity of the nigrostriatal pathway, ameliorating the behavioral deficits in the Parkinson's disease rat model. Here, we demonstrated a dynamic molecular network in systematic analysis by addressing spatiotemporal molecular expression, intracellular protein-protein interaction and inhibition, imaging study, and genetic expression to explore the regulatory mechanisms of RA induction in the differentiation of human trophoblast stem cells to DA committed progenitor cells. We focused on the tyrosine receptor kinase (Trk), G proteins, canonical Wnt2B/ß-catenin, genomic and non-genomic RA signaling transductions with Tyrosine hydroxylase (TH) gene expression as the differentiation endpoint. We found that at the early stage, integration of TrkA and G protein signalings aims for axonogenesis and morphogenesis, involving the novel RXRα/Gαq/11 and RARß/Gß signaling pathways. While at the later stage, five distinct signaling pathways together with epigenetic histone modifications emerged to regulate expression of TH, a precursor of dopamine. RA induction generated DA committed progenitor cells in one day. Our results provided substantial mechanistic evidence that human trophoblast stem cell-derived neural stem cells can potentially be used for neurobiological study, drug discovery, and as an alternative source of cell-based therapy in neurodegenerative diseases like Parkinson's disease.

miR-125a-5p is a prognostic biomarker that targets HDAC4 to suppress breast tumorigenesis.

Identifying stably expressed tumor markers that can be used easily to detect cancer is currently an important area of cancer research. By using miRNA microarray, we identified 20 differentially expressed miRNAs in serum samples of breast cancer patients. Expression of miR-125a-5p was relatively lower in patients with shorter survival compared to long-term survivors. In a cohort of breast cancer patients (N = 300), serum expression of miR-125a-5p was negatively and significantly correlated with tumor grade (P = 0.004), lymph-node status (P = 0.004), and tumor size (P < 0.001). Low miR-125a-5p expression was an independent prognostic marker (OR = 0.421; 95% CI = 0.184 to 0.961; P = 0.04) associated with poor survival rates (P = 0.0062). We show that miR-125a-5p directly inhibits expression of the HDAC4 gene, resulting in tumor suppression in vitro and in vivo. Together these results demonstrate that serum miR-125a-5p level in breast cancer may be a useful prognostic biomarker and offer a novel therapeutic avenue by targeting HDAC4 in breast cancer.

An unexpected quadruplet heterotopic pregnancy after bilateral salpingectomy and replacement of three embryos.

OBJECTIVE: To report a case of combined intrauterine and interstitial twin pregnancies after bilateral salpingectomy and IVF with replacement of three embryos. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 31-year-old woman known to have bilateral salpingectomy for ectopic pregnancies who underwent IVF. INTERVENTION(S): Laparotomy. MAIN OUTCOME MEASURE(S): Postoperation intrauterine monozygotic twins survival and birth. RESULT(S): After removing the interstitial monozygotic twin pregnancy, the patient had an uneventful postoperative course and delivered two healthy girls by cesarean section at 38 weeks' gestation. CONCLUSION(S): Heterotopic pregnancy can still occur in women treated by IVF after bilateral salpingectomy. The early sonography follow-up of IVF pregnancy would be of value because of the reported higher incidence of pathological pregnancies and especially monozygotic twinning.

Chronic administration of atrial natriuretic peptide reduces testosterone production of testes in mice.

The purpose of the present study was to examine the effect of the long-term administration of human atrial natriuretic peptide (ANP) on testosterone production in male mice. Twenty-five mice received ANP (20 ng/hour/g body weight) for 7 days via mini-osmotic pump, and the other group (n = 25) received twice-daily intraperitoneal injections. After death, levels of follicle-stimulating hormone, luteinizing hormone (LH), and testosterone in plasma, pituitary gland, and testis were measured by radioimmunoassay. Five mice from each group were examined histologically. In the minipump group, pituitary and plasma levels were significantly higher than those in the control group (771.2 +/- 43.6 vs 644.8 +/- 24.9 ng/mg and 6.7 +/- 0.6 ng/mg vs 2.5 +/- 0.6 ng/mL, respectively). In the intraperitoneal group, plasma LH levels were significantly higher in the ANP-treated group than that in control mice (9.6 +/- 0.3 ng/mg vs 3.8 +/- 0.5 ng/mL), whereas pituitary levels did not differ significantly. In both studies, testicular and plasma testosterone levels were significantly lower than those in control mice (P <.02). Histological features of the testes in ANP-treated mice revealed structural disorganization and inhibition of spermatogenesis. We conclude that the chronic administration of ANP may result in reduced testosterone production due to testicular damage.

Differential expression of manganese containing superoxide dismutase in patients with breast cancer in Taiwan.

This study used Western blot analysis to measure the expression of superoxide dismutases (Mn-SOD and Cu/Zn-SOD) in breast cancer tissues from 57 patients. Mn-SOD expression in the breast cancer tissues averaged 1.5-fold higher than in the adjacent tumor-free tissues (p <0.05). There was no significant difference in Cu/Zn-SOD expression between neoplastic and tumor-free breast tissues. This study shows a significant increase of Mn-SOD expression in breast cancer tissues. The authors speculate that up-regulation of Mn-SOD expression induced by oxidative stress or local inflammation may contribute a selective growth advantage to tumor cells compared to their normal counterparts.

Medical abortion with mifepristone and misoprostol: a clinical trial in Taiwanese women.

BACKGROUND AND PURPOSE: Medical abortion was not officially approved in Taiwan until the end of 2001. We investigated the efficacy of combination mifepristone and misoprostol therapy for medical abortion (which has now been approved) in early pregnant Taiwanese women and whether the attitudes of women who received this treatment affected the clinical outcome of medical abortion. METHODS: Eighty healthy women in early pregnancy (< 49 d of gestation) were enrolled into two studies of medical abortion using mifepristone and misoprostol regimens. The outcomes were evaluated based on complete expulsion of intrauterine contents, with or without surgical intervention. Study 1 used treatment with mifepristone (200 mg or 600 mg) and misoprostol (400 micrograms), and the decision to perform surgical intervention was made mainly on the basis of the patient's request. Study 2 used treatment with mifepristone (200 mg or 600 mg) and misoprostol (600 micrograms) where the decision to perform surgical intervention was made exclusively by the physician. Serum or urinary human chorionic gonadotropin (hCG) concentration was measured serially after abortion. RESULTS: In general, the success rate was 95% as judged by complete expulsion of intrauterine contents without surgical intervention. However, the success rate in Study 1 was only 62.5%. The mean duration of bleeding after abortion was 16.7 to 21.7 days. Serum or urinary hCG concentration remained positive in one woman (1.2%) studied during 43 to 60 days after abortion. CONCLUSION: A combination of mifepristone and misoprostol for medical abortion in Taiwanese women during early pregnancy can achieve a high success rate. Our study showed that a mifepristone dose of 200 mg and a misoprostol dose of 400 micrograms were most effective. Our results suggest that sufficient physician and patient communication regarding medical abortion affects the clinical outcome.

Multilobular cyst as endosalpingiosis of uterine serosa: a case report.

A case of endosalpingiosis presented as a multilobular cyst on sonography. The tentative clinical diagnosis was an ovarian tumor; however, laparotomy revealed a degenerative cyst of the uterine myoma with a stalk connecting to the uterus. Histopathologically, it showed characteristics of endosalpingiosis. To our knowledge, such a multilobular cyst of endosalpingiosis originating solely from the uterine serosa has not been reported.

Early diagnosis of fetal sacrococcygeal teratoma: a case report.

Sacrococcygeal teratoma is a rare fetal neoplasm with an incidence of 1 in 40,000 births. Antenatal diagnosis is usually made after 22 weeks of gestation. Fetuses with this malformation are at risk of significant perinatal morbidity and mortality. Malignant components, coexisting with life-threatening anomalies, and chromosomal abnormalities are rare. Postulated causes of perinatal death include hydrops, dystocia, tumor rupture, preterm labor secondary to polyhydramnios, and anemia due either to hemorrhage or hemolysis within the tumor. Herein, we present a case of fetal sacrococcygeal teratoma diagnosed as early as 17 weeks of gestation.

Ectopic pregnancy-derived human trophoblastic stem cells regenerate dopaminergic nigrostriatal pathway to treat parkinsonian rats.

BACKGROUND: Stem cell therapy is a potential strategy to treat patients with Parkinson's disease (PD); however, several practical limitations remain. As such, finding the appropriate stem cell remains the primary issue in regenerative medicine today. We isolated a pre-placental pluripotent stem cell from the chorionic villi of women with early tubal ectopic pregnancies. Our objectives in this study were (i) to identify the characteristics of hTS cells as a potential cell source for therapy; and (ii) to test if hTS cells can be used as a potential therapeutic strategy for PD. METHODS AND FINDINGS: hTS cells expressed gene markers of both the trophectoderm (TE) and the inner cell mass (ICM). hTS cells exhibited genetic and biological characteristics similar to that of hES cells, yet genetically distinct from placenta-derived mesenchymal stem cells. All-trans retinoic acid (RA) efficiently induced hTS cells into trophoblast neural stem cells (tNSCs) in 1-day. Overexpression of transcription factor Nanog was possibly achieved through a RA-induced non-genomic c-Src/Stat3/Nanog signaling pathway mediated by the subcellular c-Src mRNA localization for the maintenance of pluripotency in tNSCs. tNSC transplantation into the lesioned striatum of acute and chronic PD rats not only improved behavioral deficits but also regenerated dopaminergic neurons in the nigrostriatal pathway, evidenced by immunofluorescent and immunohistological analyses at 18-weeks. Furthermore, tNSCs showed immunological advantages for the application in regenerative medicine. CONCLUSIONS: We successfully isolated and characterized the unique ectopic pregnancy-derived hTS cells. hTS cells are pluripotent stem cells that can be efficiently induced to tNSCs with positive results in PD rat models. Our data suggest that the hTS cell is a dynamic stem cell platform that is potentially suitable for use in disease models, drug discovery, and cell therapy such as PD.

Phthalates stimulate the epithelial to mesenchymal transition through an HDAC6-dependent mechanism in human breast epithelial stem cells.

Phthalates are environmental hormone-like molecules that are associated with breast cancer risk and are involved in metastasis, a process that requires the epithelial-mesenchymal transition (EMT). However, few studies have addressed the potential effects of phthalates on stem cells. Here we tested the hypothesis that phthalates such as butyl benzyl phthalate and di-n-butyl phthalate induce EMT in R2d cells, a stem cell-derived human breast epithelial cell line that is responsive to estradiol for tumor development. We observed that phthalates induced EMT as evidenced by morphological changes concomitant with increased expression of mesenchymal markers and decreased expression of epithelial markers. Molecular mechanism studies revealed that histone deacetylase 6 (HDAC6) is required for phthalate-induced cell migration and invasion during EMT in vitro and metastasis into the lungs of nude mice. We also constructed a series of mutant HDAC6 promoter fragments and found that the transcription factor AP-2a plays a novel role in regulating the HDAC6 promoter. Furthermore, phthalates stimulated estrogen receptors and triggered the downstream EGFR-PKA signaling cascade, leading to increased expression of AP-2a in the nucleus. We also observed that phthalates increased expression of the PP1/HDAC6 complex and caused Akt activation and GSK3ß inactivation, leading to transcriptional activation of vimentin through the ß-catenin-TCF-4/LEF1 pathway. Understanding the signaling cascades of phthalates that activate EMT through HDAC6 in breast epithelial stem cells provides the identification of novel therapeutic target for human breast cancer.