Jau-Nan Lee

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 (EC50: 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 β‐catenin‐LEF1/TCF4 transcriptional complex and transactivation of the c‐Myc oncogene. This nongenomic pathway emanated from the phthalateinduced 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.—Hsieh, T.‐H., Tsai, C.‐F., Hsu, C.‐Y., Kuo, P.‐L., Lee, J.‐N., Chai, C.‐Y., Wang, S.‐C., Tsai, E.‐M. Phthalates induce proliferation and invasiveness of estrogen receptor‐negative breast cancer through the AhR/HDAC6/c‐Myc signaling pathway. FASEB J. 26, 778–787 (2012). www.fasebj.org

Comparative study of human eutopic and ectopic endometrial mesenchymal stem cells and the development of an in vivo endometriotic invasion model

OBJECTIVE To elucidate the role of endometrial stem-progenitor cells in the etiology of endometriosis and to develop an animal model to study the invasion ability of endometrial cells. DESIGN Gene expression and cell function studies were designed. SETTING Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. PATIENT(S) Human endometrial mesenchymal stem cells (MSCs) were isolated from 22 different endometrium biopsies after surgery for treatment of endometriosis. INTERVENTION(S) Endometrial MSCs developed from eutopic and ectopic endometrial tissues. MAIN OUTCOME MEASURE(S) Characterization of MSC phenotypes (i.e., differentiation induction and gene expression by flow cytometric analysis); comparative study of cell functions (i.e., cell growth, migration, and invasion assays). The invasion of implants in an animal model was examined by histologic staining. RESULT(S) We compared the characteristics of eutopic and ectopic endometrial MSCs from the same endometrial donor. Although both showed similar mesenchymal cell phenotypes, ectopic endometrial MSCs showed distinctly greater ability of cell migration and invasion. Furthermore, in an in vivo cell invasion model using cells grown in scaffold and transplantation in immune-deficient mice, the ectopic endometrial MSCs were found to form many new blood vessels and to invade surrounding tissue. CONCLUSION(S) These results indicate unique invasion and angiogenesis characteristics of ectopic endometrial MSCs that may underlie the pathogenesis of ectopic endometriosis. The animal invasion model will be useful for future characterization of endometrial MSCs.

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. Copyright

Glabridin, an isoflavan from licorice root, inhibits migration, invasion and angiogenesis of MDA-MB-231 human breast adenocarcinoma cells by inhibiting focal adhesion kinase/Rho signaling pathway

SCOPE In this study we first report the antimigration, antiinvasive effect of glabridin, a flavonoid obtained from licorice, in MDA-MB-231 human breast adenocarcinoma cells. METHODS AND RESULTS Glabridin exhibited effective inhibition of cell metastasis by decreasing cancer cell migration and invasion of MDA-MB-231 cells. In addition, glabridin also blocked human umbilical vein endothelial cells (HUVEC) migration and decreased MDA-MB-231-mediated angiogenesis. Further investigation revealed that the inhibition of cancer angiogenesis by glabridin was also evident in a nude mice model. Blockade of MDA-MB-231 cells and HUVEC migration was associated with an increase of αγβ3 integrin proteosome degradation. Glabridin also decreased the active forms of FAK and Src, and enhanced levels of inactivated phosphorylated Src (Tyr 416), decreasing the interaction of FAK and Src. Inhibition of the FAK/Src complex by glabridin also blocked AKT and ERK1/2 activation, resulting in reduced activation of RhoA as well as myosin light chain phosphorylation. CONCLUSION This study demonstrates that glabridin may be a novel anticancer agent for the treatment of breast cancer in three different ways: inhibition of migration, invasion and angiogenesis.

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

BackgroundCancer 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).ResultsIn 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.ConclusionsHER2 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.

n-Butyl Benzyl Phthalate Promotes Breast Cancer Progression by Inducing Expression of Lymphoid Enhancer Factor 1

Environmental hormones play important roles in regulating the expression of genes involved in cell proliferation, drug resistance, and breast cancer risk; however, their precise role in human breast cancer cells during cancer progression remains unclear. To elucidate the effect of the most widely used industrial phthalate, n-butyl benzyl phthalate (BBP), on cancer progression, we evaluated the results of BBP treatment using a whole human genome cDNA microarray and MetaCore software and selected candidate genes whose expression was changed by more than ten-fold by BBP compared with controls to analyze the signaling pathways in human breast cancer initiating cells (R2d). A total of 473 genes were upregulated, and 468 were downregulated. Most of these genes are involved in proliferation, epithelial-mesenchymal transition, and angiogenesis signaling. BBP induced the viability, invasion and migration, and tube formation in vitro, and Matrigel plug angiogenesis in vivo of R2d and MCF-7. Furthermore, the viability and invasion and migration of these cell lines following BBP treatment was reduced by transfection with a small interfering RNA targeting the mRNA for lymphoid enhancer-binding factor 1; notably, the altered expression of this gene consistently differentiated tumors expressing genes involved in proliferation, epithelial-mesenchymal transition, and angiogenesis. These findings contribute to our understanding of the molecular impact of the environmental hormone BBP and suggest possible strategies for preventing and treating human breast cancer.

Phthalates Stimulate the Epithelial to Mesenchymal TransitionThrough an HDAC6-Dependent Mechanism in Human BreastEpithelial 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 GSK3β inactivation, leading to transcriptional activation of vimentin through the β-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.

Androgen receptor-mediated apoptosis in bovine testicular induced pluripotent stem cells in response to phthalate esters.

The androgen receptor (AR) has a critical role in promoting androgen-dependent and -independent apoptosis in testicular cells. However, the molecular mechanisms that underlie the ligand-independent apoptosis, including the activity of AR in testicular stem cells, are not completely understood. In the present study, we generated induced pluripotent stem cells (iPSCs) from bovine testicular cells by electroporation of octamer-binding transcription factor 4 (OCT4). The cells were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4, which maintained and stabilized the expression of stemness genes and pluripotency. The iPSCs were used to assess the apoptosis activity following exposure to phthalate esters, including di (2-ethyhexyl) phthalates, di (n-butyl) phthalate, and butyl benzyl phthalate. Phthalate esters significantly reduced the expression of AR in iPSCs and induced a higher ratio of BAX/BCL-2, thereby favoring apoptosis. Phthalate esters also increased the expression of cyclin-dependent kinase inhibitor 1 (p21Cip1) in a p53-dependent manner and enhanced the transcriptional activity of p53. The forced expression of AR and knockdown of p21Cip1 led to the rescue of the phthalate-mediated apoptosis. Overall, this study suggests that testicular iPSCs are a useful system for screening the toxicity of environmental disruptors and examining their effect on the maintenance of stemness and pluripotency, as well as for identifying the iPSC signaling pathway(s) that are deregulated by these chemicals.

A functional promoter polymorphism in interleukin-10 gene influences susceptibility to endometriosis.

OBJECTIVE To investigate the involvement of inflammation in the development of endometriosis. DESIGN Case-control study to investigate the association between endometriosis and four inflammation-related genes: interleukin (IL)-6, IL-10, IL-1 beta, and cyclooxygenase-2. SETTING University hospital. PATIENT(S) We had 196 cases with pathologically proved endometriosis and 397 disease-free women as control subjects. INTERVENTION(S) A total of 12 single nucleotide polymorphisms (SNPs) were selected for genotyping, including functional SNPs and common tagging SNPs. MAIN OUTCOME MEASURE(S) Logistic regression and haplotype analyses were performed to evaluate the genetic effect with adjustment for other covariates. RESULT(S) Genotypes at each SNP were in Hardy-Weinberg equilibrium in either case or control subjects, except for rs1800871 at IL-10 in the case subjects (P=.04). We found that the individuals carrying minor allele C of a functional promoter SNP rs1800871 at IL-10 was associated with a reduced risk by approximately twofold compared with the common TT genotype. The T allele was reported to have a lower gene expression level than the C allele, suggesting inadequate suppression of inflammation leading to endometriosis development. Haplotype analysis of the IL-10 gene did not yield a better result. Other genes were not associated with endometriosis. CONCLUSION(S) This study suggests that the functional promoter polymorphism at IL-10 may play a role in the development of endometriosis.

The concentrations of visfatin in the follicular fluids of women undergoing controlled ovarian stimulation are correlated to the number of oocytes retrieved

OBJECTIVE To compare the concentrations of visfatin in the plasma with those in follicular fluid of women undergoing controlled ovarian stimulation and to discover their correlation to the number of oocytes retrieved. Further, to examine whether FSH or hCG affects the expression of visfatin and whether visfatin affects COX-2 expression in cultured granulosa luteal (GL) cells. DESIGN A clinical and in vitro study. SETTING University hospital. PATIENT(S) Women subjected to IVF procedures were enrolled in the study. INTERVENTION(S) Plasma and follicular fluid visfatin levels were analyzed using ELISA. HCG, FSH, PGE2 and visfatin were added to cultured GL cells. MAIN OUTCOME MEASURE(S) Enzyme immunoassay and RT-PCR were performed. RESULT(S) There was no correlation between follicular fluid and plasma visfatin levels (r = 0.443). The number of oocytes retrieved was significantly correlated to follicular visfatin levels in multiple linear regression analysis (r = 0.891, r(2) = 0.794). In vitro experiments on GL cells revealed that hCG and PGE(2) considerably increased visfatin mRNA expression. FSH did not affect visfatin mRNA expression. Treatment with visfatin caused an induction of COX-2 mRNA. CONCLUSIONS The follicular fluid visfatin concentrations are correlated to the number of oocytes retrieved. Human GL cells produce visfatin, and visfatin synthesis is increased by hCG and PGE2 treatment. Visfatin can induce expression of COX-2 mRNA in GL cells.