A system for detecting high impact-low frequency mutations in primary tumors and metastases.

Tumor complexity and intratumor heterogeneity contribute to subclonal diversity. Despite advances in next-generation sequencing (NGS) and bioinformatics, detecting rare mutations in primary tumors and metastases contributing to subclonal diversity is a challenge for precision genomics. Here, in order to identify rare mutations, we adapted a recently described epithelial reprograming assay for short-term propagation of epithelial cells from primary and metastatic tumors. Using this approach, we expanded minor clones and obtained epithelial cell-specific DNA/RNA for quantitative NGS analysis. Comparative Ampliseq Comprehensive Cancer Panel sequence analyses were performed on DNA from unprocessed breast tumor and tumor cells propagated from the same tumor. We identified previously uncharacterized mutations present only in the cultured tumor cells, a subset of which has been reported in brain metastatic but not primary breast tumors. In addition, whole-genome sequencing identified mutations enriched in liver metastases of various cancers, including Notch pathway mutations/chromosomal inversions in 5/5 liver metastases, irrespective of cancer types. Mutations/rearrangements in FHIT, involved in purine metabolism, were detected in 4/5 liver metastases, and the same four liver metastases shared mutations in 32 genes, including mutations of different HLA-DR family members affecting OX40 signaling pathway, which could impact the immune response to metastatic cells. Pathway analyses of all mutated genes in liver metastases showed aberrant tumor necrosis factor and transforming growth factor signaling in metastatic cells. Epigenetic regulators including KMT2C/MLL3 and ARID1B, which are mutated in >50% of hepatocellular carcinomas, were also mutated in liver metastases. Thus, irrespective of cancer types, organ-specific metastases may share common genomic aberrations. Since recent studies show independent evolution of primary tumors and metastases and in most cases mutation burden is higher in metastases than primary tumors, the method described here may allow early detection of subclonal somatic alterations associated with metastatic progression and potentially identify therapeutically actionable, metastasis-specific genomic aberrations.

NF-κB-HOTAIR axis links DNA damage response, chemoresistance and cellular senescence in ovarian cancer.

The transcription factor nuclear factor kappa B (NF-κB) and the long non-coding RNA (lncRNA) HOTAIR (HOX transcript antisense RNA) have diverse functional roles in cancer. In this study, we show that upregulation of HOTAIR induced platinum resistance in ovarian cancer, and increased HOTAIR levels were observed in recurrent platinum-resistant ovarian tumors vs primary ovarian tumors. To investigate the role of HOTAIR during DNA damage induced by platinum, we monitored double-strand breaks and show that HOTAIR expression results in sustained activation of DNA damage response (DDR) after platinum treatment. We demonstrate that ectopic expression of HOTAIR induces NF-κB activation during DDR and interleukin-6 and interleukin-6 expression, both key NF-κB target genes. We show that HOTAIR regulates activation of NF-κB by decreasing Iκ-Bα (NF-κB inhibitor) and establish that by inducing prolonged NF-κB activation and expression of NF-κB target genes during DNA damage, HOTAIR has a critical role in cellular senescence and platinum sensitivity. Our findings suggest that an NF-κB-HOTAIR axis drives a positive-feedback loop cascade during DDR and contributes to cellular senescence and chemotherapy resistance in ovarian and other cancers.

Regulation of triple-negative breast cancer cell metastasis by the tumor-suppressor liver kinase B1.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), has been identified as a tumor suppressor in many cancers including breast. Low LKB1 expression has been associated with poor prognosis of breast cancer patients, and we report here a significant association between loss of LKB1 expression and reduced patient survival specifically in the basal subtype of breast cancer. Owing to the aggressive nature of the basal subtype as evidenced by high incidences of metastasis, the purpose of this study was to determine if LKB1 expression could regulate the invasive and metastatic properties of this specific breast cancer subtype. Induction of LKB1 expression in basal-like breast cancer (BLBC)/triple-negative breast cancer cell lines, MDA-MB-231 and BT-549, inhibited invasiveness in vitro and lung metastatic burden in an orthotopic xenograft model. Further analysis of BLBC cells overexpressing LKB1 by unbiased whole transcriptomics (RNA-sequencing) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). In addition, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating reversal of the EMT phenotype in the MDA-MB-231 cells. We further demonstrated marked inhibition of matrix metalloproteinase 1 expression and activity via regulation of c-Jun through inhibition of p38 signaling in LKB1-expressing cells. Taken together, these data support future development of LKB1 inducing therapeutics for the suppression of invasion and metastasis of BLBC.

Epithelial ovarian cancer experimental models.

Epithelial ovarian cancer (OvCa) is associated with high mortality and, as the majority (>75%) of women with OvCa have metastatic disease at the time of diagnosis, rates of survival have not changed appreciably over 30 years. A mechanistic understanding of OvCa initiation and progression is hindered by the complexity of genetic and/or environmental initiating events and lack of clarity regarding the cell(s) or tissue(s) of origin. Metastasis of OvCa involves direct extension or exfoliation of cells and cellular aggregates into the peritoneal cavity, survival of matrix-detached cells in a complex ascites fluid phase and subsequent adhesion to the mesothelium lining covering abdominal organs to establish secondary lesions containing host stromal and inflammatory components. Development of experimental models to recapitulate this unique mechanism of metastasis presents a remarkable scientific challenge, and many approaches used to study other solid tumors (for example, lung, colon and breast) are not transferable to OvCa research given the distinct metastasis pattern and unique tumor microenvironment (TME). This review will discuss recent progress in the development and refinement of experimental models to study OvCa. Novel cellular, three-dimensional organotypic, and ex vivo models are considered and the current in vivo models summarized. The review critically evaluates currently available genetic mouse models of OvCa, the emergence of xenopatients and the utility of the hen model to study OvCa prevention, tumorigenesis, metastasis and chemoresistance. As these new approaches more accurately recapitulate the complex TME, it is predicted that new opportunities for enhanced understanding of disease progression, metastasis and therapeutic response will emerge.

CpG island shore methylation regulates caveolin-1 expression in breast cancer.

Caveolin-1 (Cav1) is an integral membrane, scaffolding protein found in plasma membrane invaginations (caveolae). Cav1 regulates multiple cancer-associated processes. In breast cancer, a tumor suppressive role for Cav1 has been suggested; however, Cav1 is frequently overexpressed in aggressive breast cancer subtypes, suggesting an oncogenic function in advanced-stage disease. To further delineate Cav1 function in breast cancer progression, we evaluated its expression levels among a panel of cell lines representing a spectrum of breast cancer phenotypes. In basal-like (the most aggressive BC subtype) breast cancer cells, Cav1 was consistently upregulated, and positively correlated with increased cell proliferation, anchorage-independent growth, and migration and invasion. To identify mechanisms of Cav1 gene regulation, we compared DNA methylation levels within promoter 'CpG islands' (CGIs) with 'CGI shores', recently described regions that flank CGIs with less CG-density. Integration of genome-wide DNA methylation profiles ('methylomes') with Cav1 expression in 30 breast cancer cell lines showed that differential methylation of CGI shores, but not CGIs, significantly regulated Cav1 expression. In breast cancer cell lines having low Cav1 expression (despite promoter CGI hypomethylation), we found that treatment with a DNA methyltransferase inhibitor induced Cav1 expression via CGI shore demethylation. In addition, further methylome assessments revealed that breast cancer aggressiveness associated with Cav1 CGI shore methylation levels, with shore hypermethylation in minimally aggressive, luminal breast cancer cells and shore hypomethylation in highly aggressive, basal-like cells. Cav1 CGI shore methylation was also observed in human breast tumors, and overall survival rates of breast cancer patients lacking estrogen receptor α (ERα) negatively correlated with Cav1 expression. Based on this first study of Cav1 (a potential oncogene) CGI shore methylation, we suggest this phenomenon may represent a new prognostic marker for ERα-negative, basal-like breast cancer.

MicroRNA-221/222 confers breast cancer fulvestrant resistance by regulating multiple signaling pathways.

Fulvestrant is a selective estrogen receptor downregulator (SERD) and highly effective antagonist to hormone-sensitive breast cancers following failure of previous tamoxifen or aromatase inhibitor therapies. However, after prolonged fulvestrant therapy, acquired resistance eventually occurs in the majority of breast cancer patients, due to poorly understood mechanisms. To examine a possible role(s) of aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant and -sensitive breast cancer cells, revealing the overexpression of miR-221/222 in the SERD-resistant cell lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in estrogen receptor-α (ERα)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death, thus also conferring hormone-independent growth and fulvestrant resistance. In cells with acquired resistance to fulvestrant, miR-221/222 expression was essential for cell growth and cell cycle progression. To identify possible miR-221/222 targets, miR-221- or miR-222- induced alterations in global gene expression profiles and target gene expression at distinct time points were determined, revealing that miR-221/222 overexpression resulted in deregulation of multiple oncogenic signaling pathways previously associated with drug resistance. Activation of ß-catenin by miR-221/222 contributed to estrogen-independent growth and fulvestrant resistance, whereas TGF-ß-mediated growth inhibition was repressed by the two miRNAs. This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance, a clinically important problem, demonstrates that these two 'oncomirs' may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.

A conditionally replicative, Wnt/beta-catenin pathway-based adenovirus therapy for anaplastic thyroid cancer.

Thyroid cancer affects between 10,000 and 15,000 people per year in the US. Typically, this disease can be controlled with surgical resection and radioiodide treatment. However, resistance to these conventional therapies is observed in some patients, who develop intractable anaplastic thyroid cancer (ATC), for which no effective therapies exist. Recently, a sizable fraction of undifferentiated or poorly differentiated thyroid cancers were shown to contain mutations in beta-catenin, an oncogenic protein involved in the etiology of cancers of many tissues. We developed a conditionally replicative adenovirus (named 'HILMI') which, by virtue of TCF response elements drives E1A and E1B expression, replicates specifically in cells with an active Wnt/beta-catenin pathway. We show that several thyroid cancer cell lines, derived from undifferentiated or anaplastic tissues and possessing an active Wnt/beta-catenin pathway, are susceptible to cell killing by HILMI. Furthermore, viral replication in ATC cells as xenograft tumors in nude mice was observed, and prolonged survival of mice with ATC tumors was observed following administration of the HILMI therapeutic vector. The results warrant further development of this therapeutic approach for ATC patients.

DNA methylation and ovarian cancer. I. Analysis of CpG island hypermethylation in human ovarian cancer using differential methylation hybridization.

OBJECTIVE: The aim of this study was to examine CpG island methylation patterns in ovarian cancer and determine whether epigenetic information can be related to clinical data of patients. CpG island (CpGI) hypermethylation is commonly associated with cancer progression, but little is currently known about the role of methylation in ovarian cancer. METHODS: Differential methylation hybridization (DMH) analysis at 742 loci was performed to determine methylation signatures for 20 primary epithelial ovarian carcinomas (Stages II, III, and IV adenocarcinomas, serous papillary), 6 ovarian cancer cell lines, and normal ovarian surface epithelial cells. RESULTS: Between 23 and 108 methylated CpGIs were seen in the ovarian carcinomas. Fewer (P < 0.05) methylated CpGIs were observed in the ovarian cancer cell lines; however, a number of CpGIs were commonly hypermethylated in both the cell lines and the tumor samples. A methylation signature, consisting of frequently (P < 0.05) methylated CpGIs, was determined for the samples. The observed pattern of methylation in ovarian cancers included several (11) CpGI tags that were previously reported to be hypermethylated in human breast cancer. CONCLUSIONS: Epigenetic signatures in ovarian cancer were determined using DMH. This proof-of-concept study lays the foundation for genome-wide screening of methylation to examine epigenotype-phenotype relationships in ovarian cancer.

DNA methylation in ovarian cancer. II. Expression of DNA methyltransferases in ovarian cancer cell lines and normal ovarian epithelial cells.

OBJECTIVE: The aim of this study was to investigate whether expression of the enzymes that catalyze cytosine CpG island methylation, DNA methyltransferases, DNMT1, DNMT3a, and DNMT3b is altered in human ovarian cancer. Aberrations in DNA methylation are common in cancer and have important roles in tumor initiation and progression. Tumors that display frequent and concurrent inactivation of multiple genes by methylation are designated as having a CpG Island methylator phenotype, or CIMP. To date, colon, gastric, and most recently ovarian cancers meet the CIMP criteria for cancer. We hypothesized that altered expression of DNA methyltransferases can result in hypermethylation events seen in CIMP cancers. METHODS: DNMT1, DNMT3a, and DNMT3b mRNA levels in eight ovarian cancer cells lines (Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5) were compared to DNMT expression in normal ovarian surface epithelial cells using semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: In HeyA8 and HeyC2 ovarian cancer cells, DNMT1 expression levels were up to threefold higher (P < 0.05) than in normal ovarian surface epithelial cells. SK-OV-3 and PA-1 displayed increased DNMT3b expression (P < 0.05) compared to normal ovarian surface epithelial cells. Transcript levels for DNMT3a, however, were similar in cancer and normal ovarian cells. CONCLUSIONS: We observed differential expression of the DNMT genes in some ovarian cancer cell lines and conclude that alterations in DNMT expression might contribute to the CIMP phenotype in ovarian cancer. However, based on the lack of aberrant DNMT expression in some of the cancer cell lines examined, we further suggest that another mechanism(s), in addition to DNMT overexpression, accounts for methylation anomalies commonly observed in ovarian cancer.

Evidence for expression of estrogen receptor cofactor messenger ribonucleic acid in the ovary and uterus of domesticated animals (sheep, cow and pig).

Expression levels of estrogen receptor cofactors (coactivators or corepressors) in specific tissue compartments and cells are thought to influence the expression of estrogen responsive genes and thereby influence overall hormonal responsiveness of target tissues. To date, the presence of cofactors has been reported in tissues from humans, rats and mice. We analyzed the presence and distribution of messenger ribonucleic acids (mRNAs) encoding several transcriptional cofactors in the ovary and uterus of three domestic animal species, the sheep, cow and pig. Northern analysis for cofactors SRC-1, GRIP1, RAC3, p300, RIP140, and SPA showed expression in ovaries from all three species. In addition, lower expression of SRC-1, GRIP1, RAC3, p300, and RIP140 mRNAs was observed during the luteal phase (day 10-12 of the estrous cycle) than at estrus (day 0); however, SPA transcript levels remained unchanged. We then examined expression of mRNAs for changing (SRC-1, RIP140) and constitutively expressed (SPA) cofactors in ovine ovaries. SRC-1 and RIP140 transcripts in corpus luteum were lower compared to the surrounding ovarian tissue. SPA mRNA expression, however, was similar in corpus luteum and surrounding tissues. To determine which ovarian cell types express SRC-1, RIP140, and SPA, in situ hybridization was performed on sheep ovaries. Silver grains corresponding to these cofactors were seen in ovarian granulosa, theca and stromal cells, but appeared to be most abundant in the granulosa cells. Expression of SRC-1 and RIP140 in corpus luteum, however, was reduced compared to expression in follicular cells. Finally, we examined cofactor expression in ovine, bovine, and porcine uterus. Northern blot analysis for SRC-1, GRIP1, RAC3, p300, and RIP140 mRNAs showed higher expression in extracts of the endometrium compared to whole uterus. We provide the first evidence for the presence of estrogen receptor cofactor mRNAs in the ovary and uterus of three domestic animal species. We suggest that coactivators are conserved among species and associated with hormonal responsiveness of reproductive tract tissues in sheep, cow and pig.

Effects of the xenoestrogen bisphenol A on expression of vascular endothelial growth factor (VEGF) in the rat.

Bisphenol-A (BPA) is used to produce polymers for production of polycarbonate and epoxy resins that are used in food containers and dental appliances. BPA binds to estrogen receptors and induces estrogenic activity in a number of biological systems. We recently reported that although Fisher 344 (F344) and Sprague-Dawley (S-D) rat strains exhibit different sensitivities to BPA at the level of vaginal epithelial cell proliferation, there was no difference in immediate early proto-oncogene expression between the two animal strains. In the present study we investigated the effects of BPA on expression of another estrogen-target gene, vascular endothelial growth factor (VEGF), in the uterus, vagina, and pituitary of F344 and S-D rats. Adult rats were ovariectomized and treated with BPA by intraperitoneal injection at concentrations of 0.02 to 150 mg/kg body wt. Expression of VEGF was monitored by RNase protection assay at 2 hr after treatment. There was a significant effect of dose of BPA on the type of VEGF isoform expressed in the uterus, vagina, and pituitary. BPA induced greater (P < 0.01) levels of VEGF164 and VEGF120+188 than VEGF110 levels. The lowest BPA dose that had a significant (P< 0.05) effect on VEGF expression compared with vehicle treatment was 37.5 mg/kg body wt.; dose-response curves did not differ between strains. This is the first report that the primary response of the uterus, vagina, and pituitary to BPA includes rapid induction of VEGF expression. Due to the capacity of VEGF to engage pleiotropic signaling pathways in other cellular systems, we suggest that modulation of VEFG may play a role in establishing the response of estrogen-target organs to estrogenic xenobiotics.

Estrogen receptor beta in the sheep ovary during the estrous cycle and early pregnancy.

Objectives were to sequence and examine the expression of the estrogen receptor beta (ERbeta) in the sheep ovary. The sequence of the ovine ERbeta (oERbeta) was determined using reverse-transcription polymerase chain reaction (RT-PCR) and cloning techniques. The reading frame of oERbeta contained 527 amino acids and exhibited high overall homology with cow (98%), rat (88%), and human (88%) ERbeta. In addition, an oERbeta isoform having a 139-base pair deletion (oERbeta1) was identified. The predicted amino acid sequence of this isoform is lacking the ligand-binding and carboxyl-terminal transactivation domains. The oERbeta protein and mRNA were determined in ovaries obtained from ewes on Days 0 (first day of estrus), 2, 6, and 10 of the estrous cycle and Day 30 of gestation. Immunohistochemistry showed that oERbeta protein was located in granulosa cells, the ovarian surface epithelium, endothelium, and Day 2 corpus luteum (CL). Weak immunostaining for ERbeta was detected in the theca interna. Relative steady-state amounts of oERbeta mRNA in the CL were determined using semiquantitative RT-PCR. Amounts of oERbeta mRNA were greater (P < 0.05) during CL formation (Day 2) than at later stages. The oERbeta to oERbeta1 mRNA ratio was lower (P < 0.05) on Day 2 than on Day 10 or Day 30 due to a decrease in amounts of oERbeta1. Results indicate that the oERbeta is a 527-amino acid protein expressed in specific cells of the ovary. Changes in relative amounts of full-length oERB and a deletion isoform in CL occurred during the estrous cycle, suggesting that these two types of ERbeta might regulate estrogen actions during early CL development in sheep.

Expression of estrogen receptor coactivators in the rat uterus.

Nuclear receptor coactivators associate in a ligand-dependent manner with estrogen receptors (ER) and other nuclear receptors, and they enhance ligand-dependent transcriptional activation. This study examined basal coactivator expression in rat uterus to investigate if expression of these genes is regulated by estradiol-17 beta or tamoxifen. Ovariectomized mature and immature rats were injected with estradiol-17 beta, tamoxifen, or vehicle (i.e., sesame oil) alone. Uteri were collected and analyzed for changes in coactivator mRNA expression using Northern blot and in situ hybridization analyses. Constitutive uterine mRNA expression of switch protein for antagonist (SPA), SRC-1, GRIP1, RAC3, RIP140, and p300 mRNAs was observed in control uteri, and treatment with ER ligands did not alter coactivator mRNA levels. The data suggest that expression of these coactivator genes is not sensitive to estradiol or tamoxifen in the rat uterus. No cell type-specific pattern of expression was apparent in uterine sections from mature and immature rats; however, silver grains were more abundant in luminal and glandular epithelial cells compared with the stroma and myometrium, indicating that coactivator mRNA levels vary among the uterine compartments. Thus, to our knowledge, we show for the first time that there is constitutive expression of several uterine nuclear receptor coactivators in a physiological setting that remains insensitive to estrogenic regulation. Furthermore, we speculate that higher constitutive levels of coactivator expression in glandular and luminal epithelial cells may be associated with increased hormonal responsiveness by these uterine compartments.

Immunohistochemical detection of estrogen receptor alpha in male rat spinal cord during development.

The alpha subtype of the estrogen receptor (ERalpha) is present in nociceptive and parasympathetic regions of the adult rat spinal cord. The pattern of ERalpha expression in the rat spinal cord during development, however, is unknown. We used a polyclonal antibody (ER-21) to examine the expression of ERalpha in male rat lumbosacral spinal cords at embryonic day (E) 17, E21 (the day before birth), postnatal day (P) 1 (the day of birth), P8, P17, P21, and P36. At E17, ERalpha immunoreactivity (ERalpha-ir) was observed predominantly in ependymal cells. Perinatally, ERalpha-ir was also present in neurons in dorsal root ganglia and in fibers capping and within laminae I and II. By P8, ERalpha-ir was absent in ependymal cells, but ERalpha-ir fibers were dense in laminae I and II and in sympathetic and parasympathetic areas. ERalpha-ir was also present in neurons in the dorsal horns. To determine whether ERalpha-ir fibers in laminae I and II were processes of spinal neurons or primary afferents, dorsal rhizotomies were performed on P17 and P21 animals. Unilateral transection of the lumbosacral dorsal roots virtually eliminated ERalpha-ir fibers in the ipsilateral superficial laminae, demonstrating that the majority of ERalpha-ir fibers in these laminae were primary afferents. We show for the first time that ERalpha-ir is present in neurons and fibers of male prenatal and postnatal spinal cord. The presence of ERalpha in neuronal nuclei and processes may reflect diverse roles and novel mechanisms of action for 17 beta-estradiol in development of spinal sensory and autonomic circuitry.

Strain differences in vaginal responses to the xenoestrogen bisphenol A.

Bisphenol A (BPA) is the monomer component of polycarbonate plastics and epoxy resins; human exposure derives from leachate in foodstuffs packaged in certain plastics or from epoxy-based dental appliances. BPA stimulates prolactin secretion in Fischer 344 (F344) rats but not in Sprague-Dawley (S-D) rats. The present studies were performed to determine if another classic estrogen target tissue, the rat vagina, responds to BPA in a strain-specific manner. In F344 rats BPA increased DNA synthesis in vaginal epithelium with a median effective dose (ED(50)) of 37.5 mg/kg body weight; DNA synthesis was not stimulated in S-D rats by any dose tested. Clearance of (3)H-BPA from blood followed the same time course in both strains of rats, with a half-life of 90 min. Scatchard analysis of [(3)H]estradiol binding showed no strain differences in concentration or affinity of the vaginal estrogen receptor. BPA increased the level of mRNA for the immediate early gene, c-fos, with similar dose-response curves in both rat strains. Thus, F344 and S-D rats exhibit differences in sensitivity to BPA at the level of cell proliferation in the vaginal epithelium. However, metabolic clearance of BPA and the early events that lead to the proliferative response, receptor-ligand interaction and induction of immediate early genes, show no strain differences. These observations suggest that differences in intermediate effects must account for the difference in sensitivity of the proliferative response to the xenoestrogen. Furthermore, these results point to the need for caution in choosing a suitable end point and animal model when seeking to test the estrogenic effects of xenobiotics.

Effects of oral administration of tamoxifen, toremifene, dehydroepiandrosterone, and vorozole on uterine histomorphology in the rat.

Tamoxifen, toremifene, DHEA, and vorozole inhibit tumor growth in rodent mammary carcinoma models and are promising chemotherapeutic agents for use against breast cancer development. In the present study, the effect of these agents on uterine histomorphology following oral administration to mature ovary-intact rats (n = 380) was examined. Animals received diet only (control), tamoxifen (0.4 and 1 mg/kg of diet; 10 mg/kg BW by daily gavage), toremifene (3-30 mg/kg of diet), DHEA (24-2000 mg/kg of diet), or vorozole (0.08-1.25 mg/kg BW by daily gavage) for 28 days and were either sacrificed or returned to a basal diet and then sacrificed 21 days later. Treatment with toremifene (all doses) or tamoxifen (1 and 10 mg/kg) for 28 days produced a decrease (P<0.05) in overall uterine size and myometrial thickness; however, uterine luminal and glandular epithelia cell height increased (P<0.05) compared with control. These compartmentalized uterotrophic and antiestrogenic effects of toremifene and tamoxifen were still apparent after 21 days post-treatment. Administration of DHEA (2000 mg/kg of diet) for 28 days had dramatic uterotrophic effects, increasing (P<0.05) overall uterine size and stimulating all three uterine compartments (epithelia, stroma, and myometrium). The other doses of DHEA, however, were not uterotrophic. Interestingly, after removal of DHEA from the diet, uterine weight and myometrial thickness decreased (P<0.05). Vorozole (1.25 mg/kg) administration for 28 days had differential, compartmentalized uterine effects, producing an increase (P<0.05) in epithelial cell height, a decrease (P<0.05) in stromal size, but no change in myometrial thickness. After 21 days postadministration of vorozole, luminal epithelial cell height was increased (P<0.05) compared with control. The data suggest that oral administration of tamoxifen, toremifene, DHEA, and vorozole results in differential, compartmentalized effects in the uterus that are highly dependent on treatment dose. The data may have implications for risk assessment of these agents prior to administration to healthy, cancer-free women.

Activation of transcription factors activator protein-1 and nuclear factor-kappaB by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin), the prototype agonist of the aromatic hydrocarbon (Ah) receptor, is a potent tumor promoter as well as a complete liver carcinogen that produces an oxidative stress response in rodents and in cultured cell lines. It has been proposed that TCDD promotes neoplastic transformation through oxidative signal transduction pathways, which results in activation of immediate-early response transcription factors. To set the stage for a test of this hypothesis, we evaluated the effect of TCDD treatment on the activation of several transcription factors, including those in the nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) families, which are activated by changes in the redox state of cells. In an extension of prior results, we found that TCDD treatment produced a sustained overexpression of AP-1 for at least 72 hr in wild-type mouse hepatoma Hepa-1 cells, but not in the Ah receptor-deficient derivative c35 or in cytochrome P450-1A1 (CYP1A1)-negative c37 cells. In addition, TCDD treatment caused a significant increase in the DNA binding activity of NF-kappaB, but not in the activities of the other transcription factors tested. AP-1 and NF-kappaB activation were blocked by the thiol antioxidant N-acetylcysteine and by nordihydroguaiaretic acid, an antioxidant and lipooxygenase inhibitor and an inhibitor of the epoxygenase activity of CYP1A1, and did not take place in c35, c37, or in Ah nuclear translator-deficient c4 cells. Hence, sustained activation of these two transcription factors by TCDD is likely to result from a CYP1A1-dependent and Ah receptor complex-dependent oxidative signal. Electrophoretic mobility supershift analyses with specific antibodies showed that most of the increase in NF-kappaB binding activity could be accounted for by increases in p50/p50 complexes. Since these complexes are known to repress NF-kappaB-dependent gene transcription, our results delineate a second molecular mechanism, in addition to the recently found block of tumor necrosis factor-alpha-mediated p50/p65 activation, that may be responsible for the immunosuppresive effects of TCDD.

Expression of fos and jun proto-oncogenes in benign versus malignant human uterine tissue.

OBJECTIVE: The objective of this study was to evaluate expression of fos and jun proto-oncogenes in benign human uterine tissue compared with malignant uterine tissue. METHODS: Forty-two endometrial tissue specimens were obtained at the time of hysterectomy. Tissue samples from different phases of the menstrual cycle and from postmenopausal patients were stained using immunohistochemical methods to detect Fos and Jun proteins, estrogen and progesterone receptor status, and Ki67 (detects a nuclear antigen associated with proliferating cells). Tissue was examined microscopically for nuclear staining in endometrial epithelium and stroma. The endometrium was based on the patient's last menstrual period, pathologic dating, and proliferative versus nonproliferative status as determined by Ki67. Benign and malignant specimens were subjected to Northern blot analysis to evaluate levels of expression of c-fos, c-jun, and jun-B mRNA. The pattern of c-fos mRNA expression in malignant samples was further evaluated using in situ hybridization. RESULTS: In proliferative, secretory, postmenopausal, and progesterone-influenced, uterine specimens immunohistochemically stained and examined, the endometrial and stromal nuclei stained for both Fos and Jun in varying intensities. However, no pattern was found in the variation of intensity according to the phase of the endometrium. Similarly, in malignant and benign endometrial tissue examined by Northern blot and in situ hybridization analyses, expression of proto-oncogene mRNAs was readily detectable, but no statistical correlation between type of tissue examined, grade of adenocarcinoma, and stage of endometrial cancer was found in this study. CONCLUSIONS: In rodent models, control of uterine cell proliferation is related to change in expression of fos and jun proto-oncogenes. Our results indicate that hormonal control is likely to be different in human endometrium and probably involves genes other than the proto-oncogenes under study. Expression of Fos and Jun do not correlate with endometrial cancer stage and grade.

Effect of estradiol on estrogen receptor expression in rat uterine cell types.

In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERalpha) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERalpha. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERalpha expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17beta. Uteri were collected and analyzed for changes in ERalpha mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERalpha. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERalpha. Expression of ERalpha in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERalpha mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERalpha mRNA occurs independent of new protein synthesis. A decrease in ERalpha mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERalpha expression. The results indicate that down-regulation of ERalpha in the epithelia and up-regulation of stromal ERalpha play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.

Studies of dehydroepiandrosterone (DHEA) with the human estrogen receptor in yeast.

Dehydroepiandrosterone (DHEA) is a C19 adrenal steroid synthesized in the human adrenal cortex and serving as a biosynthetic precursor to testosterone and 17beta-estradiol. Despite the fact that it is one of the most abundant steroid hormones in circulation, the physiological role of DHEA in humans remains unclear. The action of DHEA itself, such as its interactions with receptors and nuclear transcription factors, is not well understood, and a specific DHEA receptor has yet to be identified. Although the activity of DHEA can be due to its metabolism into androgens and estrogens, DHEA has been shown to interact with the androgen receptor and the estrogen receptor (ER) in vitro. We demonstrate in this study that DHEA (3beta-Hydroxy-5alpha-androstan-17-one) inhibits 17beta-estradiol (E2) binding to its receptor in vivo in yeast. DHEA stimulates human ER dimerization in yeast, as determined by ER fusion protein interactions, GAL4 reconstitution and subsequent measurement of increased beta-galactosidase activity. DHEA causes an increase in estrogen response element-dependent beta-galactosidase activity, demonstrating that the ER dimer induced by DHEA is transcriptionally active, but at a concentration of DHEA about 1000 times greater than E2. Inclusion of the nuclear receptor co-activator RIP140 in the yeast enhances ER transactivation by DHEA or E2 in a ligand-dependent manner; moreover, only in the presence of RIP140 is DHEA able to stimulate beta-galactosidase activity to levels similar to those achieved by E2. Ligand-receptor interaction for other C19-steroids was also examined. While 5-androstene-3beta, 17beta-diol (ADIOL) displayed estrogenic activity in this system, 4-androstene-17-dione (androstenedione) and 4-androstene-17beta-ol,3-one (testosterone) did not. We have investigated whether DHEA can interact with the human ER in vivo. Our findings demonstrate a mechanism by which DHEA interacts directly with estrogen signaling systems; however, because DHEA is several orders of magnitude less potent than E2 in this system, we conclude that it essentially is not an estrogen agonist.