Yoko Omoto

HDAC6 expression is correlated with better survival in breast cancer

Purpose: The structure and function of chromatin can be altered by modifications to histone. Histone acetylation in vivo is a dynamic reversible process governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC6 is a unique isoform among the HDACs, and a gene expression pattern study, with cDNA microarray in MCF-7 cells, showed the HDAC6 gene to be late responsive, estrogen induced, and up-regulated. This led us to hypothesize that there was a link between levels of HDAC6 expression and the metastatic potential of breast cancer and also, therefore, the prognosis of these patients. Experimental Design: In the present study, the level of HDAC6 mRNA expression was analyzed with quantitative real-time reverse transcription-PCR, in 135 female patients with invasive breast cancer. HDAC6 protein expression was also determined by immunohistochemistry. An association was sought between HDAC6 expression and various clinicopathologic factors. Results: HDAC6 mRNA was expressed at significantly higher levels in breast cancer patients with small tumors measuring less than 2 cm, with low histologic grade, and in estrogen receptor α- and progesterone receptor-positive tumors. By contrast, no relationship was found between HDAC6 mRNA expression and any of the other clinicopathologic factors, namely, age, menopausal status, and axillary lymph node involvement. Patients expressing high levels of HDAC6 mRNA and protein had a better prognosis than those expressing low levels, in terms of disease-free survival. However, multivariate analysis failed to show that HDAC6 mRNA and protein are an independent prognostic factors for disease-free survival and overall survival. Furthermore, the patients with high levels of HDAC6 mRNA tended to be more responsive to endocrine treatment than those with low levels. Specific HDAC6 staining was found in the nucleus of some normal epithelial cells and in the cytoplasm of the majority of cancer cells. Although postmenopausal patients showed higher HDAC6 protein expression, there were no relationship between protein expression and any other clinicopathologic factors. Conclusions: We conclude that the levels of HDAC6 mRNA expression may have potential both as a marker of endocrine responsiveness and also as a prognostic indicator in breast cancer. Additional investigations are warranted concerning the relationship between HDAC6 expression and response to endocrine therapy.

Role of estrogen receptor β in uterine stroma and epithelium: Insights from estrogen receptor β−/− mice

In this study, we compared the uterine tissue of estrogen receptor (ER)β−/− mice and their WT littermates for differences in morphology, proliferation [the percentage of labeled cells 2 h after BrdUrd injection and EGF receptor (EGFR) expression], and differentiation (expression of progesterone receptor, E-cadherin, and cytokeratins). In ovariectomized mice, progesterone receptor expression in the uterine epithelium was similar in WT and ERβ−/− mice, but E-cadherin and cytokeratin 18 expression was lower in ERβ−/− mice. The percentage of cells in S phase was 1.5% in WT mice and 8% in ERβ−/− mice. Sixteen hours after injection of 17β-estradiol (E2), the number of BrdUrd-labeled cells increased 20-fold in WT mice and 80-fold in ERβ−/− mice. Although ERα was abundant in intact mice, after ovariectomy, ERα could not be detected in the luminal epithelium of either WT or ERβ−/− mice. In both untreated and E2-treated mice, ERα and ERβ were colocalized in the nuclei of many stromal and glandular epithelial cells. However, upon E2 + progesterone treatment, ERα and ERβ were not coexpressed in any cells. In WT mice, EGFR was located on the membranes and in the cytoplasm of luminal epithelium, but not in the stroma. In ERβ−/− mice, there was a marked expression of EGFR in the nuclei of epithelial and stromal cells. Upon E2 treatment, EGFR on cell membranes was down-regulated in WT but not in ERβ−/− mice. These findings reveal an important role for ERβ in response to E2 and in the organization, growth, and differentiation of the uterine epithelium.

Prediction of prognosis of estrogen receptor‐positive breast cancer with combination of selected estrogen‐regulated genes

Estrogen receptor (ER)‐positive breast cancer is a distinct subpopulation of breast cancer exhibiting a high response rate to endocrine therapy. However, not all ER‐positive patients respond to the therapy, and a subgrouping of ER‐positive patients based on the physiology of estrogen signaling is expected to be useful for predicting the prognosis. This study has revealed that selected estrogen‐regulated genes (ERGs) are useful in identification of a poor‐prognosis population among ER‐positive breast cancer patients. First, the expression levels of 11 ERGs, selected based on our earlier microarray study in cultured cells, were analyzed by means of real‐time reverse transcription‐PCR in 14 ER‐positive human breast cancer tissues. The patients were clearly divided into two groups in cluster analysis. Then, we examined the expression levels of two representative ERGs, histone deacetylase 6 (HDAC6) and insulin‐like growth factor binding protein 4 (IGFBP‐4), In 62 ER‐positive patients with immunohistochemistry to assess the impact of ERG expression on prognosis (median follow‐up 4409 days). Positive HDAC6 staining was significantly correlated with a lower disease‐free survival rate. Moreover, when the expression level of HDAC6 was assessed in combination with IGFBP‐4 expression in the nucleus, the poor‐prognosis patients were more accurately identified. This study has identified new candidate ERGs for prediction of prognosis, and we suggest that combined assessment of the expression levels of these ERGs will contribute to the clinically useful stratification of ER‐positive breast cancer patients.

The Expression of ERβcx in Human Breast Cancer and the Relationship to Endocrine Therapy and Survival

Purpose: Estrogen receptor (ER) α-positive breast cancer is often treated with endocrine therapy using either antiestrogens or aromatase inhibitors. However, 30% of patients who receive endocrine therapy will derive no benefit from such treatments and may indeed suffer adverse effects. Currently, there are no ways to predict response to such treatments. ERβcx, a variant of ERβ, has a dominant-negative effect over ERα, and its expression thought to modulate response to endocrine treatment may represent a predictor of response to endocrine therapy. Experimental Design: We investigated the expression of the ERβcx in 82 frozen breast samples (8 benign, 1 ductal carcinoma in situ, and 73 malignant) by Western blot analysis. The relationship between the expression of ERβcx variants with prognosis and outcome of endocrine therapy was examined. Results: There was a statistically significant association between the presence of ERβcx and the response to endocrine therapy (Fisher’s exact test, P = 0.04). We also examined the influence of the ERβcx status of a tumor on time to progression and death. There was a relationship between the presence of ERβcx and survival, with patients whose tumors express ERβcx having a longer survival rate (P = 0.05). Cell-type specificity of expression was assessed by immunohistochemistry on a selection of histological samples. Conclusions: On the basis of this small group of patients, we conclude that the expression of ERβcx correlated with favorable response to endocrine therapy. A larger study involving the staining of archival material is currently underway to confirm these preliminary results.

Estrogen receptor β represses Akt signaling in breast cancer cells via downregulation of HER2/HER3 and upregulation of PTEN: implications for tamoxifen sensitivity

IntroductionThe inhibition of estrogen receptor (ER) α action with the ER antagonist tamoxifen is an established treatment in the majority of breast cancers. De novo or acquired resistance to this therapy is common. Expression of ERβ in breast tumors has been implicated as an indicator of tamoxifen sensitivity. The mechanisms behind this observation remain largely uncharacterized. In the present study, we investigated whether ERβ can modulate pathways implicated in endocrine resistance development.MethodsT47-D and MCF-7 ERα-expressing breast cancer cells with tetracycline-regulated expression of ERβ were used as a model system. Expression levels and activity of known regulators of endocrine resistance were analyzed by performing quantitative polymerase chain reaction assays, Western blot analysis and immunostaining, and sensitivity to tamoxifen was investigated by using a cell proliferation kit.ResultsExpression of ERβ in ERα-positive T47-D and MCF-7 human breast cancer cells resulted in a decrease in Akt signaling. The active form of an upstream regulator of Akt, proto-oncogene c-ErbB-2/receptor tyrosine kinase erbB-3 (HER2/HER3) receptor dimer, was also downregulated by ERβ. Furthermore, ERβ increased expression of the important inhibitor of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Importantly, ERβ expression increased the sensitivity of these breast cancer cells to tamoxifen.ConclusionsOur results suggest a link between expression of ERβ and endocrine sensitivity by increasing PTEN levels and decreasing HER2/HER3 signaling, thereby reducing Akt signaling with subsequent effects on proliferation, survival and tamoxifen sensitivity of breast cancer cells. This study supports initiatives to further investigate whether ERβ presence in breast cancer samples is an indicator for endocrine response. Current therapies in ERα-positive breast cancers aim to impair ERα activity with antagonists or by removal of endogenous estrogens with aromatase inhibitors. Data from this study could be taken as indicative for also using ERβ as a target in selected groups of breast cancer.

Differential expression of oestrogen receptors in human secondary lymphoid tissues.

Many autoimmune diseases including rheumatoid arthritis (RA), Sjögrens syndrome (SS) and systemic lupus erythematosus (SLE) occur much more frequently in women than in men. There is much evidence that oestrogen is the major cause of this gender difference. Interestingly, oestrogen relieves the symptoms of RA and SS but it exacerbates SLE. This contradictory effect of oestrogen on autoimmune diseases is not well understood. Most of the effects of oestrogen are mediated by two receptors: oestrogen receptor α and β (ERα and ERβ). To determine whether these contradictory effects of oestrogen relate to the involvement of distinct effects of the two ERs, we investigated expression of ERα and ERβ in human secondary lymphoid tissues. We observed that, in tonsils, ERβ is expressed in lymphocytes of germinal centres (GC) and the follicular mantle zone as well as in granulocytes, while ERα is expressed only in activated germinal centres but not in the follicular zone. ERβ is the predominant ER in human leucocytes from peripheral blood, spleen and in leucocytes infiltrating cancers in both males and females. In addition, in different human lymphoma cell lines including Hodgkin lymphoma, Burkitt lymphoma, and multiple myeloma, ERβ is abundant while ERα is not detectable. Our results indicate that ERβ is the predominant type of ER in mature lymphocytes. We suggest that ERα and ERβ have distinct roles in secondary lymphoid tissues and that further studies with ERβ‐specific agonists will help to elucidate the role of ERβ in these tissues. Copyright

Estrogen Receptor-Beta Regulates Epithelial Cell Differentiation in the Mouse Ventral Prostate

The epithelium of the ventral prostate expresses high levels of ERβ (but no ERα). ERα is expressed in the stroma. Thus, in addition to the indirect effects of estrogen on the epithelium which are reported to be mediated by ERα, there is a direct estrogenic influence on prostatic epithelium mediated by ERβ. We have reported that loss of ERβ results in epithelial hypercellularity in the ventral prostate. In ERβ–/– mouse prostates, there is overexpression of the androgen receptor and of the antiapoptotic factor Bcl-2 in the prostate. Bcl-2 is an estrogen-regulated gene. It is normally expressed only in the basal cells in the prostate. This apparent expansion of the ‘stem cell-like population’ in the ERβ–/– mouse prostate has been further examined. We found a higher expression of cytokeratin 5 in ERβ–/– mouse prostates so that the ratio of cytokeratin 5 to that of 19 is much higher in ERβ–/– than in wild-type littermates. In addition, labeling of DNA with BrdU showed a 3.5-fold higher proliferation rate in ERβ–/– mouse prostate. Despite these clear differences, the piling up of epithelial cells never progressed to high-grade PIN-like lesions. Hyperplastic foci in ERβ–/– mice show accumulation of cells without signs of atypia, resembling low-grade PIN in humans. The reason for this appeared to be a high rate of cellular detachment and subsequent fall off into the lumen in ERβ–/– mice. The fall off phenomenon is possibly related to the finding that the expression of the cell adhesion molecule E-cadherin was very reduced. We conclude that in ERβ–/– mouse prostates, the epithelial cell population contains more epithelial cells in the intermediate stage of differentiation, possessing both the ability to proliferate as the basal cells and the ability to secrete as the highly differentiated luminal epithelium.

Estrogen Signalling and the Metabolic Syndrome: Targeting the Hepatic Estrogen Receptor Alpha Action

An increasing body of evidence now links estrogenic signalling with the metabolic syndrome (MS). Despite the beneficial estrogenic effects in reversing some of the MS symptoms, the underlying mechanisms remain largely undiscovered. We have previously shown that total estrogen receptor alpha (ERα) knockout (KO) mice exhibit hepatic insulin resistance. To determine whether liver-selective ablation of ERα recapitulates metabolic phenotypes of ERKO mice we generated a liver-selective ERαKO mouse model, LERKO. We demonstrate that LERKO mice have efficient reduction of ERα selectively within the liver. However, LERKO and wild type control mice do not differ in body weight, and have a comparable hormone profile as well as insulin and glucose response, even when challenged with a high fat diet. Furthermore, LERKO mice display very minor changes in their hepatic transcript profile. Collectively, our findings indicate that hepatic ERα action may not be the responsible factor for the previously identified hepatic insulin resistance in ERαKO mice.

Generation of ERα-floxed and knockout mice using the Cre/LoxP system.

Estrogen receptor alpha (ERα) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ERα mouse line that can be used to knock out ERα in selected tissues by using the Cre/LoxP system. In this study, we established a new ERα knockout mouse line by crossing the floxed ERα mice with Cre deleter mice. Here we show that genetic disruption of the ERα gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ERα is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

Estrogen receptor β exon 3-deleted mouse: The importance of non-ERE pathways in ERβ signaling

Significance Estrogen receptor β (ERβ) is thought to be the predominant nuclear receptor that regulates estrogen signaling in a wide variety of tissues. In several circumstances, it can oppose the effects of ERα. Here, we generated a novel ERβ exon 3-deleted mouse model (ERβ-Δex3) in which the first zinc finger of the DNA binding domain is missing but the remaining C-terminus part of the receptor is present. We observed that mutant male mice were normal but females were anovulatory. Thus, this work demonstrates that most of the physiological functions of ERβ do not involve estrogen response element binding. In 1998, an estrogen receptor β (ERβ) knockout (KO) mouse was created by interrupting the gene at the DNA binding domain (DBD) with a neocassette. The mutant females were subfertile and there were abnormalities in the brain, prostate, lung, colon, and immune system. In 2008, another ERβ mutant mouse was generated by deleting ERβ exon 3 which encodes the first zinc finger in the DBD. The female mice of this strain were unable to ovulate but were otherwise normal. The differences in the phenotypes of the two KO strains, have led to questions about the physiological function of ERβ. In the present study, we created an ERβ exon 3-deleted mouse (ERβ-Δex3) and confirmed that the only observable defect was anovulation. Despite the two in-frame stop codons introduced by splicing between exons 2 and 4, an ERβ protein was expressed in nuclei of prostate epithelial cells. Using two different anti-ERβ antibodies, we showed that an in-frame ligand binding domain and C terminus were present in the ERβ-Δex3 protein. Moreover, with nuclear extracts from ERβ-Δex3 prostates, there was an ERβ-dependent retardation of migration of activator protein-1 response elements in EMSA. Unlike the original knockout mouse, expression of Ki67, androgen receptor, and Dachshund-1 in prostate epithelium was not altered in the ERβ-Δex3 mouse. We conclude that very little of ERβ transcriptional activity depends on binding to classical estrogen response elements (EREs).