Increased ER Expression Research Articles

The value of imaging combined with clinicopathological features in the diagnosis of high-risk breast lesions.

BackgroundThe upgrade of high-risk breast lesions (HRLs) is closely related to subsequent treatment, but the current predictors for upgrade are limited to intratumoral features of single imaging mode.MethodsWe retrospectively reviewed 230 HRLs detected by mammography, ultrasound, and magnetic resonance imaging (MRI) before biopsy at the Fudan University Cancer Hospital from January 2017 to March 2018. The clinical features, imaging data according to the Breast Imaging Reporting and Data System (BI-RADS) lexicon, and tumor upgrade situation were received. Based on the different risks of upgrade reported, the lesions were classified into high-risk I [HR-I, with atypical hyperplasia (AH)] and high-risk II (HR-II, without AH). We analyzed the association between clinicopathological and imaging factors and upgrade. We used the receiver operating characteristic (ROC) curve to compare the efficacy of three imaging modes for predicting upgrade.ResultsWe included 230 HRLs in 230 women in the study, and the overall upgrade rate was 20.4% (47/230). The upgrade rate was higher in HR-I compared to HR-II (38.5% vs. 4.1%, P<0.01). In patients with AH, estrogen receptor-positive (ER+) patients accounted for 81.0% (64/79). For all HRLs and HR-I, in clinical characteristics, age, maximum size of lesion, and menopausal status were significantly associated with upgrade (P<0.05). In imaging factors, MRI background parenchymal enhancement (BPE), signs of MRI and ultrasound were significantly correlated with upgrade (P<0.05). Patients with negative MRI or ultrasound manifestations had lower upgrade rates (P<0.01). For HR-II, only BPE showed a significant difference between groups (P=0.001). Multifactorial analysis of all HRLs showed that age and BPE were independent predictors of upgrade (P<0.01). The areas under the ROC cure (AUCs) for predicting upgrade in mammography, ultrasound, and MRI were 0.606, 0.590, and 0.913, respectively, indicating that MRI diagnosis was significantly better than mammography and ultrasound (P<0.001).ConclusionsHRLs with AH had a higher rate of upgrade and increased ER expression. Among three imaging modes, MRI was more effective than ultrasound and mammography in diagnosing the upgrade of HRLs. Older age and moderate to marked BPE can indicate malignant upgrade. MRI can provide a certain value for the diagnosis and follow-up of HRLs.

Clinical evaluation of BCL-2/XL levels pre- and post- HER2-targeted therapy.

Our previous pre-clinical work defined BCL-2 induction as a critical component of the adaptive response to lapatinib-mediated inhibition of HER2. To determine whether a similar BCL-2 upregulation occurs in lapatinib-treated patients, we evaluated gene expression within tumor biopsies, collected before and after lapatinib or trastuzumab treatment, from the TRIO-B-07 clinical trial (NCT#00769470). We detected BCL2 mRNA upregulation in both HER2+/ER- as well as HER2+/ER+ patient tumors treated with lapatinib or trastuzumab. To address whether mRNA expression correlated with protein expression, we evaluated pre- and post-treatment tumors for BCL-2 via immunohistochemistry. Despite BCL2 mRNA upregulation within HER2+/ER- tumors, BCL-2 protein levels were undetectable in most of the lapatinib- or trastuzumab-treated HER2+/ER- tumors. BCL-2 upregulation was evident within the majority of lapatinib-treated HER2+/ER+ tumors and was often coupled with increased ER expression and decreased proliferation. Comparable BCL-2 upregulation was not observed within the trastuzumab-treated HER2+/ER+ tumors. Together, these results provide clinical validation of the BCL-2 induction associated with the adaptive response to lapatinib and support evaluation of BCL-2 inhibitors within the context of lapatinib and other HER2-targeted receptor tyrosine kinase inhibitors.

Endometrial estrogen and progesterone receptors in infertility and abnormal uterine bleeding

Introduction: Evaluation of endometrial histology can be made more meaningful with the study of Estrogen and Progesterone receptor (ER and PR) status. Present study aimed at studying ER and PR status of normal endometrium and endometrium in infertility and abnormal uterine bleeding and elucidating its relevance and utility. Materials and Methods: This case control study was carried out on prospectively registered patients. It included 12 cases of infertility, 24 cases of abnormal uterine bleeding, 14 cases of uterine leiomyoma and 22 cases of age matched controls undergoing endometrial sampling for causes other than above mentioned conditions. The cases were subjected to thorough clinical evaluation. The study of endometrium was done with routine histology. Endometrial ER and PR expression was studied immunohistochemically. The data collected was analyzed to find out the variability of expression of endometrial ER and PR in normal and abnormal endometrium with student’s t-test and correlation t-test (SPSS software version 11). Results: The ER and PR expression in normal endometrium showed cyclical variation. It was highest in the proliferative phase followed by the periovulatory phase followed by a significant decline in the late secretory phase. In cases of infertility and abnormal uterine bleeding with endometrial dysfunction; late secretory endometrium showed increased ER expression as compared to controls. Conclusion: Variations noted in endometrial ER and PR expression in infertility and abnormal uterine bleeding provided insight in its pathogenesis and helped in contemplating treatment options. Keywords: ER, PR, Endometrium, Infertility, Abnormal uterine bleeding, Uterine leiomyoma

Abstract P6-05-05: The estrogen switch: Estrogen receptor alpha levels determine the proliferative response to estrogen

Abstract Background: The premise that estrogen promotes the growth of all estrogen receptor positive (ER) + breast cancers has led to anti-estrogen therapies as the standard of care for all ER+ tumors. Paradoxically, estrogen (E2) has demonstrated a therapeutic effect in the adjuvant, palliative and prevention of breast cancers in some women suggesting that there is a subset of ER+ breast cancers that may be growth-suppressed by E2. Our companion window-of-opportunity trial (abstract submitted) found E2 is growth suppressive for some post-menopausal women with high ER expressing tumors. We utilized an in vitro cell model to investigate whether differential growth responses to E2 could be due to transcriptional differences mediated by the level of the ER. Method: We generated a stable MCF-7 transfectant (MCF7-ER) using a fluorescently labelled lentiviral plasmid with a doxycycline inducible ER and compared the response to E2 against an MCF-7 mock transfectant (MCF7-EM). We assessed changes in proliferation (S-phase fraction), gene regulation (RNA-Seq) and differential ER-DNA binding (chromatin immunoprecipitation (ChIP-Seq)). To determine if high levels of ER were causing changes in the underlying chromatin configuration, we compared our ChIP-Seq profiles against previously mapped DNA loop anchor regions obtained by chromatin interaction analysis by paired end-tag sequencing (ChIA-PET) on the MCF-7 parental cell line treated with E2 (Fullwood M et al. Nature 2009). Changes in DNA loop formation were assessed by fluorescence in situ hybridization (FISH) and chromatin conformation capture (3C). Results: We confirmed previously published findings that increased ER expression in the presence of E2 leads to significant decreases in S-phase fraction (P = 0.003) with impaired transition at the G1/S and G2/M checkpoints. RNA-Seq studies defined 342 basally-upregulated genes in the MCF7-ER cells that became down-regulated after E2 treatment indicative of inverse regulation compared to the effect of E2 in the MCF7-EM mock transfectants. In E2-deprived conditions, there was an increased number of ER-DNA binding peaks in the high ER expressing cells (2,776 in MCF7-ER vs. 1,906 in MCF7-EM), suggesting unliganded ER may mediate basal gene transcription. Only 17 (5%) of the basally upregulated genes had ER binding at a proximal or distant anchor associated with previously mapped ER-DNA loops. This suggests that high ER expressing cells may develop a novel chromatin configuration that is distinct from those of parental MCF-7 cells. FISH analysis confirmed a significant increase in loop formation at the promoter region of the TFF1 gene in the MCF7-ER cells compared to MCF7-EM mock transfectants (P = 0) in the absence of E2. This DNA loop was maintained, though slightly decreased, after E2 treatment. Conclusions: These results support a theoretical model where increased ER expression may enable a novel chromatin configuration which mediates a basal level of gene expression that switches to a growth-suppressive transcriptional response upon E2 treatment. Further elucidation of an anti-proliferative DNA configuration signature using MCF7-ER cells may generate a novel set of interactions that could serve as a strategy to predict a patient’s response to estrogens and endocrine therapies. Citation Format: Lacey Haddon, Xiuying Hu, Hosna Jabbari, Judith Hugh. The estrogen switch: Estrogen receptor alpha levels determine the proliferative response to estrogen [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-05.

Antiosteoporotic activity of Du–Zhong–Wan water extract in ovariectomized rats

Context Eucommiae Cortex and Radix Dipsaci, occurring in a ratio of 1:1 in Du-Zhong-Wan (DZW), a Chinese herbal medicine, is available as a water extract followed by ethanol precipitation for the treatment of osteoporosis, fractures and menopausal syndrome.Objective This study investigates the protective effects of DZW in ovariectomy (OVX)-induced bone loss in a rat osteopenia model.Materials and methods Sixty Sprague–Dawley rats were randomly divided into the sham-operated group (SHAM) and five OVX subgroups: OVX with vehicle (OVX), 17β-estradiol (E2) and with three graded doses of DZW. Daily oral administration of the different samples started on the fifth week and lasted for 12 weeks, respectively. The body weight, uterus wet weight, serum biochemical parameters, bone mineral density (BMD), bone biomechanical properties, bone microarchitecture and immunohistochemistry were examined.Results Compared with the SHAM group, the DZW treatment significantly reversed the osteoporotic changes in OVX rats. The DZW-H group showed that serum tartrate-resistant acid phosphatase 5b (TRACP-5b) levels reduced by 152.25% (p < 0.01) and osteocalein (OCN) levels dose dependently increased by 118.43% (p < 0.01) as compared with the OVX group. Compared with the OVX group, the DZW at different three dosages of DZW evidently increased the right femur BMD by 112.43, 114.56 and 116.45%, and dramatically promoted bone quality and bone strength (p < 0.05). Further, immunohistochemical evaluation also showed that DZW administration increased ER expression in uteri (p < 0.01).Conclusions DZW exhibits an anti-osteoporotic effect, probably mediated via phyto-estrogenic effects. It might be a potential herbal alternative for the management of postmenopausal osteoporosis.

Effects of steroid hormones on differentiated glandular epithelial and stromal cells in a three dimensional cell culture model of the canine endometrium

BackgroundOestrogens and progesterone have a significant impact on the endometrium during the canine oestrous cycle. Their receptors mediate plasma steroid hormone levels and are expressed in several endometrial cell types. Altered steroid receptor expression patterns are involved in serious uterine diseases; however the mechanisms of hormone action during pathogenesis in these tissues remain unclear. The development of 3D culture systems of canine endometrial cells provides an opportunity for the effects of steroid hormones to be quantitatively assessed in a more in vivo-like setting. The present study aimed to determine the effects of the steroid hormones 17β-estradiol (E) and progesterone (P) on the expression of the oestrogen and progesterone receptors (ER and PR), and on proliferative activity, in a 3D co-culture system of canine uterine origin, comprising differentiated endometrial glands, and stromal cells (SCs).ResultsMorphology, differentiation, and apical-basolateral polarity of cultured glandular epithelial cells (GECs) were comparable to those in native uterine tissue as assessed by immunohistochemistry using differentiation markers (β-catenin, laminin), lectin histochemistry, and transmission electron microscopy. Supplementation of our 3D-culture system with E (at 15, 30 and 100 pg/mL) resulted in constant levels of ER expression in GECs, but reduced expression levels in SCs. PR expression was reduced in both GECs and SCs following treatment with E. 3 ng/mL P resulted in increased ER expression in GECs, but a decrease in SCs. PR expression in GECs increased in all P-treated groups, whereas PRs in SCs decreased with the lowest and highest doses, but increased with the middle dose of treatment. Proliferative activity, assessed by Ki67 staining, remained below 1% in all assays and cell types.ConclusionsThe present study demonstrates the applicability of our 3D organotypic canine endometrium-derived culture system for cellular-level studies. 3D cultures represent near-physiological systems allowing reproducible quantitative experimentation, thus reducing the need to experiment on living animals. The results of the present investigation emphasize the importance of co-culture of the uterine glands with SCs, as it was shown that the responsiveness of the different cell types to steroid hormones were divergent in the 3D cell culture model.

Abstract P3-14-03: Combining or Sequencing Targeted Therapies in ER+/HER2 Amplified Breast Cancer (BC): In Vitro and In Vivo Studies of Letrozole and Lapatinib in an ER+/HER2+ Aromatase-Transfected BC Model

Abstract Rationale: ER+/HER2+ BC may be resistant to endocrine therapies, but sensitive to HER2 inhibitors such as lapatinib. There is evidence that lapatinib may reactivate ERα expression1,2 raising the possibility that lapatinib may sensitize BC to subsequent endocrine therapy. Whether targeting the ER and HER2 in combination or in sequence in ER+/HER2+ BC may enhance anti-tumour effect is unknown. Aims: (1) To determine in ER+/HER2+ aromatase-expressing breast cancer models in vitro and in vivo.whether the combination of lapatinib with letrozole or tamoxifen is synergistic, and (2) to investigate whether using lapatinib and letrozole in sequence rather than in combination may provide a superior strategy. Methods: ER+/HER2+ BT474 cells were stably transfected with aromatase (BTArom). Combination growth studies, immunoblots and gene expression assays were conducted in BTArom cells. BTArom xenograft studies were performed over 18 weeks under androstenedione support and mice randomized to daily treatment with vehicle, lapatinib, letrozole, or lapatinib and letrozole (L+L). In addition, mice in the lapatinib alone arm, were randomized at 6 weeks to continued lapatinib, vs. switch to letrozole, vs switch to L+L. Results: Formal combination studies in aromatase-transfected BT474 (BTArom) cells demonstrated synergy between lapatinib and letrozole (combination index, CI=0.4, p&amp;lt;10-5), but antagonism between lapatinib and 4-OH-tamoxifen (CI=1.34, p=0.05). In BTArom cells, lapatinib increased ESR1 2.8-fold (P&amp;lt;0.0001) and ERα protein expression 2- fold. As expected, androstenedione upregulated both ER-controlled genes, PGR and GREB1, however lapatinib treatment increased PGR and GREB1 further, by 4.6- and 1.8—fold over androstenedione alone (p=0.0001 and p=0.045, respectively). In long-term BTArom xenografts the combination of lapatinib and letrozole was no more effective at controlling tumour growth than either treatment alone (Kruskall-Wallis comparing treatment arms, P&amp;gt;0.05). However for the group of mice treated with lapatinib for 6 weeks followed by the combination of lapatinib and letrozole for 12 weeks, tumour volume increased only 2.2-fold, compared to 4.5-fold for those continuing on lapatinib, 5.7-fold for those switching to letrozole and 5.2-fold for those on lapatinib and letrozole from the start. Conclusion: We have demonstrated that HER2 targeted therapy enhanced responsiveness to aromatase inhibition in an ER+/HER2+ breast cancer model in vitro and in vivo. In contrast, the interaction between lapatinib and 4-OH-tamoxifen was antagonistic. Lapatinib increased ER expression and function, and the most effective tumour control was achieved with lapatinib alone followed by the combination of lapatinib and letrozole. Maximizing benefit from ER and HER2 targeted therapies in ER+/HER2+ BC may involve sequential rather than concurrent treatment to exploit lapatinib-induced ER reactivation. 1 Leary et al, Clin Canc Res 2010 2Xia et al, PNAS 2006 Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-14-03.

Estrogens modulate intracellular Ca2+ in human airway smooth muscle

Asthma is a disease characterized by airway hyperresponsiveness and inflammation. One‐third of pregnant women with pre‐existing disease experience a decrease in symptoms during the third trimester, when estrogen levels are highest. Previous studies have shown that estrogens relax vascular smooth muscle, however, little is known about estrogen effects on airway smooth muscle (ASM). We tested the hypothesis that estrogen modulates human ASM contractility. Western blot analysis and immunohistochemical studies showed the presence of both full‐length and truncated isoforms of ERα and β. Furthermore, ER isoforms were localized to caveolae. Caveolin‐1 siRNA downregulated ERα isoform in human ASM. Exposure to the pro‐inflammatory cytokine TNFα substantially increased ERα expression in ASM. In fura‐2 loaded human ASM cells, acute exposure to biologically‐active 17β‐estradiol decreased the intracellular Ca2+ response to contractile agonists such as histamine and bradykinin; this effect was blunted by the ER antagonist ICI 182, 780. Overall, these data suggest that estrogens may have a bronchodilatory effect. Accordingly, increased ER expression with airway inflammation may actually be protective during pregnancy in that bronchodilation resulting from higher estrogen levels (acting via ERs) may alleviate the increased ASM contractility of asthma.Supported by the Mayo Graduate School and NIH grant UL1RR024150‐01(Mayo Clinic CTSA awards to YSP and CMP)

Aromatase and breast cancer

Several aromatase inhibitors and also new antiestrogens are now available for treating breast cancer. We have developed a model to compare the antitumor efficacy of these agents and to explore strategies for their optimal use. Results from the model have been predictive of clinical outcome. In this model, tumors are grown in ovariectomized, immunodeficient mice from MCF-7 human breast cancer cells transfected with the aromatase gene (MCF-7Ca). The possibility that blockade of estrogen action and estrogen synthesis may be synergistic was explored by treating mice with the aromatase inhibitor letrozole and the antiestrogen tamoxifen alone and in combination. The results indicated that letrozole alone was better than all other treatments. In addition, when tamoxifen treatment was no longer effective, tumor growth was significantly reduced in mice switched to letrozole treatment. However, tumors ultimately began to grow during continued treatment. To investigate the mechanisms by which tumors eventually adapt and grow during letrozole treatment, we determined the expression of signaling proteins in tumors during the course of letrozole treatment compared to the tumors of control mice. Tumors initially up-regulated the ER while responding to treatment, but subsequently receptor levels decreased in tumors unresponsive to letrozole. Also, Her-2 and adapter proteins (p-Shc and Grb-2) as well as all of the signaling proteins in the MAPK cascade (p-Raf, p-Mekl/2, and p-MAPK), but not in the Pl3/Akt pathway, were increased in tumors no longer responsive to letrozole. To investigate whether sensitivity to letrozole could be regained, cells were isolated from the letrozole resistant tumors (LTLT) and treated with inhibitors of the MAPKinase pathway (PD98059 and UO126). These compounds reduced MAPK activity and increased ER expression. EGFR/Her-2 inhibitors, gefitinib and AEE78S although not effective in the parental MCF-70a cells, restored the sensitivity of LTLT cells to letrozole. In xenografts, beginning treatment with letrozole and faslodex to down regulate the ER prevented increases in Her-2 and activation of MAPK and was highly effective in inhibiting tumor growth throughout 29 weeks of treatment. These results suggest that blocking both ER- and growth factor-mediated transcription may delay development of resistance and maintain growth inhibition of ER+ breast cancer.