ER Pathway Research Articles

SILAC-Based Mass Spectrometry Analysis Reveals That Epibrassinolide Induces Apoptosis via Activating Endoplasmic Reticulum Stress in Prostate Cancer Cells.

Epibrassinolide (EBR) is a polyhydroxylated sterol derivative and biologically active compound of the brassinosteroids. In addition to well-described roles in plant growth, EBR induces apoptosis in the LNCaP prostate cancer cells expressing functional androgen receptor (AR). Therefore, it is suggested that EBR might have an inhibitory potential on androgen receptor signaling pathway. However, the mechanism by which EBR exerts its effects on LNCaP is poorly understood. To address this gap in knowledge, we used an unbiased global proteomics approach, i.e., stable-isotope labeling by amino acids in cell culture (SILAC). In total, 964 unique proteins were identified, 160 of which were differentially expressed after 12 h of EBR treatment. The quantification of the differentially expressed proteins revealed that the expression of the unfolded protein response (UPR) chaperone protein, calreticulin (CALR), was dramatically downregulated. The decrease in CALR expression was also validated by immunoblotting. Because our data revealed the involvement of the UPR in response to EBR exposure, we evaluated the expression of the other UPR proteins. We demonstrated that EBR treatment downregulated calnexin and upregulated BiP and IRE1α expression levels and induced CHOP translocation from the cytoplasm to nucleus. The translocation of CHOP was associated with caspase-9 and caspase-3 activation after a 12 h EBR treatment. Co-treatment of EBR with rapamycin, an upstream mTOR pathway inhibitor, prevented EBR-induced cell viability loss and PARP cleavage in LNCaP prostate cancer cells, suggesting that EBR could induce ER stress in these cells. In addition, we observed similar results in DU145 cells with nonfunctional androgen receptor. When proteasomal degradation of proteins was blocked by MG132 co-treatment, EBR treatment further induced PARP cleavage relative to drug treatment alone. EBR also induced Ca2+ sequestration, which confirmed the alteration of the ER pathway due to drug treatment. Therefore, we suggest that EBR promotes ER stress and induces apoptosis.

Abstract 5291: Prognosis within different breast cancer subtypes using functional activity of signaling pathways

Abstract Introduction Gene signatures to assess prognosis of breast cancer patients are generally restricted to ER-positive/luminal breast cancers, and provide limited insight into the reason of a good or poor prognosis. Based on our computational models that determine functional activity of oncogenic signaling pathways [1], we are able to assess prognosis across all breast cancer subtypes, and explain the results in terms of tumor driving pathway. Methods Previously, we developed models to determine functional activity [1] of the ER, Wnt, Hedgehog (HH), AR, PI3K and TGFβ pathways, based on gene expression levels of the respective pathway's target genes. Here, we report results of applying these models on 1294 breast cancer samples from public data sets GSE6532, GSE9195, GSE20685, GSE21653 and E-MTAB-365 (1169 with survival data), in relation to breast cancer subtype. The pathways’ individual risk association was assessed by univariate Cox proportional hazards regression. Next, a multi-pathway score (MPS) was derived using Cox regression coefficients on 164 training samples, and its prognostic value was tested on the remaining 1005 samples. Hazard ratios on different test subsets were calculated after scaling MPS to yield a unit length 95%-confidence interval, to enable fair comparison. Results In 749 (58%) of the 1294 breast cancer samples, at least one of the six pathways was found active, and in 1026 (79%) at least one moderately active. Of the 749, 167 showed two or more active pathways. Different distributions of active pathways were observed across different subtypes. For instance, ER was active in most luminal A samples, less often in luminal B samples, and not in HER2 and basal samples. Conversely, Wnt, HH and TGFβ were more often active in HER2 and basal samples. PI3K was mostly active in luminal B and HER2 samples. ER pathway activity was associated with a better prognosis (HR = 0.42, p = 9.8e-7), while Wnt, HH, PI3K and TGFβ were associated with worse prognosis (HR = 1.46 - 3.56, p = 4e-10 - 7.1e-2); AR was not associated with prognosis and left out of the MPS. On the 1005 test samples, MPS was highly associated with prognosis (HR = 4.90, p = 7.3e-15). Not only can it distinguish good from poor prognosis cases among luminal cancers (HR = 4.11, p = 2.1e-7), but also within the luminal A and B groups (HR = 5.15 & 2.43, p = 4.7e-5 & 1.3e-2, respectively). Furthermore, MPS can identify HER2 cases with a very poor prognosis (HR = 4.81, p = 3.2e-5), and basal cases with a fairly good prognosis (HR = 3.40, p = 3.7e-3). Conclusion We have demonstrated that we can assess prognosis of breast cancer patients based on functional activity of oncogenic signaling pathways. A combined multi-pathway score (MPS) clearly distinguishes good from poor prognosis cases, even within each breast cancer subtype. Furthermore, the underlying pathway activities can give relevant information for therapy selection. [1] Verhaegh W et al. Cancer Res. 2014;74:2936-45. Citation Format: Henk van Ooijen, Márcia A. Inda, Ralf Hoffmann, Paul van de Wiel, Anja van de Stolpe, Wim Verhaegh. Prognosis within different breast cancer subtypes using functional activity of signaling pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5291. doi:10.1158/1538-7445.AM2015-5291

Abstract 4717: In vitro potency of dual PI3K/mTORC1/C2 inhibitor, GDC-0980 in ER+ and HER2 overexpressing breast cancer cells

Abstract Background: Persistent activation of the PI3K-AKT-mTORC1/C2 signaling pathway drives aberrant cell growth and proliferation in a variety of tumor types including breast tumors. Recent works have demonstrated a strong association between mutational activation of PIK3CA or loss of function of PTEN and resistance to therapies targeted against the ER or HER2 pathway. Purpose: Since the PI3K-AKT-mTORC1/C2 is one of the major cell survival pathways responsible for the progression of breast cancer and appears to be upregulated during development of the resistance to endocrine and trastuzumab treatments suppression of this pathway by administration of GDC-0980 may therefore be efficacious in ER+ and HER2+ breast cancer models. Experimental Design: To examine this possibility, GDC-0980 was tested in ER+ (MCF7, BT-483, T-47D and MDA-MB-415), trastuzumab-sensitive HER2+ (BT-474, SK-BR-3), trastuzumab-resistant (BT-474/HerR), and PIK3CA mutated/HER2+ (HCC1954 and MDA-MB-453) breast cancer cells. We have examed the effects of GDC-0980 on the (a) cell survival/proliferation, (b) downstream signaling pathways for proliferation and apoptosis and (c) 3D ON-TOP-colony formation. Results: Treatment of ER+ and HER2+ cell lines with GDC-0980 in vitro showed that 1) cell lines were sensitive to single agent GDC-0980 with 50% inhibitory concentration ranging from 50 nM - 150 nM in ER+ cell lines and 100 nM - 700 nM in HER2+ cell lines 2) 70-80% anti-proliferative activities were observed on 3D-ON TOP colony formation assay in both ER+ and HER2+ cells 3) GDC-0980 dose- and time-dependently blocked downstream effectors of the PI3K-AKT-mTORC1/C2 pathways i.e. p-AKT (Ser 473, The 308), p-p70S6K, p-S6 ribosomal protein, p-4E-BP1 (only at higher concentration) 4) phosphorylation of ERK was downregulated in ER+/PIK3CA helical domain mutated cells (MCF7) but it was upregulated in ER+/PTEN mutated (loss-of-function) cells (MDA-MB-415) 5) interestingly upstream HER3 was upregulated following the treatment of GDC-0980 and 6) both ER+ and HER2+ cell lines exhibited an increase in annexin V positivity following GDC-0980 treatment. Conclusion: Our preclinical in vitro data demonstrate that concurrent inhibition of PI3K and mTORC1/C2 is likely to be more effective in both ER+ and HER2+ breast cancer cells than the inhibition of mTORC1 alone. We are currently studying the effect of GDC-0980 on cell cycle analysis in ER+ and HER2+ cell lines, the results of which will be presented in the meeting. Citation Format: Yuliang Sun, Pradip De, Jennifer H. Carlson, Lori Friedman, Nandini Dey, Brian Leyland-Jones. In vitro potency of dual PI3K/mTORC1/C2 inhibitor, GDC-0980 in ER+ and HER2 overexpressing breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4717. doi:10.1158/1538-7445.AM2015-4717

Abstract 1097: Knockdown of splicing factor ESRP1 affects multiple splicing factors

Abstract Introduction: Tissue-specific alternative splicing (AS) is an important mechanism for regulating gene expression. Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2), two RNA-binding proteins (RBPs) that promote splicing, are potential candidates that contribute to breast cancer recurrence and resistance to therapies. In our prior studies, we have shown that ESRPs are associated with endocrine resistance. In this study, we seek to investigate the impact of the ESRP1 knockdown in endocrine resistant breast cancer cells. Methods: Expression of ESRP1 was analyzed in endocrine resistant-LCC2 and LCC9 cells. Knockdown of ESRP1 expression in LCC2 and LCC9 cells was achieved using lenti-viral based shRNA approach (Mission TRC human shRNA constructs, Sigma). We next performed paired-end RNA-sequencing of LCC2 and LCC9 cells transfected with control and two clones representative of shRNA knockdown (Illumina HiSeq platform, SeqWright Genomic Services) according to the manufacturer's instructions. After processing and quality check of raw FASTQ data (Illumina CASAVA, FASTQ and FASTX toolkits), reads were aligned, and quantified using bioinformatics software tools (Tophat/Cufflinks and Sailfish). Results: Expression of ESRP1 is significantly elevated in ERα-positive endocrine -resistant cells (LCC2 and LCC9 cells). ESRP1 knockdown was confirmed using qRT-PCR analysis in LCC2 cell lines (clone1 - 80% reduction and clone 3- 95% reduction) and in LCC9 cells (clone 2- 90% reduction and clone 3- 50% reduction). RNA-seq analysis identified 5153 differentially regulated genes between ESRP1 knockdown and control LCC2 cell lines. Of the 5153 genes, 1117 and 2109 genes were unique to clone 1 and clone 3, respectively, while 1927 genes were common in both clones. Targeted RNA-seq analysis also revealed that ESRP1 regulates the expression of several genes including other splicing factors. Furthermore, a functional enrichment analysis of these genes revealed a possible role of altering important pathways such as cell-cell adhesion and cell signaling. Additionally, it was observed that neither ESR1 nor any of its downstream genes were affected by the knockdown of ESRP1. This data was similar to that obtained in knockdown of LCC9 cells. Conclusions: ESRP1 regulates splicing of multiple genes in ER+ breast cancer. The process is independent of ER pathway. Targeting splicing could be a novel modality for treating endocrine therapy resistant breast cancer. Citation Format: Yesim Gökmen-Polar, Yaseswini Neelamraju, Xiaoping Gu, Gouthami Nallamothu, Sarath C. Janga, Sunil Badve. Knockdown of splicing factor ESRP1 affects multiple splicing factors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1097. doi:10.1158/1538-7445.AM2015-1097

Activation of PI3K/AKT/mTOR pathway in ER+ breast cancer: Analysis of paired primary and metastatic tumor samples.

577 Background: The activation of the mammalian target of rapamycin (mTOR) pathway is associated with resistance to endocrine therapy in breast cancer. Our aim was to evaluate changes in activation...

Abstract OT1-1-02: PI3K-Akt-mTOR pathway analysis to obtain further insight in the efficacy of everolimus in combination with exemestane in metastatic, ER-positive breast cancer: A Dutch breast cancer research group (BOOG) study

Abstract Background In patients with hormone receptor-positive breast cancer, activation of the PI3K-Akt-mTOR pathway is associated with resistance against endocrine therapy. Previous research has shown that genetic aberrations in this pathway occur frequently, including mutation and/or amplification in PI3K subunits or PI3K effectors as well as loss of lipid phosphatases (Fu X, et al. The Breast; 2013). Central review of the BOLERO-2 randomized phase III trial in which patients refractory to a non-steroidal aromatase inhibitor were randomized between exemestane combined with the mTOR inhibitor everolimus versus exemestane and placebo has shown a progression-free survival (PFS) of, respectively, 11.0 and 4.1 months [hazard ratio = 0.38 (95% confidence interval 0.31-0.48; log-rank P <0.0001)] (Yardley DA, et al. Adv Ther; 2013). The combination, however, is known to cause more adverse events and is associated with additional costs as compared to exemestane alone. In the present study we will explore whether there are biomarkers that might indicate which patients most likely benefit from co-targeting PI3K and ER pathways. Trial design/Aims This is a Dutch prospective, open-label, single-arm, investigator-initiated, multicenter trial in which approximately 30 hospitals will participate. A total of 175 patients will be included for baseline blood sampling and archival tumor tissue collection. From 50 patients, a fresh tumor biopsy is required at baseline and from 30 out of 50, another tumor biopsy will be collected upon progressive disease. Exploratory biomarker assessment includes immunohistochemistry (total and phosphorylated PI3K, AKT, mTOR, p70S6K and 4EBP1), tissue phosphoproteomics and circulating tumor DNA (mutations). The results of the biomarker analysis will be compared with clinicopathological characteristics and PFS. Eligibility Postmenopausal patients with hormone receptor-positive, HER2-negative, locally advanced or metastatic breast cancer, refractory to anastrozole or letrozole will be included. No previous treatment with exemestane or mTOR inhibitor for advanced disease is allowed and Informed consent must be signed before enrollment. Statistical methods Since the majority of the tests involve the use of new techniques, the study will be mainly explorative in design. The association between potential biomarkers and clinicopathological characteristics will be tested using Fisher exact test or the Mann-Whitney test. PFS curves will be drawn using the Kaplan-Meier method. PFS in association with potential biomarkers will be tested using Cox proportional hazard regression analysis. Present and target accrual Recently, the study has been opened for inclusion. A period of 2 years is planned for patient enrollment. Up to May 2014, two patients were included. ClinicalTrials.gov identifier NCT02109913 Financial support is received from Novartis, the Netherlands. Citation Format: Dinja T Kruger, Karin Beelen, Connie R Jimenez, Maurice PHM Jansen, Stefan Sleijfer, Sabine C Linn, Epie Boven. PI3K-Akt-mTOR pathway analysis to obtain further insight in the efficacy of everolimus in combination with exemestane in metastatic, ER-positive breast cancer: A Dutch breast cancer research group (BOOG) study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-1-02.

Estrogen pathway mutations and cancer

Cancer is caused by an accumulation of mutations in a stem cell. A defective mismatch repair (MMR) system can lead to such an accumulation of mutations. MMR defects are found in a cancer syndrome called Lynch Syndrome, and tumors of this syndrome are indeed characterized by such an accumulation of mutations, particularly in short repetitive DNA sequences, called microsatellites. When such mutated microsatellites are located in the coding sequences of genes with essential roles for tumorigenesis, we speak of ‘target genes’. Many such target genes have been found and in this review we focus the possible involvement of target genes involved in the estrogen-receptor pathway (ER). We recently identified NRIP1, encoding the nuclear receptor-interacting protein 1, as the most frequently mutated gene in microsatellite instable (MSI) endometrial cancer (EC). NRIP1 is a known corepressor of the ER pathway, the pathway essential in regulating the concentrations of estrogens, a hormone for which the endometrium is highly responsive. This in combination with the fact that high exposure to estrogens is currently considered the major risk factor for EC - approximately 80% of all sporadic EC tumors are estrogen dependent carcinomas - make NRIP1 the perfect target gene for EC. Interestingly, mutations in NRIP1 were also detected in MSI colorectal carcinoma (CRC) samples. Finding mutations in an estrogen receptor signaling protein in colorectal tissue might not be that expected, as colon is not typically associated with being responsive to estrogens. However, evidence is accumulating to better understand this finding. For instance, it was shown that NRIP1 , in colon tissue, stimulates APC gene transcription and inhibits β-catenin activation. Moreover, some studies suggested that estrogens can increase the expression of MLH1 in colon cancer cells, highlighting the implications of estrogen protecting against colon cancer, by regulating the MMR system. All in all we conclude that genes involved in the estrogen pathway are the perfect candidates to be studied in MMR-deficient tumors, especially those developing in hormonal responsive tissues.

The apoptotic pathways in the curcumin analog MHMD-induced lung cancer cell death and the essential role of actin polymerization during apoptosis

As a mode of cell death, apoptosis could be triggered by the extrinsic, intrinsic mitochondrial and intrinsic endoplasmic reticulum pathways and actin rearrangement is needed during apoptosis. We previously found that one curcumin analog MHMD could induce A549 lung cancer cells apoptosis. But the apoptotic pathways and the actin dynamics during apoptosis are not known. Here, we detected the activation of caspase-3, -8, -9, -12, PARP and the increase ratio of Bax/Bcl-2 by western blotting in MHMD-exposed A549 cells. Alternatively, caspases inhibitors could lead to the disappearance of MHMD-eliciting nuclei fragmentation by Hoechst 33342 staining. Besides, JC-1 and DCFH-DA staining showed the fall of mitochondrial membrane potential and the release of ROS. Moreover, wound healing assay confirmed the MHMD anti-migration ability, which was much more effective than curcumin. Importantly, unlike other anticarcinogenic drugs, MHMD might induce the actin polymerization but not depolymerization in the process of A549 cell apoptosis by phalloidin-FITC staining, which is essential to MHMD-induced extrinsic, intrinsic mitochondrial and intrinsic ER pathways of cell apoptosis.

P049 Genomic landscape of the PI3K pathway and cell-cycle pathway in ER+ BC: a treatment strategy

P049 Genomic landscape of the PI3K pathway and cell-cycle pathway in ER+ BC: a treatment strategy

220 THE ESTROGEN-LIKE EFFECT OF 2-METHOXYESTRADIOL (2-ME) IN IN VITRO AND IN VIVO MODELS

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-oestradiol, interacts with oestrogen receptors and microtubules and has a low affinity for oestrogen receptors (ER). It has attracted considerable interest due to its potential anti-cancer therapeutic effects. 2-ME is also recognised for its unique and profound actions on various tumour cell lines and cancer independent of the hormone receptor status. Regardless of differences in function, 2-ME has an affinity for ER, however, the exact mechanisms of 2-ME action via the ER are not fully understood. In the current study, we examined the estrogenic effect of 2-ME on mRNA levels of CaBP-9k, ER, and progesterone receptor (PR) in the absence or presence of the 17β-oestradiol (E2) and progesterone (P4) in both in vivo and in vitro models by real-time RT–PCR. In vitro, cells (n = 3 per group) were exposed to a single dose of E2 (10–9 M), P4 (10–6 M), 2-ME (10–8 M, 10–7 M, 10–6 M). The mechanism of CaBP-9k induction by these chemicals pre-treated with 10–7 M ICI 182, 780 and 10–6 M RU 486 for 30 min before exposure to E2 and 2-ME were analysed. In vivo, 35 female ICR mice (PND 14 days) were divided into 7 groups (n = 5 per group), and each group was administered subcutaneously with 24% DMSO, 38% ethanol, and 38% sterile saline as a vehicle, E2 [40 μg kg–1 of body weight (BW)] a physiological dose level), 2-ME (4, 40, and 80 mg kg–1 of BW) for 3 days. The mice were killed 24 h after the final injection. To investigate the effect of antagonism, 10 mice were injected SC with ICI 182 780 (10 mg kg–1 of BW) and RU 486 (10 mg kg–1 of BW) at 30 min before injection with 2-ME (40 mg kg–1 of BW) for 3 days and killed 24 h after the final injection. Results are presented as mean ± s.e.m.; P-values were calculated using one-way ANOVA. In GH3 cells, the mRNA level of CaBP-9k was induced in the E2 (10–9 M) treatment group, and expression of CaBP-9k was also up-regulated in the 2-ME (10–7 M)-treated group. Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2-ME (40 mg kg–1 of BW) group, similar to the response with E2 (40 μg kg–1 of BW) in mice. As a blocker for ER and PR activity, ICI 182 780 and RU 486 reversed the E2 or 2-ME mediated increase of CaBP-9k and Ltf mRNA expression. We found that 2-ME significantly increased the levels of ERa and PR transcripts. In parallel with in vitro results, the mRNA levels of ERa and PR were induced by treatment with E2 and 2-ME. Taken together, our findings demonstrated that expression of estrogenic markers, CaBP-9k and Ltf, was regulated by 2-ME in both in vitro and in vivo, which may increase their estrogenic activities in female during the cycle through ER and/or PR-mediated pathway.

Survival rates between premenopausal and postmenopausal women with lung cancer: US SEER database

In the United State, lung cancer remains the leading cause of cancer death in both men and women. Several reports have suggested a role for estrogens in the development and/or progression of lung cancer, especially in women. Data from the national SEER registry between the years of 1990-2011 was analyzed, women between the ages 31-50 years old were chosen as representative of the premenopausal group (n=1595) and 51-70-year-old women represented the postmenopausal group (n=7075) as defined by the American College of Obstetricians and Gynecologists. For comparison, men were divided into two categories: younger men (n=2233) aged 31- 50 years and older men (n=10908) aged 51-70 years. Survival rates were analyzed by Kaplan-Meier method and compared by Z-test through SEER*Stat software version 7.0.9. The adenocarcinoma had a significant difference between premenopausal and postmenopausal groups (62% vs. 51%) respectively. Furthermore, the survival rate in premenopausal inferior to postmenopausal women in both SCC and BAC P<0.05. Premenopausal women more commonly underwent curative surgery, 42%, and 19% of postmenopausal women treated palliative. Additionally, for every stage of disease, 55% of postmenopausal women have radiotherapy. The results suggest varying estrogen effects between the histology sub-types of NSCLC and support clinical strategies need to block the ER pathway for the treatment of NSCLC.

Trastuzumab-induced HER2 phosphorylation: exploring the mechanisms and implications

Cancer is caused by an accumulation of mutations in a stem cell. A defective mismatch repair (MMR) system can lead to such an accumulation of mutations. MMR defects are found in a cancer syndrome called Lynch Syndrome, and tumors of this syndrome are indeed characterized by such an accumulation of mutations, particularly in short repetitive DNA sequences, called microsatellites. When such mutated microsatellites are located in the coding sequences of genes with essential roles for tumorigenesis, we speak of ‘target genes’. Many such target genes have been found and in this review we focus the possible involvement of target genes involved in the estrogen-receptor pathway (ER). We recently identified NRIP1, encoding the nuclear receptor-interacting protein 1, as the most frequently mutated gene in microsatellite instable (MSI) endometrial cancer (EC). NRIP1 is a known corepressor of the ER pathway, the pathway essential in regulating the concentrations of estrogens, a hormone for which the endometrium is highly responsive. This in combination with the fact that high exposure to estrogens is currently considered the major risk factor for EC - approximately 80% of all sporadic EC tumors are estrogen dependent carcinomas - make NRIP1 the perfect target gene for EC. Interestingly, mutations in NRIP1 were also detected in MSI colorectal carcinoma (CRC) samples. Finding mutations in an estrogen receptor signaling protein in colorectal tissue might not be that expected, as colon is not typically associated with being responsive to estrogens. However, evidence is accumulating to better understand this finding. For instance, it was shown that NRIP1, in colon tissue, stimulates APC gene transcription and inhibits β-catenin activation. Moreover, some studies suggested that estrogens can increase the expression of MLH1 in colon cancer cells, highlighting the implications of estrogen protecting against colon cancer, by regulating the MMR system. All in all we conclude that genes involved in the estrogen pathway are the perfect candidates to be studied in MMR-deficient tumors, especially those developing in hormonal responsive tissues.

Abstract 5345: DMBA promotes erbB-2 mediated carcinogenesis through activation of estrogen receptor and receptor tyrosine kinase pathways

Abstract ErbB-2 is a receptor tyrosine kinase (RTK) that is amplified/overexpressed in 20-30% of all invasive breast malignancies; it has been associated with poor prognosis and therapeutic resistance in breast cancer patients. 7,12-dimethylbenz anthracene (DMBA) is a polycyclic aromatic hydrocarbon known for its carcinogenic effects. The objective of this study was to investigate the effect of DMBA exposure on erbB-2 mediated carcinogenesis. This represents a combination of genetic predisposition and environmental assault that synergistically promote carcinogenesis. In this study, female MMTV-erbB-2 transgenic mice at 6-weeks of age were treated weekly by oral gavage with either 1.0 mg DMBA in peanut oil or vehicle alone for 6 weeks. In the control group, palpable tumors appeared at week 25, with an average latency of 36.0 weeks. All mice in the DMBA group developed mammary tumors between week 16 and 26 with an average latency of 20 weeks. By the endpoint (first tumor ∼1.5cm3), the average number of palpable tumors per mouse were 1.15 and 2 for control and DMBA groups, respectively. Lung metastasis in the DMBA group was significantly higher than control mice (40% DMBA vs. 5% control). These data show that DMBA exposure significantly promoted erbB-2 mediated carcinogenesis. Whole mount analysis showed that at 14 weeks of age there was marked expansion of the ducts, whereas lateral branching of mammary glands from DMBA mice appeared to be inhibited, suggesting that DMBA stimulates glandular growth while inhibiting differentiation in this model. Western blot analysis of mammary tissues at 14 weeks of age showed that ERα, p-ERα, cyclin D1, Bcl-2, c-myc, EGFR, erbB-2 and erbB-3 protein levels were significantly increased in DMBA treated mice, which was accompanied by increased phosphorylation/activation of erbB-2, EGFR, ERK and Akt1. These data suggest that DMBA induced activation of both ER and receptor tyrosine kinase (RTK) pathways. Interestingly, tumor histology between tumors from control and DMBA treated mice was similar. However, analysis of tumor karotypes from control and DMBA mice showed that tumors from DMBA mice displayed more chromosomal alterations; an addition to chromosome 4 and trisomy 5 in the control mouse, trisomy 2, trisomy 3 and a deletion to chromosome 4 were detected from tumors of the DMBA mice, suggesting that DMBA induced chromosomal instability contributed to tumor promotion in this model. Taken together, our data suggest that DMBA promoted erbB-2 mediated carcinogenesis. In addition to its well known mutational effect, DMBA induced deregulation of ER and erbB-2 pathways plays a critical role in this process. Citation Format: Zhikun Ma, Stanley Kosanke, Morgan Carrington, Xiaohe Yang. DMBA promotes erbB-2 mediated carcinogenesis through activation of estrogen receptor and receptor tyrosine kinase pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5345. doi:10.1158/1538-7445.AM2014-5345

Estrogen as Jekyll and Hyde: regulation of cell death

Sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary. Unexpectedly, physiological level of estrogen together with a novel IKKα inhibitor BAY11-7082 could effectively induce cell apoptosis in ER-positive breast cancer cells, suggesting combining estrogen with IKKα inhibition may be beneficial for breast cancer patients. This opinion article touches upon the dual role estrogen played in inducing cancer cell death and asks whether use of estrogen in combination with IKKα-targeted therapy would be possible reconsider the newly identified crosstalk between ER and NFκB pathway which can be utilized to switch the effects of estrogen on cell death.

Estrogen as Jekyll and Hyde: regulation of cell death

Sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary. Unexpectedly, physiological level of estrogen together with a novel IKKα inhibitor BAY11-7082 could effectively induce cell apoptosis in ER-positive breast cancer cells, suggesting combining estrogen with IKKα inhibition may be beneficial for breast cancer patients. This opinion article touches upon the dual role estrogen played in inducing cancer cell death and asks whether use of estrogen in combination with IKKα-targeted therapy would be possible reconsider the newly identified crosstalk between ER and NFκB pathway which can be utilized to switch the effects of estrogen on cell death.

Tyrosine phosphatase Shp2 mediates the estrogen biological action in breast cancer via interaction with the estrogen extranuclear receptor.

The extranuclear estrogen receptor pathway opens up novel perspectives in many physiological and pathological processes, especially in breast carcinogenesis. However, its function and mechanisms are not fully understood. Herein we present data identifying Shp2, a SH2-containing tyrosine phosphatase, as a critical component of extranuclear ER pathway in breast cancer. The research checked that the effect of Shp2 on the tumor formation and growth in animal model and investigated the regulation of Shp2 on the bio-effect and signaling transduction of estrogen in breast cancer cell lines. The results showed that Shp2 was highly expressed in more than 60% of total 151 breast cancer cases. The inhibition of Shp2 activity by PHPS1 (a Shp2 inhibitor) delayed the development of dimethylbenz(a)anthracene (DMBA)-induced tumors in the rat mammary gland and also blocked tumor formation in MMTV-pyvt transgenic mice. Estradiol (E2) stimulated protein expression and phosphorylation of Shp2, and induced Shp2 binding to ERα and IGF-1R around the membrane to facilitate the phosphorylation of Erk and Akt in breast cancer cells MCF7. Shp2 was also involved in several biological effects of the extranuclear ER-initiated pathway in breast cancer cells. Specific inhibitors (phps1, phps4 and NSC87877) or small interference RNAs (siRNA) of Shp2 remarkably suppressed E2-induced gene transcription (Cyclin D1 and trefoil factor 1 (TFF1)), rapid DNA synthesis and late effects on cell growth. These results introduced a new mechanism for Shp2 oncogenic action and shed new light on extranuclear ER-initiated action in breast tumorigenesis by identifying a novel associated protein, Shp2, for extranuclear ER pathway, which might benefit the therapy of breast cancer.

Estrogen as Jekyll and Hyde: regulation of cell death.

Estrogen has profound effects on growth, differentiation and function in male and female reproductive systems, and it is important for bone density, brain function and cholesterol mobilization. Despite beneficial estrogen functions, sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary. This opinion article touches upon the dual role estrogen played in cancer and asks whether the use of estrogen in combination with other targeted therapy would be possible, considering the newly identified crosstalk pathway which can switch the effects of estrogen.

HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer.

miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA-target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA-mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.

PA.07 Enhanced recovery programme for elective caesarean section

IntroductionEnhanced recovery (ER) for elective caesarean sections (CS) provides an opportunity to aid the time for patients to return to normal function.1 After trialling a pilot ER pathway for elective...

PA.15 Enhanced Recovery in Elective Caesarean section: early experience suggests reduced length of stay

IntroductionEnhanced recovery (ER) after surgery is an integrated care pathway based on the principles of best peri-operative patient care.This study looked at potential benefits of introducing an ER pathway for...