The epithelial sodium channel has a role in breast cancer cell proliferation.

PURPOSE: Breast cancer is the most common cancer affecting women worldwide with half a million associated deaths annually. Despite a huge global effort, the pathways of breast cancer progression are not fully elucidated. Ion channels have recently emerged as novel regulators of cancer cell proliferation and metastasis. The epithelial sodium channel, ENaC, made up of α, ß and γ subunits is well known for its role in Na+ reabsorption in epithelia, but a number of novel roles for ENaC have been described, including potential roles in cancer. A role for ENaC in breast cancer, however, has yet to be described. Therefore, the effects of ENaC level and activity on breast cancer proliferation were investigated. METHODS: Through the publicly available SCAN-B dataset associations between αENaC mRNA expression and breast cancer subtypes, proliferation markers and epithelial-mesenchymal transition markers (EMT) were assessed. αENaC expression, through overexpression or siRNA-mediated knockdown, and activity, through the ENaC-specific inhibitor amiloride, were altered in MCF7, T47D, BT549, and MDAMB231 breast cancer cells. MTT and EdU cell proliferation assays were used to determine the effect of these manipulations on breast cancer cell proliferation. RESULTS: High αENaC mRNA expression was associated with less aggressive and less proliferative breast cancer subtypes and with reduced expression of proliferation markers. Decreased αENaC expression or activity, in the mesenchymal breast cancer cell lines BT549 and MDAMB231, increased breast cancer cell proliferation. Conversely, increased αENaC expression decreased breast cancer cell proliferation. CONCLUSION: αENaC expression is associated with a poor prognosis in breast cancer and is a novel regulator of breast cancer cell proliferation. Taken together, these results identify ENaC as a potential future therapeutic target.

Correction to: Expression of quiescin sulfhydryl oxidase 1 is associated with a highly invasive phenotype and correlates with a poor prognosis in luminal B breast cancer.

After the publication of this work [1], an error was noticed in Fig. 4a. The micrograph image sh528 was accidentally duplicated.

Androgen receptor-positive triple negative breast cancer: a unique breast cancer subtype.

BACKGROUND: The significance of androgen receptor (AR) expression in triple-negative breast cancer (TNBC) is unclear, and published studies so far have been inconclusive. METHODS: A tissue microarray was constructed using tissue obtained from 119 patients with primary TNBC and stained for AR expression. Other tissue types obtained included recurrent TNBC, normal breast tissue, adjacent ductal carcinoma-in situ (DCIS), lymph node (LN) and distant metastases. Positive AR expression was defined as ≥10% nuclear staining. RESULTS: Epithelial tissue was present and evaluable in 94 TNBC patients with a total of 177 tissue cores. AR expression in TNBC was 22 of 94 (23%). AR expression was higher in normal breast tissue (88%) and adjacent DCIS (73% overall). All LN metastases from AR-positive TNBC patients were also AR positive; in addition, no AR-negative TNBC patient had AR-positive LNs. AR expression was associated with older patient age (63 vs. 57 years, respectively, p = 0.051) and LN metastases (p = 0.033). Locoregional recurrence and overall/disease-specific survival were similar between AR-positive and AR-negative patients, although AR-positive patients had more advanced disease. On multivariate analysis, the presence of LN metastases was associated with poorer recurrence-free survival in AR-positive patients (hazard ratio, 4.34) (p = 0.031). CONCLUSIONS: The AR is expressed in normal breast tissue, and expression decreases with advancement to DCIS and invasive cancer. AR-positive TNBC was more common in older patients and had a higher propensity for LN metastases. AR-positive TNBC may represent a breast cancer subtype with unique features that may be amenable to treatment with alternative targeted therapies.

ERß1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer.

BACKGROUND: The role and clinical value of ERß1 expression is controversial and recent data demonstrates that many ERß antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERß1 expression across all sub-types of breast cancer using a validated antibody and determine the roles of this receptor in mediating response to multiple forms of endocrine therapy both in the presence and absence of ERα expression. METHODS: Nuclear and cytoplasmic expression patterns of ERß1 were analyzed in three patient cohorts, including a retrospective analysis of a prospective adjuvant tamoxifen study and a triple negative breast cancer cohort. To investigate the utility of therapeutically targeting ERß1, we generated multiple ERß1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERß-specific agonist exposure. RESULTS: Nuclear ERß1 was shown to be expressed across all major sub-types of breast cancer, including 25% of triple negative breast cancers and 33% of ER-positive tumors, and was associated with significantly improved outcomes in ERα-positive tamoxifen-treated patients. In agreement with these observations, ERß1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs). However, in the absence of ERα expression, ERß-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective. CONCLUSIONS: Using a validated antibody, we have confirmed that nuclear ERß1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERß appears to be a novel therapeutic target. However, the efficacy of SERMs and ERß-specific agonists differ as a function of ERα expression.

Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time.

In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17ß-estradiol (E(2)) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E(2) paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E(2)-induced apoptosis by analysis of gene expression across time (2-96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E(2)-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E(2) in 5C cells compared with both WS8 and 2A cells and hence, were associated with E(2)-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E(2) inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E(2)-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E(2)-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E(2) interacted to superadditively induce apoptosis. Therefore, these data indicate that E(2) induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.

Regulation of mesenchymal-to-epithelial transition by PARAXIS during somitogenesis.

BACKGROUND: Dynamic alterations in cell shape, migration, and adhesion play a central role in tissue morphogenesis during embryonic development and congenital disease. The mesenchymal-to-epithelial transition that occurs during vertebrate somitogenesis is required for proper patterning of the axial musculoskeletal system. Somitic MET is initiated in the presomitic mesoderm by PARAXIS-dependent changes in cell adhesion, cell polarity, and the composition of the extracellular matrix. However, the target genes downstream of the transcription factor PARAXIS remain poorly described. RESULTS: A genome-wide comparison of gene expression in the anterior presomitic mesoderm and newly formed somites of Paraxis(-/-) embryos resulted in a set of deregulated genes enriched for factors associated with extracellular matrix and cytoskeletal organization and cell-cell and cell-ECM adhesion. The greatest change in expression was seen in fibroblast activation protein alpha (Fap), encoding a dipeptidyl peptidase capable of increasing fibronectin and collagen fiber organization in extracellular matrix. Further, downstream genes in the Wnt and Notch signaling pathways were downregulated, predicting that PARAXIS participates in positive feedback loops in both pathways. CONCLUSIONS: These data demonstrate that PARAXIS initiates and stabilizes somite epithelialization by integrating signals from multiple pathways to control the reorganization of the ECM, cytoskeleton, and adhesion junctions during MET.

Expression of quiescin sulfhydryl oxidase 1 is associated with a highly invasive phenotype and correlates with a poor prognosis in Luminal B breast cancer.

INTRODUCTION: Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. Tumor specific expression of QSOX1 has been reported for numerous tumor types. In this study, we investigate QSOX1 as a marker of breast tumor progression and evaluate the role of QSOX1 as it relates to breast tumor growth and metastasis. METHODS: Correlation of QSOX1 expression with breast tumor grade, subtype and estrogen receptor (ER) status was gathered through informatic analysis using the "Gene expression based Outcome for Breast cancer Online" (GOBO) web-based tool. Expression of QSOX1 protein in breast tumors tissue microarray (TMA) and in a panel of breast cancer cell lines was used to confirm our informatics analysis. To investigate malignant cell mechanisms for which QSOX1 might play a key role, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in ER+ Luminal A-like MCF7, ER+ Luminal B-like BT474 and ER- Basal-like BT549 breast cancer cell lines. RESULTS: GOBO analysis revealed high levels of QSOX1 RNA expression in ER+ subtypes of breast cancer. In addition, Kaplan Meyer analyses revealed QSOX1 RNA as a highly significant predictive marker for both relapse and poor overall survival in Luminal B tumors. We confirmed this finding by evaluation of QSOX1 protein expression in breast tumors and in a panel of breast cancer cell lines. Expression of QSOX1 in breast tumors correlates with increasing tumor grade and high Ki-67 expression. Suppression of QSOX1 protein slowed cell proliferation as well as dramatic inhibition of MCF7, BT474 and BT549 breast tumor cells from invading through Matrigel™ in a modified Boyden chamber assay. Inhibition of invasion could be rescued by the exogenous addition of recombinant QSOX1. Gelatin zymography indicated that QSOX1 plays an important role in the function of MMP-9, a key mediator of breast cancer invasive behavior. CONCLUSIONS: Taken together, our results suggest that QSOX1 is a novel biomarker for risk of relapse and poor survival in Luminal B breast cancer, and has a pro-proliferative and pro-invasive role in malignant progression partly mediated through a decrease in MMP-9 functional activity.

Activin B and Activin C Have Opposing Effects on Prostate Cancer Progression and Cell Growth.

Current prognostic and diagnostic tests for prostate cancer are not able to accurately distinguish between aggressive and latent cancer. Members of the transforming growth factor-ß (TGFB) family are known to be important in regulating prostate cell growth and some have been shown to be dysregulated in prostate cancer. Therefore, the aims of this study were to examine expression of TGFB family members in primary prostate tumour tissue and the phenotypic effect of activins on prostate cell growth. Tissue cores of prostate adenocarcinoma and normal prostate were immuno-stained and protein expression was compared between samples with different Gleason grades. The effect of exogenous treatment with, or overexpression of, activins on prostate cell line growth and migration was examined. Activin B expression was increased in cores containing higher Gleason patterns and overexpression of activin B inhibited growth of PNT1A cells but increased growth and migration of the metastatic PC3 cells compared to empty vector controls. In contrast, activin C expression decreased in higher Gleason grades and overexpression increased growth of PNT1A cells and decreased growth of PC3 cells. In conclusion, increased activin B and decreased activin C expression is associated with increasing prostate tumor grade and therefore have potential as prognostic markers of aggressive prostate cancer.

Context-specific gene regulatory networks subdivide intrinsic subtypes of breast cancer.

BACKGROUND: Breast cancer is a highly heterogeneous disease with respect to molecular alterations and cellular composition making therapeutic and clinical outcome unpredictable. This diversity creates a significant challenge in developing tumor classifications that are clinically reliable with respect to prognosis prediction. RESULTS: This paper describes an unsupervised context analysis to infer context-specific gene regulatory networks from 1,614 samples obtained from publicly available gene expression data, an extension of a previously published methodology. We use the context-specific gene regulatory networks to classify the tumors into clinically relevant subgroups, and provide candidates for a finer sub-grouping of the previously known intrinsic tumors with a focus on Basal-like tumors. Our analysis of pathway enrichment in the key contexts provides an insight into the biological mechanism underlying the identified subtypes of breast cancer. CONCLUSIONS: The use of context-specific gene regulatory networks to identify biological contexts from heterogenous breast cancer data set was able to identify genomic drivers for subgroups within the previously reported intrinsic subtypes. These subgroups (contexts) uphold the clinical relevant features for the intrinsic subtypes and were associated with increased survival differences compared to the intrinsic subtypes. We believe our computational approach led to the generation of novel rationalized hypotheses to explain mechanisms of disease progression within sub-contexts of breast cancer that could be therapeutically exploited once validated.

Genomic alterations in histopathologically normal breast tissue from BRCA1 mutation carriers may be caused by BRCA1 haploinsufficiency.

Multiple biopsies of normal breast tissue from 10 BRCA1 mutation carriers have been analyzed using array-based comparative genomic hybridization. Normal breast tissue from five age-matched control subjects without a family history of breast cancer was included for reference purposes. We repeatedly found multiple low copy number aberrations at a significantly higher frequency in histopathologically normal tissue from BRCA1 mutation carriers than in normal control tissue. Some of these aberrations were similar across samples from different patients and linked to biological functions such as transcriptional regulation and DNA binding. We also observed a high degree of genomic heterogeneity between samples from the same patient, suggestive of tissue heterogeneity and etiological clonality in the breast epithelium. We show that neither loss of heterozygosity nor promoter methylation of the wild-type BRCA1 allele is the predominant mechanistic origin of the observed genomic instability. Instead, we propose that haploinsufficiency of BRCA1 might be the underlying cause responsible for initiation of breast cancer in these predisposed women, making cells vulnerable to mitotic recombination. We also propose that loss of ERalpha expression is preceded by genetic instability in the initiation of BRCA1-dependent tumorigenesis, indicating that the breast epithelium of BRCA1 mutation carriers may initially be estrogen-responsive. Our results imply that genomic instability instigated by BRCA1 haploinsufficiency may be required for breast cancer initiation in BRCA1 mutation carriers. Finding molecular markers of tumor initiation and progression, for the potential use in early disease detection, may be of great clinical importance for the improved management of at-risk women.

Dephosphorylation of YB-1 is Required for Nuclear Localisation During G2 Phase of the Cell Cycle.

Elevated levels of nuclear Y-box binding protein 1 (YB-1) are linked to poor prognosis in cancer. It has been proposed that entry into the nucleus requires specific proteasomal cleavage. However, evidence for cleavage is contradictory and high YB-1 levels are prognostic regardless of cellular location. Here, using confocal microscopy and mass spectrometry, we find no evidence of specific proteolytic cleavage. Doxorubicin treatment, and the resultant G2 arrest, leads to a significant increase in the number of cells where YB-1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Live cell imaging reveals that the location of YB1 is linked to progression through the cell cycle. Primarily perinuclear during G1 and S phases, YB-1 enters the nucleus as cells transition through late G2/M and exits at the completion of mitosis. Atomistic modelling and molecular dynamics simulations show that dephosphorylation of YB1 at serine residues 102, 165 and 176 increases the accessibility of the nuclear localisation signal (NLS). We propose that this conformational change facilitates nuclear entry during late G2/M. Thus, the phosphorylation status of YB1 determines its cellular location.

CART-WHEEL.org: An Ethically Approved Online Database for Patient-Entered Data to Facilitate Rare Cancer Research.

PURPOSE: Rare cancers are challenging for researchers, as clinicians and scientists have difficulty recruiting sufficient patient cases to power studies appropriately. Likewise, patients often are frustrated by a lack of specific information or evidence base for their cancer and, although eager to participate in research, have limited opportunities. We established CART-WHEEL.org, an online patient-entered database, to directly engage patients in the research process, collect rare cancer data, and facilitate their entry into additional research. PATIENTS AND METHODS: Patients access CART-WHEEL.org directly online. Clinical information is collected from users via a streamlined questionnaire developed collaboratively with consumer groups to ensure accessibility and relevance. Data collected include the following: patient demographics, comorbidities, and risk factors and tumor diagnostic, biomarker, and treatment history. Patients can download a medical summary for personal use; consent for research use of data; and indicate willingness to be contacted about other research or clinical trials. We describe data collected to date and its validation, and we provide examples of how CART-WHEEL.org can facilitate rare cancer research. RESULTS: From January 2010 to March 2018, 558 patients provided consent and entered their rare cancer data. One hundred distinct rare tumor types and patients from 22 countries were included. Validation of data entered by 21 patients with sarcoma against a hospital database demonstrated accuracy sufficient to facilitate future research in key fields, such as tumor site (95%) and histopathologic diagnosis (90%). Examples of CART-WHEEL-based disease-specific projects, subsequent recruitment to other rare cancer projects, and rare cancer patient cases of interest are described. CONCLUSIONS: Online platforms like CART-WHEEL.org can engage consumers directly, facilitating collection of patient-entered rare cancer data for hypothesis generation, and connect patients with researchers to enable specific rare cancer research and clinical trials.

The fibroblast growth factor-inducible 14 receptor is highly expressed in HER2-positive breast tumors and regulates breast cancer cell invasive capacity.

Genomic characterization is beginning to define a molecular taxonomy for breast cancer; however, the molecular basis of invasion and metastasis remains poorly understood. We report a pivotal role for the fibroblast growth factor-inducible 14 (Fn14) receptor in this process. We examined whether Fn14 and its ligand tumor necrosis factor-like weak inducer of apoptosis (TWEAK) were expressed in breast tumors and whether deregulation of Fn14 levels affected malignant behavior of breast cancer cell lines. Analysis of TWEAK and Fn14 in publicly available gene expression data indicated that high Fn14 expression levels significantly correlated with several poor prognostic indicators (P < 0.05). Fn14 expression was highest in the HER2-positive/estrogen receptor-negative (HER2(+)/ER(-)) intrinsic subtype (P = 0.0008). An association between Fn14 and HER2 expression in breast tumors was confirmed by immunohistochemistry. Fn14 levels were elevated in invasive, ER(-) breast cancer cell lines. Overexpression of Fn14 in weakly invasive MCF7 and T47D cells resulted in a marked induction of invasion and activation of nuclear factor-kappaB (NF-kappaB) signaling. Ectopic expression of Fn14tCT, a Fn14 deletion mutant that cannot activate NF-kappaB signaling, was not able to induce invasion. Moreover, ectopic expression of Fn14tCT in highly invasive MDA-MB-231 cells reduced their invasive capability. RNA interference-mediated inhibition of Fn14 expression in both MDA-MB-231 and MDA-MB-436 cells reduced invasion. Expression profiling of the Fn14-depleted cells revealed deregulation of NF-kappaB activity. Our findings support a role for Fn14-mediated NF-kappaB pathway activation in breast tumor invasion and metastasis.

Long Non-coding RNA ANRIL in the Nucleus Associates With Periostin Expression in Breast Cancer.

The long non-coding RNA (LncRNA) antisense RNA in the INK4 locus (ANRIL) is overexpressed in several cancers including breast cancer. To better understand the role of ANRIL in breast cancer this study investigated where ANRIL was expressed in breast tumors using in situ hybridization by RNAscope. Additional RNAscope assays for IL6, CCL2, and POSTN were used to establish whether ANRIL correlated with increased tumor promoting cytokines. Breast tumors with ANRIL over expressed from real-time quantitative (RT-q) PCR assays were selected for analysis using RNAscope. All tumors showed ANRIL expression in malignant cells, but amongst tumors ANRIL showed different subcellular locations with 56% of tumors with ANRIL only in the nucleus, 16% with ANRIL only in the cytoplasm and 28% with ANRIL in both the nucleus and cytoplasm. Cases with nuclear ANRIL were positively correlated with POSTN expression in malignant cells (ρ = 0.57, P = 0.0086), and no correlation was found between ANRIL and IL6 or CCL2. Reduced POSTN was also found using siRNA to ANRIL in MDA-MB-231 and MCF7 breast cancer cells. These data indicate that ANRIL is expressed in malignant breast cells, and suggest its subcellular location may indicate its function in cancer progression.

Role of TWEAK and Fn14 in tumor biology.

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TWEAK) was initially described in a 1997 publication co-authored by investigators from the biotechnology company Biogen (now Biogen-Idec) and the University of Geneva. Four years later, researchers at the biotechnology company Immunex (now part of Amgen) reported the cloning and characterization of the human TWEAK receptor. A sequence database search revealed that the predicted TWEAK receptor amino acid sequence was identical to that of fibroblast growth factor-inducible 14 (Fn14), a small transmembrane protein described one year earlier in a publication from investigators at the American Red Cross Holland Laboratory. Recent studies have revealed that the TWEAK-Fn14 ligand-receptor pair likely plays an important role in a variety of cellular processes and in the pathogenesis of several human diseases, including atherosclerosis, stroke, rheumatoid arthritis and cancer. In this paper, we first summarize the general properties of these two proteins and then review the available data implicating TWEAK and Fn14 in multiple aspects of tumor biology.

Microarrays in breast cancer research and clinical practice--the future lies ahead.

Molecular profiling for classification and prognostic purposes has demonstrated that the genetic signatures of tumors contain information regarding biological properties as well as clinical behavior. This review highlights the progress that has been made in the field of gene expression profiling of human breast cancer. Breast cancer has become one of the most intensely studied human malignancies in the genomic era; several hundred papers over the last few years have investigated various clinical and biological aspects of human breast cancer using high-throughput molecular profiling techniques. Given the grossly heterogeneous nature of the disease and the lack of robust conventional markers for disease prediction, prognosis, and response to treatment, the notion that a transcriptional profile comprising multiple genes, rather than any single gene or other parameter, will be more predictive of tumor behavior is both appealing and reasonable. Promising results have emerged from these studies, correlating gene expression profiles with prognosis, recurrence, metastatic potential, therapeutic response, as well as biological and functional aspects of the disease. Clearly, the integration of genomic approaches into the clinic lies in the near future, but prospective studies based on larger patient cohorts representing the whole spectrum of breast cancer, oncogenic pathway-based studies, attendant care in bioinformatic analyses and validation studies are needed before the full promise of gene expression profiling can be realized in the clinical setting.

The gene expression response of breast cancer to growth regulators: patterns and correlation with tumor expression profiles.

The effects of hormone and growth factor signaling on gene expression contribute significantly to breast tumorigenesis and disease progression; however, the targets of signaling networks associated with deregulated growth are not well understood. We defined the dynamic transcriptional effects elicited in MCF7, T-47D, and MDA-MB-436 breast cancer cell lines by nine regulators of growth and differentiation (17beta-estradiol, antiestrogens fulvestrant and tamoxifen, progestin R5020, antiprogestin RU486, all-trans-retinoic acid, epidermal growth factor, mitogen-activated protein/extracellular signal-regulated kinase 1/2 inhibitor U0126 and phorbol ester 12-O-tetradecanoylphorbol-13-acetate) and compared the patterns of gene regulation to published tumor expression profiles. The complex pattern of response to these agents revealed unexpected relationships between their effects, including a profound overlap in genes regulated by both steroids and epidermal growth factor, and striking overlaps between fulvestrant and all-trans-retinoic acid. Estrogen-responsive genes could be divided into two major clusters, only one of which is associated with cell proliferation. Gene ontology analysis was used to highlight functionally distinct biological responses to different mitogens. Significant correlations were identified between several clusters of drug-responsive genes and genes that discriminate estrogen receptor status or disease outcome in patient samples. The majority of estrogen receptor status discriminators were not responsive in our dataset and are therefore likely to reflect underlying differences in histogenesis and disease progression rather than growth factor signaling. This article highlights the overall impact at the gene expression level of diverse regulators of breast cancer growth and links the behavior of breast cancer cells in culture to important clinical properties of human breast tumors.

Integration of Downstream Signals of Insulin-like Growth Factor-1 Receptor by Endoplasmic Reticulum Stress for Estrogen-Induced Growth or Apoptosis in Breast Cancer Cells.

UNLABELLED: Estrogen (E2) exerts a dual function on E2-deprived breast cancer cells, with both initial proliferation and subsequent induction of stress responses to cause apoptosis. However, the mechanism by which E2 integrally regulates cell growth or apoptosis-associated pathways remains to be elucidated. Here, E2 deprivation results in many alterations in stress-responsive pathways. For instance, E2-deprived breast cancer cells had higher basal levels of stress-activated protein kinase, c-Jun N-terminal kinase (JNK), compared with wild-type MCF-7 cells. E2 treatment further constitutively activated JNK after 24 hours. However, inhibition of JNK (SP600125) was unable to abolish E2- induced apoptosis, whereas SP600125 alone arrested cells at the G2 phase of the cell cycle and increased apoptosis. Further examination showed that inhibition of JNK increased gene expression of TNFα and did not effectively attenuate expression of apoptosis-related genes induced by E2. A notable finding was that E2 regulated both JNK and Akt as the downstream signals of insulin-like growth factor-1 receptor (IGFIR)/PI3K, but with distinctive modulation patterns: JNK was constitutively activated, whereas Akt and Akt-associated proteins, such as PTEN and mTOR, were selectively degraded. Endoplasmic reticulum-associated degradation (ERAD) was involved in the selective protein degradation. These findings highlight a novel IGFIR/PI3K/JNK axis that plays a proliferative role during the prelude to E2-induced apoptosis and that the endoplasmic reticulum is a key regulatory site to decide cell fate after E2 treatment. IMPLICATIONS: This study provides a new rationale for further exploration of E2-induced apoptosis to improve clinical benefit.