Search

The International Tamoxifen Pharmacogenomics Consortium was established to address the controversy regarding cytochrome P450 2D6 (CYP2D6) status and clinical outcomes in tamoxifen therapy. We performed a meta-analysis on data from 4,973 tamoxifen-treated patients (12 globally distributed sites). Using strict eligibility requirements (postmenopausal women with estrogen receptor-positive breast cancer, receiving 20 mg/day tamoxifen for 5 years, criterion 1); CYP2D6 poor metabolizer status was associated with poorer invasive disease-free survival (IDFS: hazard ratio = 1.25; 95% confidence interval = 1.06, 1.47; P = 0.009). However, CYP2D6 status was not statistically significant when tamoxifen duration, menopausal status, and annual follow-up were not specified (criterion 2, n = 2,443; P = 0.25) or when no exclusions were applied (criterion 3, n = 4,935; P = 0.38). Although CYP2D6 is a strong predictor of IDFS using strict inclusion criteria, because the results are not robust to inclusion criteria (these were not defined a priori), prospective studies are necessary to fully establish the value of CYP2D6 genotyping in tamoxifen therapy.

Tamoxifen (TAM) has both cytostatic and cytotoxic properties for breast cancer. TAM engaged mitochondrial estrogen receptor beta (ERβ) as an antagonist in MCF7-BK cells, increasing reactive oxygen species (ROS) concentrations from the mitochondria that were required for cytotoxicity. In part, this derived from TAM downregulating manganese superoxide dismutase (MnSOD) activity by causing the nitrosylation of tyrosine 34, thereby increasing ROS. ROS-activated protein kinase C delta and c-jun N-terminal kinases, resulting in the mitochondrial translocation of Bax and cytochrome C release. Interestingly, TAM failed to cause high ROS levels or induce cell death in MCF7-BK-TR cells due to stimulation of MnSOD activity through agonistic effects at mitochondrial ERβ. In several mouse xenograft models, lentiviral shRNA-induced knockdown of MnSOD caused tumors that grew in the presence of TAM to undergo substantial apoptosis. Tumor MnSOD and mitochondrial ERβ are therefore targets for therapeutic intervention to reverse TAM resistance and enhance a cell death response.

Methylation has been shown to play an important role in the down-regulation of oestrogen receptors (ER) in breast cancer cells. One critical question that remains unclear is whether methylation can account for the loss of ER expression in cells derived from an ER-positive cell line. This laboratory has established an in vitro cell system using long-term growth of human ER-positive breast cancer cell line T47D in oestrogen-free medium. A clonal cell line, T47D:C4:2 (C4:2), has been characterized. Unlike T47D:A18 (A18), which is a T47D line maintained in oestrogen medium, C4:2 has lost the expression of ER and hormone responsiveness. DNA fingerprinting and restriction fragment length polymorphism (RFLP) analysis results confirmed that C4:2 was of the same lineage as A18. These cell lines provide an invaluable system to study the mechanism of ER expression and regulatory pathways leading to hormone-independent growth. The results here clearly demonstrate that the ER CpG island in C4:2 cells remains unmethylated. The loss of ER in the cell line must be due to mechanisms other than methylation. We also evaluated the ER CpG island in the MDA-MB-231:10A (10A) cell line, which is a clone from the MDA-MB-231 line obtained from ATCC and the DNA from the MDA-MB-231 cell line used in the original report. Unlike the cell line from the report, which showed a full methylation pattern in the island, the 10A line only showed a partial methylation pattern in the CpG island. Possible mechanisms pertaining to the heterogeneous methylation pattern of the ER CpG island in the breast cancer cells are discussed.

Background & Aims: Many liver diseases are driven by inflammation, but imaging to non-invasively diagnose and quantify liver inflammation has been underdeveloped. The inflammatory liver microenvironment is aberrantly oxidising owing in part to reactive oxygen species generated by myeloid leucocytes. We hypothesised that magnetic resonance imaging using the oxidatively activated probe Fe-PyC3A will provide a non-invasive biomarker of liver inflammation., Methods: A mouse model of drug-induced liver injury was generated through intraperitoneal injection of a hepatoxic dose of acetaminophen. A mouse model of steatohepatitis was generated via a choline-deficient, l-amino acid defined high-fat diet (CDAHFD). Images were acquired dynamically before and after intravenous injection of Fe-PyC3A. The contrast agent gadoterate meglumine was used as a non-oxidatively activated negative control probe in mice fed CDAHFD. The (post-pre) Fe-PyC3A injection change in liver vs. muscle contrast-to-noise ratio (ΔCNR) recorded 2 min post-injection was correlated with liver function test values, histologic scoring assigned using the NASH Clinical Research Network criteria, and intrahepatic myeloid leucocyte composition determined by flow cytometry., Results: For mice receiving i.p. injections of acetaminophen, intrahepatic neutrophil composition correlated poorly with liver test values but positively and significantly with ΔCNR (r = 0.64, p <0.0001). For mice fed CDAHFD, ΔCNR generated by Fe-PyC3A in the left lobe was significantly greater in mice meeting histologic criteria strongly associated with a diagnosis NASH compared to mice where histology was consistent with likely non-NASH ( p  = 0.0001), whereas no differential effect was observed using gadoterate meglumine. In mice fed CDAHFD, ΔCNR did not correlate strongly with fractional composition of any specific myeloid cell subpopulation as determined by flow cytometry., Conclusions: Magnetic resonance imaging using Fe-PyC3A merits further evaluation as a non-invasive biomarker for liver inflammation., Impact and Implications: Non-invasive tests to diagnose and measure liver inflammation are underdeveloped. Inflammatory cells such as neutrophils release reactive oxygen species which creates an inflammatory liver microenvironment that can drive chemical oxidation. We recently invented a new class of magnetic resonance imaging probe that is made visible to the scanner only after chemical oxidation. Here, we demonstrate how this imaging technology could be applied as a non-invasive biomarker for liver inflammation., Competing Interests: EMG holds equity in, and receives consulting income from Reveal Pharmaceuticals, and receives consulting income from Collagen Medical, LLC. PC holds equity in, and receives consulting income from Collagen Medical LLC, holds equity in Reveal Pharmaceuticals, and has research funding from Takeda, Pliant Therapeutics, and Janssen. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Authors.)

Many factors, including reproductive hormones, have been linked to a woman's risk of developing breast cancer (BC). We reviewed the literature regarding the relationship between ovulatory menstrual cycles (MCs) and BC risk. Physiological variations in the frequency of MCs and interference with MCs through genetic variations, pathological conditions and or pharmaceutical interventions revealed a strong link between BC risk and the lifetime number of MCs. A substantial reduction in BC risk is observed in situations without MCs. In genetic or transgender situations with normal female breasts and estrogens, but no progesterone (P4), the incidence of BC is very low, suggesting an essential role of P4. During the MC, P4 has a strong proliferative effect on normal breast epithelium, whereas estradiol (E2) has only a minimal effect. The origin of BC has been strongly linked to proliferation associated DNA replication errors, and the repeated stimulation of the breast epithelium by P4 with each MC is likely to impact the epithelial mutational burden. Long-lived cells, such as stem cells, present in the breast epithelium, can carry mutations forward for an extended period of time, and studies show that breast tumors tend to take decades to develop before detection. We therefore postulate that P4 is an important factor in a woman's lifetime risk of developing BC, and that breast tumors arising during hormonal contraception or after menopause, with or without menopausal hormone therapy, are the consequence of the outgrowth of pre-existing neoplastic lesions, eventually stimulated by estrogens and some progestins., (© 2023. The Author(s).)

Antiestrogen therapy of breast cancer has been a "gold standard" of treatment of estrogen receptor (ER)-positive breast cancer for decades. Resistance to antiestrogen therapy may develop, however, a vulnerability in long-term estrogen deprived (LTED) breast cancer cells was discovered. LTED breast cancer cells may undergo estrogen-induced apoptosis within a week of treatment with estrogen in vitro . This phenomenon has been also validated in vivo and in the clinic. The molecular ER-mediated mechanism of action of estrogen-induced apoptosis was deciphered, however, the relationship between the structure of estrogenic ligands and the activity of the ER in LTED breast cancer cells remained a mystery until recently. In this review we provide an overview of the structure-activity relationship of various estrogens with different chemical structures and the modulation of estrogen-induced apoptosis in LTED breast cancer cells resistant to antihormone therapy. We provide analysis of evidence gathered over more than a decade of structure-activity relationship studies by our group on the role of the change in the conformation of the estrogen receptor and the biological activities of different classes of estrogens and the receptor as well in LTED breast cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Maximov, Fan, Abderrahman, Curpan and Jordan.)

The discovery of 17β-estradiol (E 2 )-induced apoptosis has clinical relevance. Mechanistically, E 2 over activates nuclear estrogen receptor α that results in stress responses. The unfolded protein response (UPR) is initiated by E 2 in the endoplasmic reticulum after hours of treatment in endocrine-resistant breast cancer cells, thereby activating three UPR sensors-PRK-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6) with different functions. Specifically, PERK plays a critical role in induction of apoptosis whereas IRE1α and ATF6 are involved in the endoplasmic reticulum stress-associated degradation (ERAD) of PI3K/Akt/mTOR pathways. In addition to attenuating protein translation, PERK increases the DNA-binding activity of NF-κB and subsequent TNFα expression. In addition, PERK communicates with the mitochondria to regulate oxidative stress at mitochondria-associated endoplasmic reticulum membranes (MAM). Furthermore, PERK is a component enriched in MAMs that interacts with multifunctional MAM-tethering proteins and integrally modulates the exchange of metabolites such as lipids, reactive oxygen species (ROS), and Ca 2+ at contact sites. MAMs are also critical sites for the initiation of autophagy to remove defective organelles and misfolded proteins through specific regulatory proteins. Thus, PERK conveys signals from nucleus to these membrane-structured organelles that form an interconnected network to regulate E 2 -induced apoptosis. Herein, we address the mechanistic progress on how PERK acts as a multifunctional molecule to commit E 2 to inducing apoptosis in endocrine-resistant breast cancer., (©2021 The Authors; Published by the American Association for Cancer Research.)

Background & Aims: During liver fibrosis, tissue repair mechanisms replace necrotic tissue with highly stabilized extracellular matrix proteins. Extracellular matrix stabilization influences the speed of tissue recovery. Here, we studied the expression and function of peroxidasin (PXDN), a peroxidase that uses hydrogen peroxide to cross-link collagen IV during liver fibrosis progression and regression., Methods: Mouse models of liver fibrosis and cirrhosis patients were analyzed for the expression of PXDN in liver and serum. Pxdn -/- and Pxdn +/+ mice were either treated with carbon tetrachloride for 6 weeks to generate toxin-induced fibrosis or fed with a choline-deficient L-amino acid-defined high-fat diet for 16 weeks to create nonalcoholic fatty liver disease fibrosis. Liver histology, quantitative real-time polymerase chain reaction, collagen content, flowcytometry and immunostaining of immune cells, RNA-sequencing, and liver function tests were analyzed. In vivo imaging of liver reactive oxygen species (ROS) was performed using a redox-active iron complex, Fe-PyC3A., Results: In human and mouse cirrhotic tissue, PXDN is expressed by stellate cells and is secreted into fibrotic areas. In patients with nonalcoholic fatty liver disease, serum levels of PXDN increased significantly. In both mouse models of liver fibrosis, PXDN deficiency resulted in elevated monocyte and pro-fibrolysis macrophage recruitment into fibrotic bands and caused decreased accumulation of cross-linked collagens. In Pxdn -/- mice, collagen fibers were loosely organized, an atypical phenotype that is reversible upon macrophage depletion. Elevated ROS in Pxdn -/- livers was observed, which can result in activation of hypoxic signaling cascades and may affect signaling pathways involved in macrophage polarization such as TNF-a via NF-kB. Fibrosis resolution in Pxdn -/- mice was associated with significant decrease in collagen content and improved liver function., Conclusion: PXDN deficiency is associated with increased ROS levels and a hypoxic liver microenvironment that can regulate recruitment and programming of pro-resolution macrophages. Our data implicate the importance of the liver microenvironment in macrophage programming during liver fibrosis and suggest a novel pathway that is involved in the resolution of scar tissue., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Patients with long-term estrogen-deprived breast cancer, after resistance to tamoxifen or aromatase inhibitors develops, can experience tumor regression when treated with estrogens. Estrogen's antitumor effect is attributed to apoptosis via the estrogen receptor (ER). Estrogen treatment can have unpleasant gynecologic and nongynecologic adverse events; thus, the development of safer estrogenic agents remains a clinical priority. Here, we study synthetic selective estrogen mimics (SEM) BMI-135 and TTC-352, and the naturally occurring estrogen estetrol (E 4 ), which are proposed as safer estrogenic agents compared with 17β-estradiol (E 2 ), for the treatment of endocrine-resistant breast cancer. TTC-352 and E 4 are being evaluated in breast cancer clinical trials. Cell viability assays, real-time PCR, immunoblotting, ERE DNA pulldowns, mass spectrometry, X-ray crystallography, docking and molecular dynamic simulations, live cell imaging, and Annexin V staining were conducted in 11 biologically different breast cancer models. Results were compared with the potent full agonist E 2 , less potent full agonist E 4 , the benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. We report ERα's regulation and coregulators' binding profiles with SEMs and E 4 We describe TTC-352's pharmacology as a weak full agonist and antitumor molecular mechanisms. This study highlights TTC-352's benzothiophene scaffold that yields an H-bond with Glu353, which allows Asp351-to-helix 12 (H12) interaction, sealing ERα's ligand-binding domain, recruiting E 2 -enriched coactivators, and triggering rapid ERα-induced unfolded protein response (UPR) and apoptosis, as the basis of its anticancer properties. BPTPE's phenolic OH yields an H-Bond with Thr347, which disrupts Asp351-to-H12 interaction, delaying UPR and apoptosis and increasing clonal evolution risk., (©2020 American Association for Cancer Research.)

Long-term estrogen deprivation (LTED) with tamoxifen (TAM) or aromatase inhibitors leads to endocrine-resistance, whereby physiologic levels of estrogen kill breast cancer (BC). Estrogen therapy is effective in treating patients with advanced BC after resistance to TAM and aromatase inhibitors develops. This therapeutic effect is attributed to estrogen-induced apoptosis via the estrogen receptor (ER). Estrogen therapy can have unpleasant gynecologic and nongynecologic adverse events. Here, we study estetrol (E 4 ) and a model Selective Human ER Partial Agonist (ShERPA) BMI-135. Estetrol and ShERPA TTC-352 are being evaluated in clinical trials. These agents are proposed as safer estrogenic candidates compared with 17 β -estradiol (E 2 ) for the treatment of endocrine-resistant BC. Cell viability assays, real-time polymerase chain reaction, luciferase reporter assays, chromatin immunoprecipitation, docking and molecular dynamics simulations, human unfolded protein response (UPR) RT 2 PCR profiler arrays, live cell microscopic imaging and analysis, and annexin V staining assays were conducted. Our work was done in eight biologically different human BC cell lines and one human endometrial cancer cell line, and results were compared with full agonists estrone, E 2 , and estriol, a benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. Our study shows the pharmacology of E 4 and BMI-135 as less-potent full-estrogen agonists as well as their molecular mechanisms of tumor regression in LTED BC through triggering a rapid UPR and apoptosis. Our work concludes that the use of a full agonist to treat BC is potentially superior to a partial agonist given BPTPE's delayed induction of UPR and apoptosis, with a higher probability of tumor clonal evolution and resistance. SIGNIFICANCE STATEMENT: Given the unpleasant gynecologic and nongynecologic adverse effects of estrogen treatment, the development of safer estrogens for endocrine-resistant breast cancer (BC) treatment and hormone replacement therapy remains a priority. The naturally occurring estrogen estetrol and Selective Human Estrogen-Receptor Partial Agonists are being evaluated in endocrine-resistant BC clinical trials. This work provides a comprehensive evaluation of their pharmacology in numerous endocrine-resistant BC models and an endometrial cancer model and their molecular mechanisms of tumor regression through the unfolded protein response and apoptosis., Competing Interests: Authors disclose no conflicts of interest., (Copyright © 2020 The Author(s).)

The Women's Health Initiative (WHI) was designed to evaluate the benefits of hormone replacement therapy. The primary goal was to establish the value of synthetic progestin and estrogen or estrogen alone to reduce the risk of coronary heart disease (CHD). The estrogen/synthetic progestin trial was stopped at 5.2 years and the estrogen trial was stopped after 6.8 years. Although the estrogen/synthetic progestin trial was stopped for the anticipated rise in the risk of breast cancer, the estrogen trial was stopped for elevation of strokes. Women taking estrogen/synthetic progestin or estrogen alone had no benefit from a reduction in CHD. Paradoxically, there was a decrease in breast cancer incidence in the estrogen trial. The decrease in breast cancer was sustained. The elevation of breast cancer in the estrogen/synthetic progestin trial was also sustained a decade after stopping treatment. Evidence is presented to explain the paradoxical sustained decrease in breast cancer with estrogen and the mechanism for the reversal of breast cancer incidence and mortality with the mixed synthetic progestin/glucocorticoid actions of the synthetic progestin used with estrogen in women with an intact uterus. The fact that the WHI study had an estrogen deprivation gap of at least 5 years, introduced an experimental biological dimension not observed in medical practice using progestin/estrogen hormone replacement. The evidence presented confirms the known human cancer biology of estrogen action., (©2020 American Association for Cancer Research.)

Purpose: To evaluate the prognostic value of posttreatment fibrosis in human PDAC patients, and to compare a type I collagen targeted MRI probe, CM-101, to the standard contrast agent, Gd-DOTA, for their abilities to identify FOLFIRINOX-induced fibrosis in a murine model of PDAC., Experimental Design: Ninety-three chemoradiation-treated human PDAC samples were stained for fibrosis and outcomes evaluated. For imaging, C57BL/6 and FVB mice were orthotopically implanted with PDAC cells and FOLFIRINOX was administered. Mice were imaged with Gd-DOTA and CM-101., Results: In humans, post-chemoradiation PDAC tumor fibrosis was associated with longer overall survival (OS) and disease-free survival (DFS) on multivariable analysis (OS P = 0.028, DFS P = 0.047). CPA increased the prognostic accuracy of a multivariable logistic regression model comprised of previously established PDAC risk factors [AUC CPA (-) = 0.76, AUC CPA (+) = 0.82]. In multiple murine orthotopic PDAC models, FOLFIRINOX therapy reduced tumor weight ( P < 0.05) and increased tumor fibrosis by collagen staining ( P < 0.05). CM-101 MR signal was significantly increased in fibrotic tumor regions. CM-101 signal retention was also increased in the more fibrotic FOLFIRINOX-treated tumors compared with untreated controls ( P = 0.027), consistent with selective probe binding to collagen. No treatment-related differences were observed with Gd-DOTA imaging., Conclusions: In humans, post-chemoradiation tumor fibrosis is associated with OS and DFS. In mice, our MR findings indicate that translation of collagen molecular MRI with CM-101 to humans might provide a novel imaging technique to monitor fibrotic response to therapy to assist with prognostication and disease management., (©2020 American Association for Cancer Research.)

High-dose synthetic estrogen therapy was the standard treatment of advanced breast cancer for three decades until the discovery of tamoxifen. A range of substituted triphenylethylene synthetic estrogens and diethylstilbestrol were used. It is now known that low doses of estrogens can cause apoptosis in long-term estrogen deprived (LTED) breast cancer cells resistant to antiestrogens. This action of estrogen can explain the reduced breast cancer incidence in postmenopausal women over 60 who are taking conjugated equine estrogens and the beneficial effect of low-dose estrogen treatment of patients with acquired aromatase inhibitor resistance in clinical trials. To decipher the molecular mechanism of estrogens at the estrogen receptor (ER) complex by different types of estrogens-planar [17 β -estradiol (E 2 )] and angular triphenylethylene (TPE) derivatives-we have synthesized a small series of compounds with either no substitutions on the TPE phenyl ring containing the antiestrogenic side chain of endoxifen or a free hydroxyl. In the first week of treatment with E 2 the LTED cells undergo apoptosis completely. By contrast, the test TPE derivatives act as antiestrogens with a free para-hydroxyl on the phenyl ring that contains an antiestrogenic side chain in endoxifen. This inhibits early E 2 -induced apoptosis if a free hydroxyl is present. No substitution at the site occupied by the antiestrogenic side chain of endoxifen results in early apoptosis similar to planar E 2 The TPE compounds recruit coregulators to the ER differentially and predictably, leading to delayed apoptosis in these cells. SIGNIFICANCE STATEMENT: In this paper we investigate the role of the structure-function relationship of a panel of synthetic triphenylethylene (TPE) derivatives and a novel mechanism of estrogen-induced cell death in breast cancer, which is now clinically relevant. Our study indicates that these TPE derivatives, depending on the positioning of the hydroxyl groups, induce various conformations of the estrogen receptor's ligand-binding domain, which in turn produces differential recruitment of coregulators and subsequently different apoptotic effects on the antiestrogen-resistant breast cancer cells., (Copyright © 2020 The Author(s).)

Aim: Tamoxifen (TAM) resistance remains a clinical issue in breast cancer. The authors previously reported that 15-hydroxyprostaglandin dehydrogenase ( HPGD ) was significantly downregulated in tamoxifen-resistant (TAMr) breast cancer cell lines. Here, the authors investigated the relationship between HPGD expression, TAM resistance and prediction of outcome in breast cancer., Methods: HPGD overexpression and silencing studies were performed in isogenic TAMr and parental human breast cancer cell lines to establish the impact of HPGD expression on TAM resistance. HPGD expression and clinical outcome relationships were explored using immunohistochemistry and in silico analysis., Results: Restoration of HPGD expression and activity sensitised TAMr MCF-7 cells to TAM and 17β-oestradiol, whilst HPGD silencing in parental MCF-7 cells reduced TAM sensitivity. TAMr cells released more prostaglandin E 2 (PGE 2 ) than controls, which was reduced in TAMr cells stably transfected with HPGD . Exogenous PGE 2 signalled through the EP4 receptor to reduce breast cancer cell sensitivity to TAM. Decreased HPGD expression was associated with decreased overall survival in ERα-positive breast cancer patients., Conclusions: HPGD downregulation in breast cancer is associated with reduced response to TAM therapy via PGE 2 -EP4 signalling and decreases patient survival. The data offer a potential target to develop combination therapies that may overcome acquired tamoxifen resistance., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest.

Our clinically relevant finding is that glucocorticoids block estrogen (E 2 )-induced apoptosis in long-term E 2 -deprived (LTED) breast cancer cells. However, the mechanism remains unclear. Here, we demonstrated that E 2 widely activated adipose inflammatory factors such as fatty acid desaturase 1 (FADS1), IL6, and TNFα in LTED breast cancer cells. Activation of glucocorticoid receptor (GR) by the synthetic glucocorticoid dexamethasone upregulated FADS1 and IL6, but downregulated TNFα expression. Furthermore, dexamethasone was synergistic or additive with E 2 in upregulating FADS1 and IL6 expression, whereas it selectively and constantly suppressed TNFα expression induced by E 2 in LTED breast cancer cells. Regarding regulation of endoplasmic reticulum stress, dexamethasone effectively blocked activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) by E 2 , but it had no inhibitory effects on inositol-requiring protein 1 alpha (IRE1α) expression increased by E 2 Consistently, results from reverse-phase protein array (RPPA) analysis demonstrated that dexamethasone could not reverse IRE1α-mediated degradation of PI3K/Akt-associated signal pathways activated by E 2 Unexpectedly, activated GR preferentially repressed nuclear factor-κB (NF-κB) DNA-binding activity and expression of NF-κB-dependent gene TNFα induced by E 2 , leading to the blockade of E 2 -induced apoptosis. Together, these data suggest that trans-suppression of NF-κB by GR in the nucleus is a fundamental mechanism thereby blocking E 2 -induced apoptosis in LTED breast cancer cells. This study provided an important rationale for restricting the clinical use of glucocorticoids, which will undermine the beneficial effects of E 2 -induced apoptosis in patients with aromatase inhibitor-resistant breast cancer., (©2019 American Association for Cancer Research.)

Approximately 30% of aromatase-inhibitor-resistant, estrogen receptor-positive patients with breast cancer benefit from treatment with estrogen. This enigmatic estrogen action is not well understood and how it occurs remains elusive. Studies indicate that the unfolded protein response and apoptosis pathways play important roles in mediating estrogen-triggered apoptosis. Using MCF7:5C cells, which mimic aromatase inhibitor resistance, and are hypersensitive to estrogen as evident by induction of apoptosis, we define increased global protein translational load as the trigger for estrogen-induced apoptosis. The protein kinase RNA-like endoplasmic reticulum kinase pathway was activated followed by increased phosphorylation of eukaryotic initiation factor-2 alpha (eIF2α). These actions block global protein translation but preferentially allow high expression of specific transcription factors, such as activating transcription factor 4 and C/EBP homologous protein that facilitate apoptosis. Notably, we recapitulated this phenotype of MCF7:5C in two other endocrine therapy-resistant cell lines (MCF7/LCC9 and T47D:A18/4-OHT) by increasing the levels of phospho-eIF2α using salubrinal to pharmacologically inhibit the enzymes responsible for dephosphorylation of eIF2α, GADD34, and CReP. RNAi-mediated ablation of these genes induced apoptosis that used the same signaling as salubrinal treatment. Moreover, combining 4-hydroxy tamoxifen with salubrinal enhanced apoptotic potency. IMPLICATIONS: These results not only elucidate the mechanism of estrogen-induced apoptosis but also identify a drugable target for potential therapeutic intervention that can mimic the beneficial effect of estrogen in some breast cancers., (©2019 American Association for Cancer Research.)

The recent past has seen impressive progress in the treatment of various malignancies using immunotherapy. One of the most promising approaches involves immune checkpoint inhibitors. However, the clinical results with these agents have demonstrated variability in the response. Pancreatic cancer, in particular, has proven resistant to initial immunotherapy approaches. Here, we describe an alternative strategy that relies on combining gemcitabine and a novel programmed death-ligand 1 (PD-L1) inhibitor, termed MN-siPDL1. MN-siPDL1 incorporates small interfering RNA against PD-L1 (siPDL1) conjugated to a magnetic nanocarrier (MN). We show that noninvasive magnetic resonance imaging (MRI) could be used to monitor therapeutic response. Combination therapy consisting of gemcitabine and MN-siPDL1 in a syngeneic murine pancreatic cancer model resulted in a significant reduction in tumor growth and an increase in survival. Following optimization, a 90% reduction in tumor volume was achieved 2 weeks after the beginning of treatment. Whereas 100% of the control animals had succumbed to their tumors by week 6 after the beginning of treatment, there was no mortality in the experimental group by week 5, and 67% of the experimental animals survived for 12 weeks. This method could provide therapeutic benefit against an intractable disease for which there are no effective treatments and which is characterized by a mere 1% 5-year survival.

The translational research strategy of targeting estrogen receptor α (ERα) positive breast cancer and then using long term anti-hormone adjuvant therapy (5-10 years) has reduced recurrences and mortality. However, resistance continues to occur and improvements are required to build on the success of tamoxifen and aromatase inhibitors (AIs) established over the past 40 years. Further translational research has described the evolution of acquired resistance of breast cancer cell lines to long term estrogen deprivation that parallels clinical experience over years. Additionally, recent reports have identified mutations in the ERα obtained from the recurrences of AI treated patients. These mutations allow the ERα to activate without ligands and auto stimulate metastatic tumor growth. Furthermore, the new biology of estrogen-induced apoptosis in acquired resistant models in vitro and in vivo has been interrogated and applied to clinical trials. Inflammation and stress are emerging concepts occurring in the process of acquired resistance and estrogen-induced apoptosis with different mechanisms. In this review, we will present progress in the understanding of acquired resistance, focus on stress and inflammatory responses in the development of acquired resistance, and consider approaches to create new treatments to improve the treatment of breast cancer with endocrine resistance., Competing Interests: Conflicts of interest Both authors declared that there are no conflicts of interest.

Peroxisome proliferator-activated receptor γ (PPARγ) is an important transcription factor that modulates lipid metabolism and inflammation. However, it remains unclear whether PPARγ is involved in modulation of estrogen (E 2 )-induced inflammation, thus affecting apoptosis of E 2 -deprived breast cancer cells, MCF-7:5C and MCF-7:2A. Here, we demonstrated that E 2 treatment suppressed the function of PPARγ in both cell lines, although the suppressive effect in MCF-7:2A cells was delayed owing to high PPARγ expression. Activation of PPARγ by a specific agonist, pioglitazone, selectively blocked the induction of TNFα expression by E 2 , but did not affect other adipose inflammatory genes, such as fatty acid desaturase 1 and IL6. This suppression of TNFα expression by pioglitazone was mainly mediated by transrepression of nuclear factor-κB (NF-κB) DNA-binding activity. A novel finding was that NF-κB functions as an oxidative stress inducer in MCF-7:5C cells but an antioxidant in MCF-7:2A cells. Therefore, the NF-κB inhibitor JSH-23 displayed effects equivalent to those of pioglitazone, with complete inhibition of apoptosis in MCF-7:5C cells, but it increased E 2 -induced apoptosis in MCF-7:2A cells. Depletion of PPARγ by siRNA or the PPARγ antagonist T0070907 accelerated E 2 -induced apoptosis, with activation of NF-κB-dependent TNFα and oxidative stress. For the first time, we demonstrated that PPARγ is a growth signal and has potential to modulate NF-κB activity and oxidative stress in E 2 -deprived breast cancer cell lines. All of these findings suggest that anti-PPARγ therapy is a novel strategy to improve the therapeutic effects of E 2 -induced apoptosis in E 2 -deprived breast cancer., (©2018 American Association for Cancer Research.)

Predictive tests, to refine the estrogen receptor assay, for the adjuvant treatment of breast cancer with tamoxifen and oral selective estrogen receptor degraders (SERD) are required. A splice variant of the corepressor NCOR2, BQ2313636.1 predicts tamoxifen resistance to adjuvant tamoxifen and AZ9496, the first oral SERD, completes phase I studies. Clin Cancer Res; 24(15); 3480-2. ©2018 AACR See related articles by Gong et al., p. 3681 and Hamilton et al., p. 3510 ., (©2018 American Association for Cancer Research.)

The signing of the National Cancer Act in 1971 was designed to take laboratory discoveries rapidly from the bench to the bedside. A "war on cancer" had been declared. Combination cytotoxic chemotherapy was predicted to cure all cancers, based on the stunning success in treating childhood leukemia. Breast cancer treatments were primitive; radical mastectomy and radiation were standard of care for disease that had not spread. Ablative endocrine surgery (oophorectomy, hypophysectomy, and adrenalectomy) was a palliative last option for metastatic breast cancer. However, only 30% responded, surviving for only 1 or 2 years: every patient soon died. The discovery of the estrogen receptor (ER) and translation to breast cancer treatment triggered a revolution in women's health. Two important but interconnected events occurred in 1972 at the Worcester Foundation for Experimental Biology (WFEB) that would exploit the breast tumor ER as the first target to save lives and prevent breast cancer development. Two new groups of medicines-selective ER modulators (SERMs) and aromatase inhibitors (AIs)-would continue the momentum of research at the WFEB to improve women's health. Here, we recount the important progress made in women's health based on knowledge of the endocrinology of breast cancer. We propose future opportunities in SERM therapeutics to "refresh" the current standards of care for breast cancer treatment. The opportunity is based on emerging knowledge about acquired resistance to long-term adjuvant AI therapy used to treat breast cancer.

Stress responses are critical for estrogen (E 2 )-induced apoptosis in E 2 -deprived breast cancer cells. Nuclear factor-kappa B (NF-κB) is an important therapeutic target to prevent stress responses in chronic inflammatory diseases including cancer. However, whether E 2 activates NF-κB to participate in stress-associated apoptosis in E 2 -deprived breast cancer cells is unknown. Here, we demonstrated that E 2 differentially modulates NF-κB activity according to treatment time. E 2 initially has significant potential to suppress NF-κB activation; it completely blocks tumor necrosis factor alpha (TNFα)-induced activation of NF-κB. We found that E 2 preferentially and constantly enhances the expression of the adipogenic transcription factor CCAAT/enhancer binding protein beta (C/EBPβ), which is responsible for the suppression of NF-κB activation by E 2 in MCF-7:5C cells. Interestingly, NF-κB p65 DNA-binding activity is increased when E 2 is administered for 48 h, leading to the induction of TNFα and associated apoptosis. Blocking the nuclear translocation of NF-κB can completely prevent the induction of TNFα and apoptosis induced by E 2 . Further examination revealed that protein kinase RNA-like endoplasmic reticulum kinase (PERK), a stress sensor of unfolded protein response (UPR), plays an essential role in the late activation of NF-κB by E 2 . This modulation between PERK and NF-κB is mainly mediated by a stress responsive transcription factor, transducer and activator of transcription 3 (STAT3), independently of the classic canonical IκBα signaling pathway. Thus, inhibition of PERK kinase activity completely blocks the DNA binding of both STAT3 and NF-κB, thereby preventing induction of NF-κB-dependent genes and E 2 -induced apoptosis. All of these findings suggest that PERK is a key regulator to convey stress signals from the endoplasmic reticulum to the nucleus and illustrate a crucial role for the novel PERK/STAT3/NF-κB/TNFα axis in E 2 -induced apoptosis in E 2 -deprived breast cancer cells., Competing Interests: The authors declare that they have no competing interests.

Prostate and breast cancer are the two cancers with the highest incidence in men and women, respectively. Here, we focus on the known biology of acquired resistance to antihormone therapy of prostate and breast cancer and compare laboratory and clinical similarities in the evolution of the disease. Laboratory studies and clinical observations in prostate and breast cancer demonstrate that cell selection pathways occur during acquired resistance to antihormonal therapy. Following sex steroid deprivation, both prostate and breast cancer models show an initial increased acquired sensitivity to the growth potential of sex steroids. Subsequently, prostate and breast cancer cells either become dependent upon the antihormone treatment or grow spontaneously in the absence of hormones. Paradoxically, the physiologic sex steroids now kill a proportion of selected, but vulnerable, resistant tumor cells. The sex steroid receptor complex triggers apoptosis. We draw parallels between acquired resistance in prostate and breast cancer to sex steroid deprivation. Clinical observations and patient trials confirm the veracity of the laboratory studies. We consider therapeutic strategies to increase response rates in clinical trials of metastatic disease that can subsequently be applied as a preemptive salvage adjuvant therapy. The goal of future advances is to enhance response rates and deploy a safe strategy earlier in the treatment plan to save lives. The introduction of a simple evidence-based enhanced adjuvant therapy as a global healthcare strategy has the potential to control recurrence, reduce hospitalization, reduce healthcare costs and maintain a healthier population that contributes to society., (© 2018 The authors.)

Aromatase inhibitor (AI) resistance during breast cancer treatment is mimicked by MCF-7:5C (5C) and MCF-7:2A (2A) cell lines that grow spontaneously. Survival signaling is reconfigured but cells are vulnerable to estradiol (E2)-inducible apoptosis. These model systems have alterations of stress related pathways including the accumulation of endoplasmic reticulum, oxidative, and inflammatory stress that occur prior to E2-induced apoptosis. We investigated miRNA expression profiles of 5C and 2A to characterize their AI resistance phenotypes. Affymetrix GeneChip miRNA2.0 arrays identified 184 miRNAs differentially expressed in 2A and 5C compared to E2-free wild-type MCF-7:WS8. In 5C, 34 miRNAs of the DLK1-DIO3 locus and miR-31 were overexpressed, whereas miR-222 was low. TCGA data revealed poor and favorable overall survival for low miR-31 and miR-222 levels, respectively (HR=3.0, 95% CI:1.9-4.8; HR=0.3, 95% CI:0.1-0.6). Targets of deregulated miRNAs were identified using CLIP-confirmed TargetScan predictions. KEGG enrichment analyses for 5C- and 2A-specific target gene sets revealed pathways associated with cell proliferation including insulin, mTOR, and ErbB signaling as well as immune response and metabolism. Key genes overrepresented in 5C- and 2A-specific pathway interaction networks including EGFR, IGF1R and PIK3R1 had lower protein levels in 5C compared to 2A and were found to be differentially modulated by respective miRNA sets. Distinct up-regulated miRNAs from the DLK1-DIO3 locus may cause these attenuative effects as they are predicted to interact with corresponding 3' untranslated regions. These new miRNA profiles become an important regulatory database to explore E2-induced apoptotic mechanisms of clinical relevance for the treatment of resistant breast cancer.

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., (©2015 American Association for Cancer Research.)

While rare compared to female breast cancer the incidence of male breast cancer (MBC) has increased in the last few decades. Without comprehensive epidemiological studies, the explanation for the increased incidence of MBC can only be speculated. Nevertheless, one of the most worrying global public health issues is the exponential rise in the number of overweight and obese people, especially in the developed world. Although obesity is not considered an established risk factor for MBC, studies have shown increased incidence among obese individuals. With this observation in mind, this article highlights the correlation between the increased incidence of MBC and the current trends in obesity as a growing problem in the 21(st) century, including how this may impact treatment. With MBC becoming more prominent we put forward the notion that, not only is obesity a risk factor for MBC, but that increasing obesity trends are a contributing factor to its increased incidence.

The consistent reports of mutations at Asp538 and Tyr537 in helix 12 of the ligand-binding domain (LBD) of estrogen receptors (ERs) from antihormone-resistant breast cancer metastases constitute an important advance. The mutant amino acids interact with an anchor amino acid, Asp351, to close the LBD, thereby creating a ligand-free constitutively activated ER. Amino acids Asp 538, Tyr 537, and Asp 351 are known to play a role in either the turnover of ER, the antiestrogenic activity of the ER complex, or the estrogen-like actions of selective ER modulators. A unifying mechanism of action for these amino acids to enhance ER gene activation and growth response is presented. There is a range of mutations described in metastases vs low to zero in primary disease, so the new knowledge is of clinical relevance, thereby confirming an additional mechanism of acquired resistance to antihormone therapy through cell population selection pressure and enrichment during treatment. Circulating tumor cells containing ER mutations can be cultured ex vivo, and tumor tissues can be grown as patient-derived xenografts to add a new dimension for testing drug susceptibility for future drug discovery., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)