Alcohol-Specific Inpatient Diagnoses in Germany: A Retrospective Cross-Sectional Analysis of Primary and Secondary Diagnoses from 2012 to 2021.

AIMS: Our study aimed to a) describe the distribution of hospital discharges with primary and secondary alcohol-specific diagnoses by sex and age group, and b) describe how the number of hospital discharges with primary and secondary alcohol-specific diagnoses have changed across different diagnostic groups (categorized by primary International Classification of Diseases, 10th Revision [ICD-10] diagnosis) over time. DESIGN: Retrospective cross-sectional analysis. SETTING: German hospital settings between 2012 and 2021. PARTICIPANTS: All persons aged 15-69 admitted to hospitals as registered in a nationwide data set. MEASUREMENTS: We counted a) the number of all hospital discharges and b) the number of hospital discharges with at least one alcohol-specific secondary diagnosis (secondary alcohol-specific diagnosis) by year, sex, age group, and diagnostic group. One diagnostic group included all primary alcohol-specific diagnoses, while 13 additional groups aligned with ICD-10 chapters (e.g., neoplasms). Alcohol-involvement was defined as either a primary or secondary alcohol-specific diagnosis. FINDINGS: Of 95 417 204 recorded hospital discharges between 2012 and 2021, 3 828 917 discharges (4.0%; 2 913 903 men (6.4%); 915 014 women (1.8%)) involved either a primary or at least one secondary diagnosis related to alcohol. Of all alcohol-involved hospital discharges, 56.8% (1 654 736 discharges) had no primary but only a secondary alcohol-specific diagnosis. Secondary alcohol-specific diagnoses were particularly prevalent in hospital discharges due to injuries. With rising age, alcohol-involvement in hospital discharges due to digestive or cardiovascular diseases increased. Between 2012 and 2021, the rate of alcohol-involved hospital discharges has decreased more in younger as compared with older adults (average change between 2012 and 2021: 15-24: -55%; 25-34: -41%; 35-44: -23%; 45-54: -31%; 55-64: -21%; 65-69: -8%). CONCLUSIONS: The number of alcohol-involved hospital discharges in Germany from 2012 to 2021 more than doubles (from 1 654 736 to 3 828 917) when including secondary alcohol-specific diagnoses. More pronounced declines among younger adults may be attributed to unequal changes in alcohol consumption patterns across the population and to the hazardous effects of long-term alcohol use.

Restricting alcohol marketing to reduce alcohol consumption: A systematic review of the empirical evidence for one of the 'best buys'.

BACKGROUND AND AIMS: Even though a ban of alcohol marketing has been declared a 'best buy' of alcohol control policy, comprehensive systematic reviews on its effectiveness to reduce consumption are lacking. The aim of this paper was to systematically review the evidence for effects of total and partial bans of alcohol marketing on alcohol consumption. METHODS: This descriptive systematic review sought to include all empirical studies that explored how changes in the regulation of alcohol marketing impact on alcohol consumption. The search was conducted between October and December 2022 considering various scientific databases (Web of Science, PsycINFO, MEDLINE, Embase) as well as Google and Google Scholar. The titles and abstracts of a total of 2572 records were screened. Of the 26 studies included in the full text screening, 11 studies were finally included in this review. Changes in consumption in relation to marketing bans were determined based on significance testing in primary studies. Four risk of bias domains (confounding, selection bias, information bias and reporting bias) were assessed. RESULTS: Seven studies examined changes in marketing restrictions in one location (New Zealand, Thailand, Canadian provinces, Spain, Norway). In the remaining studies, between 17 and 45 locations were studied (mostly high-income countries from Europe and North America). Of the 11 studies identified, six studies reported null findings. Studies reporting lower alcohol consumption following marketing restrictions were of moderate, serious and critical risk of bias. Two studies with low and moderate risk of bias found increasing alcohol consumption post marketing bans. Overall, there was insufficient evidence to conclude that alcohol marketing bans reduce alcohol consumption. CONCLUSIONS: The available empirical evidence does not support the claim of alcohol marketing bans constituting a best buy for reducing alcohol consumption.

Riluzole Suppresses Growth and Enhances Response to Endocrine Therapy in ER+ Breast Cancer.

Background: Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a significant clinical problem. Riluzole is FDA-approved for the treatment of amyotrophic lateral sclerosis. A benzothiazole-based glutamate release inhibitor with several context-dependent mechanism(s) of action, riluzole has shown antitumor activity in multiple malignancies, including melanoma, glioblastoma, and breast cancer. We previously reported that the acquisition of tamoxifen resistance in a cellular model of invasive lobular breast cancer is accompanied by the upregulation of GRM mRNA expression and growth inhibition by riluzole. Methods: We tested the ability of riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated invasive lobular breast cancer patient-derived xenograft model HCI-013EI. Results: Single-agent riluzole suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing a histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). Riluzole induced apoptosis and ferroptosis and reduced phosphorylation of multiple prosurvival signaling molecules, including Akt/mTOR, CREB, and Fak/Src family kinases. Riluzole, in combination with either fulvestrant or 4-hydroxytamoxifen, additively suppressed ER+ breast cancer cell growth in vitro. Single-agent riluzole significantly inhibited HCI-013EI patient-derived xenograft growth in vivo, and the combination of riluzole plus fulvestrant significantly reduced proliferation in ex vivo primary breast tumor explant cultures. Conclusion: Riluzole may offer therapeutic benefits in diverse ER+ breast cancers, including lobular breast cancer.

The impact of legal cannabis availability on cannabis use and health outcomes: A systematic review.

BACKGROUND: For alcohol, regulating availability is an effective way to reduce consumption and harm. Similarly, the higher availability of medical cannabis dispensaries has been linked to increased cannabis consumption and harm. For recreational cannabis markets, such a link is suspected but still poorly understood. METHODS: A systematic literature review (PROSPERO registration number 342357) was conducted on 1 July 2022 in common libraries (Medline, Web of Science, PsycInfo, Psyndex, CINAHL, Embase, SCOPUS, Cochrane) for publications since 2012. Studies linking variations in the availability of legal cannabis products to behavioral outcomes (cannabis use or related health indicators) were included, while studies focusing solely on the legalization of medical cannabis were excluded. The risk of bias was assessed using an adapted version of the Newcastle-Ottawa-Scale. RESULTS: After screening n = 6,253 studies, n = 136 were selected for full-text review, out of which n = 13 met the inclusion criteria, reporting on n = 333,550 study participants and n = 855,630 presentations to emergency departments. All studies were conducted in North America, with the majority from Western US states. Using longitudinal (n = 1), cross-sectional (n = 4), or repeated cross-sectional (n = 8) study designs, an increased availability of legal cannabis was linked to increased current cannabis use and health-related outcomes (vomiting, psychosis, or cannabis-involved pregnancies), regardless of the indicator employed to measure availability (proximity or density) among both adults and adolescents. The positive correlation between cannabis availability and consumption is most pronounced among those groups who have been less exposed to cannabis before legalization. The association between the availability of legal cannabis and risky use indicators was less consistent. CONCLUSIONS: Groups who have been least exposed to cannabis before legalization may be most susceptible to increased availability. In jurisdictions with legal cannabis markets, restrictions on the number of legal cannabis retailers, especially in densely populated areas, appear warranted.

The Universal and Primary Prevention of Foetal Alcohol Spectrum Disorders (FASD): A Systematic Review.

Foetal alcohol spectrum disorder (FASD) comprises multiple neurodevelopmental disorders caused by alcohol consumption during pregnancy. With a global prevalence rate of 7.7 per 1000 population, FASD is a leading cause of prenatal developmental disorders. The extent of physical, mental, and social consequences for individuals with FASD can be vast and negatively affect their social environment, daily life, school, relationships, and work. As treatment for FASD is labour- and cost-intensive, with no cure available, prevention is key in reducing FASD prevalence rates. As most systematic reviews conducted so far have focused on specific FASD risk groups, we investigated the effectiveness of universal FASD prevention and primary preventive strategies. We identified a total of 567 potentially pertinent records through PubMed, Cochrane Library, EBSCO, PubPsych, and DAHTA published from 2010 to May 2020, of which 10 studies were included in this systematic review. Results showed a substantial heterogeneity in the studies' quality, although all preventive measures, except one, proved effective in both increasing knowledge and awareness of FASD, as well as decreasing the risk of an alcohol exposed pregnancy. Limiting factors such as small sample sizes and a lack of behavioural change testing require further studies to support existing evidence for FASD prevention and its implementation, as well as detecting the best course of action for FASD prevention when creating and implementing prevention and intervention approaches.

Substitution treatment for opioid dependence with slow-release oral morphine: Retention rate, health status, and substance use after switching to morphine.

INTRODUCTION: Since April 2015, slow-release oral morphine (SROM) has been approved for opioid agonist treatment (OAT) in Germany. Experimental studies show that benefits of SROM over methadone include less heroin craving, better tolerability, and higher patient satisfaction and mental stability. The SROMOS study (Efficacy and Tolerability of Slow-Release Oral Morphine in Opioid Substitution Treatment) aims to investigate the long-term effects (effectiveness and safety) of morphine substitution under routine care in Germany. MATERIAL AND METHODS: This is a prospective, noninterventional, naturalistic, observational study. Between July 2016 and November 2017, this study recruited patients in OAT who decided to switch to SROM from 23 outpatient addiction treatment centers in Germany. The study collected data on mental health (Brief Symptom Inventory - BSI-18), substance use, somatic health (Opiate Treatment Index Health-Symptoms-Scale - OTI-HSS), opioid craving (visual analogue scale), and withdrawal symptoms (Short Opiate Withdrawal Scale) at baseline (t0) and after 3 (t3), 6 (t6) and 12 (t12) months. Physicians documented side effects as adverse events (AEs) and adverse drug reactions (ADRs). RESULTS: Three-quarters of the enrolled study participants (N = 180) were male. The average age was 44.4 years. Patients were opioid-dependent for 23 years and had been in OAT for almost seven years on average. After 12 months, 60.6% were still being treated with SROM. Mental health improved significantly under SROM treatment between t0 and t12. The intention-to-treat (ITT), as well as the per-protocol (PP) analysis, shows a statistically significant improvement of the mean Global Severity Index (GSI) of the BSI-18 value of 20% (ITT) and 24% (PP). Physical health also improved significantly under SROM treatment. There were no statistically significant changes in the use of cannabis, cocaine, amphetamines, and tranquillizers in the past 30 days, but heroin use, intravenous consumption, and the number of drinking days significantly decreased. CONCLUSIONS: This study provides some of the first long-term data on OAT with SROM under routine care conditions. SROM treatment is an effective alternative for a subgroup of opioid-dependent patients with an unsatisfactory course of OAT or in cases where undesirable side effects due to alternative substances have occurred. ETHICAL STATEMENT: The study protocol was approved by the Ethics Committee of the Chamber of Physicians in Hamburg in March 2016 (No. PV5222). The study was conducted by following the Declaration of Helsinki and is registered with the German Register of Clinical Trials (DRKS, ID: DRKS00010712).

Steroid Hormone Receptor and Infiltrating Immune Cell Status Reveals Therapeutic Vulnerabilities of ESR1-Mutant Breast Cancer.

Mutations in ESR1 that confer constitutive estrogen receptor alpha (ER) activity in the absence of ligand are acquired by ≥40% of metastatic breast cancers (MBC) resistant to adjuvant aromatase inhibitor (AI) therapy. To identify targetable vulnerabilities in MBC, we examined steroid hormone receptors and tumor-infiltrating immune cells in metastatic lesions with or without ER mutations. ER and progesterone receptor (PR) were significantly lower in metastases with wild-type (WT) ER compared with those with mutant ER, suggesting that metastases that evade AI therapy by mechanism(s) other than acquiring ER mutations lose dependency on ER and PR. Metastases with mutant ER had significantly higher T regulatory and Th cells, total macrophages, and programmed death ligand-1 (PD-L1)-positive immune-suppressive macrophages than those with WT ER. Breast cancer cells with CRISPR-Cas9-edited ER (D538G, Y537S, or WT) and patient-derived xenografts harboring mutant or WT ER revealed genes and proteins elevated in mutant ER cells, including androgen receptor (AR), chitinase-3-like protein 1 (CHI3L1), and IFN-stimulated genes (ISG). Targeting these proteins blunted the selective advantage of ER-mutant tumor cells to survive estrogen deprivation, anchorage independence, and invasion. Thus, patients with mutant ER MBC might respond to standard-of-care fulvestrant or other selective ER degraders when combined with AR or CHI3L1 inhibition, perhaps with the addition of immunotherapy. SIGNIFICANCE: Targetable alterations in MBC, including AR, CHI3L1, and ISG, arise following estrogen-deprivation, and ER-mutant metastases may respond to immunotherapies due to elevated PD-L1+ macrophages.See related article by Arnesen et al., p. 539.

Exogenous Thyroid Hormone Is Associated with Shortened Survival and Upregulation of High-Risk Gene Expression Profiles in Steroid Receptor-Positive Breast Cancers.

PURPOSE: Thyroid disease is a frequent comorbidity in women with breast cancer, and many require thyroid hormone replacement therapy (THRT). We postulated that THRT has a deleterious clinical effect mechanistically through hormonal interactions, nuclear receptor cross-talk, and upregulation of high-risk breast cancer genes. EXPERIMENTAL DESIGN: Observational studies of patients with lymph node-negative (LN-) breast cancer (n = 820 and n = 160) were performed to test interactions between THRT and clinical, histologic, outcome, and treatment variables. Differences between the two cohorts include but are not limited to patient numbers, decades of treatment, duration of follow-up/treatment, tumor sizes, incidence, and type and dose/regimen of antihormonal and/or chemotherapeutic agents. In vivo and vitro models, in silico databases, and molecular methods were used to study interactions and define mechanisms underlying THRT effects. RESULTS: THRT significantly and independently reduced disease-free and breast cancer-specific overall survival of only the steroid receptor (SR)-positive (as compared with SR-negative) node-negative patients in both long-term observational studies. Patients with SR+ LN- breast cancer who received THRT and tamoxifen experienced the shortest survival of all treatment groups. A less potent interaction between THRT and aromatase inhibitors was noted in the second patient cohort. Using in vivo and in vitro models, TH administration enhanced estrogen and TH-associated gene expression and proliferation, nuclear colocalization of estrogen receptor and thyroid hormone receptor, and activation of genes used clinically to predict tumor aggression in SR+ breast cancer, including the IGF-IR, WNT, and TGFß pathways. CONCLUSIONS: We show clinically significant adverse interactions between THRT, estrogenic, and oncogenic signaling in patients with SR+ LN- breast cancer.

New generation breast cancer cell lines developed from patient-derived xenografts.

BACKGROUND: Breast cancer is a highly heterogeneous disease characterized by multiple histologic and molecular subtypes. While a myriad of breast cancer cell lines have been developed over the past 60 years, estrogen receptor alpha (ER)+ disease and some mutations associated with this subtype remain underrepresented. Here we describe six breast cancer cell lines derived from patient-derived xenografts (PDX) and their general characteristics. METHODS: Established breast cancer PDX were processed into cell suspensions and placed into standard 2D cell culture; six emerged into long-term passageable cell lines. Cell lines were assessed for protein expression of common luminal, basal, and mesenchymal markers, growth assessed in response to estrogens and endocrine therapies, and RNA-seq and oncogenomics testing performed to compare relative transcript levels and identify putative oncogenic drivers. RESULTS: Three cell lines express ER and two are also progesterone receptor (PR) positive; PAM50 subtyping identified one line as luminal A. One of the ER+PR+ lines harbors a D538G mutation in the gene for ER (ESR1), providing a natural model that contains this endocrine-resistant genotype. The third ER+PR-/low cell line has mucinous features, a rare histologic type of breast cancer. The three other lines are ER- and represent two basal-like and a mixed ductal/lobular breast cancer. The cell lines show varied responses to tamoxifen and fulvestrant, and three were demonstrated to regrow tumors in vivo. RNA sequencing confirms all cell lines are human and epithelial. Targeted oncogenomics testing confirmed the noted ESR1 mutation in addition to other mutations (i.e., PIK3CA, BRCA2, CCND1, NF1, TP53, MYC) and amplifications (i.e., FGFR1, FGFR3) frequently found in breast cancers. CONCLUSIONS: These new generation breast cancer cell lines add to the existing repository of breast cancer models, increase the number of ER+ lines, and provide a resource that can be genetically modified for studying several important clinical breast cancer features.

FGF2 induces breast cancer growth through ligand-independent activation and recruitment of ERα and PRBΔ4 isoform to MYC regulatory sequences.

Progression to hormone-independent growth leading to endocrine therapy resistance occurs in a high proportion of patients with estrogen receptor alpha (ERα) and progesterone receptors (PR) positive breast cancer. We and others have previously shown that estrogen- and progestin-induced tumor growth requires ERα and PR interaction at their target genes. Here, we show that fibroblast growth factor 2 (FGF2)-induces cell proliferation and tumor growth through hormone-independent ERα and PR activation and their interaction at the MYC enhancer and proximal promoter. MYC inhibitors, antiestrogens or antiprogestins reverted FGF2-induced effects. LC-MS/MS identified 700 canonical proteins recruited to MYC regulatory sequences after FGF2 stimulation, 397 of which required active ERα (ERα-dependent). We identified ERα-dependent proteins regulating transcription that, after FGF2 treatment, were recruited to the enhancer as well as proteins involved in transcription initiation that were recruited to the proximal promoter. Also, among the ERα-dependent and independent proteins detected at both sites, PR isoforms A and B as well as the novel protein product PRBΔ4 were found. PRBΔ4 lacks the hormone-binding domain and was able to induce reporter gene expression from estrogen-regulated elements and to increase cell proliferation when cells were stimulated with FGF2 but not by progestins. Analysis of the Cancer Genome Atlas data set revealed that PRBΔ4 expression is associated with worse overall survival in luminal breast cancer patients. This discovery provides a new mechanism by which growth factor signaling can engage nonclassical hormone receptor isoforms such as PRBΔ4, which interacts with growth-factor activated ERα and PR to stimulate MYC gene expression and hence progression to endocrine resistance.

SNAIL is induced by tamoxifen and leads to growth inhibition in invasive lobular breast carcinoma.

PURPOSE: Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer that is predominantly estrogen receptor alpha (ER)-positive (+) and is thus treated with endocrine therapies. Herein, we sought to understand the molecular underpinnings of the 4-hydroxytamoxifen (4OHT) resistance in ILC by assessing the potential role of the epithelial-to-mesenchymal transition transcription factor (EMT-TF) SNAIL (SNAI1). METHODS: Using a series of breast cancer cell lines, we measured the basal, estrogen and 4OHT-induced expression of SNAIL and other EMT-TF family members by quantitative reverse transcription-polymerase chain reaction and immunoblotting. Chromatin immunoprecipitation experiments were performed to assess ER binding to the SNAIL promoter. Cell proliferation, cell cycle and apoptosis were assessed in 2D cultures. 3D growth was assessed in Matrigel and Collagen I cultures. RESULTS: Estrogen and 4OHT induced SNAIL expression, but not that of the other EMT-TF family members SLUG (SNAI2) and SMUC (SNAI3), with the 4OHT effect being specific to the lobular but not the ductal subtype. We observed estrogen and 4OHT-induced ER recruitment to the SNAI1 promoter and high endogenous basal levels of SNAIL and several EMT-TFs in ILC cell lines. While SNAIL knockdown had a minor impact on the 4OHT partial agonism in estrogen-depleted conditions, it led to a surprising increase in cell proliferation in full serum. In complementary experiments, inducible SNAI1 overexpression caused decreased proliferation, associated with a cell cycle arrest in G0/G1. Additionally, apoptosis was observed in BCK4 cells. CONCLUSION: These data suggest a previously unrecognized role for SNAIL in ILC, substantiating a context-dependent behavior for this EMT-TF.

Mucin 2 (MUC2) modulates the aggressiveness of breast cancer.

PURPOSE: Tumors that secrete large volumes of mucus are chemotherapy resistant, however, mechanisms underlying this resistance are unknown. One protein highly expressed in mucin secreting breast cancers is the secreted mucin, Mucin 2 (MUC2). While MUC2 is expressed in some breast cancers it is absent in normal breast tissue, implicating it in breast cancer. However, the effects of MUC2 on breast cancer are largely unknown. This study examined the role of MUC2 in modulating breast cancer proliferation, response to chemotherapy and metastasis. METHODS: Using patient derived xenografts we developed two novel cell lines, called BCK4 and PT12, which express high levels of MUC2. To modulate MUC2 levels, BCK4 and PT12 cells were engineered to express shRNA targeted to MUC2 (shMUC2, low MUC2) or a non-targeting control (shCONT, high MUC2) and proliferation and apoptosis were measured in vitro and in vivo. BCK4 cells with shCONT or shMUC2 were labeled with GFP-luciferase and examined in an experimental metastasis model; disease burden and site specific dissemination were monitored by intravital imaging and fluorescence guided dissection, respectively. RESULTS: Proliferation decreased in BCK4 and PT12 shMUC2 cells versus control cells both in vitro and in vivo. Chemotherapy induced minimal apoptosis in control cells expressing high MUC2 but increased apoptosis in shMUC2 cells containing low MUC2. An experimental metastasis model showed disease burden decreased when breast cancer cells contained low versus high MUC2. Treatment with Epidermal Growth Factor (EGF) increased MUC2 expression in BCK4 cells; this induction was abolished by the EGF-receptor inhibitor, Erlotinib. CONCLUSIONS: MUC2 plays an important role in mediating proliferation, apoptosis and metastasis of breast cancer cells. MUC2 may be important in guiding treatment and predicting outcomes in breast cancer patients.

Comprehensive Phenotypic Characterization of Human Invasive Lobular Carcinoma Cell Lines in 2D and 3D Cultures.

Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal carcinoma (IDC) and characterized by the loss of E-cadherin-mediated adherens junctions. Despite displaying unique histologic and clinical features, ILC still remains a chronically understudied disease, with limited knowledge gleaned from available laboratory research models. Here we report a comprehensive 2D and 3D phenotypic characterization of four estrogen receptor-positive human ILC cell lines: MDA-MB-134, SUM44, MDA-MB-330, and BCK4. Compared with the IDC cell lines MCF7, T47D, and MDA-MB-231, ultra-low attachment culture conditions revealed remarkable anchorage independence unique to ILC cells, a feature not evident in soft-agar gels. Three-dimensional Collagen I and Matrigel culture indicated a generally loose morphology for ILC cell lines, which exhibited differing preferences for adhesion to extracellular matrix proteins in 2D. Furthermore, ILC cells were limited in their ability to migrate and invade in wound-scratch and transwell assays, with the exception of haptotaxis to Collagen I. Transcriptional comparison of these cell lines confirmed the decreased cell proliferation and E-cadherin-mediated intercellular junctions in ILC while uncovering the induction of novel pathways related to cyclic nucleotide phosphodiesterase activity, ion channels, drug metabolism, and alternative cell adhesion molecules such as N-cadherin, some of which were differentially regulated in ILC versus IDC tumors. Altogether, these studies provide an invaluable resource for the breast cancer research community and facilitate further functional discoveries toward understanding ILC, identifying novel drug targets, and ultimately improving the outcome of patients with ILC.Significance: These findings provide the breast cancer research community with a comprehensive assessment of human invasive lobular carcinoma (ILC) cell line signaling and behavior in various culture conditions, aiding future endeavors to develop therapies and to ultimately improve survival in patients with ILC. Cancer Res; 78(21); 6209-22. ©2018 AACR.

Biological and clinical impact of imbalanced progesterone receptor isoform ratios in breast cancer.

There is a consensus that progestins and thus their cognate receptor molecules, the progesterone receptors (PR), are essential in the development of the adult mammary gland and regulators of proliferation and lactation. However, a role for natural progestins in breast carcinogenesis remains poorly understood. A hint to that possible role came from studies in which the synthetic progestin medroxyprogesterone acetate was associated with an increased breast cancer risk in women under hormone replacement therapy. However, progestins have been also used for breast cancer treatment and to inhibit the growth of several experimental breast cancer models. More recently, PR have been shown to be regulators of estrogen receptor signaling. With all this information, the question is how can we target PR, and if so, which patients may benefit from such an approach? PR are not single unique molecules. Two main PR isoforms have been characterized, PRA and PRB, that exert different functions and the relative abundance of one isoform respect to the other determines the response of PR agonists and antagonists. Immunohistochemistry with standard antibodies against PR do not discriminate between isoforms. In this review, we summarize the current knowledge on the expression of both PR isoforms in mammary glands, in experimental models of breast cancer and in breast cancer patients, to better understand how the PRA/PRB ratio can be exploited therapeutically to design personalized therapeutic strategies.

FGFR1 underlies obesity-associated progression of estrogen receptor-positive breast cancer after estrogen deprivation.

Obesity increases breast cancer mortality by promoting resistance to therapy. Here, we identified regulatory pathways in estrogen receptor-positive (ER-positive) tumors that were shared between patients with obesity and those with resistance to neoadjuvant aromatase inhibition. Among these was fibroblast growth factor receptor 1 (FGFR1), a known mediator of endocrine therapy resistance. In a preclinical model with patient-derived ER-positive tumors, diet-induced obesity promoted a similar gene expression signature and sustained the growth of FGFR1-overexpressing tumors after estrogen deprivation. Tumor FGFR1 phosphorylation was elevated with obesity and predicted a shorter disease-free and disease-specific survival for patients treated with tamoxifen. In both human and mouse mammary adipose tissue, FGF1 ligand expression was associated with metabolic dysfunction, weight gain, and adipocyte hypertrophy, implicating the impaired response to a positive energy balance in growth factor production within the tumor niche. In conjunction with these studies, we describe a potentially novel graft-competent model that can be used with patient-derived tissue to elucidate factors specific to extrinsic (host) and intrinsic (tumor) tissue that are critical for obesity-associated tumor promotion. Taken together, we demonstrate that obesity and excess energy establish a tumor environment with features of endocrine therapy resistance and identify a role for ligand-dependent FGFR1 signaling in obesity-associated breast cancer progression.

Increased High Molecular Weight FGF2 in Endocrine-Resistant Breast Cancer.

Endocrine resistance may develop as a consequence of enhanced growth factor signaling. Fibroblast growth factor 2 (FGF2) consists of a low and several high molecular weight forms (HMW-FGF2). We previously demonstrated that antiprogestin-resistant mammary carcinomas display lower levels of progesterone receptor A isoforms (PRA) than B isoforms (PRB). Our aim was to evaluate the role of FGF2 isoforms in breast cancer progression. We evaluated FGF2 expression, cell proliferation, and pathway activation in models with different PRA/PRB ratios. We performed lentiviral infections of different FGF2 isoforms using the human hormone-responsive T47D-YA cells, engineered to only express PRA, and evaluated tumor growth, metastatic dissemination, and endocrine responsiveness. We assessed FGF2 expression and localization in 81 human breast cancer samples. Antiprogestin-resistant experimental mammary carcinomas with low PRA/PRB ratios and T47D-YB cells, which only express PRB, displayed higher levels of HMW-FGF2 than responsive variants. HMW-FGF2 overexpression in T47D-YA cells induced increased tumor growth, lung metastasis, and antiprogestin resistance compared to control tumors. In human breast carcinomas categorized by their PRA/PRB ratio, we found nuclear FGF2 expression in 55.6% of tumor cells. No differences were found between nuclear FGF2 expression and Ki67 proliferation index, tumor stage, or tumor grade. In low-grade tumor samples, moderate to high nuclear FGF2 levels were associated to carcinomas with low PRA/PRB ratio. In conclusion, we show that HMW-FGF2 isoforms are PRB targets which confer endocrine resistance and are localized in the nuclei of breast cancer samples. Hence, targeting intracellular FGF2 may contribute to overcome tumor progression.

Loss of E-cadherin Enhances IGF1-IGF1R Pathway Activation and Sensitizes Breast Cancers to Anti-IGF1R/InsR Inhibitors.

Purpose: Insulin-like growth factor 1 (IGF1) signaling regulates breast cancer initiation and progression and associated cancer phenotypes. We previously identified E-cadherin (CDH1) as a repressor of IGF1 signaling and in this study examined how loss of E-cadherin affects IGF1R signaling and response to anti-IGF1R/insulin receptor (InsR) therapies in breast cancer.Experimental Design: Breast cancer cell lines were used to assess how altered E-cadherin levels regulate IGF1R signaling and response to two anti-IGF1R/InsR therapies. In situ proximity ligation assay (PLA) was used to define interaction between IGF1R and E-cadherin. TCGA RNA-seq and RPPA data were used to compare IGF1R/InsR activation in estrogen receptor-positive (ER+) invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) tumors. ER+ ILC cell lines and xenograft tumor explant cultures were used to evaluate efficacy to IGF1R pathway inhibition in combination with endocrine therapy.Results: Diminished functional E-cadherin increased both activation of IGF1R signaling and efficacy to anti-IGF1R/InsR therapies. PLA demonstrated a direct endogenous interaction between IGF1R and E-cadherin at points of cell-cell contact. Increased expression of IGF1 ligand and levels of IGF1R/InsR phosphorylation were observed in E-cadherin-deficient ER+ ILC compared with IDC tumors. IGF1R pathway inhibitors were effective in inhibiting growth in ER+ ILC cell lines and synergized with endocrine therapy and similarly IGF1R/InsR inhibition reduced proliferation in ILC tumor explant culture.Conclusions: We provide evidence that loss of E-cadherin hyperactivates the IGF1R pathway and increases sensitivity to IGF1R/InsR targeted therapy, thus identifying the IGF1R pathway as a potential novel target in E-cadherin-deficient breast cancers. Clin Cancer Res; 24(20); 5165-77. ©2018 AACR.

Targeting FGFR with BGJ398 in Breast Cancer: Effect on Tumor Growth and Metastasis.

BACKGROUND: Endocrine resistance and metastatic dissemination comprise major clinical challenges for breast cancer treatment. The fibroblast growth factor receptor family (FGFR) consists of four tyrosine kinase transmembrane receptors, involved in key biological processes. Genomic alterations in FGFR have been identified in advanced breast cancer and thus, FGFR are an attractive therapeutic target. However, the efficacy of FGFR inhibitors on in vivo tumor growth is still controversial. OBJECTIVE: The purpose of this study was to evaluate the role of FGFR in tumor growth and breast cancer progression. METHODS: Cell proliferation was assessed by 3H-thymidine uptake and cell counting in primary cultures of endocrine resistant mammary carcinomas and a human cell line, respectively. Tumor transplants and cell injections were used to determine in vivo growth and spontaneous metastasis. FGFR1-3 and αSMA expression were evaluated on primary tumors by immunohistochemistry. RESULTS: Antiprogestin resistant murine transplants and a human xenograft express high levels of total FGFR1-3. In vitro treatment with the FGFR inhibitor, BGJ398, impaired cell proliferation of resistant variants versus vehicle. In vivo, versus control, BGJ398 treatment decreased one out of four resistant tumors, however all tumors showed a decreased epithelial/stromal ratio. Finally, in a model of hormone resistant mammary cancer that spontaneously metastasizes to the lung, BGJ398 decreased the number of mice with lung metastasis. CONCLUSION: FGFR inhibitors are promising tools that require further investigation to identify sensitive tumors. These studies suggest that targeting FGFR combined with other targeted therapies will be useful to impair breast cancer progression.

Development of Novel Patient-Derived Xenografts from Breast Cancer Brain Metastases.

Brain metastases are an increasing burden among breast cancer patients, particularly for those with HER2+ and triple negative (TN) subtypes. Mechanistic insight into the pathophysiology of brain metastases and preclinical validation of therapies has relied almost exclusively on intracardiac injection of brain-homing cells derived from highly aggressive TN MDA-MB-231 and HER2+ BT474 breast cancer cell lines. Yet, these well characterized models are far from representing the tumor heterogeneity observed clinically and, due to their fast progression in vivo, their suitability to validate therapies for established brain metastasis remains limited. The goal of this study was to develop and characterize novel human brain metastasis breast cancer patient-derived xenografts (BM-PDXs) to study the biology of brain metastasis and to serve as tools for testing novel therapeutic approaches. We obtained freshly resected brain metastases from consenting donors with breast cancer. Tissue was immediately implanted in the mammary fat pad of female immunocompromised mice and expanded as BM-PDXs. Brain metastases from 3/4 (75%) TN, 1/1 (100%) estrogen receptor positive (ER+), and 5/9 (55.5%) HER2+ clinical subtypes were established as transplantable BM-PDXs. To facilitate tracking of metastatic dissemination using BM-PDXs, we labeled PDX-dissociated cells with EGFP-luciferase followed by reimplantation in mice, and generated a BM-derived cell line (F2-7). Immunohistologic analyses demonstrated that parental and labeled BM-PDXs retained expression of critical clinical markers such as ER, progesterone receptor, epidermal growth factor receptor, HER2, and the basal cell marker cytokeratin 5. Similarly, RNA sequencing analysis showed clustering of parental, labeled BM-PDXs and their corresponding cell line derivative. Intracardiac injection of dissociated cells from BM-E22-1, resulted in magnetic resonance imaging-detectable macrometastases in 4/8 (50%) and micrometastases (8/8) (100%) mice, suggesting that BM-PDXs remain capable of colonizing the brain at high frequencies. Brain metastases developed 8-12 weeks after ic injection, located to the brain parenchyma, grew around blood vessels, and elicited astroglia activation characteristic of breast cancer brain metastasis. These novel BM-PDXs represent heterogeneous and clinically relevant models to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings.