Prolactin (PRL) is a protein hormone which in humans is secreted by pituitary lactotrophs as well as by many normal and malignant non-pituitary sites. Many lines of evidence demonstrate that both circulating and locally produced PRL increase breast cancer (BC) growth and metastases and confer chemoresistance. Our objective was to identify and then characterize small molecules that block the tumorigenic actions of PRL in BC. We employed three cell-based assays in high throughput screening (HTS) of 51,000 small molecules and identified two small molecule inhibitors (SMIs), named SMI-1 and SMI-6. Both compounds bound to the extracellular domain (ECD) of the PRL receptor (PRLR) at 1–3 micromolar affinity and abrogated PRL-induced breast cancer cell (BCC) invasion and malignant lymphocyte proliferation. SMI-6 effectively reduced the viability of multiple BCC types, had much lower activity against various non-malignant cells, displayed high selectivity, and showed no apparent in vitro or in vivo toxicity. In athymic nude mice, SMI-6 rapidly and dramatically suppressed the growth of PRL-expressing BC xenografts. This report represents a pre-clinical phase of developing novel anti-cancer agents with the potential to become effective therapeutics in breast cancer patients.

Despite recent advances in the detection and treatment of breast cancer, many shortcomings remain, providing incentives to search for new therapeutic targets. This review provides information on the expression and actions of dopamine receptor-1 (D1R) in breast cancer. D1R is overexpressed in a significant number of primary breast tumors, characterized by having an aggressive phenotype and predicting a shorter survival time for patients. Activation of D1R in breast cancer cells by selective agonists caused suppression of cell viability, stimulation of apoptosis, inhibition of cell invasion, and an increase in chemosensitivity. Instead of being linked to the cAMP/PKA system as expected, D1R in breast cancer is linked to the activation of the cGMP/protein kinase G (PKG) pathway. Fenoldopam, a peripheral D1R agonist that does not penetrate the brain, dramatically suppressed the growth of breast cancer xenografts in immune-deficient mice. A new imaging system for detecting D1R-expressing tumors and metastases was also developed. The review offers a novel concept that D1R can serve as a biomarker for prognosis in advanced breast cancer and its agonists can be used as effective and personalized therapeutics in a subpopulation of patients with D1R-expressing breast tumors. Several drugs, some of which are FDA-approved, that bypass the D1R and directly activate the cGMP/PKG apoptotic system, are also identified.

Breast cancer results from time-related complex interactions between internal and external factors. In addition to endogenous estrogens, which play an undisputed role in breast tumorigenesis, exogenous compounds which mimic the actions of estrogen and are referred to as endocrine disruptors (EDCs) or xenoestrogens have strong impacts on breast development during the perinatal period and on carcinogenesis in adults. EDCs include natural compounds such as phytoestrogens and mycoestrogens, as well as numerous man-made chemicals which are widely used by the agriculture, chemical, food, and cosmetic industries, and are included in multiple everyday consumer products. This chapter reviews the evidence on human exposure to the EDCs, their in vitro and in vivo effects on breast cancer, and their proposed mechanisms of action. Emphasis has been placed on bisphenol A (BPA), a prototypical xenoestrogen whose adverse health effects have attracted considerable attention by scientists, industry, regulatory agencies, and the public at large. The disparate positions on health hazards by BPA, which have been undertaken by the chemical and food industries, environmental advocacy groups, health organizations, and regulatory agencies, are reviewed and criticized.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive and often fatal disease. Cisplatin is the most common chemotherapeutic drug in the treatment of HNSCC, but intrinsic and acquired resistance are frequent, and severe side effects occur at high doses. The second messenger cyclic GMP (cGMP) is produced by soluble guanylate cyclase (sGC). We previously reported that activation of the cGMP signaling cascade caused apoptosis in HNSCC cells, while others found that this pathway enhances cisplatin efficacy in some cell types. Here we found that sGC stimulators reduced HNSCC cell viability synergistically with cisplatin, and enhanced apoptosis by cisplatin. Moreover, the sGC stimulators effectively reduced viability in cells with acquired cisplatin resistance, and were synergistic with cisplatin. The sGC stimulator BAY 41-2272 reduced expression of the survival proteins EGFR and β-catenin, and increased pro-apoptotic Bax, suggesting a potential mechanism for the anti-tumorigenic effects of these drugs. The sGC stimulator Riociguat is FDA-approved to treat pulmonary hypertension, and others are being studied for therapeutic use in several diseases. These drugs could provide valuable addition or alternative to cisplatin in the treatment of HNSCC.

Cisplatin is an effective anti-cancer drug which has been successfully utilized in the treatment of many cancer types. However, cisplatin has shown only limited efficacy in the majority of breast tumors, and is not generally included as a monotherapy in this disease. The potential role of hormones in chemoresistance in breast cancer has received little attention. This review focuses on two classes of hormones: a) protein hormones that include prolactin, growth hormone and leptin, all of which act via class 1 cytokine receptors and activate the Jak2/stat signaling pathway, and b) steroid hormones that include estrogens, xenoestrogens, and glucocorticoids, which generally act via ligand-regulated nuclear receptors. Accumulating evidence indicate that each of these hormone antagonizes cisplatin cytotoxicity in breast cancer cells by a variety of mechanisms. The implications of chemoresistance by these hormones to patients with hormone-sensitive breast cancer, and the potential benefits of increasing chemosensitivity by using anti-hormone treatment regimens are discussed.

In this issue, Boucher et al (1) describe the stimulatory effects of bisphenol S (BPS) on the differentiation and lipid accumulation in human adipocytes. Human exposure to bisphenol A (BPA), and very recently to its close analog BPS, has been associated with widespread health concerns for over 2 decades. Consequently, this subject deserves a brief perspective with respect to the following: 1) the prevalence of the bisphenols in the environment and the extent of human exposure; 2) their metabolic activities and putative mechanisms of action; and 3) an overview of ongoing conflicts between research scientists, environmental groups, the chemical industry, and regulatory agencies in the battle over the recognition of bisphenols as hazardous endocrine-disrupting chemicals (EDCs). BPA is a synthetic small molecule composed of 2 phenol groups. It is produced in very large quantities (over 5.4 million tons worldwide in 2015) in the manufacture of polycarbonate plastics and epoxy resins, both of which are made of repeating BPA monomers (Figure 1). BPS is an analog of BPA with a similar structure of 2 phenol groups on each side of a sulfonyl group (2). Polymers made of repeating BPS units are called polyethersulfone (PES). BPA-containing consumer products include plastic bottles, food utensils, lining of beverage and food cans, as well as dental cements, thermal receipt papers, iv medical devices, and water pipes (3). Although the carbonate linkages are rather stable and the polymers are chemically inert, BPA can leach out as a result of incomplete polymerization and/or because of some degradation of the polymer by elevated temperatures or acidic conditions (4, 5). Humans can be exposed to BPA through ingestion, skin absorption, inhalation, and through iv catheters. BPA at nanomolar concentrations has been measured in blood or urine of most individuals tested and has also been detected in the amniotic fluid and fetal plasma, indicating its passage across the placental during pregnancy, and underlying its potential to affect human fetal development (6). Given the worldwide use of polycarbonate plastics, epidemiological studies have had difficult times finding control groups that have not been exposed to BPA. BPA was first synthesized in 1891, with the first evidence for its estrogenicity coming from studies with ovariectomized rats in the 1930s. A landmark study in 1993 found an estrogen-like compound in water autoclaved in polycarbonate bottles for use in tissue culture, which was subsequently identified as BPA (7). Although BPA-based plastics have been in commercial use since the 1950s, reports on its estrogen-like properties with an environmental impact began to appear only in the early 1990s (8). Figure 2 shows an exponential increase in publications on BPA in PubMed, reaching 3000 between 2011 and 2015 and totaling over 6000. In response to the increasing pressure by concerned scientists and environmental groups to ban the use of BPA in consumer products, the chemical industry began to introduce BPS as a “safe substitute” for BPA in the mid2000s. Structural/chemical data show that PESs have excellent thermal, optical, and mechanical properties as plastics. However, soon thereafter, reports on EDC properties of BPS began to appear (Figure 2) (2), as well as initial findings on its detection in human urine (9). By now, thousands of in vitro and in vivo studies have provided evidence that BPA is a prototypical EDC that affects reproduction, neural development, behavior, cardiovascular functions, and metabolism, as well as the promotion of several types of cancer. Extensive research has revealed that there is no single mechanism by which BPA acts as an estrogen agonist. Proposed targets include clas-

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease with high mortality. Treatments, which can result in significant morbidity, have not substantially changed in three decades. The second messenger cyclic GMP (cGMP), which targets protein kinase G (PKG), is generated by guanylate cyclases (GCs), and is rapidly hydrolyzed by phosphodiesterases (PDEs). Activation of the cGMP/PKG pathway is antineoplastic in several cancer types, but its impact on HNSCC has not been fully exploited. We found differential expression of critical components of this pathway in four HNSCC cell lines. Several activators of soluble GC (sGC), as well as inhibitors of PDE5, increased intracellular cGMP, reduced cell viability, and induced apoptosis in HNSCC cells. The apoptotic effects of the sGC activator BAY 41-2272 and the PDE5 inhibitor Tadalafil (Cialis) were mediated by PKG. Furthermore, Tadalafil substantially reduced the growth of CAL27-derived tumors in athymic mice. Several drugs which either activate sGC or inhibit PDE5 are approved for treatment of nonmalignant conditions. These drugs could be repurposed as novel and effective therapeutics in patients with head and neck cancer.

Patients with advanced breast cancer often fail to respond to treatment, creating a need to develop novel biomarkers and effective therapeutics. Dopamine (DA) is a catecholamine that binds to five G protein-coupled receptors. We discovered expression of DA type-1 receptors (D1Rs) in breast cancer, thereby identifying these receptors as novel therapeutic targets in this disease. Strong to moderate immunoreactive D1R expression was found in 30% of 751 primary breast carcinomas, and was associated with larger tumors, higher tumor grades, node metastasis and shorter patient survival. DA and D1R agonists, signaling through the cGMP/protein kinase G (PKG) pathway, suppressed cell viability, inhibited invasion and induced apoptosis in multiple breast cancer cell lines. Fenoldopam, a peripheral D1R agonist that does not penetrate the brain, dramatically suppressed tumor growth in two mouse models with D1R-expressing xenografts by increasing both necrosis and apoptosis. D1R-expressing primary tumors and metastases in mice were detected by fluorescence imaging. In conclusion, D1R overexpression is associated with advanced breast cancer and poor prognosis. Activation of the D1R/cGMP/PKG pathway induces apoptosis in vitro and causes tumor shrinkage in vivo. Fenoldopam, which is FDA (Food and Drug Administration) approved to treat renal hypertension, could be repurposed as a novel therapeutic agent for patients with D1R-expressing tumors.

BACKGROUND AND OBJECTIVES:Management of patients with breast cancer often fails because of inherent or acquired resistance to chemotherapy. BRUCE (BIR repeat containing ubiquitin-conjugating enzyme) is a member of the inhibitor of apoptosis protein (IAP) family. It has various cellular functions including suppression of apoptosis and promotion of cytokinesis. Furthermore, it pays a critical role in promotion of DNA damage repair and preservation of genome stability, a new function recently reported by our group. Although BRUCE is expressed in breast cancer cell lines, its expression in human primary breast tumors and its contribution to chemoresistance in breast cancers has not been explored. Chemotherapeutic drugs are used in the treatment of breast cancer patients. However, they are not effective to all patients and patients often develop resistance. Consequently we explored if BRUCE protein level, as judged by immunohistochemistry (IHC), is higher in primary breast tumors than normal breast tissue. We also examined if depletion of BRUCE, using a lentiviral shRNA approach, enhances cell sensitivity to multiple chemotherapeutic agents, including cisplatin, an agent that induces DNA damage by generating DNA cross-links, and taxol, a microtubule stabilizer and mitotic inhibitor. The reason for including these two chemotherapeutic agents in this study is that they hit two essential cellular processes of DNA repair and cytokinesis in which BRUCE plays critical roles.RESULTS AND METHODS:IHC analysis of BRUCE revealed significantly higher levels of BRUCE in primary breast tumors than normal breast tissue. Knockdown of BRUCE protein expression by lentiviral shRNA resulted in increased sensitivity to cisplatin in the resistant breast cancer MDB-MD-231 cell line. Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone.CONCLUSIONS:Our data suggest that elevated protein levels of BRUCE in breast tumors may contribute to chemoresistance in breast cancer patients. In support of this suggestion, our data demonstrate that a reduction in BRUCE expression in breast cancer cell lines increases the toxicity of several chemotherapeutic agents. In all likelihood, the contribution of increased BRUCE levels to chemoresistance are likely due to its roles in suppression of apoptosis, promotion of cytokinesis and facilitation of DNA damage repair. These observations suggest that therapeutic suppression of BRUCE could improve chemosensitivity in chemo-resistant breast cancer patients. Therefore, future development of effective inhibitors of BRUCE could benefit patients with high BRUCE expression and chemoresistance.

New information concerning the effects of prolactin (PRL) on metabolic processes warrants reevaluation of its overall metabolic actions. PRL affects metabolic homeostasis by regulating key enzymes and transporters associated with glucose and lipid metabolism in several target organs. In the lactating mammary gland, PRL increases the production of milk proteins, lactose, and lipids. In adipose tissue, PRL generally suppresses lipid storage and adipokine release and affect adipogenesis. A specific case is made for PRL in the human breast and adipose tissues, where it acts as a circulating hormone and an autocrine/paracrine factor. Although its overall effects on body composition are both modest and species-specific, PRL may be involved in the manifestation of insulin resistance.

Introduction Several studies reported that the pregnancy-specific hormone placental lactogen (hPL) is expressed at both mRNA and protein levels in breast cancer. The overall objective was to establish hPL, the product of the CSH1 and CSH2 genes, as a biomarker for breast cancer. Methods CSH expression was determined at the mRNA level in breast cancer cell lines (BCC) and primary carcinomas by real-time and conventional PCR and the products verified as CSH1 by sequencing. Expression of hPL protein was examined by western blots and immuno-histochemistry, using commercial and custom-made polyclonal and monoclonal antibodies. Results Variable levels of CSH mRNA were detected in several BCC, and in some primary tumors. We detected a protein, slightly larger than recombinant hPL by western blotting using several antibodies, leading us to postulate that it represents an hPL variant (‘hPL’). Furthermore, some monoclonal antibodies detected ‘hPL’ by immunohistochemistry in breast carcinomas but not in normal breast. However, further examination revealed that these antibodies were non-specific, as efficient suppression of CSH mRNA by shRNA did not abolish the ‘hPL’ band. Custom-made monoclonal antibodies against recombinant hPL detected hPL of the correct size in placental lysate and hPL-overexpressing BCC, but not in unmodified cells or primary carcinomas. hPL protein was detected only when mRNA was increased several thousand fold. Conclusions We call into question previous reports of hPL expression in breast cancer which relied on mRNA levels as surrogates for protein and/or used improperly validated antibodies to measure hPL protein levels. Our data suggests that an inhibitory mechanism(s) prevents translation of CSH mRNA in breast cancer when not highly expressed. The mechanism by which translation of CSH mRNA is inhibited is intriguing and should be further investigated.