Marie E. Monaco

Endothelin‐1 production and agonist activities in cultured prostate‐derived cells: Implications for regulation of endothelin bioactivity and bioavailability in prostatic hyperplasia

Endothelin‐1 (ET‐1) interacts with specific G‐protein‐coupled receptors to initiate short‐term (contraction) and long‐term (mitogenesis) events in target cells. ET‐1 is an abundant prostate secretory protein that, in its biologically active form, elicits prostatic smooth muscle contraction. The present study was designed to determine the effects of ET‐1 on prostate cell growth and to examine the regulation of endogenous ET‐1 activity and bioavailability.

Model systems for the study of estrogen action in tissue culture

Abstract Physiologic concentrations of estrogen stimulate precursor incorporation and growth in several lines of human breast cancer cells in long term tissue culture. Antiestrogens inhibit precursor incorporation and eventually kill the cells. Estrogens reverse antiestrogen effects. Responsive cell lines contain high affinity specific cytoplasmic receptors for estrogen. When binding and stimulation are measured under similar conditions, it appears that only a small proportion of receptor sites need be occupied for maximal stimulation. Thymidine kinase specific activity is increased by estradiol and may, in part, explain enhanced thymidine incorporation. These cell lines should prove useful for the study of the mechanisms by which estrogens regulate growth in human breast cancer

Thyrotropin releasing hormone rapidly enhances [32P]orthophosphate incorporation into phosphatidic acid in cloned GH3 cells.

Summary Thyrotropin releasing hormone (TRH) enhanced incorporation of [32P]orthophosphate into phospholipids in GH3 cells, cloned rat pituitary cells that produce prolactin. [32P]orthophosphate incorporation into phosphatidic acid was significantly increased 30 sec after TRH addition and attained a steady-state level after 2 min. In contrast, [32P]orthophosphate incorporation into phosphatidylinositol was not significantly increased until 2 min after TRH. [32P]orthophosphate incorporation into phosphatidylcholine and phosphatidylethanolamine were not affected. TRH had no effect on [32P]orthophosphate uptake by GH3 cells for at least 2 min, but stimulated 45Ca2+ efflux from GH3 cells within 15 sec. Since TRH enhancement of [32P]orthophosphate incorporation into phosphatidic acid was temporally associated with its effect on 45Ca2+ efflux and Ca2+ appears to be a critical intracellular regulator of prolactin release, we suggest that acidic phospholipid metabolism may be involved in TRH-induced, Ca2+-mediated prolactin secretion.

Differentiation of the ductal epithelium and smooth muscle in the prostate gland are regulated by the Notch/PTEN-dependent mechanism.

We have shown previously that during branching morphogenesis of the mouse prostate gland, Bone morphogenetic protein 7 functions to restrict Notch1-positive progenitor cells to the tips of the prostate buds. Here, we employed prostate-specific murine bi-genic systems to investigate the effects of gain and loss of Notch function during prostate development. We show that Nkx3.1(Cre) and Probasin(Cre) alleles drive expression of Cre recombinase to the prostate epithelium and periepithelial stroma. We investigated the effects of gain of Notch function using the Rosa(NI1C) conditional allele, which carries a constitutively active intracellular domain of Notch1 receptor. We carried out the analysis of loss of Notch function in Nkx3.1(Cre/+);RBP-J(flox/flox) prostates, where RBP-J is a ubiquitous transcriptional mediator of Notch signaling. We found that gain of Notch function resulted in inhibition of the tumor suppressor PTEN, and increase in cell proliferation and progenitor cells in the basal epithelium and smooth muscle compartments. In turn, loss of Notch/RBP-J function resulted in decreased cell proliferation and loss of epithelial and smooth muscle progenitors. Gain of Notch function resulted in an early onset of benign prostate hyperplasia by three months of age. Loss of Notch function also resulted in abnormal differentiation of the prostate epithelium and stroma. In particular, loss of Notch signaling and increase in PTEN promoted a switch from myoblast to fibroblast lineage, and a loss of smooth muscle. In summary, we show that Notch signaling is necessary for terminal differentiation of the prostate epithelium and smooth muscle, and that during normal prostate development Notch/PTEN pathway functions to maintain patterned progenitors in the epithelial and smooth muscle compartments. In addition, we found that both positive and negative modulation of Notch signaling results in abnormal organization of the prostate tissue, and can contribute to prostate disease in the adult organ.

Long chain fatty Acyl-CoA synthetase 4 is a biomarker for and mediator of hormone resistance in human breast cancer.

The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4) in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2) in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC), characterized by the absence of androgen receptor (AR) and therefore referred to as quadruple negative breast cancer (QNBC). Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor α (ER)-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed in vivo in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype.

LEF1 Identifies Androgen-Independent Epithelium in the Developing Prostate

Lymphoid enhancer-binding factor (LEF)1 is a major mediator and a target in canonical Wnt/β-catenin pathway. Interactions between the androgen receptor (AR) and canonical Wnt pathways have been implicated in the development of the genitourinary organs. Here, we investigated the localization and role of LEF1-positive cells during development of the prostate gland in human and in the murine model. We show that during human prostate development, LEF1 is restricted to the basal epithelial layer of the urogenital sinus. During mouse development, Lef1 is also present in the urogenital mesenchyme in addition to the basal epithelial layer of the urogenital sinus. In the course of elongation and branching of the prostatic ducts, Lef1 is localized to the proliferating epithelium at the distal tips of the buds. Notably, during branching morphogenesis, domains of Lef1 and AR are mutually exclusive. We further employed the TOPGAL reporter strain to examine the dynamics of Wnt signaling in the context of prostate regression upon a 7-d treatment with a competitive AR inhibitor, bicalutamide. We found that Wnt/Lef1-positive basal cells are not dependent upon androgen for survival. Furthermore, upon bicalutamide treatment, Wnt/Lef1-positive basal progenitors repopulated the luminal compartment. We conclude that Wnt/Lef1 activity identifies an androgen-independent population of prostate progenitors, which is important for embryonic development and organ maintenance and regeneration in the adult.

Mitogenic activation of human prostate-derived fibromuscular stromal cells by bradykinin

Biologically active kinin peptides are released from precursor kininogens by kallikreins. Kinins act on kinin receptors to mediate diverse biological functions including smooth muscle contraction, inflammation, pain and mitogenicity. All components of the kallikrein‐kinin system exist in human male genital secretions suggesting that these molecules participate in physiological and pathophysiological genitourinary function. The objective of this study was to assess the consequences of kinin action on prostate cells. Primary cultures of prostate secretory epithelial (PE) and prostate fibromuscular stromal (PS) cells were established from human prostate tissue. Transcripts encoding both the human B1 and B2 bradykinin receptor subtypes were detected in human prostate transition‐zone tissue and in cultured cells by RT–PCR. In receptor binding assays, the B1 subtype predominated on PE cell membranes and the B2 subtype predominated on PS cell membranes. In PS cells, but not in PE cells, BK induced significant inositol phosphate accumulation and [3H]‐thymidine uptake. These responses were mediated through the B2 receptor subtype. The use of signal transduction inhibitors indicated that mitogenic activation by BK occurred through both protein kinase C (PKC) and protein tyrosine kinase dependent mechanisms. PMA (phorbol 12‐myristate 13‐acetate) produced maximal [3H]‐thymidine uptake by PS cells, resulted in cell elongation and caused the α‐actin fibres present in PS smooth muscle cells to became organized into parallel arrays along the length of the elongated cells. In summary, the prostate contains a functional kallikrein‐kinin system, which could be significant in physiological and pathophysiological prostate function.

Fatty acid metabolism in breast cancer subtypes

Dysregulation of fatty acid metabolism is recognized as a component of malignant transformation in many different cancers, including breast; yet the potential for targeting this pathway for prevention and/or treatment of cancer remains unrealized. Evidence indicates that proteins involved in both synthesis and oxidation of fatty acids play a pivotal role in the proliferation, migration and invasion of breast cancer cells. The following essay summarizes data implicating specific fatty acid metabolic enzymes in the genesis and progression of breast cancer, and further categorizes the relevance of specific metabolic pathways to individual intrinsic molecular subtypes of breast cancer. Based on mRNA expression data, the less aggressive luminal subtypes appear to rely on a balance between de novo fatty acid synthesis and oxidation as sources for both biomass and energy requirements, while basal-like, receptor negative subtypes overexpress genes involved in the utilization of exogenous fatty acids. With these differences in mind, treatments may need to be tailored to individual subtypes.

An Antibody-Toxin Conjugate Directed against a Human Mammary Cancer Antigena

A conjugate has been constructed consisting of diphtheria toxin fragment A (DTA) linked to a monoclonal antibody, BLMRL-HMFG-Mc5 (MC5), directed against a mammary cancer antigen. The conjugate retains both binding and DTA enzymatic activity when tested against target MCF-7 cells, although the conjugate binds less well than the unconjugated antibody. The conjugate is toxic to MCF-7 cells. Toxicity is both dose- and time-dependent. Half-maximal toxicity is observed with 2-5 X 10(-8) M conjugate after 5 days of incubation. However, the conjugate need only be in contact with the cells for 1 day in order to effect complete killing by 5 days. The earliest that an effect can be seen on cell growth is 2 days. When tested against WRK-1 rat mammary tumor cells, the conjugate is inactive.

ACSL4 promotes prostate cancer growth, invasion and hormonal resistance

Increases in fatty acid metabolism have been demonstrated to promote the growth and survival of a variety of cancers, including prostate cancer (PCa). Here, we examine the expression and function of the fatty acid activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), in PCa. Ectopic expression of ACSL4 in ACSL4-negative PCa cells increases proliferation, migration and invasion, while ablation of ACSL4 in PCa cells expressing endogenous ACSL4 reduces cell proliferation, migration and invasion. The cell proliferative effects were observed both in vitro, as well as in vivo. Immunohistochemical analysis of human PCa tissue samples indicated ACSL4 expression is increased in malignant cells compared with adjacent benign epithelial cells, and particularly increased in castration-resistant PCa (CRPC) when compared with hormone naive PCa. In cell lines co-expressing both ACSL4 and AR, proliferation was independent of exogenous androgens, suggesting that ACSL4 expression may lead to CRPC. In support for this hypothesis, ectopic ACSL4 expression induced resistance to treatment with Casodex, via decrease in apoptosis. Our studies further indicate that ACSL4 upregulates distinct pathway proteins including p-AKT, LSD1 and β-catenin. These results suggest ACSL4 could serve as a biomarker and potential therapeutic target for CRPC.