ACSL4: biomarker, mediator and target in quadruple negative breast cancer.

Breast cancer is a heterogeneous disease for which effective treatment depends on correct categorization of its molecular subtype. For the last several decades this determination has relied on hormone receptor status for estrogen, progesterone and HER2. More recently, gene expression data have been generated that further stratify both receptor-positive and receptor-negative cancers. The fatty acid-activating enzyme, ACSL4, has been demonstrated to play a role in the malignant phenotype of a variety of cancers, including breast. This lipid metabolic enzyme is differentially expressed as a function of subtype in breast tumors, with highest expression observed in the mesenchymal (claudin low) and basal-like subtypes. Here we review data that support the potential of utilizing ACSL4 status as both a biomarker of molecular subtype and a predictor of response to a variety of targeted and non-targeted treatment regimens. Based on these findings, we suggest 3 expanded roles for ACSL4: 1. as a biomarker for classification of breast cancer subtypes; 2. as a predictor of sensitivity to hormone-based and certain other therapies; and 3. as a target for the development of new treatment modalities.

SOM230 inhibits insulin-like growth factor-I action in mammary gland development by pituitary independent mechanism: mediated through somatostatin subtype receptor 3?

Somatostatin analogs (SAs) treat acromegaly by lowering pituitary GH secretion, which, in turn, lowers systemic IGF-I. The profound systemic effect is often greater than expected in the face of only partial GH suppression. Here we report that the SA SOM230 can also act by a nonpituitary-mediated inhibition of IGF-I action. SOM230 inhibited mammary development in intact and hypophysectomized female rats, a process requiring IGF-I. IGF-I overcame this inhibition. SOM230 also inhibited other actions of IGF-I (inhibition of apoptosis, phosphorylation of insulin receptor substrate-1, and cell division). SOM230 did not reduce IGF-I mRNA abundance in mammary gland but did stimulate IGF binding protein 5 (IGFBP5). IGFBP5 was 3.75 times higher in mammary epithelium of SOM230 than in placebo animals (P < 0.001). Administration of IGFBP-5 also inhibited GH-induced mammary development (P < 0.001). Measurement of sstr(1-5) (somatostatin subtype receptor) by real-time RT-PCR revealed that the mammary glands had an abundance of sstr(3) and lower amounts of sstr(4) and sstr(5) but no sstr(1) or sstr(2.) That mammary development was also inhibited to a lesser degree than SOM230 by octreotide, whose main action is through sstr(2), strongly suggests that sstr(3) is at least in part mediating the effects of the SAs. We conclude that 1) SAs inhibit IGF-I action in the mammary gland through a novel nonpituitary mechanism; 2) IGFBP-5, here shown to inhibit pubertal mammary development, might mediate the effect; and 3) Measurement of available sstr receptors in the mammary gland suggests that sstr(3) mediates the SA activity, but sstr(5) is also a possible mediator.

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.

Breast cancer molecular subtypes: from TNBC to QNBC.

Treatment protocols for breast cancer depend predominantly on receptor status with respect to estrogen (estrogen receptor alpha), progesterone (progesterone receptor) and human epidermal growth factor [human epidermal growth factor receptor 2 (HER2)]. The presence of one or more of these receptors suggests that a treatment targeting these pathways might be effective, while the absence of, or in the case of HER2, lack of overexpression of, all of these receptors, termed triple negative breast cancer (TNBC), indicates a need for the more toxic chemotherapy. In an effort to develop targeted therapies for TNBC, it will be necessary to differentiate among specific TNBC subtypes. The subset of TNBC that expresses androgen receptor (AR) has been determined to express genes consistent with a luminal subtype and therefore may be amenable to therapies targeting either AR, itself, or other pathways typical of a luminal subtype. Recent investigations of the AR signal pathway within breast cancer lead to AR as a significant target for breast cancer therapy with several clinical trials currently in progress. The subclass of TNBC that lacks AR, which we have termed quadruple negative breast cancer (QNBC) currently lacks a defined targetable pathway. Unlike AR-positive TNBC, QNBC predominantly exhibits a basal-like molecular subtype. Several subtypes and related pathway proteins are preferentially expressed in QNBC that may serve as effective targets for treatment, such as ACSL4, SKP2 and EGFR. ACSL4 expression has been demonstrated to be inversely correlated with expression of hormone/growth factor receptors and may thus serve as a biomarker for QNBC as well as a target for therapy. In the following review we summarize some of the current efforts to develop alternatives to chemotherapy for TNBC and QNBC.

Progesterone stimulates mammary gland ductal morphogenesis by synergizing with and enhancing insulin-like growth factor-I action.

Progestins have been implicated in breast cancer development, yet a role for progesterone (Pg) in ductal morphogenesis (DM) has not been established. To determine whether Pg could cause DM, we compared relative effects of Pg, estradiol (E2) and IGF-I on anatomical and molecular biological parameters of IGF-I-related DM in oophorectomized female IGF-I(-/-) mice. Pg had little independent effect on mammary development, but together with IGF-I, in the absence of E2, Pg stimulated an extensive network of branching ducts, occupying 92% of the gland vs. 28.3% with IGF-I alone, resembling pubertal development (P < 0.002). Its major effect was on enhancing duct length and branching (P < 0.002). Additionally, Pg enhanced phosphorylation of IRS-1, increased cell division, and increased the antiapoptotic effect of IGF-I. Pg action was inhibited by RU486 (P < 0.01). E2 also stimulated DM by enhancing IGF-I action but had a greater effect on terminal end bud formation and side branching (P < 0.002). In contrast to previous findings, long-term exposure to E2 alone, without IGF-I, caused formation of ducts and side branches, a novel finding. Both IGF-I and E2 were found necessary for Pg-induced alveolar development. In conclusion, Pg, through Pg receptor can enhance IGF-I action in DM, and E2 acts through a similar mechanism; E2 alone caused formation of ducts and side branches; there were differences in the actions of Pg and E2, the former largely affecting duct formation and extension, and the latter side branching; and both IGF-I and E2 were necessary for Pg to form mature alveoli.

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 ß-catenin. These results suggest ACSL4 could serve as a biomarker and potential therapeutic target for CRPC.

Lipid metabolism in prostate cancer.

The malignant transformation of cells requires adaptations across multiple metabolic processes to satisfy the energy required for their increased rate of proliferation. Dysregulation of lipid metabolism has been a hallmark of the malignant phenotype; increased lipid accumulation secondary to changes in the levels of a variety of lipid metabolic enzymes has been documented in a variety of tumors, including prostate. Alterations in prostate lipid metabolism include upregulation of several lipogenic enzymes as well as of enzymes that function to oxidize fatty acids as an energy source. Cholesterol metabolism and phospholipid metabolism are also affected. With respect to lipogenesis, most studies have concentrated on increased expression and activity ofthe de novo fatty acid synthesis enzyme, fatty acid synthase (FASN), with suggestions that FASN might function as an oncogene. A central role for fatty acid oxidation in supplying energy to the prostate cancer cell is supported by the observation that the peroxisomal enzyme, α-methylacyl-CoA racemase (AMACR), which facilitates the transformation of branched chain fatty acids to a form suitable for ß-oxidation, is highly overexpressed in prostate cancer compared with normal prostate. Exploitation of the alterations in lipid metabolic pathways in prostate cancer could result in the development of new therapeutic modalities as well as provide candidates for new prognostic and predictive biomarkers. AMACR has already proven to be a valuable biomarker in distinguishing normal from malignant prostate tissue, and is used routinely in clinical practice.

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/ß-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.

Expression of Long-chain Fatty Acyl-CoA Synthetase 4 in Breast and Prostate Cancers Is Associated with Sex Steroid Hormone Receptor Negativity.

Previous studies have shown that key enzymes involved in lipid metabolic pathways are differentially expressed in normal compared with tumor tissues. However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established. Fatty acid synthase is overexpressed in a variety of cancers, including breast and prostate. The purpose of the present study was to examine the expression patterns of additional lipid metabolic enzymes in human breast and prostate cancers. This was accomplished by analysis of published expression databases, with confirmation by immunoblot assays. Our results indicate that the fatty acid-activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), is differentially expressed in human breast cancer as a function of estrogen receptor alpha (ER) status. In 10 separate studies, ACSL4 messenger RNA (mRNA) was overexpressed in ER-negative breast tumors. Of 50 breast cancer cell lines examined, 17 (89%) of 19 ER-positive lines were negative for ACSL4 mRNA expression and 20 (65%) of 31 ER-negative lines expressed ACSL4 mRNA. The inverse relationship between ER expression and ACSL4 expression was also observed for androgen receptor status in both breast and prostate cancers. Furthermore, loss of steroid hormone sensitivity, such as that observed in Raf1-transfected MCF-7 cells and LNCaP-AI cells, was associated with induction of ACSL4 expression. Ablation of ACSL4 expression inMDA-MB-231 breast cancer cells had no effect on cell proliferation; however, sensitivity to the cytotoxic effects of triacsin C was increased three-fold in the cells lacking ACSL4.

Subcellular localization of phosphatidylinositol synthesis.

It is well-established that the endoplasmic reticulum is the major site of phosphatidylinositol (PtdIns) synthesis. The PtdIns synthetic ability of other organelles, such as plasma membrane and nucleus, remains controversial. In the present study, we re-examine this question by comparing PtdIns synthesis in isolated cytoplasts (enucleated cells) with that in corresponding karyoplasts (nuclei surrounded by plasma membrane but lacking most cytoplasmic components). We report that cytoplasts are competent to carry out both basal and stimulated PtdIns synthesis as well as polyphosphoinositide hydrolysis, while karyoplasts can neither synthesize PtdIns nor hydrolyze phosphoinositides in response to agonists. The karyoplasts are, however, capable of synthesizing phosphatidylcholine (PtdCho), as previously reported. From these data, we conclude that PtdIns synthesis is limited to cytoplasmic components, and cannot be sustained by either plasma membrane or nucleus under conditions that permit robust PtdCho synthesis.

Regulation of de novo phosphatidylinositol synthesis.

Mechanisms that function to regulate the rate of de novo phosphatidylinositol (PtdIns) synthesis in mammalian cells have not been elucidated. In this study, we characterize the effect of phorbol ester treatment on de novo PtdIns synthesis in C3A human hepatoma cells. Incubation of cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) initially (1-6 h) results in a decrease in precursor incorporation into PtdIns; however, at later times (18-24 h), a marked increase is observed. TPA-induced glucose uptake from the medium is not required for observation of the stimulation of PtdIns synthesis, because the effect is apparent in glucose-free medium. Inhibition of the activation of arachidonic acid substantially blocks the synthesis of PtdIns but has no effect on the synthesis of phosphatidylcholine (PtdCho). Increasing the concentration of cellular phosphatidic acid by blocking its conversion to diacylglycerol, on the other hand, enhances the synthesis of PtdIns and inhibits the synthesis of PtdCho. The TPA-induced stimulation of PtdIns synthesis is not the result of the concomitant TPA-induced G1 arrest, because G1 arrest induced by mevastatin has no effect on PtdIns synthesis. Inhibition of protein kinase C activity blocks the stimulatory action of TPA on de novo synthesis of PtdIns but has no effect on TPA-induced inhibition. Potential sites of enzymatic regulation are discussed.

Analysis of hormone-stimulated phosphatidylinositol synthesis.

Agonist-stimulated phosphoinositide turnover is accompanied by compensatory resynthesis of these lipids. Several lines of evidence suggest that resynthesis of phosphatidylinositol (PtdIns) involves phosphorylation of diacylglycerol (DG) (salvage pathway) rather than acylation of glycerol phosphate (de novo pathway), although a contribution from the de novo pathway has not been ruled out. To determine the relative contribution of the de novo and salvage pathways in stimulated PtdIns resynthesis, an inhibitor of de novo synthesis (Triacsin C) was incubated simultaneously with the hormone agonist. Results indicate that at early times (90 min), hormone-stimulated PtdIns synthesis proceeds predominantly via the salvage pathway, although some de novo synthesis is also taking place. At later times (24 h), stimulated synthesis is solely via the de novo pathway. Increasing cellular DG content by either adding exogenous DG or treating cells with bacterial phospholipase C (bPLC) results in deacylation of the DG rather than phosphorylation; however, inhibition of this deacylation fails to stimulate phosphorylation by DG kinase (DGK), suggesting channeling of the DG substrate between PLC and DG kinase. Receptor activation is not required for activation of DGK, since treatment with a calcium ionophore induces the same Triacsin C-insensitive PtdIns synthesis. Depletion of the polyphosphoinositide pools by treatment with wortmannin prevents both hormone and A23187-induced polyphosphoinositide hydrolysis; however, A23187 is still able to induce hydrolysis of PtdIns and subsequent compensatory resynthesis. The inability of R59949 to inhibit either hormone-induced or ionophore-induced PtdIns resynthesis suggests that the alpha isoform is not involved; however, its possible that the channeling phenomenon prevents the inhibitor from gaining access to the diacylglycerol kinase enzyme. Further study will be required to determine which isoform catalyzes hormone-induced resynthesis of PtdIns.

Lipid metabolism in phosphatidylinositol transfer protein alpha-deficient vibrator mice.

Mice that are homozygous for the vibrator mutation express 65-85% less phosphatidylinositol transfer protein alpha (PITPalpha) than their wild type litter mates. By postnatal day 10-12 (P10-12) they exhibit signs of neurodegeneration and die prematurely by P40. In the present study, we examine the lipid content of brain, liver, and mammary glands from these animals. Lipid-mediated signal transduction is evaluated in primary fibroblast cultures. With respect to the lipid make-up of brain and liver, we report that there is a significant increase (2- to 4-fold) in the neutral lipids present in the livers of vb/vb animals when compared with wild type (+/+) litter mates. No significant changes are observed in the brains of these animals. The mammary glands of vb/vb mice are underdeveloped with respect to ductal and alveolar structures, and the fat pad is composed of predominantly brown adipose tissue rather than the white adipose tissue characteristic of age-matched wild type litter mates. No differences are observed in any aspect of lipid-mediated signal transduction.