Intratumoral androgen levels are linked to TMPRSS2-ERG fusion in prostate cancer.

Intratumoral androgen biosynthesis is one of the mechanisms involved in the progression of prostate cancer, and an important target for novel prostate cancer therapies. Using gas chromatography-tandem mass spectrometry and genome-wide RNA sequencing, we have analyzed androgen concentrations and androgen-regulated gene expression in cancerous and morphologically benign prostate tissue specimens and serum samples obtained from 48 primary prostate cancer patients. Intratumoral dihydrotestosterone (DHT) concentrations were significantly higher in the cancerous tissues compared to benign prostate (P < 0.001). The tissue/serum ratios of androgens were highly variable between the patients, indicating individual patterns of androgen metabolism and/or uptake of androgens within the prostate tissue. An unsupervised hierarchical clustering analysis of intratissue androgen concentrations indicated that transmembrane protease, serine 2/ETS-related gene (TMPRSS2-ERG)-positive patients have different androgen profiles compared to TMPRSS2-ERG-negative patients. TMPRSS2-ERG gene fusion status was also associated with an enhanced androgen-regulated gene expression, along with altered intratumoral androgen metabolism, demonstrated by reduced testosterone concentrations and increased DHT/testosterone ratios in TMPRSS2-ERG-positive tumors. TMPRSS2-ERG-positive and -negative prostate cancer specimens have distinct intratumoral androgen profiles, possibly due to activation of testosterone-independent DHT biosynthesis via the alternative pathway in TMPRSS2-ERG-positive tumors. Thus, patients with TMPRSS2-ERG-positive prostate cancer may benefit from novel inhibitors targeting the alternative DHT biosynthesis.

Antiandrogens Reduce Intratumoral Androgen Concentrations and Induce Androgen Receptor Expression in Castration-Resistant Prostate Cancer Xenografts.

The development of castration-resistant prostate cancer (CRPC) is associated with the activation of intratumoral androgen biosynthesis and an increase in androgen receptor (AR) expression. We recently demonstrated that, similarly to the clinical CRPC, orthotopically grown castration-resistant VCaP (CR-VCaP) xenografts express high levels of AR and retain intratumoral androgen concentrations similar to tumors grown in intact mice. Herein, we show that antiandrogen treatment (enzalutamide or ARN-509) significantly reduced (10-fold, P < 0.01) intratumoral testosterone and dihydrotestosterone concentrations in the CR-VCaP tumors, indicating that the reduction in intratumoral androgens is a novel mechanism by which antiandrogens mediate their effects in CRPC. Antiandrogen treatment also altered the expression of multiple enzymes potentially involved in steroid metabolism. Identical to clinical CRPC, the expression levels of the full-length AR (twofold, P < 0.05) and the AR splice variants 1 (threefold, P < 0.05) and 7 (threefold, P < 0.01) were further increased in the antiandrogen-treated tumors. Nonsignificant effects were observed in the expression of certain classic androgen-regulated genes, such as TMPRSS2 and KLK3, despite the low levels of testosterone and dihydrotestosterone. However, other genes recently identified to be highly sensitive to androgen-regulated AR action, such as NOV and ST6GalNAc1, were markedly altered, which indicated reduced androgen action. Taken together, the data indicate that, besides blocking AR, antiandrogens modify androgen signaling in CR-VCaP xenografts at multiple levels.

Novel Lignan and stilbenoid mixture shows anticarcinogenic efficacy in preclinical PC-3M-luc2 prostate cancer model.

Prostate cancer is the most common cancer of men in the Western world, and novel approaches for prostate cancer risk reduction are needed. Plant-derived phenolic compounds attenuate prostate cancer growth in preclinical models by several mechanisms, which is in line with epidemiological findings suggesting that consumption of plant-based diets is associated with low risk of prostate cancer. The objective of this study was to assess the effects of a novel lignan-stilbenoid mixture in PC-3M-luc2 human prostate cancer cells in vitro and in orthotopic xenografts. Lignan and stilbenoid -rich extract was obtained from Scots pine (Pinus sylvestris) knots. Pine knot extract as well as stilbenoids (methyl pinosylvin and pinosylvin), and lignans (matairesinol and nortrachelogenin) present in pine knot extract showed antiproliferative and proapoptotic efficacy at ≥ 40 µM concentration in vitro. Furthermore, pine knot extract derived stilbenoids enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis already at ≥ 10 µM concentrations. In orthotopic PC-3M-luc2 xenograft bearing immunocompromized mice, three-week peroral exposure to pine knot extract (52 mg of lignans and stilbenoids per kg of body weight) was well tolerated and showed anti-tumorigenic efficacy, demonstrated by multivariate analysis combining essential markers of tumor growth (i.e. tumor volume, vascularization, and cell proliferation). Methyl pinosylvin, pinosylvin, matairesinol, nortrachelogenin, as well as resveratrol, a metabolite of pinosylvin, were detected in serum at total concentration of 7-73 µM, confirming the bioavailability of pine knot extract derived lignans and stilbenoids. In summary, our data indicates that pine knot extract is a novel and cost-effective source of resveratrol, methyl pinosylvin and other bioactive lignans and stilbenoids. Pine knot extract shows anticarcinogenic efficacy in preclinical prostate cancer model, and our in vitro data suggests that compounds derived from the extract may have potential as novel chemosensitizers to TRAIL. These findings promote further research on health-related applications of wood biochemicals.

Cadmium-induced effects on cellular signaling pathways in the liver of transgenic estrogen reporter mice.

Estrogen-like effects of cadmium (Cd) have been reported in several animal studies, and recent epidemiological findings suggest increased risk of hormone-dependent cancers after Cd exposure. The mechanisms underlying these effects are still under investigation. Our aim was to study the effects of Cd on cellular signaling pathways in vivo with special focus on estrogen signaling and to perform benchmark dose analysis on the effects. Transgenic adult ERE-luciferase male mice were exposed subcutaneously to 0.5-500 µg CdCl(2) per kg body weight (bw) or 17α-ethinylestradiol (EE2) for 3 days. These doses had no effects on organ and bw or testicular histology, indicating subtoxic exposure levels. The transgene luciferase, reporting genomic estrogen response, was significantly increased by EE2 but not by Cd. However, Cd significantly affected kinase phosphorylation and endogenous gene expression. Interestingly, gene expression changes displayed a traditional dose-response relationship, with benchmark dose levels for the expression of Mt1, Mt2, p53, c-fos, and Mdm2 being 92.9, 19.9, 7.6, 259, and 25.9 µg/kg bw, respectively, but changes in kinase phosphorylation were only detected at low exposure levels. Phosphorylation of Erk1/2 was significantly increased even in the lowest dose group, 0.5 µg/kg bw, rendering pErk1/2 a more sensitive sensor of exposure than changes in gene expression. Collectively, our data suggest that the effects triggered by Cd in vivo are markedly concentration dependent. Furthermore, we conclude that the estrogen-like effects of Cd are likely to result from a mechanism different from steroidal estrogens.

ERß1 represses FOXM1 expression through targeting ERα to control cell proliferation in breast cancer.

In this study, we investigated the effects of ectopic estrogen receptor (ER)ß1 expression in breast cancer cell lines and nude mice xenografts and observed that ERß1 expression suppresses tumor growth and represses FOXM1 mRNA and protein expression in ERα-positive but not ERα-negative breast cancer cells. Furthermore, a significant inverse correlation exists between ERß1 and FOXM1 expression at both protein and mRNA transcript levels in ERα-positive breast cancer patient samples. Ectopic ERß1 expression resulted in decreased FOXM1 protein and mRNA expression only in ERα-positive but not ERα-negative breast carcinoma cell lines, suggesting that ERß1 represses ERα-dependent FOXM1 transcription. Reporter gene assays showed that ERß1 represses FOXM1 transcription through an estrogen-response element located within the proximal promoter region that is also targeted by ERα. The direct binding of ERß1 to the FOXM1 promoter was confirmed by chromatin immunoprecipitation analysis, which also showed that ectopic expression of ERß1 displaces ERα from the endogenous FOXM1 promoter. Forced expression of ERß1 promoted growth suppression in MCF-7 cells, but the anti-proliferative effects of ERß1 could be overridden by overexpression of FOXM1, indicating that FOXM1 is an important downstream target of ERß1 signaling. Together, these findings define a key anti-proliferative role for ERß1 in breast cancer development through negatively regulating FOXM1 expression.

Stromal activation associated with development of prostate cancer in prostate-targeted fibroblast growth factor 8b transgenic mice.

Expression of fibroblast growth factor 8 (FGF-8) is commonly increased in prostate cancer. Experimental studies have provided evidence that it plays a role in prostate tumorigenesis and tumor progression. To study how increased FGF-8 affects the prostate, we generated and analyzed transgenic (TG) mice expressing FGF-8b under the probasin promoter that targets expression to prostate epithelium. Prostates of the TG mice showed an increased size and changes in stromal and epithelial morphology progressing from atypia and prostatic intraepithelial neoplasia (mouse PIN, mPIN) lesions to tumors with highly variable phenotype bearing features of adenocarcinoma, carcinosarcoma, and sarcoma. The development of mPIN lesions was preceded by formation of activated stroma containing increased proportion of fibroblastic cells, rich vasculature, and inflammation. The association between advancing stromal and epithelial alterations was statistically significant. Microarray analysis and validation with quantitative polymerase chain reaction revealed that expression of osteopontin and connective tissue growth factor was markedly upregulated in TG mouse prostates compared with wild type prostates. Androgen receptor staining was decreased in transformed epithelium and in hypercellular stroma but strongly increased in the sarcoma-like lesions. In conclusion, our data demonstrate that disruption of FGF signaling pathways by increased epithelial production of FGF-8b leads to strongly activated and atypical stroma, which precedes development of mPIN lesions and prostate cancer with mixed features of adenocarcinoma and sarcoma in the prostates of TG mice. The results suggest that increased FGF-8 in human prostate may also contribute to prostate tumorigenesis by stromal activation.

Estrogen-like effects of cadmium in vivo do not appear to be mediated via the classical estrogen receptor transcriptional pathway.

BACKGROUND: Cadmium (Cd), a ubiquitous food contaminant, has been proposed to be an endocrine disruptor by inducing estrogenic responses in vivo. Several in vitro studies suggested that these effects are mediated via estrogen receptors (ERs). OBJECTIVE: We performed this study to clarify whether Cd-induced effects in vivo are mediated via classical ER signaling through estrogen responsive element (ERE)-regulated genes or if other signaling pathways are involved. METHODS: We investigated the estrogenic effects of cadmium chloride (CdCl2) exposure in vivo by applying the Organisation for Economic Co-operation and Development (OECD) rodent uterotrophic bioassay to transgenic ERE-luciferase reporter mice. Immature female mice were injected subcutaneously with CdCl2 (5, 50, or 500 µg/kg body weight) or with 17α-ethinylestradiol (EE2) on 3 consecutive days. We examined uterine weight and histology, vaginal opening, body and organ weights, Cd tissue retention, activation of mitogen-activated protein kinase (MAPK) pathways, and ERE-dependent luciferase expression. RESULTS: CdCl2 increased the height of the uterine luminal epithelium in a dose-dependent manner without increasing the uterine wet weight, altering the timing of vaginal opening, or affecting the luciferase activity in reproductive or nonreproductive organs. However, we observed changes in the phosphorylation of mouse double minute 2 oncoprotein (Mdm2) and extracellular signal-regulated kinase (Erk1/2) in the liver after CdCl2 exposure. As we expected, EE2 advanced vaginal opening and increased uterine epithelial height, uterine wet weight, and luciferase activity in various tissues. CONCLUSION: Our data suggest that Cd exposure induces a limited spectrum of estrogenic responses in vivo and that, in certain targets, effects of Cd might not be mediated via classical ER signaling through ERE-regulated genes.

Dietary sources of lignans and isoflavones modulate responses to estradiol in estrogen reporter mice.

Dietary phytoestrogens, such as the lignan metabolite enterolactone (ENL) and the isoflavone genistein (GEN), are suggested to modulate the risk of estrogen-dependent disease (e.g., breast cancer) through regulation of estrogen signaling. However, the effects of complex food items containing lignans or isoflavones on estrogen receptor (ER) transactivation have not been assessed so far. In this study, the modulation of ER-mediated signaling by dietary sources of lignans (cereals and flaxseed) and isoflavones (soy) was studied in vivo. Adult ovariectomized 3 x ERE-luciferase (luc) reporter mice received isocaloric diets supplemented with flaxseed, rye, wheat, or soy for 40 h or two weeks, and an additional group of mice was challenged with 17beta-estradiol (E(2)) following the two-week dietary intervention. In non-E(2)-treated mice, soy diet induced luc expression in liver, mammary gland, and pituitary gland while the other diets had no effects. Interestingly, all diets modulated the E(2)-induced luc expression. In particular rye diet efficiently reduced E(2)-induced luc expression as well as uterine growth, the hallmark of estrogen action in vivo. It is concluded that dietary sources of lignans and isoflavones can modulate estrogen signaling in vivo. The results suggest intriguing possibilities for the modulation of the risk of estrogen-dependent diseases by dietary means.

Can modulation of mammary gland development by dietary factors support breast cancer prevention?

Breast cancer continues to be a major challenge for public health, since it is the most common cancer of women in the Western world, and its prevalence is still increasing. In order to achieve better results in the prevention and treatment of breast cancer it is crucial to identify the mechanisms behind its initiation, i.e. the changes and deviations that have occurred in the mammary gland growth. It has long been known that a woman's reproductive history is the strongest breast cancer risk factor if genetic background and age are excluded. The reproductive hormones, and the timing of events leading to changes in these hormones, and consequently, in the mammary gland, are the most important players. However, it has become obvious that dietary components may also contribute to breast cancer risk through their effects on the mammary gland. The past few years have added important information to our knowledge of the mechanisms behind breast cancer initiation at the level of target cells (mammary stem cells) and gene expression (genetic 'fingerprint' associated with persistent pregnancy-induced protection against breast cancer), as well as of the effects of certain dietary factors (steroid action modulators). These results and their links to breast cancer initiation and progression will be discussed.

Determination of plant and enterolignans in human serum by high-performance liquid chromatography with tandem mass spectrometric detection.

An HPLC-MS/MS method was validated for the determination of the plant lignans 7-hydroxymatairesinol (HMR), matairesinol (Mat), secoisolariciresinol (Seco), lariciresinol (Lar), and cyclolariciresinol (CLar) and for the enterolignans 7-hydroxyenterolactone (HEL), enterodiol (ED), and enterolactone (EL) in human serum. The method included sample enzymatic hydrolysis, solid-phase extraction, and lignan analysis using a triple quadrupole mass spectrometer with electrospray ionisation in the multiple-reaction monitoring mode. The serum lignans were quantified using deuterated Mat or EL as internal standards. The method met the validation criteria for selectivity, intra- and inter-assay precision, and accuracy. The method was applied to ten serum samples collected from healthy individuals (five men and five women) consuming their habitual Finnish diet. All lignans except HMR and Seco were found in quantifiable amounts in the samples. All serums contained EL; the average concentration was 34 nM. In three individuals, the serum concentration of plant lignans was higher than that of enterolignans. Using the method, common dietary plant lignans and their major metabolites can be reliably quantified in human serum at low-nanomolar concentrations in a simple and rapid way.

New lignan metabolites in rat urine.

Ten potential lignan metabolites were quantified in rat urine extracts using liquid chromatography-tandem mass spectrometry. The rats were orally administered with the plant lignans 7-hydroxymatairesinol, matairesinol, lariciresinol or secoisolariciresinol, or with the mammalian lignan enterolactone. The samples were enzymatically hydrolysed and solid-phase extracted before analysis. Of the analysed compounds, only trace amounts of 7-oxoenterolactone could be detected in the urine extracts before administration, but after administration of any of the lignans, the excretion of 7-oxoenterolactone increased and monodemethylated matairesinol and 4,4'-dihydroxyenterolactone could be detected. In addition, other novel lignan metabolites were detected, i.e., 7-oxomatairesinol, alpha-conidendrin, and alpha- and beta-conidendric acid.

Urinary excretion of lignans after administration of isolated plant lignans to rats: the effect of single dose and ten-day exposures.

The difference in urinary excretion of mammalian and plant lignans in rats was determined after oral administration of equivalent doses (25 mg/kg of body weight) of 7-hydroxymatairesinol (HMR), lariciresinol (LAR), matairesinol (MR), and secoisolariciresinol (SECO). Twenty-four hours-urine samples were collected after a single dose and after administration of one dose/day for 10 days. Eight lignans were analysed in urine extracts using a high-performance liquid chromatography-tandem mass spectrometry method showing good sensitivity and repeatability. After a single dose of HMR, LAR, MR, and SECO, the main metabolites were 7-hydroxyenterolactone (HENL), cyclolariciresinol (CLAR), enterolactone (ENL), and enterodiol (END), respectively, but after 10-day exposure ENL was the main metabolite of all the tested lignans, showing a considerably higher excretion than after a single dose. Metabolic transformations of plant lignans into each other could also be observed.

Role of estrogens in development of prostate cancer.

Estrogens have previously been extensively used in prostate cancer treatment. Serious side effects, primarily in cardiovascular system have, however, limited their use. The therapeutic effect of estrogen in preventing prostate cancer growth was mainly obtained indirectly by feedback inhibition of the hypothalamic release of LRH leading to lowered serum androgen levels and castration like effects. Prostate tissue is also most probably a target for direct regulation by estrogens. Prostate contains estrogen receptor alpha (ERalpha) and beta (ERbeta), which are localized characteristically in stroma and epithelium, respectively. The physiological function of these receptors is not known but there is evidence of the role of estrogens in prostatic carcinogenesis. Developing prostate seems particularly sensitive to increased level of endogenous and/or exogenous estrogens. Perinatal or neonatal exposure of rats and mice to estrogens leads to "imprinting" of prostate associated with increased proliferation, inflammation and dysplastic epithelial changes later in life. Prolonged treatment of adult rodents with estrogens along with androgens also leads to epithelial metaplasia, PIN-like lesions and even adenocarcinoma of prostate speaking for the role of estrogen in prostate cancer development. Recent results concerning antiestrogen inhibition of prostate cancer development beyond PIN-type lesions in transgenic mouse models further suggests a role for estrogens in prostate cancer progression. These results also suggest that direct inhibition of estrogen action at the level of prostate tissue may provide an important novel principle of development of prostate cancer therapies.

Enterolactone inhibits the growth of 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas in the rat.

The inverse association between a high enterolactone (ENL) concentration in both urine and serum, and the risk of breast cancer found in epidemiological studies suggests a chemopreventive action for ENL. However, no causal relationship has been established in clinical studies or in experimental models for breast cancer. In the present study, the potential chemopreventive action of p.o. administered ENL (1 or 10 mg/kg of body weight) was tested in 7,12-dimethylbenz(a)anthracene-induced mammary cancers of the rat. Rats were maintained on a standard open-formula chow diet. Daily p.o. administration of ENL at a dose of 10 mg/kg of body weight for 7 weeks significantly inhibited tumor growth. The growth-inhibitory effect of ENL was more pronounced on the new tumors, which developed during the treatment period, but ENL also inhibited the growth of those tumors established before the start of the lignan administration. The rat serum concentration of ENL, which illustrated a permanent positive effect on breast cancer growth, was 0.4 microM, which is >10-fold as compared with the serum concentrations found in the general human population. The effect of ENL was not restricted to any specific histological tumor type. ENL was demonstrated to act as a weak aromatase inhibitor in vitro and to reduce the relative uterine weight of the 7,12-dimethylbenz(a)anthracene-treated nonovariectomized rats. However, in a short-term assay ENL had no effect on the uterine growth of the intact or androstenedione-treated immature rats. Thus, the mechanism of the ENL action and its minimum or optimal daily dose remains to be clarified.

Structural determinants of plant lignans for the formation of enterolactone in vivo.

The quantity of mammalian lignans enterolactone (ENL) and enterodiol (END) and of plant lignans secoisolariciresinol (SECO) and 7-hydroxymatairesinol (HMR) excreted in a 24-h rat urine sample was measured after a single p.o. dose of an equivalent quantity of secoisolariciresinol diglycoside (SDG), secoisolariciresinol (SECO), matairesinol (MR), 7-hydroxymatairesinol (HMR) and ENL. Plant lignans (SECO and HMR) were partially absorbed as such. The aglycone form of SECO was more efficiently converted into mammalian lignans END and ENL than the glycosylated form, SDG. Of plant lignans, MR produced the highest quantities of ENL: the quantity was over twofold compared with HMR or SDG. The majority of the animals, which had been given SECO, excreted higher quantities of END than ENL into urine, but ENL was the main lignan metabolite after SDG. The highest quantities of ENL in urine were measured after the administration of ENL as such. The (-)SECO isolated from Araucaria angustifolia was converted into (-)ENL only. The administration of (-)SDG, which was shown to produce (+)SECO, resulted in excretion of (+)ENL only and (-)HMR was converted into (-)ENL only. This confirmed that the absolute configurations at C8 and C8' are not changed during the microbial metabolism. Whether the biological effects are enantiomer-specific, remains to be resolved.