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Several conclusions can be reached from the data presented. (1). Id-1 protein is an important biomarker for PCA. (2). The level of its expression is related to the malignancy of the PCA. (3). Id-1 expression levels in PCA tissue samples has the potential to be used as a prognostic marker since high Id-1 expression is related to poor prognosis; and low expression to good prognosis. (4). One of the possible pathways for Id-1 gene action is through the inactivation of the p16/Rb tumor suppressor pathway. (5). The activation of the MAPK signaling pathway is a cell proliferation pathways induced by Id-1. (6). Activation of the Id-1 gene may be an important step in the transition stage of PCA cells from androgen-dependent to androgen-independent state, the most malignant form of PCA. [ABSTRACT FROM AUTHOR]

Cadmium chloride (Cd) is a toxicant that has been implicated in human prostate cancer (PCA). The goal of the present study was to evaluate the immunoexpression of markers for cell proliferation, apoptosis, resistance to apoptosis, and to determine mutations on segments of the bcl-2 gene, in preneoplastic lesions induced in rat prostate after treatment with Cd alone or in combination with zinc chloride (Zn). We evaluated: 1) The % of cells positively immunostained for the proliferating cell nuclear antigen (PCNA), 2) The % of apoptotic cells (evaluated by TUNEL), 3) The volume fraction of Bcl-2 immunostaining. 4) The mutations on a segment of 253 pb of bcl-2, in the ventral prostate lobe of normal and treated rats with Cd alone or in the presence of Zn in the drinking water for 18 mo. Our results indicate that the % of PCNA positive nuclei was significantly increased in preneoplastic prostatic acini of Cd-treated rats alone and in combination with Zn, compared to the normal acini of untreated animals. No significant changes were detected on the apoptotic rate or the volume fraction of Bcl-2. Moreover, no significant changes in the band pattern of the amplified segment of bcl-2 gene were observed after Cd treatment. In summary, our data indicate that, prostate dysplasia induced in rats by Cd increases proliferative activity, without significant changes in either apoptosis or bcl-2 immunoreactivity. [ABSTRACT FROM AUTHOR]

The androgen receptor (AR) is a ligand-controlled transcription factor that is required for normal development of the prostate. It is expressed in both A-dependent and -independent prostate cancer (PCA) cells. Emerging data suggest AR regulates growth of PCA cells even in the absence of As, implying it is activated by factors other than As. Herein, we show that stimulation of A-dependent PCA LNCaP cells with reagents that promote accumulation of cyclic adenosine monophosphate (cAMP) leads to protein kinase A (PKA)-dependent activation of AR. Importantly, dihydrotestosterone (DHT)-regulated activation of AR also required PKA. Stimulation with epidermal growth factor, that robustly activates extracellular signal-regulated kinase pathway, did not promote activation of AR. These data establish a critical role for PKA in AR activation and suggest it may contribute to the growth of A-independent prostate tumors. [ABSTRACT FROM AUTHOR]

Sex Hormone Binding Globulin (SHBG), in addition to regulating free concentrations of steroid sex hormones in plasma, also participates in membrane-based steroid signaling in breast and prostate cells. In this study, we address whether the breast and prostate can synthesize their own SHBG. SHBG mRNA and protein were detected in human breast and prostate cancer cell lines, as well as in normal and cancerous breast and prostate tissue where it was prominent in epithelial cells. In cultured human LNCaP prostate cancer cells, SHBG was found both in the cytoplasm, and on the outer cell membrane where it was presumably bound to its high affinity receptor (RSHBG). When LNCaP cells were treated with 2-methoxyestradiol (2MeOE2), an antagonist of SHBG binding to RSHBG, membrane-associated SHBG was eliminated. These results demonstrate that the breast and prostate can synthesize SHBG locally, and that SHBG can bind to, and perhaps participate in autocrine and/or paracrine signaling through RSHBG. Therefore, perturbations in SHBG expression in breast and prostate tumors may affect the regulation of steroid signaling through RSHBG and target SHBG-mediated biologic properties at the cellular level. [ABSTRACT FROM AUTHOR]

Estrogen receptor-related receptors (ERRs) are closely related to the estrogen receptors (ERs) in their protein structures and consist of 3 closely related isoforms: ERRα, ERRβ and ERRγ. However, ERRs do not bind to estrogens (Es) or any other known physiological ligands, and thus they are classified as "orphan nuclear receptors". Studies on the expression pattern of ERRs in prostatic cells and prostate is scarce. As the first step towards a better understanding of the significance of ERRs in prostate, we investigated their expression profiles in human prostatic cell lines, two primary prostatic carcinoma xenografts, and prostatic tissues by RT-PCR, immunohistochemistry, and Western blot analysis. The results of RT-PCR showed that ERRα transcripts were widely detected in all tested cell lines and xenografts; ERRβ transcripts were either not detected or weakly expressed in prostatic cell lines and xenografts, while ERRγ transcripts were moderately expressed in C4-2B and PC-3 cells, and two xenografts. Similar expression patterns were also shown in human hyperplastic and neoplastic prostatic tissues. ERRγ immunohistochemistry showed that immunosignals were localized to the prostatic epithelial cells. We also compared mRNA expression of ERRs with that of ERs in prostatic cell lines. The results showed that ERRs and ERs were co-expressed in most cell lines and xenografts. Based on these observations, we speculate that the two members of nuclear receptor subfamily, ERRs and ERs, may control overlapping regulatory pathways in the prostatic cells. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCA) is the most frequently diagnosed malignancy and the second leading cause of death as a result of cancer in men in the USA and many other western countries. In the year 2003 there will be approximately 220,900 cases diagnosed and 28,900 deaths due to PCA. Steroid hormones, particularly androgens (As), play a major role in PCA, but their precise role is not clear. The majority of PCA responds to A ablation therapy by temporary remission. However, most tumors will eventually relapse in an A-refractory state. A recent study from our laboratory suggests that androgen receptor (AR) function may play a major role in the proliferation of A-refractory PCA cells. Several reports suggest that the AR co-activators are involved in A-dependent and A-independent PCA. We have shown that p300 and CBP are involved in A-independent transactivation of the AR. [ABSTRACT FROM AUTHOR]

Phosphatase and tension homology deleted on chromosome ten (PTEN) is a tumor suppressor gene with protein and lipid phosphatase activity frequently altered in several types of human cancers. Herein, we demonstrate the effect of transfected wild type (wt) PTEN and its mutants (mt) L57W, H123Y, G129R that have lost lipid and protein phosphatase activity; and G129E characterized by loss of only lipid phosphatase activity, during the cell cycle and on apoptosis. We characterized the expression of important proteins regulating the cell cycle and the PI3-K/PKB/Akt pathway, analyzed PTEN cellular localization, and performed PTEN mutation analysis in DU-145 and PC-3 cells. The transfection of wt PTEN and its mt with defective lipid phosphatase activity (G129E) inhibited S-phase entry of DU-145 and PC-3 cells. In DU-145 cells, transfection of wt PTEN induced apoptosis. An inverse expression of PTEN and phosphorylated PKB/Akt was observed. We did not find any mutations of the PTEN gene in either cell line. PTEN influenced the cell cycle of tested cells in a p53 and pRb independent manner, and the effect was cell type specific. [ABSTRACT FROM AUTHOR]

The mechanisms regulating hepatocyte proliferation are relevant to liver development, carcinogenesis, and regeneration. Studies of hepatocyte proliferation control during late foetal and postnatal development have been used as a model to understand such mechanisms. Since peroxisome proliferator-activated receptor gamma (PPARγ) ligands have been implicated in the inhibition of growth and differentiation of certain human cancers, in the present study, we have investigated the effect of englitazone (EG), a PPARγ ligand, on foetal and postnatal development. Our results indicate that, EG administered semi-chronically to pregnant rats, produced a body weight reduction on the progeny. This effect may be related to the diminished level of plasma IGF-I found in the neonates from treated-mothers. Surprisingly, despite receiving an anti-diabetic drug, foetus and neonates showed high levels of insulin, and were hyperglycemic. The plasma levels of leptin, other putative mitogenic factor, were not affected by the treatment. In the liver of neonates from mothers receiving EG, the expression of PPARα, IR, PI3K and IRS-1 was unchanged, as was the phosphorylation of MAPK. Nevertheless, an increase on Akt phosphorylation was observed on liver of neonates from treated-mothers, confirming a remarkable change on the mitogenic insulin/IGF-I pathway. In conclusion, the growth inhibitory effect reported herein may be associated with the ability of PPARγ ligands to reduce IGF-I concentrations and produce an insulin resistance state on foetus/neonates. These data strengthen the idea that PPARγ ligands have potential benefits on cancer treatment. [ABSTRACT FROM AUTHOR]

Using RAPD-PCR fingerprinting, the detection of mutations induced by estrogen (E) exposure was studied in nonmalignant- and malignant cells. Cells exposed to 17α-and 17β-estradiol, diethylstilbestrol, bisphenol A, and α-zearalanol induced mutations in different regions of the genome. They consisted of insertion/deletion as a result of point or length mutations and quantitative changes as a result of hypoploidy or polyploidy. We also detected several mutated loci in tumor-free tissues, adjacent to tumors, and DES-induced tumors. The data suggest that these mutations occurred in early stages of stilbene E-induced renal carcinogenesis, and that they may be induced as a result of stilbene E treatment. [ABSTRACT FROM AUTHOR]

Pituitary tumor-transforming gene-1 (PTTG1) has been implicated as an inhibitor of chromatid separation; its overexpression may lead to chromosomal instability. PTTG1 functions through SH3-mediated signal transduction pathways and activation of growth factors, including basic fibroblast growth factor (bFGF) mediated-angiogenesis. In order to detect micrometastasis in solid tumor patients, we developed a model system based on reverse transcriptase (RT)-PCR amplification of PTTG1 mRNA. We analyzed PTTG1 expression, using RT-PCR, in a panel of human tumors cell-lines (hTCL), including breast, gastrointestinal, small-cell lung cancer, and haematopoietic neoplasms. In addition, to newly hTCL established in our laboratory were investigated: Pancreatic, MBQ-OJC1. Small cell lung carcinoma, JCA-OJC3. Colon, JJPF-OJC4. Specific amplicon for PTTG1 was confirmed by RT-PCR in all the hTCL tested. In breast cell lines expression for PTTG1-mRNA was not related to invasiveness, tumorogenicity, or estrogen receptor (ERα) status. PTTG1-mRNA was examined by PCR amplification of cDNA obtained from normal lymph nodes, bone marrow, and peripheral blood (cellular and plasmatic mRNA). Using a hot-start PCR and different amounts of input RNA, PTTG1 transcript was detected in control bone marrow but not on lymph nodes. PTTG1-mRNA was detected in peripheral blood from 4/14 healthy donors analyzed. Our data confirms that PTTG1 is abundantly expressed in human tumors of different histologic types. PTTG1 qualitative mRNA detection may be useful as a surrogate molecular marker for angiogenic phenotype and invasive tumor, however, it is not useful as a micrometastasis marker due to its transcription in normal bone marrow and peripheral blood. [ABSTRACT FROM AUTHOR]

Cholecystokinin (CCK) is a peptide found widely distributed throughout the gut and nervous systems. There is growing evidence that it affects growth and differentiation of several types of cell, including anterior pituitary cells, via two receptor subtypes, A and B. An autocrine/paracrine role for CCK in GH3 rat pituitary tumor cells has been reported. Herein, we demonstrate that functionless human pituitary tumors also possess CCK-B receptors. Total RNA was extracted from 30 human functionless pituitary tumors, reverse transcribed into cDNA, and subjected to PCR using primers specific for CCK-A and CCK-B receptors. The primers were targeted against different exons of the receptor genes, allowing identification of cDNA amplification. PCR bands of the predicted length were observed in all tumors using CCK-B receptor primers. Restriction digestion and direct sequence analysis of the products provided further evidence that they represented CCK-B receptor mRNA. CCK-A receptor mRNA was not detected by this RT-PCR analysis. In culture, CCK and CCK octapeptide sulphate powerfully stimulated phosphatidylinositol hydrolysis, providing evidence for functional activity of the CCK-B receptors. Similar analyses on pituitary-derived fibroblasts yielded negative results, indicating that the CCK-B receptor mRNA was expressed by tumors cells. These results suggest a role for CCK in the development of human functionless pituitary tumors. [ABSTRACT FROM AUTHOR]

The human Zinc finger E-box Binding (ZEB)-1 protein belongs to a family of transcription factors involved in critical developmental processes. Yet little is known of the mechanisms by which ZEB-1 is regulated. Our lab has recently demonstrated that the expression of ZEB-1 is induced by estrogen (E) in the ovarian cancer cell line Ov266 and that it is regulated by dihydrotestosterone (DHT) in the human PC-3/AR prostate carcinoma cell line. Interestingly, a dose-response assay indicates the expression of ZEB-1 is induced by 5 nM DHT and repressed at higher dosages. Cloning and analysis of approximately 1000 bp upstream of the translation start site of hZEB-1 revealed a number of putative E and androgen (A) response elements. Transient transfection assays indicate that this region is sufficient to confer responsiveness to both steroids. To determine whether expression of ZEB-1 could serve as a marker of tumor progression, real-time PCR (rtPCR) assays were performed on staged human reproductive carcinomas. Preliminary results indicate that expression of ZEB-1 increases as the normal ovary transforms to a primary carcinoma and continues to increase as the cancer progresses to an invasive and finally a metastatic state. There is an approximate 12-fold elevation in the expression of ZEB-1 in metastatic ovarian carcinoma relative to its expression in in situ cancers. rtPCR is currently being utilized to investigate the potential changes in ZEB-1 expression in breast and prostate cancer during the progression of these diseases. These data raise the possibility that overexpression of ZEB-1 contributes to the progression of reproductive carcinomas. [ABSTRACT FROM AUTHOR]

The synthetic estrogen diethylstilbestrol (DES) causes marked abnormalities in the female hamster genital tract, after either prenatal or postnatal exposure, leading to endometrial hyperplasia and carcinoma. Acting as an initiating event, DES altering uterine development may facilitate the abnormal response of promoting agents. Tamoxifen (TAM) is an antiestrogen that competes for central and peripheral estrogen receptor (ERα). TAM exerts agonistic effects on E-dependent endometrial proliferation. N-ethyl-N-nitrosourea (ENU), a potent mutagenic agent, induces tumors in a variety of organs, predominantly in the peripheral nervous system. To test whether ENU and TAM treatment in a model of hyperestrogenism, the histopathologic endometrial alterations following exposure to DES, ENU and TAM (alone or in combination) were analyzed in ovariectomized female Syrian hamsters. Herein, the incidence and characteristics of the lesions found in the endometrium, and the progressive transformation from early alterations to malignant processes, are presented and discussed. [ABSTRACT FROM AUTHOR]

Docking or adapter proteins link signals emanating from receptors to other downstream intracellular molecules and amplify the initial signal. Therefore, the activation of a docking protein can indicate the prior activation of a receptor, such as a receptor tyrosine kinase (RTK). The Gab-1 (for Grb-2-associated binder-1) docking protein is a substrate for many different RTKs. As part of our effort to define the signaling pathways stimulated by 17β-estradiol (E2) and progesterone (P) in the uterus, we investigated the activation of uterine Gab-1 in response to these hormones. In this study, Gab-1 was immunoprecipitated from CD-1 mouse uterine extracts, followed by immunoblotting with appropriate antibodies. In cycling mice, maximal uterine Gab-1 tyrosine phosphorylation (Gab-1-pY) was observed during late diestrus/early proestrus. To determine the role for specific ovarian hormones in uterine Gab-1 activation, ovariectomized mice were treated with vehicle, E2, P, or E2 + P, and 12 hours later uteri were analyzed. Consistent with the data from cycling mice, either E2 or E2 + P stimulated tyrosine phosphorylation of Gab-1. Uterine Gab-1 from E2- and E2 + P-treated mice coimmunoprecipitated with p85, the regulatory subunit of phosphatidylinositol 3-kinase; uterine Gab-1 from E2 + P-treated mice also coimmunoprecipitated with SHP-2, a tyrosine phosphatase. These data suggest that E2 and/or P activated an RTK signaling pathway(s) that utilized Gab-1. To identify this RTK, mice were administered IGF-1 or EGF. Uterine Gab-1 was tyrosine phosphorylated by 5 minutes post-treatment in EGF-, but not IGF-1-, treated mice, suggesting that EGFR may mediate steroid hormone-induced uterine Gab-1 activation. [ABSTRACT FROM AUTHOR]

Inflammatory mammary cancer is the most aggressive spontaneous type of mammary cancer in women and dogs. Our results indicate that a special endocrine mechanism could be involved in its pathogenesis with relevant action of IGF-I and androgens. [ABSTRACT FROM AUTHOR]

The p53 null mammary epithelium transplant model has been extensively characterized at the genetic, hormonal, and biological levels. Tumors progress from ductal hyperplasias and ductal carcinoma in-situ (DCIS) and are aneuploid, metastatic and approximately 80% are estrogen receptor (ERα) negative. The normal and premalignant mammary stages of development are ERα positive and hormone responsive. Continuous estrogen (E) or progesterone (P) treatment markedly enhances tumorigenesis in p53-null mammary epithelium. Blocking E signaling by tamoxifen or blocking P signaling by deleting the PR or blocking E and P signaling by ovariectomy markedly decreases hormone-induced mammary tumorigenesis in p53-null epithelium. The tumors that do arise are ERα negative. These results suggest that this model is appropriate to examine issues regarding the timing and duration of tamoxifen treatment on premalignant progression and the use of combined prevention strategies to delay the occurrence of invasive breast cancer (BC). [ABSTRACT FROM AUTHOR]

A full-term pregnancy early in life reduces the risk of breast cancer (BC) in women. Parity protection against mammary carcinogenesis is also observed in rats and mice. Administration of high pregnancy levels of ovarian hormones to nulliparious rats induces protection from N-methyl-N-nitrosourea (MNU)-induced mammary cancers. We have demonstrated that 17β-estradiol (E2) alone or in combination with progesterone (P) are effective in preventing mammary cancer while P alone is not. We have now determined the actual daily amount of E2 required to confer protection against mammary cancer. Rats were injected with MNU at 7 weeks of age. Two weeks later the rats were treated with 10 nanograms, 100 nanograms, or 1 μgram of E2 per day for 3 weeks in mini-osmotic pumps. All the animals also received a 30 milligram P silastic capsule for 3 weeks. Serum levels of E2 immediately after treatments were 6, 30, and 118 pg/ml, respectively. Control rats had a 90% mammary cancer incidence while rats treated with 1 μicrogram of E2 per day had no mammary cancers, nine months after MNU treatment. Treatments with 10 nanograms or 100 nanograms of E2 per day did not significantly decrease the mammary cancer incidence compared to the controls; they had a cancer incidence of 78% and 65%, respectively. The blood hormone data indicates that treatment with 1 microgram of E2 per day results in high late pregnancy levels of E2 and this level of E2 is effective in conferring protection against mammary carcinogenesis. This short-term chemoprevention treatment is as effective as full-term pregnancy, ovariectomy or long-term tamoxifen treatment and there is no permanent loss of ovarian function. This treatment can be used as a paradigm for developing strategies for human BC prevention. [ABSTRACT FROM AUTHOR]

Pregnancy early in life reduces the risk of breast cancer (BC) in women and this effect is universal. This phenomenon is also observed in rodents. We have shown that a treatment with high pregnancy levels of 17β-estradiol (E2) for 7 to 21 days is effective in conferring protection against mammary carcinogenesis. We determined the difference in gene expression between hormone-protected rats and unprotected rats. Nine weeks old female Lewis rats were treated with 10 microgram (unprotected), or 200 microgram (protected) of E2 in silastic capsules for 3 weeks. Control rats received silastic capsules with no hormone. The rats were terminated 8 weeks after the removal hormone treatment. Mammary RNA was used for microarray analysis. Using Agilent Rat cDNA Microarrays, with 14,815 unique clones, we have analyzed genes from Rattus norvegicus and Rattus rattus which are annotated as "mRNA" or "gene EST" in GenBank. The genes involved in growth promotion like interleukin 18, kit oncogene, thyrotropin stimulating hormone receptor, cyclin dependent kinases etc. were down regulated in the protected group compared to the unprotected groups. In contrast, genes involved in growth inhibition like early growth response 1, insulin-like growth factor binding proteins and genes involve in apoptosis and DNA repair like T-cell death activated gene, CD47, histone acetyltransferase were up regulated in the protected animals compared to the unprotected. These findings define a pattern of gene expression that could serve to determine the efficacy of protective hormone treatments and help identify potential biomarkers for prevention of mammary cancers. [ABSTRACT FROM AUTHOR]

A high frequency of aneuploid cells in carcinoma in-situ (CIS) and in small and large mammary tumors of female ACI rats are induced by estrogen (E) treatment alone. Employing G-banding karyotype analysis, consistent numerical chromosome alterations were detected as gains in chromosome 7, 11, 13, 19, and 20, and loss of chromosome 12, when the data were analyzed by binomial distribution for a frequency of occurrence ≥ 30.0%. Recurrent chromosome gains were also detected in chromosomes 3, 4, 6, 10, 14, and 15, whereas infrequent losses were observed in chromosome 5. Regional genomic alterations were detected by comparative genomic hybridization (CGH) in eleven individual E- induced primary mammary gland tumors (MGTs) obtained from an equal number of female ACI rats. CGH analysis revealed consistent regional gains at 1p11, 1q11-q22, 4q41-q44, 7q31-q33, 11p12-q11, 13p, and 20p12. [ABSTRACT FROM AUTHOR]

We evaluated the possible effectiveness of genistein (GEN) as an inhibitor of mammary cancer in the ACI rat, a strain genetically susceptible to estrogen-induced mammary carcinogenesis. In this study, three-week old ACI rats were fed a phytoestrogen-free diet or a diet containing 250 ppm GEN. At nine weeks of age, the rats were implanted with 20 mg pellets containing either 2 mg 17β-estradiol (E2)/18 mg cholesterol or cholesterol alone. The appearance of palpable mammary tumors (MTs) was recorded. Our results show that E2-treated rats fed the GEN diet both prepubertally and as adults had the highest incidence of palpable MTs. E2-treated rats fed the GEN diet only prepubertally had delayed appearance of palpable MTs but the incidence quickly approached that of the rats fed GEN for the whole treatment period. Dietary GEN fed to adult E2-treated rats had the lowest incidence of MT. Rats implanted with the cholesterol pellet and fed either diet did not develop MTs. [ABSTRACT FROM AUTHOR]

Estradiol 17-β dehydrogenase (OE2DH) is an enzyme involved in both the activation and the inactivation of estrogen, which plays an important role in breast cancer (BC). Polyunsaturated fatty acids (PUFA's) are also implicated in the development of BC, although the mechanisms are not fully understood. The aim of this investigation was to examine the growth effects of the n-3 PUFA-eicosapentaenoic acid (EPA) on the MCF7 human BC cell line in parallel with its effects upon OE2DH. A bell shaped growth inhibition of MCF7 cells was observed at around 50 µM. A single dose of EPA significantly inhibited cell proliferation at every time point measured over a twelve-day period. In the presence of 17β—estradiol (E2) alone, cell proliferation was increased peaking at lnM E2. The effect was completely abolished by co-incubation with 50 µM EPA. OE2DH activity was measured in both the reductive [estrone (E1) → E2] and the oxidative [F2 → E1] directions. These results show that whilst 50 µM EPA weakly stimulates OE2DH activity in the reductive direction, it strongly drives the reaction in the oxidative direction. This effect was specific to EPA as the structurally related n-6 fatty acid arachidonate, had no effect. These results suggest that EPA might, in part, exert its effects in BC through driving OE2DH activity towards the production of the less biologically potent E1 as well as having a direct inhibitory effect on activity. [ABSTRACT FROM AUTHOR]

In the search for differences between estrogen receptor (ER) α and ERβ, we proved that ERα but not ERβ directly interacts with calmodulin (CaM) through the hinge region. The transcriptional activity of a mutant unable to interact with CaM becomes insensitive to inhibition by CaM antagonists (W7). These residues are acetylated by p300 and substitution of lysine 302 and 303 with other residues enhance ERα-hormone sensitivity, suggesting that ERα acetylation normally suppresses ligand sensitivity. Also, the somatic mutation K303R has been identified in early premalignant breast lesions. ERα K303R normally binds E but shows increased E-induced transcriptional activation and increased proliferation in response to E when transfected into breast cancer cell (BC) lines. Herein, we show that mutations K303R and K303A, render an ERα unable to interact with CaM and therefore insensitive to W7. K303 homodimers and K303 mutant/wt heterodimers show increased sensitivity to E. Contrary to the wt ERα, AP1 transcriptional activity is inhibited by estradiol (E2) and OH-Tamoxifen (OH-TAM) in both K303R and K303A mutants. [ABSTRACT FROM AUTHOR]

Breast cancers (BCs) often have increased mitogen-activated protein kinase (MAPK) activity. This pathway influences BC cell growth in part by targeting steroid hormone receptors. Activation of p42 and p44 MAPKs increases progesterone receptor (PR) transcriptional activity in the presence of progestins, and triggers their rapid down-regulation by the ubiquitin-proteasome pathway. In turn, progestins increase the expression of type 1 growth factor receptor tyrosine kinases that feed into MAPK activation. Recently, progestins have been shown to activate the p42/p44 MAPK module in a PR-dependent manner, but independently of their function as transcription factors. Mechanisms of bi-directional cross-talk between these two pathways are becoming well-documented. Herein, we provide an overview of the primary ways in which steroid hormone receptor and growth factor cross-talk occurs, using examples from our work with human PR as a model receptor; we demonstrate MAPK regulation of PR subcellular localization, transcriptional synergy, and regulation of cyclin D1 expression. Cross-talk between growth factor and PR-mediated signaling events is an important means by which growth regulatory genes are coordinately regulated, and may contribute to the growth of hormonally responsive normal breast tissue and to BC. [ABSTRACT FROM AUTHOR]

Catechol estrogens (CEs) are considered critical intermediates in estrogen (E)-induced carcinogenesis. Previously, we demonstrated that estradiol (E2), estrone (E1), and four of their catechol estrogens, 2- and 4-OHE2 and 2- and 4-OHE1 induced morphological transformation in Syrian hamster embryo (SHE) cells, and their transforming activities varied as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 ≧ E1, E2, which are consistent with the genetic effects, i.e., chromosome aberrations and DNA adduct formation, of each E. To further elucidate the mechanism of hormonal carcinogenesis, we studied the effect of the catechol-O-methyltransferase (COMT) inhibitor Ro41-0960 on the transforming and clastogenic activities of the CEs using SHE cells. The frequencies of transformation and chromosome aberrations induced by 4-OHE1 were not affected by co-treatment with Ro-41-0960, but those induced by 2-OHE1 were markedly enhanced. The frequency of transformation induced by 4-OHE1 was markedly decreased by E2 in a concentration dependent manner, but this decrease was not inhibited by Ro41-0960. Cell treatment with E2, 2-OHE1, or 4-OHE1 alone induced apoptosis as detected by the TUNEL method. Additive effect on the induction of apoptosis was observed in cells treated with E2 + 2-OHE1 or 4-OHE1. The % apoptotic cells induced by E2 and 4-OHE1 decreased in the presence of Ro41-0960, while those induced by E2 and 2-OHE1 did not. These results suggest an important role of both the substrate specificity of COMT and the induction of apoptosis in CE-induced carcinogenesis. [ABSTRACT FROM AUTHOR]

A comparative study of the effects of chronic 17β-estradiol (E2) treatment on microsomal oxidation and conjugation of E2 via Phase I and II enzymes in the ACI rat mammary gland (MG) and liver was performed. NADPH-dependent oxidation of E2 was not detected in the MG, but was readily measured in the liver. Oxidation was not altered by chronic E2 treatment. Ascorbic acid stimulated E2 oxidation (non-enzymatically) in MG microsomes, but had no effect in the liver. Hepatic but not MG NADP(H):quinone oxidoreductase and glutathione S-transferase activities increased 4.0- and 2.0-fold, respectively, after 6 weeks (w) of treatment. MG catalase activity was decreased 64% after 28 w of E2 treatment, when the rats had developed 100% incidence of MG adenocarcinomas. Moreover, the activities of phenolsulfotransferase SULT1A1 and fatty acyl-CoA:E2-acyltransferase ACO:E2 decreased by 95 and 80%, respectively, in the MG but not in liver. Decreases in these enzymes were maximal after 6 w and preceded induction of MG tumors. Collectively, these data indicate that E2 regulates the expression of antioxidant and E2-metabolizing enzymes differentially in the ACI rat liver and MG. The decreased activities of SULT1A1 and ACO:E2 may favor accumulation of E2 available for receptor binding and conversion to catechol estrogens, both of which are implicated in E2-induced mammary oncogenesis. [ABSTRACT FROM AUTHOR]

In human prostate cancer (PCA) cells, androgens (As) coordinately stimulate the expression of genes involved in the synthesis of fatty acids and cholesterol. Interestingly, several of these genes are overexpressed in human cancers, particularly those with a poor prognosis, and are promising targets for anti-neoplastic therapy. Exploration of the mechanism underlying the lipogenic effects of As revealed that As induce activation of Sterol Regulatory Element-Binding Proteins (SREBPs), proteolytically activated transcription factors that play a central role in the control of cellular lipid homeostasis. The exact mechanism by which As interfere with the SREBP pathway remains to be investigated. Herein, we show that A-activation of the SREBP pathway depends on the presence of the structural elements required also for its sterol-regulated activation. Detailed studies revealed that As substantially increase the expression of the transporter protein SCAP in 2 of the 3 cell lines tested. Co-transfection of an expression vector encoding SCAP lead to a marked increase in the transcription of a key gene involved in cholesterol synthesis. In support of the involvement of activated SREBPs in the observed effects, SCAP-mediated transcriptional activation of this lipogenic gene was dependent on the presence of intact SREBP binding sites in its promoter region, and was counteracted by addition of dominant-negative SREBP forms. These data suggest that As activate the SREBP pathway by inducing a change in the normal cellular balance between SCAP and its retention protein complex. [ABSTRACT FROM AUTHOR]

A screening for androgen-regulated genes in prostate cancer (PCA) cells revealed that androgenes, apart from their well known effects on cell survival, proliferation, and differentiation, stimulate the expression of several lipogenic genes, including the gene encoding fatty acid synthase (FAS). FAS, a key enzyme in the biosynthesis of fatty acids, is markedly overexpressed in many epithelial cancers including cancer of the prostate, breast, ovary and endometrium. To gain more insight into the role of FAS in cancer cells and to explore its potential as a novel target for antineoplastic therapy, we have used the potent and highly sequence-specific technique of RNA interference (RNAi) to silence FAS in LNCaP prostate cancer cells. RNAi-mediated down-regulation of FAS expression resulted in a major decrease in the synthesis of triglycerides and of phospholipids partitioning into detergent-resistant membrane microdomains. These effects were accompanied by marked morphological changes, including a reduction in cell volume, a loss of cell-cell contacts, and the formation of spider-like extrusions. Furthermore, silencing of the FAS gene by RNAi significantly inhibited LNCaP cell growth and ultimately resulted in induction of apoptosis. In striking contrast with LNCaP cells, RNAi-mediated inhibition of FAS did not influence viability of nonmalignant fibroblasts. The data presented herein suggest that overexpression of FAS induced by hormones, growth factors, or other mechanisms may play an important role in cancer cell biology, and that RNA interference, particularly targeting lipogenic genes, constitutes a promising tool for the development of new cancer treatments. [ABSTRACT FROM AUTHOR]

Green tea is widely accepted to lower the risk of developing cancer, including hormone-responsive cancers, but the precise mechanism of its cancer-preventive effect is not fully understood. Recently, the green tea component epigallocatechin-3-gallate (EGCG) was demonstrated to inhibit in-vitro enzymatic activity of chicken liver fatty acid synthase (FAS), an enzyme that is frequently overexpressed in many epithelial tumors. Since chemical FAS inhibitors such as cerulenin and C75 are known to inhibit growth and to induce apoptosis of several cancer cell lines in vitro and tumor xenografts in vivo, it was investigated whether EGCG also inhibited FAS activity in cultured prostate cancer (PCA) cells in vivo and how this inhibition affected lipogenesis, cell proliferation, and cell viability. EGCG significantly inhibited FAS activity in PCA cells (with high FAS expression levels). This FAS inhibition was paralleled by decreased lipogenesis, growth inhibition, and apoptosis. In contrast, epicatechin, another closely related catechin that does not influence FAS activity, had no effect on PCA cell proliferation or survival. EGCG also inhibited FAS activity and proliferation of normal fibroblasts (with low FAS expression), but did not induce fibroblast apoptosis. Taken together, it can be concluded that EGCG efficiently inhibits FAS in cultured cells, and specifically induces apoptosis in PCA cells but not in normal fibroblasts, thereby providing interesting perspectives for using EGCG in antineoplastic therapies. [ABSTRACT FROM AUTHOR]

Human androgen receptor (AR) mutation is a possible alternative used by prostate adenocarcinoma (PCA) cells to escape androgen dependence. These mutations may broaden the specificity and/or the sensitivity of the AR to other hormones, resulting in the inappropriate receptor activation, and thus, affecting the PCA response to hormonal therapies. We have developed a yeast-based functional assay to detect mutant ARs in PCA by analyzing their transactivation capacities in response to different ligands. We report herein the detection of two different mutant ARs within the same metastatic tumor sample. Concomitantly to the T877A mutant AR, we identified an additional double mutant AR harboring the nonsense mutation Q640Stop, just downstream of the DNA binding domain together with the T877A point mutation. This mutation leads to a C-terminal truncated AR. This study is the first description of this type of mutation in PCA. We demonstrated that this truncated AR exhibited constitutive transactivation properties. In conclusion, our data suggest that mutation-induced constitutive activation of the AR may be an alternative mechanism used by PCA cells to escape androgen deprivation. [ABSTRACT FROM AUTHOR]

Prostatic interepithelial neoplasia (PIN) is considered a precursor of prostate cancer (PCA). In the majority of the PINs, the atypia is more marked in basal than luminal nuclei. The aim of this study was to quantitate the differences in the stereological estimation of mean nuclear volume weighted by volume (vv nuc) between basal and luminal cells of PIN, in relation to normal prostate epithelium, and PCA. Ten PIN cases were compared with 50 PCA, and 5 normal prostates. All the specimens were routinely processed and stained with haematoxylin-eosin. The mean ± SD of vv nuc was evaluated for all the groups. The epithelium of both normal and PIN specimens was segmented in basal and luminal compartments, and the vv nuc measured in both strata. Comparisons among groups were performed by ANOVA (p < 0.05). The value of vv nuc was significantly lower in normal epithelium compared to both PIN and PCA. Basal layer PIN vv nuc was significantly higher in comparison to the luminal stratum. Luminal PIN vv nuc was similar to vv nuc of PCA. The similarities in nuclear size between PIN and PCA are according to the pre-malignant character commonly assigned to PIN. The increase of basal layer PIN vv nuc may indicate that the changes heralding the progression from PIN to PCA are produced in this layer, whereas the nuclear features of luminal layer are the same to those of the carcinoma. [ABSTRACT FROM AUTHOR]

Men with high grade prostatic intraepithelial neoplasia (HG PIN) on prostate biopsy are at high risk for prostate cancer (PCA). The ability to reverse HG PIN, a precursor of PCA, may reduce the incidence or delay the development of PCA. ACAPODENE™ (toremifene) is a selective estrogen receptor modulator (SERM) that has been shown in preclinical models to both eliminate HG PIN and reduce the incidence of PCA. Toremifene was evaluated for safety and efficacy in men diagnosed with HG PIN. An open labeled, Phase IIa clinical trial enrolled 21 men (mean age 64.7 years) who had HG PIN only on biopsy within 6 mo of entry into the study. From those men, 18 (86%) completed toremifene treatment (60 mg/day PO for 4 mo) followed by a prostate biopsy to determine HG PIN status. Serum prostate specific antigen (PSA), % free PSA, testosterone (T), estradiol (E2), and quality of life were measured. Following toremifene treatment, 72% of the HG PIN men compared to 17.9% of historical controls had no HG PIN on subsequent prostate biopsies. Mean PSA was trended higher and % free PSA was elevated. Quality of life was not significantly affected, and there were no serious adverse events. Toremifene appears to reduce HG PIN, and was well tolerated in this small, exploratory trial. A double blind, dose finding, randomized, and placebo controlled Phase IIb/III study is currently in progress in 516 men with HG PIN to further study toremifene's activity against PCA incidence. [ABSTRACT FROM AUTHOR]

The recent cloning of the estrogen receptor β (ERβ) has been followed by the discovery of a variety of its isoforms. Since endometrial cancer (ECA) is an estrogen-dependent cancer, an analysis of its expression of ERα and ERβ isoforms may contribute to our understanding of the mechanism of its development. Using the real-time RT-PCR assay, the quantitative expression of ERα, ERβ1, ERβ2 (βcx), ERβ3, ERβ4, and ERβ5, as well as androgen (AR) and progesterone receptors (PR) was determined and compared in normal and neoplastic endometrium. Our data demonstrates the co-expression of all the ERβ isoforms in the normal endometrium, and an up-regulation of the ERβ5 transcript in malignant endometrium. A decrease in the levels of AR mRNA between normal and neoplastic endometrium was also noted. With respect to clinical parameters, the decreased expression of PR mRNA correlates inversely with endometrium tumor grade. The expression profiles of ERα and AR were different between pre and postmenopausal endometrium, with a significant increase during menopause. [ABSTRACT FROM AUTHOR]

Progesterone receptor (PR) antagonists are a potential new therapeutic strategy for the treatment of patients with invasive breast cancer (BC). One such progesterone antagonist is Onapristone. Onapristone has been reported to have strong anti-progestational and anti-tumor activity. A tumor remission rate of approximately 66% was found in a phase II clinical trial of Onapristone as a first-line endocrine therapy in post-menopausal patients with primary BC. However, during this trial, a minority of the patients developed abnormal liver function test results. As a consequence, the further therapeutic use off this drug has stop. Although the mechanism of action of Onapristone has been suggested by animal model experiments, the genes regulated by the drug in the breast are not known. In the present study, the BC cell line T47D was treated with Onapristone and examined with Affymetrix microarrays and real-time RT-PCR to identify genes which were differentially expressed. Cells were treated with Onapristone either alone or in the presence of progesterone, and microarray analysis carried out upon the RNA extracted from the cells. The expression of a selection of up- or down-regulated genes observed was further investigated using real-time RT-PCR. In addition, the expression of these genes was investigated in the PR− cell line, MDA-MB-231 treated with Onapristone, to ensure that any changes in gene expression observed, were due to Onapristone effects via the PR. [ABSTRACT FROM AUTHOR]

Recently, our laboratory and others have shown that estrogen receptor (ER)α, negative (−) breast cancer (BC) cells respond to estrogenic compounds via rapid signaling mechanisms. To understand the role of soy phytoestrogens in BC cell invasion, we analyzed focal complex formation and focal adhesion kinase (FAK) activity, as detected by phospho-specific anti-FAK antibodies to tyrosine residues 397 or 925. Stimulation of ERα+ T47D and ERα− MDA-MB-231 BC cell lines for 10 min with 17β-estradiol (E2), daidzein, or genistein (GEN) increased the number of focal adhesions and FAK activity. This data implicate E2 and soy phytoestrogens in the regulation of cellular interactions with the ECM via novel signaling mechanisms. [ABSTRACT FROM AUTHOR]

Previously, we reported that glycine N-methyltransferase (GNMT) expression levels were diminished in human liver cancer. Herein, the polymorphisms of GNMT in 5 hepatocellular carcinoma (HCC) and 2 hepatoblastoma (HBL) cell lines were analyzed. In the HCC cell lines, 4/5 had homozygous genotypes in all the GNMT polymorphism markers expect PLC/PRF/5 which had a G/A genotype of SNP3. Among 6 GNMT polymorphism markers, HA22T/VGH had 3 homo and 3 heterozygous genotypes. In contrast, both Huh 6 and Hep G2 HBL cell lines had heterozygous genotypes in most of their GNMT polymorphism markers. The INS/DEL genotypes of GNMT in peripheral blood mononuclear cells of 308 HCC patients were analyzed. The data indicate that the rates of homozygous insertion (INS/INS), heterozygous (INS/DEL), and homozygous deletion (DEL/DEL) genotypes were 11.59%, 48.19% and 40.11%, respectively. When the SNP1 genotype was analyzed, we found that the genotype rates of T/T, T/C, and C/C in HCC patients were 1.30%, 25.65% and 73.05%, respectively. The frequency distribution of both INS/DEL and SNP1 genotypes in HCC patients was not statistically different from those of the 304 normal controls. When the interaction between GNMT genotypes and HBV infection were analyzed, the data showed that among patients without HBV infection, those with C/C genotype had a 1.55-fold higher risk of developing HCC. Further studies in the interaction between GNMT and risk factors for HCC besides HBV are needed. [ABSTRACT FROM AUTHOR]

Microsatellite polymorphism may influence gene expression or receptor signaling function. In cancer, microsatellite sequences, even when located in non-coding regions of a gene, are capable of modifying the susceptibility to carcinogenic factors. The estrogen-receptors (ER) α and β are involved in breast oncogenesis, with different prognosis significance. They mediate different and complex pathways. Recently, the expression of the ERβ in breast tumors has been shown to be a marker for good prognosis for breast cancer (BC). Although this piece of information needs further testing, the involvement of the ERβ in BC signaling processes has been clearly demonstrated. The goal of the present study was to gain insight into the genetic polymorphism of ERβ, and its possible relationship to BC pathogenesis regarding estrogen dependence. We conducted a case-control study comprising 27 BC samples and 30 control samples, matched by age and hormonal status. Our results indicate that the length of the ERβ genotype variants oscillates between 149-169 bp. Clearly, the overall ERβ distribution had a bimodal distribution with a majority of alleles ranging between 161 and 165 pb. A group of ERβ alleles was significantly shorter (below 159 bp) with a predominant peak at 155 pb. A comparison of the length of the ERβ genotypes revealed that short alleles [below 21 cytosine (C) and adenosine (A) repeats - 159 bp] were significantly more frequent in BC patients (42.6%) compared to controls (18.3%; t = −2.50; p = 0.013). In conclusion, (CA)n polymorphism at the ERβ locus may represent a modulator factor of BC risk. [ABSTRACT FROM AUTHOR]

An association between sex steroids and mammographic density (MD), a predictor of breast cancer (BC) risk, is supported by findings of increased densities after hormone therapy and reduced densities after tamoxifen treatment. Herein, ethnic differences in urinary hormone levels and their relation to MDs were investigated. Because 2-hydroxyestrone (2-OH-E1) is considered less carcinogenic than 16α-hydroxyestrone(16α-OH-E1), we hypothesized an inverse relation between the 2-OH-E1/16α-OH-E1 (2/16) ratio and breast densities. Women recruited completed a questionnaire and donated urine during the luteal phase. Urinary estrone (E1), estradiol (E2), testosterone (T), and 5α-androstane-3α, 17β-diol (ADIOL) were measured by indirect radioimmunoassay (RIA) and 16α- and 2-OH-E1 by competitive immunoassays. MDs were assessed with a computer-assisted method and applied multiple linear regressions. The total number of subjects was 305 (35-75 years, $$ \bar X $$ = 47.2 years). Their ethnic distribution was Caucasian (110), Japanese (86), Hawaiian (35), Chinese (28), and mixed/other ethnicity (46). The data indicate that the body mass index (BMI), 2-OH-E1, androgens (A), total hormones, and the 2/16 ratio were significantly lower in Asians than in Caucasians, but the % MDs were 22% higher in Asians. None of the individual hormones was associated with MDs. However, contrary to the initial hypothesis, the 2/16 ratio was directly related to MDs. The ratio was 25% lower in the lowest MD category as compared to the highest category. The data suggest that the effects of endogenous hormones on BC risk may not be mediated through MDs in adult women. [ABSTRACT FROM AUTHOR]

Progestogens are given to diminish the risk of endometrial cancer (ECA) observed with postmenopausal estrogen therapy. The pattern of progestogen administration and number of days that the progestogen is given per month appear to affect ECA risk. We studied the impact of dose of progestogen, specifically medroxyprogesterone acetate (MPA), on ECA risk in a population-based case control study of 647 cases and 1,209 controls, ages 45-74 years. Among women who took a combined hormone regimen with MPA for less than 10 days/mo, the overall adjusted risk of ECA (relative to that of hormone non-users) was 4.2 [95%, Confidence Interval (CI) 2.0, 8.9] in those with an MPA dose less than 70 mg/mo; and the adjusted risk of ECA was 2.8 (95%, CI 0.9, 8.6) in those with a higher monthly MPA dose. Among women who took MPA cyclically for 10-24 days/mo or as continuous combined therapy, the risk of ECA was similar to non-users, irrespective of the monthly dose. Our findings showed that among the combined hormone regimens most commonly used by postmenopausal women in the USA today, monthly dose of MPA bears little or no relation to risk of ECA. [ABSTRACT FROM AUTHOR]

In addition to its well-documented role in the reproductive hormone cascade, GnRH may play a role as an autocrine/paracrine regulator in the inhibition of cell proliferation and/or induction of apoptosis in normal and neoplastic OSE cells. The use of GnRH and its analogs is being considered for pre-clinical trials as a possible therapeutic agent in gynecological tumors such as ovarian and endometrial cancers. The exact mechanisms of GnRH action in the inhibition of cell growth need to be delineated using in-vitro and in-vivo models for ovarian cancer. The recent discovery of a second form of GnRH (GnRH-II) is posing a new challenge in understanding the role of the GnRH/GnRH-R system in normal OSE and its neoplastic counterpart. It appears that GnRH-II may have a more potent effect in the inhibition of cell proliferation when compared to that of GnRH-I in ovarian cancer cells. On the other hand, E and ER may play a role in ovarian carcinogenesis as indicated by epidemiological and experimental observations. Further studies are needed to elucidate the influence of E and other hormones such as P4, gonadotropins and growth factors in normal and neoplastic OSE. Interestingly, it appears that E may be involved in the regulation of GnRH and GnRH-R genes in normal OSE and ovarian cancer cells. This potential cross-talk between the E and GnRH systems in the ovary may be important in the multi-faceted regulation of cell proliferation, apoptosis and/or differentiation of normal OSE and its neoplastic counterparts. [ABSTRACT FROM AUTHOR]

In summary, our data demonstrate that following androgen withdrawal, a majority of LNCaP androgen-sensitive PCA cells did not undergo apoptosis, but rather transient neuroendocrine differentiation, and eventually relapsed to an androgen-refractory phenotype. The anti-apoptotic protein Bcl-2 was overexpressed during this complex process. Additionally, the PI3K/Akt survival pathway was further activated due to androgen withdrawal. Moreover, we found that loss of PTEN and overexpression of Bcl-2 are inversely correlated in primary prostate tumors. Ectopic expression of PTEN resulted in decreased expression of Bcl-2 in PTEN-null PCA cells by negatively regulating the Akt and CREB signaling pathway. Furthermore, expression of Bcl-2 in PCA cells is androgen-regulated. Androgen treatment of PCA cells downregulates Bcl-2 expression in an E2F1-dependent manner. Therefore, our data suggest that deregulation of PI3K/Akt/CREB and E2F1 pathways caused by loss of PTEN function and/or androgen deprivation contributes to the overexpression of Bcl-2 in androgen-refractory prostate tumors. [ABSTRACT FROM AUTHOR]

The latest generation of aromatase inhibitors comprises highly potent and specific endocrine agents. They have a central role in the management of hormone-dependent BC, and their potential as preventative agents in women at high risk of the disease is currently being explored in clinical trials. [ABSTRACT FROM AUTHOR]

Rapid advances in technology provide a good platform for the systemic cataloging and characterization of the normal and cancerous phenotype and underlying genotype of PCA. This should set the stage for the development of new prognostic and therapeutic strategies. The understanding of genetic events which occur during the progression of PIN and other preneoplastic lesions to PCA would be useful for prevention, early detection, and treatment of this common disease. [ABSTRACT FROM AUTHOR]