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Many estrogen receptor alpha (ERα)-positive breast cancers initially respond to aromatase inhibitors (AIs), but eventually acquire resistance. Here, we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives acquired AI resistance. SGK3 is up-regulated and essential for endoplasmic reticulum (EnR) homeostasis through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function in AI-resistant cells. We have further found that EnR stress response down-regulates ERα expression through the protein kinase RNA-like EnR kinase (PERK) arm, and SGK3 retains ERα expression and signaling by preventing excessive EnR stress. Our study reveals regulation of ERα expression mediated by the EnR stress response and the feed-forward regulation between SGK3 and ERα in breast cancer. Given SGK3 inhibition reduces AI-resistant cell survival by eliciting excessive EnR stress and also depletes ERα expression/function, we propose SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer.

Both androgen and phosphatidylinositol 3-kinase (PI3K) signaling are critical for cell proliferation of androgen receptor (AR)–positive prostate cancer cells, but the underlying mechanisms are still not fully understood. Here we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a Ser/Thr kinase functioning downstream of PI3K, is an AR transcriptional target and promotes prostate cancer cell proliferation. SGK3 expression is up-regulated by androgen DHT via AR. We identified an AR-binding region at the sgk3 locus, which confers androgen responsiveness of sgk3 promoters. Interestingly, we found that androgen/AR-dependent SGK3 expression requires estrogen receptor (ER) (including both isoforms, ERα and ERβ). Depletion of ER blocked DHT-induced SGK3 expression. Functionally, knockdown of SGK3 expression significantly decreased LNCaP prostate cancer cell proliferation by inhibiting G1 to S phase cell cycle progression. We further provided evidence that SGK3 promotes p70 S6 kinase (p70S6K) activation and increases cyclin D1 levels. In summary, our study identifies SGK3 as an AR target and provides a novel androgen-induced cell proliferation mechanism mediated by the AR-SGK3-p70S6K-cyclin D1 pathway in prostate cancer cells.

Serum- and glucocorticoid-inducible kinase 3 (SGK3) is a protein kinase of the AGC family of protein kinase A, protein kinase G, and protein kinase C and functions downstream of phosphatidylinositol 3-kinase (PI3K). Recent study revealed that SGK3 plays a pivotal role in Akt/protein kinase B independent signaling downstream of oncogenic PI3KCA mutations in breast cancer. Here we report that SGK3 is an estrogen receptor (ER) transcriptional target and promotes estrogen-mediated cell survival of ER-positive breast cancer cells. Through a meta-analysis on 22 microarray studies of breast cancer in the Oncomine database, we found that the expression of SGK3 is significantly higher (5.7-fold, P < 0.001) in ER-positive tumors than in ER-negative tumors. In ER-positive breast cancer cells, SGK3 expression was found to be induced by 17β-estradiol (E2) in a dose- and time-dependent manner, and the induction of SGK3 mRNA by E2 is independent of newly synthesized proteins. We identified two ERα-binding regions at the sgk3 locus through chromatin immunoprecipitation with massively parallel DNA sequencing. Promoter analysis revealed that ERα stimulates the activity of sgk3 promoters by interaction with these two ERα-binding regions on E2 treatment. Loss-of-function analysis indicated that SGK3 is required for E2-mediated cell survival of MCF-7 breast carcinoma cells. Moreover, overexpression of SGK3 could partially protect MCF-7 cells against apoptosis caused by antiestrogen ICI 182,780. Together, our study defines the molecular mechanism of regulation of SGK3 by estrogen/ER and provides a new link between the PI3K pathway and ER signaling as well as a new estrogen-mediated cell survival mechanism mediated by SGK3 in breast cancer cells.

PNRC (Proline-rich Nuclear Receptor Coactivator) is a novel coactivator for multiple nuclear receptors. PNRC was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. To understand the molecular mechanisms that regulate the expression of human PNRC gene, in this study, functional analysis of the 5′ flanking region of the human PNRC gene revealed that the −123/+27 region is the minimal promoter of the human PNRC gene. Gel shift and ChIP analyses demonstrated the specific binding of RFX1 (Regulatory Factor X) protein to the human PNRC promoter region. In co-transfection experiments RFX1 was shown to repress promoter activity of PNRC gene in a dose-dependent manner. These results indicate that r RFX1 specifically bind to promoter region and negatively regulate the transcription of the human PNRC gene.

Nuclear receptor (NR) dependent transcriptional action requires recruitment of diverse factors characterized as coregulators. PNRC (proline-rich nuclear receptor coregulatory protein) is a member of coregulators that are capable of potentiating the transcriptional activity of NRs. Here we identified three human PNRC splicing variants designated PNRC1c, PNRC1d and PNRC1f. PNRC1c and PNRC1f are generated through alternative recognition of the 3'-splice site in exon 1, leading to in-frame deletion of 79 amino acids (aa) and an altered reading frame, respectively. PNRC1d is generated through the alternate promoter usage and forms a truncated protein containing C-terminus 142 aa of full-length PNRC. These isoforms differ in their abilities to bind NRs and potentiate NR mediated transcriptions. Moreover, PNRC1d can modulate the activity of full-length PNRC in enhancing ER mediated transcription. Our results suggest that PNRC exists as functionally distinct isoforms and alternative splicing serves as a regulatory mechanism of PNRC coactivator activity.

Third generation aromatase inhibitors (AI) have shown good clinical efficacy in comparison to the anti-estrogen tamoxifen. The steroidal AI, exemestane (EXE) has previously been shown to act as an androgen, but this report demonstrates the estrogen-like activity of EXE. Based on genome-wide microarray analysis, high correlation was seen between EXE-Only (EXE O, hormone-free) and hormone-containing AI-resistant lines. In addition, the top regulated genes in the EXE O lines were mostly estrogen-responsive genes. This estrogen-like activity of EXE was further validated using estrogen receptor (ER) activity assays, where in comparison to 17beta-estradiol (E2), EXE was able to induce ER activity, though at a higher concentration. Also, this EXE-mediated ER activity was blocked by the ER antagonist ICI as well as the ERalpha-specific antagonist methyl-piperidino-pyrazole (MPP). Similarly, EXE was able to induce proliferation of breast cancer cell lines, MCF-7 and MCF-7aro, as well as activate transcription of known estrogen-responsive genes, i.e., PGR, pS2 and AREG. These results suggest that EXE does have weak estrogen-like activity.

ERRalpha (estrogen receptor-related receptor alpha) is a member of the nuclear receptor superfamily. To further our understanding of the detailed molecular mechanism of transcriptional regulation by ERRalpha, we searched for ERRalpha-interacting proteins using a yeast two-hybrid system by screening a human mammary gland cDNA expression library with the ligand-binding domain (LBD) of ERRalpha as the "bait". Fast skeletal muscle troponin I (TNNI2), along with several known nuclear receptor co-activators, were isolated. We demonstrated that TNNI2 localizes to the cell nucleus and interacts with ERRalpha in co-immunoprecipitation experiments. GST pull-down assays also revealed that TNNI2 interacts directly with ERRalpha. Through luciferase reporter gene assays, TNNI2 was found to enhance the transactivity of ERRalpha. Combining mutagenesis and yeast two-hybrid assays, we mapped the ERRalpha-interacting domain on TNNI2 to a region encompassing amino acids 1-128. These findings reveal a new function for TNNI2 as a co-activator of ERRalpha.

In situ estrogen synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms especially in postmenopausal women. Several recent studies demonstrated activity of aromatase, an enzyme that plays a critical role in estrogen synthesis in breast tumors. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1/MNAR) is an estrogen receptor (ER) coregulator, and its expression is deregulated in breast tumors. In this study, we examined whether PELP1 promotes tumor growth by promoting local estrogen synthesis using breast cancer cells (MCF7) that stably overexpress PELP1. Immunohistochemistry revealed increased aromatase expression in MCF7-PELP1-induced xenograft tumors. Real-time PCR analysis showed enhanced activation of the aromatase promoter in MCF7-PELP1 clones compared with MCF7 cells. Using a tritiated-water release assay, we demonstrated that MCF7-PELP1 clones exhibit increased aromatase activity compared with control MCF-7 cells. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II, and growth factor signaling enhanced PELP1 activation of aromatase. PELP1-mediated induction of aromatase requires functional Src and phosphatidylinositol-3-kinase pathways. Mechanistic studies revealed that PELP1 interactions with ER-related receptor-α and proline-rich nuclear receptor coregulatory protein 2 lead to activation of aromatase. Immunohistochemistry analysis of breast tumor array showed increased expression of aromatase in ductal carcinoma in situ and node-positive tumors compared with no or weak expression in normal breast tissue. Fifty-four percent (n = 79) of PELP1-overexpressing tumors also overexpressed aromatase compared with 36% (n = 47) in PELP1 low-expressing tumors. Our results suggest that PELP1 regulation of aromatase represents a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.

PNRC (proline-rich nuclear receptor co-activator) was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC has been demonstrated to be a novel co-activator for multiple nuclear receptors. To understand the molecular mechanisms that regulate the expression of human PNRC gene, in this study, potential transcriptional start site was determined by 5' RACE analysis. Functional analysis of the 5' flanking region of the human PNRC gene by deletion mutagenesis, transient transfection and luciferase assays revealed that the -123/+27 region is the minimal promoter of the human PNRC gene. Within this promoter region, there is one putative binding site for the transcription factor NFY (nuclear factor Y). EMSA and ChIP analyses demonstrated the specific binding of NFY protein to the human PNRC promoter. Transient transfection and luciferase assays further revealed that over-expression of NFY represses promoter activity of PNRC gene in a dose-dependent manner. These results indicate that the transcription factor NFY specifically binds to promoter region of PNRC and negatively regulates the transcription of the human PNRC gene.

MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene translation during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete. We isolated CD34+CD38− hematopoietic stem cells (HSCs) from human umbilical cord blood, on the basis of cell-surface markers using fluorescence-activated cell sorting (FACS). Also, CD34+ subpopulation was FACS isolated as the control. Next, using microarray containing oligonucleotides corresponding to 517 miRNAs from human, mouse, and rat genomes, we obtained miRNA gene expression profiles of both subpopulations. We focused on the HSCs correlative miRNAs with comparison to the control. The miRNAs of particular interest were confirmed by real-time RT-PCR. HSCs-overexpressed hsa-miR-520h and underexpressed hsa-miR-129 were selected for target prediction and analysis. The result showed that EIF2C3 and CAMTA1, genes related to miRNAs processing or transcription regulation, were proved to be real targets for hsa-miR-129. And ABCG2, involved in stemness maintaining, a real target for hsa-miR-520h. Finally, we chose hsa-miR-520h, enriched in HSCs but low in CD34+ cells, for functional characterization, because of its possible role in differentiation of HSCs by regulating ABCG2. As a result, hsa-miR-520h transduction into CD34+ cells greatly increased number of different progenitor colonies in Colony-Forming-Cell assays, suggesting that hsa-miR-520h may promote differentiation of HSCs into progenitor cells by inhibiting ABCG2 expression. This study paves the way for identifying HSC-specific miRNAs and their roles in HSC development. J. Cell. Biochem. 104: 805–817, 2008. © 2008 Wiley-Liss, Inc.

PNRC and PNRC2 are members of a new family of nuclear receptor coactivators. We systematically determined the molecular basis and the structure/function relationship for the PNRC-ERalpha interaction. PNRC was found to interact with ERalpha mainly through its C-terminus region, amino acids 270-327, and an SH3-binding motif within this region was shown to be essential for PNRC to interact with and function as coactivator of ERalpha. The importance of the flanking sequences of SH3-binding motif in the interaction between PNRC and ERalpha was also investigated. The PNRC-interacting domain(s) on ERalpha was also mapped. PNRC was found to interact with both AF1 and LBD of ERalpha, and to function as a coactivator for both AF1 and AF2 transactivation functions. The interaction of ERalpha mutants, I358R, K362A, V376R, L539R and E542K, with PNRC/PNRC2 was further investigated. ERalpha/HBD/V376R could bind to PNRC or PNRC2, with similar affinity as wild-type ERalpha/HBD, and the transactivation activity of ERalpha/V376R was enhanced 5-fold by PNRC. Since GRIP1, a well-characterized coactivator, was found not to be able to enhance the transactivation function of this mutant, our results indicate that the PNRC-ERalpha interaction interface is not exactly identical to that of GRIP1-ERalpha interaction.

It has been demonstrated that proline-rich nuclear receptor coregulatory protein (PNRC) is a nuclear receptor coactivator that interacts with nuclear receptors through an SH3-binding motif located in its C-terminus. In the present report, a physical interaction between PNRC and Grb2 (an adapter protein involved in growth factor/Ras-mediated pathways) has been demonstrated using the GST pull-down assay, the yeast two-hybrid assay, as well as by coimmunoprecipitation. Cotransfection and fluorescence imaging have also confirmed the colocalization of PNRC and Grb2 in mammalian cells. Transient transfection experiments have demonstrated that, by interacting with each other, Grb2 decreases the coactivator activity of PNRC for nuclear receptors, and that PNRC suppresses Grb2-mediated Ras/MAP-kinase activation. Furthermore, it was discovered that HeLa cells overexpressing PNRC grew more slowly when compared to matched controls. Additionally, using a RT-PCR analysis of mRNA on six pairs of cancer/noncancer tissues, PNRC expression was found to be significantly lower in breast cancer tissue than in noncancer tissue. Based on these findings, we believe that PNRC and Grb2, by interacting with each other, can suppress nuclear receptor-mediated regulation and growth factor-mediated regulation in human breast tissue. This is a newly identified crosstalk mechanism for modulating these two important types of regulatory pathways.

Aromatase (CYP19) is the estrogen synthetase that converts androgen to estrogen. The expression of aromatase in breast cancer cells and surrounding stromal cells is up regulated compared to non-cancerous cells. In situ estrogen synthesis is thought to stimulate breast cancer growth in both an autocrine and a paracrine manner. A complex mechanism is involved in the control of human aromatase expression, in that seven promoters have been identified and found to be utilized in a tissue-selective manner. Increased aromatase expression in breast tumors is, in part, attributed to changes in the transcriptional control of aromatase expression. While promoter I.4 is the main promoter that controls aromatase expression in non-cancer breast tissue, promoters II and I.3 are the dominant promoters that drive aromatase expression in breast cancer tissue. During the last several years, our laboratory performed a series of studies to examine the transcription regulatory mechanism of aromatase expression in breast cancer cells. We functionally characterized promoters II and I.3, and carried out DNase 1 footprinting analysis that identified two regulatory elements, S1 and CREaro. Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we found that four orphan/nuclear receptors, ERR alpha-1, EAR-2, COUP-TFI and RAR gamma, bind to the S1 element, and that CREB1, Snail (SnaH) and Slug proteins bind to the CREaro element. Studies from this and other laboratories have revealed that in cancer tissue versus normal tissue, several positive regulatory proteins (e.g. ERR alpha-1 and CREB1) are present at higher levels and several negative regulatory proteins (e.g. EAR-2, COUP-TFI, RAR gamma, Snail and Slug proteins) are present at lower levels. This may explain why the activity of promoters II and I.3 is up regulated in cancer tissue. An understanding of the molecular mechanisms of aromatase expression between non-cancerous and cancerous breast tissue, at the transcriptional level, may help in the design of a therapy based on the suppression of aromatase expression in breast cancer tissue.

Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we have found that four orphan/nuclear receptors, ERRalpha-1, EAR-2, COUP-TFI (EAR-3), and RARgamma, bind to the silencer (S1) region of the human aromatase gene. S1 down regulates promoters I.3 and II of the human aromatase gene. In this study, the interaction of EAR-2, COUP-TFI, and RARgamma with S1 was confirmed by DNA mobility shift analysis. In contrast to the findings that ERRalpha-1 behaves as a positive regulatory factor, these three nuclear receptors were found, by mammalian cell transfection experiments, to act as negative regulatory factors by binding to S1. Furthermore, the negative action of these three nuclear receptors could override the positive effect of ERRalpha-1. RT-PCR analysis of 11 cell lines and 55 human breast tumor specimens has shown that these nuclear receptors are expressed in human breast tissue. Since EAR-2, COUP-TFI, and RARgamma are expressed at high levels, it is likely that S1 is a negative regulatory element that suppresses aromatase promoters I.3 and II in normal breast tissue. In cancer tissue, S1 may function as a positive element since ERRalpha-1 is expressed, but EAR-2 and RARgamma are only present in a small number of tumor specimens. This hypothesis is sustained by the finding that there is a weak inverse correlation between the expression of COUP-TFI and that of aromatase in breast tumor tissue. Our studies have revealed that estrogen receptor alpha (ERalpha) can also bind to S1, in a ligand-dependent manner. By binding to S1, ERalpha down-regulates the aromatase promoter activity. These results demonstrate that nuclear receptors play important roles in modulating aromatase expression in human breast tissue.

Aromatase deficiency is a rare disorder resulting in estrogen insufficiency in humans. It has been reported in remarkably few men with loss-of-function mutations in the CYP19A1 gene encoding the aromatase, a cytochrome P450 enzyme that plays a crucial role in the biosynthesis of estrogens from androgens. We investigated a non-consanguineous family including an adult man with clinical features of aromatase deficiency, and studied the effects of estrogen replacement in the man.We investigated the clinical and biochemical phenotype, performed CYP19A1 mutational analysis in the family and 50 unrelated persons, studied the effects of CYP19A1 mutations on aromatase protein structure, functionally characterized the mutations by cell-based aromatase activity assays, and studied the effects of estrogen replacement on the bone, lipid, liver and glucose metabolism.The man with clinical features of aromatase deficiency had novel compound heterozygous CYP19A1 mutations (Y81C and L451P) that were not found in 50 unrelated persons. Three-dimensional modeling predicted that Y81C and L451P mutants disrupted protein structure. Functional studies on the basis of in vitro expression showed that Y81C and L45P mutants significantly decreased the aromatase activity and catalytic efficiency. Estrogen replacement in the man increased bone mineral density, accelerated bone maturation, improved lipid profile and liver steatosis, and improved glucose levels but not insulin resistance.We have identified two novel CYP19A1 missense mutations in an aromatase-deficient man. Estrogen replacement in the man shows great impact on recovering the impairments in the bone, lipid, liver and glucose metabolism, but fails to improve insulin resistance.

Serum- and glucocorticoid-inducible kinase 3 (SGK3) mediates a variety of cellular processes including membrane transport, cell proliferation, and survival, and it has been implicated in Akt-independent signaling downstream of oncogenic PIK3CA mutations (activating mutations in the α catalytic subunit of PI3K) in human cancers. However, the regulation of SGK3 is poorly understood. Here we report that SGK3 stability and kinase activation are regulated by the Hsp90-Cdc37 chaperone complex. Hsp90-Cdc37 associates with the kinase domain of SGK3 and acts in concert with a C-terminal hydrophobic motif of SGK3 to prevent Hsp70 association and ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein)-mediated degradation. Phosphorylation of hydrophobic motif triggers release of Cdc37 and concomitant association of 3-phosphoinositide dependent kinase 1 (PDK1) to activate SGK3. Our study provides new insights into regulation of SGK3 stability and activation and the rationale for application of Hsp90 inhibitors in treating SGK3-dependent cancers.

PNRC (proline-rich nuclear receptor coregulatory protein) was identified using bovine SF1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC is unique in that it has a molecular mass of 35 kDa, significantly smaller than most of the coregulatory proteins reported so far, and it is proline-rich. PNRC's nuclear localization was demonstrated by immunofluorescence and Western blot analyses. In the yeast two-hybrid assays, PNRC interacted with the orphan receptors SF1 and ERRalpha1 in a ligand-independent manner. PNRC was also found to interact with the ligand-binding domains of all the nuclear receptors tested including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR) in a ligand-dependent manner. Functional AF2 domain is required for nuclear receptors to bind to PNRC. Furthermore, in vitro glutathione-S-transferase pull-down assay was performed to demonstrate a direct contact between PNRC and nuclear receptors such as SF1. Coimmunoprecipitation experiment using Hela cells that express PNRC and ER was performed to confirm the interaction of PNRC and nuclear receptors in vivo in a ligand-dependent manner. PNRC was found to function as a coactivator to enhance the transcriptional activation mediated by SF1, ERR1 (estrogen related receptor alpha-1), PR, and TR. By examining a series of deletion mutants of PNRC using the yeast two-hybrid assay, a 23-amino acid (aa) sequence in the carboxy-terminal region, aa 278-300, was shown to be critical and sufficient for the interaction with nuclear receptors. This region is proline rich and contains a SH3-binding motif, S-D-P-P-S-P-S. Results from the mutagenesis study demonstrated that the two conserved proline (P) residues in this motif are crucial for PNRC to interact with the nuclear receptors. The exact 23-amino acid sequence was also found in another protein isolated from the same yeast two-hybrid screening study. These two proteins belong to a new family of nuclear receptor coregulatory proteins.

Aromatase (CYP19) converts androgens to estrogens. By synthesizing estrogens, aromatase (especially tumor aromatase) is thought to play an important role in promoting breast cancer growth in postmenopausal women. By inhibiting estrogen biosynthesis, aromatase inhibitors, including phytochemicals, are potential chemopreventive agents for breast cancer. Our recent aromatase inhibition experiments have revealed that grape juice contains compounds that inhibit aromatase. Inhibition kinetic analysis indicates that the active components in grape juice inhibit aromatase by competing for the binding of the substrate androstenedione. Results from cell culture experiments suggest that chemicals in grape juice can act as weak agonists/antagonists of estrogen receptor and as aromatase inhibitors. Finally, the breast cancer-protective action of grape juice was demonstrated with a nude mouse model using MCF7aro, an aromatase-transfected MCF-7 cell line. It was found that the tumor size in mice fed (by gavage) with 0.5 ml of grape juice/day for 5 weeks is reduced 70% by comparing to the tumor size in the animals not fed with grape juice. Our finding suggests that grape juice may be useful in breast cancer prevention by inhibiting in situ aromatase/estrogen biosynthesis.

The expression of aromatase in human breast tumors was studied using the reverse transcription-polymerase chain reaction (RT-PCR) method on 70 breast tissue specimens. An RT-PCR analysis using two oligonucleotide primers derived from exon II of the human aromatase gene revealed that aromatase mRNA was detected in all but three tissue specimens. Furthermore, primer-directed RT-PCR was performed to determine the exon I usage in aromatase mRNA in these breast tumor specimens. The analysis revealed that exons I.3 and PII are the two major exons I present in aromatase mRNA isolated from breast tumors, suggesting that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer and surrounding adipose stromal cells (ASCs). Recently, the regulatory properties of a 696-base pair region that contains promoter II, and is situated immediately upstream of exon II of the human aromatase gene, were investigated. Detailed DNase 1 footprinting analysis, DNA mobility shift assays, and chloramphenicol acetyltransferase (CAT) functional studies of this genomic region were performed and led to the identification of a segment (B1) that could act as a promoter (probably promoter I.3) in adipose stromal and breast cancer cells. The study further revealed that the B1 region could be divided into two domains which were designated RE1 and RE2. RE1 was found to have the promoter activity, and RE2 was found to regulate the promoter activity of RE1, but in different manners in MCF-7 cells (as an example of breast cancer cells) and in ASCs. RE2 was found to function as a positive regulatory element in MCF-7 cells and as a negative regulatory element in ASCs, respectively. It was also found that in several breast cancer cell lines, including MCF-7, the promoter activities of both promoter II and promoter I.3 were found to be suppressed by a negative regulatory element, a silencer, present in the 162 bp fragment which is located upstream from promoter II and downstream from promoter I.3. The precise position of the silencer element (termed S1) was localized by deletion mutation and DNase 1 footprinting analysis, and the silencing activity of S1 on promoter I.3 (in B1 fragment) was confirmed by CAT plasmid transfection experiments. UV crosslinking experiments are being performed to examine the regulatory proteins interacting with the silencer element. These studies serve as the basis for the further characterization of the regulatory mechanism of aromatase expression in human breast cancer and ASCs.

Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogens. In this study, the regulatory properties of a 696-base pair region, that contains the promoter II and is situated immediately upstream of exon II of the human aromatase gene, were investigated. Chloramphenicol acetyltransferase (CAT) functional studies with DNA segments derived from this genomic region and primer-extension analysis revealed the presence of a second promoter which is functional in adipose stromal cells and in breast cancer cells. Detailed DNase-1 footprinting analysis, DNA mobility shift assays, and CAT functional studies of this genomic region were performed and led to the identification of a segment (B1) that could act as a promoter (probably promoter I.3) in adipose stromal and breast cancer cells. The study revealed further that the B1 region could be divided into two domains which were designated RE1 and RE2. RE1 was found to have the promoter activity, and RE2 was found to regulate the promoter activity of RE1, but in different manners in MCF-7 cells (as an example of breast cancer cells) and in adipose stromal cells. RE2 was found to function as a positive regulatory element in MCF-7 cells and as a negative regulatory element in adipose stromal cells, respectively. DNA mobility shift and UV-cross-linking experiments with BrUrd-substituted B1 fragment and nuclear extracts isolated from two types of cells were performed. The experiments identified DNA-bound proteins with molecular masses around 50 kDa. These findings serve as the basis for further examination of the regulatory mechanism of aromatase expression in human breast cancer and adipose stromal cells.

Aromatase is present in human breast tumors and in breast cancer cell lines suggesting the possibility of in-situ estrogen production via the androstenedione to estrone and estradiol pathway. However, proof of the biologic relevance of aromatase in breast cancer tissue requires the demonstration that this enzyme mediates biologic effects on cell proliferation. Accordingly, we studied the effects of the aromatase substrate, androstenedione, on the rate of proliferation of wild-type and aromatase-transfected MCF-7 breast cancer cells. Androstenedione did not increase cell growth in wild-type MCF-7 cells which contained relatively low aromatase activity and produced 4-fold more estrone than estradiol. In contrast, aromatase-transfected cell contained higher amounts of aromatase, produced predominantly estradiol, and responded to androstenedione with enhanced growth. An aromatase inhibitor fadrozole hydrochloride, blocked the proliferative effects of androstenedione providing evidence for the role of aromatase in this process. As further evidence of the requirement for aromatase, cells transfected with the neomycin resistance expression plasmid but lacking the aromatase cDNA did not respond to androstenedione. These studies provide evidence that aromatase may have a biologic role for in-situ synthesis of estrogens of breast cancer tissue.

Mutant forms of aromatase cytochrome P -450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The K m for androstendione for expressed wild type (11.0 ± 0.3 nM SEM, n = 3) increased 4-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. 4-Hydroxyandrostenedione (4-OHA) a strong inhibitor of wild type aromatase activity (IC 50 = 21 nM and K i = 10 nM), was even more effective against mutant Pro308Phe (IC 50 = 13 nM and K i = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC 50 = 345 and 330 nM and K i = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 16949A (IC 50 = 4.0 and 4.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC 50 = 140 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced K i = 0.4 nM for CGS 16949A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for androstenedione aromatization. The substitution of Pro308 with Phe produces weaker affinity for androstenedione in the substrate pocket, but this alteration favors 4-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase.

Aromatase, a cytochrome P-450, catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. Using the x-ray structure of cytochrome P-450cam as the model, seven mutants of human aromatase were designed and expressed in Chinese hamster ovary cells by a stable expression method. They are His-128→Gln, His-128→Ala, Cys-299→Ala, Glu-302→Leu, Asp-309→Asn, Asp-309→Ala, and Ser-312→Cys. The presence of the aromatase mutants in the transfected Chinese hamster ovary cells were confirmed by immunoprecipitation analysis. The kinetic parameters of these mutants using [1 beta,2 beta-3H] androstenedione (or [1 beta-3H]androstenedione), and [1 beta,2 beta-3H]testosterone as substrates were determined. In addition, inhibition profiles for these mutants with two aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide were obtained. Furthermore, the reactions catalyzed by these mutants were examined by evaluating the levels of the product estrone, and two intermediates, 19-hydroxyandrostenedione and 19-oxoandrostenedione by reverse phase high performance liquid chromatography using [7-3H]androstenedione as the substrate. Our results indicate that among the positions we modified, Asp-309 appears to be very important for the enzyme catalysis.

PNRC2 was identified in our laboratory as a general cofactor for nuclear receptors. To better characterize the physiological function of PNRC2, we used gene-targeting technology to generate PNRC2-null mice (PNRC2(-/-) mice). These PNRC2(-/-) mice are viable and fertile. PNRC2-null mice, especially male mice, are lean and are resistant to high fat diet-induced obesity but without the induction of insulin resistance. Male mice devoid of PNRC2 protein have a higher metabolic rate than wild-type mice. They consume more oxygen and produce more heat. Consistent with reduced adipose mass, the levels of leptin are lower in PNRC2(-/-) mice. This study provides evidence that PNRC2 plays one or more important roles in controlling the energy balance between energy storage and energy expenditure. PNRC2 may be a new target in the treatment of obesity and related metabolic diseases.

Background PNRC transcriptionally regulates a wide range of RNA polymerase (pol) II-transcribed genes by functioning as a nuclear receptor coactivator. To search for additional PNRC-interacting proteins other than nuclear receptors, a PNRC fragment was used as bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. Results RNA pol III/RPC39 fragments were repeatedly identified as PNRC-interacting partners in two independent screenings. The interaction between these RPC39 fragments and PNRC was further confirmed in the independent yeast two-hybrid assays. The association of endogenous PNRC and RPC39 in MCF7 cells was demonstrated by co-immunoprecipitation. Furthermore, ChIP analysis detected co-recruitment of PNRC and RPC39 to tRNA and U6 RNA promoters. The biological consequence of the interaction between PNRC and RPC39 was further studied. Overexpression of PNRC, either by transient or stable transfection, increased RNA pol III-dependent transcription in MCF7 cells, while a decrease in transcription in MCF7 cells treated with PNRC/siRNA was observed. Conclusion Here, we demonstrate that human PNRC stimulates RNA pol III transcription through its interaction with the subunit RPC39 of RNA pol III. PNRC is a unique coactivator that has profound effects on many aspects of cellular function by directly influencing both RNA pol II- and RNA pol III-dependent transcription.

DT-diaphorase (EC 1.6.99.2), also referred to as NAD(P)H:(quinone-acceptor) oxidoreductase, is involved in the reductive activation process of several cytotoxic antitumor quinones and nitrobenzenes. It has been observed in our and other laboratories that the rat enzyme is significantly more effective in activating these drugs than the human and mouse enzymes. These results indicate that the available cytotoxic drugs are better substrates for the rat enzyme and are not the most ideal prodrugs for activation by DT-diaphorase in human tumors. In this study, using site-directed mutagenesis to replace residues in the rat enzyme with the human sequences and residues in the human enzyme with the rat sequences, we have found that residue 104 (Tyr in the rat enzyme and Gln in the human and mouse enzymes) is an important residue responsible for the catalytic differences between the rat and the human (and mouse) enzymes. With an exchange of a single amino acid, the rat mutant Y104Q behaved like the wild-type human enzyme, and the human mutant Q104Y behaved like the wild-type rat enzyme in their ability to reductively activate the cytotoxic drug CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The study also confirms the conclusion of the x-ray structural analysis of rat enzyme that residue 130 (Thr in the rat enzyme and Ala in the human and mouse enzymes) is positioned near the binding region of the nicotinamide portion of NAD(P)H. This structural information is very important for designing suitable drugs and approaches for human cancer chemotherapy mediated by DT-diaphorase.

By performing primer-specific RT-PCR analyses, three laboratories including ours have found that exons I.3 and PII are the two major exon Is present in aromatase mRNAs isolated from breast tumors. These results suggest that promoters I.3 and II are the major promoters directing aromatase expression in breast tumors. The characterization of transcription factors that interact with the two elements near promoters I.3 and II, i.e., S1 and CREaro, helps us better understand the mechanism of the switch of promoter usage between normal breast tissue and cancer tissue. The positions of the two regulatory regions were mapped by DNase I footprinting and DNA deletion analyses. We applied the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library for genes encoding the proteins binding to these regions. Our results suggest that in normal breast tissue, the function of promoters I.3 and II is suppressed through the binding of EAR-2, COUP-TFI, and RARgamma to S1, and through the binding of Snail/Slug proteins to their binding site that quenches the CREaro activity. In cancer tissue, the expression levels of EAR-2, COUP-TF1, EARgamma, Snail, and Slug decrease, and aromatase expression is then up-regulated through the binding of ERRalpha to S1 and the binding of CREB1 or related factors to CREaro. In a separate study, we found that estrogen could up-regulate aromatase expression in breast cancer cells by a non-genomic action of ERalpha via cross-talk with growth factor-mediated pathways. Our preliminary results suggest that protein kinase C delta participates in this ERalpha-growth factor mediated regulation. To further understand the regulatory mechanism, we have recently initiated an in vivo footprinting analysis of the -260/+76 bp region of promoter I.3. The experiments were conducted with both MCF-7 and MDA-MB-231 breast cancer cells. Our results revealed several footprinted sites. Five regions (sites 1-5) were then selected for functional analysis through DNA site-directed mutagenesis experiments. This analysis has also confirmed the promoter I.3 TATA site and Snail/Slug binding site. These mutants showed higher luciferase activity when compared to the wild-type, indicating that the proteins binding to these sites were acting as repressors. By reviewing findings from our laboratory and other laboratories, a detailed mechanism for the transcriptional regulation of aromatase expression in breast cancer tissue is summarized and discussed.

PNRC2 (Proline-rich Nuclear Receptor Coactivator 2) was previously identified through its interaction with SF1 (steroidogenic factor 1) and has been demonstrated to be a novel coactivator for multiple nuclear receptors. In this study, PNRC2 was found to be widely expressed in mouse tissues with a strong expression in lung, spleen, ovary, thymus, and colon. Alignment of mouse genomic sequence with mouse cDNA sequence (BC006598), using mouse genome browser, defines that PNRC2 gene, located on chromosome 4, contains 3 exons: 166 bp-exon I, 205 bp-exon II, and 1526 bp-exon III. The translational start site is located in exon III. The first two exons are not translated. The 420 bp coding sequence in exon III encodes a 140 amino acid protein. To understand the molecular mechanisms that regulate the expression of PNRC2 gene, we have cloned and characterized the 5'-flanking region of the gene. Potential transcriptional start sites were determined by 5' RACE analysis. Functional analysis of the 5' flanking region of the mPNRC2 gene by deletion mutagenesis, transient transfection and luciferase assays revealed that the -67/+53 region is the minimal promoter of the mouse PNRC2 gene in HeLa cells. Within this sequence we identified two putative binding sites (inverted CCAAT box) for the transcription factor NFY (nuclear factor Y), a factor mediating cell type-specific and cell-cycle regulated expression of genes, and one binding site for E2F1, a founding member of the E2F family that displays the properties of both an oncogene and a tumor suppressor gene. Mutating each individual CCAAT site or changing the orientation of the CAATT box led to a 5-fold decrease in PNRC2 promoter activity in transient transfection experiments. Gel shift, supershift assay, and ChIP analysis demonstrated the specific binding of NFY and E2F1 proteins to the mouse PNRC2 promoter. Transient transfections and luciferase assays further revealed that overexpression of NFY enhanced-promoter activity of PNRC2 gene in a dose-dependent manner while overexpression of E2F1 strongly repressed the activity of the PNRC2 promoter. Since most genes regulated by E2F1 or NFY play a regulatory role in the cell cycle, the finding that the PNRC2 promoter is activated by NFY and repressed by E2F1 indicates that in addition to functioning as nuclear receptor coactivator, PNRC2 may also play a role in the cell cycle.

Estrogen stimulates the proliferation of estrogen receptor (ER)-positive breast cancer cells. Aromatase is the enzyme responsible for the conversion of androgens into estrogens, and synthetic aromatase inhibitors such as letrozole, anastrozole, and exemestane have proven to be effective endocrine regimens for ER-positive breast cancer. In a recent study, we have found that 4-benzyl-3-(4'-chlorophenyl)-7-methoxycoumarin is a potent competitive inhibitor of aromatase with respect to the androgen substrate. Its K(i) value was determined to be 84 nm, significantly more potent than several known aromatase inhibitors. The specific interaction of this compound with aromatase was further demonstrated by the reduction of its binding by several mutations at the active site region of aromatase and evaluated by computer modeling analysis. The structure-activity studies have revealed that three functional groups (i.e. 3-(4'-chlorophenyl), 4-benzyl, and 7-methoxyl) of this coumarin are important in its inhibition of aromatase. In addition, through a matrigel thread three-dimensional cell culture, this compound was shown to behave like known aromatase inhibitors that suppress the proliferation of aromatase and estrogen receptor positive MCF-7aro breast cancer cells. This coumarin has been shown not to be cytotoxic at up to 40 mum. It was found not to be an inhibitor of steroid 5alpha-reductase that also utilizes androgen as the substrate and not to be a ligand of ERalpha, ERbeta, estrogen-related receptors, or androgen receptor. These results demonstrate that coumarins (a common type of phytochemical) or their derivatives can be potent inhibitors of aromatase and may be useful in suppressing aromataseand ER-positive breast tumors.

The utilization of computer modeling, site-directed mutagenesis, inhibition kinetic analysis and reaction metabolite analysis allows us to better understand the structure-function relationship between aromatase and its inhibitors. Our results have helped in determining how steroidal and nonsteriodal aromatase inhibitors bind to the active site of the enzyme. This information has also aided in the understanding of the reaction mechanism of aromatase. Furthermore, our structure-function studies of aromatase have generated important information for predicting how environmental chemicals interact with the enzyme. During the last 2 years, a new aromatase computer model based on the X-ray structure of rabbit cytochrome P450 2C5 has been generated and used to evaluate the results obtained from new aromatase mutants produced in this laboratory. In addition, we have succeeded in the expression and purification of functionally active aromatase using an Escherichia coli expression method. The catalytic properties of this recombinant aromatase are similar to those properties exhibited by the human placental aromatase preparation and the mammalian cell-expressed enzyme. The E. coli expressed aromatase will be very useful for further structure-function studies of aromatase. Our laboratory has also evaluated the growth-inhibiting activity of aromatase inhibitors in estrogen receptor-positive breast cancer using three-dimensional cell cultures of aromatase-over expressing MCF-7 and T-47D cell lines (i.e. MCF-7aro and T-47Daro). Our results demonstrate that these three-dimensional cultures are valuable approaches to assess the growth-inhibiting activity of aromatase inhibitors. Finally, we have identified several phytochemicals to be potent inhibitors of aromatase. To demonstrate the impact of the phytochemicals on estrogen formation in vivo, we showed that the intake of anti-aromatase chemicals from red wine was capable of suppressing MCF-7aro-mediated tumor formation in nude mice and aromatase-induced hyperplasia in a transgenic mouse model in which aromatase is over-expressed in the mammary tissue.

Aromatase plays an important role in breast cancer development through its role in the synthesis of estrogen. Aromatase expression in breast tissue can be regulated by several mechanisms. The major promoter usage for aromatase expression in breast tumors (i.e. cAMP-stimulated promoters I.3 and II) is different from that in normal breast tissue (i.e. glucocorticoid-stimulated promoter I.4). Recent characterization of transcription factors that interact with the two important regulatory elements near promoters I.3 and II, i.e. S1 and CREaro, helps us better understand the mechanism of the switch of promoter usage between normal breast tissue and cancer tissue. It is thought that in normal breast tissue, the function of promoters I.3 and II is suppressed through the binding of EAR-2, COUP-TFI, and EARgamma to S1, and through the binding of Snail/Slug proteins to their binding site that quenchs the CREaro activity. In cancer tissue, the expression levels of EAR-2, COUP-TFI, EARgamma, Snail, and Slug decrease, and aromatase expression is then up regulated through the binding of ERRalpha-1 to S1 and the binding of CREB or related factors to CREaro. Results from this and other laboratories reveal that aromatase activity in aromatase expressing cells can also be modified by treatment with aromatase inhibitors and the antiestrogen ICI 182, 780. While aromatase inhibitors are used to treat breast cancer, the treatment has been found to increase the level of aromatase in the breast tissue of some patients. The enhancement of aromatase activity by aromatase inhibitors is thought to be due to a decrease of aromatase protein degradation by enzyme-inhibitor complex formation, up-regulation of the aromatase gene transcription through a cAMP-mediated mechanism, and an induction of aromatase expression by gonadtropins that are released from the pituitary in response to a reduction of estrogen levels in circulation in premenopausal women. Antiestrogen ICI 182, 780 has been found to suppress aromatase expression, but the mechanism has not yet been determined. In addition, aromatase activity and expression can be affected by environmental chemicals. A detailed structure-function study has revealed that flavones, but not isoflavones, are inhibitors of aromatase. It was found that flavones bind to the active site of aromatase in an orientation in which their rings-A and -C mimic rings-D and -C of the androgen substrate. The modulation of aromatase expression by endocrine disrupting chemicals is exemplified by two organochlorine pesticides (i.e. toxaphene and chlordane) that have been found to be antagonists of ERRalpha-1 orphan receptor. These compounds reduce ERRalpha-1 activity, resulting in a suppression of aromatase expression.

PNRC2 (proline-rich nuclear receptor co-regulatory protein 2) was identified using mouse steroidogenic factor 1 (SF1) as bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC2 is an unusual coactivator in that it is the smallest coactivator identified so far, with a molecular weight of 16 kDa, and interacts with nuclear receptors using a proline-rich sequence. In yeast two-hybrid assays PNRC2 interacted with orphan receptors SF1 and estrogen receptor-related receptor alpha1 in a ligand-independent manner. PNRC2 was also found to interact with the ligand-binding domains of estrogen receptor, glucocorticoid receptor, progesterone receptor, thyroid receptor, retinoic acid receptor and retinoid X receptor in a ligand-dependent manner. A functional activation function 2 domain is required for nuclear receptors to interact with PNRC2. Using the yeast two-hybrid assay, the region amino acids 85-139 was found to be responsible for the interaction with nuclear receptors. This region contains an SH3 domain-binding motif (SEPPSPS) and an NR box-like sequence (LKTLL). A mutagenesis study has shown that the SH3 domain-binding motif is important for PNRC2 to interact with all the nuclear receptors tested. Our results reveal that PNRC2 has a structure and function similar to PNRC, a previously characterized coactivator. These two proteins represent a new type of nuclear receptor co-regulatory proteins.

Some orphan nuclear receptors, including estrogen-related receptor alpha-1 (ERRalpha-1), can activate gene transcription in a constitutive manner. Little is known about the molecular basis of the constitutive activity of these receptors. Our results from site-directed mutagenesis experiments have revealed that Phe-329 (analogous to Ala-350 in estrogen receptor alpha (ERalpha)) is responsible for the constitutive activity of ERRalpha-1. The ERRalpha-1 mutant F329A lost the transactivation activity and acted as a dominant negative mutant. The mammalian cell transfection experiments revealed that the ERRalpha-1 mutant F329A, like wild-type ERalpha, recognized toxaphene (an organochlorine pesticide) as an agonist. This compound was previously shown to be an antagonist of wild-type ERRalpha-1. On the other hand, like wild-type ERRalpha-1, the ERalpha mutant A350F was found to be constitutively active (as demonstrated by mammalian cell transfection and yeast two-hybrid assays). These results indicate that Phe-329 in ERRalpha-1 and Ala-350 in ERalpha play important roles in both ligand binding and transactivation function.

Estrogens (Es) play a crucial role in breast cancer (BC) development. Aromatase (CYP19), a cytochrome P450, is the enzyme that synthesizes Es. Using enzyme activity measurements (1–3), immunocytochemistry (4–7), and RT-PCR analysis (8, 9), aromatase is expressed at a higher level in human BC tissue than in normal breast tissue. Cell culture (10,11), animal experiments using aromatase-transfected BC cells (12, 13), and transgenic mouse studies (14) have demonstrated that in-situ produced E plays a more important role than circulating Es in breast tumor promotion. In addition, tumor aromatase has been shown to stimulate BC growth in both an autocrine and aparacrine manner (15). RT-PCR and gene transcriptional studies have revealed that in normal tissue, the aromatase promoter switches from a glucocorticoid-stimulated promoter, 1.4, to the cAMP-stimulated promoters, 1.3 and II, in cancerous tissue (9, 16, 17). Suppression of in-situ E biosynthesis can be achieved by the prevention of aromatase expression, or by the inhibition of aromatase activity in breast tumors. Our laboratory has devoted significant effort to understand the regulatory mechanism of aromatase expression in BC tissue and to determine the structure- function relationship of aromatase. The information obtained from our studies will help in developing approaches to repress aromatase/E biosynthesis in BC tissue.

Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogens. The control of human aromatase expression is complex in that several promoters drive aromatase expression in a tissue-specific manner. Promoters I.3 and II are situated within the 700-bp region immediately upstream of exon II of the human aromatase gene, and these promoters have been shown to drive aromatase expression in breast tumors. This paper reports the characterization of a negative regulatory element that is situated between promoters I.3 and II and down regulates the action of these promoters. This negative regulatory element is thought to be a silencer element (S1) because it acts in an orientation- and promoter-independent manner. The position of S1 (5′-CCAA GG TCA GAA A TGC T GCA A TTC AAG CC A-3′) was mapped by DNase I footprinting and DNA deletion analyses. The region contains three pairs of inverted repeats, as indicated by an underline, probably explaining why S1 functions in both orientations. Chloramphenicol acetyltransferase functional analyses indicated that the transcriptional activity of promoter I.3 was suppressed two- to threefold by S1. Mutations of two inverted repeat segments (i.e., TTC and CC) in S1 destroyed its silencing action and modified the protein binding patterns in DNA mobility shift analysis. UV cross-linking experiments with 32 P-labeled bromodeoxyuridine-substituted S1 as the probe and nuclear extracts from MCF-7 breast cancer cells and skin fibroblasts revealed that four major nuclear proteins, with molecular masses of approximately 150, 45, 30, and 25 kDa, bound to this element. Interestingly, two smaller proteins could be competed by an unlabeled fragment which contains promoter I.3. In addition, mutation of S1 at the region CC destroyed the ability to compete with the wild-type S1 for the binding of 30- and 25-kDa proteins. These results led us to propose that S1 down regulates the action of promoter I.3, and the 30- and 25-kDa proteins present in the nuclear extract of MCF-7 cells and skin fibroblasts are involved in the silencer action.

Stable aromatase-expressing MCF-7 and T-47D cell lines (i.e. MCF-7aro and T-47Daro) have been prepared by aromatase cDNA transfection and G418 (neomycin) selection. MCF-7aro was further subjected to a clonal purification. Aromatase activity in the transfected MCF-7 and T-47D cell lines was determined to be 73 ± 6 pmol/mg/h and 48 ± 4 pmol/mg/h, respectively. It is thought that these cell lines express aromatase in a stable manner, as demonstrated by a steady expression of the enzyme during culture in the absence of G418. The growth of these cells could be stimulated by androgens (1–10 nM) as demonstrated through a spheroid culture method. The androgen-stimulated growth could be suppressed by 4-hydroxyandrostenedione (4-OHA) (0.01–0.1 mM) or tamoxifen (50 nM-1 μM). In order to test the hypothesis that tumor aromatase can affect breast tumor growth in a paracrine manner, we have carried out cell culture experiments by co-culturing MCF-7 cells with either MCF-7aro or T-47Daro cells. Testosterone (1 nM) increased cell growth to a similar degree for MCF-7/MCF-7aro co-culture (0.75 × 10 6 cells each type) as with MCF-7aro only (2- to 3-fold). In addition, the enzyme activities remained unchanged for MCF-7/MCF-7aro co-culture samples with and without androgen treatment, indicating that estrogen produced by transfected cells can also stimulate the growth of untransfected cells. The androgen response could be inhibited by an addition of 4-OHA (0.01–0.1 mM). For MCF-7/T-47Daro co-culture experiments, a clear induction of cell growth by androgen was observed, and the level of the increase was similar to that on T-47Daro only. However, for either culture with T-47D only or with MCF-7/T-47Daro co-culture, the aromatase activity was found to increase significantly after testosterone treatment. T-47Daro cells were not subjected to a clonal purification, and it is therefore thought that the androgen treatment may selectively stimulate the growth of high aromatase-expressing T-47Daro cells. These results indicate that estrogen synthesized by tumor aromatase can stimulate breast tumor growth in both an autocrine and a paracrine manner

The expression of aromatase in human breast tumors has been studied by the reverse-transcription polymerase chain reaction (RT-PCR) method on 70 breast tissue specimens. An RT-PCR analysis using two oligonucleotide primers derived from the exon II of the human aromatase gene revealed that aromatase mRNA was detected in all but three tissue specimens. Furthermore, primer-directed RT-PCR was performed to determine the exon I usage in aromatase mRNA in these breast tumor specimens. The analysis has revealed that exons I.3 and PII are the two major exon Is present in aromatase mRNA isolated from breast tumors, suggesting that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer and surrounding adipose stromal cells. The RT-PCR analysis also detected two products, I.3A (334 bp in length) and I.3B (222 bp in length), when it was carried out using a primer derived from exon I.3 and a reverse primer derived from exon II. The nucleotide sequences of these products have been determined and indicate that I.3A contains a region which was previously thought to be an intron. In addition, RT-PCR analyses of RNA isolated from eight pairs of breast tumor and neighboring normal tissue specimens were performed to evaluate the exon I usage and the distribution of I.3A- and I.3B-containing aromatase RNA messages in breast tumor and neighboring normal tissues. The results suggest that I.3B- and I.3A-containing messages are mainly present in breast tumor and neighboring normal tissues, respectively. Finally, the exon I/promoter usage for aromatase expression in eight cell lines (skin fibroblast, MCF-7, MDA-MB-231, T-47D, SK-BR-3, JAR, OVCAR-3, and human adipose stromal cells) was examined by primer-directed RT-PCR analyses. These studies provide a basis for further evaluation of the control mechanism of aromatase expression and estrogen biosynthesis in breast tumors.

The levels of the aromatase gene and its expression in MCF-7 human breast cancer cells and seven additional cultured cells were investigated. Using normal human foreskin fibroblasts as the control, the aromatase gene appeared to be amplified in MCF-7 cells as shown by Southern and DNA slot blot analyses utilizing human placental aromatase cDNA as the probe. However, the promoter I.1 and the first exon of the aromatase gene were not amplified in MCF-7 cells based on results obtained from DNA slot blot analysis using oligonucleotide probes having sequences derived from those regions of human aromatase gene. Aromatase was expressed at a very low level in this cell line as indicated by Northern blot analysis to measure the level of aromatase mRNA, immunoprecipitation analysis to measure the level of aromatase protein, and aromatase activity measurement. Furthermore, nucleotide sequence analysis of the aromatase cDNA obtained from MCF-7 cells by PCR techniques, revealed no sequence difference from that of the enzyme expressed in placenta. These results lead us to conclude that the expression of aromatase in MCF-7 cells is under the control of an unusual promoter and aromatase gene expression is repressed at the transcriptional level in these cells.

Mutant aromatase cytochrome P -450 s , expressed in CHO cells after transfection with cDNAs, have been characterized in terms of their catalytic efficiencies. After solubilization from microsomes, specific aromatase P -450 content of wild-type and mutants Pro308Phe, Asp309Asn, Asp309Ala and Phe406Arg was quantitated by a sandwich enzyme-linked immunosorbent assay (ELISA). Microsomal aromatase activity was determined by the 3 H-water method using [1β- 3 H]androstenedione as substrate. Estimations of the actual turnover rate (catalytic efficiency) were derived from the combined data. The P -450 content in the mutants varied but was always less than that in the wild type. Hence, the decreases in the V max observed in the mutant enzymes did not correlate completely with reductions in catalytic effectiveness. In recent studies on the structure-function relationship of aromatase cytochrome P -450, the observed reduction of enzyme activity in terms of V max following site-directed mutagenesis led to the assumption that there was a corresponding loss of catalytic effectiveness. The present study reveals that a lower P -450 content can contribute significantly to decreasing catalytic activity in the mutants. In fact, in mutant Phe406Arg which exhibited virtually no catalytically active aromatase, the specific P -450 content was below the detectable level. Because of its location, the result of this latter mutation could be a major structural perturbation of the heme-binding property. Thus, interpretation of losses and reductions in aromatase activity resulting from single amino-acid replacement should take into account changes in the specific content of aromatase cytochrome P -450

Site-directed mutagenesis experiments have been carried out to determine the structure-function relationship of human aromatase. By sequence comparison, the region in aromatase that corresponds to the distal helix of cytochrome P-450cam has been identified to be Gln-298 to Val-313. Eight aromatase mutants with changes in this region, i.e. C299A, E302L, P308F, D309N, D309A, T310S, T310C, and S312C, have been generated using a mammalian cell stable-expression system. The results from site-directed mutagenesis studies indicate that the region containing Gln-298 to Val-313 is indeed a very important part of the active site of aromatase. The catalytic properties of P308F, D309N, and D309A have been examined in detail and are discussed. Active site-directed labeling is also an important approach to investigate the structure-function relationship of aromatase. HPLC-linked electrospray mass spectrometry is indicated as a useful technique for the characterization of active site-directed probe-modified enzyme. The mass spectral analysis of aromatase suggests that aromatase is glycosylated.

Several functional domains, especially the active site regions, in aromatase cytochrome P450 were inferred by alignment of amino acid sequences of the enzyme from five species, human, rat, mouse, chicken, and trout, and that of Pseudomonas putida cytochrome P450cam, whose x-ray structure has been determined (Poulos, T.L., Finzel, B.C., and Howard, A.J. (1987) J. Mol. Biol. 195, 687-700). The predicted functions of these domains have been evaluated by site-directed mutagenesis. Eighteen mutants, including seven new mutants, have been generated in this laboratory. The seven newly prepared mutants are Q123E, Q123H, T310S, T310C, R365K, R365A, and N delta 20 (a mutant without the first 20 amino acids). The preparation and characterization of these new mutants are described. The structural model described in this paper should be very useful for future structure-function studies of aromatase by site-directed mutagenesis.

Although aromatase inhibitors (AIs) are useful in the treatment of hormone-responsive breast cancer, unfortunately, resistance to such therapy still develops. Based on our published preclinical results that ER, AREG and EGFR play important roles in the acquired resistance mechanism of exemestane (EXE), a steroidal AI, intermittent use of EXE was thought to delay the development of acquired resistance by preventing up-regulation of AREG and activation of EGFR-mediated pathways. This hypothesis was verified by preclinical cell culture and animal studies. Microarray analysis was performed using RNA isolated from week-14 (EXE-resistant) MCF7aro cells that were continuously treated with EXE, week-14 (EXE-responsive) cells that were intermittently treated with EXE (2 weeks on and 1 week off), and cells without EXE treatment. A selective number of estrogen-regulated genes, including AREG and SGK3 (serum/glucocorticoid regulated kinase family member 3), were differentially transcribed between the continuously treated samples and intermittently treated samples. Treatment of EXE resistant cells with siRNAs against AREG and SGK3 suppressed cell growth, supporting the roles of these two proteins in the development of resistance to EXE. Additional results from ChIP and gene expression analyses confirm that the expression of SGK3 can be up-regulated by E2 as well as EXE, and over-expression of SGK3 suppresses apoptosis and increases cell survival. Our animal experiments revealed that intermittent treatment of EXE (250 µg/day, 2 weeks on and 1 week off) was effective at suppressing androgen-induced MCF7aro tumor growth in nude mice, and that after 21 weeks of treatment, resistance resulted in continuous EXE treatment group, while the tumor volumes in the positive control group (androgen treated only) reached 10 cm 3 at week 18. qPCR analysis indicated that SGK3 expression in tumors from continous EXE mice was two times that in tumors from the positive control mice, with lowest expression in tumors from the intermittent EXE group (30% of that of the positive control). An analysis on 11 pairs of malignant and adjacent normal tissue has revealed that SGK3 expression is significantly greater in breast tumor specimens than adjacent non-cancer specimens (P=0.006). Since results from these translational research studies provide an important mechanistic basis for intermittent EXE therapy, a clinical trial to evaluate the intermittent use of EXE on postmenopausal women with ER-positive metastatic breast cancer has been recently approved and initiated at the City of Hope NCI-designated Comprehensive Cancer Center. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 605.

Aromatase, a cytochrome P450, catalyzes the formation of aromatic C-18 estrogenic steroids from C-19 androgens. Four mutants of human aromatase have been expressed in Chinese hamster ovary cells using a stable expression method. The activities of these mutants were determined using [1 beta,2 beta-3H]androstenedione, [19-14C]androstenedione, and [1 beta,2 beta-3H]testosterone as substrates. The mutant Phe-406----Arg was completely inactive. Since there were only small changes in the Km and Vmax values for all substrates for mutants Tyr-361----Phe and Tyr-361----Leu, the residue Tyr-361 appears not to be directly involved in the substrate binding. The mutant Pro-308----Phe had altered catalytic properties; the Km values for androstenedione, but not testosterone, decreased significantly. These results, along with those obtained from inhibition studies with aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide, suggest that Pro-308 is probably situated in the active site of the enzyme and may be interacting with the D ring of the steroids.

PNRC (proline-rich nuclear receptor coregulatory protein) was primarily identified as a coactivator of nuclear receptors (NRs) by our laboratory, which enhances NR-mediated transcription by RNA polymerase II. Recent study has shown that PNRC also stimulates RNA polymerase III-dependent transcription through interaction with the subunit RPC39 of RNA polymerase III. Here, we report that PNRC accumulates in the nucleolus and its depletion by small interfering RNA (siRNA) impairs pre-rRNA transcription by RNA polymerase I. We identified the sequence at position 94–101 (94PKKRRKKK101) of PNRC as its nucleolar localization sequence (NoLS). Fusion of this sequence to GFP directed GFP to the nucleolus. Characterization of the NoLS revealed that the stretches of six successive basic residues are sufficient to function as a NoLS. Through co-immunoprecipitation assay, we demonstrated that the NoLS is necessary and sufficient to mediate the association of PNRC with B23/nucleophosmin. Moreover, B23 depletion by siRNA disrupted the accumulation of PNRC in the nucleolus. Together, our study indicates that PNRC is a novel nucleolar protein that might be involved in regulation of pre-rRNA synthesis, and it localizes to the nucleolus by interaction with B23 via its NoLS. Our study also suggests that the stretches of six successive basic residues (lysine and/or arginine) could function as NoLS.

The molecular mechanisms underlying premalignant gastrointestinal diseases, such as ulcerative colitis and Barrett's esophagus, remain unknown. For this reason, the expression of the protooncogene c-Ha- ras was studied in ulcerative colitis and Barrett's esophagus. Total cellular RNA was extracted from different regions of the gastrointestinal tract in these two diseases. Expression of c-Ha- ras was greater in proximal than in distal colon and undetectable in Barrett's esophagus. These regional differences in expression were not seen with the control gene β-actin or with the protooncogenes c- myc and p53. In order to evaluate structural factors contributing to expression, amplification and methylation of c-Ha- ras DNA were studied in these tissues by Southern and slot blotting. No amplification of c-Ha- ras or six other protooncogenes was detected. These data suggest tissue-specific regulation of c-Ha- ras expression in the gastrointestinal tract in certain premalignant disease states.