Depletion of the Transcriptional Coactivator Amplified in Breast Cancer 1 (AIB1) Uncovers Functionally Distinct Subpopulations in Triple-Negative Breast Cancer.

The transcriptional coactivator Amplified in Breast Cancer 1 (AIB1) plays a major role in the progression of hormone and HER2-dependent breast cancers but its role in triple negative breast cancer (TNBC) is undefined. Here, we report that established TNBC cell lines, as well as cells from a TNBC patient-derived xenograft (PDX) that survive chemotherapy treatment in vitro express lower levels of AIB1 protein. The surviving cell population has an impaired tube-formation phenotype when cultured onto basement membrane, a property shared with TNBC cells that survive shRNA-mediated depletion of AIB1 (AIB1LOW cells). DNA analysis by exome sequencing revealed that AIB1LOW cells represent a distinct subpopulation. Consistent with their in vitro phenotype AIB1LOW cells implanted orthotopically generated slower growing tumors with less capacity for pulmonary metastases. Gene expression analysis of cultured cells and tumors revealed that AIB1LOW cells display a distinct expression signature of genes in pro-inflammatory pathways, cell adhesion, proteolysis and tissue remodeling. Interestingly, the presence of this AIB1LOW expression signature in breast cancer specimens is associated with shorter disease free survival of chemotherapy treated patients. We concluded that TNBC cell lines contain heterogeneous populations with differential dependence on AIB1 and that the gene expression pattern of AIB1LOW cells may represent a signature indicative of poor response to chemotherapy in TNBC patients.

Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion.

Cancer cell vascular invasion is a crucial step in the malignant progression toward metastasis. Here we used a genome-wide RNA interference screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these Krtaps have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/ß4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a Krtap can serve as a switch toward malignant progression.

Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling.

Metastasis of cancer cells involves multiple steps, including their dissociation from the primary tumor and invasion through the endothelial cell barrier to enter the circulation and finding their way to distant organ sites where they extravasate and establish metastatic lesions. Deficient contact inhibition is a hallmark of invasive cancer cells, yet surprisingly the vascular invasiveness of commonly studied cancer cell lines is regulated by the density at which cells are propagated in culture. Cells grown at high density were less effective at invading an endothelial monolayer than cells grown at low density. This phenotypic difference was also observed in a zebrafish model of vascular invasion of cancer cells after injection into the yolk sac and extravasation of cancer cells into tissues from the vasculature. The vascular invasive phenotypes were reversible. A kinome-wide RNA interference screen was used to identify drivers of vascular invasion by panning small hairpin RNA (shRNA) library-transduced noninvasive cancer cell populations on endothelial monolayers. The selection of invasive subpopulations showed enrichment of shRNAs targeting the large tumor suppressor 1 (LATS1) kinase that inhibits the activity of the transcriptional coactivator yes-associated protein (YAP) in the Hippo pathway. Depletion of LATS1 from noninvasive cancer cells restored the invasive phenotype. Complementary to this, inhibition or depletion of YAP inhibited invasion in vitro and in vivo. The vascular invasive phenotype was associated with a YAP-dependent upregulation of the cytokines IL6, IL8 and C-X-C motif ligand 1, 2 and 3. Antibody blockade of cytokine receptors inhibited invasion and confirmed that they are rate-limiting drivers that promote cancer cell vascular invasiveness and could provide therapeutic targets.

The nuclear coactivator amplified in breast cancer 1 maintains tumor-initiating cells during development of ductal carcinoma in situ.

The key molecular events required for the formation of ductal carcinoma in situ (DCIS) and its progression to invasive breast carcinoma have not been defined. Here, we show that the nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is expressed at low levels in normal breast but is highly expressed in DCIS lesions. This is of significance since reduction of AIB1 in human MCFDCIS cells restored a more normal three-dimensional mammary acinar structure. Reduction of AIB1 in MCFDCIS cells, both before DCIS development or in existing MCFDCIS lesions in vivo, inhibited tumor growth and led to smaller, necrotic lesions. AIB1 reduction in MCFDCIS cells was correlated with significant reduction in the CD24-/CD44+ breast cancer-initiating cell (BCIC) population, and a decrease in myoepithelial progenitor cells in the DCIS lesions in vitro and in vivo. The loss of AIB1 in MCFDCIS cells was also accompanied by a loss of expression of NOTCH 2, 3 and 4, JAG2, HES1, GATA3, human epidermal growth factor receptor 2 (HER2) and HER3 in vivo. These signaling molecules have been associated with differentiation of breast epithelial progenitor cells. These data indicate that AIB1 has a central role in the initiation and maintenance of DCIS and that reduction of AIB1 causes loss of BCIC, loss of components of the NOTCH, HER2 and HER3 signaling pathways and fewer DCIS myoepithelial progenitor cells in vivo. We propose that increased expression of AIB1, through the maintenance of BCIC, facilitates formation of DCIS, a necessary step before development of invasive disease.

Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor.

The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) and its ligand, the growth factor pleiotrophin (PTN), are highly expressed during the development of the nervous system and have been implicated in the malignant progression of different tumor types. Here, we describe human single-chain variable fragment (scFv) antibodies that target the ligand-binding domain (LBD) in ALK and show the effect in vitro and in vivo. The ALK LBD was used as a bait in a yeast two-hybdrid system to select human scFv from a library with randomized complementarity-determining region 3 domains. Surface plasmon resonance showed high-affinity binding of the selected scFv. The anti-ALK scFv competed for binding of PTN to ALK in intact cells and inhibited PTN-dependent signal transduction through endogenous ALK. Invasion of an intact endothelial cell monolayer by U87MG human glioblastoma cells was inhibited by the anti-ALK scFv. In addition, the growth of established tumor xenografts in mice was reversed after the induction of the conditional expression of the anti-ALK scFv. In archival malignant brain tumors expression levels of ALK and PTN were found elevated and appear correlated with poor patient survival. This suggests a rate-limiting function of the PTN/ALK interaction that may be exploited therapeutically.

Recruitment of insulin receptor substrate-1 and activation of NF-kappaB essential for midkine growth signaling through anaplastic lymphoma kinase.

Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase in the insulin receptor superfamily. We recently demonstrated that the growth factors pleiotrophin (PTN) and midkine (MK) are ligands for ALK and that upon ALK activation, insulin receptor substrate-1 (IRS-1) and other substrates are phosphorylated. Here, the role of IRS-1 in ligand-mediated ALK signaling is investigated in interleukin-3 (IL-3)-dependent 32D murine myeloid cells. These cells do not express ALK and IRS family members, and do not respond to exogenously added PTN or MK. We show that expression of ALK plus IRS-1 renders these cells independent of IL-3 owing to the activation of ALK by endogenous MK. Mutational analysis reveals that this transformed phenotype of 32D cells requires kinase-active ALK as well as the interaction of ALK with IRS-1. Furthermore, 32D/IRS-1/ALK cells display an enhanced activation of mitogen-activated protein kinase and PI3-kinase pathways, and a selective transcriptional activation of nuclear factor (NF)-kappaB. Small interfering RNA-mediated knockdown of the endogenous MK or p65/NF-kappaB revealed that both these are rate limiting for the transformed phenotype induced by ALK plus IRS-1. We conclude that the recruitment of IRS-1 to activated ALK and the activation of NF-kappaB are essential for the autocrine growth and survival signaling of MK.

Ribozyme targeting of the growth factor pleiotrophin in established tumors: a gene therapy approach.

The growth and metastasis of solid tumors relies on the activities of polypeptide growth factors to recruit stromal tissue and expand the tumor mass. Pleiotrophin (PTN) is a secreted growth factor with angiogenic activity that has been found to contribute to the growth and metastasis of tumors including melanoma. Here, we present a gene therapy approach of targeting PTN in established tumors using ribozymes. Tetracycline-regulated ribozyme expression vectors were used to deplete conditionally PTN mRNA from melanoma xenograft tumors in vivo. We found that tetracycline-mediated initiation of ribozyme expression in established tumors reduced further tumor growth. Next, we generated synthetic anti-PTN ribozymes that inhibit PTN-dependent colony formation of cells in soft agar. Intraperitoneal administration of these synthetic ribozymes into nude mice inhibited growth of PTN-positive, subcutaneous melanoma. Furthermore, PTN released from the tumors into the circulation of mice was reduced after ribozyme treatment. These data show that ribozyme targeting of rate-limiting tumor growth factors could provide an efficient tool for cancer therapy and that the efficacy may be reflected in the reduction of the serum levels of the targeted protein, PTN.

Expression of the nuclear coactivator AIB1 in normal and malignant breast tissue.

The gene of the nuclear receptor coactivator AIB1 (amplified in breast cancer 1) is amplified in breast cancer cell lines as well as in breast tumor tissues. AIB1 mRNA is often highly expressed (>60%) in primary breast tumors and it has been shown that AIB1 enhances estrogen and progesterone dependent transcription in vitro. Therefore, it has been postulated that AIB1 contributes to the development of breast cancer. However, to date, it has not been shown that AIB1 amplification and overexpression correlates with elevated protein levels in breast cancer tissues. In this study we analyzed protein levels of AIB1 in normal and breast tumor tissues by immunohistochemistry. We compared 41 human breast tumor tissues with 24 normal breast tissue samples and found that AIB1 stained in the nuclei of approximately 46% of the tumors and 30% of the normal tissues. Overall, AIB1 protein levels were significantly higher in tumor tissue than in normal tissue and the highest levels of nuclear staining were found exclusively in breast tumor tissues in 9.8% of the cases. These data suggest that increased AIB1 mRNA expression does not always translate into elevated protein levels and that AIB1 most likely will be relevant to the etiology of a subset of about 10% of breast carcinomas.

An isoform of the coactivator AIB1 that increases hormone and growth factor sensitivity is overexpressed in breast cancer.

The AIB1 (amplified in breast cancer 1) protein is a coactivator that potentiates the transcriptional activity of nuclear hormone receptors, and its gene is amplified in a subset of human breast cancers. Here we report a splice variant of AIB1 mRNA that lacks the exon 3 sequence. We determined that the AIB-Delta3 mRNA encoded a 130-kDa protein that lacks the NH(2)-terminal basic helix-loop-helix and a portion of the PAS (Per-Arnt-Sim homology) dimerization domain. The 130-kDa protein was detected in MCF-7 breast cancer cells at levels that were 5-10% of the full-length protein, whereas in non-transformed mammary epithelium lines, the AIB-Delta3 protein was present at significantly lower levels compared with the full-length AIB1. Consistent with this finding, the abundance of AIB1-Delta3 mRNA was increased in human breast cancer specimens relative to that in normal breast tissue. To determine whether there were phenotypic changes associated with the overexpression of the AIB-Delta3 isoform, we performed functional reporter gene assays. These revealed that the ability of AIB1-Delta3 to promote transcription mediated by the estrogen or progesterone receptors was significantly greater than that of the full-length protein. Surprisingly, the AIB1-Delta3 isoform was also more effective than AIB1 in promoting transcription induced by epidermal growth factor. Overexpression of AIB1-Delta3 may thus play an important role in sensitizing breast tumor cells to hormone or growth factor stimulation.

Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells.

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified in breast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor alpha or progesterone receptor beta on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.

Identification of anaplastic lymphoma kinase as a receptor for the growth factor pleiotrophin.

Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.

Differential regulation of a fibroblast growth factor-binding protein by receptor-selective analogs of retinoic acid.

We have demonstrated earlier that a secreted fibroblast growth factor-binding protein (FGF-BP) can enhance angiogenesis and promote tumor growth in vivo. Furthermore, we found that FGF-BP expression in squamous cell carcinoma (SCC) is reduced by concentrations of retinoids that are effective in the treatment of SCC and that this repression can occur at the transcriptional and post-transcriptional level. To further examine the mechanism of regulation of FGF-BP by retinoids and the role played by retinoid receptor subtypes, we utilized retinoic acid receptor (RAR)-selective (TTNPB) and retinoid X receptor (RXR)-selective (LG100268) ligands. In ME-180 SCC cells, FGF-BP mRNA was down-regulated by TTNPB with an IC(50) value of 1 nM, whereas transcription was only repressed at 10,000-fold higher concentrations (IC(50) > 10 microM). This suggests that the major effects of retinoids on FGF-BP occur at the post-transcriptional level. In four additional SCC cell lines, FGF-BP was also down-regulated by TTNPB with IC(50) values of

Effects of antiandrogens on chromatin remodeling and transcription of the integrated mouse mammary tumor virus promoter.

Inhibition of the ligand-activated androgen receptor (AR) by antiandrogens plays an important role in the treatment of various hyperandrogenic disorders including prostate cancer. However, the molecular mechanisms of antiandrogen activity in vivo remain unclear. In this study we analyzed the effects of cyproterone acetate (CPA), flutamide (F), and hydroxyflutamide (OHF) on transcriptional activation and chromatin remodeling of the genomically integrated mouse mammary tumor virus (MMTV) promoter. This promoter has provided an excellent model system to study the impact of steroid hormones on transcriptional activation in the context of a defined chromatin structure. The MMTV hormone response element is positioned on a phased nucleosome, which becomes remodeled in response to steroids. We utilized this model system in mouse L-cell fibroblasts that contain a stably integrated MMTV promoter. In these cells, dihydrotestosterone (DHT) induced a large increase of AR protein levels that correlated with transcriptional activation and chromatin remodeling of the MMTV promoter. Coadministration of DHT and CPA or DHT and OHF in these cells inhibited the increase of AR levels, which resulted in a strong blockage of transcriptional activation and chromatin remodeling of the MMTV promoter. In contrast, F had no significant influence on these activities. We conclude that a major portion of the antiandrogenic effects of CPA and OHF in vivo are mediated by the reduction of AR levels.

Influence of the human endogenous retrovirus-like element HERV-E.PTN on the expression of growth factor pleiotrophin: a critical role of a retroviral Sp1-binding site.

Germ line insertion of a human endogenous retrovirus-like element (HERV-E.PTN) into the growth factor pleiotrophin (PTN) gene generated a phylogenetically new promoter driving the expression of functional HERV-PTN fusion transcripts. Here we show by in situ hybridization, that HERV-PTN fusion transcripts are expressed in malignant trophoblasts (i.e. choriocarcinoma) and in the proliferative and in the invasive trophoblasts of gestational trophoblastic tissue. Additionally, a 1.9 kb fragment of the HERV-derived PTN promoter was analysed which has strong activity when transiently transfected into choriocarcinoma JEG-3 cells in contrast to HeLa cells. Deletion of the retrovirally-derived promoter portion abolished its activity and an enhancer (+443 to +486) was identified in this region. Electrophoretic mobility shift and supershift experiments identified a Sp1 binding site in this enhancer and site specific mutation of this site abolished its activity in choriocarcinoma cells. Sp1 overexpression in Drosophila SL2 cells showed that the enhancer activity is mediated via Sp1 binding in vivo. Furthermore, mutation of the Sp1 binding site reduced the activity of a promoter test fragment in choriocarcinoma cells by 80%. Our result shows that a retroviral Sp1 binding site in the PTN promoter is important for the expression of growth factor pleiotrophin in human choriocarcinoma cells. Oncogene (2000) 19, 3988 - 3998.

Mitogen-induced expression of the fibroblast growth factor-binding protein is transcriptionally repressed through a non-canonical E-box element.

The fibroblast growth factor-binding protein (FGF-BP) stimulates FGF-2-mediated angiogenesis and is thought to play an important role in the progression of squamous cell, colon, and breast carcinomas. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induction of the FGF-BP gene occurs through transcriptional mechanisms involving Sp1, AP-1, and CCAATT/enhancer-binding protein sites in the proximal FGF-BP gene promoter. The level of TPA induction, however, is limited due to the presence of a repressor element that shows similarity to a non-canonical E-box (AACGTG). Mutation or deletion of the repressor element led to enhanced induction by TPA or epidermal growth factor in cervical squamous cell and breast carcinoma cell lines. Repression was dependent on the adjacent AP-1 site, without discernible alteration in the binding affinity or composition of AP-1. We investigated the following two possible mechanisms for E-box-mediated repression: 1) CpG methylation of the core of the E-box element, and 2) binding of a distinct protein complex to this site. Point mutation of the CpG methylation site in the E-box showed loss of repressor activity. Conversely, in vitro methylation of this site significantly reduced TPA induction. In vitro gel shift analysis revealed distinct and TPA-dependent binding of USF1 and USF2 to the repressor element that required nucleotides within the E-box. Furthermore, chromatin immunoprecipitation assay showed that USF, c-Myc, and Max proteins were associated with the FGF-BP promoter in vivo. Overall, these findings suggested that the balance between trans-activation by AP-1 and repression through the E-box is an important control mechanism for fine-tuning the angiogenic response to growth factor-activated pathways.

Induction of the angiogenic modulator fibroblast growth factor-binding protein by epidermal growth factor is mediated through both MEK/ERK and p38 signal transduction pathways.

Fibroblast growth factor-binding protein (FGF-BP) is a secreted protein that binds and activates fibroblast growth factors (FGF-1 and FGF-2) and induces angiogenesis in some human cancers. FGF-BP is expressed at high levels in squamous cell carcinoma (SCC) cell lines and tumor samples and has been shown to be rate-limiting for the growth of SCC tumors in vivo. In this study, we examine the regulation of FGF-BP by epidermal growth factor (EGF) and the signal transduction mechanisms that mediate this effect. We found that EGF treatment of the ME-180 SCC cell line caused a rapid induction of FGF-BP gene expression. This induction was mediated transcriptionally through the AP-1 (c-Fos/JunD) and CCAAT/enhancer-binding protein elements as well as through an E-box repressor site in the proximal regulatory region of the FGF-BP promoter. Pharmacological inhibition of protein kinase C and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) completely blocked EGF induction of FGF-BP mRNA, whereas inhibition of phosphatidylinositol 3-kinase had no effect. Additionally, both EGF- and anisomycin-induced FGF-BP mRNA was abrogated by inhibition of p38 mitogen-activated protein kinase, demonstrating a role for p38 in the regulation of FGF-BP. Co-transfection of the FGF-BP promoter with dominant negative forms of MEK2, extracellular signal-regulated kinase 2, and p38 significantly decreased the level of EGF induction, whereas expression of a dominant negative c-Jun N-terminal kinase mutant or expression of c-Jun N-terminal kinase inhibitory protein had no effect. Similarly, activation of the p38 pathway by overexpression of wild-type p38 or MKK6 enhanced FGF-BP transcription. These results demonstrate that EGF induction of FGF-BP occurs selectively through dual activation of the stress-activated p38 and the MEK/extracellular signal-regulated kinase mitogen-activated protein kinase pathways, which ultimately leads to activation of the promoter through AP-1 and CCAAT/enhancer-binding protein sites.

Ionizing radiation stimulates octamer factor DNA binding activity in human carcinoma cells.

In mammalian cells, the octamer motif (ATGCAAAT) binding proteins, Oct-1 and Oct-2, play an important role in the transcriptional transactivation of several ubiquitously expressed genes as well as cell-specifically expressed genes. To date, a role of the octamer binding proteins in damage-stimulated response is not known. In this report, we demonstrate that DNA-binding activity of Oct-1, as demonstrated by the electrophoretic mobility shift assay, is significantly induced in a dose-dependent manner upon treatment of human head and neck squamous carcinoma cells (PCI-04A) with ionizing radiation (5 Gy: 5-fold; 15 Gy: 11-fold). By comparison, activities of other transcription factors were modestly increased (15 Gy: AP-1, 2.5-fold; NF-kappaB, 2.6-fold; SP-1, 5-fold). Radiation stimulation of Oct-1 activity was also noted in two other human cancer cell lines, albeit to a lesser extent (MDA-MB231 breast carcinoma cells and PC-3 prostate carcinoma cells (5 Gy: approximately 2-fold). These data represent the first report of the activation of an octamer factor DNA binding activity in response to environmental cues and suggest a novel role of Oct-1 in the radiation signaling cascade in these cancer cells.

Inhibition of histone deacetylation augments dihydrotestosterone induction of androgen receptor levels: an explanation for trichostatin A effects on androgen-induced chromatin remodeling and transcription of the mouse mammary tumor virus promoter.

The integrated mouse mammary tumor virus (MMTV) promoter has provided an excellent model system with which to study the impact of steroid hormones on transcriptional activation in the context of a defined chromatin structure. The hormone response element (HRE) of this promoter is positioned on a phased nucleosome which becomes remodeled in response to steroids. One possible mechanism of chromatin remodeling by steroid receptors could involve recruitment of coactivators which alter the histone acetylation status of the HRE nucleosome. To examine how the androgen receptor (AR) influences transcription and chromatin remodeling and to assess whether changes in histone acetylation are involved in these effects, we determined whether the specific histone deacetylase inhibitor trichostatin A (TSA) influenced basal- and androgen-mediated transcriptional activation of the integrated MMTV promoter in the mouse L-cell fibroblast cell line 29+. These cells harbor the MMTV promoter integrated in the genome and express only one steroid hormone receptor subtype, i.e., the AR. Surprisingly, we found that treatment of the cells with TSA alone had virtually no effect on transcription and chromatin remodeling of the MMTV promoter nor on AR levels. However, pretreatment with TSA augmented the DHT effects on all three parameters. These results suggest that histone acetylation changes at the MMTV B nucleosome per se are not alone sufficient to induce chromatin remodeling and subsequent induction of MMTV transcription. Rather, the histone deacetylase inhibitor TSA exerts a portion of its effect on MMTV chromatin remodeling and transcriptional activation indirectly through increases in AR levels.

Comparison of chromatin remodeling and transcriptional activation of the mouse mammary tumor virus promoter by the androgen and glucocorticoid receptor.

We examined the interaction between the androgen (AR) and glucocorticoid receptor (GR) at the transcriptional level using mouse fibroblast cell lines harboring an integrated mouse mammary tumor virus (MMTV) promoter. We found that the AR, after induction with dihydrotestosterone (DHT), caused a progressive increase in MMTV-CAT reporter activity over 72 h which was correlated to an increase in chromatin remodeling of the MMTV promoter in the vicinity of the hormone response element (HRE). In contrast, stimulation of the GR by the synthetic glucocorticoid dexamethasone (Dex) caused a transient increase in MMTV transcriptional activity which returned to basal levels after 72 h. These changes were correlated to a transient increase in chromatin remodeling in the region of the HRE. Neither cotreatment nor pretreatment with Dex affected the DHT response. In fact, there was a more than additive effect of the two hormones on transcription at early time points. This suggests that the inability of GR to remodel chromatin, after 24 h of hormone treatment, is most likely related to changes in the GR itself and not the chromatin remodeling process. Consistent with this, nuclear GR levels dropped by greater than 50% after Dex treatment whereas the AR was induced fourfold after 24 h of DHT treatment. We conclude that a promoter with an ordered chromatin structure can still respond to androgens even after its glucocorticoid responsiveness is lost. This may be one mechanism cells utilize to establish target gene specificity for nuclear receptors that recognize identical DNA sequences.

Phorbol ester-induced transcription of a fibroblast growth factor-binding protein is modulated by a complex interplay of positive and negative regulatory promoter elements.

Earlier studies from our laboratory showed that a secreted binding protein for fibroblast growth factors (FGF-BP) is expressed at high levels in squamous cell carcinoma (SCC) cell lines. Overexpression studies or conversely reduced expression of FGF-BP by ribozyme targeting have elucidated a direct role of this protein in angiogenesis during tumor development. We have also observed a significant up-regulation of FGF-BP during TPA (12-O-tetradecanoylphorbol-13-acetate) promotion of skin cancer. Here we investigate the mechanism of TPA induction of FGF-BP gene expression in the human ME-180 SCC cell line. We found that TPA increased FGF-BP mRNA levels in a time- and dose-dependent manner mediated via the protein kinase C signal transduction pathway. Results from actinomycin D and cycloheximide experiments as well as nuclear transcription assays revealed that TPA up-regulated the steady-state levels of FGF-BP mRNA by increasing its rate of gene transcription independently of de novo protein synthesis. We isolated the human FGF-BP promoter and determined by deletion analysis that TPA regulatory elements were all contained in the first 118 base pairs upstream of the transcription start site. Further mutational analysis revealed that full TPA induction required interplay between several regulatory elements with homology to Ets, AP-1, and CAATT/enhancer binding protein C/EBP sites. In addition, deletion or mutation of a 10-base pair region juxtaposed to the AP-1 site dramatically increased TPA induced FGF-BP gene expression. This region represses the extent of the FGF-BP promoter response to TPA and contained sequences recognized by the family of E box helix-loop-helix transcription factors. Gel shift analysis showed specific and TPA-inducible protein binding to the Ets, AP-1, and C/EBP sites. Furthermore, distinct, specific, and TPA-inducible binding to the imperfect E box repressor element was also apparent. Overall, our data indicate that TPA effects on FGF-BP gene transcription are tightly controlled by a complex interplay of positive elements and a novel negative regulatory element.