Pin1 modulates ERα levels in breast cancer through inhibition of phosphorylation-dependent ubiquitination and degradation.

Estrogen receptor-alpha (ERα) is an important biomarker used to classify and direct therapy decisions in breast cancer (BC). Both ERα protein and its transcript, ESR1, are used to predict response to tamoxifen therapy, yet certain tumors have discordant levels of ERα protein and ESR1, which is currently unexplained. Cellular ERα protein levels can be controlled post-translationally by the ubiquitin-proteasome pathway through a mechanism that depends on phosphorylation at residue S118. Phospho-S118 (pS118-ERα) is a substrate for the peptidyl prolyl isomerase, Pin1, which mediates cis-trans isomerization of the pS118-P119 bond to enhance ERα transcriptional function. Here, we demonstrate that Pin1 can increase ERα protein without affecting ESR1 transcript levels by inhibiting proteasome-dependent receptor degradation. Pin1 disrupts ERα ubiquitination by interfering with receptor interactions with the E3 ligase, E6AP, which also is shown to bind pS118-ERα. Quantitative in situ assessments of ERα protein, ESR1, and Pin1 in human tumors from a retrospective cohort show that Pin1 levels correlate with ERα protein but not to ESR1 levels. These data show that ERα protein is post-translationally regulated by Pin1 in a proportion of breast carcinomas. As Pin1 impacts both ERα protein levels and transactivation function, these data implicate Pin1 as a potential surrogate marker for predicting outcome of ERα-positive BC.

Proteasome inhibition represses ERalpha gene expression in ER+ cells: a new link between proteasome activity and estrogen signaling in breast cancer.

Estrogen receptor-alpha (ERalpha) is a major therapeutic target of hormonal therapies in breast cancer, and its expression in tumors is predictive of clinical response. Protein levels of ERalpha are tightly controlled by the 26S proteasome; yet, how the clinical proteasome inhibitor, bortezomib, affects ERalpha regulation has not been studied. Bortezomib selectively inhibits the chymotrypsin-like activity of the proteasome. Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERalpha protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity. Unexpectedly, however, chronic bortezomib exposure caused a reduction of ERalpha levels in multiple ER+ breast cancer cell lines. This response can be explained by the fact that bortezomib induced a dramatic decrease in ERalpha mRNA because of direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERalpha gene promoter. Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related with occupancy of ERalpha and RNA polymerase II (PolII) on endogenous promoters. In addition, bortezomib inhibited estrogen-dependent growth in soft agar. These results reveal a novel link between proteasome activity and expression of ERalpha in breast cancer and uncover distinct roles of the chymotrypsin-like activity of the proteasome in the regulation of the ERalpha pathway.

Interplay between the levels of estrogen and estrogen receptor controls the level of the granzyme inhibitor, proteinase inhibitor 9 and susceptibility to immune surveillance by natural killer cells.

Estrogens promote cell proliferation and metastases in several human cancers. Here, we describe a different action of estrogens likely to contribute to tumor development-blocking immunosurveillance. In breast cancer cells, increasing concentrations of estrogen induce increasing levels of the granzyme B inhibitor, SerpinB9/proteinase inhibitor 9 (PI-9) and progressively block cell death induced by NK92 natural killer (NK) cells, but do not block killing by a second NK cell line, NKL cells. RNA interference knockdown of PI-9 abolishes estrogen's ability to block NK92 cell-induced cytotoxicity. Expressing elevated levels of estrogen receptor alpha (ERalpha) increases the induced level of PI-9, and makes tamoxifen (TAM), but not raloxifene or ICI 182,780, a potent inducer of PI-9. At elevated levels of ERalpha, induction of PI-9 by estradiol or TAM blocks killing by both NK92 and NKL cells. When the Erk pathway is activated with epidermal growth factor, the concentration of estrogen required to induce a protective level of PI-9 is reduced to 10 pM. Elevated concentrations of estrogen and ER may provide a dual selective advantage to breast cancer cells by controlling PI-9 levels and thereby blocking immunosurveillance. Expressing elevated levels of ERalpha reveals a potentially important difference in the effects of TAM, raloxifene and ICI 182,780 on immunosurveillance in breast cancer.

Proteasome-mediated proteolysis of estrogen receptor: a novel component in autologous down-regulation.

Regulation of estrogen receptor (ER) concentration is a key component in limiting estrogen responsiveness in target cells. Yet the mechanisms governing ER concentration in the lactotrope cells of the anterior pituitary, a major site of estrogen action, are undetermined. In this study, we used a lactotrope cell line, PR1, to explore regulation of ER protein by estrogen. Estrogen treatment resulted in an approximate 60% decrease in ER steady state protein levels. Suprisingly, the decline in ER protein was apparent within 1 h of estrogen treatment and occurred in the absence of protein synthesis and transcription. Direct regulation of ER protein was further confirmed by pulse chase analysis, which showed that ER protein half-life was shortened from greater than 3 h to 1 h in the presence of estrogen. The estrogen-induced degradation of ER protein could be prevented by pretreatment with peptide aldehyde inhibitors of proteasome protease whereas inhibitors of calpain and lysosomal proteases were ineffective. Inhibition of proteasome activity maintained ER protein at a level equivalent to control cells not stimulated with estrogen but increased estrogen-binding activity by 1.75-fold. Proteolytic regulation of ER by the proteasome is not limited to pituitary lactotrope cells but is also operational in MCF-7 breast cancer cells, suggesting that this may be a common regulatory pathway used by estrogen. These studies describe a nongenomic action of estrogen that involves nuclear ER: rapid proteolysis of ER protein via a proteasome-mediated pathway.

The thyrotropin beta-subunit gene is repressed by thyroid hormone in a novel thyrotrope cell line, mouse T alphaT1 cells.

TSH is expressed in two populations of thyrotropes in the pituitary: one in the pars distalis and a second in the pars tuberalis. Pars distalis thyrotropes exhibit classical endocrine inhibition of TSH by thyroid hormone, whereas pars tuberalis thyrotropes do not. The majority of our understanding of TSH subunit gene regulation has come from studies conducted in dispersed pituitary, dispersed thyrotropic tumors, or the GH3 somatolactotrope cell line. However, the dispersed pituitary model is limited because of its inherent heterogeneity, thyrotropic tumors are difficult to grow and maintain, and the GH3 cells lack endogenous TSH expression. The recent derivation of a clonal thyrotrope cell line, T alphaT1, that expresses thyrotrope-specific markers, overcomes these limitations. However, because it was not possible to distinguish whether the tumor from which the T alphaT1 cells are derived originated in the pars distalis or the pars tuberalis, it was necessary to define their cellular origin and thereby establish their status as representative thyrotrope cells for future molecular studies. In this study, we demonstrate that the T alphaT1 cells express thyroid hormone receptors (beta1 and beta2) and their heterodimeric partner, retinoid X receptor-gamma. Treatment with T3 causes a dose- and time-dependent decrease in the expression of the TSH beta-subunit messenger RNA. In contrast to previous reports in rat pituitary cultures, T3 does not alter TSH beta-subunit messenger RNA stability in the T alphaT1 cells. Based on these data and the presence of thyrotrope-specific isoforms of the transcription factor Pit-1, we conclude that the T alphaT1 cells represent differentiated thyrotropes of the pars distalis and will be a useful model system for future analysis of the cis- and trans-acting factors necessary for thyrotrope-specific and thyroid hormone-regulated TSH gene expression.

Discrete stages of anterior pituitary differentiation recapitulated in immortalized cell lines.

Targeted expression of oncogenes in transgenic mice can immortalize specific cell types to serve as valuable cultured model systems. Utilizing promoter regions from pituitary genes activated in the gonadotrope/thyrotrope lineage at discrete stages of development, we have demonstrated that targeted oncogene expression in transgenic mice can produce cell lines representing sequential stages of differentiation. Each cell line expresses a specific subset of the genes denoting differentiated function such as the subunits of the glycoprotein hormones, hormone receptors, and transcriptional regulatory proteins. These model systems have allowed detailed investigations into molecular and cellular mechanisms otherwise inaccessible in vivo or in complex primary pituitary cell cultures.

Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice.

Targeted expression of oncogenes in transgenic mice can immortalize specific cell types to serve as valuable cultured model systems. Utilizing promoter regions from a set of genes expressed at specific stages of differentiation in a given cell lineage, we demonstrate that targeted oncogenesis can produce cell lines representing sequential stages of development, in essence allowing both spatial and temporal immortalization. Our strategy was based on our production of a committed but immature pituitary gonadotrope cell line by directing expression of the oncogene SV40 T antigen using a gonadotrope-specific region of the human glycoprotein hormone alpha-subunit gene in transgenic mice. These cells synthesize alpha-subunit and gonadotropin-releasing hormone (GnRH) receptor, yet are not fully differentiated in that they do not synthesize the beta-subunits of luteinizing hormone (LH) or follicle-stimulating hormone (FSH). This observation lead to the hypothesis that targeting oncogenesis with promoters that are activated earlier or later in development might immortalize cells that were more primitive or more differentiated, respectively. To test this hypothesis, we used an LHbeta promoter to immortalize a cell that represents a subsequent stage of gonadotrope differentiation (expression of alpha-subunit, GnRH receptor, and LH beta-subunit but not FSH beta-subunit). Conversely, targeting oncogenesis with a longer fragment of the human alpha-subunit gene (which is activated earlier in development) resulted in the immortalization of a progenitor cell that is more primitive, expressing only the alpha-subunit gene. Interestingly, this transgene also immortalized cells of the thyrotrope lineage that express both alpha- and beta-subunits of thyroid-stimulating hormone and the transcription factor GHF-1 (Pit-1). Thus, targeted tumorigenesis immortalizes mammalian cells at specific stages of differentiation and allows the production of a series of cultured cell lines representing sequential stages of differentiation in a given cell lineage.

Down-regulation of the gonadotropin-releasing hormone receptor messenger ribonucleic acid by activation of adenylyl cyclase in alpha T3-1 pituitary gonadotrope cells.

Pituitary regulation of reproductive processes depends on the sensitivity of gonadotrope cells to both positive and negative regulators. Hypothalamic GnRH is the primary stimulus for gonadotropin synthesis and secretion. Therefore, the ability of the gonadotrope to respond to GnRH and the status of GnRH receptors (GnRH-R) are critical in the control of reproduction. In the present study, we address the role of GnRH and two second messenger activators, a phorbol ester (12-O-tetradecanoylphorbol-13-acetate) and forskolin, in the regulation of GnRH-R gene expression in the alpha T3-1 gonadotrope cell line. Using Northern blot analysis to monitor endogenous GnRH-R steady state messenger RNA (mRNA) levels, we found that although GnRH and 12-O-tetradecanoylphorbol-13-acetate do not change GnRH-R mRNA levels, forskolin causes a time-dependent decline. All three treatments stimulate glycoprotein alpha-subunit gene expression. To dissect the molecular mechanism of forskolin action on GnRH-R gene expression, de novo RNA synthesis was inhibited with the transcription inhibitor, actinomycin-D (act-D). Act-D alone does not change GnRH-R message levels. However, in the presence of both act-D and forskolin, GnRH-R mRNA levels decline dramatically. These data demonstrate that forskolin alters GnRH-R posttranscriptionally by destabilizing its mRNA. Our data do not, however, exclude possible direct transcriptional effects. This study suggests that activators of the protein kinase-A pathway may alter gonadotrope sensitivity to GnRH by decreasing GnRH-R gene expression and, therefore, indirectly affect reproductive status.

Keratinocyte growth factor functions in epithelial induction during seminal vesicle development.

Development of the seminal vesicle (SV) is elicited by androgens and is dependent on epithelial-mesenchymal interactions. Androgenic signal transmission from the androgen-receptor-positive mesenchyme to the epithelium has been postulated to involve paracrine factors. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is produced by stromal/mesenchymal cells and acts specifically on epithelial cells. The KGF transcript was detected by reverse transcription-polymerase chain reaction in newborn mouse SVs and by Northern blot analysis of RNA from cultured neonatal SV mesenchymal cells. Newborn SVs placed in organ culture undergo androgen-dependent growth and differentiation. Addition of a KGF-neutralizing monoclonal antibody to this system caused striking inhibition of both SV growth and branching morphogenesis. This inhibition was due to a decline in epithelial proliferation and differentiation, as the mesenchymal layer was not affected by anti-KGF treatment. When KGF (100 ng/ml) was substituted for testosterone in the culture medium, SV growth was approximately 50% that observed with an optimal dose of testosterone (10(-7) M). All of these findings suggest that KGF is present during a time of active SV morphogenesis and functions as an important mediator of androgen-dependent development.

Differential effects of an antiserum to epidermal growth factor on the development of transplanted rat embryos and fetal structures in vivo.

In order to obtain information on the possible role of epidermal growth factor (EGF) in rat prenatal development, we tested the effects of a neutralizing antiserum to rat EGF and of recombinant human EGF on the growth and development of transplanted rat embryos and fetal structures. Ten-day embryos or 16-day fetal intestines (ileum and jejunum) or paws were transplanted under the capsule of both kidneys of young adult syngeneic host rats. Osmotic minipumps were used to infuse antiserum to rat EGF or normal rabbit serum (NRS) into the renal artery of the right kidney to ascertain direct effects on development. The transplants on the contralateral side served as internal controls. Infusion of the NRS did not affect growth of any of the fetal structures or of the embryo transplants. The antiserum to rEGF did not affect growth of the fetal ileum or paw transplants, but it inhibited growth of the fetal jejunum by 38%, and suppressed differentiation of hair follicles in the paws by approximately 90%. Tissue differentiation in the two segments of the intestine was unaffected by the antiserum. By contrast, growth of embryo transplants was stimulated by approximately 60% by the anti-EGF serum. Infusion of antiserum did not affect the growth of the kidneys upon which the transplants were grown, and infusion of different doses of recombinant human EGF had no effect on growth of embryo transplants. Our data suggest that EGF may function as a negative growth regulator during the embryonic period, but it becomes a growth stimulator for specific tissues during the fetal period.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal and abnormal development of the male urogenital tract. Role of androgens, mesenchymal-epithelial interactions, and growth factors.

Androgen-dependent male urogenital development occurs via mesenchymal-epithelial interactions in which mesenchyme induces epithelial morphogenesis, regulates epithelial proliferation, and evokes expression of tissue-specific secretory proteins. Mesenchymal-epithelial interactions continue to be important into adulthood. For example, mesenchyme of the urogenital sinus (UGM) and seminal vesicle (SVM) induce dramatic morphologic and functional changes in various adult epithelia. Since adult epithelial cells are unquestionably responsive to mesenchymes that can elicit expression of alternative morphologic and functional phenotypes, established carcinomas might also be influenced by their connective tissue environment. In this regard, Dunning prostatic tumor has been induced by UGM or SVM to differentiate into tall columnar secretory epithelial cells. This change in cytodifferentiation is associated with a reduction in growth rate and loss of tumorigenesis. The role of soluble growth factors in the mechanism of mesenchymal-epithelial interactions is discussed.

Evidence suggesting that insulin-like growth factor-I is necessary for the trophic effects of insulin on cartilage growth in vivo.

The possibility that insulin-stimulated cartilage growth in hypophysectomized rats involves local production of insulin-like growth factor I (IGF-I) was investigated. Osmotic minipumps with attached catheters were used to infuse insulin into the right hindlimb of rats via the common iliac artery for 7 days starting 14 days after pituitary removal. The left, noninfused limb served as an internal control. Doses of insulin ranging from 0.25 to 50 mU/day caused significant increases in the tibial epiphyseal plate width (TEPW) of the infused limbs. The optimal dose of 1.25 mU/day increased the TEPW by 51 microns. Immunohistochemical analysis with an antiserum to human IGF-I showed that the growth response to the optimal dose was accompanied by the accumulation of IGF-I in the chondrocytes in the tibial plate. Infusion of insulin at a dose of 10 mU/day increased the TEPW by 18.6 +/- 3.0 microns. When the same dose of insulin was infused with the antiserum to human IGF-I, the growth response was completely nullified. These results indicate that cartilage cells in hypophysectomized rats are highly sensitive to the direct growth-promoting action of insulin. Furthermore, the growth effect appears to be mediated by or dependent on local production of IGF-I.

Evidence for an organ- and sex-specific role of basic fibroblast growth factor in the development of the fetal mammalian reproductive tract.

Basic fibroblast growth factor (bFGF) has been implicated as an embryonic mesoderm-inducing factor. It has also been detected in the mesodermally derived fetal and adult reproductive tracts. To address its possible role in the development of the reproductive tract, male and female fetal genital ridges and urogenital (UG) sinuses were transplanted bilaterally under the kidney capsule of syngeneic host rats; the transplants and the hosts were the same sex. Antiserum to bFGF (anti-bFGF) or normal rabbit serum (NRS) was infused into the right renal artery. The transplants on the left uninfused kidney served as internal controls. Upon vascularization, the transplants grow and differentiate essentially normally, but their overall growth after 14 days was reduced by about 40% compared to their growth in situ. Neutralization of endogenous bFGF with anti-bFGF resulted in approximately 41% inhibition of growth in the male genital ridge, but had no effect on the male UG sinus or any component of the female reproductive tract. The growth suppression of the male genital ridge was accompanied by an inhibition of the differentiation of the epididymis. After 7 days of treatment, development of the epididymis was impaired by 75%, and by 14 days, the organ was absent. Previous studies of fetal reproductive tract development have focused on the role of steroids and Mullerian inhibiting hormone. Our studies suggest that bFGF plays a role in the development of the male reproductive tract, especially the epididymis, and further suggest the possible role of this peptide growth factor in a steroid hormone-dependent process.