Patients with hypercortisolemic Cushing disease possess a distinct class of hematopoietic progenitor cells leading to erythrocytosis.

Although human cell cultures stimulated with dexamethasone suggest that the glucocorticoid receptor (GR) activates stress erythropoiesis, the effects of GR activation on erythropoiesis in vivo remain poorly understood. We characterized the phenotype of a large cohort of patients with Cushing disease, a rare condition associated with elevated cortisol levels. Results from hypercortisolemic patients with active Cushing disease were compared with those obtained from eucortisolemic patients after remission and from volunteers without the disease. Patients with active Cushing disease exhibited erythrocytosis associated with normal hemoglobin F levels. In addition, their blood contained elevated numbers of GR-induced CD163+ monocytes and a unique class of CD34+ cells expressing CD110, CD36, CD133 and the GR-target gene CXCR4. When cultured, these CD34+ cells generated similarly large numbers of immature erythroid cells in the presence and absence of dexamethasone, with raised expression of the GR-target gene GILZ. Of interest, blood from patients with Cushing disease in remission maintained high numbers of CD163+ monocytes and, although their CD34+ cells had a normal phenotype, these cells were unresponsive to added dexamethasone. Collectively, these results indicate that chronic exposure to excess glucocorticoids in vivo leads to erythrocytosis by generating erythroid progenitor cells with a constitutively active GR. Although remission rescues the erythrocytosis and the phenotype of the circulating CD34+ cells, a memory of other prior changes is maintained in remission.

Concise Review: Advanced Cell Culture Models for Diamond Blackfan Anemia and Other Erythroid Disorders.

In vitro surrogate models of human erythropoiesis made many contributions to our understanding of the extrinsic and intrinsic regulation of this process in vivo and how they are altered in erythroid disorders. In the past, variability among the levels of hemoglobin F produced by adult erythroblasts generated in vitro by different laboratories identified stage of maturation, fetal bovine serum, and accessory cells as "confounding factors," that is, parameters intrinsically wired in the experimental approach that bias the results observed. The discovery of these factors facilitated the identification of drugs that accelerate terminal maturation or activate specific signaling pathways for the treatment of hemoglobinopathies. It also inspired studies to understand how erythropoiesis is regulated by macrophages present in the erythroid islands. Recent cell culture advances have greatly increased the number of human erythroid cells that can be generated in vitro and are used as experimental models to study diseases, such as Diamond Blackfan Anemia, which were previously poorly amenable to investigation. However, in addition to the confounding factors already identified, improvement in the culture models has introduced novel confounding factors, such as possible interactions between signaling from cKIT, the receptor for stem cell factor, and from the glucocorticoid receptor, the cell proliferation potential and the clinical state of the patients. This review will illustrate these new confounding factors and discuss their clinical translation potential to improve our understanding of Diamond Blackfan Anemia and other erythroid disorders. Stem Cells 2018;36:172-179.

Genetic disarray follows mutant KLF1-E325K expression in a congenital dyserythropoietic anemia patient.

Congenital dyserythropoietic anemia type IV is caused by a heterozygous mutation, Glu325Lys (E325K), in the KLF1 transcription factor. Molecular characteristics of this disease have not been clarified, partly due to its rarity. We expanded erythroid cells from a patient's peripheral blood and analyzed its global expression pattern. We find that a large number of erythroid pathways are disrupted, particularly those related to membrane transport, globin regulation, and iron utilization. The altered genetics lead to significant deficits in differentiation. Glu325 is within the KLF1 zinc finger domain at an amino acid critical for site specific DNA binding. The change to Lys is predicted to significantly alter the target site recognition sequence, both by subverting normal recognition and by enabling interaction with novel sites. Consistent with this, we find high level ectopic expression of genes not normally present in the red cell. These altered properties explain patients' clinical and phenotypic features, and elucidate the dominant character of the mutation.

2p15-p16.1 microdeletions encompassing and proximal to BCL11A are associated with elevated HbF in addition to neurologic impairment.

Elevated fetal hemoglobin (HbF) ameliorates the clinical severity of hemoglobinopathies such as ß-thalassemia and sickle cell anemia. Currently, the only curative approach for individuals under chronic transfusion/chelation support therapy is allogeneic stem cell transplantation. However, recent analyses of heritable variations in HbF levels have provided a new therapeutic target for HbF reactivation: the transcriptional repressor BCL11A. Erythroid-specific BCL11A abrogation is now actively being sought as a therapeutic avenue, but the specific impact of such disruption in humans remains to be determined. Although single nucleotide polymorphisms in BCL11A erythroid regulatory elements have been reported, coding mutations are scarcer. It is thus of great interest that patients have recently been described with microdeletions encompassing BCL11A. These patients display neurodevelopmental abnormalities, but whether they show increased HbF has not been reported. We have examined the hematological phenotype, HbF levels, and erythroid BCL11A expression in 3 such patients. Haploinsufficiency of BCL11A induces only partial developmental γ-globin silencing. Of greater interest is that a patient with a downstream deletion exhibits reduced BCL11A expression and increased HbF. Novel erythroid-specific regulatory elements in this region may be required for normal erythroid BCL11A expression, whereas loss of separate elements in the developing brain may explain the neurological phenotype.

Activation of non-canonical TGF-ß1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by megakaryocyte hyperplasia, dysplasia and death with progressive reticulin/collagen fibrosis in marrow and hematopoiesis in extramedullary sites. The mechanism of fibrosis was investigated by comparing TGF-ß1 signaling of marrow and spleen of patients with PMF and of non-diseased individuals. Expression of 39 (23 up-regulated and 16 down-regulated) and 38 (8 up-regulated and 30 down-regulated) TGF-ß1 signaling genes was altered in the marrow and spleen of PMF patients, respectively. Abnormalities included genes of TGF-ß1 signaling, cell cycling and abnormal in chronic myeloid leukemia (EVI1 and p21(CIP)) (both marrow and spleen) and Hedgehog (marrow only) and p53 (spleen only) signaling. Pathway analyses of these alterations predict an increased osteoblast differentiation, ineffective hematopoiesis and fibrosis driven by non-canonical TGF-ß1 signaling in marrow and increased proliferation and defective DNA repair in spleen. Since activation of non-canonical TGF-ß1 signaling is associated with fibrosis in autoimmune diseases, the hypothesis that fibrosis in PMF results from an autoimmune process triggered by dead megakaryocytes was tested by determining that PMF patients expressed plasma levels of mitochondrial DNA and anti-mitochondrial antibodies greater than normal controls. These data identify autoimmunity as a possible cause of marrow fibrosis in PMF.

Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion.

Cultures of human CD34(pos) cells stimulated with erythroid growth factors plus dexamethasone, a model for stress erythropoiesis, generate numerous erythroid cells plus a few macrophages (approx. 3%; 3:1 positive and negative for CD169). Interactions occurring between erythroblasts and macrophages in these cultures and the biological effects associated with these interactions were documented by live phase-contrast videomicroscopy. Macrophages expressed high motility interacting with hundreds/thousands of erythroblasts per hour. CD169(pos) macrophages established multiple rapid 'loose' interactions with proerythroblasts leading to formation of transient erythroblastic island-like structures. By contrast, CD169(neg) macrophages established 'tight' interactions with mature erythroblasts and phagocytosed these cells. 'Loose' interactions of CD169(pos) macrophages were associated with proerythroblast cytokinesis (the M phase of the cell cycle) suggesting that these interactions may promote proerythroblast duplication. This hypothesis was tested by experiments that showed that as few as 103 macrophages significantly increased levels of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide incorporation frequency in S/G2/M and cytokinesis expressed by proerythroblasts over 24 h of culture. These effects were observed also when macrophages were co-cultured with dexamethasone directly conjugated to a macrophage-specific CD163 antibody. In conclusion, in addition to promoting proerythroblast proliferation directly, dexamethasone stimulates expansion of these cells indirectly by stimulating maturation and cytokinesis supporting activity of macrophages.

The glucocorticoid receptor elicited proliferative response in human erythropoiesis is BCL11A-dependent.

Prior evidence indicates that the erythroid cellular response to glucocorticoids (GC) has developmental specificity, namely, that developmentally more advanced cells that are undergoing or have undergone fetal to adult globin switching are more responsive to GC-induced expansion. To investigate the molecular underpinnings of this, we focused on the major developmental globin regulator BCL11A. We compared: a) levels of expression and nuclear content of BCL11A in adult erythroid cells upon GC stimulation; b) response to GC of CD34+ cells from patients with BCL11A microdeletions and reduced BCL11A expression, and; c) response to GC of two cellular models (HUDEP-2 and adult CD34+ cells) before and after reduction of BCL11A expression by shRNA. We observed that: a) GC-expanded erythroid cells from a large cohort of blood donors displayed amplified expression and nuclear accumulation of BCL11A; b) CD34+ cells from BCL11A microdeletion patients generated fewer erythroid cells when cultured with GC compared to their parents, while the erythroid expansion of the patients was similar to that of their parents in cultures without GC, and; c) adult CD34+ cells and HUDEP-2 cells with shRNA-depleted expression of BCL11A exhibit reduced expansion in response to GC. In addition, RNA-seq profiling of shRNA-BCL11A CD34+ cells cultured with and without GC was similar (very few differentially expressed genes), while GC-specific responses (differential expression of GILZ and of numerous additional genes) were observed only in controls cells with unperturbed BCL11A expression. These data indicate that BCL11A is an important participant of certain aspects of the stress pathway sustained by GC.

The dominant negative ß isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients.

Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro. To clarify the effect of the dominant negative GRß isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GRß was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRß mRNA, had greater frequency in PV (55%; n = 22; P = .0028) and myelofibrosis (35%; n = 20) patients than in NDs (9%; n = 22) or patients with essential thrombocythemia (6%; n = 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and ß-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and ß-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GRα, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GRß. These data indicate that GRß expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents.

Transcriptomic and phospho-proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera.

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRß, TGFß, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera.

A Phase Ib Trial of AVID200, a TGFß 1/3 Trap, in Patients with Myelofibrosis.

PURPOSE: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by systemic symptoms, cytopenias, organomegaly, and bone marrow fibrosis. JAK2 inhibitors afford symptom and spleen burden reduction but do not alter the disease course and frequently lead to thrombocytopenia. TGFß, a pleiotropic cytokine elaborated by the MF clone, negatively regulates normal hematopoiesis, downregulates antitumor immunity, and promotes bone marrow fibrosis. Our group previously showed that AVID200, a potent and selective TGFß 1/3 trap, reduced TGFß1-induced proliferation of human mesenchymal stromal cells, phosphorylation of SMAD2, and collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PC) with wild-type JAK2 rather than JAK2V617F. PATIENTS AND METHODS: We conducted an investigator-initiated, multicenter, phase Ib trial of AVID200 monotherapy in 21 patients with advanced MF. RESULTS: No dose-limiting toxicity was identified at the three dose levels tested, and grade 3/4 anemia and thrombocytopenia occurred in 28.6% and 19.0% of treated patients, respectively. After six cycles of therapy, two patients attained a clinical benefit by IWG-MRT criteria. Spleen and symptom benefits were observed across treatment cycles. Unlike other MF-directed therapies, increases in platelet counts were noted in 81% of treated patients with three patients achieving normalization. Treatment with AVID200 resulted in potent suppression of plasma TGFß1 levels and pSMAD2 in MF cells. CONCLUSIONS: AVID200 is a well-tolerated, rational, therapeutic agent for the treatment of patients with MF and should be evaluated further in patients with thrombocytopenic MF in combination with agents that target aberrant MF intracellular signaling pathways.

Megakaryocytes Are Regulators of the Tumor Microenvironment and Malignant Hematopoietic Progenitor Cells in Myelofibrosis.

Megakaryocytes (MKs) are multifunctional hematopoietic cells that produce platelets, serve as components of bone marrow (BM) niches that support the development of hematopoietic stem and progenitor cell (HSPC) and provide inflammatory signals. MKs can dynamically change their activities during homeostasis and following stress, thereby regulating hematopoietic stem cell (HSC) function. Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm (MPN) characterized by hyperactivation of JAK/STAT signaling and MK hyperplasia, which is associated with an aberrant inflammatory signature. Since JAK1/2 inhibitor alone is incapable of depleting the malignant HSC clones or reversing BM fibrosis, the identification of mechanisms that cooperate with MF JAK/STAT signaling to promote disease progression might help in developing combination therapies to modify disease outcomes. Chronic inflammation and MK hyperplasia result in an abnormal release of TGFß1, which plays a critical role in the pathobiology of MF by contributing to the development of BM fibrosis. Dysregulated TGFß signaling can also alter the hematopoietic microenvironment supporting the predominance of MF-HSCs and enhance the quiescence of the reservoir of wild-type HSCs. Upregulation of TGFß1 levels is a relatively late event in MF, while during the early pre-fibrotic stage of MF the alarmin S100A8/S100A9 heterocomplex promotes pro-inflammatory responses and sustains the progression of MF-HSCs. In this review, we will discuss the recent advances in our understanding of the roles of abnormal megakaryopoiesis, and the altered microenvironment in MF progression and the development of novel combined targeted therapies to disrupt the aberrant interplay between MKs, the BM microenvironment and malignant HSCs which would potentially limit the expansion of MF-HSC clones.

Under HEMA conditions, self-replication of human erythroblasts is limited by autophagic death.

The number of erythroblasts generated ex-vivo under human-erythroid massive-amplification conditions by mononuclear cells from one unit of adult blood (~10(10)) are insufficient for transfusion (~10(12) red cells), emphasizing the need for studies to characterize cellular interactions during culture to increase erythroblast production. To identify the cell populations which generate erythroblasts under human-erythroid-massive-amplification conditions and the factors that limit proliferation, day 10 non-erythroblasts and immature- and mature-erythroblasts were separated by sorting, labelled with carboxyfluorescein-diacetate-succinimidyl-ester and re-cultured either under these conditions (for proliferation, maturation and/or apoptosis/autophagy determinations) or in semisolid media (for progenitor cell determination). Non-erythroblasts contained 54% of the progenitor cells but did not grow under human-erythroid-massive-amplification conditions. Immature-erythroblasts contained 25% of the progenitor cells and generated erythroblasts under human-erythroid-massive-amplification conditions (FI at 48 h=2.57±1.15). Mature-erythroblasts did not generate colonies and died in human-erythroid-massive-amplification conditions. In sequential sorting/re-culture experiments, immature-erythroblasts retained the ability to generate erythroblasts for 6 days and generated 2-5-fold more cells than the corresponding unfractionated population, suggesting that mature-erythroblasts may limit erythroblast expansion. In co-cultures of carboxyfluorescein-diacetate-succinimidyl-ester-labelled-immature-erythroblasts with mature-erythroblasts at increasing ratios, cell numbers did not increase and proliferation, maturation and apoptotic rates were unchanged. However, Acridine Orange staining (a marker for autophagic death) increased from ~3.2% in cultures with immature-erythroblasts alone to 14-22% in cultures of mature-erythroblasts with and without immature-erythroblasts. In conclusion, these data identify immature-erythroblasts as the cells that generate additional erythroblasts in human-erythroid-massive-amplification cultures and autophagy as the leading cause of death limiting the final cellular output of these cultures.

TGF-ß1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis.

Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGF-ß plays a pivotal role in the pathobiology of MF by promoting BM fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF-MKs elaborated significantly greater levels of TGF-ß1 than TGF-ß2 and TGF-ß3 to a varying degree, and we evaluated the ability of AVID200, a potent TGF-ß1/TGF-ß3 protein trap, to block the excessive TGF-ß signaling. Treatment of human mesenchymal stromal cells with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGF-ß1 to induce collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PCs) with WT JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PCs was attributed to its ability to block TGF-ß1-induced p57Kip2 expression and SMAD2 activation, thereby allowing normal rather than MF PCs to preferentially proliferate and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1lo mice, a murine model of MF, were treated with AVID200, resulting in the reduction in BM fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short-term and long-term HSCs. Collectively, these data provide the rationale for TGF-ß1 blockade, with AVID200 as a therapeutic strategy for patients with MF.

Compensated variability in the expression of globin-related genes in erythroblasts generated ex vivo from different donors.

BACKGROUND: Ex vivo generated erythroblasts are being evaluated for transfusion. Expression of balanced levels of globin mRNA is essential for normal red blood cell function and survival but it is unknown whether the expression of the globin genes in ex vivo expanded cells is balanced. STUDY DESIGN AND METHODS: Immature erythroblasts (IEs) were expanded in human erythroid massive amplification cultures from blood mononuclear cells of 19 normal donors and four beta(0)-thalassemia patients (for comparison) and induced to mature for 4 days in the presence of erythropoietin. mRNA was prepared from IEs and mature erythroblasts to evaluate the expression of alpha-, beta-, and gamma-globin genes and of adult hemoglobin-stabilizing protein (AHSP) and BCL11A, two proteins directly controlling globin function and/or production. Results were analyzed using Pearson's correlation coefficient, the Wilcoxon signed rank, and the Mann-Whitney rank sum tests. RESULTS: The absolute levels of globin, AHSP, and BCL11A mRNA expressed by erythroblasts generated ex vivo from normal donors were distributed along a 2-log range. With maturation, the levels of gamma-globin and BCL11A mRNA did not decrease while those of alpha-globin, gamma + beta-globins, and AHSP mRNA greatly increased. In normal cells, the modest imbalance (two- to fourfold) observed between alpha- and gamma + beta-globin mRNA was fully compensated by AHSP expression. Thus, the levels of alpha-globin mRNA were correlated with those of gamma + beta-globin (R(2) = 0.93, p < 0.0001) and AHSP (R(2) = 0.86, p < 0.0001). CONCLUSIONS: Ex vivo expanded erythroblasts from normal donors express modestly imbalanced levels of alpha-globin and gamma + beta-globin fully compensated by AHSP expression, likely ensuring normal function and survival.

Shared and Tissue-Specific Expression Signatures between Bone Marrow from Primary Myelofibrosis and Essential Thrombocythemia.

Megakaryocytes have been implicated in the micro-environmental abnormalities associated with fibrosis and hematopoietic failure in the bone marrow (BM) of primary myelofibrosis (PMF) patients, the Philadelphia-negative myeloproliferative neoplasm (MPN) associated with the poorest prognosis. To identify possible therapeutic targets for restoring BM functions in PMF, we compared the expression profiling of PMF BM with that of BM from essential thrombocytopenia (ET), a fibrosis-free MPN also associated with BM megakaryocyte hyperplasia. The signature of PMF BM was also compared with published signatures associated with liver and lung fibrosis. Gene set enrichment analysis (GSEA) identified distinctive differences between the expression profiles of PMF and ET. Notch, K-Ras, IL-8, and apoptosis pathways were altered the most in PMF as compared with controls. By contrast, cholesterol homeostasis, unfolded protein response, and hypoxia were the pathways found altered to the greatest degree in ET compared with control specimens. BM from PMF expressed a noncanonical transforming growth factor ß (TGF-ß) signature, which included activation of ID1, JUN, GADD45b, and genes with binding motifs for the JUN transcriptional complex AP1. By contrast, the expression of ID1 and GADD45b was not altered and there was a modest signal for JUN activation in ET. The similarities among PMF, liver fibrosis, and lung fibrosis were modest and included activation of integrin-α9 and tropomyosin-α1 between PMF and liver fibrosis, and of ectoderm-neural cortex protein 1 and FRAS1-related extracellular matrix protein 1 between PMF and lung fibrosis, but not TGF-ß. These data identify TGF-ß as a potential target for micro-environmental therapy in PMF.

Phosphoproteomic Landscaping Identifies Non-canonical cKIT Signaling in Polycythemia Vera Erythroid Progenitors.

Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV.

Inhibition of estradiol receptor/Src association and cell growth by an estradiol receptor alpha tyrosine-phosphorylated peptide.

This report offers direct evidence that association of the estradiol receptor (ER) with Src triggered by steroid agonists or growth factors controls breast and prostate cancer cell growth. This association is abolished in whole cells and in vitro by a six-amino-acid peptide that mimics the sequence around the phosphotyrosine residue in position 537 of the human ERalpha. The phosphorylated peptide, at nanomolar concentrations, is taken up by MCF-7 and LNCaP cells derived from human mammary and prostate cancers, respectively. In addition, to block the ER/Src interaction, the phosphopeptide inhibits Src/Erk pathway, cyclin D1 expression, and DNA synthesis induced by estradiol or androgen or triggered by epidermal growth factor. In contrast, no inhibition of the Src-mediated epidermal growth factor action on DNA synthesis is detectable in human mammary cancer cells that do not express ER (MDA-MB231), indicating that the peptide specifically targets the ER-associated Src. Remarkably, the peptide, in contrast with classic steroid antagonists, does not interfere in ER- or androgen receptor-dependent transcriptional activity. Nevertheless, it markedly inhibits the growth of MCF-7 cell xenografts induced in immunodepressed and estradiol-treated mice. The present report suggests that inhibition of association of steroid receptors with Src or other signaling effectors may have therapeutic applications for patients with ER-positive tumors.

Role of atypical protein kinase C in estradiol-triggered G1/S progression of MCF-7 cells.

Expression of a dominant negative atypical protein kinase C (aPKC), PKCzeta, prevents nuclear translocation of extracellular regulated kinase 2 (ERK-2), p27 nuclear reduction, and DNA synthesis induced by estradiol in human mammary cancer-derived MCF-7 cells. aPKC action upstream of these events has been analyzed. In hormone-stimulated NIH 3T3 and Cos cells ectopically expressing human estrogen receptor alpha (hERalpha), aPKC is activated by phosphatidylinositol 3-kinase (PI 3-kinase) and, in turn, controls the Ras/MEK-1/ERK cascade. In MCF-7 and Cos cells stimulated by hormone, PI 3-kinase activates PKCzeta by Thr410 phosphorylation. Serine phosphorylation of PKCzeta is simultaneously induced. PKCzeta activation leads to recruitment of Ras to a multimolecular complex that also includes hERalpha, Src, PI 3-kinase, and aPKC. We propose that PKCzeta pushes Ras and the signaling complex close together in such a way that it facilitates the Src-dependent Ras activation. This activation is crucial for the interplay between estradiol-triggered signaling and cell cycle machinery.

Steroid receptor regulation of epidermal growth factor signaling through Src in breast and prostate cancer cells: steroid antagonist action.

Under conditions of short-term hormone deprivation, epidermal growth factor (EGF) induces DNA synthesis, cytoskeletal changes, and Src activation in MCF-7 and LNCaP cells. These effects are drastically inhibited by pure estradiol or androgen antagonists, implicating a role of the steroid receptors in these findings. Interestingly, EGF triggers rapid association of Src with androgen receptor (AR) and estradiol receptor alpha (ERalpha) in MCF-7 cells or ERbeta in LNCaP cells. Here, we show that, through EGF receptor (EGFR) and erb-B2, EGF induces tyrosine phosphorylation of ER preassociated with AR, thereby triggering the assembly of ER/AR with Src and EGFR. Remarkably, experiments in Cos cells show that this complex stimulates EGF-triggered EGFR tyrosine phosphorylation. In turn, estradiol and androgen antagonists, through the Src-associated receptors, prevent Src activation by EGF and heavily reduce EGFR tyrosine phosphorylation and the subsequent multiple effects, including DNA synthesis and cytoskeletal changes in MCF-7 cells. In addition, knockdown of ERalpha or AR gene by small interfering RNA (siRNA) almost abolishes EGFR tyrosine phosphorylation and DNA synthesis in EGF-treated MCF-7 cells. The present findings reveal that steroid receptors have a key role in EGF signaling. EGFR tyrosine phosphorylation, depending on Src, is a part of this mechanism. Understanding of EGF-triggered growth and invasiveness of mammary and prostate cancer cells expressing steroid receptors is enhanced by this report, which reveals novel aspects of steroid receptor action.