Estrogen Receptor ß (ESR2) Transcriptome and Chromatin Binding in a Mantle Cell Lymphoma Tumor Model Reveal the Tumor-Suppressing Mechanisms of Estrogens.

Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma with one of the highest male-to-female incidence ratios. The reason for this is not clear, but epidemiological as well as experimental data have suggested a role for estrogens, particularly acting through estrogen receptor ß (ESR2). To study the ESR2 effects on MCL progression, MCL cells sensitive and resistant to the Bruton tyrosine kinase inhibitor ibrutinib were grafted to mice and treated with the ESR2-selective agonist diarylpropionitrile (DPN). The results showed that the DPN treatment of mice grafted with both ibrutinib-sensitive and -resistant MCL tumors resulted in impaired tumor progression. To identify the signaling pathways involved in the impaired tumor progression following ESR2 agonist treatment, the transcriptome and ESR2 binding to target genes were investigated by genome-wide chromatin immunoprecipitation in Granta-519 MCL tumors. DPN-regulated genes were enriched in several biological processes that included cell-cell adhesion, endothelial-mesenchymal transition, nuclear factor-kappaB signaling, vasculogenesis, lymphocyte proliferation, and apoptosis. In addition, downregulation of individual genes, such as SOX11 and MALAT1, that play a role in MCL progression was also observed. Furthermore, the data suggested an interplay between the lymphoma cells and the tumor microenvironment in response to the ESR2 agonist. In conclusion, the results clarify the mechanisms by which estrogens, via ESR2, impair MCL tumor progression and provide a possible explanation for the sex-dependent difference in incidence. Furthermore, targeting ESR2 with a selective agonist may be an additional option when considering the treatment of both ibrutinib-sensitive and -resistant MCL tumors.

Mathematical modeling of immune modulation by glucocorticoids.

The cellular and molecular mechanisms of immunomodulatory actions of glucocorticoids (GC) remain to be identified. Using our experimental findings, a mathematical model based on a system of ordinary differential equations for characterization of the regulation of anti-tumor immune activity by the both direct and indirect GC effects was generated to study the effects of GC treatment on effector CD8+ T cells, GC-generated tolerogenic dendritic cells (DC), regulatory T cells and the growth of lymphoma cells. In addition, we compared the data from in vivo and in silico experiments. The mathematical simulations indicated that treatment with GCs may suppress anti-tumor immune response in a dose-dependent manner. The model simulations were in line with earlier experimental observations of inhibitory effects of GCs on T and NK cells and DCs. The results of this study might be useful for predicting clinical outcomes in patients receiving GC therapy.

Expression and Effects of Ligand-activated Estrogen Receptors in Chronic Lymphocytic Leukemia.

BACKGROUND/AIM: Recent epidemiological data indicate that lymphoid tumors may be influenced by estrogens. The effects of estrogens are mediated via nuclear estrogen receptors α (ERα) and ß (ERß). This study investigated the potential functions of ligand-activated ERs in chronic lymphocytic leukemia (CLL). MATERIALS AND METHODS: The ER mRNA expression in B lymphocytes isolated from patients with CLL was analyzed by quantitative real-time polymerase chain reaction. To evaluate the effects of ERß signaling, primary CLL cells and CLL-derived MEC1 cells were treated with selective ERß agonists. RESULTS: The mRNA expression of ERα, ERß1 and its splice variant ERß2 was detected in CLL cells. Selective ERß agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile induced apoptosis in primary CLL cells and suppressed the growth of CLL-derived MEC1 cells. CONCLUSION: A suppressive effect of ERß agonists on the growth of ERß-expressing CLL cells was found, indicating that ERß may be considered as a potential therapeutic target in CLL.

Suppression of lymphoma growth by the xenoestrogens bisphenol A and genistein.

Well-defined physiological functions of estrogens are mediated via nuclear estrogen receptors α (ESR1) and ß (ESR2). With regard to hematological malignancies, expression of ESR2 has been found in both B and T cell lymphomas. In addition to endogenous estrogens or selective ESR2 agonists, ESR2 signaling may be affected by both environmental synthetic estrogen-mimicking compounds and dietary phytoestrogens. In the present study, we demonstrate that oral exposure with either the synthetic compound bisphenol A (BPA) or the dietary phytoestrogen genistein reduced the growth of grafted murine T cell (EG7) and human B cell (Granta-519 mantle cell) lymphomas which both express ESR2. Suppression of lymphoma growth was due to reduced proliferation (BPA and genistein) and induction of apoptosis (genistein). Inhibition of lymphoma growth was seen at a BPA dose of 50 µg/kg body weight (BW)/day considered to be safe human exposure dose or a genistein dose of 1 mg/kg BW/day orally, which is reached in soy-rich diets. Thus, our study indicates that the environmental xenoestrogens BPA and genistein have anti-proliferative effects on ESR2-expressing lymphomas. Our data suggest that phytoestrogens may be considered as a dietary supplement for lymphoma patients and possibly for prevention of lymphoid malignancies.

p110α Inhibition Overcomes Stromal Cell-Mediated Ibrutinib Resistance in Mantle Cell Lymphoma.

Acquired resistance to cancer drugs is common, also for modern targeted drugs like the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, a new drug approved for the treatment of the highly aggressive and relapsing mantle cell lymphoma (MCL). The tumor microenvironment often impacts negatively on drug response. Here, we demonstrate that stromal cells protect MCL cells from ibrutinib-induced apoptosis and support MCL cell regrowth after drug removal by impairing ibrutinib-mediated downregulation of PI3K/AKT signaling. Importantly, the stromal cell-mediated ibrutinib resistance was overcome in vitro by inhibiting AKT activity using the PI3K catalytic p110α subunit-specific inhibitor BYL719. This was seen both for MCL cell lines and primary MCL cells. Furthermore, inhibition of p110α activity by BYL719 potentiated the ability of ibrutinib to inhibit MCL tumor growth in vivo in a mouse xenograft model. The stromal cell-mediated ibrutinib resistance was found to be due to a direct interaction with MCL cells and involves the integrin VLA-4, as disrupting stromal cell-MCL cell interaction using a VLA-4 blocking antibody abrogated the ibrutinib resistance. This suggests that combined treatment with ibrutinib and a p110α inhibitor, alternatively by disrupting stromal cell-MCL cell interaction, may be a promising therapeutic strategy to overcome stromal cell-mediated ibrutinib resistance in MCL. Mol Cancer Ther; 17(5); 1090-100. ©2018 AACR.

Regulatory effects of dexamethasone on NK and T cell immunity.

Glucocorticoids (GCs) act via the intracellular glucocorticoid receptor (GR), which can regulate the expression of target genes. With regard to the immune system, GCs may affect both innate and adaptive immunity. Our study analyzed the immunoregulatory effects of dexamethasone (Dex) treatment on splenic T, Treg, NK and NKT cells by treating C57Bl6 mice with various doses of Dex. We observed that treatment with Dex decreased the number of NK cells in the spleen and suppressed their activity. In particular, the expression of both Ly49G and NKG2D receptors was decreased by Dex. However, Dex did not affect the population of NKT cells. With regard to splenic T cells, our results show a dose-dependent reduction in CD3+, CD4+, CD8+, CD44+ and CD8+CD122+ T cells, but a stimulatory effect on CD4+CD25+ regulatory T cells by Dex treatment. In addition, treatment with Dex suppressed anti-tumor immune response in a mouse EG7 tumor model. We conclude that Dex may suppress both T- and NK-mediated immunity.

Modification of anti-tumor immunity by tolerogenic dendritic cells.

Immunosuppressive functions of glucocorticoids (GC) can be mediated via various mechanisms, including the modulation of dendritic cells (DC). Our study investigates the effects of tolerogenic GC-treated DCs on NK and T cell anti-tumor responses in OT-1/Rag-/- mice, expressing a transgenic TCR in CD8+ T cells. The effects caused by GC-treated DCs were compared to the responses to immunogenic, CpG-activated DCs. The effects of DCs on anti-tumor immune responses were analyzed using the EG7 tumor model, where the tumor cells express the peptide epitope recognized by OT-1 T cells. We observed that immunization with CpG and peptide-treated DCs protected against tumor growth by activation of NK cell response. Also, immunogenic DCs induced the expansion of cytotoxic CD8+OT-1 cells, expressing activation markers CD44 and CD69 and producing IFNγ. In contrast, the peptide and GC-treated DCs in OT-1 mice increased the numbers of immature Mac-1+CD27- NK cells as well as Foxp3+ and IL-10 secreting CD8+OT-1 cells with suppressive properties. We conclude that the generation of tolerogenic DCs is one of many immunosuppressive mechanisms that can be induced by GC. Our study demonstrated that tolerogenic DCs modify anti-tumor immune response by suppressing NK cell activity and stimulating the formation of IL-10-secreting CD8+ Tregs.

Estrogen receptor ß1 in diffuse large B-cell lymphoma growth and as a prognostic biomarker.

Diffuse large B-cell lymphoma (DLBCL) shows a higher incidence in males versus females. Epidemiological studies have shown that female gender is a favorable prognostic factor, which may be explained by estrogens. Here we show that when grafting human DLBCL cells to immunocompromised mice, tumor growth in males is faster. When treating mice grafted with either germinal center or activated B-cell like DLBCL cells with the selective estrogen receptor ß (ERß) agonist diarylpropionitrile, tumor growth was significantly inhibited. Furthermore, nuclear ERß1 expression analysis in primary DLBCL's by immunohistochemistry revealed expression in 89% of the cases. Nuclear ERß1 expression was in a univariate and multivariate analysis, an independent prognostic factor for adverse progression-free survival in Rituximab-chemotherapy treated DLBCL (p = 0.02 and p = 0.04, respectively). These results suggest that estrogen signaling through ERß1 is an interesting future therapeutic target for treatment of DLBCL, and that ERß1 expression can be used as a prognostic marker.

Inhibition of estrogen biosynthesis enhances lymphoma growth in mice.

Most lymphomas show higher incidence and poorer prognosis in males compared to females. However, the endocrine contribution to this gender difference is not entirely known. Here we show that castration accelerates lymphoma growth in C57BL6 male mice grafted with murine EG7 T cell lymphoma cells. However, the androgen receptor antagonist Bicalutamide did not affect lymphoma growth, suggesting no impact of androgen receptor signaling on lymphoma progression. In contrast, inhibition of androgen-to-estrogen conversion by the aromatase inhibitor (AI) Letrozole induced faster lymphoma growth in mice, suggesting that androgens impact lymphoma growth through its conversion to estrogens. This was supported by the inability of dihydrotestosterone, which is not converted to estrogens by aromatase, to influence lymphoma growth in castrated male mice. Lymphoma growth was also stimulated in immunocompromised mice grafted with human B cell lymphoma (Granta-519) and treated with either reversible or irreversible AIs, showing that the blockage of estrogen synthesis caused enhanced growth of both murine T and human B cell lymphomas and with different AIs. Additionally, AI-treated EG7 lymphomas showed accelerated growth not only in male but also in intact female mice. Altogether, our results demonstrate that aromatase inhibition accelerates lymphoma growth but not androgens per se, highlighting a protective role of estrogens in lymphoma pathogenesis. These results also raise concern that the use of AIs in women with breast cancer might enhance lymphoma progression.

The selective impact of transgenically expressed glucocorticoid receptor on T cells.

Glucocorticoids (GCs) strongly impact on different T cell subsets inducing generally immunosuppressive effects, whereas much less is known about the effect of GC on natural killer (NK) cells. The aims of this study were to investigate the effects of GC on T cell functions, including T cell-mediated anti-tumor immune response, and on NK cells. We have used lck-GR mice, which overexpress a transgenic rat GR in both T and NK cells. These mice were found to have decreased both CD4(+) and CD8(+) T cell populations in the periphery. In contrast, both NK and NKT cells were found in normal numbers in lck-GR mice. To identify genes and pathways affected by GR overexpression in our system in T cells, we have compared gene expression profiles in wild-type and lck-GR T cells. Among the genes upregulated in T cells from lck-GR mice, the microarray analysis has identified genes regulating expansion of regulatory T cells. The analysis of genes downregulated in lck-GR mice has identified genes and gene associated with the regulation of immune response. With regard to the effects on T cell functions in lck-GR mice, transgenic expression of GR had a suppressive effect on killer cell activity in vitro. In addition, lck-GR mice showed an increased tumor growth in murine tumor model in vivo, which may be a possible consequence of reduced T cell numbers and activity. We conclude that an increased expression of the GR strongly affects numbers and possibly functions of T cell subsets, but has little effect on NK cells.

Inhibition of lymphoma vascularization and dissemination by estrogen receptor ß agonists.

Most lymphomas show an increased incidence and poorer prognosis in males vs females, suggesting endocrine regulation. We have previously shown that tumor growth in vivo of a murine T-cell-derived lymphoma is repressed following activation of estrogen receptor ß (ERß, ESR2). By using ERß-deficient mice, we now demonstrate that this inhibition is mediated via a direct effect on the tumor cells and not on the microenvironment. Furthermore, we show that the growth-suppressing effects of ERß agonist are also valid for human B-cell lymphomas as demonstrated in tumors derived from Granta-519 mantle cell lymphoma (MCL) and Raji Burkitt lymphoma (BL) cells. In Granta-519 MCL tumors, activation of ERß reduced expression of BAFF and GRB7, 2 important molecules involved in B-cell proliferation and survival. Importantly, activation of ERß inhibited angiogenesis and lymphangiogenesis, possibly mediated by impaired vascular endothelial growth factor C expression. Furthermore, using disseminating Raji BL cells, we show that ERß activation reduces dissemination of grafted Raji BL tumors. We also show by immunohistochemistry that ERß is expressed in primary MCL tissue. These results suggest that targeting ERß with agonists may be valuable in the treatment of some lymphomas, affecting several aspects of the malignant process, including proliferation, vascularization, and dissemination.

Estrogen receptor α and ß in the normal immune system and in lymphoid malignancies.

Estrogens regulate various normal and pathophysiological processes including cancers. Cellular signaling by estrogens is mediated by estrogen receptor α (ERα) and ß (ERß), respectively. Binding of agonists to the ERs affects gene transcription. The main endogenous estrogen, 17ß-estradiol (E2), binds to both ERα and ERß with similar affinity. However, the ligand-binding pocket of ERα and ERß are slightly different which has allowed the development of selective ER ligands. Importantly, while estrogens via ERα stimulate proliferation, signaling via ERß inhibits proliferation and promotes apoptosis. In both normal and cancer cells the ERs are co-expressed with ER splice variants which may modify the transcriptional activity of the wild-type receptors. Estrogens have prominent effects on immune functions and both ERα and ERß are expressed in immune cells and lymphoid malignancies. With regard to lymphoid malignancies, most show estrogen influence as several epidemiological studies of lymphoid cancers demonstrate gender differences in incidence and prognosis with males being more affected. In line with these findings, recent results generated by us have shown that ERß selective agonists inhibit growth and induce apoptosis in human and murine lymphomas in vivo in xenograft experiments. This suggests that ERß selective agonists in the future may be useful in the treatment of lymphomas.

Keratinocyte growth factor (KGF) delays the onset of collagen-induced arthritis.

BACKGROUND: Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family. KGF protects the oral and intestinal mucosa against damage induced by irradiation or chemotherapy. Previous studies have found the expression of KGF in chondrocytes and suggested that KGF promotes the wound healing process in injured cartilage. KGF also has important effects on the immune system such as the regeneration of thymus tissue and the formation of regulatory T cells (T(reg)) in the periphery. AIM: Here we investigated the effect of KGF on collagen type II induced arthritis (CIA) and anti-collagen antibody induced arthritis (CAIA) in order to discriminate between immunoregulatory effect and direct protective effect on chondrocytes. METHODS: CIA was induced by immunization with CII and CAIA by treatment of mice with a cocktail of four different anti-CII antibodies. The effect of KGF on the thymus and spleen was analyzed by FACS and by immunohistochemistry. RESULTS: We have found that KGF treatment delayed the onset of CIA but had no effect on CAIA. Our results show that KGF treatment leads both to an outflow of naïve T cells from the thymus and to a statistically significant increase in the percentage of CD4(+)Foxp3(+) T(regs) in the periphery. CONCLUSIONS: We suggest that the effect of KGF on CIA depends on immunoregulatory mechanisms. KGF may delay the aging of the cellular immune system and thus improve the resilience of the immune system against autoimmune reactions.

Up-regulated estrogen receptor ß2 in chronic lymphocytic leukemia.

The estrogen receptors alpha (ERα) and beta (ERß) have been demonstrated in mouse models to be important for immune system regulation, and are differentially expressed in lymphoid organs. One ERß splice variant, ERß2, inhibits the ERα-mediated estrogen effect, and expression might predict response to selective estrogen receptor modulators. We studied the expression of ERα, ERß1 and ERß2 in peripheral blood mononuclear cells from 26 patients with chronic lymphocytic leukemia (CLL) and 30 normal controls using immunocytochemistry. ERα expression was generally low, while ERß1 was expressed in 65% of patients with CLL and in 83% of controls (NS). In contrast, nuclear staining for ERß2 was positive in 69% of patients with CLL, but in only 17% of controls (p < 0.001). In CLL, ERß2 was found in B- but not in T-lymphocytes. Our data show the expression of ERß1 and ERß2 in the majority of patients with CLL, suggesting that the ERs are important in CLL and might be used as therapeutic targets.

Borrelia burgdorferi infection regulates CD1 expression in human cells and tissues via IL1-ß.

The appearance of group 1 CD1 proteins (CD1a, CD1b and CD1c) on maturing myeloid DC is a key event that converts myeloid DC to effective lipid APC. Here, we show that Borrelia burgdorferi, the causative agent of Lyme disease, triggers appearance of group 1 CD1 proteins at high density on the surface of human myeloid DC during infection. Within human skin, CD1b and CD1c expression was low or absent prior to infection, but increased significantly after experimental infections and in erythema migrans lesions from Lyme disease patients. The induction of CD1 was initiated by borrelial lipids acting through TLR-2 within minutes, but required 3 days for maximum effect. The delay in CD1 protein appearance involved a multi-step process whereby TLR-2 stimulated cells release soluble factors, which are sufficient to transfer the CD1-inducing effect in trans to other cells. Analysis of these soluble factors identified IL-1ß as a previously unknown pathway leading to group 1 CD1 protein function. This study establishes that upregulation of group 1 CD1 proteins is an early event in B. burgdorferi infection and suggests a stepwise mechanism whereby bacterial cell walls, TLR activation and cytokine release cause DC precursors to express group 1 CD1 proteins.

Inner ear pathology and loss of hearing in estrogen receptor-beta deficient mice.

There are well known differences between males and females in hearing. In the present study, the role of estrogen receptor-beta (ER-beta; listed as ESR2 in the MGI Database) in hearing was investigated by comparing hearing and morphology of the inner ear in ER-beta knock-out mice (ER-beta(-/-)) with that of wild-type (WT) littermates. Hearing was analyzed with auditory brainstem response audiometry at 3 and 12 months. The ER-beta(-/-) mice were deaf at 1 year of age, and the morphological analysis showed absence of hair cells and loss of the whole organ of Corti initiated in the basal turn of the cochlea. Furthermore, in ER-beta(-/-), but not in WT mice, the spiral ganglion was lacking many of its neurons. Immunostaining showed the presence of both ER-alpha (listed as ESR1 in the MGI Database) and ER-beta in the nuclei of some neurons in the inner ear in WT mice, but no ER-beta was found in the ER-beta(-/-) mice as expected. ER-alpha staining was predominant in the nuclei of large neurons and ER-beta in nuclei of small neurons and fibroblasts. These results reveal that both ERs are present in the inner ear at specific localizations suggesting subtype-specific functions. It is concluded that ER-beta is important for the prevention of age-related hearing loss. These findings strengthen the hypothesis that estrogen has a direct effect on hearing functions.

Liver X receptor beta (LXRbeta): a link between beta-sitosterol and amyotrophic lateral sclerosis-Parkinson's dementia.

Administration of beta-sitosterol (42 mg/kg per day) for 3 weeks to 8-month-old male LXRbeta-/- mice resulted in the death of motor neurons in the lumbar region of the spinal cord and loss of tyrosine hydroxylase-positive dopaminergic neurons in the substantia nigra. In mice at 5 months of age, beta-sitosterol had no observed toxicity but at 16 months of age, it caused severe paralysis and symptoms typical of dopaminergic dysfunction in LXRbeta-/- mice. WT mice were not affected by these doses of beta-sitosterol. In 5-month-old mice, levels of the intestinal transporters, ABCG5/8 and Niemann-Pick C1 Like 1, were not affected by loss of liver X receptor (LXR) beta and/or treatment with beta-sitosterol nor were there changes in plasma levels of cholesterol or beta-sitosterol. In 8-month-old LXRbeta-/- mice there was activation of microglia in the substantia nigra pars reticulata and aggregates of ubiquitin and TDP-43 in the cytoplasm of large motor neurons in the lumbar spinal cord. Brain cholesterol concentrations were higher in LXRbeta-/- than in their WT counterparts, and treatment with beta-sitosterol reduced brain cholesterol in both WT and LXRbeta-/- mice. In LXRbeta-/- mice but not in WT mice levels of 24-hydrocholesterol were increased upon beta-sitosterol treatment. These data indicate that multiple mechanisms are involved in the sensitivity of LXRbeta-/- mice to beta-sitosterol. These include activation of microglia, accumulation of protein aggregates in the cytoplasm of large motor neurons, and depletion of brain cholesterol.

Estrogen receptor beta-deficient female mice develop a bladder phenotype resembling human interstitial cystitis.

Interstitial cystitis/painful bladder syndrome is a disease seen mostly in women, and symptoms tend to be worse premenopausally or during ovulation. The four cardinal symptoms of interstitial cystitis/painful bladder syndrome are bladder pain, urgency, frequency, and nocturia. Estrogen has been implicated in the etiology of this disease, but the role of the two estrogen receptors (ER), ERalpha and ERbeta, has not been investigated. We found that, in the bladders of WT mice, ERbeta is expressed in the basal cell layer of the urothelium. Bladders of male ERbeta(-/-) mice were intact and morphologically indistinguishable from those of their WT littermates. However, in female ERbeta(-/-) mice, there was ulceration and atrophy of bladder urothelium concomitant with infiltration of gammadelta T cells concentrated in the areas of atrophy and shedding of urothelium. The data support the idea that activated gammadelta T cells are causing the damage to the urothelium. The hyperactivity of T cells may be because of an imbalance between ERalpha and ERbeta signaling in female ERbeta(-/-) mice. Our data suggest that reduced ERbeta signaling might have a role in the pathogenesis of interstitial cystitis, and ERbeta could be a candidate for a target of medical therapy.

Epstein-Barr virus EBNA-3C is targeted to and regulates expression from the bidirectional LMP-1/2B promoter.

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is essential for EBV-mediated immortalization of human B lymphocytes and regulates both the cell cycle and transcription. Transient reporter gene assays have implicated a pivotal role for EBNA-3C in the regulation of transcription of the majority of latency-associated genes expressed during the EBV growth program, including the viral oncoprotein LMP-1. To examine the regulation of latency gene expression by EBNA-3C, we generated an EBV-positive cell line that inducibly expresses EBNA-3C. This cell line allowed us to examine expression from the endogenous latency gene promoters in the context of an actual latent infection and the presence of other EBNA proteins, in particular EBNA-2, which is presumed to coregulate transcription with EBNA-3C. EBNA-3C induced the expression of both LMP-1 and LMP-2B mRNAs from the bidirectional LMP-1/LMP-2B promoter. In contrast, no effect was seen on expression from the common EBNA promoter Cp, which is responsive to EBNA-3C in reporter assays. Activation of LMP expression was not the consequence of increases in EBNA-2, PU.1 or Spi-B transcription factors, all of which are believed to be critical for activation of LMP-1. Chromatin immunoprecipitation assays furthermore indicated that EBNA-3C is present at the bidirectional LMP-1/LMP-2B promoter. These results indicate that EBNA-3C directly activates the expression of LMP-1 and LMP-2B but is unlikely to significantly regulate EBNA expression via Cp under normal growth conditions.