Targeting the immune microenvironment in Waldenström macroglobulinemia via halting the CD40/CD40-ligand axis.

Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. First, we showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T cells (Tregs) relative to control mice. These findings were translated into the WM clinical setting, in which the transcriptomic profiling of Tregs derived from patients with WM unveiled a peculiar WM-devoted messenger RNA signature, with significant enrichment for genes related to nuclear factor κB-mediated tumor necrosis factor α signaling, MAPK, and PI3K/AKT, which was paralleled by a different Treg functional phenotype. We demonstrated significantly higher Treg induction, expansion, and proliferation triggered by WM cells, compared with their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-cell-to-T-cell cross talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis that supports WM cell-Tregs interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

Deep MRD profiling defines outcome and unveils different modes of treatment resistance in standard- and high-risk myeloma.

Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of âˆ¼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species-mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.

PD-1/PD-L1 immune checkpoint and p53 loss facilitate tumor progression in activated B-cell diffuse large B-cell lymphomas.

Refractory or relapsed diffuse large B-cell lymphoma (DLBCL) often associates with the activated B-cell-like (ABC) subtype and genetic alterations that drive constitutive NF-κB activation and impair B-cell terminal differentiation. Here, we show that DNA damage response by p53 is a central mechanism suppressing the pathogenic cooperation of IKK2ca-enforced canonical NF-κB and impaired differentiation resulting from Blimp1 loss in ABC-DLBCL lymphomagenesis. We provide evidences that the interplay between these genetic alterations and the tumor microenvironment select for additional molecular addictions that promote lymphoma progression, including aberrant coexpression of FOXP1 and the B-cell mutagenic enzyme activation-induced deaminase, and immune evasion through major histocompatibility complex class II downregulation, PD-L1 upregulation, and T-cell exhaustion. Consistently, PD-1 blockade cooperated with anti-CD20-mediated B-cell cytotoxicity, promoting extended T-cell reactivation and antitumor specificity that improved long-term overall survival in mice. Our data support a pathogenic cooperation among NF-κB-driven prosurvival, genetic instability, and immune evasion mechanisms in DLBCL and provide preclinical proof of concept for including PD-1/PD-L1 blockade in combinatorial immunotherapy for ABC-DLBCL.

Richter transformation driven by Epstein-Barr virus reactivation during therapy-related immunosuppression in chronic lymphocytic leukaemia.

The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukaemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In contrast to EBV- tumours, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL, and they also showed CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal centre DLBCL phenotype, EBV latency programme type II or III, and very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoural B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/- IL2γc-/- mice. Remarkably, the recipients' impaired immunosurveillance favoured the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients samples, but not from healthy donors. Eventually, these cells generated monoclonal tumours (mostly CLL-unrelated but also CLL-related), recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts showed indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and B-cell receptor signalling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Targeting the anion exchanger 2 with specific peptides as a new therapeutic approach in B lymphoid neoplasms.

Regulatory T (Treg) cells can weaken antitumor immune responses, and inhibition of their function appears to be a promising therapeutic approach in cancer patients. Mice with targeted deletion of the gene encoding the Cl-/HCO3- anion exchanger AE2 (also termed SLC4A2), a membrane-bound carrier involved in intracellular pH regulation, showed a progressive decrease in the number of Treg cells. We therefore challenged AE2 as a potential target for tumor therapy, and generated linear peptides designed to bind the third extracellular loop of AE2, which is crucial for its exchange activity. Peptide p17AE2 exhibited optimal interaction ability and indeed promoted apoptosis in mouse and human Treg cells, while activating effector T-cell function. Interestingly, this linear peptide also induced apoptosis in different types of human leukemia, lymphoma and multiple myeloma cell lines and primary malignant samples, while it showed only moderate effects on normal B lymphocytes. Finally, a macrocyclic AE2 targeting peptide exhibiting increased stability in vivo was effective in mice xenografted with B-cell lymphoma. These data suggest that targeting the anion exchanger AE2 with specific peptides may represent an effective therapeutic approach in B-cell malignancies.

A GAPDH-mediated trans-nitrosylation pathway is required for feedback inhibition of bile salt synthesis in rat liver.

BACKGROUND & AIMS: Bile salts inhibit their own production by inducing the nuclear receptor small heterodimer partner (SHP) (encoded by NR0B2), which contributes to repression of the gene encoding cholesterol 7α-hydroxylase (CYP7A1), a key enzyme for the control of bile salt synthesis. On the other hand, bile salts stimulate hepatic synthesis of nitric oxide. We investigated the role of nitric oxide signaling in the control of CYP7A1 expression and the involvement in this process of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which participates in intracellular propagation of nitric oxide signals. METHODS: We studied the effects of inhibitors of nitric oxide synthesis (L-NG-nitroarginine methyl ester [L-NAME]) or protein nitrosylation (via dithiothreitol) on bile salt homeostasis in male Wistar rats placed on a cholate-rich diet for 5 days and in cultured primary hepatocytes. S-nitrosylation of GAPDH was assessed using a biotin-switch assay. Interacions of SHP with other proteins and with the Cyp7a1 promoter sequence were studied using immunoprecipitation and chromatin immunoprecipitation (ChIP) assays. We reduced the GAPDH levels in H35 cells with small interfering RNAs. GAPDH nitrosylation was assessed in normal and cholestatic rat and human livers. RESULTS: Rats placed on cholate-rich diets and given L-NAME had increased intrahepatic and biliary levels of bile salts, and deficiency in repression of CYP7A1 (at the messenger RNA and protein levels) in liver tissue, despite preserved induction of SHP. In cultured hepatocytes, L-NAME or dithiothreitol blocked cholate-induced down-regulation of CYP7A1 without impairing SHP up-regulation. In hepatocytes, cholate promoted S-nitrosylation of GAPDH and its translocation to the nucleus, accompanied by S-nitrosylation of histone deacetylase 2 (HDAC2) and Sirtuin 1 (SIRT1), deacetylases that participate, respectively, in the formation of Cyp7a1 and Shp repressor complexes. Knockdown of GAPDH prevented repression of CYP7A1 by cholate, and blocking nuclear transport of nitrosylated GAPDH reduced cholate-induced nitrosylation of HDAC2 and SIRT1; this effect was accompanied by abrogation of Cyp7a1 repression. Cholate induced binding of SHP to HDAC2 and its recruitment to the Cyp7a1 promoter; these processes were inhibited by blocking nitric oxide synthesis. Levels of nitrosylated GAPDH and nitrosylated HDAC2 were increased in cholestatic human and rat livers reflecting increased concentrations of bile salts in these conditions. CONCLUSIONS: In rat liver, excess levels of bile salts activate a GAPDH-mediated transnitrosylation cascade that provides feedback inhibition of bile salt synthesis.

Chronic docosahexaenoic acid supplementation improves metabolic plasticity in subcutaneous adipose tissue of aged obese female mice.

This study aimed to characterize the potential beneficial effects of chronic docosahexaenoic acid (DHA) supplementation on restoring subcutaneous white adipose tissue (scWAT) plasticity in obese aged female mice. Two-month-old female C57BL/6J mice received a control (CT) or a high fat diet (HFD) for 4 months. Then, 6-month-old diet-induced obese (DIO) mice were distributed into the DIO and the DIOMEG group (fed with a DHA-enriched HFD) up to 18 months. In scWAT, the DHA-enriched diet reduced the mean adipocyte size and reversed the upregulation of lipogenic genes induced by the HFD, reaching values even lower than those observed in CT animals. DIO mice exhibited an up-regulation of lipolytic and fatty oxidation gene expressions that was reversed in DHA-supplemented mice except for Cpt1a mRNA levels, which were higher in DIOMEG as compared to CT mice. DHA restored the increase of proinflammatory genes observed in scWAT of DIO mice. While no changes were observed in total macrophage F4/80+/CD11b+ content, the DHA treatment switched scWAT macrophages profile by reducing the M1 marker Cd11c and increasing the M2 marker CD206. These events occurred alongside with a stimulation of beige adipocyte specific genes, the restoration of UCP1 and pAKT/AKT ratio, and a recovery of the HFD-induced Fgf21 upregulation. In summary, DHA supplementation induced a metabolic remodeling of scWAT to a healthier phenotype in aged obese mice by modulating genes controlling lipid accumulation in adipocytes, reducing the inflammatory status, and inducing beige adipocyte markers in obese aged mice.

Differential remodeling of subcutaneous white and interscapular brown adipose tissue by long-term exercise training in aged obese female mice.

Obesity exacerbates aging-induced adipose tissue dysfunction. This study aimed to investigate the effects of long-term exercise on inguinal white adipose tissue (iWAT) and interscapular brown adipose tissue (iBAT) of aged obese mice. Two-month-old female mice received a high-fat diet for 4 months. Then, six-month-old diet-induced obese animals were allocated to sedentarism (DIO) or to a long-term treadmill training (DIOEX) up to 18 months of age. In exercised mice, iWAT depot revealed more adaptability, with an increase in the expression of fatty acid oxidation genes (Cpt1a, Acox1), and an amelioration of the inflammatory status, with a favorable modulation of pro/antiinflammatory genes and lower macrophage infiltration. Additionally, iWAT of trained animals showed an increment in the expression of mitochondrial biogenesis (Pgc1a, Tfam, Nrf1), thermogenesis (Ucp1), and beige adipocytes genes (Cd137, Tbx1). In contrast, iBAT of aged obese mice was less responsive to exercise. Indeed, although an increase in functional brown adipocytes genes and proteins (Pgc1a, Prdm16 and UCP1) was observed, few changes were found on inflammation-related and fatty acid metabolism genes. The remodeling of iWAT and iBAT depots occurred along with an improvement in the HOMA index for insulin resistance and in glucose tolerance. In conclusion, long-term exercise effectively prevented the loss of iWAT and iBAT thermogenic properties during aging and obesity. In iWAT, the long-term exercise program also reduced the inflammatory status and stimulated a fat-oxidative gene profile. These exercise-induced adipose tissue adaptations could contribute to the beneficial effects on glucose homeostasis in aged obese mice.

Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma.

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.

Preneoplastic somatic mutations including MYD88L265P in lymphoplasmacytic lymphoma.

Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88L265P in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88L265P is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas.

In Vitro Assessment of the Role of p53 on Chemotherapy Treatments in Neuroblastoma Cell Lines.

Neuroblastoma is the most frequent malignant extracranial solid tumor of infancy. The overall objective of this work consists of determining the presence of alterations in the p53/MDM2/p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. Firstly, we characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway by analysis of TP53 point mutations, MYCN and MDM2 amplification, and p14ARF methylation, homozygous deletions, and expression. Secondly, we chose SK-N-FI (mutated at TP53) and SK-N-Be(2) (wild-type TP53) cell lines, treated them with chemotherapeutic agents (doxorubicin, etoposide, cisplatin, and melphalan) and with two isomers of retinoic acid (RA): (9-cis and all-trans). Finally, we analyzed the distribution of the cell cycle, the induction of apoptosis, and the expression levels of p53, p21, and Bcl-2 in those two cell lines. P14ARF did not present promoter methylation, homozygous deletions, and protein expression in any of the 10 neuroblastoma cell lines. One TP53 point mutation was detected in the SK-N-FI cell line. MYCN amplification was frequent, while most cell lines did not present MDM2 amplification. Treatment of SK-N-FI and SK-N-Be(2) cells with doxorubicin, etoposide, cisplatin, and melphalan increased apoptosis and blocked the cycle in G2/M, while retinoic acid isomers induced apoptosis and decreased the percentage of cells in S phase in TP53 mutated SK-N-FI cells, but not in TP53 wild-type SK-N-Be(2) cells. Treatment with cisplatin, melphalan, or 9-cis RA decreased p53 expression levels in SK-N-FI cells but not in SK-N-Be (2). The expression of p21 was not modified in either of the two cell lines. Bcl-2 levels were reduced only in SK-N-FI cells after treatment with cisplatin. However, treatments with doxorubicin, etoposide, or 9-cis-RA did not modify the levels of this protein in either of the two cell lines. In conclusion, TP53 mutated SK-N-FI cells respond better to the retinoic isomers than TP53 wild-type SK-N-Be(2) cells. Although these are in vitro results, it seems that deciphering the molecular alterations of the p53/MDM2/p14ARF signaling pathway prior to treating patients of neuroblastoma might be useful for standardizing therapies with the aim of improving survival.

Endogenous Retroelement Activation by Epigenetic Therapy Reverses the Warburg Effect and Elicits Mitochondrial-Mediated Cancer Cell Death.

For millions of years, endogenous retroelements have remained transcriptionally silent within mammalian genomes by epigenetic mechanisms. Modern anticancer therapies targeting the epigenetic machinery awaken retroelement expression, inducing antiviral responses that eliminate tumors through mechanisms not completely understood. Here, we find that massive binding of epigenetically activated retroelements by RIG-I and MDA5 viral sensors promotes ATP hydrolysis and depletes intracellular energy, driving tumor killing independently of immune signaling. Energy depletion boosts compensatory ATP production by switching glycolysis to mitochondrial oxidative phosphorylation, thereby reversing the Warburg effect. However, hyperfunctional succinate dehydrogenase in mitochondrial electron transport chain generates excessive oxidative stress that unleashes RIP1-mediated necroptosis. To maintain ATP generation, hyperactive mitochondrial membrane blocks intrinsic apoptosis by increasing BCL2 dependency. Accordingly, drugs targeting BCL2 family proteins and epigenetic inhibitors yield synergistic responses in multiple cancer types. Thus, epigenetic therapy kills cancer cells by rewiring mitochondrial metabolism upon retroelement activation, which primes mitochondria to apoptosis by BH3-mimetics. SIGNIFICANCE: The state of viral mimicry induced by epigenetic therapies in cancer cells remodels mitochondrial metabolism and drives caspase-independent tumor cell death, which sensitizes to BCL2 inhibitor drugs. This novel mechanism underlies clinical efficacy of hypomethylating agents and venetoclax in acute myeloid leukemia, suggesting similar combination therapies for other incurable cancers.This article is highlighted in the In This Issue feature, p. 995.

Transcriptional regulation of the human type 8 17beta-hydroxysteroid dehydrogenase gene by C/EBPbeta.

17beta-Hydroxysteroid dehydrogenases (17beta-HSD) regulate the intracellular concentration of active sex steroid hormones in target tissues. To date, at least 14 different isozymes have been identified. The type 8 17beta-hydroxysteroid dehydrogenase (17beta-HSD8) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To map the promoter region and to investigate its regulation, we cloned and fused a 1600 bp DNA fragment upstream of the 17beta-HSD8 transcriptional start site to a luciferase reporter gene. After transient transfection in HepG2 cells, this fragment was shown to possess promoter activity. Deletion constructs of the 5' flanking region of the 17beta-HSD8 gene led to the identification of the minimal promoter region within the first 75 bp upstream of the transcriptional start site. This region included two CCAAT boxes and sequences closely resembling the consensus Sp1 and NF-kappaB motifs. Site directed mutagenesis revealed that the CCAAT boxes were essential for transcription in HepG2. EMSA, supershift and chromatin immunoprecipitation reflected that these sequences were binding sites for C/EBPbeta. Furthermore, promoter activity was increased by the co-transfection of a C/EBPbeta expression vector, and this transactivation was through both CCAAT boxes. Our studies indicate that C/EBPbeta is essential for the transcription of the 17beta-HSD8 gene in the liver.

Changes in gene expression profiling of apoptotic genes in neuroblastoma cell lines upon retinoic acid treatment.

To determine the effect of retinoic acid (RA) in neuroblastoma we treated RA sensitive neuroblastoma cell lines with 9-cis RA or ATRA for 9 days, or for 5 days followed by absence of RA for another 4 days. Both isomers induced apoptosis and reduced cell density as a result of cell differentiation and/or apoptosis. Flow cytometry revealed that 9-cis RA induced apoptosis more effectively than ATRA. The expression profile of apoptosis and survival pathways was cell line specific and depended on the isomer used.

Transcriptional regulation of type 11 17ß-hydroxysteroid dehydrogenase expression in prostate cancer cells.

Type 11 hydroxysteroid (17-beta) dehydrogenase (HSD17B11) catalyzes the conversion of 5α-androstan-3α,17ß-diol into androsterone suggesting that it may play an important role in androgen metabolism. We previously described that overexpression of C/EBPα or C/EBPß induced HSD17B11 expression in HepG2 cells but this process was not mediated by the CCAAT boxes located within its proximal promoter region. Here, we study HSD17B11 transcriptional regulation in prostate cancer (PC) cells. Transfection experiments showed that the region -107/+18 is sufficient for promoter activity in PC cells. Mutagenesis analysis indicated that Sp1 and C/EBP binding sites found in this region are essential for promoter activity. Additional experiments demonstrated that ectopic expression of Sp1 and C/EBPα upregulated HSD17B11 expression only in PC cell lines. Through DAPA and ChIP assays, specific recruitment of Sp1 and C/EBPα to the HSD17B11 promoter was detected. These results show that HSD17B11 transcription in PC cells is regulated by Sp1 and C/EBPα.

Type 10 17ß-hydroxysteroid dehydrogenase expression is regulated by C/EBPß in HepG2 cells.

17ß-hydroxysteroid dehydrogenases (HSD17Bs) are enzymes that stereospecifically reduce or oxidize a keto- or hydroxy group at C17 of the steroid scaffold, respectively. Fourteen mammalian HSD17Bs have been identified so far. We previously described that the HSD17B8 gene is regulated by C/EBPß in the hepatocarcinoma cell line HepG2. Here, we analyze the 5'-flanking region of 14 promoters (HSD17B1-14) looking for CCAAT boxes and binding sites for CCAAT enhancer binding factors (C/EBPs). All promoters were found to have binding sites for these transcription factors except HSD17B1. Ectopic expression of C/EBPα or C/EBPß in HepG2 cells showed that HSD17B11 expression was induced by both transcription factors while HSD17B10 expression was only induced by C/EBPß. The first 500bp of the 5'-flanking region of both genes contain two putative binding sites for C/EBPs. Gene reporter assays showed that C/EBPß transactivated HSD17B10 but not HSD17B11. Additional experiments showed that several isoforms of C/EBPß are involved in HSD17B10 regulation. Mutation of the CCAAT box located at -30/-19 induced HSD17B10 promoter activity when only LIP was expressed, while impaired LAP-induced HSD17B10 transactivation in HepG2 cells when LAP isoforms are expressed. Taken together, our findings reveal that HSD17B10 is regulated by several isoforms of C/EBPß in HepG2 cells.

Estradiol induces type 8 17beta-hydroxysteroid dehydrogenase expression: crosstalk between estrogen receptor alpha and C/EBPbeta.

Hydroxysteroid (17-beta) dehydrogenase (HSD17B) are the enzymes responsible for the reversible interconversion of 17-hydroxy and 17-keto steroids. The human and mouse type 8 17beta-HSD (HSD17B8) selectively catalyze the conversion of estradiol (E2) to estrone (E1). We previously described thatHSD17B8 is transcriptionally regulated by C/EBPbeta, and that C/EBPbeta is bound to CCAAT boxes located at -5 and -46 of the transcription start site in basal conditions in HepG2 cells. Furthermore, ectopic expression of C/EBPbeta transactivated the HSD17B8 promoter activity. Here, we show that HSD17B8 expression is up-regulated in response to E2 in the estrogen receptor alpha (ERalpha) positive MCF-7 cells. Results showed that this induction is mediated by ERalpha because i) E2 did not induce HSD17B8 expression in ERalphanegative HepG2 cells, ii) ectopic expression of ERalpha restored E2-induced HSD17B8 expression, and iii) this induction was blocked by the anti-ER ICI 182,780. Additional experiments showed that no estrogen response element was necessary for this regulation. However, the CCAAT boxes located at the HSD17B8 proximal promoter were required for E2-induced transcription. Furthermore, co-immunoprecipitation studies revealed tethering of ERalphatoC/EBPbeta in response to E2 in cells expressing ERalpha. Additionally, chromatin immunoprecipitation assays demonstrated that, in response to E2, ERalpha is recruited to the CCAAT boxes in which C/EBPbeta is already bound. Taken together, our results reveal that ERalpha is involved in the transcriptional regulation of HSD17B8 gene in response to E2 through its interaction with C/EBPbeta.