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Leukemia Nov 2021T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL), where CLL cells downregulate T-cell responses through regulatory molecules including...
T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL), where CLL cells downregulate T-cell responses through regulatory molecules including programmed death ligand-1 (PD-L1) and Interleukin-10 (IL-10). Immune checkpoint blockade (ICB) aims to restore T-cell function by preventing the ligation of inhibitory receptors like PD-1. However, most CLL patients do not respond well to this therapy. Thus, we investigated whether IL-10 suppression could enhance antitumor T-cell activity and responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated analogue of the Sp1 inhibitor mithramycin (MTM32E) to suppress CLL IL-10. MTM32E treatment inhibited mouse and human CLL IL-10 production and maintained T-cell effector function in vitro. In the Eμ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden and increased CD8 T-cell proliferation, effector and memory cell prevalence, and interferon-γ production. When combined with ICB, suppression of IL-10 improved responses to anti-PD-L1 as shown by a 4.5-fold decrease in CLL cell burden compared to anti-PD-L1 alone. Combination therapy also produced more interferon-γ, cytotoxic effector KLRG1, and memory CD8 T-cells, and fewer exhausted T-cells. Since current therapies for CLL do not target IL-10, this provides a novel strategy to improve immunotherapies.
Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Case-Control Studies; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Immune Checkpoint Inhibitors; Interleukin-10; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Activation; Mice; Mice, Inbred NOD; Mice, SCID; Plicamycin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 33731852
DOI: 10.1038/s41375-021-01217-1 -
Frontiers in Cardiovascular Medicine 2021Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and organic stenosis. It has been demonstrated that endothelin-1 (ET-1) induces...
Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and organic stenosis. It has been demonstrated that endothelin-1 (ET-1) induces pulmonary vasoconstriction through the activation of RhoA. In addition, a gene mutation of activin receptor-like kinase (ACVRL)-1 is recognized in PAH patients. However, little is known about the association between ET-1 and ACVRL-1. In the present study, we aimed to investigate the effect of ET-1 on ACVRL-1 expression and delineate the involvement of the G/RhoA/Rho kinase pathway. ET-1 was added to culture medium of human pulmonary arterial endothelial cells (PAECs). Pre-treatment with pertussis toxin (PTX) or exoenzyme C3 transferase (C3T) was performed for inhibition of G or RhoA, respectively. Rho kinase was inhibited by Y27632. Mithramycin A was used for inhibition of Sp-1, which is a transcriptional factor of ACVRL-1. The active form of RhoA (GTP-RhoA) was assessed by pull-down assay. ACVRL-1 expression was increased by ET-1 in the PAECs. Pull-down assay revealed that ET-1 induced GTP-loading of RhoA, which was suppressed by pre-treatment with PTX or C3T. Further, PTX, C3T, and Y27632 suppressed the ET-1-induced ACVRL-1 expression. ET-1 increased the activity of the ACVRL-1 promoter and stabilized the ACVRL-1 mRNA. Sp-1 peaked 15 min after adding ET-1 to the PAECs. PTX and C3T prevented the increase of Sp-1 induced by ET-1. Inhibition of Sp-1 by mithramycin A suppressed ET-1-induced ACVRL-1 upregulation. The present study demonstrated that ET-1 increases ACVRL-1 expression in human PAECs the G/RhoA/Rho kinase pathway with the involvement of Sp-1.
PubMed: 33708809
DOI: 10.3389/fcvm.2021.648981 -
Biomedicines Jan 2021Ovarian cancer is a highly deadly malignancy in which recurrence is considered incurable. Resistance to platinum-based chemotherapy bodes a particularly abysmal... (Review)
Review
Ovarian cancer is a highly deadly malignancy in which recurrence is considered incurable. Resistance to platinum-based chemotherapy bodes a particularly abysmal prognosis, underscoring the need for novel therapeutic agents and strategies. The use of mithramycin, an antineoplastic antibiotic, has been previously limited by its narrow therapeutic window. Recent advances in semisynthetic methods have led to mithramycin analogs with improved pharmacological profiles. Mithramycin inhibits the activity of the transcription factor Sp1, which is closely linked with ovarian tumorigenesis and platinum-resistance. This article summarizes recent clinical developments related to mithramycin and postulates a role for the use of mithramycin, or its analog, in the treatment of platinum-resistant ovarian cancer.
PubMed: 33445667
DOI: 10.3390/biomedicines9010070 -
Oncology Reports Jan 2021The oncogenic role of Erb‑B2 Receptor Tyrosine Kinase 2 (ERBB2) has been identified in several types of cancer, but less is known on its function and mechanism of... (Observational Study)
Observational Study
The oncogenic role of Erb‑B2 Receptor Tyrosine Kinase 2 (ERBB2) has been identified in several types of cancer, but less is known on its function and mechanism of action in cervical cancer cells. The present study employed a multipronged approach to investigate the role of ERBB2 in cervical cancer. ERBB2 and microRNA (miR)‑3184‑5p expression was assessed in patient‑derived cervical cancer biopsy tissues, revealing that higher levels of ERBB2 and lower levels of miR‑3184‑5p were associated with clinicopathological indicators of cervical cancer progression. Furthermore, ERBB2 stimulated proliferation, migration and sphere‑formation of cervical cancer cells in vitro. This effect was mediated by enhanced phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α activity. Additionally, it was revealed that miR‑3184‑5p directly suppressed ERBB2 in cervical cancer cells. The p53 activator Mithramycin A stimulated p53 and miR‑3184‑5p expression, thereby lowering the levels of ERBB2 and attenuating proliferation, migration and sphere‑formation of cervical cancer cells. In conclusion, the findings of the present study suggested ERBB2 as an oncogenic protein that may promote invasiveness in cervical cancer cells. Treatment of cervical cancer cells with the p53 activator Mithramycin A restored the levels of the endogenous ERBB2 inhibitor miR‑3184‑5p and may represent a novel treatment strategy for cervical cancer.
Topics: Adult; Aged; Biopsy; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cervix Uteri; Class I Phosphatidylinositol 3-Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Hysterectomy; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Plicamycin; Receptor, ErbB-2; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms
PubMed: 33416166
DOI: 10.3892/or.2020.7862 -
EMBO Molecular Medicine Feb 2021Rhabdoid tumor (RT) is a pediatric cancer characterized by the inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. Although this deletion is...
Rhabdoid tumor (RT) is a pediatric cancer characterized by the inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. Although this deletion is the known oncogenic driver, there are limited effective therapeutic options for these patients. Here we use unbiased screening of cell line panels to identify a heightened sensitivity of rhabdoid tumor to mithramycin and the second-generation analogue EC8042. The sensitivity of MMA and EC8042 was superior to traditional DNA damaging agents and linked to the causative mutation of the tumor, SMARCB1 deletion. Mithramycin blocks SMARCB1-deficient SWI/SNF activity and displaces the complex from chromatin to cause an increase in H3K27me3. This triggers chromatin remodeling and enrichment of H3K27ac at chromHMM-defined promoters to restore cellular differentiation. These effects occurred at concentrations not associated with DNA damage and were not due to global chromatin remodeling or widespread gene expression changes. Importantly, a single 3-day infusion of EC8042 caused dramatic regressions of RT xenografts, recapitulated the increase in H3K27me3, and cellular differentiation described in vitro to completely cure three out of eight mice.
Topics: Animals; Cell Differentiation; Chromosomal Proteins, Non-Histone; Humans; Mice; Plicamycin; Rhabdoid Tumor; Transcription Factors
PubMed: 33332735
DOI: 10.15252/emmm.202012640 -
Structure (London, England : 1993) May 2021ETS family transcription factors of ERG and FLI1 play a key role in oncogenesis of prostate cancer and Ewing sarcoma by binding regulatory DNA sites and interfering with...
ETS family transcription factors of ERG and FLI1 play a key role in oncogenesis of prostate cancer and Ewing sarcoma by binding regulatory DNA sites and interfering with function of other factors. Mithramycin (MTM) is an anti-cancer, DNA binding natural product that functions as a potent antagonist of ERG and FLI1 by an unknown mechanism. We present a series of crystal structures of the DNA binding domain (DBD) of ERG/FLI1 culminating in a structure of a high-order complex of the ERG/FLI1 DBD, transcription factor Runx2, core-binding factor beta (Cbfβ), and MTM on a DNA enhancer site, along with supporting DNA binding studies using MTM and its analogues. Taken together, these data provide insight into allosteric mechanisms underlying ERG and FLI1 transactions and their disruption by MTM analogues.
Topics: Allosteric Regulation; Antibiotics, Antineoplastic; Binding Sites; Core Binding Factor Alpha 1 Subunit; Core Binding Factor beta Subunit; Humans; Molecular Docking Simulation; Plicamycin; Protein Binding; Proto-Oncogene Protein c-fli-1; Transcriptional Regulator ERG
PubMed: 33275876
DOI: 10.1016/j.str.2020.11.012 -
Journal of Medicinal Chemistry Nov 2020Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address...
Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2'-oxime (MTM) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTM analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusion-independent/dependent selectivity indices for select 2'-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified (a Phe-Trp dipeptide-based 2'-conjugate) for in vivo testing. Relative to MTM, displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.
Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Bone Neoplasms; Cell Proliferation; Female; Humans; Mice; Mice, SCID; Oximes; Plicamycin; Sarcoma, Ewing; Tissue Distribution; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 33191745
DOI: 10.1021/acs.jmedchem.0c01526 -
Fertility and Sterility Jan 2021To determine the expression and functional roles of a long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) in leiomyoma.
OBJECTIVE
To determine the expression and functional roles of a long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) in leiomyoma.
DESIGN
Experimental study.
SETTING
Academic research laboratory.
PATIENT(S)
Women undergoing hysterectomy for leiomyoma.
INTERVENTION(S)
Overexpression and underexpression of XIST; blockade of specific protein 1 (SP1).
MAIN OUTCOME MEASURE(S)
Expression of XIST in leiomyoma and its effects on microRNA 29c (miR-29c), miR-200c, and their targets.
RESULT(S)
Leiomyoma expressed statistically significantly more XIST as compared with matched myometrium, independent of race/ethnicity and menstrual cycle phase. By use of a three-dimensional spheroid culture system, we found reduced XIST levels in leiomyoma smooth muscle cells (LSMC) after treatment with 17β-estradiol, progesterone, and their combination. The expression of XIST was down-regulated by treatment with the SP1-inhibitor mithramycin A and SP1 small interfering RNA. Knockdown of XIST resulted in inhibition of cell proliferation, up-regulation of miR-29c and miR-200c, and a concomitant inhibition of the target genes of these miRNAs, namely collagen type I (COL1A1), collagen type III (COL3A1), and fibronectin (FN1). By contrast, overexpression of XIST in myometrium smooth muscle cells repressed miR-29c and miR-200c, and induced COL1A1, COL3A1, and FN1 levels. By use of RNA immunoprecipitation analysis we confirmed XIST has sponge activity over miR-29c and miR-200c, which is more pronounced in leiomyoma as compared with myometrium.
CONCLUSION(S)
Our data demonstrate that increased expression of XIST in leiomyoma results in reduced expression of miR-29c and miR-200c with a consequent up-regulation of the genes targeted by these microRNAs including COL1A1, COL3A1, and FN1, which play key roles in extracellular matrix accumulation associated with fibroids.
Topics: Adult; Cell Proliferation; Cells, Cultured; Female; Gene Expression Regulation, Neoplastic; Humans; Leiomyoma; Middle Aged; Myocytes, Smooth Muscle; Myometrium; Primary Cell Culture; RNA, Long Noncoding; Uterine Neoplasms
PubMed: 33070965
DOI: 10.1016/j.fertnstert.2020.07.024 -
Journal of Thoracic Oncology : Official... Jan 2021Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human...
INTRODUCTION
Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human cancers. To date, no studies have evaluated UHRF1 expression in malignant pleural mesothelioma (MPM). This study was undertaken to explore the therapeutic potential of targeting UHRF1 in MPM.
METHODS
Microarray, real-time quantitative reverse transcription-polymerase chain reaction, immunoblot, and immunohistochemistry techniques were used to evaluate UHRF1 expression in normal mesothelial cells (NMCs) cultured with or without asbestos, MPM lines, normal pleura, and primary MPM specimens. The impact of UHRF1 expression on MPM patient survival was evaluated using two independent databases. RNA-sequencing, proliferation, invasion, and colony formation assays, and murine xenograft experiments were performed to evaluate gene expression and growth of MPM cells after biochemical or pharmacologic inhibition of UHRF1 expression.
RESULTS
UHRF1 expression was significantly higher in MPM lines and specimens relative to NMC and normal pleura. Asbestos induced UHRF1 expression in NMC. The overexpression of UHRF1 was associated with decreased overall survival in patients with MPM. UHRF1 knockdown reversed genomewide DNA hypomethylation, and inhibited proliferation, invasion, and clonogenicity of MPM cells, and growth of MPM xenografts. These effects were phenocopied by the repurposed chemotherapeutic agent, mithramycin. Biochemical or pharmacologic up-regulation of p53 significantly reduced UHRF1 expression in MPM cells. RNA-sequencing experiments exhibited the pleiotropic effects of UHRF1 down-regulation and identified novel, clinically relevant biomarkers of UHRF1 expression in MPM.
CONCLUSIONS
UHRF1 is an epigenetic driver in MPM. These findings support the efforts to target UHRF1 expression or activity for mesothelioma therapy.
Topics: Animals; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Mice; Pleural Neoplasms; Ubiquitin-Protein Ligases
PubMed: 32927122
DOI: 10.1016/j.jtho.2020.08.024 -
Cancers Aug 2020Hepatoblastoma (HB) is the most common malignant liver tumor in childhood and it generally has a good prognosis. However, if associated with aggressive metastatic...
Hepatoblastoma (HB) is the most common malignant liver tumor in childhood and it generally has a good prognosis. However, if associated with aggressive metastatic disease, outcome is still poor. The molecular mechanisms leading to metastatic spread in HB patients are still unknown. By combining RNA-sequencing and a genome-wide methylome analysis, we identified the transcription factor SP8 and the growth factor FGF8 among the most strongly upregulated genes in metastatic HB cases, with a concomitant robust demethylation of the respective promoter regions. Of note, high expression of both candidates was associated with the aggressive C2 subtype of the 16-gene signature and poor survival. Chromatin immunoprecipitation revealed a direct transcriptional regulation of FGF8 through binding of SP8 to the promoter. Gain- and loss-of-function experiments proved promoting effects of SP8 on motility, self-renewal, migration, and the invasive potential of HB cells. Moreover, stable overexpression of SP8 in Hep3B cells resulted in the acquisition of a mesenchymal phenotype and a strong upregulation of epithelial-mesenchymal transition-associated genes. Using KRAB-mediated CRISPR-dCas9 interference directed against FGF8, we could show that FGF8 is essential for the SP8-mediated aggressive tumor behavior. Treatment of HB cell lines with the pan SP family inhibitor mithramycin A resulted in a significant inhibition of their clonogenic growth. In summary, we identified SP8 and FGF8 as key players in aggressive traits of HB and propose SP8 inhibiting drugs as a new effective treatment strategy especially for metastatic tumors.
PubMed: 32824198
DOI: 10.3390/cancers12082294