Comprehensive Genomic Analysis of Adult Burkitt Lymphoma Identifies the B-Cell Receptor Signaling Pathway As a Potential Therapeutic Target

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4095-4095
Author(s):  
Alyssa Bouska ◽  
Chengfeng Bi ◽  
Waseem Lone ◽  
Weiwei Zhang ◽  
Ambreen Kedwaii ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Burkitt Lymphoma ◽  
Copy Number ◽  
Genetic Alterations ◽  
Dependent Manner ◽  
Mirna Sponge ◽  
Bcr Signaling

Abstract Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma (NHL) in children. Although it accounts for only 1-5% of NHL in adults, approximately 60% of the BL cases diagnosed each year in western countries occur in patients >40 years of age. Although adult and pediatric BL cases are indistinguishable by molecular classification, pediatric patients have a significantly better outcome than adults. While translocation of MYC to the immunoglobulin heavy or light chain genes is characteristic of pediatric and adult BL, genetic differences may contribute to the superior clinical outcome of childhood cases. Therefore, we aimed to identify the spectrum of additional genetic abnormalities that occur in adult and pediatric BL. Copy number analysis, gene expression profiling (GEP), and targeted sequencing of ~400 genes known to be mutated in NHLs were performed on a cohort of molecularly defined BL samples. Copy number abnormalities (CNAs) were identified by the Affymetrix 250k NspI SNP array in 73 BL tumors (28 adult, 45 pediatric), and sequencing was performed on 52 BLs (21 adult, 31 pediatric). Pediatric cases had fewer CNAs than adults. The most common focal abnormality identified was a gain on 13q31.3 encompassing MIR17HG. It was more frequent in adult compared to pediatric cases (35% vs 16%, p=0.085) and was associated with increased expression of miR-17~92 cluster members; and among adults, patients with this gain trended towards worse overall survival, though the number of cases with available information was small. Gain of 8q was found in ~20% of adult cases, but in no pediatric cases. Surprisingly, cases with 8q gain had significantly lower MYC mRNA expression (p< 0.001) and lower protein expression. In cases with MYC gain 0/4 cases were positive for MYC protein expression by immunohistochemistry; in contrast,6/10 cases with no MYC gain were positive for MYC expression. This suggests that gain of 8q is driven by another gene in the region. Additional genetic alterations included gains of genomic loci encompassing MCL1 and MDM4 (1q21-24) and losses encompassing RB1, p53 and CDKN2A/CDKN2B. Pathway analysis of genes differentially expressed by CN status showed an enrichment of genes involved in cell cycle regulation, the p53 signaling pathway, and the ubiquitin proteasome pathway. The frequencies of mutations in commonly mutated genes including MYC, ID3, TP53, CCND3, DDX3X, ARID1A, and TCF3 were not significantly different in adult and pediatric BL. However, BCL2, (43%, p<0.001), ZFHX3 (24%, p<0.01), SPTBN5 (20%, p=0.02), RB1 (14%, p=0.06), BTG1 (14%, p=0.06), TCF4 (14%, p=0.06), and TNFRSF14 (14%, p=0.06), were exclusively mutated in adult BL. In contrast, mutations in CDH23 (29% vs 5%, p=0.04) and SMARCA4 (35% vs 19%, p=0.05) were more frequent in pediatric BL. When mutations were placed into oncogenic pathways, mutations in genes regulating the PI3K-AKT pathway did not shown significant differences between adult and pediatric cases. Mutations promoting tonic BCR signaling (TCF3 and ID3) by activation of the PI3K pathway had similar frequencies in the two age groups, however, BCR signaling effectors inducing chronic active NF-kB signaling (CD79A, SYK, MYD88, BCL10, CARD11) were significantly associated with adult BL (adult cases with any mutation: 19% vs 7%). Gene expression studies suggested activated BCR signaling in BL cases with CN gain of miR-17~92. In vitro analysis of miR17~92 in BL cell lines (n=4) showed that functional loss of miR-17 ~ 92 expression using a miRNA sponge led to reduced proliferation. Treatment of BL cell lines with anti-IgM induced BCR activation in a time- and dosage-dependent manner, as estimated by increased phosphorylation of downstream mediators (SYK and BLNK). This activation was reduced upon loss of functional miR-17 ~ 92 expression in cell lines. Since miR-17~92 can directly inhibit proximal negative regulators of BCR signaling, treatment with the FDA-approved BTK inhibitor, Ibrutnib, further inhibited proliferation of BL cell lines carrying the miRNA sponge. The BCR signaling pathway is one example of how unique abnormalities in adult BL can provide possible targets for therapeutic intervention. Disclosures No relevant conflicts of interest to declare.

Clonal Evolution In Patients With Chronic Lymphocytic Leukemia (CLL) Developing Resistance To BTK Inhibition

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 866-866 ◽  
Author(s):  
Jan A. Burger ◽  
Dan Landau ◽  
Julia Hoellenriegel ◽  
Carrie Sougnez ◽  
Matthias Schlesner ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Copy Number ◽  
Research Funding ◽  
Progressive Disease ◽  
Single Agent ◽  
Disease Patient ◽  
Genetic Alterations ◽  
Bcr Signaling ◽  
Trisomy 12 ◽  
Pre Treatment

Abstract The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib thwarts B cell receptor (BCR) signaling via irreversible inhibition of BTK, and induces durable remissions in relapsed/refractory CLL with a progression-free survival rate of 75% after 26 months of therapy (Byrd JC et al., NEJM 2013). However, a small fraction of patients treated with this targeted therapy develop progressive disease after an initial response. Here, we describe a longitudinal genomic investigation, utilizing whole-exome sequencing (WES) and copy number information of 3 patients who, with daily exposure to ibrutinib, achieved partial remission and later experienced CLL progression, but not Richter’s transformation. All 3 patients had advanced stage CLL (Rai stage 3-4) and were enrolled on IRB-approved phase 1/2 trials of ibrutinib (420 mg) as a single agent (Patients 1 and 2) or combined with rituximab (Patient 3). Patients 1 and 3 had relapsed diseased after prior FCR frontline therapy, while Patient 2 had received 4 prior lines of therapy. At treatment initiation, Patients 1 and 2 had known acquired TP53 deletions. Patient 1 additionally had del(13q), and Patient 2 a subclone (11.5% by FISH) carrying trisomy 12 and del(13q). Patient 3 had complex cytogenetics which included del(11q). As the best response to ibrutinib-based therapy, all three experienced partial responses. Patient 1 demonstrated normalization of hematologic parameters but experienced persistent bone marrow disease. Patient 2 achieved a >10-fold reduction but persistently elevated absolute lymphocyte count, and resolution of anemia and thrombocytopenia. Patient 3 had resolution of anemia, thrombocytopenia, and splenomegaly. Progressive disease was observed at days 1022, 554 and 206 following ibrutinib initiation for Patients 1-3, respectively. DNA was extracted from CD19-purified CLL cells before ibrutinib therapy and at the time of disease progression. Matched germline and tumor DNA from 2 timepoints underwent WES (mean coverage depth 170X) and copy number analysis (by SNP 6.0 arrays). Somatic alterations were identified through comparison with germline DNA. To examine clonal populations, we measured the allelic fractions of somatic variants and integrated this information with local copy number and purity information to infer the fraction of cancer cells (CCF) affected by the mutation. Since ibrutinib targets BTK, we searched for resistance-conferring mutations in the BTK gene in the progressing leukemias, such as the previously described C481S BTK mutation in 4 of 13 patients with acquired resistance (Chang et al., ASCO 2013, Abstract 7014). We observed that all three patients lacked mutations in BTK and for the most part, in other genes in the BCR signaling pathway. In Patient 2, we did identify a single nucleotide variant in PLCg2, a substrate of BTK previously reported to be mutated in a patient with ibrutinub resistance. However, the CCF affected by this mutation was smaller than 0.15, and therefore it is unlikely to be the main driver of relapse in this patient. All three CLLs acquired new somatic mutations at the time of progression not observed in the pre-treatment samples, involving recurrent lesions in CLL associated with poor clinical outcome. Patient 1 acquired a new clonal (>0.95 CCF) mutation in SF3B1 (K666T). Patients 2 and 3 revealed clonal deletions in chromosome 8p. Patient 2 additionally demonstrated an increase in a subclone harboring trisomy 12 with an associated MLL2 missense substitution, with CCF rising from 0.12 pre-treatment to 0.5 upon relapse. Our results confirm that clinically evident ibrutinib resistance cannot be uniformly attributed to mutations in BTK or other genes of the BCR signaling pathway. In the 3 CLLs presented herein, progressive disease was associated with the emergence of leukemic populations harboring genetic alterations with putative driver characteristics (del(8p), SF3B1 mutation) arising from a background of pre-existing 17p or 11q deletions. Our findings support the concept that CLL clones persisting during continuous therapeutic pressure can adapt to bypass BTK-related survival signaling. Ongoing studies focus on finer kinetic analysis of clonal dynamics in these patients during the period leading up to progressive disease to elucidate whether these alterations were newly acquired following ibrutinib exposure or represent selective expansions of pre-existing small subclones. Disclosures: Burger: Pharmacyclics: Research Funding. O'Brien:Pharmacyclics: Research Funding. Neuberg:Synta Pharmaceuticals: Trust owns stock; I am a Trustee Other.

The PI3K Delta Inhibitor Idelalisib Interferes With Pre-B Cell Receptor Signaling In Acute Lymphoblastic Leukemia (ALL): A New Therapeutic Concept

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2632-2632
Author(s):  
Nathalie Y. Rosin ◽  
Ekaterina Kim ◽  
Stefan Koehrer ◽  
Zhiqiang Wang ◽  
Susan O'Brien ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Differentially Expressed ◽  
B Cell Malignancies ◽  
Bcr Signaling ◽  
Dose Dependent

Abstract Phosphoinositide-3-kinases (PI3K) play an important role in transmitting signals from surface receptors such as the B-cell receptor (BCR), cytokine receptors and receptor tyrosine kinases, that result in survival and growth of normal and malignant B cells. In mature B-cell malignancies such as CLL and indolent B-NHL, the PI3K pathway is constitutively upregulated and is dependent on PI3Kδ. Idelalisib is a p110δ-isoform-specific PI3K inhibitor that is highly active in patients with CLL and indolent NHL. In contrast to mature B cell malignancies, expression and function of PI3Kδ in B-ALL has not been well characterized. RNA expression of the PI3K isoforms α, β, γ, and δ was detected in all B-ALL cell lines. Protein expression was analyzed by immunoblotting. We noted that in vitro responsiveness to idelalisib treatment was associated with protein expression of the δ isoform and presence of the Pre-BCR. We found that treatment of B-ALL cell lines with idelalisib at concentrations between 10 nM and 5 µM inhibited metabolism and growth of B-ALL cells expressing a pre-BCR, whereas only minor effects were observed in Pro-B. To monitor the expression and phosphorylation of proteins (CD72, Akt, Plcγ2, S6, Vav) involved in BCR and PI3K signaling, we prepared protein lysates of B-ALL cell lines. Cells were treated with idelalisib with/without stimulation of the µ-heavy chain of the (Pre-) BCR. We were able to show an increase of pAktSer473 and pS6Ser235/236 after stimulation, as well as a decrease in a dose dependent manner with idelalisib. The decreased amount of Akt phosphorylation was linear with the sensitivity to idelalisib treatment of the cell lines. To investigate intracellular calcium mobilization which occurs in B cells through pre-BCR stimulation, we treated cells with idelalisib and stimulated them with anti-Igµ. A dose dependent decrease in calcium flux was observed in 5 of 6 pre-B cell lines. To examine the effects of idelalisib treatment on the gene expression of pre-B ALL cells, gene expression profiling (GEP) was performed. This revealed down regulation of several genes involved in MAP-Kinase signaling, (Pre-) BCR signaling and Natural Killer cell (NK-cell) mediated cytotoxicity. For the Pre-BCR signaling pathway several genes were differentially expressed, including genes encoding the following proteins, which were found to be down regulated after 3 days of 1 µM idelalisib treatment: BCL-6, CD72, CD79a, Vav, and Plcγ2. To verify the data of the GEP, qPCR analysis was performed. To further investigate the effects of idelalisib on proteins involved in BCR signaling, six Pre-B-ALL cell lines, as well as one mature and one Pro-B cell line were treated with 1 µM idelalisib and the protein expression was quantified after immunoblotting. Most of the proteins that were differentially expressed on genomic levels have also been differentially expressed on proteomic levels and therefore confirmed the effect on Pre-BCR signaling. In summary, these experiments demonstrate inhibition of Pre-BCR signaling on both gene and protein expression levels via idelalisib treatment of Pre-B-ALL cell lines and support the importance of clinical development of the δ isoform specific PI3K inhibitor idelalisib. Disclosures: Burger: Gilead Sciences Inc: Research Funding.

scholarly journals Protein citrullination as a source of cancer neoantigens

2021 ◽  
Vol 9 (6) ◽  
pp. e002549
Author(s):  
Hiroyuki Katayama ◽  
Makoto Kobayashi ◽  
Ehsan Irajizad ◽  
Alejandro Sevillarno ◽  
Nikul Patel ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Immune Response ◽  
Protein Expression ◽  
Cell Lines ◽  
Cancer Cell ◽  
Family Members ◽  
Immunohistochemical Analysis ◽  
Cancer Cell Lines ◽  
Mhc Ii

BackgroundCitrulline post-translational modification of proteins is mediated by protein arginine deiminase (PADI) family members and has been associated with autoimmune diseases. The role of PADI-citrullinome in immune response in cancer has not been evaluated. We hypothesized that PADI-mediated citrullinome is a source of neoantigens in cancer that induces immune response.MethodsProtein expression of PADI family members was evaluated in 196 cancer cell lines by means of indepth proteomic profiling. Gene expression was assessed using messenger RNA data sets from The Cancer Genome Atlas. Immunohistochemical analysis of PADI2 and peptidyl-citrulline was performed using breast cancer tissue sections. Citrullinated 12–34-mer peptides in the putative Major Histocompatibility Complex-II (MHC-II) binding range were profiled in breast cancer cell lines to investigate the relationship between protein citrullination and antigen presentation. We further evaluated immunoglobulin-bound citrullinome by mass spectrometry using 156 patients with breast cancer and 113 cancer-free controls.ResultsProteomic and gene expression analyses revealed PADI2 to be highly expressed in several cancer types including breast cancer. Immunohistochemical analysis of 422 breast tumor tissues revealed increased expression of PADI2 in ER− tumors (p<0.0001); PADI2 protein expression was positively correlated (p<0.0001) with peptidyl-citrulline staining. PADI2 expression exhibited strong positive correlations with a B cell immune signature and with MHC-II-bound citrullinated peptides. Increased circulating citrullinated antigen–antibody complexes occurred among newly diagnosed breast cancer cases relative to controls (p=0.0012).ConclusionsAn immune response associated with citrullinome is a rich source of neoantigens in breast cancer with a potential for diagnostic and therapeutic applications.

scholarly journals Mot3, a Zn Finger Transcription Factor That Modulates Gene Expression and Attenuates Mating Pheromone Signaling in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 879-892 ◽  
Author(s):  
Anatoly V Grishin ◽  
Michael Rothenberg ◽  
Maureen A Downs ◽  
Kendall J Blumer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Signaling Pathway ◽  
G Protein ◽  
Binding Sites ◽  
Dependent Manner ◽  
Pheromone Response ◽  
Mating Pheromone ◽  
Pheromone Signaling ◽  
Yeast Genes

Abstract In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by several mechanisms that promote adaptation or desensitization. To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein β subunits. This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers. MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects. Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2. First, a Mot3-lexA fusion functions as a transcriptional activator. Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2. Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner. These findings, and the fact that consensus binding sites are found in the 5′ flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator. We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway.

scholarly journals Insulin-like Growth Factor Receptor 1 (IGF1R) Gene Copy Number Is Associated With Survival in Operable Non–Small-Cell Lung Cancer: A Comparison Between IGF1R Fluorescent In Situ Hybridization, Protein Expression, and mRNA Expression

2010 ◽  
Vol 28 (13) ◽  
pp. 2174-2180 ◽  
Author(s):  
Rafal Dziadziuszko ◽  
Daniel T. Merrick ◽  
Samir E. Witta ◽  
Adelita D. Mendoza ◽  
Barbara Szostakiewicz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Protein Expression ◽  
Squamous Cell ◽  
Copy Number ◽  
Gene Copy Number ◽  
Growth Factor Receptor ◽  
Gene Copy ◽  
Cell Versus

PurposeThe purpose of this study was to characterize insulin-like growth factor-1 receptor (IGF1R) protein expression, mRNA expression, and gene copy number in surgically resected non–small-cell lung cancers (NSCLC) in relation to epidermal growth factor receptor (EGFR) protein expression, patient characteristics, and prognosis.Patients and MethodsOne hundred eighty-nine patients with NSCLC who underwent curative pulmonary resection were studied (median follow-up, 5.3 years). IGF1R protein expression was evaluated by immunohistochemistry (IHC) with two anti-IGF1R antibodies (n = 179). EGFR protein expression was assessed with PharmDx kit. IGF1R gene expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) from 114 corresponding fresh-frozen samples. IGF1R gene copy number was assessed by fluorescent in situ hybridization using customized probes (n = 181).ResultsIGF1R IHC score was higher in squamous cell carcinomas versus other histologies (P < .001) and associated with stage (P = .03) but not survival (P = .46). IGF1R and EGFR protein expression showed significant correlation (r = 0.30; P < .001). IGF1R gene expression by qRT-PCR was higher in squamous cell versus other histologies (P = .006) and did not associate with other clinical features nor survival (P = .73). Employing criteria previously established for EGFR copy number, patients with IGF1R amplification/high polysomy (n = 48; 27%) had 3-year survival of 58%, patients with low polysomy (n = 87; 48%) had 3-year survival of 47% and patients with trisomy/disomy (n = 46; 25%) had 3-year survival of 35%, respectively (P = .024). Prognostic value of high IGF1R gene copy number was confirmed in multivariate analysis.ConclusionIGF1R protein expression is higher in squamous cell versus other histologies and correlates with EGFR expression. IGF1R protein and gene expression does not associate with survival, whereas high IGF1R gene copy number harbors positive prognostic value.

scholarly journals Fibroblast Growth Factors Regulate Prolactin Transcription via an Atypical Rac-Dependent Signaling Pathway

2003 ◽  
Vol 17 (10) ◽  
pp. 1921-1930 ◽  
Author(s):  
Twila A. Jackson ◽  
David M. Koterwas ◽  
Melissa A. Morgan ◽  
Andrew P. Bradford
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Signaling Pathway ◽  
Promoter Activity ◽  
Fibroblast Growth Factors ◽  
Dominant Negative ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Fibroblast Growth ◽  
Prl Gene

Abstract Fibroblast growth factors (FGFs) play a critical role in pituitary development and in pituitary tumor formation and progression. We have previously characterized FGF signal transduction and regulation of the tissue-specific rat prolactin (rPRL) promoter in GH4 pituitary cells. FGF induction of rPRL transcription is independent of Ras, but mediated by a protein kinase C-δ (PKCδ)-dependent activation of MAPK (ERK). Here we demonstrate a functional role for the Rho family monomeric G protein, Rac1, in FGF regulation of PRL gene expression via an atypical signaling pathway. Expression of dominant negative Rac, but not RhoA or Cdc42, selectively inhibited FGF-induced rPRL promoter activity. Moreover, expression of dominant negative Rac also attenuated FGF-2 and FGF-4 stimulation of MAPK (ERK). However, in contrast to other Rac-dependent signaling pathways, FGF activation of rPRL promoter activity was independent of the c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase/Akt cascades. FGFs failed to activate JNK1 or JNK2, and expression of dominant negative JNK or Akt constructs did not block FGF-induced PRL transcription. Consistent with the role of PKCδ in FGF regulation of PRL gene expression, activation of the rPRL promoter was blocked by an inhibitor of phospholipase Cγ (PLCγ) activity. FGF treatment also induced rapid tyrosine phosphorylation of PLCγ in a Rac-dependent manner. These results suggest that FGF-2 and FGF-4 activate PRL gene expression via a novel Rac1, PLCγ, PKCδ, and ERK cascade, independent of phosphoinositol-3-kinase and JNK.

P–543 Inhibition of cell proliferation and extracellular matrix formation in human uterine leiomyomas by 5-aza–2’-deoxycitidine via Wnt/ β-catenin pathway

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M C Carbajo-García ◽  
A Corachán ◽  
M Segura ◽  
J Monleón ◽  
J Escrig ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Hormonal Treatment ◽  
Dependent Manner ◽  
Dnmt Inhibitors ◽  
Qrt Pcr ◽  
Dose Dependent

Abstract Study question Is DNA methylation reversion through DNA methyltransferases (DNMT) inhibitors, such as 5-aza–2’-deoxycitidine, a potential therapeutic option for treatment of patients with uterine leiomyomas (UL)? Summary answer 5-aza–2’-deoxycitidine reduces proliferation and extracellular matrix (ECM) formation by inhibition of Wnt/ β-catenin pathway on UL cells, suggesting DNMT inhibitors as an option to treat UL. What is known already: UL is a multifactorial disease with an unclear pathogenesis and inaccurate treatment. Aberrant DNA methylation have been found in UL compared to myometrium (MM) tissue, showing hypermethylation of tumor suppressor genes, which contributes to the development of this tumor. The use of DNMT inhibitors, such as 5-aza–2’-deoxycytidine (5-aza-CdR), has been suggested to treat tumors in which altered methylation pattern is related to tumor progression, as occurs in UL. Based on this, we aimed to evaluate whether DNA methylation reversion through 5-aza-CdR reduces cell proliferation and ECM formation in UL cells, being a potential option for UL medical treatment. Study design, size, duration Prospective study comparing UL versus MM tissue and human uterine leiomyoma primary (HULP) cells treated with/without 5-aza-CdR at 0 µM (control), 2 µM, 5 µM and 10 µM for 72 hours. UL and MM tissue were collected from women without any hormonal treatment for the last 3 months (n = 16) undergoing myomectomy or hysterectomy due to symptomatic leiomyoma pathology. Participants were recruited between January 2019 and February 2020 at Hospital Universitario y Politecnico La Fe (Spain). Participants/materials, setting, methods Samples were collected from Caucasian premenopausal women aged 31–48 years, with a body mass index of &lt; 30 and without hormonal treatment. DNMT1 gene expression was analysed in UL vs MM tissue by qRT-PCR and activity of DNMT was measured in UL and MM tissue and cells by ELISA. 5-aza-CdR effect on proliferation was assessed by CellTiter test and Western blot (WB), apoptosis and ECM analyzed by WB and Wnt/ β-catenin pathway by qRT-PCR and WB. Main results and the role of chance: DNMT1 gene expression was increased in UL compared to MM tissue (fold change [FC]=2.49, p-value [p]=0.0295). Similarly, DNMT activity was increased in both UL compared to MM tissue and HULP cells versus MM cells (6.50 vs 3.76 OD/h/mg, p = 0.026; 211.30 vs 63.67 OD/h/mg, p = 0.284, respectively). After 5-aza-CdR treatment, cell viability of HULP cells was reduced in a dose dependent manner, being statistically significant at 10 µM (85.25%, p = 0.0001). Accordantly, PCNA protein expression was significantly decreased at 10 µM in HULP cells (FC = 0.695, p = 0.034), demonstrating cell proliferation inhibition. Additionally, 5-aza-CdR inhibited ECM protein expression in HULP cells in a dose-dependent manner being statistically significant at 10 µM for COLLAGEN I (FC = 0.654, p = 0.023) and PAI–1 (FC = 0.654, p = 0.023), and at 2 µM and 10 µM for FIBRONECTIN (FC = 0.812, p = 0.020; FC = 0.733, p = 0.035; respectively). Final targets of Wnt/ β-catenin pathway were decreased after 5-aza-CdR treatment, protein expression of WISP1 was significantly inhibited at 10 µM (FC = 0.699, p = 0.026), while expression levels of Wnt/ β-catenin target genes C-MYC (FC = 0.745, p = 0.028 at 2 µM; FC = 0.728, p = 0.019 at 10 µM) and MMP7 (FC = 0.520, p = 0.003 at 5 µM, FC = 0.577, p = 0.007 at 10 µM) were also significantly downregulated in HULP-treated cells vs untreated cells. Limitations, reasons for caution: This study has strict inclusion criteria to diminish epigenetic variability, thereby we should be cautious extrapolating our results to general population. Besides, this is a proof of concept with the inherent cell culture limitations. Further studies are necessary to determine 5-aza-CdR dose and adverse effects on UL in vivo. Wider implications of the findings: 5-aza-CdR treatment reduces cell proliferation and ECM formation through Wnt/ β-catenin pathway inhibition, suggesting that inhibition of DNA methylation could be a promising new therapeutic approach to treat UL. Trial registration number Not applicable

Abstract 555: Bone Morphogenetic Protein Modulator BMPER Induces Angiogenesis via Inhibition of Thrombospondin-1 and Activation of the Fibroblast Growth Factor Signaling Pathway

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Jennifer S Esser ◽  
Susanne Rahner ◽  
Meike Deckler ◽  
Christoph Bode ◽  
Martin Moser
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Cell Activity ◽  
Dependent Manner ◽  
Fgf Signaling ◽  
Endothelial Cell Function ◽  
Angiogenic Activity ◽  
Thrombospondin 1

Introduction: Bone morphogenetic proteins (BMP) play a key role in vascular development. Previously, we have identified BMP endothelial cell precursor-derived regulator (BMPER), an extracellular BMP modulator, to increase the angiogenic activity of endothelial cells in a concentration-dependent manner. In this project we now investigate how the BMPER effect is mediated by key molecules of angiogenesis. Methods and Results: To assess the effect of BMPER on angiogenesis-related molecules we performed an angiogenesis antibody array with BMPER-stimulated human umbilical venous endothelial cells (HUVECs) and vice versa with BMPER-silenced HUVECs compared to control conditions, respectively. We detected increased protein expression of the anti-angiogenic thrombospondin-1 (TSP-1) 48 hours after siBMPER transfection and, consistently, decreased TSP-1 expression after stimulation with BMPER (60 ng/ml; 39% ± 7.3 N=4). Furthermore, the pro-angiogenic protein bFGF was increased after BMPER stimulation, which was confirmed by realtime-PCR and western blot analysis (288.8% ± 74.8 N=3). Additionally, we detected increased FGF receptor-1 protein expression (137.7% ± 0.4 N=3) as well as FGF signaling pathway activation. Next, we investigated the interaction of BMPER and the FGF signaling pathway in endothelial cell function. BMPER stimulation increased HUVEC angiogenic activity in matrigel, migration and spheroid assays and concomitant inhibition of FGF signaling by an anti-bFGF antibody effectively inhibited the pro-angiogenic BMPER effect. Silencing of BMPER decreased the expression of FGFR1 and, accordingly, stimulation of BMPER-silenced cells with bFGF showed decreased angiogenic endothelial cell activity (65%) compared to control. The angiogenic activity of bFGF was also reduced in C57BL/6_Bmper +/- mice as assessed in the matrigel plug assay. Ex vivo aortic ring assays of C57BL/6_Bmper +/- mice confirmed a specific effect for bFGF but not for VEGF. Conclusion: In summary, BMPER inhibits the expression of the anti-angiogenic TSP-1 and increased the expression as well as activation of the pro-angiogenic FGF signaling pathway, which overall lead to the promotion of angiogenesis.

Effect of Auraptene on angiogenesis in Xenograft model of breast cancer

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mohammad Reza Shiran ◽  
Elham Mahmoudian ◽  
Abolghasem Ajami ◽  
Seyed Mostafa Hosseini ◽  
Ayjamal Khojasteh ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Animal Model ◽  
Protein Expression ◽  
Western Blot ◽  
Xenograft Model ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Abstract Objectives Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. Methods In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1 × 104 HUVEC treated with Auraptene, 20 ng/mL EGF, 20 ng/mL bFGF and 20 ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Results Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80 μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Conclusions Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

CD22 Cross-Linking Generates B-Cell Antigen Receptor-Independent Signals That Activate the JNK/SAPK Signaling Cascade

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1382-1392 ◽  
Author(s):  
Joseph M. Tuscano ◽  
Agostino Riva ◽  
Salvador N. Toscano ◽  
Thomas F. Tedder ◽  
John H. Kehrl
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Burkitt Lymphoma ◽  
B Cell Antigen Receptor ◽  
Erk Activation ◽  
Cell Antigen ◽  
Bcr Signaling ◽  
Burkitt Lymphoma Cell

Abstract CD22 is a B-cell–specific adhesion molecule that modulates BCR-mediated signal transduction. Ligation of human CD22 with monoclonal antibodies (MoAbs) that block the ligand binding site triggers rapid tyrosine phosphorylation of CD22 and primary B-cell proliferation. Because extracellular signal-regulated kinases (ERKs) couple upstream signaling pathways to gene activation and are activated by B-cell antigen receptor (BCR) signaling, we examined whether CD22 ligation also activated ERKs and/or modified BCR-induced ERK activation. Ligation of CD22 on either primary B cells or B-cell lines failed to significantly activate the mitogen activated protein kinase (MAPK) ERK-2, but did activate the stress-activated protein kinases (SAPKs; c-jun NH2-terminal kinases or JNKs). In contrast, BCR ligation resulted in ERK-2 activation without significant SAPK activation. Concurrent ligation of CD22 and BCR enhanced BCR-mediated ERK-2 activation without appreciably modulating CD22-induced SAPK activation. Consistent with its induction of SAPK activity, there was a marked increase in nuclear extracts of activator protein-1 (AP-1) and c-jun levels within 2 hours of exposure of primary B cells to the CD22 MoAb. Despite their differences in ERK activation, both CD22 and BCR ligation triggered several Burkitt lymphoma cell lines to undergo apoptosis, and the 2 stimuli together induced greater cell death than either signal alone. The pro-apoptotic effects were CD22-blocking MoAb-specific and dose-dependent. Examination of expression levels of Bcl-2 protoncogene family members (Bcl-2, Bcl-xL, Mcl-1, and Bax) showed a downregulation of Bcl-xL and Mcl-1 after CD22 ligation. This study provides a plausible mechanism to explain how CD22 and BCR signaling can costimulate B-cell proliferation and induce apoptosis in Burkitt lymphoma cell lines.

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