scholarly journals PL3.6 Targeting GSK-3 activity promotes mitotic catastrophe via centrosome destabilisation and enhances the effect of radiotherapy in glioma models

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii4-iii4
Author(s):  
A Bruning-Richardson ◽  
H Sanganee ◽  
S Barry ◽  
D Tams ◽  
T Brend ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Progression ◽  
Combination Treatment ◽  
Single Agent ◽  
Drug Penetration ◽  
In Vivo Models ◽  
Human Astrocytes ◽  
Normal Human

Abstract BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

In Vitro and in Vivo Single-Agent Efficacy of Checkpoint Kinase 1 (Chk1) and 2 (Chk2) Inhibitor PF-0477736 (Pfizer) in B- and T-Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1496-1496 ◽  
Author(s):  
Ilaria Iacobucci ◽  
Andrea Ghelli Luserna Di Rorà ◽  
Maria Vittoria Verga Falzacappa ◽  
Enrico Derenzini ◽  
Anna Ferrari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Western Blot ◽  
Cell Lines ◽  
Single Agent ◽  
Time Dependent ◽  
Leukemia Cells ◽  
In Vivo Models

Abstract Abstract 1496 Introduction: Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1- negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5–2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 > 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p < 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

scholarly journals Combining IMGN779, a Novel Anti-CD33 Antibody-Drug Conjugate (ADC), with the PARP Inhibitor, Olaparib, Results in Enhanced Anti-Tumor Activity in Preclinical Acute Myeloid Leukemia (AML) Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1645-1645 ◽  
Author(s):  
Scott Portwood ◽  
Robert A Puchalski ◽  
Russell M Walker ◽  
Eunice S. Wang
Keyword(s):  
Cell Lines ◽  
Combination Treatment ◽  
Parp Inhibitor ◽  
Single Agent ◽  
S Phase ◽  
Complex Karyotype ◽  
Anti Tumor Activity ◽  
Dose Dependent

Abstract Background: New therapies for AML are urgently needed. IMGN779 is a novel CD33-targeting ADC that utilizes as the cytotoxic agent a DNA-alkylatingindolinobenzodiazepine, DGN462. Previous data have shown that IMGN779 exerts dose-dependent activity against human CD33+ AML cells in vitro and in vivo. We hypothesized that combination treatment of AML cells with the poly (ADP-ribose) polymerase (PARP) inhibitor, olaparib, which blocks cellular DNA repair would further enhance the anti-leukemic activity of IMGN779 in preclinical human AML models. Methods:Human CD33+ AML cell lines (HEL, MV4-11, HL60) were treated in vitro with controls, IMGN779, olaparib, and IMGN779 + olaparib. Proliferation was measured by WST-8 reagent. Synergistic/additive effects were calculated using Compusyn software. Flow cytometry was performed to assess apoptosis, viability, and cell cycle effects. SCID mice were engrafted systemically with human AML (HEL-luciferase) cells followed by treatment with IMGN779, olaparib, or both drugs in combination. Changes in disease burden and possible treatment-related toxicities were determined by whole animal bioluminescent imaging, body weights, and time to morbidity, respectively. Primary cells from patients with relapsed/refractory AML characterized by complex karyotype or FLT-3 mutations were plated in methocellulose media supplemented with hematopoietic cytokines for colony formation unit assays (CFU). Vehicle, IMGN779 and/or olaparib in triplicate were added followed by quantification of leukemic CFU 15 days later with a Spot-RT3 camera mounted to an inverted microscope. Results: IMGN779 treatment induced significant growth inhibition in vitro in all CD33+ human AML cell lines tested that was dose dependent. IMGN779 cell killing was CD33 dependent. Olaparib induced cell death in human AML cell lines via reversal of DNA damage repair mechanisms. Combination treatment with IMGN779 (500 pM-1 nM) and olaparib (10-50 μM) significantly enhanced anti-leukemic effects over monotherapy in the same cell lines (representative data in Table 1). Combination indices for IMGN779 and olaparib therapy ranged from 0.7-0.9, consistent with synergistic effects. The combination markedly reduced overall cell viability, increased apoptosis, and induced almost complete S-phase cell cycle arrest as compared with controls and single-agent treatments. Exposure to a combination of IMGN779 and olaparib also significantly inhibited CFU growth of progenitor cells established from bone marrow samples of patients (n=7) with relapsed/refractory, FLT3-ITD, and/or complex karyotype AML. Statistically significant inhibition of viable CFUs was observed following combination IMGN779 (10 pM) and olaparib (1 μM) therapy as compared with monotherapy or vehicle controls (p<0.001). In vivo, IMGN779 administered as a single dose that ranged from 0.5 to 5 mg/kg, by antibody, was overall well tolerated in SCID mice bearing systemic human CD33+ AML (HEL-luciferase) xenografts. Significant dose-dependent anti-leukemic activity, as reflected by decreased leukemia burden and prolonged overall survival, was observed. Combination treatment with IMGN779 (3 mg/kg) and olaparib (100 mg/kg) further significantly decreased leukemic burden when compared with vehicle (p<0.0001), IMGN779 alone (p<0.01) and olaparib alone (p<0.05) on day 14 after dosing (Figure 1). Conclusion:The combination of the CD33-targeting ADC, IMGN779, and the PARP inhibitor, olaparib, enhanced anti-tumor activity in multiple preclinical human CD33+ AML models. The combination increased in vitro DNA damage, apoptosis, S-phase arrest, and cell death effects on human CD33+ AML cells vs. single agent therapy. Combination therapy also markedly inhibited colony formation of primary AML cells representing clinically chemoresistant disease and significantly decreased in vivo leukemic burden in systemic human AML xenograft models. Our results support the future clinical investigation of this novel combinatorial regimen for AML therapy. In vivo IMGN779 + olaparib treatment in systemic HEL AML xenografts In vivo IMGN779 + olaparib treatment in systemic HEL AML xenografts Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Walker: ImmunoGen, Inc.: Employment.

Primary glioblastoma cell lines as source for analysis of the relationship between molecular phenotype and in-vitro drug sensitivity.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13053-e13053
Author(s):  
Carl Friedrich Classen ◽  
Christina S Mullins ◽  
Julia Schubert ◽  
Piek Jürgen ◽  
Michael Linnebacher ◽  
...  
Keyword(s):  
Cell Lines ◽  
Drug Sensitivity ◽  
Combined Treatment ◽  
Single Agent ◽  
Response Analysis ◽  
Pathology Laboratory ◽  
In Vivo Models ◽  
Molecular Features

e13053 Background: Glioblastoma multiforma (GBM) still has a very poor prognosis even with today’s multimodal combination therapy of resection followed by radio-chemotherapy with temozolomide. Both new molecular inhibitors and immunotherapy may represent future therapeutical tools but due to the variability of the molecular composition in individual GBM, they probably depend on better response prediction. Methods: Based on a close cooperation and logistics between surgery, pathology, laboratory researchers and animal care facilities, we established and analysed cell lines of about 30 primary brain tumors, most of them GBM. Tumors and cell lines were characterised by morphology, growth behaviour, numerous extra- and intracellular antigens, and particularly regarding molecular features. Further, tumors were established in a subcutaneous xenograft mouse model, and growth characteristics were described. These in vitro and in vivo models represent an ideal tool for response analysis of single agent or combined treatment in early cell passages in order to predict response. Besides, relevant antigens and possible shifts of antigen-presentation in tumors can be assessed. Numerous analyses using established and new antitumor compounds – e.g. the integrin inhibitor cilengitide – were performed. Results: We found that although the specific integrin expression is individually different in the tumors analysed, cilengitide sensitivity was similar in most. Addition of temozolomide led to additional, yet not over-additive cytotoxicity. In our panel of GBM specimens we found a very heterogenous pattern of molecular alterations, going along with variations of drug sensitivity. Conclusions: This indicates that, in the future field of individualized approaches, such models are required, in order to enable future strategies to predict the efficacy of immunological, small molecular, biological-based, cytotoxic or other therapeutic strategies. The project was supported by the Förderkreis für krebskranke Kinder Rostock e.V., by a Mecklenburg-Vorpommern graduate scholarship, and by a cooperation with Merck-Serono Research and Development (gift of drug).

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

scholarly journals Long noncoding RNA LINC00941 promotes pancreatic cancer progression by competitively binding miR-335-5p to regulate ROCK1-mediated LIMK1/Cofilin-1 signaling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jie Wang ◽  
Zhiwei He ◽  
Jian Xu ◽  
Peng Chen ◽  
Jianxin Jiang
Keyword(s):  
Pancreatic Cancer ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Progression ◽  
Noncoding Rna ◽  
Epithelial Mesenchymal Transition ◽  
Loss Of Function ◽  
Mesenchymal Transition

AbstractAn accumulation of evidence indicates that long noncoding RNAs are involved in the tumorigenesis and progression of pancreatic cancer (PC). In this study, we investigated the functions and molecular mechanism of action of LINC00941 in PC. Quantitative PCR was used to examine the expression of LINC00941 and miR-335-5p in PC tissues and cell lines, and to investigate the correlation between LINC00941 expression and clinicopathological features. Plasmid vectors or lentiviruses were used to manipulate the expression of LINC00941, miR-335-5p, and ROCK1 in PC cell lines. Gain or loss-of-function assays and mechanistic assays were employed to verify the roles of LINC00941, miR-335-5p, and ROCK1 in PC cell growth and metastasis, both in vivo and in vitro. LINC00941 and ROCK1 were found to be highly expressed in PC, while miR-335-5p exhibited low expression. High LINC00941 expression was strongly associated with larger tumor size, lymph node metastasis, and poor prognosis. Functional experiments revealed that LINC00941 silencing significantly suppressed PC cell growth, metastasis and epithelial–mesenchymal transition. LINC00941 functioned as a molecular sponge for miR-335-5p, and a competitive endogenous RNA (ceRNA) for ROCK1, promoting ROCK1 upregulation, and LIMK1/Cofilin-1 pathway activation. Our observations lead us to conclude that LINC00941 functions as an oncogene in PC progression, behaving as a ceRNA for miR-335-5p binding. LINC00941 may therefore have potential utility as a diagnostic and treatment target in this disease.

scholarly journals Experimental Models for Studying HPV-Positive and HPV-Negative Penile Cancer: New Tools for An Old Disease

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 460
Author(s):  
Beatriz Medeiros-Fonseca ◽  
Antonio Cubilla ◽  
Haissa Brito ◽  
Tânia Martins ◽  
Rui Medeiros ◽  
...  
Keyword(s):  
Mouse Models ◽  
Cell Lines ◽  
Penile Cancer ◽  
Hpv Infection ◽  
Experimental Models ◽  
In Vivo Models ◽  
Recent Developments ◽  
Key Aspects

Penile cancer is an uncommon malignancy that occurs most frequently in developing countries. Two pathways for penile carcinogenesis are currently recognized: one driven by human papillomavirus (HPV) infection and another HPV-independent route, associated with chronic inflammation. Progress on the clinical management of this disease has been slow, partly due to the lack of preclinical models for translational research. However, exciting recent developments are changing this landscape, with new in vitro and in vivo models becoming available. These include mouse models for HPV+ and HPV− penile cancer and multiple cell lines representing HPV− lesions. The present review addresses these new advances, summarizing available models, comparing their characteristics and potential uses and discussing areas that require further improvement. Recent breakthroughs achieved using these models are also discussed, particularly those developments pertaining to HPV-driven cancer. Two key aspects that still require improvement are the establishment of cell lines that can represent HPV+ penile carcinomas and the development of mouse models to study metastatic disease. Overall, the growing array of in vitro and in vivo models for penile cancer provides new and useful tools for researchers in the field and is expected to accelerate pre-clinical research on this disease.

scholarly journals BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

ESMO Open ◽  
2018 ◽  
Vol 3 (6) ◽  
pp. e000387 ◽  
Author(s):  
Chiara Tarantelli ◽  
Elena Bernasconi ◽  
Eugenio Gaudio ◽  
Luciano Cascione ◽  
Valentina Restelli ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Antitumour Activity ◽  
Single Agent ◽  
Treatment Strategies ◽  
Bet Inhibitor ◽  
Mantle Cell

BackgroundThe outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials.Materials and methodsThe activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines.ResultsBirabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines.ConclusionOur data provide the rationale to evaluate birabresib in patients affected by MCL.

scholarly journals BSCI-12. Inhibition of melanoma brain metastasis by targeting miR-146a

2021 ◽  
Vol 3 (Supplement_3) ◽  
pp. iii3-iii3
Author(s):  
Jiwei Wang ◽  
Emma Rigg ◽  
Taral R Lunavat ◽  
Wenjing Zhou ◽  
Zichao Feng ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Tumor Cells ◽  
Cell Lines ◽  
Western Blots ◽  
Cell Growth And Migration ◽  
Human Astrocytes ◽  
And Migration ◽  
The Brain

Abstract Background Melanoma has the highest propensity of any cancer to metastasize to the brain, with late-stage patients developing brain metastasis (MBM) in 40% of cases. Survival of patients with MBM is around 8 months with current therapies, illustrating the need for new treatments. MBM development is likely caused by molecular interactions between tumor cells and the brain, constituting the brain metastatic niche. miRNAs delivered by exosomes released by the primary tumor cells may play a role in niche establishment, yet the mechanisms are poorly understood. Here, the aim was to identify miRNAs released by exosomes from melanomas, which may be important in niche establishment and MBM progression. Materials and Methods miRNAs from exosomes collected from human astrocytes, melanocytes, and MBM cell lines were profiled to determine differential expression. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR knockdown in MBM cell lines. An in silico docking study was performed to determine drugs that potentially inhibit transcription of miR-146a to impede MBM development. Results miR-146a was the most upregulated miRNA in exosomes from MBM cells and was highly expressed in human and animal MBM samples. miR-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. In animal studies, knockdown of miR-146a in MBM cells injected intracardially into mice reduced BM burden and increased animal survival. Based on the docking studies, deserpidine was found to be an effective inhibitor of MBM growth in vitro and in vivo. Conclusions MiR-146a may play an important role in MBM development, and deserpidine is a promising candidate for clinical use.

scholarly journals Sp1-Induced Upregulation of LBX2-AS1 Aggravates The Progression of Glioblastoma By Targeting The miR-491-5p/LIF Axis

2021 ◽  
Author(s):  
Wentao Li ◽  
Ismatullah Soufiany ◽  
Xiao Lyu ◽  
Lin Zhao ◽  
Chenfei Lu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Progression ◽  
Epithelial To Mesenchymal Transition ◽  
Luciferase Reporter ◽  
Mesenchymal Transition ◽  
Stat3 Signaling ◽  
Regulatory Effects

Abstract Background: Mounting evidences have shown the importance of lncRNAs in tumorigenesis and cancer progression. LBX2-AS1 is an oncogenic lncRNA that has been found abnormally expressed in gastric cancer and lung cancer samples. Nevertheless, the biological function of LBX2-AS1 in glioblastoma (GBM) and potential molecular mechanism are largely unclear. Methods: Relative levels of LBX2-AS1 in GBM samples and cell lines were detected by qRT-PCR and FISH. In vivo and in vitro regulatory effects of LBX2-AS1 on cell proliferation, epithelial-to-mesenchymal transition (EMT) and angiogenesis in GBM were examined through xenograft models and functional experiments, respectively. The interaction between Sp1 and LBX2-AS1 was assessed by ChIP. Through bioinformatic analyses, dual-luciferase reporter assay, RIP and Western blot, the regulation of LBX2-AS1 and miR-491-5p on the target gene leukemia Inhibitory factor (LIF) was identified. Results: LBX2-AS1 was upregulated in GBM samples and cell lines, and its transcription was promoted by binding to the transcription factor Sp1. As a lncRNA mainly distributed in the cytoplasm, LBX2-AS1 upregulated LIF, and activated the LIF/STAT3 signaling by exerting the miRNA sponge effect on miR-491-5p, thus promoting cell proliferation, EMT and angiogenesis in GBM. Besides, LBX2-AS1 was unfavorable to the progression of glioma and the survival. Conclusion: Upregulated by Sp1, LBX2-AS1 promotes the progression of GBM by targeting the miR-491-5p/LIF axis. It is suggested that LBX2-AS1 may be a novel diagnostic biomarker and therapeutic target of GBM.

P16.08 Inhibition of melanoma brain metastasis by targeting miR-146a

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii57-ii57
Author(s):  
J Wang ◽  
E K Rigg ◽  
T R Lunavat ◽  
W Zhou ◽  
Z Feng ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Tumor Cells ◽  
Cell Lines ◽  
Western Blots ◽  
Cell Growth And Migration ◽  
Human Astrocytes ◽  
And Migration ◽  
The Brain

Abstract BACKGROUND Melanoma has the highest propensity of any cancer to metastasize to the brain, with late-stage patients developing brain metastasis (MBM) in 40% of cases. Survival of patients with MBM is around 8 months with current therapies, illustrating the need for new treatments. MBM development is likely caused by molecular interactions between tumor cells and the brain, constituting the brain metastatic niche. miRNAs delivered by exosomes released from the primary tumor cells may play a role in niche establishment, yet the mechanisms are poorly understood. Here, the aim was to identify miRNAs released by exosomes from melanomas, which may be important in niche establishment and MBM progression. MATERIAL AND METHODS miRNAs in exosomes collected from human astrocytes, melanocytes, and MBM cell lines were profiled to determine differential expression. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR knockdown in MBM cell lines. An in silico docking study was performed to determine drugs that potentially inhibit transcription of miR-146a to impede MBM development. RESULTS miR-146a was the most upregulated miRNA in exosomes from MBM cells and was highly expressed in human and animal MBM samples. miR-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. In animal studies, knockdown of miR-146 in MBM cells injected intracardially into mice reduced BM burden and increased animal survival. Based on the docking studies, deserpidine was found to be an effective inhibitor of MBM growth in vitro and in vivo. CONCLUSION miR-146a may play an important role in MBM development, and deserpidine is a promising candidate for clinical use.

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