Data from Measurement of Plasma Cell-Free Mitochondrial Tumor DNA Improves Detection of Glioblastoma in Patient-Derived Orthotopic Xenograft Models
Richard MairFlorent MoulièreChristopher G. SmithDineika ChandranandaDavina GaleFrancesco MarassDana W.Y. TsuiCharles MassieAlan J. WrightColin WattsNitzan RosenfeldKevin M. Brindle
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<div>Abstract<p>The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of patients with glioblastoma (GBM) are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GBM (<i>n</i> = 64) and show that tumor size and cell proliferation, but not the integrity of the blood–brain barrier or cell death, affect the release of ctDNA in treatment-naïve GBM PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (<0.2× coverage) of host (rat) and tumor (human) circulating DNA identified a peak at 145 bp in the human DNA fragments, indicating a difference in the origin or processing of the ctDNA. The concentration of ctDNA correlated with cell death only after treatment with temozolomide and radiotherapy. Digital PCR detection of plasma tumor mitochondrial DNA (tmtDNA), an alternative to detection of nuclear ctDNA, improved plasma DNA detection rate (82% vs. 24%) and allowed detection in cerebrospinal fluid and urine. Mitochondrial mutations are prevalent across all cancers and can be detected with high sensitivity, at low cost, and without prior knowledge of tumor mutations via capture-panel sequencing. Coupled with the observation that mitochondrial copy number increases in glioma, these data suggest analyzing tmtDNA as a more sensitive method to detect and monitor tumor burden in cancer, specifically in GBM, where current methods have largely failed.</p>Significance:<p>These findings show that detection of tumor mitochondrial DNA is more sensitive than circulating tumor DNA analysis to detect and monitor tumor burden in patient-derived orthotopic xenografts of glioblastoma.</p></div>Keywords:
Temozolomide
Glioma is the most prevalent primary brain tumor in adults and has an extremely unfavorable prognosis. As a member of the lysyl oxidase (LOX) family, lysyl-oxidase-like-2 (LOXL2) is known to play different roles in different tumors. However, the role of LOXL2 in glioma has not yet been fully elucidated. In the present study, we detected that LOXL2 was considerably upregulated in glioma and that LOXL2 upregulation was evidently related to glioma WHO grade, malignant molecular subtypes, and poor prognosis in glioma patients. Additionally, we found that LOXL2 not only promoted glioma cells proliferation, migration, invasion, and induced the epithelial-to-mesenchymal transition (EMT) process, but also reduced the sensitivity of glioma cells to temozolomide (TMZ). Furthermore, we identified that LOXL2 reduced TMZ sensitivity and induced EMT in glioma via the activation of autophagy. Mechanistically, LOXL2 enhanced Atg7 expression by promoting the phosphorylation of Erk1/2, leading to the activation of autophagy and regulation of EMT process and TMZ sensitivity through autophagy. Our study describes an LOXL2-Erk1/2-Atg7 signaling axis that influences glioma EMT and chemosensitivity through autophagy; moreover, LOXL2 may serve as a promising therapeutic target in the treatment of glioma.
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Glioma is the most frequent and aggressive type of brain neoplasm, being anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM), its most malignant forms. The survival rate in patients with these neoplasms is 15 months after diagnosis, despite a diversity of treatments, including surgery, radiation, chemotherapy, and immunotherapy. The resistance of GBM to various therapies is due to a highly mutated genome; these genetic changes induce a de-regulation of several signaling pathways and result in higher cell proliferation rates, angiogenesis, invasion, and a marked resistance to apoptosis; this latter trait is a hallmark of highly invasive tumor cells, such as glioma cells. Due to a defective apoptosis in gliomas, induced autophagic death can be an alternative to remove tumor cells. Paradoxically, however, autophagy in cancer can promote either a cell death or survival. Modulating the autophagic pathway as a death mechanism for cancer cells has prompted the use of both inhibitors and autophagy inducers. The autophagic process, either as a cancer suppressing or inducing mechanism in high-grade gliomas is discussed in this review, along with therapeutic approaches to inhibit or induce autophagy in pre-clinical and clinical studies, aiming to increase the efficiency of conventional treatments to remove glioma neoplastic cells.
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Abstract Glioblastoma multiforme is characterized by high invasiveness, multidrug resistance, and inevitable recurrence, and current standard treatment regimens are not curative. Even if most glioma cells are eliminated by chemotherapy and radiotherapy, glioma stem cells can survive and differentiate into new tumor cells, thereby triggering tumor recurrence and drug resistance. Therefore, inhibiting tumor invasiveness, reversing drug resistance, and effectively ablating glioma stem cells are critical for improving the prognosis of glioblastoma multiforme. Previous studies reported that the combination of β-diketone–cobalt complexes (CoAc2) and temozolomide (TMZ) has synergistic inhibitory effects on glioma cells. Therefore, we compared cell proliferation, colony-forming capacity, cell migration, and invasion of TMZ-resistant glioma cells and corresponding glioma stem cells after treatment with CoAc2 and/or TMZ. We also explored the underlying mechanism by which CoAc2 sensitizes cells to TMZ through transcriptome sequencing and related signal pathway blockade. We found that CoAc2 significantly increased the inhibitory effect of TMZ on the proliferation, colony formation, migration, invasion, and survival of drug-resistant stem cells. By downregulating ERK pathway activity, CoAc2 inhibited the expression of O6-methylguanine-DNA methyltransferase and eventually sensitized drug-resistant glioma cells to TMZ. In conclusion, the combined use of CoAc2 and TMZ can reverse TMZ resistance and significantly enhance its inhibitory effect on the malignant phenotype of glioma cells and glioma stem cells.
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Objective To compare the curative effects and safety of temozolomide plus radiotherapy in the treatment of post-operative malignant glioma.Methods 55 postoperative patients with pathologically confirmed malignant glioma were randomly assigned to receive temozolomide plus radiotherapy(experiment group,n= 21),125I or 131I intra-radiotherapy alone(control group ;n=15) and temozolomide alone(control group;n=19).All patients were followed up in long term for observation the time to progression,over all survival time and safety.Results Median time to progression was(56.33±3.36) weeks in experiment group,(44.83±4.55) and(41.5±3.95) weeks in control group(P 0.05).Median overall survival time was(59.27 ±3.19) weeks in experiment group,compare to(50.19±4.80) and(47.65±3.97) weeks in control group(P 0.05).Only one patient experienced leukopenia and released with dose reduction treatment.Conclusions Temozolomide plus radiotherapy is more effective than intra-radiotherapy or chemotherapy alone in the treatment of malignant glioma and is well tolerated.
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Glioblastoma multiforme is the most common primary malignancy in the brain and confers a uniformly poor prognosis. MicroRNAs have been shown to activate or inhibit tumorigenesis. Abnormalities in the p53 signaling pathway are found in various cancers and correlate with tumor formation. We examined the expression of microRNA-141-3p (miR-141-3p) in glioma of different grades by analysis of expression profiling databases and clinical specimens. Cell proliferation and flow cytometry assays were performed to evaluate the promotion of miR-141-3p in proliferation, cell cycle, apoptosis, and temozolomide resistance of glioblastoma cells in vitro. Bioinformatics analyses, luciferase reporter assays, and immunoblotting showed that p53 is a target gene of miR-141-3p. A significant inverse correlation was observed between expression of miR-141-3p and p53 in glioma and normal brain tissues (R2=0.506, P<0.0001). Rescue experiments indicated that overexpression of p53 significantly reversed the alterations in proliferation, cell cycle distribution, and temozolomide resistance measured by cell apoptosis induced by miR-141-3p overexpression. In an orthotopic mouse model of human glioma, inhibition of miRNA-141-3p reduced the proliferation and growth of glioma cells in the brain and significantly prolonged the survival of glioma-bearing mice. We suggest that miR-141-3p promotes glioblastoma progression and temozolomide resistance by altering p53 expression and therefore may serve as a new diagnostic marker and therapeutic target for glioma in the future.
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Abstract Circular RNAs (circRNAs) are a kind of noncoding RNAs that have different biological functions. CircRNAs play very important parts in the progression of cancers. Nevertheless, the exact mechanism and function of many circRNAs in glioma are not clear. In our study, circKIF4A was identified as a remarkably upregulated circRNA expressed in glioma tissues and cell lines. We performed loss-off function and gain-of-function experiments to inquire into the biological function of circKIF4A in the progression of glioma. We discovered that knockdown of circKIF4A remarkably decreased the proliferation and invasion ability of glioma cells. Moreover, subcutaneous tumorigenesis model and intracranial injection of orthotopic glioma model were established to investigate the functions of circKIF4A in vivo. Suppression of circKIF4A remarkably enhanced the sensitivity of glioma to temozolomide treatment. The glycolysis rate was accelerated by circKIF4A overexpression, which promoted glioma growth and temozolomide resistance. The glycolysis regulating enzyme ALDOA was regulated by circKIF4A through the mechanism of interactivity with miR-335-5p in glioma cells. In a word, our data showed that the upregulation of circKIF4A facilitates glioma progression by means of binding miR-335-5p and upregulating ALDOA expression.
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In adults, glioma is the most commonly occurring and invasive brain tumour. For malignant gliomas, the current advanced chemotherapy includes TMZ (temozolomide). However, a sizeable number of gliomas are unyielding to TMZ, hence, giving rise to an urgent need for more efficient treatment choices. Here, we report that cyclin-dependent kinases 4 (CDK4) is expressed at significantly high levels in glioma cell lines and tissues. CDK4 overexpression enhances colony formation and proliferation of glioma cells and extends resistance to inhibition of TMZ-mediated cell proliferation and induction of apoptosis. However, CDK4 knockdown impedes colony formation and cell proliferation, and enhances sensitivity of glioma cells to TMZ. The selective inhibition of CDK4/6 impedes glioma cell proliferation and induces apoptotic induction. The selective inhibitors of CDK4/6 may enhance glioma cell sensitivity to TMZ. We further showed the possible role of RB phosphorylation mediated by CDK4 for its oncogenic function in glioma. The growth of glioma xenografts was inhibited in vivo, through combination treatment, and corresponded to enhanced p-RB levels, reduced staining of Ki-67 and enhanced activation of caspase 3. Therefore, CDK4 inhibition may be a favourable strategy for glioma treatment and overcomes TMZ resistance.
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Abstract The prognosis of glioma is generally poor and is the cause of primary malignancy in the brain. The role of microRNAs has been implicated in tumour inhibition or activation. In several cancers, the Six1 signalling pathway has been found to be aberrant and also relates to the formation of tumours. We analysed the database for expression profiles and clinical specimens of various grades of glioma to assess microRNA‐155‐3p (miR‐155‐3p) expression. The role of miR‐155‐3p in glioblastoma, cell cycle, proliferation, apoptosis and resistance to temozolomide was assessed in vitro through flow cytometry and cell proliferation assays . Bioinformatics analyses, and assays using luciferase reporter, and immunoblotting revealed that miR‐155‐3p targets Six1 and that the relationship between glioma and healthy brain tissues was significantly inverse. In rescue experiments, overexpressed Six1 revoked the changes in cell cycle distribution, proliferation and resistance to temozolomide estimated by apoptosis induced by overexpressed miR‐155‐3p. MiR‐155‐3p inhibition reduced glioma cell growth and proliferation in the brain of a mouse model and increased the survival of mice with gliomas. Thus, miR‐155‐3p modulates Six1 expression and facilitates the progression of glioblastoma and resistance to temozolomide and may act as a novel diagnostic biomarker and a target for glioma treatment.
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