Hypoxia-Inducible Factor-1? Activity as a Switch for Glioblastoma Responsiveness to Temozolomide.
ABSTRACT: Rationale:The activity of the transcription factor, hypoxia-inducible factor (HIF)-1?, is a common driver of a number of the pathways involved in the aggressiveness of glioblastomas (GBMs), and it has been suggested that the reduction in this activity observed, soon after the administration of temozolomide (TMZ), can be a biomarker of an early response in GBM models. As HIF-1? is a tightly regulated protein, studying the processes involved in its downregulation could shed new light on the mechanisms underlying GBM sensitivity or resistance to TMZ. Methods:The effect of HIF-1? silencing on cell responsiveness to TMZ was assessed in four genetically different human GBM cell lines by evaluating cell viability and apoptosis-related gene balance. LAMP-2A silencing was used to evaluate the contribution of chaperone-mediated autophagy (CMA) to the modulation of HIF-1? activity in TMZ-sensitive and TMZ-resistant cells. Results:The results showed that HIF-1? but not HIF-2? activity is associated with GBM responsiveness to TMZ: its downregulation improves the response of TMZ-resistant cells, while blocking CMA-mediated HIF-1? degradation induces resistance to TMZ in TMZ-sensitive cells. These findings are in line with the modulation of crucial apoptosis-related genes. Conclusion:Our results demonstrate the central role played by HIF-1? activity in determining the sensitivity or resistance of GBMs to TMZ, and we suggest that CMA is the cellular mechanism responsible for modulating this activity after TMZ treatment.
Project description:Glioblastoma (GBM) is the most common astrocytic-derived brain tumor in adults, characterized by a poor prognosis mainly due to the resistance to the available therapy. The study of mitochondria-derived oxidative stress, and of the biological events that orbit around it, might help in the comprehension of the molecular mechanisms at the base of GBM responsiveness to Temozolomide (TMZ). Sensitive and resistant GBM cells were used to test the role of mitochondrial ROS release in TMZ-resistance. Chaperone-Mediated Autophagy (CMA) activation in relation to reactive oxygen species (ROS) release has been measured by monitoring the expression of specific genes. Treatments with H2O2 were used to test their potential in reverting resistance. Fluctuations of cytoplasmic ROS levels were accountable for CMA induction and cytotoxic effects observed in TMZ sensitive cells after treatment. On the other hand, in resistant cells, TMZ failed in producing an increase in cytoplasmic ROS levels and CMA activation, preventing GBM cell toxicity. By increasing oxidative stress, CMA activation was recovered, as also cell cytotoxicity, especially in combination with TMZ treatment. Herein, for the first time, it is shown the relation between mitochondrial ROS release, CMA activation and TMZ-responsiveness in GBM.
Project description:Glioblastoma multiforme (GBM) is the most common brain tumour, characterized by a central and partially necrotic (i.e., hypoxic) core enriched in cancer stem cells (CSCs). We previously showed that the most hypoxic and immature (i.e., CSCs) GBM cells were resistant to Temozolomide (TMZ) in vitro, owing to a particularly high expression of O6-methylguanine-DNA-methyltransferase (MGMT), the most important factor associated to therapy resistance in GBM. Bone morphogenetic proteins (BMPs), and in particular BMP2, are known to promote differentiation and growth inhibition in GBM cells. For this reason, we investigated whether a BMP2-based treatment would increase TMZ response in hypoxic drug-resistant GBM-derived cells. Here we show that BMP2 induced strong differentiation of GBM stem-like cells and subsequent addition of TMZ caused dramatic increase of apoptosis. Importantly, we correlated these effects to a BMP2-induced downregulation of both hypoxia-inducible factor-1? (HIF-1?) and MGMT. We report here a novel mechanism involving the HIF-1?-dependent regulation of MGMT, highlighting the existence of a HIF-1?/MGMT axis supporting GBM resistance to therapy. As confirmed from this evidence, over-stabilization of HIF-1? in TMZ-sensitive GBM cells abolished their responsiveness to it. In conclusion, we describe a HIF-1?-dependent regulation of MGMT and suggest that BMP2, by down-modulating the HIF-1?/MGMT axis, should increase GBM responsiveness to chemotherapy, thus opening the way to the development of future strategies for GBM treatment.
Project description:BACKGROUND:Glioblastoma (GBM) is the most common primary malignant adult brain tumor. Temozolomide (TMZ) is the standard of care and is most effective in GBMs that lack the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Moreover, even initially responsive tumors develop a secondary resistance to TMZ and become untreatable. Since aberrant epidermal growth factor receptor (EGFR) signaling is widespread in GBM, EGFR inhibition has been tried in multiple clinical trials without success. We recently reported that inhibiting EGFR leads to increased secretion of tumor necrosis factor (TNF) and activation of a survival pathway in GBM. Here, we compare the efficacy of TMZ versus EGFR plus TNF inhibition in an orthotopic mouse model of GBM. METHODS:We use an orthotopic model to examine the efficacy of TMZ versus EGFR plus TNF inhibition in multiple subsets of GBMs, including MGMT methylated and unmethylated primary GBMs, recurrent GBMs, and GBMs rendered experimentally resistant to TMZ. RESULTS:The efficacy of the 2 treatments was similar in MGMT methylated GBMs. However, in MGMT unmethylated GBMs, a combination of EGFR plus TNF inhibition was more effective. We demonstrate that the 2 treatment approaches target distinct and non-overlapping pathways. Thus, importantly, EGFR plus TNF inhibition remains effective in TMZ-resistant recurrent GBMs and in GBMs rendered experimentally resistant to TMZ. CONCLUSION:EGFR inhibition combined with a blunting of the accompanying TNF-driven adaptive response could be a viable therapeutic approach in MGMT unmethylated and recurrent EGFR-expressing GBMs.
Project description:The management of glioblastomas (GBMs) is challenged by the development of therapeutic resistance and early disease recurrence, despite multi-modal therapy. This may be attributed to the presence of glioma stem cells (GSCs) which are known to survive radio- and chemotherapy, by circumventing death signals and inducing cell re-population. Recent findings suggest GSCs may be enriched by certain treatment modality. These necessitate the development of novel therapeutics capable of targeting GBM cell plasticity and therapy-resistant GSCs. Here, aided by computer-assisted structure characterization and target identification, we predicted that a novel 5-(2',4'-difluorophenyl)-salicylanilide derivative, LCC-09, could target dopamine receptors and oncogenic markers implicated in GBMs. Bioinformatics data have indicated that dopamine receptor (DRD) 2, DRD4, CD133 and Nestin were elevated in GBM clinical samples and correlated to Temozolomide (TMZ) resistance and increased aldehyde dehydrogenase (ALDH) activity (3.5-8.9%) as well as enhanced (2.1-2.4-fold) neurosphere formation efficiency in U87MG and D54MG GBM cell lines. In addition, TMZ-resistant GSC phenotype was associated with up-regulated DRD4, Akt, mTOR, ?-catenin, CDK6, NF-?B and Erk1/2 expression. LCC-09 alone, or combined with TMZ, suppressed the tumorigenic and stemness traits of TMZ-resistant GBM cells while concomitantly down-regulating DRD4, Akt, mTOR, ?-catenin, Erk1/2, NF-?B, and CDK6 expression. Notably, LCC-09-mediated anti-GBM/GSC activities were associated with the re-expression of tumor suppressor miR-34a and reversal of TMZ-resistance, in vitro and in vivo. Collectively, these data lay the foundation for further exploration of the clinical feasibility of administering LCC-09 as single-agent or combinatorial therapy for patients with TMZ-resistant GBMs.
Project description:Glioblastoma multiforme (GBM) is a highly invasive and chemoradioresistant brain malignancy. Temozolomide (TMZ), a DNA-alkylating agent, is effective against GBM and has become the standard first-line drug. However, the mechanism by which TMZ regulates the progression of GBM remains elusive. Here, we demonstrate that TMZ targets TAp63, a p53 family member, inducing its expression to suppress the progression of human GBM. High levels of TAp63 expression in GBM tissues after TMZ treatment was an indicator of favourable prognosis. In human GBM cells, TMZ-induced TAp63 directly repressed MYC transcription. Activation of this TAp63-MYC pathway by TMZ inhibited human GBM progression both in vitro and in vivo. Furthermore, downregulation of MYC mRNA levels in recurrent GBMs after TMZ treatment correlated with better patient survival. Therefore, our results suggest that the TAp63-mediated transcriptional repression of MYC is a novel pathway regulating TMZ efficacy in GBM.
Project description:Glioblastoma (GBM) is the most malignant glioma, with a median overall survival (OS) of 14-16 months. Temozolomide (TMZ) is the first-line chemotherapy drug for glioma, but whether TMZ should be withheld from patients with GBMs that lack O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is still under debate. DNA methylation profiling holds great promise for further stratifying the responses of MGMT promoter unmethylated GBMs to TMZ. In this study, we studied 147 TMZ-treated MGMT promoter unmethylated GBM, whose methylation information was obtained from the HumanMethylation27 (HM-27K) BeadChips (n = 107) and the HumanMethylation450 (HM-450K) BeadChips (n = 40) for training and validation, respectively. In the training set, we performed univariate Cox regression and identified that 3,565 CpGs were significantly associated with the OS of the TMZ-treated MGMT promoter unmethylated GBMs. Functional analysis indicated that the genes corresponding to these CpGs were enriched in the biological processes or pathways of mitochondrial translation, cell cycle, and DNA repair. Based on these CpGs, we developed a 31-CpGs methylation signature utilizing the least absolute shrinkage and selection operator (LASSO) Cox regression algorithm. In both training and validation datasets, the signature identified the TMZ-sensitive GBMs in the MGMT promoter unmethylated GBMs, and only the patients in the low-risk group appear to benefit from the TMZ treatment. Furthermore, these identified TMZ-sensitive MGMT promoter unmethylated GBMs have a similar OS when compared with the MGMT promoter methylated GBMs after TMZ treatment in both two datasets. Multivariate Cox regression demonstrated the independent prognostic value of the signature in TMZ-treated MGMT promoter unmethylated GBMs. Moreover, we also noticed that the hallmark of epithelial-mesenchymal transition, ECM related biological processes and pathways were highly enriched in the MGMT unmethylated GBMs with the high-risk score, indicating that enhanced ECM activities could be involved in the TMZ-resistance of GBM. In conclusion, our findings promote our understanding of the roles of DNA methylation in MGMT umethylated GBMs and offer a very promising TMZ-sensitivity predictive signature for these GBMs that could be tested prospectively.
Project description:Glioblastoma multiforme (GBM) is one of the most hypoxic tumors of the central nervous system. Although temozolomide (TMZ) is an effective clinical agent in the GBM therapy, the hypoxic microenvironment remains a major barrier in glioma chemotherapy resistance, and the underlying mechanisms are poorly understood. Here, we find hypoxia can induce the protective response to mitochondrion via HIF-1?-mediated miR-26a upregulation which is associated with TMZ resistance in vitro and in vivo. Further, we demonstrated that HIF-1?/miR-26a axis strengthened the acquisition of TMZ resistance through prevention of Bax and Bad in mitochondria dysfunction in GBM. In addition, miR-26a expression levels negatively correlate with Bax, Bad levels, and GBM progression; but highly correlate with HIF-1? levels in clinical cancer tissues. These findings provide a new link in the mechanistic understanding of TMZ resistance under glioma hypoxia microenvironment, and consequently HIF-1?/miR-26a/Bax/Bad signaling pathway as a promising adjuvant therapy for GBM with TMZ.
Project description:Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human. Temozolomide (TMZ) in combination with radiotherapy is currently used for the treatment of glioblastoma (GBM) patients, but less than half of the patients respond to therapy and chemoresistance develops rapidly. Epigenetic silencing of the O(6)-methylguanine-DNA methyltransferase (MGMT) has been associated with longer survival in GBM patients treated with TMZ, but nuclear factor ?B (NF-?B)-mediated survival signaling and TP53 mutations contribute significantly to TMZ resistance. Enhanced NF-?B is in part owing to downregulation of negative regulators of NF-?B activity, including Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and NF-?B inhibitor interacting RAS-like 2 (NKIRAS2). Here we provide a novel mechanism independent of TP53 and MGMT by which oncogenic miR-125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2. GBM cells overexpressing miR-125b showed increased NF-?B activity and upregulation of anti-apoptotic and cell cycle genes. This was significantly associated with resistance of GBM cells to TNF?- and TNF-related inducing ligand-induced apoptosis as well as resistance to TMZ. Conversely, overexpression of anti-miR-125b resulted in cell cycle arrest, increased apoptosis and increased sensitivity to TMZ, indicating that endogenous miR-125b is sufficient to control these processes. GBM cells overexpressing TNFAIP3 and NKIRAS2 were refractory to miR-125b-induced apoptosis resistance as well as TMZ resistance, indicating that both genes are relevant targets of miR-125b. In GBM tissues, high miR-125b expression was significantly correlated with nuclear NF-?B confirming that miR-125b is implicated in NF-?B signaling. Most remarkably, miR-125b overexpression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that this microRNA is an important predictor of response to therapy.
Project description:There is variation in the survival and therapeutic outcome of patients with glioblastomas (GBMs). Therapy resistance is an important challenge in the treatment of GBM patients. The aim of this study was to identify Temozolomide (TMZ) related genes and confirm their clinical relevance. The TMZ-related genes were discovered by analysis of the gene-expression profiling in our cell-based microarray. Their clinical relevance was verified by in silico meta-analysis of the Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) datasets. Our results demonstrated that BICD1 expression could predict both prognosis and response to therapy in GBM patients. First, high BICD1 expression was correlated with poor prognosis in the TCGA GBM cohort (n=523) and in the CGGA glioma cohort (n=220). Second, high BICD1 expression predicted poor outcome in patients with TMZ treatment (n=301) and radiation therapy (n=405). Third, multivariable Cox regression analysis confirmed BICD1 expression as an independent factor affecting the prognosis and therapeutic response of TMZ and radiation in GBM patients. Additionally, age, MGMT and BICD1 expression were combinedly utilized to stratify GBM patients into more distinct risk groups, which may provide better outcome assessment. Finally, we observed a strong correlation between BICD1 expression and epithelial-mesenchymal transition (EMT) in GBMs, and proposed a possible mechanism of BICD1-associated survival or therapeutic resistance in GBMs accordingly. In conclusion, our study suggests that high BICD1 expression may result in worse prognosis and could be a predictor of poor response to TMZ and radiation therapies in GBM patients.
Project description:Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during and after radiotherapy has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O(6)-methylguanine (O(6)-meG) mediates GBM cell killing. Moreover, low or absent expression of O(6)-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O(6)-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O(6)-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O(6)-meG, is mediated by the base excision repair (BER) pathway, and occurs independently of removal of O(6)-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets.