BMP2 sensitizes glioblastoma stem-like cells to Temozolomide by affecting HIF-1? stability and MGMT expression.
ABSTRACT: 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:Temozolomide (TMZ)-resistance in glioblastoma multiforme (GBM) has been linked to upregulation of O(6)-methylguanine-DNA methyltransferase (MGMT). Wild-type (wt) p53 was previously shown to down-modulate MGMT. However, p53 therapy for GBM is limited by lack of efficient delivery across the blood brain barrier (BBB). We have developed a systemic nanodelivery platform (scL) for tumor-specific targeting (primary and metastatic), which is currently in multiple clinical trials. This self-assembling nanocomplex is formed by simple mixing of the components in a defined order and a specific ratio. Here, we demonstrate that scL crosses the BBB and efficiently targets GBM, as well as cancer stem cells (CSCs), which have been implicated in recurrence and treatment resistance in many human cancers. Moreover, systemic delivery of scL-p53 down-modulates MGMT and induces apoptosis in intracranial GBM xenografts. The combination of scL-p53 and TMZ increased the antitumor efficacy of TMZ with enhanced survival benefit in a mouse model of highly TMZ-resistant GBM. scL-p53 also sensitized both CSCs and bulk tumor cells to TMZ, increasing apoptosis. These results suggest that combining scL-p53 with standard TMZ treatment could be a more effective therapy for GBM.
Project description:Glioblastoma multiforme (GBM) contains a population of cells that exhibit stem cell phenotypes. These cancer stem cells (CSCs) may be a source of therapeutic resistance, although support for this important concept is limited.We determined whether early-passage GBM CSCs respond differently than patient-matched, genotypically similar non-CSCs to clinically relevant single or serial doses of temozolomide (TMZ), radiation therapy (XRT), or alternating TMZ treatment and XRT, which is the standard of care for GBM patients.Despite the phenotypic differences, including the presence of stem cell markers and formation of intracranial tumors, the CSCs and matched non-CSCs were equally resistant to TMZ in a majority of patients, using 2 independent assays. TMZ response was consistent with methylated O(6)-DNA methylguanine-methyltransferase (MGMT) and MGMT protein levels in both culture types. In contrast, CSCs were unexpectedly more responsive to XRT compared with matched non-CSCs from 2 patients despite having relatively equal resistance to TMZ. However, for the majority of culture pairs from individual patients, responses in CSCs were indistinguishable from non-CSC cultures.In our patient-matched primary cultures, response to TMZ was tightly linked to the individual tumor's MGMT status and independent of their phenotypic differences. TMZ and XRT together revealed no additive benefit compared with monotherapy for either culture type, in contrast to the notion that the CSC population is more resistant to XRT. If the tumor cell response in vitro mirrors therapeutic response in larger patient cohorts, these rapid assays in primary cultures could allow -empirical selection of efficacious therapeutic agents on a patient-specific basis.
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:Long noncoding RNAs (lncRNAs) have emerged as new regulatory molecules implicated in diverse biological processes, including therapeutic resistance. However, the mechanisms underlying lncRNA-mediated temozolomide (TMZ) resistance in glioblastoma (GBM) remain largely unknown. To illustrate the role of lncRNA in TMZ resistance, we induce TMZ-resistant GBM cells, perform a lncRNA microarray of the parental and TMZ-resistant cells, and find an unreported lncRNA in GBM, lnc-TALC (temozolomide-associated lncRNA in glioblastoma recurrence), correlated with TMZ resistance via competitively binding miR-20b-3p to facilitate c-Met expression. A phosphorylated AKT/FOXO3 axis regulated lnc-TALC expression in TMZ-resistant GBM cells. Furthermore, lnc-TALC increased MGMT expression by mediating the acetylation of H3K9, H3K27 and H3K36 in MGMT promoter regions through the c-Met/Stat3/p300 axis. In clinical patients, lnc-TALC is required for TMZ resistance and GBM recurrence. Our results reveal that lnc-TALC in GBM could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy.
Project description: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:<h4>Background</h4>Glioblastoma multiforme (GBM) is a highly aggressive brain tumor in which cancer cells with stem cell-like features, called cancer stem cells (CSCs), were identified. Two CSC populations have been previously identified in GBM, one derived from the GBM area called enhanced lesion (GCSCs) and the other one from the brain area adjacent to the tumor margin (PCSCs) that greatly differ in their growth properties and tumor-initiating ability. To date the most effective chemotherapy to treat GBM is represented by alkylating agents such as temozolomide (TMZ), whose activity can be regulated by histone deacetylases (HDACs) inhibitors through the modulation of O6-methylguanine-DNA methyltransferase (MGMT) expression. Levetiracetam (LEV), a relatively new antiepileptic drug, modulates HDAC levels ultimately silencing MGMT, thus increasing TMZ effectiveness. However, an improvement in the therapeutic efficacy of TMZ is needed.<h4>Methods</h4>Cell proliferation was investigated by BrdU cell proliferation assay and by Western Blot analysis of PCNA expression. Apoptosis was evaluated by Western Blot and Immunofluorescence analysis of the cleaved Caspase-3 expression. MGMT and HDAC4 expression was analyzed by Western Blotting and Immunofluorescence. Statistical analysis was performed using the Student's <i>t</i> test and Mann-Whitney test.<h4>Results</h4>Here we evaluated the effect of TMZ on the proliferation rate of the IDH-wildtype GCSCs and PCSCs derived from six patients, in comparison with the effects of other drugs such as etoposide, irinotecan and carboplatin. Our results demonstrated that TMZ was less effective compared to the other agents; hence, we verified the possibility to increase the effect of TMZ by combining it with LEV. Here we show that LEV enhances the effect of TMZ on GCSCs proliferation (being less effective on PCSCs) by decreasing MGMT expression, promoting HDAC4 nuclear translocation and activating apoptotic pathway.<h4>Conclusions</h4>Although further studies are needed to determine the exact mechanism by which LEV makes GBM stem cells more sensitive to TMZ, these results suggest that the clinical therapeutic efficacy of TMZ in GBM might be enhanced by the combined treatment with LEV.
Project description:Hypoxia is a key driver of tumor adaptation promoting tumor progression and resistance to therapy. Hypoxia related pathways might represent attractive targets for the treatment of Glioblastoma Multiforme (GBM), that up to date is characterized by a poor prognosis. Primary aim of this study was to investigate the role of hypoxia and hypoxia-related modifications in the effect of temozolomide (TMZ) given alone or in association with the antidiabetic agent Metformin (MET) or the PI3K/mTOR blocker, BEZ235. The study was conducted in the TMZ responsive U251 and resistant T98 GBM cells. Our results showed that during hypoxia, TMZ plus MET reduced viability of U251 cells affecting also CD133 and CD90 expressing cells. This effect was associated with a reduction of HIF-1? activity, VEGF release and AKT activation. In T98 TMZ-resistant cells, TMZ plus MET exerted similar effects on HIF-1?. However, in this cell line, TMZ plus MET failed to reduce CD133 positive cells and AKT phosphorylation. Nevertheless, the administration of the dual PI3K/mTOR inhibitor BEZ235 potentiated the effect of TMZ plus MET on cell viability, inducing a pro-apoptotic phenotype during hypoxic condition also in T98 cells, suggesting the block of the PI3K/AKT/mTOR pathway as a complementary target to further overcome GBM resistance during hypoxia. In conclusion, we proposed TMZ plus MET as suitable treatment to revert TMZ-resistance also during hypoxia, an effect potentiated by the inhibition of PI3K/mTOR axis.
Project description:Glioblastoma multiforme (GBM) is the most common adult malignant glioma with poor prognosis due to the resistance to radiotherapy and chemotherapy, which might be critically involved in the repopulation of cancer stem cells (CSCs) after treatment. We had investigated the characteristics of cancer stem-like side population (SP) cells sorted from GBM cells, and studied the effect of Honokiol targeting on CSCs. GBM8401 SP cells possessed the stem cell markers, such as nestin, CD133 and Oct4, and the expressions of self-renewal related stemness genes, such as SMO, Notch3 and IHH (Indian Hedgehog). Honokiol inhibited the proliferation of both GBM8401 parental cells and SP cells in a dose-dependent manner, the IC50 were 5.3±0.72 and 11±1.1 ?M, respectively. The proportions of SP in GBM8401 cells were diminished by Honokiol from 1.5±0.22% down to 0.3±0.02% and 0.2±0.01% at doses of 2.5 ?M and 5 ?M, respectively. The SP cells appeared to have higher expression of O6-methylguanine-DNA methyltransferase (MGMT) and be more resistant to Temozolomide (TMZ). The resistance to TMZ could be only slightly reversed by MGMT inhibitor O6-benzylguanine (O6-BG), but markedly further enhanced by Honokiol addition. Such significant enhancement was accompanied with the higher induction of apoptosis, greater down-regulation of Notch3 as well as its downstream Hes1 expressions in SP cells. Our data indicate that Honokiol might have clinical benefits for the GBM patients who are refractory to TMZ treatment.
Project description:Abstract Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Current standard-of-care includes surgery, radiation and treatment with temozolomide (TMZ), however nearly all tumors recur and the prognosis for recurrent GBM is dismal. Resistance to TMZ is correlated with expression of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT), which is highly expressed in a majority of GBM tumors. Dianhydrogalactitol (VAL-083) is a first-in-class bi-functional DNA-targeting agent that exhibited activity against GBM in NCI-sponsored clinical trials both as a single agent and in combination with radiotherapy. VAL-083 readily crosses the blood-brain barrier and accumulates in brain tumor tissue. We have demonstrated that VAL-083 targets N7-Guanine and rapidly induces interstrand DNA cross-links, leading to DNA double-strand breaks, S/G2 cell-cycle arrest and cell death in GBM cell lines and GBM cancer stem cells (CSCs) in vitro. This unique N7-guanine targeting mechanism not only circumvents MGMT-mediated chemo-resistance but also maintains cytotoxic activity in cancer cells deficient in mismatch repair (MMR). These data suggest VAL-083 may offer a superior chemotherapeutic alternative in the treatment of MGMT-unmethylated or MMR deficient GBM. Here, we provide an update of ongoing clinical trials with VAL-083 in MGMT-unmethylated GBM: i) a single-arm, biomarker-driven, Phase II study to determine if VAL-083 treatment of MGMT-unmethylated adult GBM patients at first recurrence/progression, prior to bevacizumab improves survival compared to historical lomustine control (clinicaltrials.gov identifier: NCT02717962); ii) a single-arm, biomarker-driven, Phase II study to confirm the tolerability and efficacy of VAL-083 in combination with radiotherapy in newly diagnosed MGMT-unmethylated GBM patients (clinicaltrials.gov identifier: NCT03050736). The results of these studies may support a new treatment paradigm in for the treatment of MGMT-unmethylated GBM.
Project description:CpG methylation within the O6-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with enhanced survival of glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ). Although MGMT promoter is methylated in approximately 50% of GBM, several studies have reported a lack of correlation between MGMT methylation and protein expression levels and consequently inaccurate discrimination of TMZ sensitive and resistant patients. To understand the limitations of currently used assays, TMZ responsiveness of 13 GBM xenograft lines was correlated with MGMT protein expression and MGMT promoter methylation determined by (1) standard methylation-specific polymerase chain reaction (MS-PCR), (2) quantitative MS-PCR (qMS-PCR), and (3) bisulfite sequencing. For each xenograft line, mice with established intracranial xenografts were treated with vehicle control or TMZ (66 mg/kgx5 days), and TMZ response was defined as relative prolongation in median survival for TMZ-treated versus control-treated mice. The relative survival benefit with TMZ was inversely related to MGMT protein expression (r=-0.75; P=0.003) and directly correlated with qMS-PCR (r=0.72; P=0.006). There was a direct correlation between MGMT methylation signal by qMS-PCR and the number of methylated CpG sites within the region amplified by MS-PCR (r=0.78, P=0.002). However, bisulfite sequencing revealed heterogeneity in the extent of CpG methylation in those tumors with a robust qMS-PCR signal. Three of the 4 GBM lines with a qMS-PCR signal greater than 10% had at least 1 unmethylated CpG site, while only one line was fully methylated at all 12 CpG sites. These data highlight one potential limitation of the evaluation of MGMT methylation by MS-PCR assay and suggest that more detailed evaluation of methylation at individual CpG sites relative to TMZ response may be worth pursuing.