The evolution of alternative splicing in glioblastoma under therapy.
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE155434 | GEO | 2020/12/10
REPOSITORIES: GEO
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Project description:Glioblastoma multiforme (GBM) is the most aggressive and frequent primary brain tumor with dismal prognosis which might be due to the resistance to the current standard chemotherapeutic regimens and high recurrence rate even after total resection of GBM mass. Here, to gain the mechanistic insights on the altered phenotypic acquisition of recurrent tumors compared to the corresponding primary tumors, we performed gene expression profiling on the 43 cases of GBM tumors including 15 paired recurrent and primary GBMs. Unsupervised and consensus clustering analyses revealed two subtypes of G1 and G2, which were enriched with proliferation, and neuron-like gene expression traits, respectively. Most of the primary tumors were classified into G1, while the recurrent tumors were classified into both G1 and G2 groups. The gene expression of recurrent GBMs in G2 group have changed from proliferation type to neuron-like type, while the other recurrent GBMs in the G1 group showed no significant subtype change. In addition, we observed that the recurrent tumors in G1 subtype were characterized with the expression of stemness-related genes and DNA-repair genes. Of the G1 subtype classifier genes, AURKA/B and HOX genes were identified as key hub regulators. On the other hand, the recurrent G2 tumors have acquired the expression of MGMT but suppression of MSH6 gene, which may lead to TMZ resistance. The consistency of these findings could be validated with independent data sets. In summary, our molecular classification of recurrent GBMs could suggest that the differential mechanisms of drug resistance be involved in the tumor recurrence. Targeting the subtype-specific mechanisms might be a beneficial strategy for the treatment of recurrent GBMs.
2015-10-20 | GSE62153 | GEO
Project description:Most clinical trials for glioblastoma (GBM) enroll patients at recurrence. However, most pre-clinical animal models are of primary disease. Most studies have used tissues from primary tumors. Knowledge of GBM cellular composition at recurrence is limited. We profiled 80 human IDH-wild-type GBM specimens via single-nucleus RNA sequencing, 40 primary tumors and 40 patient-matched recurrent tumors. Select cohorts were also profiled via single-cell assay for transposase-accessible chromatin, single-cell digital-spatial, and single-cell spatial-transcriptomic assays. All patients were treated only with standard-of-care therapy: temozolomide, radiation and surgical resection. The cohort had a 1.2 male/female ratio, ages 35–76. We found a significant increase at recurrence in cells with the Verhaak mesenchymal phenotype and fewer proneural cells. Although there was an increase in the percentage of cycling cells overall, the fraction of cycling proneural cells decreased while the fraction of cycling mesenchymal cells increased. These changes were concomitant with a significant increase in the percentage of tumor-infiltrating monocytic-lineage cells derived from the periphery. Taken together, these findings support a proneural-to-mesenchymal shift at recurrence due to an increase in the birth rate of mesenchymal cells, supported by an expansion of myeloid-derived cells from peripheral blood. Although the percentages of glioblastoma-infiltrating T-cells are generally low (~1% on average in our data), we found a statistically significant increase in T-cell abundance at recurrence. We identified a cohort of T-cell outliers that demonstrate 2-to-8-fold increases in T-cells over average levels. We validated that T-cells in these tumors have extravasated beyond the perivascular space and into the cellular tumor via immunohistochemistry and spatial profiling. We present our ongoing analysis of the paracrine signals, upstream transcription-factor expression, and cis-regulatory grammars utilized in immune-outlier GBMs. We describe how these programs change under therapy. These studies shed light on the effect of standard therapy in shaping GBM composition at recurrence.
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Project description:Recent studies have identified alternative splicing (AS) as a novel source of neoantigens for immunotherapy. Surprisingly little is known about the AS milieu in recurrent glioblastoma (GBM), despite this being the venue for most clinical trials. We profiled 29 primary-recurrent paired human GBM specimens via RNA sequencing and re-analyzed RNA-sequencing data from non-malignant human brain tissues. From these data, we reconstructed the landscape of AS in GBM through recurrence and contrasted that to isoforms observed in non-malignant brain. The AS events we identified were cross-referenced with single-cell GBM atlases to determine cell-type specific splicing patterns. From this we identified novel splicing events in cell-surface proteins that are suitable targets for engineered T-cell therapies. We found recurrent-specific isoforms of mitogen-activated kinase pathway genes which are expressed exclusively by GBM stem-like cells that enhance invasiveness. These studies shed light on the effect of therapy on AS and identify novel targets for emerging immunotherapies.
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Project description:Glioblastoma multiforme (GBM) is the most lethal brain malignant neoplasm, associated with poor prognosis and high recurrence. The mechanisms involved in GBM development and recurrent remains largely unknown. Hence, it’s considerably imperative to explore the potential targets and unravel the mechanism implicated in GBM tumorigenesis and recurrence. In our study, mass spectrometry-based label-free quantitative proteomics was employed to explore the proteomic profiling in non-paired samples of primary and recurrent GBM. Bioinformatic analysis of differentially expression proteins (DEPs) were applied to screen the hub genes. Subsequently, hub genes expression was verified by western blot and immunohistochemistry (IHC) in GBM tissues, followed by its biological effect on GBM at a cellular level determined. We contoured the divergent landscape of proteome between primary and recurrent GBM, with only ten overlapped DEPs included. The several hub genes screened (e.g., CKAP4 and CANX) and enriched pathways correlated with primary GBM such as post-translation, metabolism, energy pathways and cell growth were profiled. Furthermore, CKAP4 was implicated in proliferation, migration and invasion of A172 and T98G cell lines. However, UQCRC1, together with relative enriched pathways such as oxidative phosphorylation, were screened in recurrent GBM. Establishment of the proteomics and several candidate proteins identified could pose considerable access to understand complex biochemical processes and obtain a better picture of GBM biology.
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Project description:Tumor recurrence following a standard treatment is the major cause of mortality for glioblastoma (GBM) patients. However, insights on the evolutionary process of the tumor have been limited due to the lack of longitudinally sampled cases. Here, we describe our genomic analyses of 38 GBM patients with pre- and post-treatment samples for each individual (78 tumor samples in total; aCGH data were obtained for 36 among the 78). A substantial shift in the landscape of driver alterations was associated with distant appearances of the recurrent tumors from the initial tumor, suggesting that the genomic profile of an initial tumor can mislead targeted therapies for the distant recurrent tumor. In addition, in contrast to the previous work on IDH1-R132H low-grade gliomas, our GBM patients rarely developed hypermutation following the standard treatment, supporting the safety of temozolomide for IDH1-wild type, primary GBMs under the current standard regimen.
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Project description:The objective of our study was to determine whether aberrant alternative splicing could play a role in the malignant phenotype of GBM. Patients with GBM were operated with 5-aminolevulinic fluorescence guided surgery. The fluorescent was used to take biopsies from the tumor center, and from adjacent normal-looking tissue. Three paired normal/GBM samples were analyzed in a HJAY J array. We validated our results with conventional PCR and qRT-PCR in those tumor samples and in ten additional GBMs. We performed functional studies using MTT assays (proliferation) and IF qRT-PCR (differentiation). We generated a list of seven genes with differential alternative splicing between central tumor samples and the adjacent normal-looking tissue. DPF-2(BAF45d) was one of our top candidates. Interestingly, DPF-2 is known as a tumor suppressor gene in the context of the SWI/SNF complex and plays a key role in the development of the central nervous system. The inhibition of our DPF-2 isoform resulted in a significative reduction in proliferation and in a morphology changes towards a more differentiated phenotype in GBM cell lines. Interestingly, our DPF2 tumoral isoform was the predominant transcript in early postnatal murine neural precursors and its expression disappeared as these cells were instructed towards neurons. Our results suggest that the alternative splicing of DPF-2 in GBM could participate in the maintenance of an undifferentiated cellular state. In addition, our data suggest that alternative splicing is a mechanism that could be central to GBM development We compared three paired samples of tumor (glioma)/normal like and 1 sample of pooled brain RNA from healthy donors
2016-09-01 | E-GEOD-76070 | biostudies-arrayexpress
Project description:Despite aggressive multi-modal treatment, advanced head and neck squamous cell carcinoma (HNSCC) patients are at high risk of recurrence. n=19 different selected areas of matched pairs (n=3) of primary/recurrent tumor specimens were subjected to RNA sequencing. Transcriptional subtyping (classical/CL, basal/BA, inflamed-mesenchymal/IMS) in matched primary and recurrent HNSCC specimens revealed heterogeneity.
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Project description:Glioblastoma multiforme (GBM) is the most common form of malignant glioma and is characterized by marked genetic instability, extensive intra-tumoral histopathological variability, and unpredictable variation in its clinical behavior. We investigated global gene expression in surgical samples of primary brain tumors. Gene expression profiling revealed large differences between normal brain samples and tumor tissues and between GBMs and lower grade oligodendroglial tumors. Extensive differences in gene expression were found among GBMs, particularly in genes involved in angiogenesis, immune cell infiltration, and extracellular matrix re-modeling. Significantly, we found that the global gene expression patterns in paired specimens from the same GBM invariably were more closely related to each other than to any other tumor, even when the paired specimens had strikingly divergent histologies. Survival analyses revealed a set of approximately 70 genes that were more highly expressed in rapidly progressing tumors and which stratified GBMs into two groups that differed by more than four-fold in median duration of survival. We further investigated one gene from the group, FABP7, and confirmed its association with survival in two unrelated cohorts totaling 105 patients. Expression of FABP7 enhanced the motility of glioma-derived cells in vitro. Our analyses thus provide a series of prognostic markers and suggest that their expression may enhance the invasive potential of tumor cells.
2005-11-11 | E-SMDB-2677 | biostudies-arrayexpress
Project description:Treating recurrent GBM is a clinical challenge due to its highly resistant and aggressive nature. In order to develop new therapeutic targets for recurrent GBM a better understanding of its molecular landscape is necessary. Here we used a cellular model, developed in our lab which generates paired primary and recurrent samples from GBM cell lines and primary patient samples hence allowing us to compare the molecular differences between the two populations. Total RNA seq analysis of parent and recurrent population of two cell lines and one patient sample revealed a significant upregulation of Extracellular matrix interaction in recurrent population. Since matrix stiffness plays a pivotal role in cell-ECM interaction and downstream signaling, we developed a system that mimicked the brain like substrate stiffness by using collagen coated polyacrylamide-based substrate whose stiffness can be modified from normal brain (0.5kPa) to tumorigenic (10kPa). Using these substrates, we were able to capture the morphological and physiological differences between parent and recurrent GBM which were not evident on plastic surfaces (~1 GPa). On 0.5kPa, unlike circular parent cells, recurrent GBM cells showed two morphologies (circular and elongated). The recurrent cells growing on 0.5kPa also showed higher proliferation, invasion, migration and in-vivo tumorigenicity in orthotropic GBM mouse model, compared to parent cells. Furthermore, recurrent cells exhibited elevated velocity irrespective of substrate stiffness, which indicated that recurrent cells may possess inherent differential mechanosignalling ability which was reflected by higher expression of ECM proteins like Collagen IVA, MMP2 and MMP9. Moreover, mice brain injected with recurrent cells grown on 0.5kPa substrate showed higher Young’s modulus values suggesting that recurrent cells conditioned on 0.5kPa make the surrounding ECM stiffer. Importantly, inhibition of EGFR signaling, that is amplified with tissue stiffening in GBM resulted in decreased invasion, migration and proliferation in 0.5kPa recurrent cells, but interestingly survival remained unaffected, highlighting the importance of mimicking the physiological stiffness of the brain mimicking clinical scenario. Total RNA seq analysis of parent and recurrent cells grown on plastic and 0.5kPa substrate identified PLEKHA7 as significantly upregulated gene specifically in 0.5kPa recurrent sample. Higher protein expression of PLEKHA7 in recurrent GBM as compared to primary GBM was validated in patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provides a model system that captures the differential mechanosensing signals of primary and recurrent GBM cells and identifies a novel potential target specific for recurrent GBM.
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Project description:The objective of our study was to determine whether aberrant alternative splicing could play a role in the malignant phenotype of GBM. Patients with GBM were operated with 5-aminolevulinic fluorescence guided surgery. The fluorescent was used to take biopsies from the tumor center, and from adjacent normal-looking tissue. Three paired normal/GBM samples were analyzed in a HJAY J array. We validated our results with conventional PCR and qRT-PCR in those tumor samples and in ten additional GBMs. We performed functional studies using MTT assays (proliferation) and IF qRT-PCR (differentiation). We generated a list of seven genes with differential alternative splicing between central tumor samples and the adjacent normal-looking tissue. DPF-2(BAF45d) was one of our top candidates. Interestingly, DPF-2 is known as a tumor suppressor gene in the context of the SWI/SNF complex and plays a key role in the development of the central nervous system. The inhibition of our DPF-2 isoform resulted in a significative reduction in proliferation and in a morphology changes towards a more differentiated phenotype in GBM cell lines. Interestingly, our DPF2 tumoral isoform was the predominant transcript in early postnatal murine neural precursors and its expression disappeared as these cells were instructed towards neurons. Our results suggest that the alternative splicing of DPF-2 in GBM could participate in the maintenance of an undifferentiated cellular state. In addition, our data suggest that alternative splicing is a mechanism that could be central to GBM development
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