The lncRNA TP73-AS1 is linked to aggressiveness in glioblastoma and promotes temozolomide resistance in glioblastoma cancer stem cells

Abstract

Glioblastoma multiform (GBM) is the most common brain tumor characterized by a dismal prognosis. GBM cancer stem cells (gCSC) or tumor-initiating cells are the cell population within the tumor-driving therapy resistance and recurrence. While temozolomide (TMZ), an alkylating agent, constitutes the first-line chemotherapeutic significantly improving survival in GBM patients, resistance against this compound commonly leads to GBM recurrence and treatment failure. Although the roles of protein-coding transcripts, proteins and microRNA in gCSC, and therapy resistance have been comprehensively investigated, very little is known about the role of long noncoding RNAs (lncRNAs) in this context. Using nonoverlapping, independent RNA sequencing and gene expression profiling datasets, we reveal that TP73-AS1 constitutes a clinically relevant lncRNA in GBM. Specifically, we demonstrate significant overexpression of TP73-AS1 in primary GBM samples, which is particularly increased in the gCSC. More importantly, we demonstrate that TP73-AS1 comprises a prognostic biomarker in glioma and in GBM with high expression identifying patients with particularly poor prognosis. Using CRISPRi to downregulate our candidate lncRNA in gCSC, we demonstrate that TP73-AS1 promotes TMZ resistance in gCSC and is linked to regulation of the expression of metabolism- related genes and ALDH1A1, a protein known to be expressed in cancer stem cell markers and protects gCSC from TMZ treatment. Taken together, our results reveal that high TP73-AS1 predicts poor prognosis in primary GBM cohorts and that this lncRNA promotes tumor aggressiveness and TMZ resistance in gCSC.

Fig. 1. TP73-AS1 is relevant in glioblastoma.

a TP73-AS1 expression in normal, LGG, and GBM tumor tissue. Normal tissue data were obtained from GTEx and tumor data from TCGA. Data were analyzed using GEPIA (gepia.cancer-pku.cn). b TP73-AS1 expression in tumor samples with or without IDH1 R132 mutation. Data were analyzed using R2. c Kaplan–Meier plots of patient outcome based on TP73-AS1 expression. Plots were generated using http://www.betastasis.com/ and R2. d Kaplan–Meier plots of patient outcome based on TP73-AS1 expression in GBM. Plots were generated using http://www.betastasis.com/. e Kaplan–Meier plots of patient outcome based on TP73-AS1 expression in glioma, stratified according to the annotated IDH mutation status and glioma subtype. Data, statistical evaluation, and visualization were obtained using the R2 website “R2: Genomics Analysis and Visualization Platform” (http://r2.amc.nl) and http://www.betastasis.com/

Fig. 2. TP73-AS1 kd does not induce cell death in gCSC.

a Histone K27ac of the TP73-AS1 locus in gCSC (CSC), differentiated gCSC (dCSC), and differentiated gCSC that had been reprogrammed to gCSC (iCSC). The putative promoter region of TP73-AS1 is highlighted in gray. Data were obtained from graphs generated using http://www.broadinstitute.org/epigenomics/dataportal/clonePortals/Suva_Cell_2014.html. b The expression level of the TP73-AS1 in normal brain tissue versus neuronal stem cells and gCSC. Data, statistical evaluation, and visualization were obtained using the R2 website “R2: Genomics Analysis and Visualization Platform” (http://r2.amc.nl). c Efficient knockdown of the lncRNA TP73-AS1 in GBM tumor cells using CRISPRi. gCSC cells expressing a doxycycline-inducible dCAS9-KREB fusion protein and the indicated gRNAs targeting TP73-AS1 promoter region were generated. The indicated GBM tumor cell lines were engineered to express doxycyline-inducible dCAS9-KREB and gRNA targeting the TP73-AS1 promoter or scramble controls. Cells were induced with doxycycline for 10 days, after which the levels of TP73-AS1 were measured using qRT-PCR. *p < 0.05; n = 3. dCAS9-KREB expression was induced after 10 days, and subsequently, the percentage of cell death induction was determined using Annexin V and PI staining and was measured by flow cytometry. Representative images are shown. The graph represents average ± SD; n = 3

Fig. 3. TP73-AS1 promotes self-renewal in G7 but not in G26 gCSC.

a gCSC cells expressing a doxycycline-inducible dCAS9-KREB fusion protein and the indicated gRNAs targeting TP73-AS1 promoter region were generated. dCAS9-KREB expression was induced for 10 days, after which the expression of the indicated stemness and differentiation markers was determined using immunofluorescence. Representative images are shown. Bar = 100 μM. b LDA assay measures the self-renewal capacity. dCAS9-KREB expression was induced for 10 days, after which the cells were plated in 96-well plates in the concentration of (1, 5, 10, and 20 cells per well). Spheres were counted manually 21 days after the induction. The estimate depicts the confidence interval for one per stem cell frequency. Data were analyzed using http://bioinf.wehi.edu.au/software/elda/

Fig. 4. TP73-AS1 promotes gCSC resistance to TMZ.

a Representative images of TP73-AS1 depleted and control gCSC cells treated with TMZ (500 µM) for 7 days. Images were recorded using a light microscope. b TP73-AS1 depleted and control gCSC models were treated with TMZ (500 µM) for 7 days, after which the viability was determined using Annexin V/PI staining and flow cytometry. Representative images are shown. Bar = 50 μM; *p < 0.05; n = 3; average ± SD. c TP73-AS1 depleted and control gCSC were treated with the indicated dose of TMZ for 7 days, after which viability was measured using crystal violet staining and a plate reader. Values represent the relative average OD; n ≥ 3; *p < 0.05 Student’s t test

Fig. 5. Transcriptional landscape upon TP73-AS1 depletion in gCSCs.

a Heat map depicting transcripts whose expression is affected by TP73-AS1 kd. Red represents upregulation and blue represents downregulation. b Transcripts affected by TP73-AS1 kd in each cell line and conditions were analyzed to identify significantly affected GO categories. The number of GO categories identified in each cell line, condition, and direction of gene expression change (upregulation or downregulation by TP73-AS1 kd) are shown. c Adjusted p values (average ± SD) of GO categories related to the indicated biological functions are shown. Note that there were no common categories in the list of genes that were downregulated in the kd cells. See Table S1 for a complete list

Fig. 6. TP73-AS1 depletion leads to reduced ALDH1A1 expression.

a The indicated gCSC lines were treated with TMZ and the levels of ALDH1A1 were measured using qRT-PCR. The results matched the trends found in RNAseq experiments, as indicated by the RNAseq group each condition belongs to. b G7 cells were treated with TMZ (300 μM), DEAB (200 μM), or a combination of the two compounds for 5 days, after which cell death was measured using PI and FACS. Average values and SEM are shown. n = 3; *p < 0.05 ANOVA