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A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide.

Pojo M, Gonçalves CS, Xavier-Magalhães A, Oliveira AI, Gonçalves T, Correia S, Rodrigues AJ, Costa S, Pinto L, Pinto AA, Lopes JM, Reis RM, Rocha M, Sousa N, Costa BM - Oncotarget (2015)

Bottom Line: Additionally, HOXA9 promoted the malignant transformation of human immortalized astrocytes in an orthotopic in vivo model, and caused tumor-associated death.Importantly, the pharmacological inhibition of BCL2 with the BH3 mimetic ABT-737 reverted temozolomide resistance in HOXA9-positive cells.These data establish HOXA9 as a driver of glioma initiation, aggressiveness and resistance to therapy.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar 4710-057 Braga, Portugal.

ABSTRACT
Glioblastoma is the most malignant brain tumor, exhibiting remarkable resistance to treatment. Here we investigated the oncogenic potential of HOXA9 in gliomagenesis, the molecular and cellular mechanisms by which HOXA9 renders glioblastoma more aggressive, and how HOXA9 affects response to chemotherapy and survival. The prognostic value of HOXA9 in glioblastoma patients was validated in two large datasets from TCGA and Rembrandt, where high HOXA9 levels were associated with shorter survival. Transcriptomic analyses identified novel HOXA9-target genes with key roles in cancer-related processes, including cell proliferation, DNA repair, and stem cell maintenance. Functional studies with HOXA9-overexpressing and HOXA9-silenced glioblastoma cell models revealed that HOXA9 promotes cell viability, stemness and invasion, and inhibits apoptosis. Additionally, HOXA9 promoted the malignant transformation of human immortalized astrocytes in an orthotopic in vivo model, and caused tumor-associated death. HOXA9 also mediated resistance to temozolomide treatment in vitro and in vivo via upregulation of BCL2. Importantly, the pharmacological inhibition of BCL2 with the BH3 mimetic ABT-737 reverted temozolomide resistance in HOXA9-positive cells. These data establish HOXA9 as a driver of glioma initiation, aggressiveness and resistance to therapy. In the future, the combination of BH3 mimetics with temozolomide should be further explored as an alternative treatment for glioblastoma.

No MeSH data available.


Related in: MedlinePlus

HOXA9 transcriptomes in hTERT/E6/E7 human immortalized astrocytes, and in U87MG, U251 and GBML18 glioblastoma cell lines(A) qPCR confirming the overexpression of HOXA9 in hTERT/E6/E7-HOXA9 and U87MG-HOXA9 cells and HOXA9-silencing in U251-shHOXA9 and GBML18-shHOXA9 cells, comparing to their respective control counterparts. (B) Venn diagram summarizing the number of differentially expressed transcripts in the microarray data in all cell lines. The numbers in each area represent the total number of transcripts within each intersection. (C–F) DAVID was used to query the HOXA9-transcriptome from each cell line (C, hTERT/E6/E7; D, U87MG; E, U251; F, GBML18), in order to identify enriched biological terms on the differentially expressed genes extracted from the microarray data. Statistically significant enriched GO terms are shown for each cell line.
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Figure 2: HOXA9 transcriptomes in hTERT/E6/E7 human immortalized astrocytes, and in U87MG, U251 and GBML18 glioblastoma cell lines(A) qPCR confirming the overexpression of HOXA9 in hTERT/E6/E7-HOXA9 and U87MG-HOXA9 cells and HOXA9-silencing in U251-shHOXA9 and GBML18-shHOXA9 cells, comparing to their respective control counterparts. (B) Venn diagram summarizing the number of differentially expressed transcripts in the microarray data in all cell lines. The numbers in each area represent the total number of transcripts within each intersection. (C–F) DAVID was used to query the HOXA9-transcriptome from each cell line (C, hTERT/E6/E7; D, U87MG; E, U251; F, GBML18), in order to identify enriched biological terms on the differentially expressed genes extracted from the microarray data. Statistically significant enriched GO terms are shown for each cell line.

Mentions: The endogenous expression levels of HOXA9 in a panel of glioma cell lines was evaluated by qPCR (Supplementary Figure 1). In order to provide the first characterization of HOXA9 targets on a genome-wide level in GBM, expression microarray analyses were performed in matched HOXA9-positive and HOXA9-negative human GBM cell models (U87MG-MSCV vs. U87MG-HOXA9, human immortalized astrocytes hTERT/E6/E7-MSCV vs. hTERT/E6/E7-HOXA9, U251-shControl vs. U251-shHOXA9, and a primary GBM cell line GBML18-shControl vs. GBML18-shHOXA9; Figure 2A and 2B). Due to HOXA9 expression, 3454 probes were significantly differentially expressed in U87MG cells (1537 upregulated and 1917 downregulated), 417 probes in hTERT/E6/E7 cells (166 upregulated and 251 downregulated), 2452 probes in U251 cells (1301 upregulated and 1151 downregulated), and 5886 probes in GBML18 patient-derived primary cells (2802 upregulated and 3084 downregulated; Figure 2B and Supplementary Figures 4–9; GEO accession number GSE56517). Only a small subset of probes was consistently altered in the 3 GBM cell lines (17 probes upregulated and 40 downregulated due to HOXA9 expression in U87MG, U251 and GBML18 cells), which, as expected, was even smaller when integrating the non-tumor immortalized astrocytes (1 probe upregulated and 3 downregulated; Figure 2B), indicating that the transcriptome of HOXA9 is cell-type dependent, as previously suggested in leukemia models [19]. Gene-specific expression analyses in a subset of HOXA9 targets validated the array data (Supplementary Figure 2A–2D).


A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide.

Pojo M, Gonçalves CS, Xavier-Magalhães A, Oliveira AI, Gonçalves T, Correia S, Rodrigues AJ, Costa S, Pinto L, Pinto AA, Lopes JM, Reis RM, Rocha M, Sousa N, Costa BM - Oncotarget (2015)

HOXA9 transcriptomes in hTERT/E6/E7 human immortalized astrocytes, and in U87MG, U251 and GBML18 glioblastoma cell lines(A) qPCR confirming the overexpression of HOXA9 in hTERT/E6/E7-HOXA9 and U87MG-HOXA9 cells and HOXA9-silencing in U251-shHOXA9 and GBML18-shHOXA9 cells, comparing to their respective control counterparts. (B) Venn diagram summarizing the number of differentially expressed transcripts in the microarray data in all cell lines. The numbers in each area represent the total number of transcripts within each intersection. (C–F) DAVID was used to query the HOXA9-transcriptome from each cell line (C, hTERT/E6/E7; D, U87MG; E, U251; F, GBML18), in order to identify enriched biological terms on the differentially expressed genes extracted from the microarray data. Statistically significant enriched GO terms are shown for each cell line.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4480707&req=5

Figure 2: HOXA9 transcriptomes in hTERT/E6/E7 human immortalized astrocytes, and in U87MG, U251 and GBML18 glioblastoma cell lines(A) qPCR confirming the overexpression of HOXA9 in hTERT/E6/E7-HOXA9 and U87MG-HOXA9 cells and HOXA9-silencing in U251-shHOXA9 and GBML18-shHOXA9 cells, comparing to their respective control counterparts. (B) Venn diagram summarizing the number of differentially expressed transcripts in the microarray data in all cell lines. The numbers in each area represent the total number of transcripts within each intersection. (C–F) DAVID was used to query the HOXA9-transcriptome from each cell line (C, hTERT/E6/E7; D, U87MG; E, U251; F, GBML18), in order to identify enriched biological terms on the differentially expressed genes extracted from the microarray data. Statistically significant enriched GO terms are shown for each cell line.
Mentions: The endogenous expression levels of HOXA9 in a panel of glioma cell lines was evaluated by qPCR (Supplementary Figure 1). In order to provide the first characterization of HOXA9 targets on a genome-wide level in GBM, expression microarray analyses were performed in matched HOXA9-positive and HOXA9-negative human GBM cell models (U87MG-MSCV vs. U87MG-HOXA9, human immortalized astrocytes hTERT/E6/E7-MSCV vs. hTERT/E6/E7-HOXA9, U251-shControl vs. U251-shHOXA9, and a primary GBM cell line GBML18-shControl vs. GBML18-shHOXA9; Figure 2A and 2B). Due to HOXA9 expression, 3454 probes were significantly differentially expressed in U87MG cells (1537 upregulated and 1917 downregulated), 417 probes in hTERT/E6/E7 cells (166 upregulated and 251 downregulated), 2452 probes in U251 cells (1301 upregulated and 1151 downregulated), and 5886 probes in GBML18 patient-derived primary cells (2802 upregulated and 3084 downregulated; Figure 2B and Supplementary Figures 4–9; GEO accession number GSE56517). Only a small subset of probes was consistently altered in the 3 GBM cell lines (17 probes upregulated and 40 downregulated due to HOXA9 expression in U87MG, U251 and GBML18 cells), which, as expected, was even smaller when integrating the non-tumor immortalized astrocytes (1 probe upregulated and 3 downregulated; Figure 2B), indicating that the transcriptome of HOXA9 is cell-type dependent, as previously suggested in leukemia models [19]. Gene-specific expression analyses in a subset of HOXA9 targets validated the array data (Supplementary Figure 2A–2D).

Bottom Line: Additionally, HOXA9 promoted the malignant transformation of human immortalized astrocytes in an orthotopic in vivo model, and caused tumor-associated death.Importantly, the pharmacological inhibition of BCL2 with the BH3 mimetic ABT-737 reverted temozolomide resistance in HOXA9-positive cells.These data establish HOXA9 as a driver of glioma initiation, aggressiveness and resistance to therapy.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar 4710-057 Braga, Portugal.

ABSTRACT
Glioblastoma is the most malignant brain tumor, exhibiting remarkable resistance to treatment. Here we investigated the oncogenic potential of HOXA9 in gliomagenesis, the molecular and cellular mechanisms by which HOXA9 renders glioblastoma more aggressive, and how HOXA9 affects response to chemotherapy and survival. The prognostic value of HOXA9 in glioblastoma patients was validated in two large datasets from TCGA and Rembrandt, where high HOXA9 levels were associated with shorter survival. Transcriptomic analyses identified novel HOXA9-target genes with key roles in cancer-related processes, including cell proliferation, DNA repair, and stem cell maintenance. Functional studies with HOXA9-overexpressing and HOXA9-silenced glioblastoma cell models revealed that HOXA9 promotes cell viability, stemness and invasion, and inhibits apoptosis. Additionally, HOXA9 promoted the malignant transformation of human immortalized astrocytes in an orthotopic in vivo model, and caused tumor-associated death. HOXA9 also mediated resistance to temozolomide treatment in vitro and in vivo via upregulation of BCL2. Importantly, the pharmacological inhibition of BCL2 with the BH3 mimetic ABT-737 reverted temozolomide resistance in HOXA9-positive cells. These data establish HOXA9 as a driver of glioma initiation, aggressiveness and resistance to therapy. In the future, the combination of BH3 mimetics with temozolomide should be further explored as an alternative treatment for glioblastoma.

No MeSH data available.


Related in: MedlinePlus