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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 decreases overall survival and increases resistance to temozolomide in vivo via mismatch-repair and BCL2 proteins(A) Kaplan-Meier survival curves for in vivo orthotopic intracranial GBM models. While temozolomide (TMZ) treatment successfully increased overall survival, HOXA9-positive tumors cause significantly decreased overall survival in both untreated and TMZ-treated animals (Log-rank test HOXA9 vs MSCV, p = 0.0001; HOXA9 TMZ vs MSCV TMZ, p < 0.0001). (B) Hematoxylin-eosin staining of mice brains showing a well-delimited tumor area (T  ) with GBM-like features, and surrounding non-tumor brain tissue (N). (C) RT-PCR analysis confirmed the expression of HOXA9 in brain tumors derived from U87MG-HOXA9-xenografted cells. (D) Western Blot to mismatch-repair proteins (MMR), showing that HOXA9 decreases the expression of PMS2 and MSH6 proteins in U87MG cells in vitro. (E) and (F) qPCR and Western blot analyses of BCL2 mRNA and protein levels, respectively, showing HOXA9 significantly increases BCL2 levels. Results are representative of three independent experiments for each blot. (G) Cell viability of U87MG HOXA9-negative or -positive cells after 4 days of treatment with vehicle, Temozolomide (TMZ), ABT-737 (BCL2 inhibitor), or both. HOXA9-positive cells are significantly more sensitive to the combination of TMZ and ABT-737 than HOXA9-negative cells. Results are representative of three independent experiments, performed in triplicates (data points represent mean ± standard deviation). Statistical differences calculated by Student t-tests (*p < 0.05; **p < 0.01; ***p < 0.001).
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Figure 7: HOXA9 decreases overall survival and increases resistance to temozolomide in vivo via mismatch-repair and BCL2 proteins(A) Kaplan-Meier survival curves for in vivo orthotopic intracranial GBM models. While temozolomide (TMZ) treatment successfully increased overall survival, HOXA9-positive tumors cause significantly decreased overall survival in both untreated and TMZ-treated animals (Log-rank test HOXA9 vs MSCV, p = 0.0001; HOXA9 TMZ vs MSCV TMZ, p < 0.0001). (B) Hematoxylin-eosin staining of mice brains showing a well-delimited tumor area (T  ) with GBM-like features, and surrounding non-tumor brain tissue (N). (C) RT-PCR analysis confirmed the expression of HOXA9 in brain tumors derived from U87MG-HOXA9-xenografted cells. (D) Western Blot to mismatch-repair proteins (MMR), showing that HOXA9 decreases the expression of PMS2 and MSH6 proteins in U87MG cells in vitro. (E) and (F) qPCR and Western blot analyses of BCL2 mRNA and protein levels, respectively, showing HOXA9 significantly increases BCL2 levels. Results are representative of three independent experiments for each blot. (G) Cell viability of U87MG HOXA9-negative or -positive cells after 4 days of treatment with vehicle, Temozolomide (TMZ), ABT-737 (BCL2 inhibitor), or both. HOXA9-positive cells are significantly more sensitive to the combination of TMZ and ABT-737 than HOXA9-negative cells. Results are representative of three independent experiments, performed in triplicates (data points represent mean ± standard deviation). Statistical differences calculated by Student t-tests (*p < 0.05; **p < 0.01; ***p < 0.001).

Mentions: In order to validate the prognostic value of HOXA9 observed in GBM patients (Figure 1E–1G, and [16]), and its relevance in temozolomide chemo-resistance observed in vitro (Figure 6 and Supplementary Figure 11), we established intracranial orthotopic GBM xenografts with U87MG-MSCV and U87MG-HOXA9 cells in nude mice (Figure 7A). Untreated animals bearing U87MG-HOXA9-derived tumors presented significantly lower overall survival (median 28 days) than their respective negative counterparts (median 46 days; Log rank test, p = 0.0001; Figure 7A). Additionally, while treatment with temozolomide was able to significantly extend overall survival of all animals (median OS increased from 46 to 140 days in mice bearing U87MG-MSCV tumors, p < 0.0001; and median OS from 28 to 65 days in mice bearing U87MG-HOXA9 tumors, p = 0.0002; Figure 7A), those bearing HOXA9-expressing tumors presented a significantly shorter overall survival than animals bearing HOXA9-negative tumors (p < 0.0001; Figure 7A). Histological and RT-PCR analyses confirmed tumor formation and HOXA9 expression levels in tumors excised from mice brains (Figure 7B and 7C, respectively). Taken together, our in vivo experiments specifically establish HOXA9 as a solid biomarker of prognosis in GBM and its relevance in determining temozolomide chemo-resistance.


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 decreases overall survival and increases resistance to temozolomide in vivo via mismatch-repair and BCL2 proteins(A) Kaplan-Meier survival curves for in vivo orthotopic intracranial GBM models. While temozolomide (TMZ) treatment successfully increased overall survival, HOXA9-positive tumors cause significantly decreased overall survival in both untreated and TMZ-treated animals (Log-rank test HOXA9 vs MSCV, p = 0.0001; HOXA9 TMZ vs MSCV TMZ, p < 0.0001). (B) Hematoxylin-eosin staining of mice brains showing a well-delimited tumor area (T  ) with GBM-like features, and surrounding non-tumor brain tissue (N). (C) RT-PCR analysis confirmed the expression of HOXA9 in brain tumors derived from U87MG-HOXA9-xenografted cells. (D) Western Blot to mismatch-repair proteins (MMR), showing that HOXA9 decreases the expression of PMS2 and MSH6 proteins in U87MG cells in vitro. (E) and (F) qPCR and Western blot analyses of BCL2 mRNA and protein levels, respectively, showing HOXA9 significantly increases BCL2 levels. Results are representative of three independent experiments for each blot. (G) Cell viability of U87MG HOXA9-negative or -positive cells after 4 days of treatment with vehicle, Temozolomide (TMZ), ABT-737 (BCL2 inhibitor), or both. HOXA9-positive cells are significantly more sensitive to the combination of TMZ and ABT-737 than HOXA9-negative cells. Results are representative of three independent experiments, performed in triplicates (data points represent mean ± standard deviation). Statistical differences calculated by Student t-tests (*p < 0.05; **p < 0.01; ***p < 0.001).
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Related In: Results  -  Collection

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Figure 7: HOXA9 decreases overall survival and increases resistance to temozolomide in vivo via mismatch-repair and BCL2 proteins(A) Kaplan-Meier survival curves for in vivo orthotopic intracranial GBM models. While temozolomide (TMZ) treatment successfully increased overall survival, HOXA9-positive tumors cause significantly decreased overall survival in both untreated and TMZ-treated animals (Log-rank test HOXA9 vs MSCV, p = 0.0001; HOXA9 TMZ vs MSCV TMZ, p < 0.0001). (B) Hematoxylin-eosin staining of mice brains showing a well-delimited tumor area (T  ) with GBM-like features, and surrounding non-tumor brain tissue (N). (C) RT-PCR analysis confirmed the expression of HOXA9 in brain tumors derived from U87MG-HOXA9-xenografted cells. (D) Western Blot to mismatch-repair proteins (MMR), showing that HOXA9 decreases the expression of PMS2 and MSH6 proteins in U87MG cells in vitro. (E) and (F) qPCR and Western blot analyses of BCL2 mRNA and protein levels, respectively, showing HOXA9 significantly increases BCL2 levels. Results are representative of three independent experiments for each blot. (G) Cell viability of U87MG HOXA9-negative or -positive cells after 4 days of treatment with vehicle, Temozolomide (TMZ), ABT-737 (BCL2 inhibitor), or both. HOXA9-positive cells are significantly more sensitive to the combination of TMZ and ABT-737 than HOXA9-negative cells. Results are representative of three independent experiments, performed in triplicates (data points represent mean ± standard deviation). Statistical differences calculated by Student t-tests (*p < 0.05; **p < 0.01; ***p < 0.001).
Mentions: In order to validate the prognostic value of HOXA9 observed in GBM patients (Figure 1E–1G, and [16]), and its relevance in temozolomide chemo-resistance observed in vitro (Figure 6 and Supplementary Figure 11), we established intracranial orthotopic GBM xenografts with U87MG-MSCV and U87MG-HOXA9 cells in nude mice (Figure 7A). Untreated animals bearing U87MG-HOXA9-derived tumors presented significantly lower overall survival (median 28 days) than their respective negative counterparts (median 46 days; Log rank test, p = 0.0001; Figure 7A). Additionally, while treatment with temozolomide was able to significantly extend overall survival of all animals (median OS increased from 46 to 140 days in mice bearing U87MG-MSCV tumors, p < 0.0001; and median OS from 28 to 65 days in mice bearing U87MG-HOXA9 tumors, p = 0.0002; Figure 7A), those bearing HOXA9-expressing tumors presented a significantly shorter overall survival than animals bearing HOXA9-negative tumors (p < 0.0001; Figure 7A). Histological and RT-PCR analyses confirmed tumor formation and HOXA9 expression levels in tumors excised from mice brains (Figure 7B and 7C, respectively). Taken together, our in vivo experiments specifically establish HOXA9 as a solid biomarker of prognosis in GBM and its relevance in determining temozolomide chemo-resistance.

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