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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

Functional roles of HOXA9 in GBM cell viability, death, and invasion, under basal conditions and temozolomide treatment(A and B) Determination of the half inhibitory concentration (IC50) values after 6 days of temozolomide (TMZ) treatment in HOXA9-positive or HOXA9-negative hTERT/E6/E7 and U87MG (A), and HOXA9-silenced or control U251 and GBML18 (B) cell lines. (C–F) Cell viability trypan blue assays in HOXA9-negative/low or HOXA9–positive/high hTERT/E6/E7 (C), U87MG (D), U251 (E) and GBML18 (F) cells, exposed to temozolomide or vehicle. (G) Cell death was evaluated by annexin V staining followed by flow cytometry in HOXA9-positive/high and HOXA9-negative/low hTERT/E6/E7, U87MG, U251 and GBML18 cell lines, both in basal conditions and after exposure to temozolomide (TMZ). HOXA9 expression decreases cell death of all GBM cell models, both in basal conditions and after TMZ treatment, except in basal conditions for U251 cell line. (H) Cell invasion in the same cell lines, both in basal conditions and after exposure to TMZ. HOXA9 increases the invasion of hTERT/E6/E7, U87MG, and GBML18 cells. U251 cells did not show significant differences in invasion profiles due to HOXA9 levels or TMZ treatment. Results are representative of at least three independent experiments, performed in triplicates (data points represent mean ± SEM). Statistical differences were calculated by Student's t-test (panels A, B, G, H) and two-way ANOVA (panels C–F) (*p < 0.05; **p < 0.01; ***p < 0.001).
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Figure 6: Functional roles of HOXA9 in GBM cell viability, death, and invasion, under basal conditions and temozolomide treatment(A and B) Determination of the half inhibitory concentration (IC50) values after 6 days of temozolomide (TMZ) treatment in HOXA9-positive or HOXA9-negative hTERT/E6/E7 and U87MG (A), and HOXA9-silenced or control U251 and GBML18 (B) cell lines. (C–F) Cell viability trypan blue assays in HOXA9-negative/low or HOXA9–positive/high hTERT/E6/E7 (C), U87MG (D), U251 (E) and GBML18 (F) cells, exposed to temozolomide or vehicle. (G) Cell death was evaluated by annexin V staining followed by flow cytometry in HOXA9-positive/high and HOXA9-negative/low hTERT/E6/E7, U87MG, U251 and GBML18 cell lines, both in basal conditions and after exposure to temozolomide (TMZ). HOXA9 expression decreases cell death of all GBM cell models, both in basal conditions and after TMZ treatment, except in basal conditions for U251 cell line. (H) Cell invasion in the same cell lines, both in basal conditions and after exposure to TMZ. HOXA9 increases the invasion of hTERT/E6/E7, U87MG, and GBML18 cells. U251 cells did not show significant differences in invasion profiles due to HOXA9 levels or TMZ treatment. Results are representative of at least three independent experiments, performed in triplicates (data points represent mean ± SEM). Statistical differences were calculated by Student's t-test (panels A, B, G, H) and two-way ANOVA (panels C–F) (*p < 0.05; **p < 0.01; ***p < 0.001).

Mentions: Our data implicated HOXA9 in several hallmarks of cancer, including cell proliferation, invasion, DNA repair pathways, and cancer stem cell features (Figure 3, Supplementary Figure 3 and Supplementary Figure 10 and Supplementary Table 1). Since most of these characteristics have been implicated in resistance to treatments, we performed an integrated set of functional assays to investigate HOXA9's effects on these processes, both in basal conditions and under temozolomide-treatment, the gold standard chemotherapeutic agent for GBM patients. Both hTERT/E6/E7 and U87MG cells overexpressing HOXA9 presented significantly higher temozolomide IC50 values than their respective control cell lines (Figure 6A). In agreement with the overexpression models, we observed a decrease in temozolomide IC50 values for both cell lines after HOXA9 downregulation (Figure 6B). Time-course viability assays were further evaluated by trypan blue exclusion (Figure 6C–6F) and MTS reduction (Supplementary Figure 11A–11D). Globally, both assays revealed that all HOXA9-high cell lines hTERT/E6/E7, U87MG, U251, and GBML18 (red lines) presented higher cell viability in basal conditions than their respective HOXA9-low counterparts (blue lines; Figure 6C–6F and Supplementary Figure 11A–11D), and that temozolomide-mediated cytotoxicity was significantly more pronounced in HOXA9-low cells than in HOXA9-high cells (Figure 6C–6F and Supplementary Figure 11A–11D), collectively indicating that HOXA9 contributes to temozolomide resistance in GBM cells. We next assessed the influence of HOXA9 expression in cell death by annexin V staining followed by flow cytometry. In basal conditions, most HOXA9-high cells presented significantly lower levels of cell death than their respective HOXA9-low counterparts (Figure 6G). In addition, all HOXA9-high cells presented significantly lower levels of temozolomide-mediated cell death than HOXA9-low cells (Figure 6G). An important feature of GBM is its striking ability to invade adjacent brain tissue, which is a major cause of tumor recurrence and resistance to therapy. Using invasion assays, we observed that HOXA9 significantly increases the invasive capacity of hTERT/E6/E7, U87MG, and GBML18 cells, both in basal conditions and after treatment with temozolomide. In contrast, no significant changes in invasion were observed in U251 cells (Figure 6H). Since invasion and migration are intimately-related cellular processes, we also evaluated the migration capacity of U251 cells by wound healing assays (Supplementary Figure 12). Interestingly, U251-shControl cells presented significantly higher migration rates than U251-shHOXA9 counterparts. Together, our data consistently establishes the functional relevance of HOXA9 expression in several hallmarks of GBM cells behavior, by affecting key cellular processes such as cell viability, death, invasion, and drug resistance, which together may dictate the more aggressive behavior and poorer clinical outcome of HOXA9-positive GBMs.


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)

Functional roles of HOXA9 in GBM cell viability, death, and invasion, under basal conditions and temozolomide treatment(A and B) Determination of the half inhibitory concentration (IC50) values after 6 days of temozolomide (TMZ) treatment in HOXA9-positive or HOXA9-negative hTERT/E6/E7 and U87MG (A), and HOXA9-silenced or control U251 and GBML18 (B) cell lines. (C–F) Cell viability trypan blue assays in HOXA9-negative/low or HOXA9–positive/high hTERT/E6/E7 (C), U87MG (D), U251 (E) and GBML18 (F) cells, exposed to temozolomide or vehicle. (G) Cell death was evaluated by annexin V staining followed by flow cytometry in HOXA9-positive/high and HOXA9-negative/low hTERT/E6/E7, U87MG, U251 and GBML18 cell lines, both in basal conditions and after exposure to temozolomide (TMZ). HOXA9 expression decreases cell death of all GBM cell models, both in basal conditions and after TMZ treatment, except in basal conditions for U251 cell line. (H) Cell invasion in the same cell lines, both in basal conditions and after exposure to TMZ. HOXA9 increases the invasion of hTERT/E6/E7, U87MG, and GBML18 cells. U251 cells did not show significant differences in invasion profiles due to HOXA9 levels or TMZ treatment. Results are representative of at least three independent experiments, performed in triplicates (data points represent mean ± SEM). Statistical differences were calculated by Student's t-test (panels A, B, G, H) and two-way ANOVA (panels C–F) (*p < 0.05; **p < 0.01; ***p < 0.001).
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Figure 6: Functional roles of HOXA9 in GBM cell viability, death, and invasion, under basal conditions and temozolomide treatment(A and B) Determination of the half inhibitory concentration (IC50) values after 6 days of temozolomide (TMZ) treatment in HOXA9-positive or HOXA9-negative hTERT/E6/E7 and U87MG (A), and HOXA9-silenced or control U251 and GBML18 (B) cell lines. (C–F) Cell viability trypan blue assays in HOXA9-negative/low or HOXA9–positive/high hTERT/E6/E7 (C), U87MG (D), U251 (E) and GBML18 (F) cells, exposed to temozolomide or vehicle. (G) Cell death was evaluated by annexin V staining followed by flow cytometry in HOXA9-positive/high and HOXA9-negative/low hTERT/E6/E7, U87MG, U251 and GBML18 cell lines, both in basal conditions and after exposure to temozolomide (TMZ). HOXA9 expression decreases cell death of all GBM cell models, both in basal conditions and after TMZ treatment, except in basal conditions for U251 cell line. (H) Cell invasion in the same cell lines, both in basal conditions and after exposure to TMZ. HOXA9 increases the invasion of hTERT/E6/E7, U87MG, and GBML18 cells. U251 cells did not show significant differences in invasion profiles due to HOXA9 levels or TMZ treatment. Results are representative of at least three independent experiments, performed in triplicates (data points represent mean ± SEM). Statistical differences were calculated by Student's t-test (panels A, B, G, H) and two-way ANOVA (panels C–F) (*p < 0.05; **p < 0.01; ***p < 0.001).
Mentions: Our data implicated HOXA9 in several hallmarks of cancer, including cell proliferation, invasion, DNA repair pathways, and cancer stem cell features (Figure 3, Supplementary Figure 3 and Supplementary Figure 10 and Supplementary Table 1). Since most of these characteristics have been implicated in resistance to treatments, we performed an integrated set of functional assays to investigate HOXA9's effects on these processes, both in basal conditions and under temozolomide-treatment, the gold standard chemotherapeutic agent for GBM patients. Both hTERT/E6/E7 and U87MG cells overexpressing HOXA9 presented significantly higher temozolomide IC50 values than their respective control cell lines (Figure 6A). In agreement with the overexpression models, we observed a decrease in temozolomide IC50 values for both cell lines after HOXA9 downregulation (Figure 6B). Time-course viability assays were further evaluated by trypan blue exclusion (Figure 6C–6F) and MTS reduction (Supplementary Figure 11A–11D). Globally, both assays revealed that all HOXA9-high cell lines hTERT/E6/E7, U87MG, U251, and GBML18 (red lines) presented higher cell viability in basal conditions than their respective HOXA9-low counterparts (blue lines; Figure 6C–6F and Supplementary Figure 11A–11D), and that temozolomide-mediated cytotoxicity was significantly more pronounced in HOXA9-low cells than in HOXA9-high cells (Figure 6C–6F and Supplementary Figure 11A–11D), collectively indicating that HOXA9 contributes to temozolomide resistance in GBM cells. We next assessed the influence of HOXA9 expression in cell death by annexin V staining followed by flow cytometry. In basal conditions, most HOXA9-high cells presented significantly lower levels of cell death than their respective HOXA9-low counterparts (Figure 6G). In addition, all HOXA9-high cells presented significantly lower levels of temozolomide-mediated cell death than HOXA9-low cells (Figure 6G). An important feature of GBM is its striking ability to invade adjacent brain tissue, which is a major cause of tumor recurrence and resistance to therapy. Using invasion assays, we observed that HOXA9 significantly increases the invasive capacity of hTERT/E6/E7, U87MG, and GBML18 cells, both in basal conditions and after treatment with temozolomide. In contrast, no significant changes in invasion were observed in U251 cells (Figure 6H). Since invasion and migration are intimately-related cellular processes, we also evaluated the migration capacity of U251 cells by wound healing assays (Supplementary Figure 12). Interestingly, U251-shControl cells presented significantly higher migration rates than U251-shHOXA9 counterparts. Together, our data consistently establishes the functional relevance of HOXA9 expression in several hallmarks of GBM cells behavior, by affecting key cellular processes such as cell viability, death, invasion, and drug resistance, which together may dictate the more aggressive behavior and poorer clinical outcome of HOXA9-positive GBMs.

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