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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 induces glioma initiation and tumor-associated death in intracranial orthotopic xenografts(A) Nude mice were injected with HOXA9-negative and HOXA9-positive hTERT/E6/E7 immortalized astrocytes, either subcutaneously or intracranially. No tumor formation was observed in the subcutaneous model, regardless of HOXA9 expression. In the intracranial orthotopic model, HOXA9-positive hTERT/E6/E7 cells originated tumors in the majority of tested mice (70%). (B) Mice intracranially injected with hTERT/E6/E7-HOXA9 cells display glioma-related loss of weight. (C) Kaplan-Meier survival curves showing tumor-associated death exclusively in mice bearing HOXA9-positive cells (Log-rank test, p = 0.0035). (D) Histological characterization of mice brains orthotopically-injected with hTERT/E6/E7-control or hTERT/E6/E7-HOXA9 cells. Brains from animals injected with HOXA9-negative cells display a normal, non-malignant appearance (images 1 and 2 at 20x and 200x magnification, respectively), while HOXA9-positive cells formed tumors displaying characteristic hallmarks of malignant gliomas (images 3, 4, 5, 6 and 7 at the magnification of 20x, 40x, 200x, 100x and 200x, respectively), including pleomorphic and spindle shape tumor cells (arrows), high mitotic activity (open arrowheads), prominent nuclear polymorphism (closed arrowheads), and infiltration to meninges and brain parenchyma (6 and 7). In image 4, cells infiltrating bone and soft tissues (#) presented a more sarcomatoid appearance, amplified in image 5. (E) RT-PCR analysis confirmed the expression of human GUSB housekeeping gene and HOXA9 exclusively in HOXA9-positive xenografts. Me = meninges; pa = brain parenchyma.
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Figure 5: HOXA9 induces glioma initiation and tumor-associated death in intracranial orthotopic xenografts(A) Nude mice were injected with HOXA9-negative and HOXA9-positive hTERT/E6/E7 immortalized astrocytes, either subcutaneously or intracranially. No tumor formation was observed in the subcutaneous model, regardless of HOXA9 expression. In the intracranial orthotopic model, HOXA9-positive hTERT/E6/E7 cells originated tumors in the majority of tested mice (70%). (B) Mice intracranially injected with hTERT/E6/E7-HOXA9 cells display glioma-related loss of weight. (C) Kaplan-Meier survival curves showing tumor-associated death exclusively in mice bearing HOXA9-positive cells (Log-rank test, p = 0.0035). (D) Histological characterization of mice brains orthotopically-injected with hTERT/E6/E7-control or hTERT/E6/E7-HOXA9 cells. Brains from animals injected with HOXA9-negative cells display a normal, non-malignant appearance (images 1 and 2 at 20x and 200x magnification, respectively), while HOXA9-positive cells formed tumors displaying characteristic hallmarks of malignant gliomas (images 3, 4, 5, 6 and 7 at the magnification of 20x, 40x, 200x, 100x and 200x, respectively), including pleomorphic and spindle shape tumor cells (arrows), high mitotic activity (open arrowheads), prominent nuclear polymorphism (closed arrowheads), and infiltration to meninges and brain parenchyma (6 and 7). In image 4, cells infiltrating bone and soft tissues (#) presented a more sarcomatoid appearance, amplified in image 5. (E) RT-PCR analysis confirmed the expression of human GUSB housekeeping gene and HOXA9 exclusively in HOXA9-positive xenografts. Me = meninges; pa = brain parenchyma.

Mentions: Given our data implicating HOXA9 as a key molecule in mediating several aspects of cancer aggressiveness and stem cell characteristics, we hypothesized that HOXA9 may also play an important role in the tumorigenic process, possibly facilitating carcinogenesis initiation. To test this hypothesis, we evaluated the influence of HOXA9 on the capacity of hTERT/E6/E7 cells, a non-tumorigenic cell line [28], to establish tumors in vivo. In a subcutaneous model, hTERT/E6/E7 cells did not form tumors, regardless of HOXA9 expression status (Figure 5A). Considering the relevance of the interplay between tumor cells and its microenvironment, we investigated the tumorigenic potential of these cells in an orthotopic intracranial model. Strikingly, while HOXA9-negative hTERT/E6/E7 cells did not form tumors, the expression of HOXA9 in hTERT/E6/E7 rendered these cells highly tumorigenic in the brain, accompanied by glioma-related symptomatology and death (Figure 5A–5E). Histological analyses of the brains revealed that hTERT/E6/E7-HOXA9 tumors displayed hallmark features of malignant gliomas, including pleomorphic and spindle shape tumor cells, with prominent nuclear polymorphism and mitotic activity, and were highly infiltrative throughout the meninges and brain parenchyma. In some cases, tumors grew outside the brain, infiltrating bone and soft tissues, where cells displayed a more sarcomatoid appearance (Figure 5D). These data clearly establish HOXA9 as a novel critical molecular driver in the initial steps of malignant transformation of brain gliomas.


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 induces glioma initiation and tumor-associated death in intracranial orthotopic xenografts(A) Nude mice were injected with HOXA9-negative and HOXA9-positive hTERT/E6/E7 immortalized astrocytes, either subcutaneously or intracranially. No tumor formation was observed in the subcutaneous model, regardless of HOXA9 expression. In the intracranial orthotopic model, HOXA9-positive hTERT/E6/E7 cells originated tumors in the majority of tested mice (70%). (B) Mice intracranially injected with hTERT/E6/E7-HOXA9 cells display glioma-related loss of weight. (C) Kaplan-Meier survival curves showing tumor-associated death exclusively in mice bearing HOXA9-positive cells (Log-rank test, p = 0.0035). (D) Histological characterization of mice brains orthotopically-injected with hTERT/E6/E7-control or hTERT/E6/E7-HOXA9 cells. Brains from animals injected with HOXA9-negative cells display a normal, non-malignant appearance (images 1 and 2 at 20x and 200x magnification, respectively), while HOXA9-positive cells formed tumors displaying characteristic hallmarks of malignant gliomas (images 3, 4, 5, 6 and 7 at the magnification of 20x, 40x, 200x, 100x and 200x, respectively), including pleomorphic and spindle shape tumor cells (arrows), high mitotic activity (open arrowheads), prominent nuclear polymorphism (closed arrowheads), and infiltration to meninges and brain parenchyma (6 and 7). In image 4, cells infiltrating bone and soft tissues (#) presented a more sarcomatoid appearance, amplified in image 5. (E) RT-PCR analysis confirmed the expression of human GUSB housekeeping gene and HOXA9 exclusively in HOXA9-positive xenografts. Me = meninges; pa = brain parenchyma.
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Figure 5: HOXA9 induces glioma initiation and tumor-associated death in intracranial orthotopic xenografts(A) Nude mice were injected with HOXA9-negative and HOXA9-positive hTERT/E6/E7 immortalized astrocytes, either subcutaneously or intracranially. No tumor formation was observed in the subcutaneous model, regardless of HOXA9 expression. In the intracranial orthotopic model, HOXA9-positive hTERT/E6/E7 cells originated tumors in the majority of tested mice (70%). (B) Mice intracranially injected with hTERT/E6/E7-HOXA9 cells display glioma-related loss of weight. (C) Kaplan-Meier survival curves showing tumor-associated death exclusively in mice bearing HOXA9-positive cells (Log-rank test, p = 0.0035). (D) Histological characterization of mice brains orthotopically-injected with hTERT/E6/E7-control or hTERT/E6/E7-HOXA9 cells. Brains from animals injected with HOXA9-negative cells display a normal, non-malignant appearance (images 1 and 2 at 20x and 200x magnification, respectively), while HOXA9-positive cells formed tumors displaying characteristic hallmarks of malignant gliomas (images 3, 4, 5, 6 and 7 at the magnification of 20x, 40x, 200x, 100x and 200x, respectively), including pleomorphic and spindle shape tumor cells (arrows), high mitotic activity (open arrowheads), prominent nuclear polymorphism (closed arrowheads), and infiltration to meninges and brain parenchyma (6 and 7). In image 4, cells infiltrating bone and soft tissues (#) presented a more sarcomatoid appearance, amplified in image 5. (E) RT-PCR analysis confirmed the expression of human GUSB housekeeping gene and HOXA9 exclusively in HOXA9-positive xenografts. Me = meninges; pa = brain parenchyma.
Mentions: Given our data implicating HOXA9 as a key molecule in mediating several aspects of cancer aggressiveness and stem cell characteristics, we hypothesized that HOXA9 may also play an important role in the tumorigenic process, possibly facilitating carcinogenesis initiation. To test this hypothesis, we evaluated the influence of HOXA9 on the capacity of hTERT/E6/E7 cells, a non-tumorigenic cell line [28], to establish tumors in vivo. In a subcutaneous model, hTERT/E6/E7 cells did not form tumors, regardless of HOXA9 expression status (Figure 5A). Considering the relevance of the interplay between tumor cells and its microenvironment, we investigated the tumorigenic potential of these cells in an orthotopic intracranial model. Strikingly, while HOXA9-negative hTERT/E6/E7 cells did not form tumors, the expression of HOXA9 in hTERT/E6/E7 rendered these cells highly tumorigenic in the brain, accompanied by glioma-related symptomatology and death (Figure 5A–5E). Histological analyses of the brains revealed that hTERT/E6/E7-HOXA9 tumors displayed hallmark features of malignant gliomas, including pleomorphic and spindle shape tumor cells, with prominent nuclear polymorphism and mitotic activity, and were highly infiltrative throughout the meninges and brain parenchyma. In some cases, tumors grew outside the brain, infiltrating bone and soft tissues, where cells displayed a more sarcomatoid appearance (Figure 5D). These data clearly establish HOXA9 as a novel critical molecular driver in the initial steps of malignant transformation of brain gliomas.

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