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Expression of MYCN in Multipotent Sympathoadrenal Progenitors Induces Proliferation and Neural Differentiation, but Is Not Sufficient for Tumorigenesis.

Mobley BC, Kwon M, Kraemer BR, Hickman FE, Qiao J, Chung DH, Carter BD - PLoS ONE (2015)

Bottom Line: MYCN overexpression in spheres promoted commitment to the neural lineage, evidenced by an increased frequency of neuron-containing colonies.MYCN promoted proliferation of both sympathoadrenal progenitor spheres and differentiated neurons derived from these spheres, but there was also an increase in apoptosis.We find, however, that MYCN overexpression is not sufficient for these cells to form tumors in nude mice, suggesting that additional transforming mutations are necessary for tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Microbiology, and Immunology, Division of Neuropathology, Vanderbilt University Medical Center, Nashville, Tennessee, the United States of America.

ABSTRACT
Neuroblastoma is a pediatric malignancy of the sympathetic ganglia and adrenal glands, hypothesized to originate from progenitors of the developing sympathetic nervous system. Amplification of the MYCN oncogene is a genetic marker of risk in this disease. Understanding the impact of oncogene expression on sympathoadrenal progenitor development may improve our knowledge of neuroblastoma initiation and progression. We isolated sympathoadrenal progenitor cells from the postnatal murine adrenal gland by sphere culture and found them to be multipotent, generating differentiated colonies of neurons, Schwann cells, and myofibroblasts. MYCN overexpression in spheres promoted commitment to the neural lineage, evidenced by an increased frequency of neuron-containing colonies. MYCN promoted proliferation of both sympathoadrenal progenitor spheres and differentiated neurons derived from these spheres, but there was also an increase in apoptosis. The proliferation, apoptosis, and neural lineage commitment induced by MYCN are tumor-like characteristics and thereby support the hypothesis that multipotent adrenal medullary progenitor cells are cells of origin for neuroblastoma. We find, however, that MYCN overexpression is not sufficient for these cells to form tumors in nude mice, suggesting that additional transforming mutations are necessary for tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

MYCN Overexpression Induces Neuronal Lineage Commitment.(A) Adrenal progenitor cells isolated by differential plating were infected with empty or Mycn lentivirus, and 3 to 4 days following infection the resulting primary spheres were analyzed for MYCN expression by western blot. (B) Spheres infected with Mycn lentivirus showed lower mean levels of Bmi1 and increased Dbh and Th levels, though differences did not reach statistical significance (unpaired Student’s t-test). Data are the mean values from four experiments run in triplicate. mRNA levels from primary spheres were analyzed by qRT-PCR and are shown in relation to TATA-Box binding protein-1 (TBP1). (C) MYCN expression induced neuronal lineage committment in differentiated secondary spheres with a markedly increased frequency of N-only colonies and a substantial reduction of M-only colonies (n = 5). (D) Neural lineage induction by MYCN was maintained in the subsequent passage to tertiary spheres: N-only colonies were observed with MYCN overexpression but were absent from differentiated control spheres (n = 3). Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple comparison test. *p<0.05,**p<0.01, ***p < .001.
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pone.0133897.g002: MYCN Overexpression Induces Neuronal Lineage Commitment.(A) Adrenal progenitor cells isolated by differential plating were infected with empty or Mycn lentivirus, and 3 to 4 days following infection the resulting primary spheres were analyzed for MYCN expression by western blot. (B) Spheres infected with Mycn lentivirus showed lower mean levels of Bmi1 and increased Dbh and Th levels, though differences did not reach statistical significance (unpaired Student’s t-test). Data are the mean values from four experiments run in triplicate. mRNA levels from primary spheres were analyzed by qRT-PCR and are shown in relation to TATA-Box binding protein-1 (TBP1). (C) MYCN expression induced neuronal lineage committment in differentiated secondary spheres with a markedly increased frequency of N-only colonies and a substantial reduction of M-only colonies (n = 5). (D) Neural lineage induction by MYCN was maintained in the subsequent passage to tertiary spheres: N-only colonies were observed with MYCN overexpression but were absent from differentiated control spheres (n = 3). Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple comparison test. *p<0.05,**p<0.01, ***p < .001.

Mentions: To explore the effects of MYCN overexpression on sympathoadrenal progenitor lineage committment, we delivered the oncogene to freshly harvested cells by lentiviral transduction. Primary spheres were allowed to form, and western blot confirmed overexpression of the MYCN protein by spheres (Fig 2A). Quantitative PCR showed a lower mean level of the NCSC marker Bmi1 in the setting of MYCN expression, and increased levels of the sympathetic neural lineage markers Dbh and Th (Fig 2B). These differences, however, did not reach statistical significance. Primary spheres were then dissociated for secondary sphere growth at a density of one cell per microliter, and the resulting secondary spheres were differentiated to assess the impact of MYCN expression on colony composition. Interestingly, with MYCN expression, differentiated colonies were significantly more likely to contain neurons: N-only colonies were observed at a markedly increased frequency with a substantial reduction of M-only colonies (Fig 2C). Neural lineage induction by MYCN was maintained in the subsequent passage to tertiary spheres (Fig 2D), where N-only colonies were absent from controls but constituted the majority of MYCN overexpressing colonies. Control spheres were again more likely to form M-only colonies in this passage. While MYCN expression in development and neoplasia inhibits neural differentiation to maintain cells in a precursor state [27–30], our results indicate that MYCN strongly promotes the neural lineage in multipotent SAPs. This finding is consistent with the neural lineage commitment seen in neuroblastomas.


Expression of MYCN in Multipotent Sympathoadrenal Progenitors Induces Proliferation and Neural Differentiation, but Is Not Sufficient for Tumorigenesis.

Mobley BC, Kwon M, Kraemer BR, Hickman FE, Qiao J, Chung DH, Carter BD - PLoS ONE (2015)

MYCN Overexpression Induces Neuronal Lineage Commitment.(A) Adrenal progenitor cells isolated by differential plating were infected with empty or Mycn lentivirus, and 3 to 4 days following infection the resulting primary spheres were analyzed for MYCN expression by western blot. (B) Spheres infected with Mycn lentivirus showed lower mean levels of Bmi1 and increased Dbh and Th levels, though differences did not reach statistical significance (unpaired Student’s t-test). Data are the mean values from four experiments run in triplicate. mRNA levels from primary spheres were analyzed by qRT-PCR and are shown in relation to TATA-Box binding protein-1 (TBP1). (C) MYCN expression induced neuronal lineage committment in differentiated secondary spheres with a markedly increased frequency of N-only colonies and a substantial reduction of M-only colonies (n = 5). (D) Neural lineage induction by MYCN was maintained in the subsequent passage to tertiary spheres: N-only colonies were observed with MYCN overexpression but were absent from differentiated control spheres (n = 3). Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple comparison test. *p<0.05,**p<0.01, ***p < .001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519318&req=5

pone.0133897.g002: MYCN Overexpression Induces Neuronal Lineage Commitment.(A) Adrenal progenitor cells isolated by differential plating were infected with empty or Mycn lentivirus, and 3 to 4 days following infection the resulting primary spheres were analyzed for MYCN expression by western blot. (B) Spheres infected with Mycn lentivirus showed lower mean levels of Bmi1 and increased Dbh and Th levels, though differences did not reach statistical significance (unpaired Student’s t-test). Data are the mean values from four experiments run in triplicate. mRNA levels from primary spheres were analyzed by qRT-PCR and are shown in relation to TATA-Box binding protein-1 (TBP1). (C) MYCN expression induced neuronal lineage committment in differentiated secondary spheres with a markedly increased frequency of N-only colonies and a substantial reduction of M-only colonies (n = 5). (D) Neural lineage induction by MYCN was maintained in the subsequent passage to tertiary spheres: N-only colonies were observed with MYCN overexpression but were absent from differentiated control spheres (n = 3). Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple comparison test. *p<0.05,**p<0.01, ***p < .001.
Mentions: To explore the effects of MYCN overexpression on sympathoadrenal progenitor lineage committment, we delivered the oncogene to freshly harvested cells by lentiviral transduction. Primary spheres were allowed to form, and western blot confirmed overexpression of the MYCN protein by spheres (Fig 2A). Quantitative PCR showed a lower mean level of the NCSC marker Bmi1 in the setting of MYCN expression, and increased levels of the sympathetic neural lineage markers Dbh and Th (Fig 2B). These differences, however, did not reach statistical significance. Primary spheres were then dissociated for secondary sphere growth at a density of one cell per microliter, and the resulting secondary spheres were differentiated to assess the impact of MYCN expression on colony composition. Interestingly, with MYCN expression, differentiated colonies were significantly more likely to contain neurons: N-only colonies were observed at a markedly increased frequency with a substantial reduction of M-only colonies (Fig 2C). Neural lineage induction by MYCN was maintained in the subsequent passage to tertiary spheres (Fig 2D), where N-only colonies were absent from controls but constituted the majority of MYCN overexpressing colonies. Control spheres were again more likely to form M-only colonies in this passage. While MYCN expression in development and neoplasia inhibits neural differentiation to maintain cells in a precursor state [27–30], our results indicate that MYCN strongly promotes the neural lineage in multipotent SAPs. This finding is consistent with the neural lineage commitment seen in neuroblastomas.

Bottom Line: MYCN overexpression in spheres promoted commitment to the neural lineage, evidenced by an increased frequency of neuron-containing colonies.MYCN promoted proliferation of both sympathoadrenal progenitor spheres and differentiated neurons derived from these spheres, but there was also an increase in apoptosis.We find, however, that MYCN overexpression is not sufficient for these cells to form tumors in nude mice, suggesting that additional transforming mutations are necessary for tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Microbiology, and Immunology, Division of Neuropathology, Vanderbilt University Medical Center, Nashville, Tennessee, the United States of America.

ABSTRACT
Neuroblastoma is a pediatric malignancy of the sympathetic ganglia and adrenal glands, hypothesized to originate from progenitors of the developing sympathetic nervous system. Amplification of the MYCN oncogene is a genetic marker of risk in this disease. Understanding the impact of oncogene expression on sympathoadrenal progenitor development may improve our knowledge of neuroblastoma initiation and progression. We isolated sympathoadrenal progenitor cells from the postnatal murine adrenal gland by sphere culture and found them to be multipotent, generating differentiated colonies of neurons, Schwann cells, and myofibroblasts. MYCN overexpression in spheres promoted commitment to the neural lineage, evidenced by an increased frequency of neuron-containing colonies. MYCN promoted proliferation of both sympathoadrenal progenitor spheres and differentiated neurons derived from these spheres, but there was also an increase in apoptosis. The proliferation, apoptosis, and neural lineage commitment induced by MYCN are tumor-like characteristics and thereby support the hypothesis that multipotent adrenal medullary progenitor cells are cells of origin for neuroblastoma. We find, however, that MYCN overexpression is not sufficient for these cells to form tumors in nude mice, suggesting that additional transforming mutations are necessary for tumorigenesis.

No MeSH data available.


Related in: MedlinePlus