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Role of MYCN in retinoblastoma: From carcinogenesis to tumor progression
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neurooncology and neurodegeneration.
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

Retinoblastoma, a pediatric malignancy of the retina, is primarily driven by the bi-allelic inactivation of the RB1gene. However, a subset of cases are characterized by proficient RB1 functions but with MYCN copy number mutations, suggesting an alternative oncogenic mechanism in the absence of RB1 mutations. The aim of this thesis is to investigate the intricate molecular and cellular pathways implicated in retinoblastoma, with a particular focus on the role of MYCN expression and its interplay with the cell cycle and apoptotic pathways.

In Paper I, we explored the regulatory mechanisms underpinning MYCN-induced retinoblastoma using aRB1-proficient MYCN-overexpressing in vivo model in embryonic chicken retina and MYCN-transformed cells in culture. Our findings revealed that MYCN overexpression led to a significant upregulation of E2F levels, thereby dysregulating the cell cycle and mimicking the mechanistic phenotype of RB1-deficient tumors. Inhibition on E2f DNA-binding activity efficiently normalized growth and apoptosis in MYCN-transformed cells in culture. Despite RB1 proficiency, the elevated E2F levels induced a neoplastic behavior in retinal cells, indicating a novel mechanism of retinoblastoma carcinogenesis independent of RB1 inactivation.

Paper II employed single-cell RNA sequencing to dissect the cellular composition of MYCN-driven retinoblastoma in chicken in vivo model, revealing a predominant origin in cone photoreceptor progenitors. This finding suggested a cell-type-specific vulnerability to MYCN-induced transformation. The research further identifies a notable heterogeneity within the MYCN-transformed cells, with a subset of cells exhibiting non-cone photoreceptor features but features of other neurons like ganglion cells. A cluster was also identified withelevated expression of genes related to malignancy and tumor progression, including UBE2C and TOP2A. This suggested a link between MYCN overexpression and tumor development, potentially mediated through the E2F pathway.

In Paper III, the focus shifted to the interplay between MYCN expression, E2f activity, and the p53 pathway in human retinoblastoma cell lines exhibiting both RB1 deficiency and MYCN amplification. By modulating E2f and p53 pathway activities using chemical inhibitors, we demonstrated the essential role of MYCN expression level in mediating p53-driven growth inhibition and highlighted the independent effects of E2f inhibition and p53 activation by a Mdm2 inhibitor.

Together, these studies illuminate the intricate molecular pathways involved in MYCN-amplified retinoblastoma, emphasizing the pivotal role of MYCN in disrupting cell cycle regulation and promoting tumorigenesis. These insights not only advance our understanding of retinoblastoma pathogenesis but also provide potential therapeutic targets within the MYCN-E2F axis, offering novel treatment strategies in MYCN-amplified retinoblastoma.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2024. , p. 51
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 2019
Keywords [en]
MYCN, retinoblastoma, RNA sequencing, cancer, avian, E2F, carcinogenesis
National Category
Cancer and Oncology
Research subject
Developmental Neurosciences
Identifiers
URN: urn:nbn:se:uu:diva-523595ISBN: 978-91-513-2041-0 (print)OAI: oai:DiVA.org:uu-523595DiVA, id: diva2:1839714
Public defence
2024-04-10, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds Väg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2024-03-19 Created: 2024-02-21 Last updated: 2024-03-19
List of papers
1. Inhibition of high level E2F in a RB1 proficient MYCN overexpressing chicken retinoblastoma model normalizes neoplastic behaviour.
Open this publication in new window or tab >>Inhibition of high level E2F in a RB1 proficient MYCN overexpressing chicken retinoblastoma model normalizes neoplastic behaviour.
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2023 (English)In: Cellular Oncology, ISSN 2211-3428, E-ISSN 2211-3436Article in journal (Refereed) Epub ahead of print
Abstract [en]

PURPOSE: Retinoblastoma, a childhood cancer, is most frequently caused by bi-allelic inactivation of RB1 gene. However, other oncogenic mutations such as MYCN amplification can induce retinoblastoma with proficient RB1. Previously, we established RB1-proficient MYCN-overexpressing retinoblastoma models both in human organoids and chicken. Here, we investigate the regulatory events in MYCN-induced retinoblastoma carcinogenesis based on the model in chicken.

METHODS: MYCN transformed retinal cells in culture were obtained from in vivo MYCN electroporated chicken embryo retina. The expression profiles were analysed by RNA sequencing. Chemical treatments, qRT-PCR, flow cytometry, immunohisto- and immunocytochemistry and western blot were applied to study the properties and function of these cells.

RESULTS: The expression profile of MYCN-transformed retinal cells in culture showed cone photoreceptor progenitor signature and robustly increased levels of E2Fs. This expression profile was consistently observed in long-term culture. Chemical treatments confirmed RB1 proficiency in these cells. The cells were insensitive to p53 activation but inhibition of E2f efficiently induced cell cycle arrest followed by apoptosis.

CONCLUSION: In conclusion, with proficient RB1, MYCN-induced high level of E2F expression dysregulates the cell cycle and contributes to retinoblastoma carcinogenesis. The increased level of E2f renders the cells to adopt a similar mechanistic phenotype to a RB1-deficient tumour.

Keywords
Animal model, Chicken, E2F, Intraocular cancer, MYCN, RB1 proficient, Retinoblastoma
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-522867 (URN)10.1007/s13402-023-00863-0 (DOI)37606819 (PubMedID)
Available from: 2024-02-12 Created: 2024-02-12 Last updated: 2024-02-21
2. Single-cell RNA sequencing reveals cellular properties and carcinogenesis of a RB1-proficient MYCN-overexpressing retinoblastoma model from embryonic chicken retina
Open this publication in new window or tab >>Single-cell RNA sequencing reveals cellular properties and carcinogenesis of a RB1-proficient MYCN-overexpressing retinoblastoma model from embryonic chicken retina
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-523113 (URN)
Available from: 2024-02-14 Created: 2024-02-14 Last updated: 2024-02-21
3. Characterization of MYCN amplified retinoblastoma lines with respect to effects of E2f and p53 activity
Open this publication in new window or tab >>Characterization of MYCN amplified retinoblastoma lines with respect to effects of E2f and p53 activity
(English)Manuscript (preprint) (Other academic)
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-523107 (URN)
Available from: 2024-02-14 Created: 2024-02-14 Last updated: 2024-02-21

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Zhang, Hanzhao

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