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Role of STIL overexpression in supernumerary centriole formation, chromosomal instability and cancer development in mice
Role of STIL overexpression in supernumerary centriole formation, chromosomal instability and cancer development in mice
Centrosome is the major microtubule-organizing center in mammalian cell and consists of a pair of centrioles embedded in pericentriolar material. Centrosomes are important for bipolar spindle formation and correct chromosome segregation during mitosis. Disruption of normal centrosome function leads to aneuploidy and chromosome missegregation. Many cancer cells harbor supernumerary centrosomes, which were shown to correlate to chromosomal instability, clinical aggressiveness, and poor prognosis. Nevertheless, the question, whether amplified centrosomes can drive tumorigenesis in vivo remains unresolved. The process of centrosome duplication is tightly controlled by a small set of proteins including the kinase protein PLK4 and the structural centriole proteins as STIL and SAS6. Depletion of any one of these proteins’ blocks centrosome duplication and, conversely, overexpression causes centrosome amplification. In different PLK4 overexpression in vivo models, it remains arguable, whether extra centrosomes could derive tumorigenesis and whether the generated tumors might be induced by the additional serine/threonine kinase functions of PLK4 besides its role in regulating centriole duplication. On the other hand, the structural centrosome protein STIL is involved only in centriole replication without any other known functions up to now and its overexpression leads to the formation of supernumerary centrioles in tissue culture. Therefore, investigating the role of centrosome aberrations in chromosomal instability and tumor formation in vivo using STIL overexpression (STILOE) mouse model will add important information to the conflicting data generated by PLK4 overexpressing mice. Accordingly, we generated a new transgenic Cre-LoxP mouse model, B6-STIL, that overexpresses STIL when bred with a Cre-deleter line leading to STOP cassette excision. These mice were used for (i) generation and characterization of mice with ubiquitous STIL overexpression (STILOE) for the assessment of spontaneous centrosome amplification-driven tumor development; (ii) generation and characterization of mouse embryonic fibroblasts (MEFs) derived from these STILOE mice to determine STIL overexpression levels and the development of centrosome amplification, mitotic aberrations; (iii) generation and characterization of mice with tamoxifen-inducible epithelium-specific STIL overexpression (K14(CreERT2);STILOE) with and without active TP53, which were used in skin carcinogenesis assays, to determine the relative contribution of supernumerary centrosomes and chromosomal instability to chemical tumor induction and progression. Our results showed a graded overexpression of STIL mRNA and protein in early MEFs passages and tissues from heterozygous STILOE and homozygous STILOE mice, leading to significant centrosome amplification and chromosomal aberrations via aberrant mitoses. Importantly, MEFs with high levels of STIL-induced centrosome amplification showed a proliferative disadvantage with a strong selective pressure to eliminate cells overexpressing STIL by apoptosis and senescence. In line, rates of both, spontaneous tumor formation in STILOE mice and chemically induced skin tumors in K14(CreERT2);STILOE animals are largely reduced as compared to controls. Thus, centrosome amplification induced by STIL overexpression seems to inhibit tumor formation rather than enhancing it in vivo in mammals.
STIL Overexpression, STIL protein, PLK4, PLK4 overexpression, domain negative TP53, B6-STIL mice, STILOE mice, STILOE MEFs, Cell cycle, Mitosis, abnormal mitosis, metaphase spreads, M-FISH, Centrosome, Centriole, Supernumerary Centriole Formation, Centrosome amplification, Centriole rosettes, Chromosomal Instability, aneuploidy, Chromosome missegregation, Tetraploiy, Polyploidy, Cancer, Tumorigenesis, Tumor, Transgenic mice, p53 R172H mice, K14(CreERT2) mice, tamoxifen, skin carcinogenesis assay, spontaneous tumorigenesis, chemically induced tumorigenesis
Moussa, Amira-Talaat
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Moussa, Amira-Talaat (2022): Role of STIL overexpression in supernumerary centriole formation, chromosomal instability and cancer development in mice. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

Centrosome is the major microtubule-organizing center in mammalian cell and consists of a pair of centrioles embedded in pericentriolar material. Centrosomes are important for bipolar spindle formation and correct chromosome segregation during mitosis. Disruption of normal centrosome function leads to aneuploidy and chromosome missegregation. Many cancer cells harbor supernumerary centrosomes, which were shown to correlate to chromosomal instability, clinical aggressiveness, and poor prognosis. Nevertheless, the question, whether amplified centrosomes can drive tumorigenesis in vivo remains unresolved. The process of centrosome duplication is tightly controlled by a small set of proteins including the kinase protein PLK4 and the structural centriole proteins as STIL and SAS6. Depletion of any one of these proteins’ blocks centrosome duplication and, conversely, overexpression causes centrosome amplification. In different PLK4 overexpression in vivo models, it remains arguable, whether extra centrosomes could derive tumorigenesis and whether the generated tumors might be induced by the additional serine/threonine kinase functions of PLK4 besides its role in regulating centriole duplication. On the other hand, the structural centrosome protein STIL is involved only in centriole replication without any other known functions up to now and its overexpression leads to the formation of supernumerary centrioles in tissue culture. Therefore, investigating the role of centrosome aberrations in chromosomal instability and tumor formation in vivo using STIL overexpression (STILOE) mouse model will add important information to the conflicting data generated by PLK4 overexpressing mice. Accordingly, we generated a new transgenic Cre-LoxP mouse model, B6-STIL, that overexpresses STIL when bred with a Cre-deleter line leading to STOP cassette excision. These mice were used for (i) generation and characterization of mice with ubiquitous STIL overexpression (STILOE) for the assessment of spontaneous centrosome amplification-driven tumor development; (ii) generation and characterization of mouse embryonic fibroblasts (MEFs) derived from these STILOE mice to determine STIL overexpression levels and the development of centrosome amplification, mitotic aberrations; (iii) generation and characterization of mice with tamoxifen-inducible epithelium-specific STIL overexpression (K14(CreERT2);STILOE) with and without active TP53, which were used in skin carcinogenesis assays, to determine the relative contribution of supernumerary centrosomes and chromosomal instability to chemical tumor induction and progression. Our results showed a graded overexpression of STIL mRNA and protein in early MEFs passages and tissues from heterozygous STILOE and homozygous STILOE mice, leading to significant centrosome amplification and chromosomal aberrations via aberrant mitoses. Importantly, MEFs with high levels of STIL-induced centrosome amplification showed a proliferative disadvantage with a strong selective pressure to eliminate cells overexpressing STIL by apoptosis and senescence. In line, rates of both, spontaneous tumor formation in STILOE mice and chemically induced skin tumors in K14(CreERT2);STILOE animals are largely reduced as compared to controls. Thus, centrosome amplification induced by STIL overexpression seems to inhibit tumor formation rather than enhancing it in vivo in mammals.