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Kinase-independent role of cyclin D1 in chromosomal instability and mammary tumorigenesis.

Casimiro MC, Di Sante G, Crosariol M, Loro E, Dampier W, Ertel A, Yu Z, Saria EA, Papanikolaou A, Li Z, Wang C, Addya S, Lisanti MP, Fortina P, Cardiff RD, Tozeren A, Knudsen ES, Arnold A, Pestell RG - Oncotarget (2015)

Bottom Line: Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1.ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN.We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cancer Biology, Thomas Jefferson University & Hospital, Philadelphia, PA 19107, USA.

ABSTRACT
Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

Cyclin D1 induces CIN genes in vivo and mouse mammary tumorigenesis independent of its kinase activity(A) Western blot using anti-FLAG of mammary gland protein lysates from Tet-CCND1WT and Tet-CCND1KE mice treated with doxycycline compared to control (Left panel). (B) Venn diagram representing genes differentially regulated by Tet-CCND1WT (n = 3) and Tet-CCND1KE (n = 3) (Right panel). 1-way ANOVA was used to evaluate the significance of differential expression between biological conditions. Data represents p < 0.05 and fold change in gene expression >1.5. (C) The most highly differentially regulated genes (Fold >2, p < 0.05) for Tet-CCND1WT (red line) and Tet-CCND1KE (green line) induced genes [16] are enriched for high CIN score (p < 0.0001). (D) Tumor incidence was markedly increased in MMTV-CCND1WT mice and MMTV-CCND1KE mice compared to WT mice. (E) Kaplan–Meier survival curves from mammary tumors of MMTV-CCND1WT (red line) and MMTV-CCND1KE (green line). (F) The most highly differentially regulated genes (Fold >2, B >3) for MMTV-CCND1WT (red line) and MMTV-CCND1KE (green line) induced genes are enriched for high CIN score (p < 0.0001). mRNA from the mammary glands of ponasterone A inducible cyclin D1 antisense mice [12] were subjected to microarray analysis demonstrated reduced CIN gene expression for cyclin D1 induced genes (p < 0.0001).
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Figure 3: Cyclin D1 induces CIN genes in vivo and mouse mammary tumorigenesis independent of its kinase activity(A) Western blot using anti-FLAG of mammary gland protein lysates from Tet-CCND1WT and Tet-CCND1KE mice treated with doxycycline compared to control (Left panel). (B) Venn diagram representing genes differentially regulated by Tet-CCND1WT (n = 3) and Tet-CCND1KE (n = 3) (Right panel). 1-way ANOVA was used to evaluate the significance of differential expression between biological conditions. Data represents p < 0.05 and fold change in gene expression >1.5. (C) The most highly differentially regulated genes (Fold >2, p < 0.05) for Tet-CCND1WT (red line) and Tet-CCND1KE (green line) induced genes [16] are enriched for high CIN score (p < 0.0001). (D) Tumor incidence was markedly increased in MMTV-CCND1WT mice and MMTV-CCND1KE mice compared to WT mice. (E) Kaplan–Meier survival curves from mammary tumors of MMTV-CCND1WT (red line) and MMTV-CCND1KE (green line). (F) The most highly differentially regulated genes (Fold >2, B >3) for MMTV-CCND1WT (red line) and MMTV-CCND1KE (green line) induced genes are enriched for high CIN score (p < 0.0001). mRNA from the mammary glands of ponasterone A inducible cyclin D1 antisense mice [12] were subjected to microarray analysis demonstrated reduced CIN gene expression for cyclin D1 induced genes (p < 0.0001).

Mentions: To directly determine the role of cyclin D1-mediated kinase activity in promoting mammary tumorigenesis, transgenic mouse models were deployed using either the tetracycline-inducible cyclin D1 transgenic mice (rtTA/CCND1), the Ponasterone inducible mammary epithelial cell targeted cyclin D1-antisense or the MMTV-cyclin D1 transgenic mouse model [12, 22] (Supplementary Figures S6A and S6C). Mammary-targeted expression of cyclin D1 was achieved by crossing transgenic mice carrying a mammary gland targeted recombinant Tetracycline transcription factor (rtTA-Tet ON system) to transgenic mice bearing an rtTA-responsive promoter driving either cyclin D1WT or cyclin D1KE (PTet-CCND1WT and PTet-CCND1KE). The resulting offspring double positive for the transgenes were designated rtTA/CCND1WT and rtTA/CCND1KE (Supplementary Figure S6B). Pregnant females (14 days post coitus) were treated with tetracycline for 7 days, followed by sacrifice of the animals and removal of the thoracic mammary glands for further studies. Western blot analysis verified the induction of the cyclin D1 transgene (Figure 3A). Microarray analysis for gene expression profiles of the mammary glands identified gene clusters regulated by cyclin D1WT and cyclin D1KE (Figure 3B, Supplementary Figure S7A and Dataset S1). There was significant overlap between the gene expression profile regulated by cyclin D1WT and cyclin D1KE (p < 1 × 10−10). Pathway analysis of the genes in common between rtTA/CCND1WT and rtTA/CCND1KE revealed many functional terms previously identified as being cyclin D1 regulated including cell cycle and mitosis (Supplementary Figure S7B). Notably, the rtTA/CCND1WT gene profile was enriched for high CIN score genes to a similar level as the rtTA/CCND1KE gene profile (Figure 3C). Therefore, acute expression of cyclin D1KE was sufficient to induce CIN gene expression profiles within 7 days.


Kinase-independent role of cyclin D1 in chromosomal instability and mammary tumorigenesis.

Casimiro MC, Di Sante G, Crosariol M, Loro E, Dampier W, Ertel A, Yu Z, Saria EA, Papanikolaou A, Li Z, Wang C, Addya S, Lisanti MP, Fortina P, Cardiff RD, Tozeren A, Knudsen ES, Arnold A, Pestell RG - Oncotarget (2015)

Cyclin D1 induces CIN genes in vivo and mouse mammary tumorigenesis independent of its kinase activity(A) Western blot using anti-FLAG of mammary gland protein lysates from Tet-CCND1WT and Tet-CCND1KE mice treated with doxycycline compared to control (Left panel). (B) Venn diagram representing genes differentially regulated by Tet-CCND1WT (n = 3) and Tet-CCND1KE (n = 3) (Right panel). 1-way ANOVA was used to evaluate the significance of differential expression between biological conditions. Data represents p < 0.05 and fold change in gene expression >1.5. (C) The most highly differentially regulated genes (Fold >2, p < 0.05) for Tet-CCND1WT (red line) and Tet-CCND1KE (green line) induced genes [16] are enriched for high CIN score (p < 0.0001). (D) Tumor incidence was markedly increased in MMTV-CCND1WT mice and MMTV-CCND1KE mice compared to WT mice. (E) Kaplan–Meier survival curves from mammary tumors of MMTV-CCND1WT (red line) and MMTV-CCND1KE (green line). (F) The most highly differentially regulated genes (Fold >2, B >3) for MMTV-CCND1WT (red line) and MMTV-CCND1KE (green line) induced genes are enriched for high CIN score (p < 0.0001). mRNA from the mammary glands of ponasterone A inducible cyclin D1 antisense mice [12] were subjected to microarray analysis demonstrated reduced CIN gene expression for cyclin D1 induced genes (p < 0.0001).
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Figure 3: Cyclin D1 induces CIN genes in vivo and mouse mammary tumorigenesis independent of its kinase activity(A) Western blot using anti-FLAG of mammary gland protein lysates from Tet-CCND1WT and Tet-CCND1KE mice treated with doxycycline compared to control (Left panel). (B) Venn diagram representing genes differentially regulated by Tet-CCND1WT (n = 3) and Tet-CCND1KE (n = 3) (Right panel). 1-way ANOVA was used to evaluate the significance of differential expression between biological conditions. Data represents p < 0.05 and fold change in gene expression >1.5. (C) The most highly differentially regulated genes (Fold >2, p < 0.05) for Tet-CCND1WT (red line) and Tet-CCND1KE (green line) induced genes [16] are enriched for high CIN score (p < 0.0001). (D) Tumor incidence was markedly increased in MMTV-CCND1WT mice and MMTV-CCND1KE mice compared to WT mice. (E) Kaplan–Meier survival curves from mammary tumors of MMTV-CCND1WT (red line) and MMTV-CCND1KE (green line). (F) The most highly differentially regulated genes (Fold >2, B >3) for MMTV-CCND1WT (red line) and MMTV-CCND1KE (green line) induced genes are enriched for high CIN score (p < 0.0001). mRNA from the mammary glands of ponasterone A inducible cyclin D1 antisense mice [12] were subjected to microarray analysis demonstrated reduced CIN gene expression for cyclin D1 induced genes (p < 0.0001).
Mentions: To directly determine the role of cyclin D1-mediated kinase activity in promoting mammary tumorigenesis, transgenic mouse models were deployed using either the tetracycline-inducible cyclin D1 transgenic mice (rtTA/CCND1), the Ponasterone inducible mammary epithelial cell targeted cyclin D1-antisense or the MMTV-cyclin D1 transgenic mouse model [12, 22] (Supplementary Figures S6A and S6C). Mammary-targeted expression of cyclin D1 was achieved by crossing transgenic mice carrying a mammary gland targeted recombinant Tetracycline transcription factor (rtTA-Tet ON system) to transgenic mice bearing an rtTA-responsive promoter driving either cyclin D1WT or cyclin D1KE (PTet-CCND1WT and PTet-CCND1KE). The resulting offspring double positive for the transgenes were designated rtTA/CCND1WT and rtTA/CCND1KE (Supplementary Figure S6B). Pregnant females (14 days post coitus) were treated with tetracycline for 7 days, followed by sacrifice of the animals and removal of the thoracic mammary glands for further studies. Western blot analysis verified the induction of the cyclin D1 transgene (Figure 3A). Microarray analysis for gene expression profiles of the mammary glands identified gene clusters regulated by cyclin D1WT and cyclin D1KE (Figure 3B, Supplementary Figure S7A and Dataset S1). There was significant overlap between the gene expression profile regulated by cyclin D1WT and cyclin D1KE (p < 1 × 10−10). Pathway analysis of the genes in common between rtTA/CCND1WT and rtTA/CCND1KE revealed many functional terms previously identified as being cyclin D1 regulated including cell cycle and mitosis (Supplementary Figure S7B). Notably, the rtTA/CCND1WT gene profile was enriched for high CIN score genes to a similar level as the rtTA/CCND1KE gene profile (Figure 3C). Therefore, acute expression of cyclin D1KE was sufficient to induce CIN gene expression profiles within 7 days.

Bottom Line: Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1.ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN.We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cancer Biology, Thomas Jefferson University & Hospital, Philadelphia, PA 19107, USA.

ABSTRACT
Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.

No MeSH data available.


Related in: MedlinePlus