Limits...
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 induction of centrosome amplification and mitotic spindle disorganization is independent of cyclin D1 kinase activity(A) Representative confocal maximum Z projections of mitotic cells from cyclin D1−/−Control, cyclin D1−/−D1 Rescue and cyclin D1−/−KE Rescue. Cells were immunostained for α-tubulin (red), γ-tubulin (yellow), crest (green), and Hoechst (blue). Scalebar 5 μm. (B) Frequencies of mitotic cells with multiple polar spindles (**p = 0.0051, ***p = 0.0004; calculated by Fisher contingency test). (C) Frequency of cells with multiple chromosomes (*p = 0.021, ***p = 0.0007; calculated by Fisher contingency test). (D and E) Spindle measurements on maximum Z projections of metaphase cyclin D1−/−, cyclin D1−/−D1 Rescue and cyclin D1−/−KE Rescue cells. Measurement of metaphase plate dimensions (DAPI): ChL, chromatin length; ChW, chromatin width (**p = 0.0087, ***p < 0.001). Measurement of spindle dimensions (tubulin): SpW, spindle width; SpL, spindle length (*p = 0.0486; data are mean of ± SEM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496164&req=5

Figure 1: Cyclin D1 induction of centrosome amplification and mitotic spindle disorganization is independent of cyclin D1 kinase activity(A) Representative confocal maximum Z projections of mitotic cells from cyclin D1−/−Control, cyclin D1−/−D1 Rescue and cyclin D1−/−KE Rescue. Cells were immunostained for α-tubulin (red), γ-tubulin (yellow), crest (green), and Hoechst (blue). Scalebar 5 μm. (B) Frequencies of mitotic cells with multiple polar spindles (**p = 0.0051, ***p = 0.0004; calculated by Fisher contingency test). (C) Frequency of cells with multiple chromosomes (*p = 0.021, ***p = 0.0007; calculated by Fisher contingency test). (D and E) Spindle measurements on maximum Z projections of metaphase cyclin D1−/−, cyclin D1−/−D1 Rescue and cyclin D1−/−KE Rescue cells. Measurement of metaphase plate dimensions (DAPI): ChL, chromatin length; ChW, chromatin width (**p = 0.0087, ***p < 0.001). Measurement of spindle dimensions (tubulin): SpW, spindle width; SpL, spindle length (*p = 0.0486; data are mean of ± SEM).

Mentions: Prior to engaging in studies to question whether the induction of aneuploidy by cyclin D1 is kinase-independent we verified the relative abundance and nuclear localization of cyclin D1KE. In cyclin D1−/− cells rescued with either cyclin D1WT or cyclin D1KE, the protein abundance was similar between the two cell lines (Supplementary Figure S1B). In addition there was no difference in the abundance within the nuclear compartment (Supplementary Figures S1C and S1D). Next we, determined the subcellular compartmentalization of cyclin D1KE and cyclin D1WT. We compared 3T3 wild type cells to 3T3 wild type cells transduced with MSCV-Cyclin D1KE and the localization of exogenous cyclin D1KE and endogenous cyclin D1WT protein monitored during aphidocoline block in G1 to release into S phase. Cyclin D1KE, like endogenous cyclin D1WT, was exported from the nucleus to the cytoplasm (Supplementary Figure S2). Next, to determine whether the alterations in mitotic abnormalities were induced by cyclin D1WT via its CDK-activating function, we performed immunofluorescence followed by high resolution confocal imaging of cyclin D1−/− 3T3 cells, rescued with either cyclin D1WT or cyclin D1KE (Figure 1A). The number of cells with multi-polar spindles was increased 28% in the cyclin D1−/−D1 Rescue cells and 31% in the cyclin D1−/−KE Rescue cells compared to control (p = 0.0051 and p = 0.0004 respectively) (Figures 1A and 1B). The generation of multi-polar spindle cells arising from abnormalities in centrosome number and distribution were quantitatively assessed using α-tubulin staining in conjunction with γ-tubulin. The cyclin D1−/−D1 Rescue and the cyclin D1−/−KE Rescue increased the percentage of prometaphase/metaphase cells with multiple centrosomes by 20% (p = 0.0021) and 28% (p = 0.0007) respectively compared to control cells (Figures 1A and 1C). The alteration of spindle architecture associated with metaphase plate disruption was measured by assessing metaphase plate length and width (ChL, Chw) and spindle length and width (SpL, SpW) (Figures 1D and 1E). Consistent with the increase in spindle/centrosome abnormalities, the ChW and SpL were significantly increased in cyclin D1−/−D1 Rescue and cyclin D1−/−KE Rescue cells compared with cyclin D1−/−Control cells.


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 induction of centrosome amplification and mitotic spindle disorganization is independent of cyclin D1 kinase activity(A) Representative confocal maximum Z projections of mitotic cells from cyclin D1−/−Control, cyclin D1−/−D1 Rescue and cyclin D1−/−KE Rescue. Cells were immunostained for α-tubulin (red), γ-tubulin (yellow), crest (green), and Hoechst (blue). Scalebar 5 μm. (B) Frequencies of mitotic cells with multiple polar spindles (**p = 0.0051, ***p = 0.0004; calculated by Fisher contingency test). (C) Frequency of cells with multiple chromosomes (*p = 0.021, ***p = 0.0007; calculated by Fisher contingency test). (D and E) Spindle measurements on maximum Z projections of metaphase cyclin D1−/−, cyclin D1−/−D1 Rescue and cyclin D1−/−KE Rescue cells. Measurement of metaphase plate dimensions (DAPI): ChL, chromatin length; ChW, chromatin width (**p = 0.0087, ***p < 0.001). Measurement of spindle dimensions (tubulin): SpW, spindle width; SpL, spindle length (*p = 0.0486; data are mean of ± SEM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496164&req=5

Figure 1: Cyclin D1 induction of centrosome amplification and mitotic spindle disorganization is independent of cyclin D1 kinase activity(A) Representative confocal maximum Z projections of mitotic cells from cyclin D1−/−Control, cyclin D1−/−D1 Rescue and cyclin D1−/−KE Rescue. Cells were immunostained for α-tubulin (red), γ-tubulin (yellow), crest (green), and Hoechst (blue). Scalebar 5 μm. (B) Frequencies of mitotic cells with multiple polar spindles (**p = 0.0051, ***p = 0.0004; calculated by Fisher contingency test). (C) Frequency of cells with multiple chromosomes (*p = 0.021, ***p = 0.0007; calculated by Fisher contingency test). (D and E) Spindle measurements on maximum Z projections of metaphase cyclin D1−/−, cyclin D1−/−D1 Rescue and cyclin D1−/−KE Rescue cells. Measurement of metaphase plate dimensions (DAPI): ChL, chromatin length; ChW, chromatin width (**p = 0.0087, ***p < 0.001). Measurement of spindle dimensions (tubulin): SpW, spindle width; SpL, spindle length (*p = 0.0486; data are mean of ± SEM).
Mentions: Prior to engaging in studies to question whether the induction of aneuploidy by cyclin D1 is kinase-independent we verified the relative abundance and nuclear localization of cyclin D1KE. In cyclin D1−/− cells rescued with either cyclin D1WT or cyclin D1KE, the protein abundance was similar between the two cell lines (Supplementary Figure S1B). In addition there was no difference in the abundance within the nuclear compartment (Supplementary Figures S1C and S1D). Next we, determined the subcellular compartmentalization of cyclin D1KE and cyclin D1WT. We compared 3T3 wild type cells to 3T3 wild type cells transduced with MSCV-Cyclin D1KE and the localization of exogenous cyclin D1KE and endogenous cyclin D1WT protein monitored during aphidocoline block in G1 to release into S phase. Cyclin D1KE, like endogenous cyclin D1WT, was exported from the nucleus to the cytoplasm (Supplementary Figure S2). Next, to determine whether the alterations in mitotic abnormalities were induced by cyclin D1WT via its CDK-activating function, we performed immunofluorescence followed by high resolution confocal imaging of cyclin D1−/− 3T3 cells, rescued with either cyclin D1WT or cyclin D1KE (Figure 1A). The number of cells with multi-polar spindles was increased 28% in the cyclin D1−/−D1 Rescue cells and 31% in the cyclin D1−/−KE Rescue cells compared to control (p = 0.0051 and p = 0.0004 respectively) (Figures 1A and 1B). The generation of multi-polar spindle cells arising from abnormalities in centrosome number and distribution were quantitatively assessed using α-tubulin staining in conjunction with γ-tubulin. The cyclin D1−/−D1 Rescue and the cyclin D1−/−KE Rescue increased the percentage of prometaphase/metaphase cells with multiple centrosomes by 20% (p = 0.0021) and 28% (p = 0.0007) respectively compared to control cells (Figures 1A and 1C). The alteration of spindle architecture associated with metaphase plate disruption was measured by assessing metaphase plate length and width (ChL, Chw) and spindle length and width (SpL, SpW) (Figures 1D and 1E). Consistent with the increase in spindle/centrosome abnormalities, the ChW and SpL were significantly increased in cyclin D1−/−D1 Rescue and cyclin D1−/−KE Rescue cells compared with cyclin D1−/−Control cells.

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