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Consequence of the tumor-associated conversion to cyclin D1b.

Augello MA, Berman-Booty LD, Carr R, Yoshida A, Dean JL, Schiewer MJ, Feng FY, Tomlins SA, Gao E, Koch WJ, Benovic JL, Diehl JA, Knudsen KE - EMBO Mol Med (2015)

Bottom Line: Extensive analyses uncovered overlapping but non-redundant functions of cyclin D1b compared to cyclin D1 on developmental phenotypes, and illustrated the importance of the transcriptional regulatory functions of cyclin D1b in vivo.Data obtained identify cyclin D1b as an oncogene, wherein cyclin D1b expression under the endogenous promoter induced cellular transformation and further cooperated with known oncogenes to promote tumor growth in vivo.Collectively, these data are the first to define the consequence of cyclin D1b expression on normal cellular function, present evidence for cyclin D1b as an oncogene, and provide pre-clinical evidence of effective methods to thwart growth of cells dependent upon this oncogenic variant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA.

No MeSH data available.


Related in: MedlinePlus

Genetic evidence for cyclin D1b as a credentialed oncogeneMurine adult fibroblasts (MAFs) were harvested from the peritoneum of Ccnd1+/+ and Ccnd1KI/KI mice, and stable cell lines were generated utilizing a 3T3 protocol.Indicated passage-matched MAF lines were grown to 70% confluency and then harvested for RNA and protein extraction. Top: qPCR analysis of cyclin D1 transcript using primer pairs common to both transcript a and transcript b. Bottom: Immunoblot of cyclin D1 levels in Ccnd1+/+ and Ccnd1KI/KMAF lines using an antibody common to both isoforms.Percentage of tumor-free mice post-injection of 1 million cells mixed with Matrigel (1:1 ratio) into the flanks of nude mice over a period of 10 months (n = 10 per genotype).Indicated tumors were harvested 3 weeks after detectable tumor formation, fixed in formalin, and stained with H&E. Tumors were analyzed for features of malignancy by a board-certified veterinary pathologist. Arrows indicate the specific tumor-associated features noted. Top panels were taken at 40× magnification, and lower panels were taken at 400× magnification.Ccnd1+/+ or Ccnd1KI/KI MAF lines were stably transfected with either cyclin D1a (+/+), cyclin D1b (KI/KI) or vector control constructs. After selection with puromycin, individual lines were assessed for the induction of cyclin D1a in the +/+ line and cyclin D1b in KI/KI lines via immunoblot.One million cells from the indicated stable cell lines were injected subcutaneously into the flanks of nude mice and monitored for tumor formation over a period of 365 days. Indicated time points represent time of palpable tumor detection and are plotted as % of tumor-free mice over time.Sections from KI/KI vec and KI/KI-D1b tumors were stained for the proliferative marker Ki67. Three random fields from each tumor were quantified for Ki67 positivity, and are plotted as the mean ratio of Ki67-positive cells/total cell number for each individual tumor.Data information: Error bars represent ± SEM.
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fig03: Genetic evidence for cyclin D1b as a credentialed oncogeneMurine adult fibroblasts (MAFs) were harvested from the peritoneum of Ccnd1+/+ and Ccnd1KI/KI mice, and stable cell lines were generated utilizing a 3T3 protocol.Indicated passage-matched MAF lines were grown to 70% confluency and then harvested for RNA and protein extraction. Top: qPCR analysis of cyclin D1 transcript using primer pairs common to both transcript a and transcript b. Bottom: Immunoblot of cyclin D1 levels in Ccnd1+/+ and Ccnd1KI/KMAF lines using an antibody common to both isoforms.Percentage of tumor-free mice post-injection of 1 million cells mixed with Matrigel (1:1 ratio) into the flanks of nude mice over a period of 10 months (n = 10 per genotype).Indicated tumors were harvested 3 weeks after detectable tumor formation, fixed in formalin, and stained with H&E. Tumors were analyzed for features of malignancy by a board-certified veterinary pathologist. Arrows indicate the specific tumor-associated features noted. Top panels were taken at 40× magnification, and lower panels were taken at 400× magnification.Ccnd1+/+ or Ccnd1KI/KI MAF lines were stably transfected with either cyclin D1a (+/+), cyclin D1b (KI/KI) or vector control constructs. After selection with puromycin, individual lines were assessed for the induction of cyclin D1a in the +/+ line and cyclin D1b in KI/KI lines via immunoblot.One million cells from the indicated stable cell lines were injected subcutaneously into the flanks of nude mice and monitored for tumor formation over a period of 365 days. Indicated time points represent time of palpable tumor detection and are plotted as % of tumor-free mice over time.Sections from KI/KI vec and KI/KI-D1b tumors were stained for the proliferative marker Ki67. Three random fields from each tumor were quantified for Ki67 positivity, and are plotted as the mean ratio of Ki67-positive cells/total cell number for each individual tumor.Data information: Error bars represent ± SEM.

Mentions: The model of endogenous cyclin D1b characterized above revealed unique contributions of cyclin D1b to developmental processes and illuminated the first evidence of biological distinctions between the two isoforms. In human cancer, high cyclin D1b is associated with the poor outcome (Li et al, 2008; Millar et al, 2009), and cyclin D1b overexpression transforms cells of mesenchymal origin (Lu et al, 2003). However, the oncogenic capacity of cyclin D1b in the absence of endogenous cyclin D1a or via the endogenous CCND1 promoter had not been demonstrated. Murine models of forced cyclin D1a overexpression show limited oncogenic capacity (Wang et al, 1994); moreover, tumor development is not fully penetrant and is quite latent. Similar results were observed herein utilizing models of cyclin D1b expression driven from the endogenous promoter. Ccnd1KI/KI mice up to 9 months of age showed no significant difference in the number hyperplastic or neoplastic lesions compared to age-matched wild-type controls, though animals are currently being aged further to quantify tumor formation rates. To expedite generation time and define the potential of an endogenous shift to cyclin D1b on tumor formation, murine adult fibroblasts (MAFs) were generated from the peritoneum of Ccnd1+/+ and Ccnd1KI/KI mice (Fig3A). Similar models that modulated the expression of various oncogenes and tumor suppressors have demonstrated that MAF lines are an effective tool with which to study both biochemical and transformation phenotypes (de Napoles et al, 2004; Powers et al, 2004; Dean et al, 2010; Bourgo et al, 2011) and as such, each line was assayed for evidence of cellular transformation. Exclusive expression of respective cyclin D1 isoforms was initially confirmed by immunoblot using an antibody common to both cyclin D1a and cyclin D1b (Fig3B). Individual Ccnd1+/+ and Ccnd1KI/KI lines were mixed with Matrigel and injected subcutaneously into the flanks of nude mice. As is shown in Fig3C, Ccnd1+/+ control cells were non-tumorigenic. By contrast, Ccnd1KI/KI cells formed tumors by ~15 weeks with high penetrance (80%). H&E staining confirmed the tumors were of mesenchymal origin, and displayed hallmarks of neoplasia including (1) invasion into the subcutaneous fat, (2) nuclear and cytoplasmic atypia, and (3) prevalence of high mitotic figures (Fig3D, left). These data are the first to demonstrate that physiological levels of cyclin D1b are sufficient to promote tumorigenesis in immortalized cells, and provide evidence to support its role as an oncogene.


Consequence of the tumor-associated conversion to cyclin D1b.

Augello MA, Berman-Booty LD, Carr R, Yoshida A, Dean JL, Schiewer MJ, Feng FY, Tomlins SA, Gao E, Koch WJ, Benovic JL, Diehl JA, Knudsen KE - EMBO Mol Med (2015)

Genetic evidence for cyclin D1b as a credentialed oncogeneMurine adult fibroblasts (MAFs) were harvested from the peritoneum of Ccnd1+/+ and Ccnd1KI/KI mice, and stable cell lines were generated utilizing a 3T3 protocol.Indicated passage-matched MAF lines were grown to 70% confluency and then harvested for RNA and protein extraction. Top: qPCR analysis of cyclin D1 transcript using primer pairs common to both transcript a and transcript b. Bottom: Immunoblot of cyclin D1 levels in Ccnd1+/+ and Ccnd1KI/KMAF lines using an antibody common to both isoforms.Percentage of tumor-free mice post-injection of 1 million cells mixed with Matrigel (1:1 ratio) into the flanks of nude mice over a period of 10 months (n = 10 per genotype).Indicated tumors were harvested 3 weeks after detectable tumor formation, fixed in formalin, and stained with H&E. Tumors were analyzed for features of malignancy by a board-certified veterinary pathologist. Arrows indicate the specific tumor-associated features noted. Top panels were taken at 40× magnification, and lower panels were taken at 400× magnification.Ccnd1+/+ or Ccnd1KI/KI MAF lines were stably transfected with either cyclin D1a (+/+), cyclin D1b (KI/KI) or vector control constructs. After selection with puromycin, individual lines were assessed for the induction of cyclin D1a in the +/+ line and cyclin D1b in KI/KI lines via immunoblot.One million cells from the indicated stable cell lines were injected subcutaneously into the flanks of nude mice and monitored for tumor formation over a period of 365 days. Indicated time points represent time of palpable tumor detection and are plotted as % of tumor-free mice over time.Sections from KI/KI vec and KI/KI-D1b tumors were stained for the proliferative marker Ki67. Three random fields from each tumor were quantified for Ki67 positivity, and are plotted as the mean ratio of Ki67-positive cells/total cell number for each individual tumor.Data information: Error bars represent ± SEM.
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fig03: Genetic evidence for cyclin D1b as a credentialed oncogeneMurine adult fibroblasts (MAFs) were harvested from the peritoneum of Ccnd1+/+ and Ccnd1KI/KI mice, and stable cell lines were generated utilizing a 3T3 protocol.Indicated passage-matched MAF lines were grown to 70% confluency and then harvested for RNA and protein extraction. Top: qPCR analysis of cyclin D1 transcript using primer pairs common to both transcript a and transcript b. Bottom: Immunoblot of cyclin D1 levels in Ccnd1+/+ and Ccnd1KI/KMAF lines using an antibody common to both isoforms.Percentage of tumor-free mice post-injection of 1 million cells mixed with Matrigel (1:1 ratio) into the flanks of nude mice over a period of 10 months (n = 10 per genotype).Indicated tumors were harvested 3 weeks after detectable tumor formation, fixed in formalin, and stained with H&E. Tumors were analyzed for features of malignancy by a board-certified veterinary pathologist. Arrows indicate the specific tumor-associated features noted. Top panels were taken at 40× magnification, and lower panels were taken at 400× magnification.Ccnd1+/+ or Ccnd1KI/KI MAF lines were stably transfected with either cyclin D1a (+/+), cyclin D1b (KI/KI) or vector control constructs. After selection with puromycin, individual lines were assessed for the induction of cyclin D1a in the +/+ line and cyclin D1b in KI/KI lines via immunoblot.One million cells from the indicated stable cell lines were injected subcutaneously into the flanks of nude mice and monitored for tumor formation over a period of 365 days. Indicated time points represent time of palpable tumor detection and are plotted as % of tumor-free mice over time.Sections from KI/KI vec and KI/KI-D1b tumors were stained for the proliferative marker Ki67. Three random fields from each tumor were quantified for Ki67 positivity, and are plotted as the mean ratio of Ki67-positive cells/total cell number for each individual tumor.Data information: Error bars represent ± SEM.
Mentions: The model of endogenous cyclin D1b characterized above revealed unique contributions of cyclin D1b to developmental processes and illuminated the first evidence of biological distinctions between the two isoforms. In human cancer, high cyclin D1b is associated with the poor outcome (Li et al, 2008; Millar et al, 2009), and cyclin D1b overexpression transforms cells of mesenchymal origin (Lu et al, 2003). However, the oncogenic capacity of cyclin D1b in the absence of endogenous cyclin D1a or via the endogenous CCND1 promoter had not been demonstrated. Murine models of forced cyclin D1a overexpression show limited oncogenic capacity (Wang et al, 1994); moreover, tumor development is not fully penetrant and is quite latent. Similar results were observed herein utilizing models of cyclin D1b expression driven from the endogenous promoter. Ccnd1KI/KI mice up to 9 months of age showed no significant difference in the number hyperplastic or neoplastic lesions compared to age-matched wild-type controls, though animals are currently being aged further to quantify tumor formation rates. To expedite generation time and define the potential of an endogenous shift to cyclin D1b on tumor formation, murine adult fibroblasts (MAFs) were generated from the peritoneum of Ccnd1+/+ and Ccnd1KI/KI mice (Fig3A). Similar models that modulated the expression of various oncogenes and tumor suppressors have demonstrated that MAF lines are an effective tool with which to study both biochemical and transformation phenotypes (de Napoles et al, 2004; Powers et al, 2004; Dean et al, 2010; Bourgo et al, 2011) and as such, each line was assayed for evidence of cellular transformation. Exclusive expression of respective cyclin D1 isoforms was initially confirmed by immunoblot using an antibody common to both cyclin D1a and cyclin D1b (Fig3B). Individual Ccnd1+/+ and Ccnd1KI/KI lines were mixed with Matrigel and injected subcutaneously into the flanks of nude mice. As is shown in Fig3C, Ccnd1+/+ control cells were non-tumorigenic. By contrast, Ccnd1KI/KI cells formed tumors by ~15 weeks with high penetrance (80%). H&E staining confirmed the tumors were of mesenchymal origin, and displayed hallmarks of neoplasia including (1) invasion into the subcutaneous fat, (2) nuclear and cytoplasmic atypia, and (3) prevalence of high mitotic figures (Fig3D, left). These data are the first to demonstrate that physiological levels of cyclin D1b are sufficient to promote tumorigenesis in immortalized cells, and provide evidence to support its role as an oncogene.

Bottom Line: Extensive analyses uncovered overlapping but non-redundant functions of cyclin D1b compared to cyclin D1 on developmental phenotypes, and illustrated the importance of the transcriptional regulatory functions of cyclin D1b in vivo.Data obtained identify cyclin D1b as an oncogene, wherein cyclin D1b expression under the endogenous promoter induced cellular transformation and further cooperated with known oncogenes to promote tumor growth in vivo.Collectively, these data are the first to define the consequence of cyclin D1b expression on normal cellular function, present evidence for cyclin D1b as an oncogene, and provide pre-clinical evidence of effective methods to thwart growth of cells dependent upon this oncogenic variant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA.

No MeSH data available.


Related in: MedlinePlus