Inactivation of the retinoblastoma protein (pRb) is one of the most frequent events in cancer and is often the consequence of hyperactivation of regulatory cyclin D/cdk complexes. Examples of this include overexpression of cyclin D1 in breast tumors and loss of p16r[INK4] in melanoma and many other tumor types. Indeed tumors that frequently overexpress cyclin D1 show infrequent pRB loss, suggesting that cyclin D1 overexpression provides an oncogenic advantage that extends beyond loss of pRB. This might derive from tissue-specific functions of cyclin D1. For example, cyclin D1 has been reported to activate transcription in complex with the estrogen receptor, directly regulate the transcription factors DMP-1, v-myb, and STAT-3, and, with cdks, to act as a """"""""sink"""""""" for p21 and p27, inhibitory proteins that prevent the activity of cyclin E/cdk2. Moreover, it has been proposed based on experiments in cells lacking p21 and p27 and in mice expressing cyclin E in place of cyclin D1 that cyclin D1's kinase activity is dispensable for proliferation. Because of these observations, it is now unclear if kinase activation by cyclin D1 underlies its role in tumorigenesis, and any efforts to inhibit cyclin D1-dependent tumors specifically are compromised by a lack of certainty as to the biochemical role of cyclin D1 in dysregulated proliferation. To address this, we have produced alleles of cyclin D1 that retain catalytic activity but lack the LXCXE """"""""pRb binding"""""""" domain or bind cdk4/6 without ability to activate the kinase. This latter mutant, K112E, cannot reverse pRb-mediated proliferation arrest, but does interfere with expression of senescence markers, suggesting retention of a noncatalytic function capable of interrupting aspects of pRb-induced cell cycle exit. We have moved these analyses in vivo, exploiting the fact that mice lacking cyclin D1 are viable but smaller than wild-type mice and show profound defects in breast and retinal development. Further, cyclin D1-nullizygotes resist experimental induction of cancer. We have produced mice expressing the K112E or the LXCXE-deleted allele from the endogenous cyclin D1 locus. Our initial studies indicate that K112E homozygotes display essentially normal retinal and breast development, but are small as are cyclin D1 knockouts, suggesting kinase activation may be important in some tissues but not others. Here, it is proposed to (1) fully characterize """"""""knock-in"""""""" mice expressing mutant alleles of cyclin D1; (2) produce primary cell cultures from these animals to analyze the biochemical properties of endogenously expressed cyclin D1 mutants and to analyze the impact of such mutants on normal cell cycle and transformation; (3) mate the knock-in animals to pre-existing transgenic animals prone to develop breast cancer to determine cyclin D1's function in this disease, and (4) study the kinase-dependent role of cyclin D1 in organism size control.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA096527-07
Application #
6851645
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Mietz, Judy
Project Start
1997-05-01
Project End
2007-06-30
Budget Start
2004-02-25
Budget End
2004-06-30
Support Year
7
Fiscal Year
2003
Total Cost
$208,490
Indirect Cost
Name
Tufts University
Department
Type
DUNS #
079532263
City
Boston
State
MA
Country
United States
Zip Code
02111
Hinds, Philip W (2014) A little pRB can lead to big problems. Cancer Discov 4:764-5
Luo, Chi; Sheng, Jinghao; Hu, Miaofen G et al. (2013) Loss of ARF sensitizes transgenic BRAFV600E mice to UV-induced melanoma via suppression of XPC. Cancer Res 73:4337-48
Jeselsohn, Rinath; Brown, Nelson E; Arendt, Lisa et al. (2010) Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis. Cancer Cell 17:65-76
Hu, Miaofen G; Deshpande, Amit; Enos, Miriam et al. (2009) A requirement for cyclin-dependent kinase 6 in thymocyte development and tumorigenesis. Cancer Res 69:810-8
Piscopo, Denise M; Hinds, Philip W (2008) A role for the cyclin box in the ubiquitin-mediated degradation of cyclin G1. Cancer Res 68:5581-90
Landis, Mark W; Brown, Nelson E; Baker, Gregory L et al. (2007) The LxCxE pRb interaction domain of cyclin D1 is dispensable for murine development. Cancer Res 67:7613-20
Grossel, Martha J; Hinds, Philip W (2006) Beyond the cell cycle: a new role for Cdk6 in differentiation. J Cell Biochem 97:485-93
Grossel, Martha J; Hinds, Philip W (2006) From cell cycle to differentiation: an expanding role for cdk6. Cell Cycle 5:266-70
Landis, Mark W; Pawlyk, Basil S; Li, Tiansen et al. (2006) Cyclin D1-dependent kinase activity in murine development and mammary tumorigenesis. Cancer Cell 9:13-22
Deshpande, Amit; Sicinski, Peter; Hinds, Philip W (2005) Cyclins and cdks in development and cancer: a perspective. Oncogene 24:2909-15

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