[Cell Cycle 5:21, 2440-2442 1 November 2006]; ©2006 Landes Bioscience
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Cyclin D1 Functions in Cell Migration Zhiping Li1 Chenguang Wang1 George C. Prendergast2,3 Richard G. Pestell1
Abstract
*Correspondence to: Richard G. Pestell; Kimmel Cancer Center; Department of Cancer Biology; Thomas Jefferson University; Bluemle Life Sciences Building, Rm 1050; 233 South 10th St; Philadelphia, Pennsylvania 19107 USA; Tel.: 215.503.5649; Fax: 215.503.9334; Email:
[email protected]
Cyclin D1 is best known as the regulatory subunit of a dimeric holoenzyme including the cell cycle‑dependent kinase CDK4, which phosphorylates and inactivates the retinoblastoma protein Rb to promote progression through the G1‑S phase of the cell cycle. While there are three D‑type cyclins, only cyclin D1 is observed to be overexpressed frequently in cancer. Notably, cyclin D1 overexpression is closely associated with malignant progression and metastatic disease. Overexpression of the cyclin D1 gene has been reported in a variety of human cancers including breast, colon, prostate, and hematopoietic malignancies.1,2 In invasive breast cancers, cyclin D1 is overexpressed in up to 50% of cases.3 Functional analyses indicate that disruption of cyclin D1 can limit HER2/neu‑induced mammary tumor growth.4 Analysis of mouse strains that are genetically deficient in cyclin D1 have provided key insights into the functions of this protein in cell division and survival.5 Cyclin D1‑deficient mice are resistant to breast cancers induced by the HER2/neu or ras oncogenes4,6,7 and to gastrointestinal tumors induced by mutated forms of the Apc gene.8 In addition to regulating the cell cycle, cyclin D1 regulates angiogenesis, lipogenesis, and mitochondrial function.9‑12 Notably, cyclin D1 has a function in transcriptional control that does not involve CDKs.13 This function of cyclin D1, which has been reviewed recently,2 involves promoter recruitment of histone deacetylases (HDACs) and histone methyltransferases including SUV39 and HP1.14 Given clinical observations that cyclin D1 overexpression in human cancers is correlated with metastasis, we hypothesized that cyclin D1 may control cell migration in cancer. ��������������������������������������������� In support of this hypothesis, we found that cyclin D1‑/‑ mouse embryo fibroblasts (MEFs) displayed increased cellular adherence, defective motility, and an impaired wound healing response.15 Investigating how cyclin D1 drives cell migration led us to define two critical events in repression of thrombospondin‑1 (TSP‑1) and ROCK II, an important effector kinase for Rho small GTPases.
Cyclin D1, migration, TSP-1, Rho, ROCK
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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3428
Abbreviations
Cyclin D1 Promotes Cell Migration
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Original manuscript submitted: 09/21/06 Manuscript accepted: 09/22/06
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3Lankenau Institute for Medical Research; Wynnewood, Pennsylvania USA
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of Cancer Biology; Medical Oncology and 2Pathology Anatomy and Cell Biology; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, Pennsylvania USA
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Cell migration is essential for developmental morphogenesis, tissue repair, and tumor metastasis. A recent study reveals that cyclin D1 acts to promote cell migration by inhib‑ iting Rho/ROCK signaling and expression of thrombospondin‑1 (TSP‑1), an extracellular matrix protein that regulates cell migration in many settings including cancer. Given the frequent overexpression of cyclin D1 in cancer cells, due to its upregulation by Ras, Rho, Src, and other genes that drive malignant development, the new findings suggest that cyclin D1 may have a central role in mediating invasion and metastasis of cancer cells by controlling Rho/ROCK signaling and matrix deposition of TSP‑1.
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ROCK Rho-associated coiled-coil forming kinase TSP-1 thrombospondin 1
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Cyclin D1 Inhibits Rho‑ROCK Signaling ROCK II activity is increased in cyclin D1‑/‑ cells as evidenced by increased phosphorylation of the ROCK II substrates LIM kinase, cofilin and myosin light chain (MLC), the activation of which mediates cell migration. Restoring cyclin D1 in the null cells rescued normal regulation of ROCK II, restoring it to wild‑type levels. The specificity of these effects was confirmed by demonstrating that knocking cyclin E into cyclin D1‑/‑ cells rescued their defect in DNA synthesis but not their defects in ROCK II control or cell migration. Together, these findings broaden the functional connections of cyclin D1 and Rho small GTPase signaling pathways, the latter of which are known to regulate cyclin Cell Cycle
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Cyclin D1 Functions in Cell Migration
D1 itself as well as cell migration.16 For example, induction of cyclin D1 by the Rho family members RhoA, Rac1 and Cdc42 is associated with their requirements in neoplastic cell transformation by Ras or HER2/neu.17 While older studies have identified cyclin D1 as a downstream target of Rho proteins, the new work shows that cyclin D1 also functions upstream of Rho by suppressing ROCK II activity, thereby acting as a negative feedback on Rho signaling. The finding that cyclin D1 directly regulates cell migration are consistent with recent evidence that the cyclin D1‑binding proteins p21CIP1 and p27KlP1 also regulate migration. p27KlP1 has been shown to either inhibit18 or promote cell migration,19,20 perhaps depending on cell type, context, or other factors. The pro‑migratory function of p27KIP1 has been defined by showing that genetic deletions of p27KIP1 cause Rho hyperactivation. Cyclin D1 has been identified in the cell membrane, where it may interact with Rho signaling proteins, and in transgenic mice cyclin D1 and p27KIP1 act collaboratively. Collectively these studies raise the possibility that physical interaction between cyclin D1 and p27KIP1 may promote cellular migration and invasiveness involving RhoA.21 Anti‑migratory effects of p27KIP1 localize to its C‑terminal region, which binds the cytosolic protein stathmin, a microtubule destabilizing protein implicated in migration of germ cells and neurons.22 By activating Rac or inactivating CDK1, p27KIP1 may also inactivate stathmin indirectly by inducing its phosphorylation‑dependent inactivation.23 Stathmin is expressed at very high levels in many cancer cells making the stoichiometry of its interaction with p27KIP1 of interest to future studies. ROCK is an important effector kinase for Rho GTPases, which control cell motility by regulating cytoskeletal actin organization and cell adhesion.24,25 Major ROCK substrates include myosin light chain kinase (MLCK) and LIM kinase. MLCK phosphorylates myosin light chain �������������������������������������������������� but also inhibits myosin light chain phosphatase. LIM kinase phosphorylates the actin‑depolymerizing protein cofilin and inhibits its ability to sever actin filaments, thereby stabilizing actin stress fibers. Additionally, ROCK promotes stabilization of actin stress fibers by enhancing the binding of adducin to actin filaments and by facilitating PIP2‑dependent activation of the ezrin/ rodixin/moesin (ERM) family of actin‑binding proteins. ��������� Although Rho activity negatively influences cell migration by increasing stress fiber‑dependent adhesions to substratum, Rho activity is also required for actin‑myosin contractility needed to drive cell body retraction at the rear of the cell.26 Rho GTPases also regulate assembly of actin stress fibers and focal adhesions through the Diaphanous effector proteins (mDia or DRF). Rac and its effector kinase PAK control cell motility by regulating formation of lamellipodia and new focal contacts.27 Activation and inactivation of Rho proteins is tightly coordinated and insufficient or excessive Rho GTPase activity will prevent cell migration.28-31 Rac and Rho activities are geographically coordinated in the cell, conveying distinct functions at different sites. Thus, Rac activity facilitates protrusive activity at the front of a moving cell, while Rho activity prevents protrusive activity at the rear of the cell. A large number of cytokines, chemokines, growth factors, ECM proteins and ECM‑degrading proteins influence cell migration by regulating Rho and Rac, which in turn regulate the expression of these diverse factors. With regard to ROCK signaling, while most current understanding is derived from studies of fibroblasts cultured on fibronectin, the presence of other ECM components may alter signaling patterns significantly. For example, the ECM protein tenascin‑C, which is highly expressed during development and metaswww.landesbioscience.com
tasis, attenuates fibronectin‑induced Rho activity. Similarly, the ECM protein TSP‑1 modifies cell spreading responses, causing induction of actin‑containing microspikes and disassembly of Rho‑dependent focal adhesions.32
TSP‑1 Inhibition Mediates Cell Migration by Cyclin D1 The ECM protein TSP‑1—a known suppressor of cell migration —was identified initially as a target for inhibition by cyclin D1 through an expression array analysis that employed cyclin D1‑/‑ cells.15 In considering targets of cyclin D1 that mediated effects on migration, the role of TSP‑1 was highlighted by the ability of neutralizing antibodies to ablate the effects of cyclin D1 loss on motility.15 TSP‑1 inhibits cellular metastasis and is repressed by oncogenic Ras.33 TSP‑1 was the first protein to be recognized as a naturally occurring inhibitor of angiogenesis.34 Overexpression of TSP‑1 limits wound healing as well as tumorigenesis.33,35-37 Adhesion of cells to TSP‑1 leads to activation of Rac and Cdc42, both of which are required for extension of fascin‑containing microspikes and cell migration.38 TSP‑1 also induces FAK‑dependent inactivation of RhoA,32 consistent with a role for TSP‑1 in regulating cell migration and adhesion. Several recent studies place cyclin D1 at the center of a set of converging pathways that include TSP‑1, oncogenes, and the proangiogenic growth factor VEGF. Cyclin D1 has been shown previously to promote VEGF production and enhance angiogenesis.12,15,39 TSP‑1 is repressed by Myc, Src, Jun and Id1 as well as Ras.40 Conversely, the tumor suppressors PTEN and p53 induce expression of TSP‑1. Ras repression of TSP‑1 is associated with induction of RhoA and ROCK.41 Transgenic mouse studies support and extend the role of TSP‑1 in limiting cell migration, cancer, and angiogenesis. TSP‑1 knockout mice have defective wound healing and they develop lung hyperplasia and pneumonitis, but not any apparent vascular phenotype.42 In HER2/ErbB2‑induced mammary tumors, TSP‑1 levels are elevated in the reactive stroma of the tumor as compared to the normal mammary gland, and deleting TSP‑1 accelerates breast tumor onset and vascularity.35 Blood vessels in tumors from TSP‑1 null mice were also relatively more dilated than control animals. Interestingly, the abundance of VEGF mRNA and protein were unchanged but the distribution of VEGF was altered: in mammary tumors in TSP‑1 null mice, more VEGF was bound to its receptor VEGFR2. Conversely, overexpression of TSP‑1 in transgenic mice correlated with an increase in unbound VEGF, which remained largely associated with the ECM or the surface of tumor cells. These studies offer compelling in vivo evidence for tight control of availability of VEGF for receptor binding by TSP‑1.
Conclusions Over the last decade it has become increasingly clear that cyclins and cyclin‑binding proteins have cell cycle‑independent roles. Cyclin D1 and the cyclin binding protein p27kip1 clearly play important roles in promoting cellular migration in fibroblasts. Recent studies suggest cyclin D1 also plays a key role in promoting cellular migration of epithelial cells. The foundations provided by these studies prompt further analyses of these important new roles of cyclin D1 in tumor progression and metastasis. Both the pro‑migratory function of cyclin D1 and its recently reported role in regulating mitochondrial metabolism are independent of Rb binding. Moreover, a growing body of evidence argues that cyclin D1 promotes oncogenesis in an Rb‑independent manner. Secreted factors and perhaps
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angiogenic promoters represent tractable targets for therapeutic intervention. Given the clinical importance of metastasis in cancer, focusing further studies on the mechanisms by which the cyclins and their binding proteins regulate these heterotypic signals may be of practical value in the future. References 1. Arnold A, Papanikolaou A. Cyclin d1 in breast cancer pathogenesis. J Clin Oncol 2005; 23:4215‑24. 2. Fu M, Wang C, Li Z, Sakamaki T, Pestell RG. Minireview: Cyclin D1: Normal and abnormal functions. Endocrinology 2004; 145:5439‑47. 3. van Diest PJ, Michalides RJ, Jannink L, van der Valk P, Peterse HL, de Jong JS, Meijer CJ, Baak JP. Cyclin D1 expression in invasive breast cancer. Correlations and prognostic value. Am J Pathol 1997; 150:705‑11. 4. Lee RJ, Albanese C, Fu M, D’Amico M, Lin B, Watanabe G, Haines IIIrd GK, Siegel PM, Hung MC, Yarden Y, Horowitz JM, Muller WJ, Pestell RG. 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