Cyclin D1 is part of a cell cycle control node consistently deregulated in most human cancers. However, studies with cyclin D1-null mice indicate that it is dispensable for normal mouse development as well as cell growth in culture. Here, we provide evidence that ras-mediated tumorigenesis depends on signaling pathways that act preferentially through cyclin D1. Cyclin D1 expression and the activity of its associated kinase are up-regulated in keratinocytes in response to oncogenic ras. Furthermore, cyclin D1 deficiency results in up to an 80% decrease in the development of squamous tumors generated through either grafting of retroviral ras-transduced keratinocytes, phorbol ester treatment of ras transgenic mice, or twostage carcinogenesis.

In a previous study, we reported that overexpression of cyclin-dependent kinase-4 (CDK4) in mouse epidermis results in epidermal hyperplasia, hypertrophy and severe dermal fibrosis. In this study, we have investigated the susceptibility to skin tumor formation by forced expression of CDK4. Skin tumors from transgenic mice showed a dramatic increase in the rate of malignant progression to squamous cell carcinomas (SCC) in an initiation-promotion protocol. Histopathological analysis of papillomas from transgenic mice showed an elevated number of premalignant lesions characterized by dysplasia and marked atypia. Interestingly, transgenic mice also developed tumors in initiated but not promoted skin, demonstrating that CDK4 replaced the action of tumor promoters. These results suggest that expression of cyclin D1 upon ras activation synergizes with CDK4 overexpression. However, cyclin D1 transgenic mice and double transgenic mice for cyclin D1 and CDK4 did not show increased malignant progression in comparison to CDK4 transgenic mice. Biochemical analysis of tumors showed that CDK4 sequesters the CDK2 inhibitors p27 Kip1 and p21 Cip1 , suggesting that indirect activation of CDK2 plays an important role in tumor development. These results indicate that, contrary to the general assumption, the catalytic subunit, CDK4, has higher oncogenic activity than cyclin D1, revealing a potential use of CDK4 as therapeutic target.

The proto-oncogene c-myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis and that has also been found to be deregulated in several forms of human and experimental tumors. We have shown that forced expression of c-myc in epithelial tissues of transgenic mice (K5-Myc) resulted in keratinocyte hyperproliferation and the development of spontaneous tumors in the skin and oral cavity. Although a number of genes involved in cancer development are regulated by c-myc, the actual mechanisms leading to Myc-induced neoplasia are not known. Among the genes regulated by Myc is the cyclin-dependent kinase 4 (CDK4) gene. Interestingly, previous studies from our laboratory showed that the overexpression of CDK4 led to keratinocyte hyperproliferation, although no spontaneous tumor development was observed. Thus, we tested the hypothesis that CDK4 may be one of the critical downstream genes involved in Myc carcinogenesis. Our results showed that CDK4 inhibition in K5-Myc transgenic mice resulted in the complete inhibition of tumor development, suggesting that CDK4 is a critical mediator of tumor formation induced by deregulated Myc. Furthermore, a lack of CDK4 expression resulted in marked decreases in epidermal thickness and keratinocyte proliferation compared to the results obtained for K5-Myc littermates. Biochemical analysis of the K5-Myc epidermis showed that CDK4 mediates the proliferative activities of Myc by sequestering p21Cip1 and p27 Kip1 and thereby indirectly activating CDK2 kinase activity. These results show that CDK4 mediates the proliferative and oncogenic activities of Myc in vivo through a mechanism that involves the sequestration of specific CDK inhibitors.

Most human tumors have mutations that result in deregulation of the cdk4/cyclin-Ink4-Rb pathway. Overexpression of D-type cyclins or cdk4 and inactivation of Ink4 inhibitors are common in human tumors. Conversely, lack of cyclin D1 expression results in significant reduction in mouse skin and mammary tumor development. However, complete elimination of tumor development was not observed in these models, suggesting that other cyclin/cdk complexes play an important role in tumorigenesis. Here we described the effects of cdk4 deficiency on mouse skin proliferation and tumor development. Cdk4 deficiency resulted in a 98% reduction in the number of tumors generated through the two-stage carcinogenesis model. The absence of cdk4 did not affect normal keratinocyte proliferation and both wild-type and cdk4 knockout epidermis are equally affected after topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in epidermal hyperplasia. In similar fashion, cdk4 knockout keratinocytes proliferated well in an in vivo model of wound-induced proliferation. Biochemical studies in mouse epidermis showed that cdk6 activity increased twofold in cdk4-deficient mice compared to wild-type siblings. These results suggest that therapeutic approaches to inhibit cdk4 activity could provide a target to inhibit tumor development with minimal or no effect in normal tissue. The cyclin-dependent kinases (cdks) are a family of key cell-cycle regulators that function by association with cyclins, the regulatory subunits, at specific points of the cell cycle to phosphorylate various proteins during cell cycle progression.1,2 cdk4 and cdk6 form complexes with D-type cyclins (cyclin D1, D2, and D3) during the G 1 phase of the cell cycle.3,4 A key substrate for G 1 cyclin/ cdk complexes is the retinoblastoma protein, pRb. Phosphorylation of pRb, a tumor suppressor gene product, has been attributed to cyclin/cdk complexes and implicated in the regulation of proliferation in keratinocytes and other cell types.5,6 cdk4,6/D-type cyclins complex formation is induced during the middle of the G 1 phase and performs the first step of pRb phosphorylation. Then cyclin E binds and activates cdk2 at the G 1 /S phase transition and the second step of pRb phosphorylation is carried out on a different pRb motif.7 Thus, phosphorylation of pRb blocks its ability to suppress the activity of S-phase promoting transcription factors such as E2F. 5,6 Phosphorylation of the C-terminal region of pRb by cdk4,6 triggers sequential intramolecular interactions that progressively block pRb functions as cells move through G 1 . 8,9 The first phosphorylation facilitates a second interaction that leads to phosphorylation by cdk2 and further S phase progression. 9 cdk4-Deficient mice were generated and showed normal development, although the mutant mice show defects associated with growth retardation such as testicular atrophy, insulin-deficient diabetes and perturbed corpus luteum formation.10,11 Consistent with this observation, Rane et al hav...

Activation of c-Met signaling and B-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated B-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of B-catenin. To determine the importance of CCND1 as a mediator of c-Met-and B-catenin-induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, B-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by B-catenin/c-Met. In addition, when activated B-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of B-catenin during HCC pathogenesis, although other molecules may be required to fully propagate B-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and Bcatenin. [Cancer Res 2009;69(1):253-61]

No abstract

In a previous report we have described the effects of expression of D-type cyclins in epithelial tissues of transgenic mice. To study the involvement of the Dtype cyclin partner cyclin-dependent kinase 4 (CDK4) in epithelial growth and differentiation, transgenic mice were generated carrying the CDK4 gene under the control of a keratin 5 promoter. As expected, transgenic mice showed expression of CDK4 in the epidermal basal-cell layer. Epidermal proliferation increased dramatically and basal cell hyperplasia and hypertrophy were observed. The hyperproliferative phenotype of these transgenic mice was independent of D-type cyclin expression because no overexpression of these proteins was detected. CDK4 and CDK2 kinase activities increased in transgenic animals and were associated with elevated binding of p27 Kip1 to CDK4. Expression of CDK4 in the epidermis results in an increased spinous layer compared with normal epidermis, and a mild hyperkeratosis in the cornified layer. In addition to epidermal changes, severe dermal fibrosis was observed and part of the subcutaneous adipose tissue was replaced by connective tissue. Normal cell growth and differentiation requires precise control of the mechanisms that govern the entry into, passage through, and exit from the cell cycle. Progress through the G 1 phase of the mammalian cell cycle is regulated by the ordered synthesis, assembly, and activation of distinct cyclin-dependent kinase (CDK)-cyclin holoenzymes.1,2 This process is mediated by the D-type cyclins (D1, D2, and D3), whose expression is modulated by growth-stimulatory signals. These cyclins associate with the closely related CDK4 and CDK6 kinases, resulting in their catalytic activation and substrate recognition. A key substrate for G 1 cyclin/CDK complexes is the retinoblastoma protein, pRb. Phosphorylation of pRb, a tumor suppressor gene product, has been attributed to cyclin/CDK complexes and implicated in the regulation of proliferation of keratinocytes and other cell types.

It is now evident that several genes encoding regulatory activities that control the mammalian cell cycle, particularly some that control the progression of quiescent cells through G1 and into S phase, are targets for alterations that underlie the development of neoplasms. Here, we made a sequential study of alterations in cell cycle protein expression and complex formation among cyclin, cyclin dependent kinases (CDKs) and CDK inhibitors (CKIs) during premalignant progression in SENCAR mouse skin tumors. Changes in the level of expression were observed in positive (cyclin D1, D2, and E2F family members) and negative regulators (p16 Ink4a , p57 Kip2 ) of the cell cycle. Also, we observed the formation of cyclin/CDK/CKI complexes. The amounts of these proteins and complexes increased substantially at speci®c times during promotion but not during malignant conversion to carcinomas. These data show that deregulation of growth control occurs in benign tumors and that subsequent mutations not involved cell-cycle regulation are probably necessary to induce invasive behavior.