Cyclin D1 in Breast Cancer Pathogenesis
http://www.100md.com
▲還散笫雖悝◎
the Center for Molecular Medicine, University of Connecticut School of Medicine, Farmington, CT
ABSTRACT
Taking a perspective on available evidence that emphasizes relevance to human disease, cyclin D1 is solidly established as an oncogene with an important pathogenetic role in breast cancer and other human tumors. However, the precise cellular mechanisms through which aberrant cyclin D1 expression drives human neoplasia are less well established. Indeed, emerging evidence suggests that cyclin D1 might act, predominantly or at least in part, through pathways that do not involve its widely accepted function as a cell cycle regulator. Although therapeutic exploitation of the role of cyclin D1 as a molecular driver of breast cancer carries great promise, it is also suggested that direct targeting of the cyclin D1 gene or gene products may prove more successful than approaches that rely on arguably incomplete knowledge of the oncogenic mechanisms of cyclin D1.
INTRODUCTION
We have attempted to review the role of cyclin D1 in human neoplasia, with a specific focus on breast cancer pathogenesis, using a hierarchical approach that gives strongest weight to evidence derived from or validated in primary human tumors. This emphasis is intended to recognize appropriately the complexity of human cancer pathogenesis, and to highlight aspects that are, or are likely to become, clinically relevant. Our approach is not meant to deny the potential value of work in cultured cells, cell extracts, transfections with enforced gene overexpression, or genetically engineered mice
indeed, the latter in particular has much appeal as a mammalian whole-organism in vivo model. However, such systems bear the disadvantage of potentially obscuring the difference between "what can it do" versus "what does it do" in actual human tumorigenesis. Work in these in vitro or nonhuman in vivo systems is most valuable when explaining or supporting the pathogenetic significance of recurrent clonal lesions found in primary human tumors.
CYCLIN D1 AS A HUMAN ONCOGENE
Recurrent clonal DNA alterations observed in primary human tumors are the end result of in vivo selection, and therefore constitute exceedingly powerful functional as well as structural evidence for a gene's importance in the development of neoplasia.1-4 The cyclin D1 gene, located on human chromosome band 11q13, is an established oncogene for which genomic rearrangement or amplification leading to overexpression is commonly found as a clonal lesion in multiple types of human cancer. These tumor-specific alterations in the cyclin D1 gene reflect the critical selective advantage conferred by its overexpressed gene product and emphasize the importance of cyclin D1 as a driver of the neoplastic process. Indeed, the advantage of defining an oncogene as a gene in which specific DNA alterations (activating/gain of function) have been recurrently identified in human tumors is that any such gene is virtually certain to be pathogenetically important.1-4 In contrast to the strong and convincing evidence that cyclin D1 is a human oncogene, there is considerable uncertainty about the precise downstream cellular mechanisms by which dysregulated cyclin D1 leads to neoplasia in vivo, its widely recognized role in cell cycle control notwithstanding (Fig 1).
Gene Discovery and Gene Rearrangements
The cyclin D1 gene, designated as CCND1 or PRAD1, was originally cloned by three independent groups,5-7 and in one instance the mode of discovery directly identified cyclin D1 as an oncogene.5 Specifically, a subset of parathyroid adenomas contains a chromosome 11 inversion, the clonality of which reflected the selective advantage it conferred. Molecular cloning and sequencing of the DNA immediately adjacent to the tumor-specific breakpoint resulted in the identification of the activated oncogene, which was called PRAD1 (for parathyroid adenoma 1).5,8 This rearrangement activates PRAD1/CCND1 by juxtaposing it with a strong parathyroid tissue-specific regulatory element, analogous to a number of important human oncogenes that were identified at clonal translocation breakpoints (eg, BCL2).
The PRAD1 gene product was immediately recognized to have a domain of structural similarity to known eukaryotic cyclins,5-7 was able to function like a cyclin (eg, by activating members of the cyclin-dependent kinase [cdk] family),9 and became more commonly known as cyclin D1. Indeed, although the recognized membership of the oncoprotein in the cyclin family was tremendously helpful in focusing experimental studies of its function into the cell cycle arena, this focus may have also delayed recognition of other functions of cyclin D1 that could be important in its ability to drive human neoplasia.10,11
In another particularly direct demonstration of its primary role in human cancer, cyclin D1 has proven to be the BCL-1 oncogene, located at the breakpoint of the characteristic t(11;14)(q13;q32) clonal translocation in mantle-cell lymphomas12-15 and in a subset of multiple myeloma.16 Importantly, breakpoints have been found within 1 to 2 kb of cyclin D1, and on both sides of the gene, essentially eliminating the alternative possibility that some nearby gene is the true BCL-1 oncogene. Thus, tumor-specific rearrangements of cyclin D1 clearly show that its activation confers a clinically significant selective advantage in human B-cell neoplasia and is important in tumor pathogenesis. It is, however, also worth noting that even in mantle-cell lymphoma (in which cyclin D1 rearrangement/activation is so prevalent as to suggest its near-necessity for yielding the mantle-cell phenotype),15 additional independent genetic lesions are also selected. This point emphasizes the general principle that development of a human malignancy requires multiple genetic hits, with no single lesion being both necessary and sufficient.
Cyclin D1 As a Breast Cancer Oncogene
Strong evidence implicates cyclin D1 amplification and overexpression as a driving force in human breast cancer. Approximately 13% to 20% of breast cancers possess three- to more than 10-fold amplification of DNA on 11q13.4 to 11q13.5.17,18 Such amplification, or tandem extra copies, of a limited chromosomal region (the amplicon) initially occurs in one cell as an error in DNA replication (Fig 1). In the rare circumstances when a DNA amplification event confers a selective advantage on the cell and its progeny, the amplification will eventually emerge as an observable clonal alteration in the ensuing tumor's genome. A gene, the amplification of which contributes to the tumorigenic selective advantage, by definition, is an oncogene. However, amplicons are often large, containing many genes that are passively coamplified and provide no selective advantage
the difficulty of identifying the tumorigenically relevant driver oncogene(s) on a large amplicon is widely recognized. Indeed, for the gene-rich 11q13 region, evidence suggests that more than one distinct and independent amplicon, each with its own driver, may exist.17-20 That said, cyclin D1 is the only gene on an 11q13 amplicon that has, to date, fulfilled all the criteria expected of such a pathogenetically key driver oncogene in breast cancer. First, across multiple tumors, cyclin D1 is consistently found within the borders of an amplicon (ie, it is a core gene).21 Second, with great regularity, cyclin D1 is strongly overexpressed when amplified.21-26 Third, cyclin D1 overexpression causes mammary cancer in a physiologically relevant in vivo model, namely transgenic mice.27 Notably, these mice required a long latency before developing mammary cancers, which some have interpreted to mean that cyclin D1 has less than robust strength as an oncogene.28 To the contrary, however, the need for cooperativity with other oncogenic hits is entirely expected for an authentic human oncogene, is quite consistent with the role of cyclin D1 as a contributor in human cancer pathogenesis, and makes these mice an excellent model for key aspects of human breast cancer.
Furthermore, frequent and equally consistent involvement of cyclin D1 on an 11q13 amplicon in other cancers (eg, 30% to 35% of head and neck squamous cell and 35% of esophageal carcinomas),24,29,30 plus its clear role as a clonally disturbed oncogene in other primary human tumors (eg, lymphoid and parathyroid), add support to its designation as a major driver oncogene in breast cancer. As discussed, other amplified genes on 11q13 may also contribute, but their ability to drive mammary cancer in a mammalian in vivo system remains to be demonstrated
some interesting candidates include EMS1 and EMSY.18,19,31
If we take this human disease-oriented perspective, the interpretation of evidence from genetically engineered mice warrants elaboration. Such evidence can be tremendously valuable, and even crucial when distinguishing driver oncogenes from uninvolved passengers on tumor amplicons. However, because there are important differences between mice and humans, it must be re-emphasized that results from these models provide only secondary evidence that runs the risk of misinterpretation if not cross validated through analysis of human tissue and genetics. For example, the importance and significance of demonstrating that cyclin D1 can induce mammary cancer in transgenic mice depends strongly on the initial demonstration of the gene's central presence on clonal DNA amplicons in primary human breast cancers. In contrast, a number of overexpressed and/or mutant genes that can occasionally (eg, cyclin E)32 or even potently (eg, RAS)33 cause mammary neoplasia in transgenic mice lack this validation in human tumors. To follow the given examples, the cyclin E gene is not amplified or rearranged,34 nor are activating RAS mutations recurrently found in human breast cancers,35 which suggests strongly that primary dysregulation of these genes does not provide a clinically important selective advantage in mammary tumorigenesis, the results from mouse modeling notwithstanding.
Cyclin D1 Overexpression Without Gene Amplification in Breast Cancer
Cyclin D1 protein overexpression is found in up to 50% of human breast cancers. In many of these tumors the overexpression cannot be explained by increased gene copy number, suggesting that pathogenic activation of cyclin D1 can occur via additional mechanisms, including transcriptional and post-transcriptional dysregulation.22,23,25,26,36-44
The pattern of cyclin D1 overexpression in tissues along the spectrum from normal epithelium to invasive breast cancer37,45-47 also suggests the involvement of cyclin D1 in the earliest stages of mammary carcinogenesis, and in both ductal and lobular subtypes.37,38,45-47 One cannot be as certain that cyclin D1 is driving breast tumorigenesis in cancers with cyclin D1 protein overexpression but no gene amplification, compared with breast cancers in which cyclin D1 is overexpressed due to clonally selected gene amplification. On one hand, it is plausible that different, primary clonal lesions could secondarily lead to cyclin D1 activation and thus provide the cell with an alternate route toward achieving the same neoplastic common denominator. On the other hand, it is possible that such tumors, containing different primary oncogenic mutations, are not pathogenetically dependent on cyclin D1 despite its overexpression. The major significance of this unsettled issue, from a clinical perspective, relates to whether a future anti每cyclin D1 therapy will prove equally effective against overexpression-positive/amplification-negative tumors as it might against overexpression-positive/amplification-positive tumors.
STRUCTURE AND EXPRESSION OF THE CYCLIN D1 GENE
Products of Alternative Splicing
The human PRAD1/CCND1 gene spans approximately 15 kb and includes five exons.48 The gene encodes a 295-amino acid protein with molecular weight about 34 kd.5 Whereas most functional studies have been performed with this gene product, an alternatively spliced cyclin D1 transcript, termed transcript [b], has also been identified.49-52 Unlike the originally reported gene product, now called transcript [a], transcript [b] results from failure of splicing at the exon 4-intron 4 junction. Therefore, the 3' end of the transcript [b] mRNA sequence continuing downstream into what would otherwise have been intron 4 while lacking exon 5.49-52 Thus, the cyclin D1 isoform encoded by transcript [b], cyclin D1b, contains only 274 amino acids and has a different C-terminus from the transcript [a] -derived protein cyclin D1a (Fig 2).
Because the C-terminal proline, glutamic acid, serine, and threonine (PEST) domain of cyclin D1a, associated with protein stability and ubiquitin-mediated degradation, is lacking in cyclin D1b, it has been predicted that cyclin D1b would be more stable, and therefore perhaps more tumorigenic, than cyclin D1a.49-52 However, Solomon et al52 showed that the two cyclin D1 isoforms exhibited similar stability and cyclin D1b did not accumulate to higher levels. Another residue in cyclin D1a but lacking in cyclin D1b, threonine 286, has been shown in vitro to promote nuclear export and protein turnover through its phosphorylation, and engineered mutation at this site can alter the oncogenicity of cyclin D1 in vitro.53,54 Codon 286 mutations have not been identified in primary human breast cancers or other human tumors, so the significance of these findings to human neoplasia remain uncertain. Somatic mutation of proline codon 287 has been reported in a subclone of two endometrial carcinomas with cyclin D1 protein overexpression
another showed deletion of codons 289 to 292.55 Such mutations might well have similar effects on protein degradation, resulting in oncoprotein activation
however, none were detected in a parallel study of breast cancers55 and in an independent study.50 Cyclin D1b, which also lacks these residues, exhibits enhanced oncogenicity in vitro,52,56 and its expression has been demonstrated in some human tumors,56,57 so it conceivably may play an important role in vivo.
For human breast cancer, transcript [b] is expressed in some primary tumors and breast cancer cell lines but at dramatically lower levels than transcript [a], and overexpression of cyclin D1 in breast cancer samples is primarily accounted for by transcript [a].51 Relative levels of cyclin D1a and D1b protein isoforms in primary breast cancers have not yet been reported, although cyclin D1b was not found in the ZR-75-1 breast cancer cell line known to bear cyclin D1 gene amplification and overexpression.57 To date, only transcript [a] has been examined and found to drive mammary tumorigenesis in transgenic mice.27 Still, cyclin D1b could also be making a significant contribution to breast oncogenesis,52 and its role deserves additional investigation in human tumors and in complex in vivo systems.
Exon 4 Polymorphism
A common polymorphism within cyclin D1, A/G at nucleotide 870 in exon 4, does not alter its encoded amino acid but has been reported to influence the ratio of alternatively spliced transcripts [a] and [b].58 Results have been inconsistent, however, with the A allele favoring production of transcript [b] or transcript [a] in different studies and different tissues.58 In addition, the A/A genotype has been associated with increased predisposition to a number of specific cancers, and/or to poor prognosis. However, the genotype at the 870A/G polymorphism does not appear to influence a woman's risk of breast cancer,59,60 and it has not been determined whether the 870A/G genotype importantly alters the ratios of the cyclin D1 protein isoforms in breast cancer.
The Cyclin Box
Both isoforms of the cyclin D1 protein contain the so-called cyclin box sequence, a region of about 100 amino acids near the amino terminus. The sequence is fairly well conserved among all cyclins and is required for binding to their respective partner cdk. This sequence conservation does suggest that some aspect of cyclin D1 function relies on its ability to bind and activate a cdk. Whether such cdk dependence relates to the role of cyclin D1 in development, normal postnatal physiology, and/or tumorigenesis is still at issue.
HOW DOES CYCLIN D1 ACTIVATION CAUSE BREAST CANCER
Although the importance of cyclin D1 activation as a primary molecular cause of breast cancer is solidly established, the precise mechanisms through which cyclin D1 exerts its oncogenic action still require better definition. Resolving this issue carries important implications for devising new therapeutic strategies for breast cancer patients.
cdk-Mediated Effects on Cell Cycle Progression
Numerous studies, generally employing synchronized cultured cells and other in vitro systems, have led to the widely accepted view that the primary way in which cyclin D1 functions is by activation, as a regulatory subunit, of cdk4 and/or cdk6.9 Biochemically, cyclin D1每cdk complexes, followed in the cell cycle by analogous cyclin E-containing complexes, cause phosphorylation of retinoblastoma protein (pRB), the product of the retinoblastoma susceptibility gene, thereby inactivating the G1-maintaining function of pRB. A noncatalytic function of cyclin D每cdk4/6 complexes in sequestering cdk inhibitor proteins p21 and p27 from the cyclin E每cdk2 complex has also been described.61 The ability of cyclin D1 to activate its cdk partner is antagonized by the p16INK4a tumor suppressor. Although a huge body of evidence indicates that the main (and perhaps only) important role of cyclin D1 in the proliferation of cultured cells does indeed involve cdk activation and pRB inactivation, the same cannot be stated as definitively for the role of activated cyclin D1 in breast cancer or other primary human tumors.
The emphasis on cdk-dependent cell cycle actions as the major mechanism of cyclin D1 in human tumorigenesis has been argued on the basis that individual tumors tend to have a specific alteration of only one component of the cyclin D1/cdk/pRB/p16 pathway, suggesting a oncogenically relevant functional redundancy. On the other hand, there are many exceptions to this rule, and the functional equivalence of these lesions is further called into question by the major differences in their relative distributions across distinct types of primary tumors. It is also worth noting that cyclin D1 overexpression has not been associated with markers of tumor cell cycle activity in human breast cancers.38 If we examine this problem more broadly, there are many genes that have been identified as key regulators of the cell cycle, but few have been established through recurrent clonal derangements to be human oncogenes or tumor suppressors. It is likely, therefore, that achieving a selective advantage requires something more than cell cycle misregulation. Thus the small subset designated cell cycle regulators with proven oncogenic significance contribute to tumorigenesis through special mechanisms in addition to (or possibly instead of) their recognized roles in the cell cycle. Beyond this theoretical argument, considerable evidence for specific non每cdk-dependent functions of cyclin D1 now exists.
Does Cyclin D1 Activation Contribute to Breast Cancer Through cdk-Independent Mechanisms
As discussed, the recent accumulation of evidence that cyclin D1 may contribute to neoplasia through cdk (or cell cycle) -independent mechanisms is a departure from the widely accepted paradigm but in principle, is not surprising to those with a clinical perspective. A sampling of this intriguing evidence is described here (also see Coqueret44), but it must also be emphasized that no definitive answers currently exist for the important questions about exactly how cyclin D1 exerts its oncogenicity in human neoplasia.
Evidence for cdk-independent mechanisms, in addition to the tissue type每specific skewing of distribution of mutations among cyclin D1, RB, and p16 genes in human tumors, includes in vitro observations of non每cell cycle effects of cyclin D1 on genomic instability,62,63 an influence on DNA replication and repair in UV-treated fibroblasts,64 a report of direct binding of cyclin D1 to proliferating cell nuclear antigen (PCNA),65 and a link to the apoptotic pathway in neurons66 and in cyclin D1每overexpressing MCF7 breast cancer cells.67 Furthermore, in a series of breast cancer cell lines, overexpression of cyclin D1 was not related to cdk4 activity.68 D-type cyclins bind to and repress the activity of a myb-like transcription factor, DMP1, by interacting with the DNA binding domain of DMP1, which in turn prevents its proper binding to target promoters.69 A mutant cyclin D1 that is unable to activate cdk4 can still interact with DMP1 and inhibit its activity. Of great interest, DMP1 expression in transfected cells prevented S phase entry, and this DMP1-induced growth arrest could be overcome by the same mutant cyclin D1, indicating that the reversal of DMP1-induced growth arrest does not occur through its role as a cdk regulatory subunit.70 Cyclin D1 strongly inhibits activity of the B-Myb transcription factor, not through cdk-dependent phosphorylation of B-Myb, but through formation of a specific complex between cyclin D1 and B-Myb.71 In addition, in cultured cells, the effect of cyclin D1 on transformation does not always rely on inactivation of pRB.44,72,73 A variety of other transcriptional targets of cyclin D1 have been described, including the androgen receptor,74 PPAR (peroxisome proliferator-activated receptor gamma),75 and histone acetylases and deacetylases.44
In breast cancer cell lines, overexpressed cyclin D1 has been shown to bind directly and activate the estrogen receptor alpha (ER) in a cdk- and pRB-independent fashion.76,77 This fascinating finding, that overexpressed cyclin D1 can activate the ER in a hormonally independent fashion in vitro, and might thereby drive all of the key mitogenic effects of estrogen on breast epithelium, has the potential to add a crucial dimension to our understanding of mammary tumorigenesis. Although cyclin D1 overexpression is present across multiple histologic subtypes of breast cancer, a theme of commonality has been that the large majority of cyclin D1每overexpressing breast cancers are ER positive.25,78,79 This strong association has been difficult to explain, given the prevalent concept that cyclin D1 expression is only a downstream consequence of estrogen-induced activation of the ER, a nuclear hormone receptor which functions as a transcription factor.80-82 In that scenario, one might expect that tumors with more autonomous cyclin D1 expression due to gene amplification could bypass the need for functional ER, predicting an association between cyclin D1 overexpression and ER-negative breast cancer, which in fact is not observed. For example, almost all (94%) of the cyclin D1每positive breast cancers in one study were ER positive
in contrast, only 61% of cyclin D1每nonoverexpressing tumors were ER positive.25 Such results raised doubt that the action of cyclin D1 as a downstream mediator of sex steroid mitogen-induced proliferation in cultured cell lines would fully explain its oncogenicity in breast cancer. The subsequent finding that cyclin D1 may act upstream or as a coregulator of ER activity,44 as opposed to solely being a downstream target of ER-mediated activation, would provide an appealing explanation for the typical coexpression of ER in cyclin D1每positive breast cancers. In other words, cyclin D1 overexpression would bestow a selective advantage mainly in the presence of a serviceable ER.
Specifically, Zwijsen et al76 and Neuman et al77 tested the effect of cyclin D1 on ER transactivation by transfecting ER-positive breast cancer cells with a cyclin D1 expression vector, together with a reporter gene construct. Cyclin D1 directly interacted with liganded and unliganded ER to trigger binding of ER to an estrogen receptor response element and thereby increased reporter target gene transcription. Furthermore, the effect was not dependent on cyclin D1 interacting with cdk4 or pRB, given that it was reproduced with cyclin D1 mutants incapable of binding to cdk4 or pRb. Subsequent work showed that cyclin D1 not only interacted with the ER in cell lines but also with certain steroid receptor coactivators.83,84 This raises the possibility that cyclin D1 may act as a bridge, recruiting essential coactivators to the ER even in the absence of estrogen. It has been suggested that cyclin D1 may exert its cdk-independent, transcriptional effects only when overexpressed, as in pathologic situations.10
A recent study has added considerable weight to the potential tumorigenic relevance of cdk-independent transcriptional functions of cyclin D1.11 Lamb et al11 determined the global patterns of gene expression in cultured breast cancer cells after enforced overexpression of either wild-type cyclin D1 or a mutant form unable to activate cdk4 (KE mutant). Strikingly, both wild-type and mutant cyclin D1 effected the same signature of major changes in downstream gene expression, suggesting that the observed effects of cyclin D1 overexpression were cdk4 independent. The expression of this set of cyclin D1 target genes was then examined across a large panel of primary human tumors, and a positive correlation with cyclin D1 expression was found. Thus, the expression signature demonstrated in vitro was cross validated in vivo, providing support for the presence of cdk-independent actions of cyclin D1 in human tumors. Furthermore, a focused look at whether expression of known E2F target genes (downstream in the cyclin D/p16/cdkpRBE2F pathway, driving the G1 to S transition) would correlate with cyclin D1 expression in these human tumors proved negative, casting more doubt on the idea that cyclin D1 acts primarily as a cell cycle regulator in cancers. Interestingly, and in marked contrast, cyclin D3 expression correlated nicely with the E2F target genes, emphasizing the idea that cyclin D1 in vivo has unique functional attributes not shared by other cyclins, including other D-type cyclins. Indeed, analysis of these differences may uncover the key features that make cyclin D1, unlike other cyclins, a robust human oncoprotein. Finally, this study provided evidence that overexpression of the transcription factor C/EBP is highly correlated with cyclin D1 expression in the same human cancer database, and that cyclin D1 acts on or with C/EBP in regulating cyclin D1 target genes in vitro.11
Clearly, the relevance of these alternative mechanisms of cyclin D1 action carry tremendous potential significance to human cancer, and the direct role of cdk-independent actions in tumorigenesis must be further examined in appropriately complex in vivo systems.
In Addition to Its Primary Role in Tumorigenesis, Does Cyclin D1 Mediate the Action of Other Oncogenes in Causing Breast Cancer
Because cyclin D1 overexpression in breast cancers cannot always be attributed to clonally selected cyclin D1 DNA alterations such as gene amplification, it is conceivable that primary activation of other oncogenic/mitogenic pathways could provide an alternate route to tumorigenic cyclin D1 dysregulation. Consistent with this possibility, in vitro experiments have shown direct or indirect regulation of the cyclin D1 promoter, or of cyclin D1 expression, by -catenin, STAT5, STAT3, nuclear factor kappa B, ets-2, cyclic adenosine monophosphate每response element, tissue-coding factor/leukokinesis-enhancing factor, c-Jun, E2F-1, PPAR, calveolin-1, Ras signaling, and others.44 It will be important to sort out the relevance of these cyclin D1 regulatory mechanisms to primary human neoplasia.
An interesting series of in vivo experiments have exploited genetically engineered cyclin D1每null mice. These animals exhibited normal development in most respects, a major exception being defective postnatal mammary development revealed by a lack of proliferation of alveolar epithelial cells in response to the sex steroid milieu of pregnancy.85,86 Crossing of cyclin D1每null mice with transgenic mice that develop mammary cancer in response to oncogenes (Wnt1, v-Ha-ras, Neu/Erbb2, or Myc) overexpressed in mammary tissue showed that the cyclin D1每null background blocked mammary tumorigenesis driven by Ras or Neu, but not by Wnt1 or Myc.87 These results have been widely interpreted to mean that mammary cancers driven by Neu or Ras pathway activation are dependent on cyclin D1 as an essential oncogenic intermediary in mice, and likely in humans. Similarly, it has been speculated that inhibitors of cyclin D1 could therefore effectively treat human breast cancers that overexpress NEU or RAS.
There are reasons, however, to view the common interpretation of these experiments with caution if not skepticism. First, cyclin D1 knockout mice are fundamentally experiments in development rather than tumorigenesis. Because these mice lack cyclin D1 throughout development, the specific mammary cell that is susceptible later in life to the action of a particular oncogene may not have developed or may be different from it would have been had development been normal. Second, even if Neu-driven mammary cancer could be shown to be crucially dependent on cyclin D1 in a background of normal development in mice, this may not be true in humans. Indeed, many or most NEU-activated human breast cancers are ER negative and would not be expected to overexpress cyclin D1, given that most cyclin D1每overexpressing breast cancers are ER positive
furthermore, Bieche et al26 observed no link between cyclin D1 alterations and NEU overexpression in primary breast cancers.
Does Cyclin D1 Activation Cause Breast Cancer Solely by Leading to Increased Cyclin E Expression
The action of cyclin D1 in fostering cell cycle progression has been viewed as preliminary to and necessary for the subsequent expression of cyclin E, but does cyclin D1 activation cause human breast cancer solely through a downstream activation of cyclin E expression An affirmative answer to this question has been suggested by recent studies using genetically engineered mice. Cyclin E每encoding DNA under the control of the regulatory region of cyclin D1 was knocked in to cyclin D1每null mice, and the mammary developmental defect characteristic of cyclin D1每null mice was thereby abrogated.88 This cyclin E knock-in also eliminated the resistance to Ras- or Neu-induced mammary cancer otherwise imparted by the cyclin D1每null background.87 If ultimately shown to apply in human breast cancer, these results would suggest that the efficacy of an anticyclin D1 therapy might be enhanced by simultaneous targeting of cyclin E.
Again, however, there are strong reasons for caution before embracing these conclusions as relevant to human breast cancer. Although a compensatory upregulation of cyclin E was sometimes eventually able to overcome the blockade to an engineered mutant Neu-induced tumorigenesis imposed by the cyclin D1每null background in mice, this was not the case for the wild-type Neu sequence typically amplified in cancers.89 Next, just as noted, these cyclin E knock-in experiments similarly fail to isolate the role of cyclin D1 in tumorigenesis from the effects of its loss during development. In addition, the ability of transgenically overexpressed cyclin E to drive mammary tumorigenesis in mice appears to be weaker than cyclin D1,27 with a penetrance of only 10%.32
Finally, and perhaps of most significance to the clinician, the roles of cyclin D1 and cyclin E in human breast cancer may well differ markedly from their roles in the mouse. The crucial point is that if cyclin E activation could drive human breast cancer as effectively as cyclin D1, or even at a less effective but still clinically relevant level, then robust evidence for cyclin E as a human breast oncogene should exist. To the contrary, however, such evidence is strikingly lacking in primary human breast cancers, nor has cyclin E core amplification, rearrangement, or other clonal activating mutation been recurrently identified in other human tumors. The strong implication is that cyclin E activation fails to confer a clinically important selective advantage on a mammary cell in the development of human breast cancer.
PATHOGENESIS VERSUS PROGNOSIS
It is important to emphasize that the clear involvement of a gene in tumor pathogenesis is no guarantee of its utility as a prognostic marker, and vice versa. For example, involvement of a given gene may be so crucial to the development of a given type of tumor that interindividual differences in tumor behavior and prognosis will necessarily depend on other factors. For a given type of tumor, it can also be true that there are multiple alternative molecular means to the common end of tumor development. Perhaps for these reasons, cyclin D1 has both a clear, central role in breast cancer pathogenesis and an apparent but certainly less clear role as a clinically useful prognostic marker. Conversely, cyclin E (or E1) has been suggested to have major value as a prognostic marker,90 although there is strikingly little evidence from primary human tumors that cyclin E is an oncogene.
The prognostic value of a marker depends on the available therapeutic armamentarium. For example, when some (but not all) studies defined cyclin D1 amplification or overexpression as marking a subset of ER-positive breast cancers as having a poorer prognosis than other ER-positive breast cancers,91,92 a standard approach to treatment of the patients is assumed. However, molecular alteration in cyclin D1 could become an essential prognostic marker, if an effective therapy that specifically targeted cyclin D1 existed.
CYCLIN D1 AS A THERAPEUTIC TARGET
The large number of breast (and other) cancers for which cyclin D1 activation is a pathogenetic cornerstone has appropriately attracted much attention in the search for improved therapeutics. Based in part on the widely accepted paradigm that the entire contribution of cyclin D1 to tumorigenesis is achieved through its activation of cdk4/6, much effort has been directed toward finding small molecule inhibitors of cdk, or activators of endogenous cdk inhibitors. For example, flavopiridol is a cdk-inhibitory agent in clinical trials.93
However, if an important component of the oncogenic action of cyclin D1 is accomplished through cdk-independent mechanisms, therapeutic cdk inhibition might not be expected to be especially efficacious in cyclin D1每driven cancers. By the same reasoning, blocking cyclin D1 expression specifically and directly (by targeting the cyclin D1 gene, RNA, or protein) should increase the chances for therapeutic success in these tumors while advantageously avoiding the need for complete knowledge of the downstream effectors of cyclin D1 (Fig 1). Cell culture studies have raised the possibility that certain compounds might act in this way,93-95 and approaches to blocking cyclin D1 expression using antisense, siRNA or related molecules also have conceptual appeal in specifically targeting the driving molecular lesion itself,96-98 assuming in vivo use of these technologies becomes feasible.
Tumors bearing clonal genetic alterations in the cyclin D1 gene should be maximally susceptible to a highly specific anticyclin D1 therapy, akin to the efficacy of imatinib,99 trastuzumab,100 and gefitinib101,102 against tumors with analogous clonal DNA lesions (BCR-ABL fusion,99 HER2/neu amplification,100 and EGFR mutation,101,102 respectively) that speak to their central pathogenetic role in selection and, evidently, the ongoing viability of the tumor cell.103 Earlier concern about potentially broad toxicity of a cell cycle agent has been diminished by the meager phenotype of cyclin D1每null animals and the new information on the cdk-independent actions of cyclin D1
nonetheless, achieving the ability to deliver an anti每cyclin D1 agent with high selectivity to tumor cells should certainly be advantageous. Finally, it might be expected that any successful anticyclin D1 therapy will find optimal use in a combination protocol, together with an agent that targets a different clonally selected cooperating gene (Fig 1) in the tumor, to eradicate the rare tumor cells that could escape a single agent through fortuitous mutation and subsequent selection.
In summary, because of its established role as a major human oncogene, the therapeutic exploitation of cyclin D1 holds tremendous promise and should be vigorously pursued. For many breast cancers, strong evidence of the role of cyclin D1 as a pathogenetic cornerstone suggests that appropriately selected tumors will be highly susceptible to future anti每cyclin D1 therapy.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by a grant CA55909 (to A.A.) from the National Institutes of Health and a postdoctoral fellowship from the Susan G. Komen Breast Cancer Foundation (to A.P).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
Taking a perspective on available evidence that emphasizes relevance to human disease, cyclin D1 is solidly established as an oncogene with an important pathogenetic role in breast cancer and other human tumors. However, the precise cellular mechanisms through which aberrant cyclin D1 expression drives human neoplasia are less well established. Indeed, emerging evidence suggests that cyclin D1 might act, predominantly or at least in part, through pathways that do not involve its widely accepted function as a cell cycle regulator. Although therapeutic exploitation of the role of cyclin D1 as a molecular driver of breast cancer carries great promise, it is also suggested that direct targeting of the cyclin D1 gene or gene products may prove more successful than approaches that rely on arguably incomplete knowledge of the oncogenic mechanisms of cyclin D1.
INTRODUCTION
We have attempted to review the role of cyclin D1 in human neoplasia, with a specific focus on breast cancer pathogenesis, using a hierarchical approach that gives strongest weight to evidence derived from or validated in primary human tumors. This emphasis is intended to recognize appropriately the complexity of human cancer pathogenesis, and to highlight aspects that are, or are likely to become, clinically relevant. Our approach is not meant to deny the potential value of work in cultured cells, cell extracts, transfections with enforced gene overexpression, or genetically engineered mice
indeed, the latter in particular has much appeal as a mammalian whole-organism in vivo model. However, such systems bear the disadvantage of potentially obscuring the difference between "what can it do" versus "what does it do" in actual human tumorigenesis. Work in these in vitro or nonhuman in vivo systems is most valuable when explaining or supporting the pathogenetic significance of recurrent clonal lesions found in primary human tumors.
CYCLIN D1 AS A HUMAN ONCOGENE
Recurrent clonal DNA alterations observed in primary human tumors are the end result of in vivo selection, and therefore constitute exceedingly powerful functional as well as structural evidence for a gene's importance in the development of neoplasia.1-4 The cyclin D1 gene, located on human chromosome band 11q13, is an established oncogene for which genomic rearrangement or amplification leading to overexpression is commonly found as a clonal lesion in multiple types of human cancer. These tumor-specific alterations in the cyclin D1 gene reflect the critical selective advantage conferred by its overexpressed gene product and emphasize the importance of cyclin D1 as a driver of the neoplastic process. Indeed, the advantage of defining an oncogene as a gene in which specific DNA alterations (activating/gain of function) have been recurrently identified in human tumors is that any such gene is virtually certain to be pathogenetically important.1-4 In contrast to the strong and convincing evidence that cyclin D1 is a human oncogene, there is considerable uncertainty about the precise downstream cellular mechanisms by which dysregulated cyclin D1 leads to neoplasia in vivo, its widely recognized role in cell cycle control notwithstanding (Fig 1).
Gene Discovery and Gene Rearrangements
The cyclin D1 gene, designated as CCND1 or PRAD1, was originally cloned by three independent groups,5-7 and in one instance the mode of discovery directly identified cyclin D1 as an oncogene.5 Specifically, a subset of parathyroid adenomas contains a chromosome 11 inversion, the clonality of which reflected the selective advantage it conferred. Molecular cloning and sequencing of the DNA immediately adjacent to the tumor-specific breakpoint resulted in the identification of the activated oncogene, which was called PRAD1 (for parathyroid adenoma 1).5,8 This rearrangement activates PRAD1/CCND1 by juxtaposing it with a strong parathyroid tissue-specific regulatory element, analogous to a number of important human oncogenes that were identified at clonal translocation breakpoints (eg, BCL2).
The PRAD1 gene product was immediately recognized to have a domain of structural similarity to known eukaryotic cyclins,5-7 was able to function like a cyclin (eg, by activating members of the cyclin-dependent kinase [cdk] family),9 and became more commonly known as cyclin D1. Indeed, although the recognized membership of the oncoprotein in the cyclin family was tremendously helpful in focusing experimental studies of its function into the cell cycle arena, this focus may have also delayed recognition of other functions of cyclin D1 that could be important in its ability to drive human neoplasia.10,11
In another particularly direct demonstration of its primary role in human cancer, cyclin D1 has proven to be the BCL-1 oncogene, located at the breakpoint of the characteristic t(11;14)(q13;q32) clonal translocation in mantle-cell lymphomas12-15 and in a subset of multiple myeloma.16 Importantly, breakpoints have been found within 1 to 2 kb of cyclin D1, and on both sides of the gene, essentially eliminating the alternative possibility that some nearby gene is the true BCL-1 oncogene. Thus, tumor-specific rearrangements of cyclin D1 clearly show that its activation confers a clinically significant selective advantage in human B-cell neoplasia and is important in tumor pathogenesis. It is, however, also worth noting that even in mantle-cell lymphoma (in which cyclin D1 rearrangement/activation is so prevalent as to suggest its near-necessity for yielding the mantle-cell phenotype),15 additional independent genetic lesions are also selected. This point emphasizes the general principle that development of a human malignancy requires multiple genetic hits, with no single lesion being both necessary and sufficient.
Cyclin D1 As a Breast Cancer Oncogene
Strong evidence implicates cyclin D1 amplification and overexpression as a driving force in human breast cancer. Approximately 13% to 20% of breast cancers possess three- to more than 10-fold amplification of DNA on 11q13.4 to 11q13.5.17,18 Such amplification, or tandem extra copies, of a limited chromosomal region (the amplicon) initially occurs in one cell as an error in DNA replication (Fig 1). In the rare circumstances when a DNA amplification event confers a selective advantage on the cell and its progeny, the amplification will eventually emerge as an observable clonal alteration in the ensuing tumor's genome. A gene, the amplification of which contributes to the tumorigenic selective advantage, by definition, is an oncogene. However, amplicons are often large, containing many genes that are passively coamplified and provide no selective advantage
the difficulty of identifying the tumorigenically relevant driver oncogene(s) on a large amplicon is widely recognized. Indeed, for the gene-rich 11q13 region, evidence suggests that more than one distinct and independent amplicon, each with its own driver, may exist.17-20 That said, cyclin D1 is the only gene on an 11q13 amplicon that has, to date, fulfilled all the criteria expected of such a pathogenetically key driver oncogene in breast cancer. First, across multiple tumors, cyclin D1 is consistently found within the borders of an amplicon (ie, it is a core gene).21 Second, with great regularity, cyclin D1 is strongly overexpressed when amplified.21-26 Third, cyclin D1 overexpression causes mammary cancer in a physiologically relevant in vivo model, namely transgenic mice.27 Notably, these mice required a long latency before developing mammary cancers, which some have interpreted to mean that cyclin D1 has less than robust strength as an oncogene.28 To the contrary, however, the need for cooperativity with other oncogenic hits is entirely expected for an authentic human oncogene, is quite consistent with the role of cyclin D1 as a contributor in human cancer pathogenesis, and makes these mice an excellent model for key aspects of human breast cancer.
Furthermore, frequent and equally consistent involvement of cyclin D1 on an 11q13 amplicon in other cancers (eg, 30% to 35% of head and neck squamous cell and 35% of esophageal carcinomas),24,29,30 plus its clear role as a clonally disturbed oncogene in other primary human tumors (eg, lymphoid and parathyroid), add support to its designation as a major driver oncogene in breast cancer. As discussed, other amplified genes on 11q13 may also contribute, but their ability to drive mammary cancer in a mammalian in vivo system remains to be demonstrated
some interesting candidates include EMS1 and EMSY.18,19,31
If we take this human disease-oriented perspective, the interpretation of evidence from genetically engineered mice warrants elaboration. Such evidence can be tremendously valuable, and even crucial when distinguishing driver oncogenes from uninvolved passengers on tumor amplicons. However, because there are important differences between mice and humans, it must be re-emphasized that results from these models provide only secondary evidence that runs the risk of misinterpretation if not cross validated through analysis of human tissue and genetics. For example, the importance and significance of demonstrating that cyclin D1 can induce mammary cancer in transgenic mice depends strongly on the initial demonstration of the gene's central presence on clonal DNA amplicons in primary human breast cancers. In contrast, a number of overexpressed and/or mutant genes that can occasionally (eg, cyclin E)32 or even potently (eg, RAS)33 cause mammary neoplasia in transgenic mice lack this validation in human tumors. To follow the given examples, the cyclin E gene is not amplified or rearranged,34 nor are activating RAS mutations recurrently found in human breast cancers,35 which suggests strongly that primary dysregulation of these genes does not provide a clinically important selective advantage in mammary tumorigenesis, the results from mouse modeling notwithstanding.
Cyclin D1 Overexpression Without Gene Amplification in Breast Cancer
Cyclin D1 protein overexpression is found in up to 50% of human breast cancers. In many of these tumors the overexpression cannot be explained by increased gene copy number, suggesting that pathogenic activation of cyclin D1 can occur via additional mechanisms, including transcriptional and post-transcriptional dysregulation.22,23,25,26,36-44
The pattern of cyclin D1 overexpression in tissues along the spectrum from normal epithelium to invasive breast cancer37,45-47 also suggests the involvement of cyclin D1 in the earliest stages of mammary carcinogenesis, and in both ductal and lobular subtypes.37,38,45-47 One cannot be as certain that cyclin D1 is driving breast tumorigenesis in cancers with cyclin D1 protein overexpression but no gene amplification, compared with breast cancers in which cyclin D1 is overexpressed due to clonally selected gene amplification. On one hand, it is plausible that different, primary clonal lesions could secondarily lead to cyclin D1 activation and thus provide the cell with an alternate route toward achieving the same neoplastic common denominator. On the other hand, it is possible that such tumors, containing different primary oncogenic mutations, are not pathogenetically dependent on cyclin D1 despite its overexpression. The major significance of this unsettled issue, from a clinical perspective, relates to whether a future anti每cyclin D1 therapy will prove equally effective against overexpression-positive/amplification-negative tumors as it might against overexpression-positive/amplification-positive tumors.
STRUCTURE AND EXPRESSION OF THE CYCLIN D1 GENE
Products of Alternative Splicing
The human PRAD1/CCND1 gene spans approximately 15 kb and includes five exons.48 The gene encodes a 295-amino acid protein with molecular weight about 34 kd.5 Whereas most functional studies have been performed with this gene product, an alternatively spliced cyclin D1 transcript, termed transcript [b], has also been identified.49-52 Unlike the originally reported gene product, now called transcript [a], transcript [b] results from failure of splicing at the exon 4-intron 4 junction. Therefore, the 3' end of the transcript [b] mRNA sequence continuing downstream into what would otherwise have been intron 4 while lacking exon 5.49-52 Thus, the cyclin D1 isoform encoded by transcript [b], cyclin D1b, contains only 274 amino acids and has a different C-terminus from the transcript [a] -derived protein cyclin D1a (Fig 2).
Because the C-terminal proline, glutamic acid, serine, and threonine (PEST) domain of cyclin D1a, associated with protein stability and ubiquitin-mediated degradation, is lacking in cyclin D1b, it has been predicted that cyclin D1b would be more stable, and therefore perhaps more tumorigenic, than cyclin D1a.49-52 However, Solomon et al52 showed that the two cyclin D1 isoforms exhibited similar stability and cyclin D1b did not accumulate to higher levels. Another residue in cyclin D1a but lacking in cyclin D1b, threonine 286, has been shown in vitro to promote nuclear export and protein turnover through its phosphorylation, and engineered mutation at this site can alter the oncogenicity of cyclin D1 in vitro.53,54 Codon 286 mutations have not been identified in primary human breast cancers or other human tumors, so the significance of these findings to human neoplasia remain uncertain. Somatic mutation of proline codon 287 has been reported in a subclone of two endometrial carcinomas with cyclin D1 protein overexpression
another showed deletion of codons 289 to 292.55 Such mutations might well have similar effects on protein degradation, resulting in oncoprotein activation
however, none were detected in a parallel study of breast cancers55 and in an independent study.50 Cyclin D1b, which also lacks these residues, exhibits enhanced oncogenicity in vitro,52,56 and its expression has been demonstrated in some human tumors,56,57 so it conceivably may play an important role in vivo.
For human breast cancer, transcript [b] is expressed in some primary tumors and breast cancer cell lines but at dramatically lower levels than transcript [a], and overexpression of cyclin D1 in breast cancer samples is primarily accounted for by transcript [a].51 Relative levels of cyclin D1a and D1b protein isoforms in primary breast cancers have not yet been reported, although cyclin D1b was not found in the ZR-75-1 breast cancer cell line known to bear cyclin D1 gene amplification and overexpression.57 To date, only transcript [a] has been examined and found to drive mammary tumorigenesis in transgenic mice.27 Still, cyclin D1b could also be making a significant contribution to breast oncogenesis,52 and its role deserves additional investigation in human tumors and in complex in vivo systems.
Exon 4 Polymorphism
A common polymorphism within cyclin D1, A/G at nucleotide 870 in exon 4, does not alter its encoded amino acid but has been reported to influence the ratio of alternatively spliced transcripts [a] and [b].58 Results have been inconsistent, however, with the A allele favoring production of transcript [b] or transcript [a] in different studies and different tissues.58 In addition, the A/A genotype has been associated with increased predisposition to a number of specific cancers, and/or to poor prognosis. However, the genotype at the 870A/G polymorphism does not appear to influence a woman's risk of breast cancer,59,60 and it has not been determined whether the 870A/G genotype importantly alters the ratios of the cyclin D1 protein isoforms in breast cancer.
The Cyclin Box
Both isoforms of the cyclin D1 protein contain the so-called cyclin box sequence, a region of about 100 amino acids near the amino terminus. The sequence is fairly well conserved among all cyclins and is required for binding to their respective partner cdk. This sequence conservation does suggest that some aspect of cyclin D1 function relies on its ability to bind and activate a cdk. Whether such cdk dependence relates to the role of cyclin D1 in development, normal postnatal physiology, and/or tumorigenesis is still at issue.
HOW DOES CYCLIN D1 ACTIVATION CAUSE BREAST CANCER
Although the importance of cyclin D1 activation as a primary molecular cause of breast cancer is solidly established, the precise mechanisms through which cyclin D1 exerts its oncogenic action still require better definition. Resolving this issue carries important implications for devising new therapeutic strategies for breast cancer patients.
cdk-Mediated Effects on Cell Cycle Progression
Numerous studies, generally employing synchronized cultured cells and other in vitro systems, have led to the widely accepted view that the primary way in which cyclin D1 functions is by activation, as a regulatory subunit, of cdk4 and/or cdk6.9 Biochemically, cyclin D1每cdk complexes, followed in the cell cycle by analogous cyclin E-containing complexes, cause phosphorylation of retinoblastoma protein (pRB), the product of the retinoblastoma susceptibility gene, thereby inactivating the G1-maintaining function of pRB. A noncatalytic function of cyclin D每cdk4/6 complexes in sequestering cdk inhibitor proteins p21 and p27 from the cyclin E每cdk2 complex has also been described.61 The ability of cyclin D1 to activate its cdk partner is antagonized by the p16INK4a tumor suppressor. Although a huge body of evidence indicates that the main (and perhaps only) important role of cyclin D1 in the proliferation of cultured cells does indeed involve cdk activation and pRB inactivation, the same cannot be stated as definitively for the role of activated cyclin D1 in breast cancer or other primary human tumors.
The emphasis on cdk-dependent cell cycle actions as the major mechanism of cyclin D1 in human tumorigenesis has been argued on the basis that individual tumors tend to have a specific alteration of only one component of the cyclin D1/cdk/pRB/p16 pathway, suggesting a oncogenically relevant functional redundancy. On the other hand, there are many exceptions to this rule, and the functional equivalence of these lesions is further called into question by the major differences in their relative distributions across distinct types of primary tumors. It is also worth noting that cyclin D1 overexpression has not been associated with markers of tumor cell cycle activity in human breast cancers.38 If we examine this problem more broadly, there are many genes that have been identified as key regulators of the cell cycle, but few have been established through recurrent clonal derangements to be human oncogenes or tumor suppressors. It is likely, therefore, that achieving a selective advantage requires something more than cell cycle misregulation. Thus the small subset designated cell cycle regulators with proven oncogenic significance contribute to tumorigenesis through special mechanisms in addition to (or possibly instead of) their recognized roles in the cell cycle. Beyond this theoretical argument, considerable evidence for specific non每cdk-dependent functions of cyclin D1 now exists.
Does Cyclin D1 Activation Contribute to Breast Cancer Through cdk-Independent Mechanisms
As discussed, the recent accumulation of evidence that cyclin D1 may contribute to neoplasia through cdk (or cell cycle) -independent mechanisms is a departure from the widely accepted paradigm but in principle, is not surprising to those with a clinical perspective. A sampling of this intriguing evidence is described here (also see Coqueret44), but it must also be emphasized that no definitive answers currently exist for the important questions about exactly how cyclin D1 exerts its oncogenicity in human neoplasia.
Evidence for cdk-independent mechanisms, in addition to the tissue type每specific skewing of distribution of mutations among cyclin D1, RB, and p16 genes in human tumors, includes in vitro observations of non每cell cycle effects of cyclin D1 on genomic instability,62,63 an influence on DNA replication and repair in UV-treated fibroblasts,64 a report of direct binding of cyclin D1 to proliferating cell nuclear antigen (PCNA),65 and a link to the apoptotic pathway in neurons66 and in cyclin D1每overexpressing MCF7 breast cancer cells.67 Furthermore, in a series of breast cancer cell lines, overexpression of cyclin D1 was not related to cdk4 activity.68 D-type cyclins bind to and repress the activity of a myb-like transcription factor, DMP1, by interacting with the DNA binding domain of DMP1, which in turn prevents its proper binding to target promoters.69 A mutant cyclin D1 that is unable to activate cdk4 can still interact with DMP1 and inhibit its activity. Of great interest, DMP1 expression in transfected cells prevented S phase entry, and this DMP1-induced growth arrest could be overcome by the same mutant cyclin D1, indicating that the reversal of DMP1-induced growth arrest does not occur through its role as a cdk regulatory subunit.70 Cyclin D1 strongly inhibits activity of the B-Myb transcription factor, not through cdk-dependent phosphorylation of B-Myb, but through formation of a specific complex between cyclin D1 and B-Myb.71 In addition, in cultured cells, the effect of cyclin D1 on transformation does not always rely on inactivation of pRB.44,72,73 A variety of other transcriptional targets of cyclin D1 have been described, including the androgen receptor,74 PPAR (peroxisome proliferator-activated receptor gamma),75 and histone acetylases and deacetylases.44
In breast cancer cell lines, overexpressed cyclin D1 has been shown to bind directly and activate the estrogen receptor alpha (ER) in a cdk- and pRB-independent fashion.76,77 This fascinating finding, that overexpressed cyclin D1 can activate the ER in a hormonally independent fashion in vitro, and might thereby drive all of the key mitogenic effects of estrogen on breast epithelium, has the potential to add a crucial dimension to our understanding of mammary tumorigenesis. Although cyclin D1 overexpression is present across multiple histologic subtypes of breast cancer, a theme of commonality has been that the large majority of cyclin D1每overexpressing breast cancers are ER positive.25,78,79 This strong association has been difficult to explain, given the prevalent concept that cyclin D1 expression is only a downstream consequence of estrogen-induced activation of the ER, a nuclear hormone receptor which functions as a transcription factor.80-82 In that scenario, one might expect that tumors with more autonomous cyclin D1 expression due to gene amplification could bypass the need for functional ER, predicting an association between cyclin D1 overexpression and ER-negative breast cancer, which in fact is not observed. For example, almost all (94%) of the cyclin D1每positive breast cancers in one study were ER positive
in contrast, only 61% of cyclin D1每nonoverexpressing tumors were ER positive.25 Such results raised doubt that the action of cyclin D1 as a downstream mediator of sex steroid mitogen-induced proliferation in cultured cell lines would fully explain its oncogenicity in breast cancer. The subsequent finding that cyclin D1 may act upstream or as a coregulator of ER activity,44 as opposed to solely being a downstream target of ER-mediated activation, would provide an appealing explanation for the typical coexpression of ER in cyclin D1每positive breast cancers. In other words, cyclin D1 overexpression would bestow a selective advantage mainly in the presence of a serviceable ER.
Specifically, Zwijsen et al76 and Neuman et al77 tested the effect of cyclin D1 on ER transactivation by transfecting ER-positive breast cancer cells with a cyclin D1 expression vector, together with a reporter gene construct. Cyclin D1 directly interacted with liganded and unliganded ER to trigger binding of ER to an estrogen receptor response element and thereby increased reporter target gene transcription. Furthermore, the effect was not dependent on cyclin D1 interacting with cdk4 or pRB, given that it was reproduced with cyclin D1 mutants incapable of binding to cdk4 or pRb. Subsequent work showed that cyclin D1 not only interacted with the ER in cell lines but also with certain steroid receptor coactivators.83,84 This raises the possibility that cyclin D1 may act as a bridge, recruiting essential coactivators to the ER even in the absence of estrogen. It has been suggested that cyclin D1 may exert its cdk-independent, transcriptional effects only when overexpressed, as in pathologic situations.10
A recent study has added considerable weight to the potential tumorigenic relevance of cdk-independent transcriptional functions of cyclin D1.11 Lamb et al11 determined the global patterns of gene expression in cultured breast cancer cells after enforced overexpression of either wild-type cyclin D1 or a mutant form unable to activate cdk4 (KE mutant). Strikingly, both wild-type and mutant cyclin D1 effected the same signature of major changes in downstream gene expression, suggesting that the observed effects of cyclin D1 overexpression were cdk4 independent. The expression of this set of cyclin D1 target genes was then examined across a large panel of primary human tumors, and a positive correlation with cyclin D1 expression was found. Thus, the expression signature demonstrated in vitro was cross validated in vivo, providing support for the presence of cdk-independent actions of cyclin D1 in human tumors. Furthermore, a focused look at whether expression of known E2F target genes (downstream in the cyclin D/p16/cdkpRBE2F pathway, driving the G1 to S transition) would correlate with cyclin D1 expression in these human tumors proved negative, casting more doubt on the idea that cyclin D1 acts primarily as a cell cycle regulator in cancers. Interestingly, and in marked contrast, cyclin D3 expression correlated nicely with the E2F target genes, emphasizing the idea that cyclin D1 in vivo has unique functional attributes not shared by other cyclins, including other D-type cyclins. Indeed, analysis of these differences may uncover the key features that make cyclin D1, unlike other cyclins, a robust human oncoprotein. Finally, this study provided evidence that overexpression of the transcription factor C/EBP is highly correlated with cyclin D1 expression in the same human cancer database, and that cyclin D1 acts on or with C/EBP in regulating cyclin D1 target genes in vitro.11
Clearly, the relevance of these alternative mechanisms of cyclin D1 action carry tremendous potential significance to human cancer, and the direct role of cdk-independent actions in tumorigenesis must be further examined in appropriately complex in vivo systems.
In Addition to Its Primary Role in Tumorigenesis, Does Cyclin D1 Mediate the Action of Other Oncogenes in Causing Breast Cancer
Because cyclin D1 overexpression in breast cancers cannot always be attributed to clonally selected cyclin D1 DNA alterations such as gene amplification, it is conceivable that primary activation of other oncogenic/mitogenic pathways could provide an alternate route to tumorigenic cyclin D1 dysregulation. Consistent with this possibility, in vitro experiments have shown direct or indirect regulation of the cyclin D1 promoter, or of cyclin D1 expression, by -catenin, STAT5, STAT3, nuclear factor kappa B, ets-2, cyclic adenosine monophosphate每response element, tissue-coding factor/leukokinesis-enhancing factor, c-Jun, E2F-1, PPAR, calveolin-1, Ras signaling, and others.44 It will be important to sort out the relevance of these cyclin D1 regulatory mechanisms to primary human neoplasia.
An interesting series of in vivo experiments have exploited genetically engineered cyclin D1每null mice. These animals exhibited normal development in most respects, a major exception being defective postnatal mammary development revealed by a lack of proliferation of alveolar epithelial cells in response to the sex steroid milieu of pregnancy.85,86 Crossing of cyclin D1每null mice with transgenic mice that develop mammary cancer in response to oncogenes (Wnt1, v-Ha-ras, Neu/Erbb2, or Myc) overexpressed in mammary tissue showed that the cyclin D1每null background blocked mammary tumorigenesis driven by Ras or Neu, but not by Wnt1 or Myc.87 These results have been widely interpreted to mean that mammary cancers driven by Neu or Ras pathway activation are dependent on cyclin D1 as an essential oncogenic intermediary in mice, and likely in humans. Similarly, it has been speculated that inhibitors of cyclin D1 could therefore effectively treat human breast cancers that overexpress NEU or RAS.
There are reasons, however, to view the common interpretation of these experiments with caution if not skepticism. First, cyclin D1 knockout mice are fundamentally experiments in development rather than tumorigenesis. Because these mice lack cyclin D1 throughout development, the specific mammary cell that is susceptible later in life to the action of a particular oncogene may not have developed or may be different from it would have been had development been normal. Second, even if Neu-driven mammary cancer could be shown to be crucially dependent on cyclin D1 in a background of normal development in mice, this may not be true in humans. Indeed, many or most NEU-activated human breast cancers are ER negative and would not be expected to overexpress cyclin D1, given that most cyclin D1每overexpressing breast cancers are ER positive
furthermore, Bieche et al26 observed no link between cyclin D1 alterations and NEU overexpression in primary breast cancers.
Does Cyclin D1 Activation Cause Breast Cancer Solely by Leading to Increased Cyclin E Expression
The action of cyclin D1 in fostering cell cycle progression has been viewed as preliminary to and necessary for the subsequent expression of cyclin E, but does cyclin D1 activation cause human breast cancer solely through a downstream activation of cyclin E expression An affirmative answer to this question has been suggested by recent studies using genetically engineered mice. Cyclin E每encoding DNA under the control of the regulatory region of cyclin D1 was knocked in to cyclin D1每null mice, and the mammary developmental defect characteristic of cyclin D1每null mice was thereby abrogated.88 This cyclin E knock-in also eliminated the resistance to Ras- or Neu-induced mammary cancer otherwise imparted by the cyclin D1每null background.87 If ultimately shown to apply in human breast cancer, these results would suggest that the efficacy of an anticyclin D1 therapy might be enhanced by simultaneous targeting of cyclin E.
Again, however, there are strong reasons for caution before embracing these conclusions as relevant to human breast cancer. Although a compensatory upregulation of cyclin E was sometimes eventually able to overcome the blockade to an engineered mutant Neu-induced tumorigenesis imposed by the cyclin D1每null background in mice, this was not the case for the wild-type Neu sequence typically amplified in cancers.89 Next, just as noted, these cyclin E knock-in experiments similarly fail to isolate the role of cyclin D1 in tumorigenesis from the effects of its loss during development. In addition, the ability of transgenically overexpressed cyclin E to drive mammary tumorigenesis in mice appears to be weaker than cyclin D1,27 with a penetrance of only 10%.32
Finally, and perhaps of most significance to the clinician, the roles of cyclin D1 and cyclin E in human breast cancer may well differ markedly from their roles in the mouse. The crucial point is that if cyclin E activation could drive human breast cancer as effectively as cyclin D1, or even at a less effective but still clinically relevant level, then robust evidence for cyclin E as a human breast oncogene should exist. To the contrary, however, such evidence is strikingly lacking in primary human breast cancers, nor has cyclin E core amplification, rearrangement, or other clonal activating mutation been recurrently identified in other human tumors. The strong implication is that cyclin E activation fails to confer a clinically important selective advantage on a mammary cell in the development of human breast cancer.
PATHOGENESIS VERSUS PROGNOSIS
It is important to emphasize that the clear involvement of a gene in tumor pathogenesis is no guarantee of its utility as a prognostic marker, and vice versa. For example, involvement of a given gene may be so crucial to the development of a given type of tumor that interindividual differences in tumor behavior and prognosis will necessarily depend on other factors. For a given type of tumor, it can also be true that there are multiple alternative molecular means to the common end of tumor development. Perhaps for these reasons, cyclin D1 has both a clear, central role in breast cancer pathogenesis and an apparent but certainly less clear role as a clinically useful prognostic marker. Conversely, cyclin E (or E1) has been suggested to have major value as a prognostic marker,90 although there is strikingly little evidence from primary human tumors that cyclin E is an oncogene.
The prognostic value of a marker depends on the available therapeutic armamentarium. For example, when some (but not all) studies defined cyclin D1 amplification or overexpression as marking a subset of ER-positive breast cancers as having a poorer prognosis than other ER-positive breast cancers,91,92 a standard approach to treatment of the patients is assumed. However, molecular alteration in cyclin D1 could become an essential prognostic marker, if an effective therapy that specifically targeted cyclin D1 existed.
CYCLIN D1 AS A THERAPEUTIC TARGET
The large number of breast (and other) cancers for which cyclin D1 activation is a pathogenetic cornerstone has appropriately attracted much attention in the search for improved therapeutics. Based in part on the widely accepted paradigm that the entire contribution of cyclin D1 to tumorigenesis is achieved through its activation of cdk4/6, much effort has been directed toward finding small molecule inhibitors of cdk, or activators of endogenous cdk inhibitors. For example, flavopiridol is a cdk-inhibitory agent in clinical trials.93
However, if an important component of the oncogenic action of cyclin D1 is accomplished through cdk-independent mechanisms, therapeutic cdk inhibition might not be expected to be especially efficacious in cyclin D1每driven cancers. By the same reasoning, blocking cyclin D1 expression specifically and directly (by targeting the cyclin D1 gene, RNA, or protein) should increase the chances for therapeutic success in these tumors while advantageously avoiding the need for complete knowledge of the downstream effectors of cyclin D1 (Fig 1). Cell culture studies have raised the possibility that certain compounds might act in this way,93-95 and approaches to blocking cyclin D1 expression using antisense, siRNA or related molecules also have conceptual appeal in specifically targeting the driving molecular lesion itself,96-98 assuming in vivo use of these technologies becomes feasible.
Tumors bearing clonal genetic alterations in the cyclin D1 gene should be maximally susceptible to a highly specific anticyclin D1 therapy, akin to the efficacy of imatinib,99 trastuzumab,100 and gefitinib101,102 against tumors with analogous clonal DNA lesions (BCR-ABL fusion,99 HER2/neu amplification,100 and EGFR mutation,101,102 respectively) that speak to their central pathogenetic role in selection and, evidently, the ongoing viability of the tumor cell.103 Earlier concern about potentially broad toxicity of a cell cycle agent has been diminished by the meager phenotype of cyclin D1每null animals and the new information on the cdk-independent actions of cyclin D1
nonetheless, achieving the ability to deliver an anti每cyclin D1 agent with high selectivity to tumor cells should certainly be advantageous. Finally, it might be expected that any successful anticyclin D1 therapy will find optimal use in a combination protocol, together with an agent that targets a different clonally selected cooperating gene (Fig 1) in the tumor, to eradicate the rare tumor cells that could escape a single agent through fortuitous mutation and subsequent selection.
In summary, because of its established role as a major human oncogene, the therapeutic exploitation of cyclin D1 holds tremendous promise and should be vigorously pursued. For many breast cancers, strong evidence of the role of cyclin D1 as a pathogenetic cornerstone suggests that appropriately selected tumors will be highly susceptible to future anti每cyclin D1 therapy.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by a grant CA55909 (to A.A.) from the National Institutes of Health and a postdoctoral fellowship from the Susan G. Komen Breast Cancer Foundation (to A.P).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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