Overexpression of cyclin E protein is associated with specific mutation types in the p53 gene and poor survival in human breast cancer
Thomas Lindahl5,
Göran Landberg1,
Johan Ahlgren2,
Hans Nordgren3,
Torbjörn Norberg,
Sigrid Klaar,
Lars Holmberg4 and
Jonas Bergh
Department of Oncology and Pathology, Radiumhemmet, Karolinska Hospital and Institute R8:03, S-171 76 Stockholm, Sweden, 1 Division of Pathology, Department of Laboratory Medicine, Lund University, Malmö University Hospital, Malmö, Sweden, 2 Department of Oncology, Uppsala UniversityGävle County Hospital, Gävle, Sweden, 3 Department of Pathology, Uppsala University Hospital, Uppsala, Sweden and 4 Uppsala-Örebro Regional Oncologic Center, Uppsala, Sweden
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Abstract
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Cyclin E is one of the key regulators of the G1/S transition in the cell cycle. Overexpression of cyclin E has been observed in several malignancies and is associated with high proliferation, aberrant expression of other cell cycle regulators and chromosomal instability in vitro. To explore potential associations between cyclin E deregulation and inactivation of the p53 tumor suppressor gene in human breast cancer, we investigated the immunohistochemical expression of cyclin E in paraffin embedded breast cancers from 270 women with known p53 status by cDNA based sequencing of the p53 gene. The breast cancers were divided into three subgroups according to the percentage of cyclin E-positive cells. One hundred and seventy-one patients (63%) had low cyclin E, 72 (27%) medium and 27 (10%) had high cyclin E content. Fifty-six percent (15/27) of the breast cancers with high cyclin E had p53 gene mutations, compared with 14% (24/171) of those with low cyclin E content (P < 0.0001). In p53 mutated breast cancers high cyclin E content was associated with insertions, deletions and nonsense point mutations in the p53 tumor suppressor gene, whereas low cyclin E was linked to p53 missense point mutations. We also observed statistically significant associations between a high cyclin E content and aneuploidy, high S phase, larger tumor size, estrogen receptor negativity, presence of axillary node metastases and high tumor grade. High cyclin E content was associated with poor overall survival in univariate and multivariate analysis (hazard ratio 2.4, 95% confidence interval 1.34.5). In summary, our findings demonstrate that overexpression of cyclin E is associated with an aggressive tumor phenotype and specific types of p53 mutations.
Abbreviations: CDK2, cyclin-dependent kinase-2; ER, estrogen receptor; IHC, immunohistochemistry/immunohistochemical; LOH, loss of heterozygosity; LMW, low molecular weight; RB, retinoblastoma gene
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Introduction
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Cyclin E is a cell cycle regulatory protein involved in several important processes in the cell cycle. With its catalytic subunit cyclin-dependent kinase-2 (CDK2), cyclin E is a key factor in the G1 checkpoint, promoting transition into S phase (1). Cyclin E/CDK-2 also plays a role in the initiation of DNA replication (2) and centrosome duplication (3,4). Cyclin E is normally induced by E2F1 at the transition from G1 into S phase and is rapidly degraded in early S phase (5,6). Under normal conditions cyclin E is therefore only present in low cellular amounts, and it has been shown that non-malignant breast cells lack cyclin E immunoreactivity (7). Cyclin E can also be overexpressed in tumor tissue as biologically hyperactive low molecular weight (LMW) isoforms which lack the normal N-terminus (8). Induction of the p53 tumor suppressor gene after DNA damage inhibits the G1 cyclins/cyclin-dependent kinase activity via the p53 downstream mediator p21 (WAF1/Cip1) (9,10). This inhibition causes cell cycle arrest to facilitate DNA repair (1113).
Deregulated expression of cyclin E has been correlated with aggressive tumor characteristics in breast cancer. Nielsen and colleagues observed a high proliferation rate and inactivation of the retinoblastoma (RB) tumor suppressor gene in human breast cancers overexpressing cyclin E (14,15). In vitro, deregulated cyclin E expression is linked to aneuploidy in immortalized rat embryo fibroblasts and human breast epithelial cells (16). Overexpression of cyclin E in breast cancer has been correlated with shorter survival in retrospective studies of patient outcome (17,18). Keyomarsi et al. reported that the hazard ratio for dying from breast cancer was more than 13 times higher for patients with high total cyclin E levels (low molecular weight isoforms and full-length protein) compared with those with low cyclin E levels (19). The findings above indicate that overexpression of cyclin E may accelerate tumor progression through increased proliferation, increased genetic instability and tumor suppressor gene inactivation. A functional p53 is potentially of importance in counterbalancing an uncontrolled CDK activation in malignancies caused by cyclin E overexpression, but theoretically may also frequently be inactivated due to the assumed increased mutagenic pressure in tumors overexpressing cyclin E. To clarify the associations between these two important events in the transformation process, we have characterized cyclin E protein content and p53 gene status, determined by immunohistochemistry (IHC) and cDNA-based sequencing, respectively, in 270 breast cancer patients receiving primary therapy during 19871989. The prognostic properties of cyclin E in comparison with p53 and several other known prognostic markers in human breast cancer were also investigated.
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Material and methods
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The IHC expression of cyclin E was investigated in 270 paraffin embedded primary breast cancers derived from a population-based cohort of 315 patients, operated on between 1987 and 1989 at Uppsala University Hospital. The clinical characteristics of the patients and the methods for p53 mutation analysis have been described in detail in previous reports (20,21). In brief, fresh frozen tumor tissue from 315 breast cancers was analyzed for p53 mutations using cDNA-based sequencing of the entire coding region of the p53 gene (exons 211) and comparison of the wild-type p53 sequence with the sample sequence. Breast cancers with possible mutations were re-analyzed for verification. Four patients had incomplete p53 sequencing results (20). All breast cancers were also analyzed by p53 IHC using the mouse monoclonal antibody pAb 1801 on formalin-fixed, paraffin-embedded breast cancer tissue. Breast cancers with positive staining of any degree were considered positive cases (21).
Of the 311 patients with known p53 status from the sequencing analysis, 270 had additional paraffin-embedded primary tumor material available for IHC determination of cyclin E expression. The paraffin sections were deparaffinized and microwave treated. Staining was performed using the mouse monoclonal anti-cyclin E antibody HE 12 (Santa Cruz Biotechnology, Santa Cruz, CA) and an automated IHC staining machine (Ventana 320-202; Ventana, AZ). The HE 12 antibody targets the cyclin E protein C-terminus (22). It therefore recognizes both the long and shorter forms of the cyclin E protein without distinction. The IHC reactivity was divided into three levels according to the percentage of tumor cells stained; low (04%), medium (549%) and high (50100%). The cut-off levels were chosen in order to achieve distinct separation between patients with high and low cyclin E expression. This selection was done before any statistical analyses were performed. For the distribution of cyclin E immunostaining see Figure 1. All slides were read without knowledge of previously determined tumor characteristics or patient outcome. IHC determination of cyclin E expression can be considered a valid method for detecting tumors overexpressing cyclin E since non-malignant breast cells lack cyclin E immunoreactivity (7).
Clinical patient information, such as age at diagnosis, menopausal status, axillary node status and adjuvant therapy, were gathered from the patient records before the previously published p53 studies (20,21). Of the 270 patients included in this study information on node status, menopausal status and adjuvant therapy was missing for 10, 67 and 3 patients, respectively. S phase, DNA content (number of chromosomes) and estrogen receptor (ER) status were determined at the time of diagnosis as part of the routine clinical management of breast cancer at Uppsala University Hospital. S phase and DNA content were determined by flow cytometry on fresh tumor material. The cut-off values for high S phase were 7% in diploid tumors and 12% in aneuploid tumors (23). S phase data were missing for 15 patients. ER status was determined by an immunoassay. Seven patients lacked data for ER. The histopathological tumor grade was classified by an experienced pathologist into three levels (low, medium and high) according to the method described by Elston and Ellis (24). Three patients were not classified due to lack of available breast cancer material.
The median follow-up for overall survival was 122 months (maximum 155). The data on overall survival were obtained from the Swedish population registry of late 1999.
Univariate survival analyses were performed using the KaplanMeier method and statistical testing was done with the log-rank test (MantelCox). The cumulative probabilities and corresponding standard errors (SE) for 5 and 10 year overall and breast cancer-specific survival were obtained from the KaplanMeier analyses. Ninety-five percent confidence intervals (95% CI) around each estimate were calculated using the standard error of the cumulative probability (formula: ±1.96 x SE). The distribution of dichotomous variables in different patient groups were tested with the
2 test and the distribution of continuous variables were tested with the unpaired ANOVA test. Multivariate survival analyses were performed using the Cox proportional hazards method using two models with different sets of variables. One model included cyclin E and other molecular prognostic variables: p53, S phase, DNA content and ER status. The other model contained cyclin E and clinical prognostic variables: axillary node status, tumor size and histopathological tumor grade. Both models contained patient age at diagnosis.
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Results
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High cyclin E protein content (detailed in Material and methods) was observed in 10% (27/270) of investigated breast cancers and was significantly associated with mutated p53, aneuploidy, high S phase, negative ER status, presence of axillary node metastases, higher tumor grade and larger tumor size. For complete results see Table I. The prevalence of p53 mutations was higher in tumors with a high cyclin E content compared with the breast cancers with lower cyclin E content; 56% (15/27) versus 14 (24/171) to 33% (24/72) (P < 0.0001) (Table I). The predominant types of p53 gene mutations in breast cancers with a high cyclin E content were insertions or deletions (P = 0.0046) (Table I and Figure 2). The prevalence of an insertion or deletion in the p53 gene increased in a dose-dependent manner with increasing cyclin E content (Table I). All but one of seven tumors with a high cyclin E content and p53 insertions/deletions had undetectable p53 protein content by IHC, suggesting that these mutation types have a strong negative effect on p53 protein expression, as previously described (21). The majority of these mutations were found in the peripheral parts of the p53 gene (exons 24 or 911) (Table I and Figure 2). Stop codon point mutations were also more commonly observed in high cyclin E tumors (Table I).

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Fig. 2. Location and frequency of p53 mutations by codon number. The upper pie charts display the distribution of cyclin E expression divided by the location of the corresponding p53 mutation. The lower pie charts display the distribution of mutation types by gene location.
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The overall occurrence of an aneuploid DNA content in this material was 74% (200/270). All tumors with a high cyclin E expression and a mutated p53 gene were aneuploid (n = 15). Both wild-type p53/high cyclin E tumors and p53 mutated/low cyclin E tumors displayed high frequencies of aneuploidy; 92 (1/11) and 96% (1/23), respectively. High S phase was positively correlated with cyclin E expression, but not significantly different in breast cancers with medium (30%, 20/67) versus high cyclin E (32%, 8/25) (Table I).
None of the breast cancers with high cyclin E displayed low histopathological tumor grade (Table I). The percentage of breast cancers with high tumor grade differed significantly between the high cyclin E group compared with low or medium cyclin E; 85% (23/27) versus 11 (18/169) and 23% (16/71), respectively (Table I).
Cyclin E expression was inversely correlated with ER expression. Eighty-nine percent (150/168) of patients in the low cyclin E group were ER-positive versus 36% (9/25) in the high cyclin E group (Table I).
Patients with high cyclin E breast cancer had a statistically significantly reduced overall survival in univariate analysis (P = 0.0002) (Figure 3). The 5 year cumulative probability of overall survival for the low cyclin E group was 82 compared with 71% in the medium group and 41% for the breast cancers with high cyclin E (detailed in Table II and Figure 3). At 10 years follow-up, differences in overall survival rates were still present (Table II and Figure 3). A high cyclin E content remained a strong negative prognostic factor for overall survival when all p53 mutated breast cancers were excluded from the analysis (Figure 4). Both multivariate Cox proportional hazard analyses (molecular and clinical variables, Table III) showed a high cyclin E content to be an independent risk factor for shorter overall survival (relative hazards 2.1 and 2.4, with corresponding 95% CI of 1.14.1 and 1.34.5, respectively).
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Discussion
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The main finding in this study is the association between a high cyclin E content and insertions, deletions and nonsense point mutations in the p53 gene. Furthermore, we found that a high cyclin E content correlated with aneuploidy and was strongly associated with patient outcome. The p53 mutation pattern in low cyclin E tumors in this study corresponded well with the reported overall frequency of different mutation types in the p53 gene, with missense point mutations being predominant (25). However, the observed mutation pattern in breast cancer cells overexpressing cyclin E differed markedly from the common frequencies of different p53 mutation types in breast cancer. In these patients we saw disproportionately high numbers of insertions, deletions and stop codon point mutations. These types of mutations should theoretically result in a more malignant phenotype, due to a complete loss of p53 function. However, we cannot confirm a worse outcome for this group of patients compared with those patients with missense point mutations. In theory, missense p53 proteins might bind and inactivate functional p53 from a wild-type allele (reviewed in 25). In reality, this phenomenon is probably not of great importance. In a study by Williams et al. on a subset of patients from our material, all informative cases (n = 15) with missense point p53 mutations displayed loss of heterozygosity (LOH) (28).
Cyclin E is involved in early events in DNA replication and replication control during S phase (29,30). Ectopic expression of cyclin E affects the G1/S transition resulting in an uncoupling of DNA replication and cell cycle progression (31). It is therefore likely that overexpression of cyclin E affects the fidelity of DNA replication. We have earlier observed over-representation of inactivation of the retinoblastoma (RB) protein, including frequent LOH of the RB gene in tumors with a high cyclin E protein content as determined by western blotting (15). Inactivation of two of the major suppressor genes in breast cancer through gross DNA damage is thus observed in tumors with high cyclin E protein, whereas inactivation of p53 through missense mutations seems to be a separate phenomenon not linked to high cyclin E protein. In the context of cyclin E inducing genetic stability, it is therefore tempting to speculate whether cyclin E overexpression is the primary event causing genetic instability and extensive DNA damage, with inactivation of suppressor genes being secondary events. To clarify this, the exact order of genetic events from a premalignant lesion to a fully transformed cell needs to be clarified. It can be stated, however, that cyclin E overexpression fulfills the criteria for a candidate key aberration in breast cancer, with potential effects on both cell proliferation and genetic stability, and therefore on tumor progression.
Spruck and colleagues reported an increased frequency of aneuploidy in vitro in immortalized rat fibroblasts and human breast epithelial cells with constitutive cyclin E overexpression (16). Our results using primary breast cancer samples support and strengthen the link between cyclin E overexpression and aneuploidy. The exact mechanisms causing genetic instability in cyclin E overexpressing tumors have not been clarified, but high cyclin E protein content, potentially throughout the active cell cycle, most likely affects the fidelity of DNA synthesis, causing disturbances in various checkpoint systems (2931). Both cyclin E and p53 have also been shown to be important for proper centrosome duplication (3,4,32,33) and our observation that both high cyclin E protein as well as p53 inactivation are separately linked to aneuploidy could support the idea that centrosomes might be the central link between cyclin E, p53 and genetic instability.
Our observation that a high S phase fraction was equally common in the breast cancers with medium or high cyclin E protein content, in contrast to tumors with low cyclin E, also indicates that high cyclin E expression conveys additional negative consequences for the malignant cell besides high proliferation.
Another observation that is in line with the hypothesis of cyclin E as a promoter of accelerated tumor progression is the negative impact on patient outcome observed for cyclin E overexpressing tumors with wild-type p53, suggesting that cyclin E overexpression is linked to aggressive tumor behavior even without inactivation of p53. Cyclin E was an independent prognostic factor in the multivariate analysis for overall survival. One should be cautious, however, when interpreting the multivariate models since it is not clear how the covariates relate to each other in terms of effect modification, confounding or even being on the same causal pathway.
Overexpression of low molecular weight isoforms of cyclin E may have an additional negative effect on patient outcome compared with overexpression of the full-length protein. IHC detection of cyclin E with the HE 12 antibody does not discriminate between these proteins. Our observed association between cyclin E overexpression and poor outcome could therefore be an underestimate. However, using IHC and the HE 12 antibody should yield the total cyclin E level, which according to the findings of Keyomarsi et al., is the most important marker for outcome (19).
In conclusion, we have observed that a high IHC cyclin E content is associated with insertions, deletions and nonsense point mutations in the p53 gene, likely to have a strong negative impact on p53 protein expression. We also observed independent associations between cyclin E, mutated p53 and aneuploidy. Our results suggest that IHC determination of cyclin E expression may very well be of clinical value, but warrant further evaluation.
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Notes
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5 To whom correspondence should be addressed Email: thomas.lindahl{at}cck.ki.se 
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Acknowledgments
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Jonas Nilsson at the Uppsala-Örebro Regional Oncologic Center is acknowledged for expert statistical assistance. The study was supported by grants from the Swedish Cancer Society, the Stockholm Cancer Society, the Swedish Society of Medicine, the King Gustav the V Jubilee Foundation and the Centre for Research and Development (FoU-forum), Uppsala UniversityGävleborg County Council.
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Received July 9, 2003;
revised October 24, 2003;
accepted October 28, 2003.