Low Molecular Weight Forms Of Cyclin Research Articles

Cyclin overexpression inhibits Smad 3 tumor suppression in breast cancer cells.

Abstract Abstract #5033 Introduction: The transcription factor Smad 3, a member of the TGFβ signaling cascade, contributes to G1 cell cycle arrest in breast cancer cell lines. Overexpression of the cell cycle regulatory protein cyclin E, and its low molecular weight (LMW) counterparts, has been associated with poor prognosis in breast cancer. Cyclin E has been shown to mediate phosphorylation within or near the linker region of Smad 3 by cyclin dependent kinase 2 (CDK 2), and to inhibit Smad 3 activity in normal mouse fibroblasts and epithelial cells. We hypothesize that overexpression of cyclin E may exert tumorigenic effects in breast cancer cell lines through the functional inhibition of Smad 3 mediated by CDK 2 phosphorylation.
 Methods: Endogenous levels of Smad 3 and phosphorylated Smad 3 in parental, vector only control, full length (FL), and two LMW forms of cyclin E overexpressing MCF-7 breast cancer cell lines were determined by western blotting. To elucidate the impact of cyclin E overexpression on Smad 3 function, constructs containing an empty vector, WT Smad 3, Smad 3 with a CDK phosphorylation site mutation within the linker region (Thr178), and outside the linker region (Thr8) were co-transfected with a Smad 3 reporter into the panel of MCF-7 cells. CDK2 or scrambled control siRNA was also transfected into the study panel of cells. Smad 3 function was then evaluated by luciferase reporter assays.
 Results: MCF-7 cells overexpressing FL or LMW cyclin E had a higher level of P- SMAD 3 as compared to the parental and vector only control cells. Total SMAD 3 levels were similar in all cells examined. Transfection of WT Smad 3 elicited an increase in reporter activity in parental and vector control cells, while both FL and LMW forms of cyclin E overexpressing cells were resistant to WT Smad 3 reporter induction. In parental and vector control MCF-7 cells, expression of the Smad 3 Thr178 mutation resulted in a three-fold induction in Smad 3 reporter activity as compared to the WT Smad 3 response. In FL and LMW cyclin E overexpressing cells, induction by the Thr178 mutation was more pronounced, as seen by a 5-fold and 8-fold induction of reporter activity in FL and LMW cyclin E cells, respectively. Transient transfection of the Thr8 mutation failed to evoke a reporter response in any of the panel of MCF-7 cells. MCF-7, FL, and LMW cyclin E cells demonstrated the greatest fold induction of Smad 3 reporter activity when transfected with CDK 2 siRNA.
 Conclusions: Overexpression of cyclin E may circumvent the growth inhibitory effects of Smad 3 signal transduction through the modulation of Smad 3 function. Specifically, this work indicates that cyclin E mediates Smad 3 inhibition in a CDK 2 dependent fashion in breast cancer cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5033.

QS287. HER2 Regulates Expression of Low Molecular Weight Cyclin E Isoforms in Breast Cancer

Objective: HER2 amplification and cyclin E overexpression have each been shown to be important prognostic indicators in breast cancer. In addition to overexpressing the full length (FL) cyclin E protein, some breast cancer cell lines and human breast cancers express up to 5 low molecular weight (LMW) isoforms which are generated by proteolytic processing of FL cyclin E by the serine protease elastase. These LMW forms have been demonstrated to be more tumorigenic than the FL protein. We hypothesized that HER2 overexpression may modulate cyclin E regulation in breast cancer, including generation of the LMW forms. Methods: HER2 expression was downregulated in the HER2-overepressing breast cancer cell lines MCF-7-HER-18 (HER18), SKBr3 and BT474 using 2 different mechanisms; treatment with trastuzumab dosed at 10 and 20 μg/ml or transfection with HER2 siRNA. The low-HER2 expressing cell line MCF-7 was used as a negative control. The effect on cyclin E expression was investigated using confocal immunofluorescence microscopy and western blot analysis. The effect of HER2 downregulation on cyclin E transcription was assessed using quantitative RT-PCR (qRT-PCR). To determine if there may be a clinically relevant relationship between these 2 proteins, HER2 and LMW cyclin E levels were assessed in 25 patient samples collected as part of an ongoing prospective protocol. Results: Confocal immunofluorescence microscopy demonstrated that HER2 downregulation using siRNA or inactivation by treatment with trastuzumab resulted in decreased total cyclin E expression. Because cyclin E is tightly regulated at the level of transcription, qRT-PCR was performed to determine the effects of HER2 downregulation on cyclin E mRNA synthesis. There was no difference (p=.18) in cyclin E mRNA synthesis for cells transfected with HER2 siRNA versus those transfected with random sequence and mock transfected controls suggesting that HER2 affects post-transcriptional processing of cyclin E. Having shown that the effect of HER2 on cyclin E expression is not at the level of transcription, western blot analysis was used to investigate the effects of HER2 downregulation on the LMW forms. Western blot demonstrated that the decrease in cyclin E was primarily in the LMW forms (figure) confirming the effect of HER2 to be post-transcriptional. Western blot analysis was then performed on lysates obtained from 25 patient samples. Nine (35%) patients had HER2-overexpressing tumors. Densitometry was used to quantify FL and LMW cyclin E expression. There was no significant difference in expression of FL cyclin E in HER2+ versus HER2- tumors (p=.87). In contrast, there was significantly higher expression of LMW cyclin E in HER2+ tumors (p=.04). Conclusions: HER2 downregulation affects post-transcriptional processing of cyclin E resulting in decreased expression of the more tumorigenic LMW forms. LMW forms of cyclin E are overexpressed in a subset of patients with HER2-overexpressing tumors. Further study into the mechanism by which HER2 effects post-transcriptional processing of FL cyclin E to its LMW isoforms may identify a role for LMW cyclin E as a second therapeutic target in patients with HER2 and LMW cyclin E-overexpressing breast cancer.

Low‐molecular weight forms of cyclin E differentiate ovarian carcinoma from cells of mesothelial origin and are associated with poor survival in ovarian carcinoma

The authors recently reported on the role of cyclin E in differentiating ovarian/primary peritoneal carcinoma from malignant peritoneal mesothelioma using gene expression arrays. In the current study, they analyzed the expression of low-molecular weight (LMW) forms of cyclin E in ovarian carcinoma, malignant mesothelioma, and benign reactive effusions. Cyclin E protein expression was analyzed in 98 effusions (72 ovarian carcinomas, 14 malignant mesotheliomas, and 12 reactive specimens) using immunoblotting. Sixty-two ovarian carcinoma effusions were studied further for cyclin E expression using immunohistochemistry. The correlations between cyclin E expression in ovarian carcinoma and clinical parameters, including chemotherapy response, were analyzed. LMW forms of cyclin E were identified in 54 of 72 ovarian carcinoma effusions (75%) compared with 1 of 14 malignant mesothelioma effusions (7%) and 1 of 12 reactive effusions (8%) (P < .001). Their presence in ovarian carcinoma was associated with a higher percentage of cyclin E-positive cells (P = .001) and increased staining intensity (P < .001) using immunohistochemistry. The presence of LMW forms of cyclin E was correlated with shorter overall survival (P = .021) and progression-free survival (P = .020). The presence of a higher percentage of cyclin E-positive cells using immunohistochemistry was correlated with shorter progression-free survival (P = .026). No association with chemotherapy response was observed. LMW forms of cyclin E differentiated ovarian carcinoma from benign and malignant mesothelial cells and were associated with increased protein expression using immunohistochemistry. The expression of LMW cyclin E forms was not associated with chemotherapy response, although it may be a marker of aggressive disease in patients with metastatic ovarian carcinoma.

Targeting the cell cycle for prognosis and therapy of breast cancer

Cyclin E is a G1-cyclin that plays a key role in the G1 to S transition of the cell cycle. Cyclin E is processed in tumor cells by an elastase-like protease into low-molecular-weight (LMW) isoforms that are biochemically hyperactive. The LMW isoforms of cyclin E are unique to cancer cells. In breast cancer, such alteration of cyclin E is a very strong predictor of poor patient outcome. Alterations in the binding properties of these LMW isoforms to CDK2 and the CDK inhibitors (CKIs), p21 and p27, result in their functional hyperactivity. The LMW forms of cyclin E are several-fold more effective at binding to CDK2. Additionally, compared with the full-length cyclin E–CDK2 complexes, the LMW cyclin E–CDK2 complexes are significantly more resistant to inhibition by p21 and p27, despite equal binding of the CKIs to the LMW complexes. When both the full-length and the LMW cyclin E are co-expressed, p27 preferentially binds to the LMW forms yet is unable to inhibit the CDK2 activity. When overexpressed in breast cancer cells, the LMW forms of cyclin E, but not the full-length form, result in their hyperactivity due to increased affinity for cdk2 and resistance to inhibition by the CDK inhibitors p21 and p27, result in resistance to the growth inhibiting effects of anti-estrogens, and result in chromosomal instability. Finally, tumors from breast cancer patients overexpressing the LMW forms of cyclin E are polyploid in nature and are resistant to endocrine therapy. To assess the oncogenic role of cyclin E-LMW as compared with full-length cyclin E, we examined the consequences of overexpressing these isoforms in the mammary glands of transgenic mice using the MMTV promoter. Four constructs were generated: MMTV-M46A coding for the full-length cyclin E (EL1), MMTV-EL1/EL4 coding for EL1 and the isoform translated at methionine 46 (EL4), and MMTV-T1 and MMTV-T2 coding for the isoforms generated by elastase cleavage at the first site (EL2 + EL3) and at the second site (EL5 and EL6), respectively. For each construct at least two transgenic lines were established. Transgene expression was demonstrated by RT-PCR, northern blotting and western blotting. Overexpression of cyclin E was seen in more than 90% of ductal and lobular cells of the mammary glands for each independent line. Mammary-specific LMW cyclin E overexpression induced extensive abnormalities at 2 months, including perturbed architecture, polyploidy, anysocytosis and apoptosis. Whole-mount preparations of mammary glands at different development stages showed that overexpression of EL1/EL4 and cyclin E-T1 induced growth delay, while at 6 months of age an increased proportion of cells in the S phase was found (25.6 ± 5.6% for EL1/EL4, 9.0 ± 2.7% for T1 compared with 3.9 ± 1.9% for non-transgenic animals). We observed a 34% (13/38) incidence of mammary adenocarcinomas in the EL1/EL4 transgenic lines with a mean latency of 18.3 months, and observed a 20% (5/25) incidence in the T1 transgenic lines with a mean latency of 17.1 months. The tumor incidence rate of the other transgenic lines, M46A and T2, are still unknown due to the young age of the mice (all under 7 months of age) and the long latency of cyclin E-mediated tumor generation. Thirty percent (4/13) of the EL1/EL4 and 40% (2/5) of the T1 tumor-bearing animals developed lung metastasis. The tumors induced by the EL1/EL4 and T1 transgenes were mainly solid adenocarcinomas with very little differential to glandular for EL1/EL4 and mostly glandular for T1. Since p53 alterations are common in human breast carcinomas, we bred a T1 line with p53+/- mice. The T1 × p53+/- cross generated tumors that are much more malignant than the T1 tumors; the incidence increased to 100%, with a much shorter latency of 11 months. Biochemical analysis of the tumors revealed that 64% (9/14) retained cyclin E expression and that, on average, the cyclin E-overexpressing tumors had threefold higher cyclin E kinase activity than the non-cyclin E-expressing tumors. Taken together, these data indicate that tumor progression in cyclin E transgenic mice follow sequential steps of dysplasia, mammary intraepithelial neoplasia and invasive/metastatic tumors. Collectively, the biochemical and biological differences between the full-length and the LMW isoforms of cyclin E provide a molecular mechanism for the poor clinical outcome observed in breast cancer patients harboring tumors expressing high levels of the LMW forms of cyclin E. The transgenic mouse model system can serve as a useful system in which to study the mechanisms responsible for LMW cyclin E-induced genetic instability and may help identify those factors that promote tumor progression and metastasis. The properties of the LMW forms of cyclin E suggest that they are not just surrogate markers of poor outcome, but that they are bona fide mediators of aggressive disease and potential therapeutic targets for patients whose tumors overexpress these forms.

The Tumor-specific Hyperactive Forms of Cyclin E Are Resistant to Inhibition by p21 and p27

The low molecular weight (LMW) isoforms of cyclin E are unique to cancer cells. In breast cancer, such alteration of cyclin E is a very strong predictor of poor patient outcome. Here we show that alteration in binding properties of these LMW isoforms to CDK2 and the CDK inhibitors (CKIs), p21 and p27, results in their functional hyperactivity. The LMW forms of cyclin E are severalfold more effective at binding to CDK2. Additionally, compared with the full-length cyclin E-CDK2 complexes, the LMW cyclin E-CDK2 complexes are significantly more resistant to inhibition by p21 and p27, despite equal binding of the CKIs to the LMW complexes. When both the full-length and the LMW cyclin E are co-expressed, p27 preferentially binds to the LMW forms yet is unable to inhibit the CDK2 activity. Thus, the LMW forms of cyclin E may contribute to tumorigenesis through their resistance to the inhibitory activities of p21 and p27 while sequestering these CKIs from the full-length cyclin E.

The Low Molecular Weight Isoforms of Cyclin E Deregulate the Cell Cycle of Mammary Epithelial Cells

Cyclin E is a positive regulator of the G1 to S phase transition of the cell cycle. In complex with CDK2 it is responsible for cells passing the restriction point, committing the cell to another round of cell division. Cyclin E is overexpressed and proteolytically cleaved into low molecular weight (LMW) isoforms in breast cancer cell lines and tumor tissues compared to normal cells and tissues. These alterations in cyclin E are linked to poor prognosis in breast cancer patients. Our laboratory has determined that the LMW forms of cyclin E are generated post-translationally, via elastase mediated cleavage at 2 specific sites in the amino-terminus of the full length cyclin E. In order to evaluate the biological effects of the LMW cyclin E, immortalized mammary epithelial cells, 76NE6, were stably transfected with each of the three cyclin E constructs. Our results reveal that the LMW forms of cyclin E (T1 and T2) are biologically functional, as their overexpression in the immortalized cells increases the ability of these cells to enter S and G2/M phase by 2 fold over full length or vector-alone transfected cells, concomitant with an increased rate of cell proliferation. In addition, these LMW isoforms are biochemically hyperactive, shown by their ability to phosphorylate substrates such as histone H1 4 fold more in cells transfected with T1 or T2 versus cells transfected with the EL form. These results suggest that overexpression of the LMW forms of cyclin E is mitogenic, stimulating the cells to progress through the cell cycle much more efficiently than the full length cyclin E.

Cyclin E in breast tumors is cleaved into its low molecular weight forms by calpain.

Abundant levels of the hyperactive low molecular weight (LMW) forms of cyclin E contribute to deregulation of Cdk2 in breast tumors, but the mechanism through which they arise is not fully understood. Here, we explored the hypothesis that post-translational processing by a protease generates the LMW forms of cyclin E in breast tumors. In ZR75 tumor cell lysates, calcium-induced cyclin E truncation into peptides corresponding in size with LMW forms of cyclin E in tumor tissues. Calpeptin inhibited calcium-stimulated cyclin E truncation, indicating that cleavage resulted from activity of the calcium-dependent protease, calpain. Consistently, calcium+calpain caused truncation of cyclin E immunoprecipitated from tumor cells and tissues. Calcium also caused truncation of the calpain regulatory subunit in tumor cell lysates, indicating that elevated calpain activity accompanies cyclin E truncation. Increased levels of the calpain small subunit were also observed in breast tumors, and significant amounts of its proteolyzed forms indicated increased calpain activity. While elastase also caused cyclin E truncation, the cleavage pattern was distinct from that generated by calpain, suggesting discrete mechanisms in regulating the formation of LMW cyclin E in breast tumors. Treatment of ZR75 cultures with calcium+A23187 recapitulated the formation of the calcium/calpain-induced LMW forms of cyclin E. Altered calcium homeostasis and/or inability of the endogenous calpain inhibitor to control the activity of high levels of the calpain small subunit may contribute to increased calpain activity in breast tumors, causing abundant levels of LMW cyclin E.