Cyclin E2 is the predominant E-cyclin associated with NPAT in breast cancer cells
© Rogers et al.; licensee BioMed Central. 2015
Received: 24 September 2014
Accepted: 2 February 2015
Published: 19 February 2015
The cyclin E oncogene activates CDK2 to drive cells from G1 to S phase of the cell cycle to commence DNA replication. It coordinates essential cellular functions with the cell cycle including histone biogenesis, splicing, centrosome duplication and origin firing for DNA replication. The two E-cyclins, E1 and E2, are assumed to act interchangeably in these functions. However recent reports have identified unique functions for cyclins E1 and E2 in different tissues, and particularly in breast cancer.
Cyclins E1 and E2 localise to distinct foci in breast cancer cells as well as co-localising within the cell. Both E-cyclins are found in complex with CDK2, at centrosomes and with the splicing machinery in nuclear speckles. However cyclin E2 uniquely co-localises with NPAT, the main activator of cell-cycle regulated histone transcription. Increased cyclin E2, but not cyclin E1, expression is associated with high expression of replication-dependent histones in breast cancers.
The preferential localisation of cyclin E1 or cyclin E2 to distinct foci indicates that each E-cyclin has unique roles. Cyclin E2 uniquely interacts with NPAT in breast cancer cells, and is associated with higher levels of histones in breast cancer. This could explain the unique correlations of high cyclin E2 expression with poor outcome and genomic instability in breast cancer.
KeywordsCyclin E1 Cyclin E2 CDK2 Centrosome Cajal bodies Histone Locus bodies (HLB) Spliceosomes NPAT Histones Breast cancer
The canonical function of cyclin E is the activation of CDK2 (cyclin dependent kinase 2) to phosphorylate Rb, hence promoting the release of E2F transcription factors and progression of the cell cycle from G1 to S phase . However there are other functions for cyclin E that may be CDK2 dependent or independent, including transcriptional processing, origin firing, and centrosome duplication . The wide range of cyclin E functions may explain the necessity for two cyclin E proteins: E1 and E2. Both these proteins activate CDK2, but are encoded by genes on different chromosomes (cyclin E1: CCNE1 at 19q12; cyclin E2: CCNE2 at 8q22.1). Cyclin E1 and E2 have differences in tissue expression, transcription and post-transcriptional regulation, and have distinct affinities for other proteins, e.g. p107 [1,3]. In this study we examined the localisation of cyclin E1 and E2 and report unique sites of localisation in breast cancer cells.
Cyclins E1 and E2 have cytoplasmic, nuclear and chromatin associated functions [1,2]. Cell fractionation showed that both cyclin E1 and E2 were predominantly nuclear and a large proportion was extracted with chromatin (Figure 1B). However a significant proportion of cyclin E1 was nucleolar and not chromatin associated (18.5%) compared to a smaller proportion of cyclin E2 (4.2%), and both proteins occurred at only very low levels in the cytoplasm (Figure 1B). Thus the majority of cyclin E1 and E2 is located on chromatin, but there is a small but significant proportion of cyclin E1 that is localized to non-chromatin foci.
As a positive control for PLA analysis we examined cyclin E1-CDK2 and cyclin E2-CDK2 interactions. We observed that both cyclin E1 and cyclin E2 had predominantly nuclear interactions with CDK2 (Figure 4C and D). A proportion of both cyclin E1-CDK2 and cyclin E2-CDK2 foci were cytoplasmic (Figure 4C and D) which is consistent with nuclear-cytoplasmic shuttling of these complexes . Cyclin E1-CDK2 interactions were 2-fold more abundant than cyclin E2-CDK2 (Figure 4E), which again suggests that it is unlikely that excess cyclin E2 prevents cyclin E1 from interacting with other binding partners such as NPAT.
Previous publications describe binding of “cyclin E” to NPAT, whereas we here identify that cyclin E2 is the major E-cyclin within HLBs in breast cancer cells. The previous studies were performed prior to the development of specific cyclin E1 and E2 antibodies, and relied upon the cyclin E HE67 (cyclin E1 aa366-381) and HE11 (full-length protein) antibodies which are raised using epitopes that may not effectively discriminate cyclin E1 and cyclin E2 [8,9]. While cyclin E1 may not influence histone transcription in breast cells via NPAT it could influence it via other pathways. Cyclin E/CDK2 indirectly controls histone transcription via E2F-mediated transcription of NPAT , and by phosphorylation of the HIRA protein which is a repressor of histone transcription that operates outside S phase .
Cyclin E1 has been recognised as an important oncogene for 20 years . The high degree of sequence homology between cyclin E1 and E2 suggests that many of their functions may be interchangeable, but recent publications in cancer and liver biology show that these proteins have unique regulation and function [15,16]. Our re-examination of cyclin E function has identified that cyclin E2 is likely to have particular role in histone regulation in breast cancer via its unique interaction with NPAT. Cyclin E2 has a strong prognostic role in breast cancer , and induces genomic instability that is associated with defects in chromosome condensation . This could be in part due to excessive histone production, as disruption of histone equilibrium is a predicted cause of genomic instability .
Our identification of multiple foci that contained only cyclin E1 or E2 indicates that there are other unique interactions. This is not surprising given that the low molecular weight derivatives of cyclin E1 also has unique binding and function in cancer cells compared to the full length protein . Future studies should carefully differentiate cyclin E1 and E2 and their isoforms, especially since each protein has unique expression patterns and their expression has distinct correlation with patient outcome in cancer .
Cell lines were authenticated by STR profiling (CellBank Australia, Westmead, NSW, Australia) and cultured for <6 months after authentication. Cyclin E1 and E2 siRNA treatment was performed and validated by western blotting as described in .
Immunoblotting and immunoprecipitation
Collection of whole cell lysates , chromatin  and sucrose gradient fractions of centrosomes  were performed as described. Lysates were separated using NuPage polyacrylamide gels (Invitrogen) prior to transfer to PVDF membranes. Western blotting, immunofluorescence and PLA antibodies are: Cdc6 (180.2), CDK2 (M2, D12), centrin-2 (S-19), cyclin E1 (HE12), estrogen receptor α (HC20), NPAT (C-19, 27), SAP145 (A-20), γ-tubulin (C-11) (Santa Cruz Biotechnology); cyclin E2 (Epitomics); p21 (610234) and p27 (610242) (BD Biosciences); αGST . Immunoprecipitation antibodies are: CDK2 (C-19), cyclin E1 (C-19), NPAT (C-19, 27), non-immune IgG (Santa Cruz Biotechnology), and cyclin E2 (Epitomics). Specificity of cyclin E1 and E2 antibodies was demonstrated in [15,19]. Additionally, we show specific loss of cyclin E1 and cyclin E2 immunofluorescence signal with siRNA treatment to cyclin E1 (Additional file 4) and cyclin E2 (Figure 3).
Immunofluorescence and microscopy
Cells were fixed with 4% PFA/PBS for 20 min at room temperature, with or without methanol post-fixation ( -20°C for 20 min). Samples were blocked with 1% BSA/PBS, stained with the indicated antibodies and counterstained with ToPro3/DAPI (Jackson ImmunoResearch Laboratories). Co-localisation was quantitated by detecting overlapping pixels with Imaris v8.0 (Bitplane) and analysed with Pearson’s Correlation Coefficient . For PLA, PFA fixed cells were subjected to the Duolink Proximity Ligation Assay (Sigma) as described by the manufacturer. Confocal microscopy was performed on Leica DMRBE/DMIRE2. Images were analysed with Imaris where individual spots were defined with a variable and initial size estimate of 0.5 μm. Images were processed with Adobe Photoshop, and adjusted for optimal brightness/contrast. Minimal gamma changes were made to enable visualisation of overlaid signals.
Expression values in 526 breast cancer samples of CCNE1, CCNE2 and representative replication-dependent and -independent histones (Additional file 5) were accessed from the cBioPortal  using the CGDSR package  in R . For each sample the number of histones with high expression (> median across patients) was established for histone subsets. Samples were grouped according to the number of histones having above median expression. For each group, the expression level of CCNE1 and CCNE2 was normalised to 100% of the median expression in the patient group with zero highly expressed histones.
Cyclin dependent kinase 2
Histone Locus Body
Proximity Ligation Assay
The Cancer Genome Atlas
We gratefully acknowledge the assistance of Dr Marco Nousch in the collection of centrosome fractions derived from the sucrose gradients. BSG and CEC are supported by Cancer Institute NSW Fellowships and AB is a Cancer Institute NSW Future Leader Fellow. EAM was suppported by a Cancer Institute NSW Fellowship and is now supported by Cancer Research UK (C596/A18076).
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