DNA replication licensing and cell cycle kinetics of normal and neoplastic breast

Abstract

Mcm2 -7 (MCM) proteins are part of the origin licensing machinery that regulates initiation of DNA replication. Geminin is a licensing repressor and prevents reinitiation of DNA replication during S -G2 -M phase by blocking reloading of Mcm2 -7 at replication origins. Here, we have analysed these replication licensing factors (RLFs) to determine whether the pathway becomes deregulated during mammary carcinogenesis, and have assessed their potential value as prognostic markers. Protein expression profiles were generated for Ki67, Mcm2, geminin, HER-2, ER and PR in a series of reduction mammoplasty (n ¼ 18) and breast cancer specimens (n ¼ 120), and compared to clinicopathological parameters. A large proportion of epithelial cells of the terminal duct lobular unit reside in a primed 'replication licensed' but not proliferating state. This state is characterised by Mcm2 expression and absence of Ki67 and the S/G2/M marker geminin. In breast cancers, increasing tumour grade is associated with increased Ki67, Mcm2 and geminin expression. The Mcm2/Ki67 ratio decreases through the grades, indicating a shift from a predominantly licensed state to an actively proliferating state. This shift is associated with an increase in the geminin/Ki67 ratio, signifying a shortening of G1 phase in breast cancer cells. Ki67, Mcm2 and the Mcm2/Ki67 ratio are statistically significantly associated with the Nottingham Prognostic Index (NPI), but geminin and the geminin/Ki67 ratio are not. Ki67, Mcm2 and Mcm2/Ki67 are highly correlated with one another, with Mcm2 being the single most important predictor of NPI score (Po0.001). However, only 12% of variation in NPI is explained by Mcm2, as the labelling index for this marker is approaching 100% for many of the high-grade tumours. The origin licensing phenotypes of normal breast and breast cancers therefore relate to their cellular differentiation status, and high-level MCM expression in more poorly differentiated tumours severely constrains their use as prognostic markers in breast cancer.

Mitogenic growth signalling, DNA replication licensing, and survival are linked in prostate cancer

“…The largest growth fraction is identified by Mcm2, reflecting identification of both cycling and non-cycling cells with proliferative potential. The lower growth fraction identified by geminin compared with Ki67 is in keeping with its expression being restricted to S-G2-M phase Wharton et al, 2004;Dudderidge et al, 2005;Shetty et al, 2005). In vitro, increased MEK5/ERK5 signalling leads to increased Mcm2, geminin, and Ki67 protein levels.…”

“…RLFs, which form the core of the initiation machinery, act as relay stations coupling growth regulatory and DNA damage response pathways to chromosomal replication. We and others have demonstrated that dysregulation of the replication Ki67 Geminin P C 3 -P a r e n t a l P C 3 -e m p t y v e c t o r P C 3 -E R K 5 P C 3 -P a r e n t a l P C 3 -e m p t y v e c t o r initiation machinery is an early event in tumourigenesis and that the Mcm2-7 RLFs are powerful diagnostic and prognostic markers in a wide range of tumour types (Williams et al, 1998Stoeber et al, 1999;Wharton et al, 2001Wharton et al, , 2004Davies et al, 2002;Going et al, 2002;Stoeber et al, 2002;Gonzalez et al, 2003;Kruger et al, 2003;Padmanabhan et al, 2004;Dudderidge et al, 2005;Korkolopoulou et al, 2005;Shetty et al, 2005). Recent reports have shown that the MEK5/ERK5 pathway is implicated in the regulation of cell proliferation, and that increased MEK5/ERK5 signalling results in increased cell proliferation, invasion and metastatic spread during prostate carcinogenesis Mehta et al, 2003).…”

“…These MCM but not Ki67-expressing cells can be found, for example, in resting tissues such as premenopausal breast or primary oocytes, which retain proliferative capacity and can rapidly respond to growth stimuli Eward et al, 2004). Their failure to initiate DNA synthesis and progress into S-G2-M phase is further reflected by lack of geminin expression (Shetty et al, 2005). Expression profiling of normal prostate shows only occasional basal Ki67, Mcm2, and geminin-positive cells, in keeping with repression of origin licensing during engagement of the differentiation programme in human tissues Eward et al, 2004).…”

“…The Mcm2 LI minus Ki67 LI (Mcm2ÀKi67) identifies the replication licensed but non-proliferating population of tumour cells, whereas the Ki67 LI minus geminin LI (Ki67Àgeminin) represents the proportion of tumour cells in G1 phase Dudderidge et al, 2005;Obermann et al, 2005). Geminin identifies the proportion of tumour cells traversing through S-G2-M phase, and therefore the geminin LI to Ki67 LI ratio (geminin/ Ki67) provides a measure of the relative length of G1 in dynamic cell populations, with higher geminin/Ki67 ratios corresponding to a shortening in the length of G1 phase Wharton et al, 2004;Shetty et al, 2005). In this study, a statistically significant link between increasing Gleason grade and Ki67-geminin (P ¼ 0.001; Table 1) was observed, indicating an increase in the proportion of cells entering G1.…”

“…The constituents of the pre-RC can be regarded as relay stations coupling growth regulatory pathways with DNA replication . We and others have demonstrated in a range of different tumour types that dysregulation of the MCM proteins is an early event in tumourigenesis and have exploited these biomarkers in primary diagnosis, tumour surveillance and prognosis (Williams et al, 1998Stoeber et al, 1999Stoeber et al, , 2002Wharton et al, 2001Wharton et al, , 2004Davies et al, 2002;Going et al, 2002;Gonzalez et al, 2003;Kruger et al, 2003;Padmanabhan et al, 2004;Dudderidge et al, 2005;Korkolopoulou et al, 2005;Shetty et al, 2005). These studies have established that the superior sensitivity of MCM proteins over the standard proliferation marker Ki67 resides in the fact that these biomarkers identify not only cycling cells but also non-cycling cells with proliferative potential ).…”