The prognostic significance of Cdc6 and Cdt1 in breast cancer

Abstract

DNA replication is a critical step in cell proliferation. Overexpression of MCM2-7 genes correlated with poor prognosis in breast cancer patients. However, the roles of Cdc6 and Cdt1, which work with MCMs to regulate DNA replication, in breast cancers are largely unknown. In the present study, we have shown that the expression levels of Cdc6 and Cdt1 were both significantly correlated with an increasing number of MCM2-7 genes overexpression. Both Cdc6 and Cdt1, when expressed in a high level, alone or in combination, were significantly associated with poorer survival in the breast cancer patient cohort (n = 1441). In line with this finding, the expression of Cdc6 and Cdt1 was upregulated in breast cancer cells compared to normal breast epithelial cells. Expression of Cdc6 and Cdt1 was significantly higher in ER negative breast cancer, and was suppressed when ER signalling was inhibited either by tamoxifen in vitro or letrozole in human subjects. Importantly, breast cancer patients who responded to letrozole expressed significantly lower Cdc6 than those patients who did not respond. Our results suggest that Cdc6 is a potential prognostic marker and therapeutic target in breast cancer patients.

Figure 1

The expression levels of Cdc6, Cdt1 and Orc1 in breast cancer patients expressing various levels of MCM genes. Histograms showing percentage of specimens that expressed different levels of (A) Cdc6, (B) Cdt1 and (C) Orc1 in six groups of specimens stratified by the number of MCM2-7 genes they overexpress.

Figure 2

The associations between the expression levels of Cdc6, Cdt1 and Orc1 in breast cancer specimens. Histograms showing percentage of specimens that expressed different levels of (A) Cdc6 vs. Cdt1, (B) Cdc6 vs. Orc1 and (C) Cdt1 and Orc1, in breast cancer specimens.

Figure 3

Survival analysis of patients who express different levels of Cdc6, Cdt1 and Orc1. Kaplan-Meier curves showing survival time of patients who express different levels of (A) Cdc6, (B), Cdt1 and (C) Orc1 in the combined breast cancer patient cohort. (D) Kaplan Meier curves showing survival time of patients who express different levels of Cdc6 and Cdt1 analyzed in combination in the whole breast cancer patient cohort. Kaplan Meier curves showing survival time of patients expressing different levels of Cdc6 in breast cancer cohort stratified based on the number of MCM genes they overexpress (E ≤ 3 MCMs overexpressed) and (F ≥ 4 MCMs overexpressed).

Figure 4

The association between expression of Cdc6, Cdt1 and Orc1 with different ER status and survival. Histograms showing the percentage of patients with various expression levels of (A) Cdc6, (B) Cdt1 and (C) Orc1 in patients stratified by ER status. Kaplan-Meier curves showing survival time of patients who express different levels of Cdc6 in patients with (D) ER-negative breast cancer and (E) ER-positive breast cancer.

Figure 5

The expression of Cdc6 and Cdt1 in breast cell lines. Histograms showing (A) Cdc6 and (B) Cdt1 mRNA expression as measured by qPCR in normal breast epithelial cell line MCF10A, and two breast cancer cell lines, MCF7 and MDA MB231 in medium without Fetal Bovine Serum (FBS). (C) Western blot analysis of Cdc6, Cdt1 and β-actin in MCF10A, MCF7 and MDA MB231 cells in normal culture media (left panel) and in media without Fetal Bovine Serum (FBS; right panel).

Figure 6

The expression of Cdc6 and Cdt1 upon inhibition of ER signalling. (A) Histogram and (B) Western blot showing the expression of Cdc6 in MCF7 cells treated with tamoxifen at 0, 12 and 24 hours post-treatment. (C) Histogram and (D) Western blot showing the expression of Cdt1 in MCF7 cells treated with tamoxifen at 0, 12 and 24 hours post-treatment.

Figure 7

The expression of Cdc6 and Cdt1 in breast cancer specimens treated with letrozole. Error plots showing the expression levels of (A) Cdc6 and (B) Cdt1 in breast cancer specimens pre- and post-treatment with letrozole. Error plots showing the impact of ER signalling inhibition on the expression of Cdc6 in breast cancer specimens (C) pre-treatment and (D) post-treatment with letrozole.