Role of the Cdc25A phosphatase in human breast cancer

Abstract

The phosphatase Cdc25A plays an important role in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases, and it has been shown to transform diploid murine fibroblasts in cooperation with activated Ras. Here we show that Cdc25A is overexpressed in primary breast tumors and that such overexpression is correlated with higher levels of cyclin-dependent kinase 2 (Cdk2) enzymatic activity in vivo. Furthermore, in the breast cancer cell line MCF-7, Cdc25A activity is necessary for both the activation of Cdk2 and the subsequent induction of S-phase entry. Finally, in a series of small (< 1 cm) breast carcinomas, overexpression of Cdc25A was found in 47% of patients and was associated with poor survival. These data suggest that overexpression of Cdc25A contributes to the biological behavior of primary breast tumors and that both Cdc25A and Cdk2 are suitable therapeutic targets in early-stage breast cancer.

Figure 1

Cdc25A expression levels are directly correlated with Cdk2 activity. (a) Cdc25A and Gsα mRNA levels were determined by semiquantitative RT-PCR analysis (upper panel) on microdissected frozen tissue samples of breast carcinomas and on MCF-7 cells. Four representative cases of high expressers (lanes 1 and 2) and low expressers (lanes 3 and 4) for Cdc25A are shown. Cdk2 enzymatic activity and Cdk2 protein level for the four tissue samples of breast carcinoma (lanes 1–4), for MCF-7 cells, and for the control sample HeLa cells are shown in the lower panel. A direct correlation between Cdc25A levels and Cdk2 enzymatic activity is evident, whereas Cdk2 levels remain unchanged. (b) Bar graph representing cumulative data of Cdc25A levels compared with Cdk2 enzymatic activity in 21 patients. The ratio of the densitometric values of Cdc25A to those of Gsα was calculated, and the median value was used as a cutoff for the categorization of cases into high and low/nonexpressers. High expressers (+) and low/nonexpressers (–) for Cdc25A were correlated with the corresponding Cdk2 enzymatic activity. To normalize densitometric values of Cdk2 activity, a ratio of the percent area of the tested samples to the one obtained with the HeLa control in each gel was used. To account for variability from gel to gel, this ratio was, in turn, related as a ratio to the total area under all curves in a given gel.

Figure 2

Cdc25A is overexpressed in human breast cancer. (a) Hematoxylin-and-eosin stain of invasive ductal carcinoma of the breast (T). Normal ducts are seen in the lower left corner (N). (b) In situ hybridization with digoxigenin-labeled, antisense riboprobe for Cdc25A demonstrates high expression levels of the phosphatase in tumor cells. Scant to no expression is seen in the adjacent normal breast tissue. (c) Representative example of a nonexpresser: no expression of Cdc25A is seen in the infiltrating ductal carcinoma cells. Scattered residual normal ducts (N) show weak expression of the phosphatase. (d) Representative example of a low expresser for Cdc25A: most infiltrating ductal carcinoma cells do not express Cdc25A mRNA. Rare ducts (arrowhead) show weak expression of the phosphatase. (e and f) Representative examples of high expressers for cdc25A: all infiltrating ductal carcinoma cells show strong staining with the antisense riboprobe for Cdc25A (Panels b, c, d, e, and f: alkaline phosphatase: nitro blue tetrazolium cytoplasmic reaction product, section counterstained with methyl green). Sense controls were negative (not shown).

Figure 3

(a and b) Antisense oligonucleotides directed to Cdc25A decrease Cdk2 activity and inhibit S-phase entry in MCF-7 cells. MCF-7 cells transfected with antisense (AS) or scrambled (S) oligonucleotides for Cdc25A were analyzed at 24 hours and 48 hours after transfection. Untransfected, proliferating MCF-7 cells represent base-line values (Control). (a) Immunoblot and RT-PCR for Cdc25A show significant decreases in Cdc25A protein and mRNA levels at 24 hours and 48 hours after transfection with AS but not S oligonucleotides. The bar graph is the densitometric quantitation of Cdc25A protein levels in three separate experiments. A 61% decrease in Cdc25A protein level was noted at 48 hours following AS transfection. No change is noted in Gsα mRNA levels after transfection. (b) Histone H1 and Rb kinases for Cdk2 and Cdk4 after immunoprecipitation with the respective antibodies and corresponding immunoblot. Cdk2 enzymatic activity decreases by 73% at 24 hours and by 88% at 48 hours after AS but not S transfection. In contrast, Cdk4 activity remains unchanged. No change in either Cdk2 or Cdk4 protein levels (Cdk2 WB and Cdk4 WB) is seen. WB, Western blot. (c and d) Antisense oligonucleotides against Cdc25A result in increased Cdk2 phosphorylation and decreased complexed p27. MCF-7 cells were transfected with antisense (AS) or scrambled (S) CDC25A oligonucleotides and analyzed 48 hours after transfection. Untransfected proliferating MCF-7 cells represent base-line values (Control). (c) Immunoprecipitated lysates with unrelated rabbit IgG (left) and Cdk2 were blotted with phosphotyrosine, Cdk2, cyclin E, and p27 antibodies. AS CDC25A results in a marked increase in Cdk2 phosphorylation. Concomitant to this, a decrease in complexed p27 is seen. (d) Total lysate immunoblots for Cdk2, cyclin E, and p27. No change in total levels of Cdk2, cyclin E, and p27 is seen.

Figure 4

Inactivation of Cdc25A inhibits S-phase entry in breast carcinoma cells. (a) S phase in MCF-7 cells is measured by FACS analysis of BrdU-stained cells. Antisense oligonucleotides inhibit S-phase entry by 31% at 24 hours and 43% at 48 hours after AS transfection. No change is noted after transfection with scrambled oligonucleotides. S-phase entry is restored by the cotransfection of the nonphosphorylatable mutant of Cdk2, Cdk2AF, and antisense oligonucleotides to Cdc25A. (b) Nonphosphorylatable Cdk2 catalytic mutant (Cdk2AF) abolishes Cdc25A antisense effects on MCF-7. MCF-7 cells were transfected with antisense Cdc25A (AS), wild-type Cdk2 (Cdk2WT), or Cdk2AF, or cotransfected with AS and Cdk2AF or AS and Cdk2WT. Untransfected proliferating MCF-7 cells represent base-line values of Cdk2 activity and S-phase fraction (Control). At 48 hours after transfection, Cdk2 kinase activity (a) and BrdU incorporation as a measure of S-phase fraction (b) show that Cdk2AF, but not Cdk2WT, abolishes the Cdc25A AS effect.

Figure 5

Cdc25A overexpression, with or without concomitant loss of p27, is associated with poor survival. (a) High levels of Cdc25A are associated with decreased disease-free survival in the 144 patients studied. (b) When Cdc25A is analyzed together with p27, stratification of disease-related mortality risk is based on four different combinations of Cdc25A and p27 levels. The group with the highest mortality is the one in which breast carcinomas overexpress Cdc25A and have low p27 levels (black curve).