Expression of Erk5 in early stage breast cancer and association with disease free survival identifies this kinase as a potential therapeutic target

Abstract

Background: Breast cancer is the most common neoplasia in women. Even though advances in its treatment have improved disease outcome, some patients relapse. Therefore, attempts to better define the molecular determinants that drive breast cancer cell proliferation may help in defining potential therapeutic targets. Mitogen-activated protein kinases (MAPK) play important roles in tumorigenesis. One of them, Erk5, has been linked to the proliferation of breast cancer cells in vitro. Here we have investigated the expression and prognostic value of Erk5 in human breast cancer.

Methodology/principal findings: Animal and cellular models were used to study Erk5 expression and function in breast cancer. In 84 human breast tumours the expression of Erk5 was analyzed by immunohistochemistry. Active Erk5 (pErk5) was studied by Western blotting. Correlation of Erk5 with clinicopathological parameters and with disease-free survival in early stage breast cancer patients was analyzed. Expression of Erk5 was detected in most patients, and overexpression was found in 20%. Active Erk5 was present in a substantial number of samples, as well as in tumours from an animal breast cancer model. Overexpression of Erk5 was associated with a decrease in disease-free survival time, which was independent of other clinicopathological parameters of prognosis. Transient transfection of a short hairpin RNA (shRNA) targeting Erk5, and a stable cell line expressing a dominant negative form of Erk5 (Erk5(AEF)), were used to investigate the influence of Erk5 on drugs used in the clinic to treat breast tumours. We found that inhibition of Erk5 decreased cancer cell proliferation and also sensitized these cells to the action of anti-HER2 therapies.

Conclusions/significance: Overexpression of Erk5 is an independent predictor of disease-free survival in breast cancer, and may represent a future therapeutic target.

Figure 1. Patterns of Erk5 staining in human breast cancer.

Immunohistochemical staining of Erk5, showing samples scored as low (panel A), or high expression level (panel B). Two patterns of Erk5 staining in breast cancer are shown in panels C and D. Magnification: for A and B, 200×; for C and D, 1000×.

Figure 2. Molecular forms of Erk5 in human breast cancer.

A, Schematic representation of Erk5 depicting the region where it is dually phosphorylated at the TEY microdomain. B, Identification of the bands recognized by the anti-Erk5 antibodies in BT474 cells, and in the breast cancer samples BT49, and BT1080. The immunoprecipitates and Westerns were probed with the indicated antibodies. Some samples were immunoprecipitated with the antibodies but in the presence of an excess of the peptide used for the raising of the antibody, the peptide excess prevented immunoprecipitation of both Erk5 bands. C, Molecular forms of Erk5 detected in breast cancer samples, and expression of HER2 and pHER2. Calnexin was used as a control for protein loading. D, Expression of Erk5, pErk5, and HER2 in the breast of transgenic (FVB/neu) tumoral breast tissue, or normal (FVB) breast tissue from mice. The amount of epithelial cytokeratins is shown.

Figure 3. Relationship between Erk5 expression and disease-free survival.

A, Kaplan-Meier plots of disease-free survival with respect to Erk5 levels in 84 early stage breast cancer patients. Patients with high levels of Erk5 (n = 17) had a worse disease-free survival (DFS) time (14.13 months; 95% CI: 3.78–24.48) compared with patients (n = 67) with low levels (34.33 months; 95% CI: 18.52–50.14). The difference was statistically significant in the log-rank test (p = 0.0014). B, Kaplan-Meier plots of disease-free survival with respect to Erk5 levels in 24 HER2 positive early stage breast cancer patients. Patients with high levels of Erk5 (n = 8) had a worse disease-free survival (DFS) time (9.70 months; 95% CI: 6.58–12.95) compared to patients (n = 16) with low levels (28 months; 95% CI: 15.53–41.40). The difference was statistically significant in the log-rank test (p = 0.012).

Figure 4. Inhibition of Erk5 sensitizes to the action of Trastuzumab.

A, Action of lapatinib and trastuzumab on Erk5, pErk5, and pHER2 in BT474 cells. The drugs were added for 24 hours and then cell extracts prepared to be immunoprecipitated with anti-Erk5, anti-pErk5, or anti-HER2. B, Effect of lapatinib (100 nM) or trastuzumab (10 nM) on the MTT uptake of BT474 cells. The results are reported as the mean±SD of quadruplicates. C, Action of Erk5^AEF or Erk5 shRNA on cell proliferation. Erk5^AEF expression was measured by Western blot using an anti-HA tag antibody, as the Erk5^AEF form is tagged with an HA epitope. GAPDH was used as a control for protein loading. BT474 cells transfected with the indicated vectors were treated with trastuzumab and their proliferation measured using MTT uptake.