Low levels of Stat5a protein in breast cancer are associated with tumor progression and unfavorable clinical outcomes

Abstract

Introduction: Signal transducer and activator of transcripton-5a (Stat5a) and its close homologue, Stat5b, mediate key physiological effects of prolactin and growth hormone in mammary glands. In breast cancer, loss of nuclear localized and tyrosine phosphorylated Stat5a/b is associated with poor prognosis and increased risk of antiestrogen therapy failure. Here we quantify for the first time levels of Stat5a and Stat5b over breast cancer progression, and explore their potential association with clinical outcome.

Methods: Stat5a and Stat5b protein levels were quantified in situ in breast-cancer progression material. Stat5a and Stat5b transcript levels in breast cancer were correlated with clinical outcome in 936 patients. Stat5a protein was further quantified in four archival cohorts totaling 686 patients with clinical outcome data by using multivariate models.

Results: Protein levels of Stat5a but not Stat5b were reduced in primary breast cancer and lymph node metastases compared with normal epithelia. Low tumor levels of Stat5a but not Stat5b mRNA were associated with poor prognosis. Experimentally, only limited overlap between Stat5a- and Stat5b-modulated genes was found. In two cohorts of therapy-naïve, node-negative breast cancer patients, low nuclear Stat5a protein levels were an independent marker of poor prognosis. Multivariate analysis of two cohorts treated with antiestrogen monotherapy revealed that low nuclear Stat5a levels were associated with a more than fourfold risk of unfavorable outcome.

Conclusions: Loss of Stat5a represents a new independent marker of poor prognosis in node-negative breast cancer and may be a predictor of response to antiestrogen therapy if validated in randomized clinical trials.

Figure 1

Nuclear localization of Stat5a (Nuc-Stat5a) is lost during breast cancer progression. (A) Detection of nuclear-localized Stat5a in Material I with immunofluorescence and quantified with AQUA revealed a significant reduction in Nuc-Stat5a protein in invasive ductal carcinoma (IDC; n = 66) and lymph node metastases (n = 19) when compared with normal breast tissue (n = 23) and ductal carcinoma in situ (DCIS; n = 18). (B) Levels of nuclear localized Stat5b (Nuc-Stat5b) remained unchanged between normal (n = 24), DCIS (n = 12), IDC (n = 67), and lymph node metastases (n = 13) in the same breast progression array (Material I). (C) Representative images of Stat5a, Stat5b, and pY-Stat5a/b detected with immunofluorescence in normal human breast tissue and lymph node metastases (Material I). Stat5a, Stat5b, pY-Stat5a/b, red (Cy5); cytokeratin, green (FITC); nuclei, blue (DAPI). (D) Stat5a protein (upper panels) translocated to the nucleus after ex vivo prolactin stimulation of human breast tissue explants, whereas prolactin induced only minimal nuclear localization of Stat5b protein (middle panels). Corresponding with enrichment of nuclear Stat5a protein in response to prolactin, tyrosine-phosphorylated Stat5a/b (pY-Stat5a/b) was induced in the nuclei of the same breast tissue after ex vivo stimulation with prolactin (lower panels). (E, F) Levels of total cellular Stat5a protein (E), but not total cellular Stat5b protein (F), were significantly reduced over breast cancer progression (Material I). (G, H) Stat5a mRNA expression levels in human breast tumor tissue (Material II) were associated with relapse-free survival (G), whereas Stat5b mRNA expression levels were not associated with relapse-free survival (H). Statistical differences (ANOVA, Dunnett T3 post hoc test) in levels of nuclear and total cellular Stat5a or Stat5b over breast cancer progression in relation to normal histologic type are indicated. ***p < 0.001; *p < 0.05. AQUA, Automated Quantitative Analysis; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; IQR, interquartile range; LN Met, lymph node metastasis.

Figure 2

Loss of nuclear Stat5a (Nuc-Stat5a) predicts unfavorable breast cancer prognosis. (A, B) Nuclear localization of Stat5a was detected with standard DAB IHC and pathologist review of whole-tissue sections in Material III. Kaplan-Meier analysis indicated that patients with low levels of Nuc-Stat5a had (A) reduced time-to-recurrence (TTR) of breast cancer and (B) poor breast cancer-specific survival (CSS). (C, D) Nuclear localization of immunofluorescently labeled Stat5a was quantified by AQUA analysis in a tissue microarray of node-negative breast cancer. (C) Representative immunofluorescent images of high and low Stat5a expression in invasive ductal carcinoma. (D) Quantitation of nuclear levels of Stat5a by AQUA revealed by Kaplan-Meier analysis that loss of Nuc-Stat5a is prognostic of poor breast cancer-specific survival (CSS) in Material IV. Censored cases (+) and number of patients per group are indicated.

Figure 3

Low levels of nuclear Stat5a (Nuc-Stat5a) predict poor response to antiestrogen therapy. (A, B) Nuc-Stat5a was detected by DAB-chromogen IHC and pathologist scoring of whole-tissue sections from node-negative breast cancer patients treated with antiestrogen monotherapy (Material V). The lack of nuclear localization of Stat5a predicted (A) poor breast cancer-specific survival (CSS) and (B) reduced time-to-recurrence (TTR) of breast cancer. (C, D) Nuc-Stat5a expression levels were measured with immunofluorescence and quantified with AQUA in node-negative and -positive breast cancer patients treated with antiestrogen monotherapy (Material VI). Low levels of Nuc-Stat5a were predictive of (C) poor cancer-specific survival (CSS) and (D) reduced TTR of breast cancer. Kaplan-Meier plots with censored cases (+) and number of patients per group indicated.