Microtubule-associated protein tau: a marker of paclitaxel sensitivity in breast cancer

Abstract

Breast cancers show variable sensitivity to paclitaxel. There is no diagnostic test to identify tumors that are sensitive to this drug. We used U133A chips to identify genes that are associated with pathologic complete response (pCR) to preoperative paclitaxel-containing chemotherapy in stage I-III breast cancer (n = 82). Tau was the most differentially expressed gene. Tumors with pCR had significantly lower (P < 0.3 x 10(-5)) mRNA expression. Tissue arrays from 122 independent but similarly treated patients were used for validation by immunohistochemistry. Seventy-four percent of pCR cases were tau protein negative; the odds ratio for pCR was 3.7 (95% confidence interval, 1.6-8.6; P = 0.0013). In multivariate analysis, nuclear grade (P < 0.01), age <50 (P = 0.03), and tau-negative status (P = 0.04) were independent predictors of pCR. Small interfering RNA experiments were performed to examine whether down-regulation of tau increases sensitivity to chemotherapy in vitro. Down-regulation of tau increased sensitivity of breast cancer cells to paclitaxel but not to epirubicin. Tubulin polymerization assay was used to assess whether tau modulates binding of paclitaxel to tubulin. Preincubation of tubulin with tau resulted in decreased paclitaxel binding and reduced paclitaxel-induced microtubule polymerization. These data suggest that low tau expression renders microtubules more vulnerable to paclitaxel and makes breast cancer cells hypersensitive to this drug. Low tau expression may be used as a marker to select patients for paclitaxel therapy. Inhibition of tau function might be exploited as a therapeutic strategy to increase sensitivity to paclitaxel.

Fig. 1.

Tau protein expression by IHC. (a-d) Tau expression in normal breast epithelium (a) and invasive breast cancer with 1+ (b), 2+ (c), and 3+ (d) staining (magnification ×40). (e) The proportion of patients with pCR and residual disease as a function of tau IHC scores (n = 122).

Fig. 2.

Tau down-regulation sensitizes ZR75.1 breast cancer cells to paclitaxel but not to epirubicin. (a and b) Baseline tau expression in 12 breast cancer cell lines (a) and in ZR75.1 cells 36-72 h after tau siRNA transfection (b). (c and d) Dose-response curves after 48-h exposure to paclitaxel (c) and epirubicin (d) in parental, lamin siRNA-transfected, and tau siRNA-transfected ZR75.1 cells indicated increased sensitivity to paclitaxel but not to epirubicin in tau knock-down cells. Error bars indicate 95% confidence intervals of triplicate measurements.

Fig. 3.

Fluorescent paclitaxel uptake is increased in tau knock-down ZR75.1 cells. (a and b) FACS analysis of cells transfected with lamin siRNA (a) and tau siRNA (b) after exposure to Oregon Green fluorescent paclitaxel. (c) The percentage of cells (±95% confidence interval) with >10 arbitrary fluorescent units 20, 50, and 80 min after incubation with 1 μM Oregon Green paclitaxel. (d and e) FACS analysis after exposure (80 min) to spontaneously fluorescent epirubicin in lamin (d) and tau (e) knocked-down cells.

Fig. 4.

Tau partially protects tubulin from maximal paclitaxel-induced polymerization in vitro.(a) Paclitaxel and tau each promoted microtubule polymerization with modest additive effect when combined. (b) Preincubation of tubulin with tau for 30 min before adding paclitaxel (20 mM) decreased the maximum paclitaxel-induced polymerization in a dose-dependent manner. (c) Fluorescence emission of BODIPY-paclitaxel is enhanced when it binds to increasing concentrations of tubulin. (d) When tubulin was preincubated with regular paclitaxel as competitor (10 and 20 mM) or with tau (15 mM) before BODIPY-paclitaxel (5 mM) was added, the increase in fluorescence was reduced, which suggests that tau inhibits BODIPY-paclitaxel binding to microtubules.