PTEN activity could be a predictive marker of trastuzumab efficacy in the treatment of ErbB2-overexpressing breast cancer

Abstract

Trastuzumab is the only HER2/neu-directed therapy to have received Food and Drug Administration approval for the treatment of patients with metastatic breast cancer. The efficacy of trastuzumab depends on the HER2/neu status of the tumour and the patient's prior treatment, but even when patients are selected on the basis of HER2/neu gene amplification, the single-agent response rate ranges from 12 to 30% and few patients respond to trastuzumab monotherapy. Here, we propose PTEN as a predictive biomarker for trastuzumab efficacy. Human breast cancer SKBR3 and drug-resistant SKBR3/R cells were investigated. We also examined clinical samples from patients who had been treated with trastuzumab and analysed the relationship between trastuzumab efficacy and PTEN level. The PI3K/Akt signalling pathway was observed to be highly active in the drug-resistant cells, and their level of PTEN was low. Delivery of antisense PTEN duplex siRNA significantly decreased the trastuzumab chemosensitivity of parental SKBR3 cells, and marked activation of Akt signalling pathway was also recognised. Moreover, immunohistochemical investigation revealed that trastuzumab treatment was remarkably successful in cells with elevated PTEN expression. Along with the immune-system-associated cytotoxic mechanism, several mechanisms have been proposed for the effect of trastuzumab. PTEN activity might play an important and major role in its HER2/PI3K/Akt-mediated antitumour effect, and could be a useful biomarker for predicting the efficacy of trastuzumab in the treatment of breast cancer.

Figure 1

(A) Growth inhibition assays revealed that chemosensitivity to trastuzumab was decreased in drug-resistant SKBR3/4R and /8R cells (120 h incubation with trastuzumab after 24 h incubation with medium). (B) Chemosensitivity of paclitaxel was not significantly different in drug-resistant and parental SKBR3 cells (12 h incubation with paclitaxel after 24 h incubation with medium).

Figure 2

The IC₅₀ value of trastuzumab in drug-resistant SKBR3/4R cells was statistically significantly higher than in parental cells.

Figure 3

(A) Western blotting analysis revealed that the level of expression of the ErbB2/PI3K/Akt signal transduction pathway is the same in trastuzumab-resistant and parental cells. (B) Phosphorylated Akt activity was increased in drug-resistant SKBR3/R cells; PTEN expression was decreased in these cells.

Figure 4

Western blotting analysis of antisense-delivered SKBR3/WT cells showed that expression of PTEN was inhibited by duplex antisense-siRNA delivery.

Figure 5

The growth inhibition curve of duplex antisense-siRNA-delivered SKBR3/WT cells shows decreased trastuzumab sensitivity (120 h incubation of trastuzumab 72 h postplating).

Figure 6

Western blotting analysis of antisense-delivered SKBR3/WT cells showed that expression of phosphorylated Akt was increased. This suggests that Akt activity is partly due to the level of PTEN in ErbB2-overexpressing SKBR3 cells.

Figure 7

The growth inhibition rate of paclitaxel in antisense-delivered SKBR3/WT cells. No significant difference was observed for the growth inhibition effect of paclitaxel between antisense- and control oligonucleotide-delivered cells (12 h incubation of paclitaxel 72 h postplating).

Figure 8

PTEN expression pattern in human breast tumours. ErbB2-overexpressing primary breast carcinomas from 17 patients who subsequently received trastuzumab plus pacliataxel were analysed. PTEN expression of these tumours was examined by IHC and semiquantified using immunoreactive scores. Representative tumour PTEN stainings (IRS 3+, 2+, 1+, and −).

Figure 9

Immunohistochemical investigation of PTEN expression from patients with erbB2-overexpressing breast cancer treated with trastuzumab. We analysed 17 breast cancer tissue samples by immunohistochemistry. In those cases with increased PTEN expression (3+, 2+), the efficacy of trastuzumab-containing chemotherapy was remarkable and the response rate statistically significant (statistical analysis was performed with Fisher's test).