Prolyl isomerase Pin1 is highly expressed in Her2-positive breast cancer and regulates erbB2 protein stability

Abstract

Overexpression of HER-2/Neu occurs in about 25-30% of breast cancer patients and is indicative of poor prognosis. While Her2/Neu overexpression is primarily a result of erbB2 amplification, it has recently been recognized that erbB2 levels are also regulated on the protein level. However, factors that regulate Her2/Neu protein stability are less well understood. The prolyl isomerase Pin1 catalyzes the isomerization of specific pSer/Thr-Pro motifs that have been phosphorylated in response to mitogenic signaling. We have previously reported that Pin1-catalyzed post-phosphorylational modification of signal transduction modulates the oncogenic pathways downstream from c-neu. The goal of this study was to examine the expression of prolyl isomerase Pin1 in human Her2+ breast cancer, and to study if Pin1 affects the expression of Her2/Neu itself.

Methods: Immunohistochemistry for Her2 and Pin1 were performed on two hundred twenty-three human breast cancers, with 59% of the specimen from primary cancers and 41% from metastatic sites. Pin1 inhibition was achieved using siRNA in Her2+ breast cancer cell lines, and its effects were studied using cell viability assays, immunoblotting and immunofluorescence.

Results: Sixty-four samples (28.7%) stained positive for Her2 (IHC 3+), and 54% (122/223) of all breast cancers stained positive for Pin1. Of the Her2-positive cancers 40 (62.5%) were also Pin1-positive, based on strong nuclear or nuclear and cytoplasmic staining. Inhibition of Pin1 via RNAi resulted in significant suppression of Her2-positive tumor cell growth in BT474, SKBR3 and AU565 cells. Pin1 inhibition greatly increased the sensitivity of Her2-positive breast cancer cells to the mTOR inhibitor Rapamycin, while it did not increase their sensitivity to Trastuzumab, suggesting that Pin1 might act on Her2 signaling. We found that Pin1 interacted with the protein complex that contains ubiquitinated erbB2 and that Pin1 inhibition accelerated erbB2 degradation, which could be prevented by treatments with the proteasome inhibitor ALLnL.

Conclusion: Pin1 is a novel regulator of erbB2 that modulates the ubiquitin-mediated degradation of erbB2. The overexpression of Pin1 in a majority of Her2-overexpressing breast cancer may contribute to maintain erbB2 levels. Pin1 inhibition alone and in conjunction with mTOR inhibition suppresses the growth of Her2+ breast cancer cells.

Figure 1

Examples of immunohistochemistry stains for Pin1 and Her2 on 3 individual tumors (in rows A through F), IHC for Her2 (A, B, C) and Pin1 (D, E, F) performed using standard procedures on consecutive slides.

Figure 2

Pin1 depletion inhibits the growth of Her2+ breast cancer cell lines. Cells were transfected with control RNAi (black bars) or Pin1 siRNA (grey bars), and seeded on day 3 after transfection at 5000 cells/well in 96-well plates. Cells were allowed to grow and the resulting cultures were subjected to an MTT-based viability assay and read in a 96 well reader after 7 days (A) or at the indicated time points (B). Assays were done in triplicates.

Figure 3

Pin1 depletion sensitizes erbB2-amplified cells to Rapamycin but not to Trastuzumab treatments. SKBR3 cells (A, D), BT474 cells (B, E) and AU565 cells (C, F) were transfected with control RNAi (dark symbols) or Pin1 siRNA (open symbols), and seeded on day 3 after transfection at 5000 cells/well in 96-well plates, and treated with the indicated concentrations of Rapamycin or Trastuzumab. Cells were allowed to grow for 5 days and the resulting cultures were subjected to an MTT-based viability assay and read in a 96 well reader. Assays were done in triplicates.

Figure 4

Pin1 inhibition leads to the down-regulation of erbB2. A. Immunofluorescence of BT474 or AU565 cells stained with anti-Pin1 (red), anti Her2 (green) and nuclear DAPI stain, and a merged image. Cells were analyzed with a Zeiss Confocal Microscope 3 days after transfection of either control or Pin1 siRNA. B, C Immunoblotting of Her2 in cells transfected with Pin1 siRNA or control siRNA (B), or dominant-negative Pin1 (S16A mutation) or control vector (C). Actin levels (lower panel) remained stable while Pin1 levels decreased in response to siRNA treatment (B, middle panel) or increased after DN Pin1 expression (C, middle panel). Her2 levels decreased both in response to Pin1 siRNA treatment (B, upper panel) and in response to expression of DN Pin1 (C, upper panel). Cells were transfected with either vector or the mutant, and lysates collected for immunoblotting 72 hrs later.

Figure 5

Decreased stability of Her2 after Pin1 inhibition. Her2-positive breast cancer cells (SKBR3) were treated with Pin1 control or siRNA for 3 days, and then treated with Cycloheximide at 100 mcg/ml, and lysates were obtained at the indicated time points and prepared for immunoblotting (A, B). B The intensity of the bands was quantified using ImageJ software and plotted as time versus ratio of Her2/Actin intensity for SKBR3.

Figure 6

ErbB2 degradation induced by Pin1 inhibition can be rescued by proteasome inhibition. A. SKBR3 cells were transfected with control or Pin1 siRNA for 72 hours. 6 hours prior to protein lysis cells were treated with 100 μM ALLnL. B. Cell lysates were immunoprecipitated with anti-erbB2 antibodies and immunoblotting was done using antibodies against erbB2, Ubiquitin, Tubulin and Pin1. C. SKBR3, AU565 and BT474 were transfected with control or Pin1 siRNA for 72 hours. RNA was extracted using Trizol reagent and RT-PCR was performed using erbB2 and GAPDH specific primers.