Impaired Pten expression in human malignant peripheral nerve sheath tumours

Abstract

Malignant peripheral nerve sheath tumours (MPNST) are aggressive sarcomas that develop in about 10% of patients with the genetic disease neurofibromatosis type 1 (NF1). Molecular alterations contributing to MPNST formation have only partially been resolved. Here we examined the role of Pten, a key regulator of the Pi3k/Akt/mTOR pathway, in human MPNST and benign neurofibromas. Immunohistochemistry showed that Pten expression was significantly lower in MPNST (n=16) than in neurofibromas (n=16) and normal nervous tissue. To elucidate potential mechanisms for Pten down-regulation or Akt/mTOR activation in MPNST we performed further experiments. Mutation analysis revealed absence of somatic mutations in PTEN (n=31) and PIK3CA (n=38). However, we found frequent PTEN promotor methylation in primary MPNST (11/26) and MPNST cell lines (7/8) but not in benign nerve sheath tumours. PTEN methylation was significantly associated with early metastasis. Moreover, we detected an inverse correlation of Pten-regulating miR-21 and Pten protein levels in MPNST cell lines. The examination of NF1-/- and NF1+/+Schwann cells and fibroblasts showed that Pten expression is not regulated by NF1. To determine the significance of Pten status for treatment with the mTOR inhibitor rapamycin we treated 5 MPNST cell lines with rapamycin. All cell lines were sensitive to rapamycin without a significant correlation to Pten levels. When rapamycin was combined with simvastatin a synergistic anti-proliferative effect was achieved. Taken together we show frequent loss/reduction of Pten expression in MPNST and provide evidence for the involvement of multiple Pten regulating mechanisms.

Figure 1. Pten expression in MPNST and neurofibroma.

a) Proportion of Pten-positive tumour cells as determined by immunohistochemistry. Each dot represents one tumour. Sporadic MPNST are depicted in grey b) MPNST 29250 with a transition to pNF. Note stronger Pten expression in the pNF (lower part). c) Higher magnification of pNF. Original magnification 200x and 400x. d) Western blot of MPNST and neurofibromas. VEGF concentrations in tumour lysates are indicated.

Figure 2. Pten expression in nerve tissue (a-c) and Schwann cell cultures (d).

a) Standard DAB staining. b) Double staining for Pten (green) and neurofilament (red). c) Double staining for Pten (green) and S100 (red). Original magnification 400x.

Figure 3. Western blot analysis of Pten and p-p70S6 kinase (isoform p-p70 of the S6 kinase, indicates mTOR activation) and examination of miR-21 expression by real time PCR a) Pten analysis of MPNST cell lines, dermal fibroblasts and neurofibroma derived Schwann cell cultures (SC1-4).

The grey line indicates mean Pten expression of 4 Schwann cell cultures. b) Quantification of Pten expression (normalized with β-actin). c) Expression of Pten and p-p70S6 kinase in NF1 positive (+/+) and NF1 negative (−/−) Schwann cells. d) Relative expression of miR-21 as determined by real time PCR. * Endogeneous control RNU44 was not detectable. This cell line was thus omitted from analysis. SC = neurofibroma derived Schwann cells.

Figure 4. Effect of rapamycin on MPNST cell lines and fibroblasts.

a) Dose dependent inhibition of proliferation after 4 days of treatment. b) Pten protein levels correlate with rapamycin sensitivity.