Activation of PDGFR and EGFR promotes the acquisition of a stem cell-like phenotype in schwannomas.

OBJECTIVES: Vestibular schwannomas (VS) are benign tumors that arise from unregulated growth of Schwann cells. Both benign and malignant tumors are believed to contain tumor stem cells that are hypothesized to originate from dysregulation of tumor suppressors and oncogenes. We aimed to determine if schwannoma cells express stem cell genes and markers and if activation of the proto-oncogenes epidermal growth factor receptor and platelet-derived growth factor receptor would regulate the stem cell properties of these cells. METHODS: Immunohistochemical staining was used to determine the expression of stem cell genes in archived VS tissue, immunofluorescence was used to investigate the expression in cell lines, and Western blot analysis was used to measure PDGFR expression in vestibular schwannoma tissue. Upon activation of PDGFR or EGFR in schwannoma cell lines using specific ligands, flow cytometry was used to quantify the side population (SP), stem cell genes were measured using quantitative PCR, and tumorsphere-forming ability was determined. RESULTS: Stem cell genes are expressed in vestibular schwannoma tissue and schwannoma cell lines. Activation of both EGFR and PDGFR resulted in increase in the induction of the expression of the stem cell genes Oct-4 and Nanog and marked increase in tumorsphere-forming ability, but only PDGFR activation resulted in an increase in the side population in JS1 cells. CONCLUSION: Dysregulation of EGFR and PDGFR promotes the acquisition of a stem cell-like phenotype in schwannnoma cells that may be critical in vestibular schwannoma tumorigenesis.

Merlin knockdown in human Schwann cells: clues to vestibular schwannoma tumorigenesis.

HYPOTHESIS: To investigate the early events in molecular progression toward schwannoma tumorigenesis, we developed an in vitro model of human Schwann cell tumorigenesis by merlin knockdown. BACKGROUND: Neurofibromatosis 2 (NF2)-related and sporadic vestibular schwannoma (VS) exhibit loss of functional merlin (schwannomin). After loss of merlin expression in the Schwann cell, the initial steps toward VS tumorigenesis are unknown. Merlin, a putative tumor suppressor protein, interacts with many cellular proteins, regulating their function. Among these are receptor tyrosine kinases, including the epidermal growth factor receptor family B (ErbB) family receptors epidermal growth factor receptor and ErbB2. Functional merlin interacts with and internalizes these growth factor receptors, silencing their proliferation and survival signaling. Deregulation of CD44, the cell adhesion/signaling molecule and cancer stem cell marker, has also been implicated in VS tumorigenesis. METHODS: Merlin knockdown was performed using small interfering RNA transfection into human Schwann cell primary cultures. Knockdown was confirmed by real-time quantitative PCR, immunofluorescence, and Western analysis. Expression profiles of ErbB, merlin, and the stem cell markers nestin and CD44 were examined in knockdowns. Proliferation rate was assessed with bromodeoxyuridine incorporation, and radiation sensitivity was assessed using the Annexin assay in knockdowns versus controls. RESULTS: Merlin knockdowns demonstrated increased proliferation rate, upregulation of epidermal growth factor receptor, ErbB2, and ErbB3, CD44, and nestin. Short-term merlin depletion had no effect on gamma irradiation sensitivity compared with controls. CONCLUSION: Merlin depletion results in deregulation of ErbB receptor signaling, promotes a dedifferentiated state, and increases Schwann cell proliferation, suggesting critical steps toward schwannoma tumorigenesis.