Abstract
The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.
Highlights
The median survival for patients with glioblastoma (GBM) is 41 weeks and the 2-year survival rate is less than 30% [1]
The results suggest that siRNA mediated co-silencing of epidermal growth factor receptor (EGFR) and rapamycin-insensitive companion of mTOR (Rictor) inhibits tumor cell migration in U251MG and LN229
The results demonstrate that silencing of EGFR or Rictor alone had no significant effect on tumor growth in the orthotopic U251MG GBM model, but the dual silencing of EGFR and Rictor in vivo results in eradication of the tumor
Summary
The median survival for patients with glioblastoma (GBM) is 41 weeks and the 2-year survival rate is less than 30% [1]. It is recognized that advances in GBM treatment will continue to rely on current treatment modalities, agents targeting proteins or pathways known to be critical to the progression and infiltration of GBM cells are being evaluated in patients. These agents, used alone and in combination with established treatments, will hopefully improve treatment outcomes for individuals diagnosed with this devastating cancer. Targeted therapeutics must be used in combination settings in order to prevent the activation of compensating mechanisms in tumors This approach will only be valuable for the treatment of GBM if the targeted agents are highly selective and capable of crossing the blood-brain barrier. The therapeutic potential of simultaneous silencing of the epidermal growth factor receptor (EGFR) and the rapamycin-insensitive companion of mTOR (Rictor) was assessed in vitro and in vivo and the rationale for selecting these proteins as therapeutic targets has been outlined below
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