Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

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.

Effects of temozolomide (TMZ) on the expression and interaction of heat shock proteins (HSPs) and DNA repair proteins in human malignant glioma cells

“…On the basis of these results, considering the particular biological behavior observed in Gli36 and since little is known on this line (Han et al 2013;Verreault et al 2013), we focused our studies in these cells. Surprisingly, a nuclear colocalization between HSPB1 and MSH2 was observed before and after TMZ treatment in a minor subpopulation of Gli36 cells when they were examined by confocal microscopy (Fig.…”

The Potential and Challenges of siRNA-Based Targeted Therapy for Treatment of Patients with Glioblastoma

“…The most striking evidence of the validity of this combined silencing came from the in vivo aspect of this study, which was done by intracranial inoculation in mice brains of U251MG cells expressing small hairpin RNA (shRNA) specific to each target. Silencing of EGFR or Rictor alone had no significant effect on tumor growth, but the dual silencing resulted in the eradication of the tumor (Verreault et al, 2011a). Also, tumor growth block in response to the combined suppression of EGFR and PI3K/AKT pathway was reported previously using the SMIs gefitinib and LY294002 in a GBM xenograft model (Fan et al, 2003), while monotherapy of each inhibitor had no impact on tumor burden.…”

“…Rictor and EGFR proteins were silenced alone and in combination by siRNA in vitro transfection in a panel of three human GBM lines (U251MG, U118MG and LN229). It was found that the co-silencing of Rictor and EGFR exerted effects on cell migration and sensitivity to chemotherapeutic drugs that were not observed by the single silencing of either target (Verreault et al, 2011a). The most striking evidence of the validity of this combined silencing came from the in vivo aspect of this study, which was done by intracranial inoculation in mice brains of U251MG cells expressing small hairpin RNA (shRNA) specific to each target.…”

“…The therapeutic value of targeting two different pathways is exemplified by some research data obtained by our laboratory (Verreault et al, 2011a). One of the most commonly reported molecular defects in GBM is the aberrant activation of the PI3K/AKT pathway, which is associated with increased proliferation rate, invasion, metastasis and poor prognosis (Ermoian et al, 2002;Haas-Kogan et al, 1998;Li X. Y. et al, 2010).…”

Overexpression of Rictor protein in colorectal cancer is correlated with tumor progression and prognosis

“…Therefore, identifying therapeutic targets for CRC and improving the 5-year survival rate of patients with CRC has important theoretical and practical significance. Rictor, as a member of mTORC2, is important in the process of tumor proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) and poor prognosis (12)(13)(14)(15)(16). For example, Akt activated by Rictor phosphorylation induced the expression of c-Myc and cyclin E, and this process could regulate the proliferation and cell cycle of colon cancer cells.…”