Experimental Neurooncology, Brain and Bone Marrow Institute Research Center, Pitie-Salpetriere Hospital, Paris, France.
PLoS One. 2013;8(3):e59597. doi: 10.1371/journal.pone.0059597. Epub 2013 Mar 15.
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.
PI3K/AKT/mTOR 通路在胶质母细胞瘤（GBM）中通常过度激活，Rictor 被证明是该通路下游的重要调节因子。表皮生长因子受体（EGFR）过度表达也经常在 GBM 肿瘤中发现，EGFR 和 Rictor 都与增殖、侵袭、转移和预后不良有关。本研究评估了 EGFR 和 Rictor 联合沉默是否会在体外和体内产生治疗益处。还评估了针对这些蛋白与在 GBM 患者中具有已知活性的常规药物联合靶向治疗的潜在疗效。在一组三种市售的人胶质母细胞瘤细胞系（包括两种 PTEN 突变系（U251MG 和 U118MG）和一种 PTEN 野生型系（LN229））中，使用基于 siRNA 的基因沉默方法进行了体外验证研究。确定了 EGFR 和/或 Rictor 沉默对细胞迁移和体外化疗药物敏感性的影响。这些研究的体内验证集中在使用能够诱导型表达短发夹 RNA（shRNA）的 U251MG 细胞进行 EGFR 和/或 Rictor 沉默上，这些细胞原位植入 Rag2M 小鼠脑内。通过定量免疫荧光染色标记物评估了靶基因沉默、肿瘤大小和肿瘤细胞增殖。siRNA 介导的 EGFR 和 Rictor 沉默降低了 U251MG 细胞的迁移，并增加了细胞对伊立替康、替莫唑胺和长春新碱的敏感性。在 LN229 中，EGFR 和 Rictor 的共沉默导致细胞迁移减少，并增加了对长春新碱和替莫唑胺的敏感性。在 U118MG 中，单独沉默 Rictor 足以增加该细胞系对长春新碱和替莫唑胺的敏感性。在体内，虽然单独沉默 EGFR 或 Rictor 对 U251MG 肿瘤生长没有显著影响，但 EGFR 和 Rictor 的联合沉默导致肿瘤完全消除。这些数据表明，联合沉默 EGFR 和 Rictor 应该是治疗 GBM 的有效手段。
