S-Space College of Medicine/School of Medicine (의과대학/대학원) Program in Cancer Biology (협동과정-종양생물학전공) Theses (Ph.D. / Sc.D._협동과정-종양생물학전공)
Targeting EGFR Signaling Pathway in NSCLC and Malignant Glioma Cells for Radiosensitization
- 의과대학 협동과정 종양생물학전공
- Issue Date
- 서울대학교 대학원
- Non-small cell lung cancer (NSCLC); Glioblastoma multiform (GBM); Epidermal growth factor receptor (EGFR); K-RAS; Temozolomide (TMZ); Radiosensitization
- 학위논문 (박사)-- 서울대학교 대학원 : 협동과정 종양생물학 전공, 2013. 2. 김인아.
- Epidermal growth factor receptor (EGFR) signalings play important roles in the pathogenesis of lung cancer and malignant glioma, and therefore, molecular inhibition of the EGFR signaling pathway represents a promising strategy to enhance the anti-tumor activity of radiation. However, therapeutic resistance has emerged as an important clinical issue. Therefore, we investigated whether inhibitors targeting downstream molecules in pathways associated with EGFR signaling would radiosensitize a panel of non-small cell lung cancer (NSCLC) and malignant glioma cell lines showing activated EGFR.
In the first part, we evaluated the efficacy of targeting EGFR-associated downstream signaling in NSCLC with activated EGFR, mutant K-RAS, or both. Specific inhibition of K-RAS attenuated downstream signaling and increased radiosensitivity of A549 and H460 cells, while inhibition of EGFR did not. A549 cells having a K-RAS mutation at codon V12 were radiosensitized by small interfering RNA (siRNA) targeting this codon. H460 cells harboring mutation at codon V61 were radiosensitized by siRNA targeting this codon. K-RAS siRNA did not radiosensitize H1299 cells possessing wild-type K-RAS. Inhibition of the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin pathway led to significant radiosensitization of the two cell lines, while selective inhibition of extracellular signal-regulated kinase signaling did not. Inhibitors targeting PI3K-AKT-mTOR pathway also abrogated G2 arrest following irradiation and induced delay of γH2AX foci formation. A dual inhibitor of Class I PI3K and mTOR increased radiosensitivity of A549 and H460 cells effectively. Inhibition of PI3K-AKT signaling was associated with down-regulation of p-DNA-PK, respectively. While apoptosis was the major mode of cell death when the cells were pretreated with LY294002 or AKT inhibitor VIII, the cells were pretreated by rapamycin or PI103 showed mixed mode of cell death including autophagy.
In the second part, we evaluated whether targeting EGFR-associated signaling would radiosensitize EGFR-activated glioma cells and further increase the radiosensitizing effect of temozolomide (TMZ). Although the result of radiotherapy (RT) for high grade glioma has significantly improved by concurrent and adjuvant TMZ, glioblastoma multiform (GBM) still has a dismal prognosis. Therefore, we tried to identify the effective targeting strategies to improve the therapeutic outcome of concurrent RT and TMZ. We first investigated the efficacy of targeting downstream molecules in EGFR-associated signaling pathway, such as Src, PI3K-AKT-mTOR, and STAT3, in glioma cell lines U251 and T98G. Next, we tested the effect of ligand-independent modulation using an HSP90 inhibitor and epigenetic modulation using a histone deacetylase (HDAC) inhibitor. U251 cells showing a high proportion of methylated methyl guanine transferase (MGMT) were highly responsive to the radiosensitizing effect of TMZ. Treatment with a Src inhibitor, PP2
a dual inhibitor of Class I PI3K and mTOR, PI103
a STAT3 inhibitor, Cpd188
a HSP90 inhibitor, 17-DMAG
or a HDAC inhibitor, LBH589, further increased the cytotoxic effect of RT plus TMZ in this cell line. Conversely, T98G cells showing a high proportion of unmethylated MGMT had a lower response to the radiosensitizing effect of TMZ although treatment with PI103, 17-DMAG, or LBH-589 increased the cytotoxic effect of radiotherapy plus TMZ. The mechanism of enhanced radiosensitizing effects of TMZ was multifactorial, involving impaired DNA damage repair and induction of autophagy or apoptosis.
Taken together, these results suggest possible mechanisms for counteracting EGFR prosurvival signaling implicated in radioresistance of NSCLC and malignant glioma cells, and offer a potential strategy for overcoming resistance to EGFR inhibitors combined with irradiation.