Study of Mechanisms of Resistance to EGFR-TKI in Non-small Cell Lung Cancer with EGFR L858R Mutation

Autor:	Hsieh, Jia-Juan, 謝佳娟
Rok vydání:	2019
Druh dokumentu:	學位論文 ; thesis
Popis:	107 Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are effective therapies for patients with non-small cell lung cancer (NSCLC) bearing EGFR mutations. It is reported that the response rate is 60-80% in Taiwan. Despite bearing EGFR active mutations, 20-40% of patients did not respond to TKIs. Moreover, in certain patients responding to TKIs initially, drug resistance will develop in 8-12 months after treatment. An acquired T790M mutation was detected in half of TKI-resistant tumors and the third-generation EGFR inhibitors, osimertinib, was demonstrated to show activity against this mutation. Nevertheless, the resistant mechanisms are still not fully understood. We have successfully cultivated an NSCLC cell line, JLR10, bearing L858R mutation and exhibiting resistance to gefitinib. JLR10 expressed wild type in KRAS and BRAF genes; no mutation in EGFR other than TK domain was found, but a variant of exon 13 (R521K) was observed. JLR10 expressed MET gene amplification, accompanied with constitutive activation of both PI3K and MAPK pathways. This cell line could serve as a good model to explore novel resistant genes in NSCLC. In searching for potential genes relevant to EGFR-TKI resistance, the genetic profiles of JLR10 (L858R, gefitinib-resistant) and H3255 (L858R, gefitinib-sensitive) were analyzed with Affymetrix CytoScan HD/750k array and compared. The results showed that while both miR221 and miR222 were highly expressed in JLR10, they were negligible in the latter, a good correlation with MET expression in these 2 cell lines. Upregulation of miR221/222 has been reported to be associated with TRAIL resistance. Our drug sensitivity experiments, however, demonstrated that while H3255 was completely resistant to TRAIL, it induced a moderate cell death in JLR10, suggesting the existence of differential resistant mechanisms in these two cell models. The antitumor activity of combining TRAIL with available small molecular inhibitors was then evaluated. MET inhibitor PHA665257 significantly inhibited autophosphorylation of MET, AKT and ERK1/2 in JLR10 but did not synergize with TRAIL to induce tumor cell death. Unexpectedly, sorafenib, a multikinase inhibitor, although not exhibiting tumor inhibitory activity alone, demonstrated a high synergistic effect with TRAIL to induce cell apoptosis in JLR10; whereas, all available EGFR mutated NSCLC cell lines were resistant to this combined treatment. The synergistic effect can even be observed shortly (6-h) after treatment. These results suggest that combined sorafenib and TRAIL treatment could represent a potential therapeutic in NSCLC. Previously we have reported that a four-gene signature (LCN2, PTHLH, FJX1, and RAB38) was associated with survival among patients with early-stage NSCLC. Our subsequent studies showed that higher RAB38 expression in tumor specimens was associated with higher frequency of tumor recurrence in these patients; RAB expression was also inversely correlated with disease-free survival and overall patient survival, suggesting that it may serve as a prognostic factor in NSCLC. Using NSCLC cell lines, we further demonstrated that tumor cells with EGFR mutations expressed higher levels of RAB38 compared with those of wild-type. Furthermore, following specific knockdown of RAB38 by shRNA transfection, substantially reduced Matrigel invasiveness was detected in HCC827 cells (Exon 19 deletion) compared to those transfected with empty vector. These results indicate that RAB38 may be associated with EGFR status and could be targeted to reduce tumor metastasis in NSCLC. Using clinical specimens and in vitro cell models, we have shown that RAB38 could serve as a potential target in NSCLC and combination of sorafenib and TRAIL treatment induced a rapid and substantial cell apoptosis in NSCLC cell line. Although the mechanisms associated with these observations remain to be explored, these studies may pave the way toward effective treatment for this deadly disease.
Databáze:	Networked Digital Library of Theses & Dissertations
Externí odkaz:	http://ndltd.ncl.edu.tw/handle/4q4af5 Zobrazit plný text záznamu