CRIPTO1 expression in EGFR-mutant NSCLC elicits intrinsic EGFR-inhibitor resistance
Kang-Seo Park, … , Yisong Wang, Giuseppe Giaccone
Kang-Seo Park, … , Yisong Wang, Giuseppe Giaccone
Published June 9, 2014
Citation Information: J Clin Invest. 2014;124(7):3003-3015. https://doi.org/10.1172/JCI73048.
View: Text | PDF
Research Article Oncology

CRIPTO1 expression in EGFR-mutant NSCLC elicits intrinsic EGFR-inhibitor resistance

Abstract

The majority of non–small cell lung cancer (NSCLC) patients harbor EGFR-activating mutations that can be therapeutically targeted by EGFR tyrosine kinase inhibitors (EGFR-TKI), such as erlotinib and gefitinib. Unfortunately, a subset of patients with EGFR mutations are refractory to EGFR-TKIs. Resistance to EGFR inhibitors reportedly involves SRC activation and induction of epithelial-to-mesenchymal transition (EMT). Here, we have demonstrated that overexpression of CRIPTO1, an EGF-CFC protein family member, renders EGFR-TKI–sensitive and EGFR-mutated NSCLC cells resistant to erlotinib in culture and in murine xenograft models. Furthermore, tumors from NSCLC patients with EGFR-activating mutations that were intrinsically resistant to EGFR-TKIs expressed higher levels of CRIPTO1 compared with tumors from patients that were sensitive to EGFR-TKIs. Primary NSCLC cells derived from a patient with EGFR-mutated NSCLC that was intrinsically erlotinib resistant were CRIPTO1 positive, but gained erlotinib sensitivity upon loss of CRIPTO1 expression during culture. CRIPTO1 activated SRC and ZEB1 to promote EMT via microRNA-205 (miR-205) downregulation. While miR-205 depletion induced erlotinib resistance, miR-205 overexpression inhibited CRIPTO1-dependent ZEB1 and SRC activation, restoring erlotinib sensitivity. CRIPTO1-induced erlotinib resistance was directly mediated through SRC but not ZEB1; therefore, cotargeting EGFR and SRC synergistically attenuated growth of erlotinib-resistant, CRIPTO1-positive, EGFR-mutated NSCLC cells in vitro and in vivo, suggesting that this combination may overcome intrinsic EGFR-inhibitor resistance in patients with CRIPTO1-positive, EGFR-mutated NSCLC.

Authors

Kang-Seo Park, Mark Raffeld, Yong Wha Moon, Liqiang Xi, Caterina Bianco, Trung Pham, Liam C. Lee, Tetsuya Mitsudomi, Yasushi Yatabe, Isamu Okamoto, Deepa Subramaniam, Tony Mok, Rafael Rosell, Ji Luo, David S. Salomon, Yisong Wang, Giuseppe Giaccone

×

Figure 4

CRIPTO1 activates SRC and EMT pathways through downregulation of miR-205.

Options: View larger image (or click on image) Download as PowerPoint
CRIPTO1 activates SRC and EMT pathways through downregulation of miR-205...
(A) Inverse correlation between CRIPTO1 and miR-205 expression in patient NSCLC samples. The graph illustrates a nonlinear regression analysis of CRIPTO1 protein and miR-205 expression in 17 lung adenocarcinoma samples. (B) Real-time PCR analysis of miR-205 expression. miR-205 expression is downregulated by CRIPTO1 in HCC827 and H3255 cells. (C) Real-time PCR analysis of ectopic miR-205 expression in HCC827/CRIPTO1/miR-205 and HCC827/CRIPTO1/mock stable cells. (D) Effect of ectopic miR-205 expression on ZEB1, Vimentin, pSRC, total SRC, and pAKT in HCC827/CRIPTO1 revealed by Western blot analysis. (E) Downregulation of total Src mRNA in miR-205–transfected HCC827/CRIPTO1 cells by real-time PCR analysis. (F) Effect of ectopic miR-205 expression on EMT morphology, migration, and invasion of HCC827/CRIPTO1 cells. Quantitation of migration and invasion is shown at the bottom. Original magnification, ×200. (G) Ectopic miR-205 expression renders HCC827/CRIPTO1 cells sensitive to erlotinib. MTS assays were performed 72 hours after erlotinib treatment of the indicated cells at the different concentrations. Data represent mean ± SD of triplicate experiments relative to untreated cells. (H) Real-time PCR analysis of miR-205 expression in HCC827 and H4006 cells treated with miR-205 inhibitor (AM11015; Ambion). Note that miR-205 knockdown efficiency by miR-205 inhibitor was comparable to CRIPTO1-induced miR-205 downregulation (see Figure 4B). (I and J) miR-205 inhibition renders HCC827 cells resistant to erlotinib. (K) Real-time PCR analysis of SRC expression in miR-205 inhibitor–treated HCC827 and H4006 cells.