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.
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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

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Figure 2

CRIPTO1 renders EGFR-mutated NSCLC cells resistant to EGFR-TKIs in vitro, in vivo, and in primary cultures.

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CRIPTO1 renders EGFR-mutated NSCLC cells resistant to EGFR-TKIs in vitro...
(A) Western blot of CRIPTO1 in CRIPTO1-transfected NSCLC cells. MT, mutant. Exogenous CRIPTO1 expression in HCC827/CRIPTO1 cells is the highest among the 3 transfected cell lines and comparable to that in NSCLC samples (Figure 1A), indicating that exogenous CRIPTO1 expression in all the 3 CRIPTO1-transfected cells should be similar to that in the NSCLC samples. (B) Effect of CRIPTO1 on erlotinib and dacomitinib sensitivity of EGFR-mutant NSCLC cells. MTS assays were performed 72 hours after drug treatment. Data represent mean ± SD of triplicate measurements relative to untreated cells. P values were calculated using paired 2-tailed t test. IC50s are reported in parenthesis in graphs. (C) Effect of CRIPTO1 expression on erlotinib sensitivity of HCC827 and H4006 cells in vivo. See Methods for details. P values were significant between vehicle and all the treatment groups (ANOVA). Bars indicate SEM. (D) Western blot of CRIPTO1, pSRC, and pAKT in xenograft tumors. Tumor cells were harvested at day 16 after erlotinib treatment. Lanes were run on the same gel but were noncontiguous. (E) Progressive loss of CRIPTO1 expression during in vitro culture of human primary NSCLC cells. Western blot of CRIPTO1 of primary cells derived from an intrinsic erlotinib-resistant NSCLC patient carrying L858R EGFR (MP41) and an NSCLC patient carrying WT EGFR (MP08) at the indicated days of primary culture. (F and G) Correlation between progressive loss of CRIPTO1 expression and erlotinib sensitivity in MP08 and MP41 cells.