Yin Min Thu, Ken Suzawa, Shuta Tomida, Kosuke Ochi, Shimpei Tsudaka, Fumiaki Takatsu, Keiichi Date, Naoki Matsuda, Kazuma Iwata, Kentaro Nakata, Kazuhiko Shien, Hiromasa Yamamoto, Mikio Okazaki, Seiichiro Sugimoto, Shinichi Toyooka
Abstract
Mechanisms underlying primary and acquired resistance to MET tyrosine kinase inhibitors (TKIs) in managing non-small cell lung cancer remain unclear. In this study, we investigated the possible mechanisms acquired for crizotinib in MET-amplified lung carcinoma cell lines. Two MET-amplified lung cancer cell lines, EBC-1 and H1993, were established for acquired resistance to MET-TKI crizotinib and were functionally elucidated. Genomic and transcriptomic data were used to assess the factors contributing to the resistance mechanism, and the alterations hypothesized to confer resistance were validated. Multiple mechanisms underlie acquired resistance to crizotinib in MET-amplified lung cancer cell lines. In EBC-1-derived resistant cells, the overexpression of SERPINE1, the gene encoding plasminogen activator inhibitor-1 (PAI-1), mediated the drug resistance mechanism. Crizotinib resistance was addressed by combination therapy with a PAI-1 inhibitor and PAI-1 knockdown. Another mechanism of resistance in different subline cells of EBC-1 was evaluated as epithelial-to-mesenchymal transition with the upregulation of antiapoptotic proteins. In H1993-derived resistant cells, MEK inhibitors could be a potential therapeutic strategy for overcoming resistance with downstream mitogen-activated protein kinase pathway activation. In this study, we revealed the different mechanisms of acquired resistance to the MET inhibitor crizotinib with potential therapeutic application in patients with MET-amplified lung carcinoma.
Introduction
As a leading cause of the incidence and mortality of cancer prevalence worldwide, lung cancer requires a comprehensive understanding of the nature and appropriate management of the disease, which are essential to reduce its global burden [1]. Among the incidences of lung cancer, non-small cell lung cancer (NSCLC) comprises approximately 80%–85%, which has a poor prognosis and requires complex strategies [2]. With the development of personalized medical treatment, molecular-targeted therapies have been used in various types of cancers [2]. MET proto-oncogene alteration, including the exon 14 skipping mutation (METex14) and amplification, comprises about 6.5% of lung adenocarcinoma, and this alteration is the driver oncogene [3]. METex14 is the common alteration of MET, which is often mutually exclusive with other oncogenic drivers (EGFR, KRAS, or ALK). At present, some MET tyrosine kinase inhibitors (TKIs) have been demonstrated to be highly effective in this molecular subgroup of patients [4–6]. MET amplification comprises 1.7% of all early and 2.5% of metastatic lung adenocarcinomas, and high-level MET amplification is thought to be an oncogenic driver [7–9].
Materials and methods
Cell lines and reagents
Two MET-amplified cell lines, namely, EBC-1 (MET-amplified lung squamous cell carcinoma) and NCI-H1993 (MET-amplified adenocarcinoma), were purchased from RIKEN Cell Bank, Tsukuba, Japan and ATCC, American Type Culture Collection (Manassas, Virginia), respectively. All cells were cultured in RPMI-1640 medium supplemented with 10% FBS under a culture condition in a 5% CO2-supplied humidified incubator at 37°C. Crizotinib-resistant cell lines were established using two methods: high-concentration exposure method using crizotinib 1 μM from the start of culture or stepwise escalation method using the concentration of crizotinib from 0.1 to 1 μM to the parental cells. Crizotinib stepwise resistant cell lines (CRS) were established by treating the cells with increasing doses of crizotinib (from 0.1 μM to 1 μM) for 6 months. The cells were first exposed to crizotinib 0.1μM until they were damaged at around 30% confluence. They were then passaged to reach about 80% confluence in a drug-free state. When these cells reached 80% confluence, the drug was exposed again. The cells were repeatedly treated with the same concentration of crizotinib until almost all the cells survived the treatment. When the cells survived the treatment, the drug exposure was increased with a higher dose of 0.2 μM. This process was repeated with a stepwise dose of crizotinib until the cells survived the 1 μM concentration. In the high-concentration exposure method establishment for crizotinib resistant high-dose (CRH) cell lines, the cells were exposed directly to the high dose of 1000nM crizotinib from the start until about 30% confluence of the cells were left after exposure.
Results
Establishment of crizotinib-resistant cell lines
Two types of crizotinib-resistant cell lines were established in two parental cell lines with MET amplification, EBC-1 and H1993, using two different methods: the high-dose concentration method and the stepwise escalation method. Cell viability assays were performed to confirm crizotinib resistance. The IC50 value of crizotinib in EBC-1 parental, EBC-1 CRH, and EBC-1 CRS were 0.043 ± 0.05, 2.115 ± 0.04 and 1.731 ± 0.11 μM ± SD, respectively. The IC50 value of H1993 parental, H1993 CRH, and H1993 CRS were 0.283 ± 0.13, 3.564 ± 0.08 and 4.376 ± 0.05 μM ± SD, respectively. We also found that the crizotinib-resistant cell lines conferred cross-resistance to tepotinib, and the IC50 values were 0.006 ± 0.01, 8.062 ± 0.00 and 7.635 ± 0.06 μM ± SD in EBC-1 parental, CRH, and CRS cells, respectively, and 0.065 ± 0.09, 9.375 ± NA and 5.05 ± 0.07 μM ± SD in H1933 parental, CRS, and CRH cells, respectively. Similarly, the IC50 value of cabozantinib was 0.089 ± 0.06, 3.618 ± 0.04 and 9.09 ± NA μM ± SD in EBC-1 parental, CRH, and CRS; 2.179 ± 0.09, 13 ± 0.05 and 10.31 ± 0.06 in H1993 parental, CRH and CRS μM ± SD respectively (Fig 1A). Compared with parental cells, morphological changes in resistant cells could be observed on examination under a light microscope. For EBC-1 CRH, EBC-1 CRS, and H1993 CRH cell lines, a gain of spindle-shaped formation and elongated structure was detected, suggesting the acquisition of epithelial-to-mesenchymal transition (EMT) features (Fig 1B).
Discussion
Our study revealed several molecular mechanisms occurring in different cell lines with different approach backgrounds of acquired resistance to crizotinib. Interestingly, the crizotinib-resistant cell line established in our study showed resistance to crizotinib and other MET TKIs, such as tepotinib and cabozantinib. In the EBC-1 CRH cell line, we found that the overexpression of PAI-1 was associated with crizotinib resistance. The gene SERPINE1 encodes the Serpin Family E Member 1 protein, which is also known as PAI-1, and this gene has been reported to be involved in tumor growth, angiogenesis, cancer cell survival, and metastasis through the regulation of several factors [22–24]. PAI-1 has been reported to confer resistance to various chemotherapeutic agents in cancer. High PAI-1 expression enhanced migration and apoptosis resistance, and such expression was associated with poor outcomes in head and neck carcinoma via the PI3K–AKT–mTOR pathway [25]. PAI-1 promotes colony formation and cell viability and decreases cisplatin-induced apoptosis via AKT-ERK signaling in esophageal squamous cell carcinoma [26]. PAI-1 also promotes actin cytoskeleton reorganization, glycolytic metabolism, migration and invasive phenotype, and orthotopic tumor growth via ERK signaling [27]. PAI-1 DNA promoter methylation is reported to be involved in carboplatin-induced EMT in epithelial ovarian cancer [28].
Acknowledgments
We thank Ms. Fumiko Isobe (General Thoracic Surgery and Breast and Endocrinological Surgery Department, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan) for her technical assistance.
Citation: Thu YM, Suzawa K, Tomida S, Ochi K, Tsudaka S, Takatsu F, et al. (2024) PAI-1 mediates acquired resistance to MET-targeted therapy in non-small cell lung cancer. PLoS ONE 19(5): e0300644. https://doi.org/10.1371/journal.pone.0300644
Editor: Abeer El Wakil, Alexandria University, EGYPT
Received: December 20, 2023; Accepted: March 3, 2024; Published: May 17, 2024
Copyright: © 2024 Thu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP19K18216.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Shinichi Toyooka received research funding from Eli Lilly Japan, Taiho (Japan) and Chugai (Japan), and lecture fees from Chugai. All other authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0300644#abstract0
