Adenocarcinoma is the most common type of lung cancer, and can be classified into various histologic subtypes. However, little is known about the subtype-dependent variations in lipid metabolism processes. We performed dual lipidomic analyses using liquid chromatography–mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to identify possible biomarkers to distinguish adenocarcinoma specimens from normal lung specimens, and to determine if there are any differences in lipid metabolism among the histologic subtypes (lepidic, acinar, papillary, micropapillary, solid, and mucinous). LC-MS was used to characterize the lipid profiles of lung adenocarcinoma and normal lung tissue, and MALDI-IMS analysis was performed to confirm the results with information on lipid localization within the lung. LC-MS analysis found significant differences in the relative abundances of phosphatidylcholine (PC)(16:0/16:0) ( P = 0.0432) and sphingomyelin (SM)(42:2) ( P < 0.0001) between adenocarcinoma and normal lung specimens. The ratios of PC(16:0/16:1)/PC(16:0/16:0), PC(16:0/18:1)/PC(16:0/16:0), and PC(16:0/18:1)/PC(16:0/18:0) were significantly higher in adenocarcinoma specimens ( P = 0.02221, P = 0.0004, and P = 0.0215, respectively). MALDI-IMS analysis confirmed that these ratios were significantly higher in adenocarcinoma regions of the lung. The ratio of PC(16:0–18:1)/PC(16:0–18:0) was significantly lower in solid subtypes than in other subtypes ( P = 0.0028). The monounsaturated/saturated PC ratios may have applications in adenocarcinoma diagnoses and subtyping.

Adjuvant cisplatin-vinorelbine is a standard therapy for stage II/III lung cancer. However, a poor survival rate of patients with lung cancer is attributed to vinorelbine resistance arising from ATP-binding cassette (ABC) sub-family B member 1 (ABCB1) and phosphorylated Fyn (p-Fyn) overexpression. However, the underlying mechanisms remain unclear. NF-E2-related factor 2 (Nrf2) regulates the ABC family and activates the nuclear transport of Fyn. The present study evaluated the roles of the Nrf2/p-Fyn/ABCB1 axis in vinorelbine-resistant (VR) cells and clinical samples. To establish VR cells, H1299 cells were exposed to vinorelbine, and the intracellular reactive oxygen species (ROS) level in the H1299 cells was determined using a DCFH-DA assay. The total and subcellular expression of Nrf2, ABCB1 and p-Fyn in VR cells was evaluated. Immunofluorescence was used to detect the subcellular localization of p-Fyn in VR cells. A cell viability assay was used to examine whether the sensitivity of VR cells to vinorelbine is dependent on Nrf2 activity. Immunohistochemistry was performed on 104 tissue samples from patients with lung cancer who underwent surgery followed by cisplatin-vinorelbine treatment. The results revealed that persistent exposure to vinorelbine induced intracellular ROS formation in H1299 cells. p-Fyn was localized in the nucleus, and ABCB1 and Nrf2 were overexpressed in VR cells. ABCB1 expression was dependent on Nrf2 downstream activation. The decreased expression of Nrf2 restored the sensitivity of VR cells to vinorelbine. In the surgical samples, Nrf2 and ABCB1 were associated with disease-free survival, and p-Fyn was associated with overall survival (P<0.05). On the whole, the present study demonstrates that Nrf2 upregulates ABCB1 and, accompanied by the nuclear accumulation of p-Fyn, induces vinorelbine resistance. These findings may facilitate the development of drug resistance prevention strategies or new drug targets against non-small cell lung cancer.

Introduction: Adjuvant cisplatin-vinorelbine is standard therapy for stage II/III lung cancer. Poor survival in lung cancer can be attributed to vinorelbine resistance arising from ABC sub-family B member 1 (ABCB1) and phosphorylated Fyn (pFyn) overexpression. However, the underlying mechanism remains unclear. Nrf2 regulates the cell-protective ABC family and activates nuclear transport of Fyn. Here, we aimed to evaluate the ROS-Nrf2-ABCB1 axis in both vinorelbine-resistant (VR) cells and clinical samples. Methods: We established VR cells by exposing H1299 cells to vinorelbine, and determined intracellular reactive oxygen species (ROS) level of H1299 cells induced by vinorelbine using DCFH-DA. We evaluated the total and subcellular expression of Nrf2, ABCB1, and pFyn in VR cells and performed immunofluorescence of VR cells to confirm the precise subcellular localization of pFyn. Cell viability assay was used to examine whether the sensitivity of VR cells to vinorelbine depends on Nrf2 activity. For immunohistochemistry, we used 104 tissue samples from lung cancer patients who underwent surgery followed by cisplatin-vinorelbine treatment between December 2006 and June 2018. Results: Persistent exposure to vinorelbine induced intracellular ROS formation of H1299 cells. Both ABCB1 and Nrf2 were overexpressed in VR cells, and ABCB1 expression was dependent on Nrf2 downstream activation. Subcellular fractionation and immunofluorescence revealed nuclear accumulation of pFyn in VR cells. Decreased expression of Nrf2 restored the sensitivity of VR cells to vinorelbine. In the 104 surgical samples, Nrf2 and ABCB1 were significantly associated with disease-free survival (p = 0.029 and 0.035, respectively), and pFyn was associated with overall survival (p = 0.040). Conclusions: ROS-Nrf2 upregulates ABCB1 and accompanied by nuclear accumulation of pFyn, induces vinorelbine resistance. Our findings may facilitate drug resistance prevention strategies or new drug target development against non-small cell lung cancer. Citation Format: Shigeyuki Tamari, Toshi Menju, Toshiya Toyazaki, Hideaki Miyamoto, Naohisa Chiba, Misa Noguchi, Hiroshi Date. ROS-Nrf2-ABCB1 axis accompanied with pFyn nuclear accumulation plays pivotal roles in vinorelbine resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6075.

OBJECTIVES It is unclear whether the movement and function of the regenerated cilia on collagen-conjugated artificial trachea are the same as those of normal cilia. This study assessed the ciliary beat frequency (CBF) and ciliary transport functions (CTFs) of regenerated cilia in a canine model. METHODS A tracheal defect introduced into the anterior portion of the cervical trachea of an adult beagle dog was covered with a collagen-conjugated prosthesis. Two months later, the trachea was harvested along the long axis, both from normal and regenerated regions. The cilia were stained with isothiocyanate-conjugated wheat germ agglutinin, and their movement was monitored with a high-speed camera to analyse CBF and CTF. Four samples each were obtained from the regenerated and normal regions for CBF analysis and 7 samples each were obtained for CTF analysis. RESULTS The wheat germ agglutinin-stained cells showed well-regulated beats in both the regenerated and normal regions of the trachea. Mean CBF in the regenerated and normal regions did not differ significantly (7.11 ± 0.41 vs 7.14 ± 1.09 Hz; P = 981). By contrast, CTF was significantly lower in the regenerated region than in the normal region (30.0 ± 6.62 vs 7.43 ± 0.58 μm/s; P = 0.005). CONCLUSIONS Mean CBF in the regenerated and normal regions did not differ significantly at 2 months. The CTF in the regenerated region recovered partially but remained lower than those in the normal region. Methods are needed to improve the CTF of regenerated cilia.