NOTCH3 Overexpression and Posttranscriptional Regulation by miR-150 Were Associated With EGFR-TKI Resistance in Lung Adenocarcinoma

Abstract

Acquired resistance remains a key challenge in epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) therapy in lung adenocarcinoma (LUAD). Recent studies have shown that Notch signaling is associated with drug resistance. However, its role and possible mechanisms in EGFR-TKI resistance are not yet clear. In our study, we found that among four members of NOTCH1-4, only NOTCH3 was upregulated in LUAD tissues and TKI-resistant cell line (HCC827GR6). Knockdown of NOTCH3 by siRNA significantly inhibited proliferative ability, and decreased colony and sphere formation in HCC827GR6 cells. Then miR-150 was identified as a posttranscriptional regulator of NOTCH3. Its expression was downregulated in LUAD tissues and negatively correlated with NOTCH3 mRNA. The cell proliferation and IC₅₀ of gefitinib were decreased in HCC827GR6 cells transfected with miR-150 mimic, but was reversed when cotransfected with NOTCH3 overexpressed vector. Moreover, we also enrolled 20 patients with advanced LUAD who have taken TKIs as first-line therapy in this study. We found that collagen 1A1 (COL1A1) expression was increased significantly in LUAD tissues both at mRNA and protein levels, and positively correlated with NOTCH3 expression verified in our data and TCGA data. Univariate survival analysis showed that patients with high protein expression of NOTCH3 or COL1A1 were associated with shorter overall survival (OS). Taken together, these results suggest that miR-150/NOTCH3/COL1A1 axis contributed to EGFR-TKI resistance in LUAD, which provide a potential therapeutic target for LUAD treatment.

Figure 1

Expression of NOTCH3 in lung adenocarcinoma (LUAD) tissues and tyrosine kinase inhibitor (TKI)-resistant cell line. (A) Expression levels of NOTCH3 mRNA were significantly higher in LUAD tissues, compared to matched normal tissues, while NOTCH1,2,4 levels were significantly lower in LUAD tissues than normal tissues (20 pairs). (B) Immunohistochemical staining of NOTCH3 in LUAD and matched normal tissues. Scale bar: 50 μm. Positive staining is located in the cell membrane. (C) NOTCH3 mRNA was upregulated in gefitinib-resistant HCC827GR6 cells compared to parental HCC827 cells, while other NOTCH members showed no significant difference. (D) Meta-analysis of NOTCH3 gene expression from nine Oncomine databases. Colored squares indicate the median rank for NOTCH3 (lung adenocarcinoma vs. normal tissue) across nine analyses. Beer lung (1), Bhattacharjee lung (2), Garber lung (3), Hou lung (4), Landi lung (5), Okayama lung (6), Selamat lung (7), Stearman lung (8), Su lung (9). The p value is given for the median rank analysis. (E) NOTCH3 protein expression was upregulated in HCC827GR6 cells compared to parental HCC827 cells.

Figure 2

Effects of NOTCH3 siRNA on the growth and stemness of TKI-resistant cells. (A) Quantitation real-time polymerase chain reaction (qRT-PCR) and (B) Western blot showed NOTCH3 expression was decreased in HCC827GR6 cells transfected with siNOTCH3-1 and siNOTCH3-2. (C) The cell proliferation and (D) IC₅₀ of gefitinib were decreased in HCC827GR6 cells transfected with siNOTCH3-1 and siNOTCH3-2. (E) Representative photographs for the colony formation and the sphere formation in HCC827GR6 cells transfected with siScramble and siNOTCH3-1. (F) Knockdown of NOTCH3 reduced colony number and sphere formation efficiency with significant difference.

Figure 3

Validation of candidate miRNAs targeting NOTCH3. (A) Among four putative miRNAs, cotransfection of miR-150 reduced NOTCH3 luciferase activity in HEK293T cells. (B) miR-150 levels were significantly lower in lung adenocarcinoma tissues compared with adjacent normal tissues. miR-150 levels were negatively correlated with NOTCH3 mRNA expression (C), NCID3 protein (D), and HES1 protein (E) expression, both at lung adenocarcinoma tissues and normal tissues (n = 40). (F) qRT-PCR detection of miR-150 expression in HCC827GR6 cells transfected with miR-NC (group 1) or miR-150 mimic (group 2). (G) qRT-PCR and Western blot detection of NOTCH3 expression in HCC827GR6 cells transfected with miR-NC (group 1), miR-150 mimic (group 2), or cotransfected with miR-150 mimic and pEGFP-NOTCH3 (group 3). (H) The cell proliferation and (I) IC₅₀ of gefitinib were decreased in HCC827GR6 cells transfected with miR-150 mimic, but was reversed when cotransfected with pEGFP-NOTCH3.

Figure 4

NOTCH3 correlated with COL1A1 as predictors for poor outcome of lung adenocarcinoma (LUAD) patients treated with TKIs. (A) Immunohistochemical staining of COL1A1 in LUAD and matched normal tissues. Scale bar: 50 μm. Positive staining is located in the cytoplasm. (B) COL1A1 mRNA expression was increased significantly in LUAD tissues compared with normal tissues (20 pairs). The level of COL1A1 mRNA expression in LUAD tissues was positively correlated with NOTCH3 in our cohort (n = 20; C) and TCGA data (n = 445; D). Patients with high expression of NOTCH3 protein (E) or COL1A1 protein (F) were associated with shorter overall survival.