BPI-9016M, a c-Met inhibitor, suppresses tumor cell growth, migration and invasion of lung adenocarcinoma via miR203-DKK1

Abstract

Activation of c-Met plays a critical role in tumorigenesis, migration and invasion in lung cancer. Here, we explored the therapeutic efficacy of a novel small-molecule c-Met inhibitor (BPI-9016M) in lung adenocarcinoma and investigated the underlying molecular mechanisms. Method: BPI-9016M, a c-Met tyrosine kinase receptor inhibitor, was used to treat patient-derived xenografts (PDX) from lung adenocarcinoma in NOD/SCID mice. Immunohistochemistry and Western blot analysis were used to determine the expression of c-Met and its downstream signaling molecules. CCK8, wound healing, and trans-well assays were used to analyze cell proliferation, spreading, migration and invasion. RNA sequencing and quantitative real-time PCR (qPCR) was used to screen and validate the expression of downstream genes in lung adenocarcinoma cells treated with BPI-9016M. Luciferase reporter assay was used to detect the interaction between miRNA and the targeted gene. Results: BPI-9016M significantly suppressed growth in three out of four lung adenocarcinoma PDX models, particularly in the tumors with high expression of c-Met. In lung adenocarcinoma cell lines, BPI-9016M treatment resulted in increased miR203, which reduced migration and invasion and also repressed Dickkopf-related protein 1 (DKK1) expression. Forced overexpression of DKK1 or down-regulation of miR203 reversed the inhibitory effect of BPI-9016M on migration and invasion. C-Met was verified to positively and negatively associate with DKK1 and miR203, respectively. High expression of c-Met/DKK1 or low expression of miR203 related to poor outcome of lung adenocarcinoma patients. Furthermore, we observed significantly enhanced tumor cell growth inhibition upon combining BPI-9016M treatment with miR203 mimics or DKK1 siRNA. Conclusion: Our data indicated that BPI-9016M is an effective agent against lung adenocarcinoma, particularly in tumors with c-Met activation, and likely functions through upregulation of miR203 leading to reduced DKK1 expression.

Keywords: BPI-9016M; Dickkopf-1 (DKK1); c-Met; lung adenocarcinoma; miR203.

Figure 1

BPI-9016M suppresses tumor growth in lung adenocarcinoma. (A) Two primary tumor cells and six cell lines of lung adenocarcinoma were treated with serially diluted BPI-9016M for 48 h and IC50 was calculated as shown in the bar graph. (B) QPCR (top panel) and Western blot (bottom panel) analyses show c-Met expression in lung adenocarcinoma cell lines including H1299, A549, H1975, PC-9, H1650 and HCC827. (C) Tumor growth inhibition in PDX1, PDX2, PDX3 and PDX4 models following BPI-9016M treatment. (D) Tumor volume in the PDX1, PDX2, PDX3 and PDX4 models with BPI-9016M or vehicle treatment. * p<0.05, ** p<0.01, *** p<0.001 at indicated time point. (E) Representative images (×200 magnification) of c-Met by immunohistochemical staining of the BPI-9016M-treated PDX1 and PDX4 tissues. (F) H-score to evaluate c-Met expression and tumor growth inhibition (TGI) in the four PDX models after administration of BPI-9016M. (G) Representative images (×200 magnification) of Ki67 staining of the PDX1 tumors with vehicle or BPI-9016M treatment.

Figure 2

BPI-9016M inhibits downstream signals of c-Met, colony formation and cell cycle. (A) C-Met, ERK, AKT, and phosphorylation of these protein were detected by Western blotting in the PDX tissues following vehicle or BPI-9016M treatment. (B) C-Met, ERK, AKT, and phosphorylation of these protein were detected by Western blotting in the A549 and H1299 cells after treatment with serial doses of BPI-9016M treatment. (C-D) Colony formation rate (C) and colony size (D) of A549 and H1299 cells treated with BPI-9016M. (E) Cell cycle distribution of A549 and H1299 cells treated with BPI-9016M. (F) Cyclin D1 and CDK4 were analyzed by Western blotting of cells after treatment with BPI-9016M. (C- E) * p<0.05, ** p<0.001, *** p<0.0001.

Figure 3

BPI-9016M suppresses spreading, migration and invasion of lung adenocarcinoma. (A) Spreading ability assessed by the wound-healing assay of A549 and H1299 cells treated with BPI-9016M for 12, 24 and 36 h. (B) The spreading ability of A549 and 1299 cells treated with BPI-9016M, HGF or a combination of these two chemicals for 12, 24 and 36 h. (C) Representative images (×100 magnification) of migrated and invasive A549 and 1299 cells stained with crystal violet after treatment with vehicle or BPI-9016M. (D) The number of migrated and invasive cells treated with vehicle or BPI-9016M. (E) Representative images (×100 magnification) of migrated and invasive A549 and 1299 cells stained with crystal violet after treatment with HGF, BPI-9016M, or a combination of these two chemicals. (F) The number of migrated and invasive cells treated with HGF, BPI-9016M, or a combination of these two chemicals. (G) EMT-associated molecules including E-cadherin, N-cadherin and vimentin were assessed by Western blotting of A549 and 1299 cells after BPI-9016M treatment. (H) E-cadherin, N-cadherin and vimentin were detected by Western blotting in A549 and 1299 cells treated with HGF, BPI-9016M, or a combination of these two chemicals. (A-B, D, F) * p<0.05, ** p<0.001, *** p<0.0001.

Figure 4

BPI-9016M inhibits DKK1 expression in lung adenocarcinoma cells. (A) Volcano plot depicting a log transformation plot of the fold difference (x-axis) and the p value (y-axis) of indicated genes between BPI-9016M-treated cells and control cells (vehicle treatment). (B) Signaling pathways were enriched in the BPI-9016M-treated A549 cells over the vehicle control by KEGG analysis using RNA sequencing. (C) FPKM values of nine genes associated with metastasis in the BPI-9016M-treated A549 cells by RNA sequencing analysis. ***P<0.0001. (D) The nine genes were investigated in the BPI-9016M-treated A549 cells by qPCR analysis. (E) Western blotting analysis shows the DKK1 expression level in BPI-9016M-treated A549 and H1299 cells. (F) Western blotting analysis shows the DKK1 expression level in cells treated with HGF and BPI-9016M alone or in combination.

Figure 5

BPI-9016M inhibits tumor migration and invasion through DKK1. (A) Western blots showing DKK1, p-AKT, AKT, ERK, p-ERK, E-cadherin, N-cadherin and vimentin expressions in A549 and 1299 cells transfected with DKK1 siRNA. (B) Wound-healing analysis of spreading of A549 and 1299 cells transfected with DKK1 siRNA for 12, 24 and 36 h. (C) Representative images (×100 magnification) of migrated and invasive A549 and 1299 cells stained with crystal violet that were transfected with DKK1 siRNA. (D) The number of migrated and invasive cells transfected with DKK1 siRNA. (E) Western blots showing DKK1 expression in A549 and 1299 cells overexpressing DKK1 with or without BPI-9016M treatment. (F) Wound-healing analyses of DKK1-overexpressing A549 and 1299 cells treated with BPI-9016M for 12, 24 and 36 h. (G) Representative images (×100 magnification) of migrated and invasive DKK1-overexpressing A549 and 1299 cells with or without BPI-9016M treatment. (H) The number of migrated and invasive cells in DKK1-overexpressing A549 and 1299 cells with or without BPI-9016M treatment. (B, D, F, H) * p<0.05, ** p<0.001, *** p<0.0001.

Figure 6

BPI-9016M increases miR203 and decreases DKK1. (A) miR103, miR203, miR200c, miR30b and miR30c expression levels by qPCR in A549 cells treated with BPI-9016M. U6 was used as the internal control. (B) Wound healing analyses of spreading miR203 inhibitor-transfected A549 and 1299 cells with BPI-9016M treatment. (C) Transwell assays to assess cell migration and invasion in miR203 inhibitor-transfected A549 and H1299 cells with or without BPI-9016M treatment. (D) Western blotting analyses demonstrating DKK1 expression in A549 and H1299 cells with miR103 or mi203 mimics (50 nM) and miR103 or miR203 inhibitors (50 nM). (E) The binding site of miR203 in the DKK1 3'-UTR is shown. 3'-UTR wild-type; 3'-UTR mutation and deletion sequences of the DKK1 3'-UTR. (F) Firefly luciferase activity between miR203 and wild-type, mutated, and deleted constructs of DKK1 3'-UTR. (G) Western blotting analysis showing DKK1 expression in A549 and H1299 cells with or without BPI-9016M/miR203 inhibitor. (H) A schematic illustration showing that BPI-9016M-inhibited proliferation, migration and invasion of lung adenocarcinoma is associated with ERK activation and miR203-DKK1-AKT signaling. (B-C, F) * p<0.05, ** p<0.001, *** p<0.0001.

Figure 7

Clinical significance of BPI-9016M targeted to c-Met, DKK1 and miR203. (A-B) QPCR analyses showing DKK1 (A) and miR203 (B) expressions in the four cases of PDX tissues of lung adenocarcinoma. (C-E) Correlations between c-Met and DKK1 (C), c-Met and miR203 (D) and DKK1 and miR203 (E). (F-G) Kaplan-Meier plots showing overall survival of lung adenocarcinoma patients with high or low expression of c-Met (G) and DKK1 (H) from the database (http://www.kmplot.com). (H-J) Kaplan-Meier plots showing overall survival of lung adenocarcinoma patients from Peking University Cancer Hospital & Institute with high or low expression of c-Met (H), DKK1 (I) and miR203 (J). (K) Tumor cell growth inhibition (%) of DKK1 siRNA-transfected cells treated with variable doses of BPI-9016M. (L) Tumor cell growth inhibition (%) of miR203 mimics-transfected cells treated with variable doses of BPI-9016M. (K-L) n.s.: no statistically significant difference; *p<0.05, ** p<0.001, *** p<0.0001; HR: hazard ratio.