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. 2022 Oct 31;23(21):13237.
doi: 10.3390/ijms232113237.

LMNA Reduced Acquired Resistance to Erlotinib in NSCLC by Reversing the Epithelial-Mesenchymal Transition via the FGFR/MAPK/c-fos Signaling Pathway

Chunsheng Hu  1   2 , Anting Zhou  1 , Xin Hu  1 , Yu Xiang  1 , Mengjun Huang  1 , Jiuhong Huang  3 , Donglin Yang  3 , Yan Tang  1   3
Affiliations

LMNA Reduced Acquired Resistance to Erlotinib in NSCLC by Reversing the Epithelial-Mesenchymal Transition via the FGFR/MAPK/c-fos Signaling Pathway

Chunsheng Hu et al. Int J Mol Sci. .

Abstract

For patients exhibiting non-small-cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are a first-line treatment. However, most patients who initially responded to EGFR-TKIs eventually developed acquired resistance, limiting the effectiveness of therapy. It has long been known that epithelial-mesenchymal transition (EMT) leads to acquired resistance to EGFR-TKIs in NSCLC. However, the mechanisms underlying the resistance dependent on EMT are unknown. This research aimed to reveal the effects of LMNA in the regulation of acquired resistance to erlotinib by EMT in NSCLC. The acquired erlotinib-resistant cells (HCC827/ER) were induced by gradual increase of concentrations of erlotinib in erlotinib-sensitive HCC827 cells. RNA sequencing and bioinformatics analysis were performed to uncover the involvement of LMNA in the EMT process that induced acquired resistance to erlotinib. The effect of LMNA on cell proliferation and migration was measured by clone-formation, wound-healing, and transwell assays, respectively. The EMT-related protein, nuclear shape and volume, and cytoskeleton changes were examined by immunofluorescence. Western blot was used to identify the underlying molecular mechanism of LMNA regulation of EMT. HCC827/ER cells with acquired resistance to erlotinib underwent EMT and exhibited lower LMNA expression compared to parental sensitive cells. LMNA negatively regulated the expression of EMT markers; HCC827/ER cells showed a significant up-regulation of mesenchymal markers, such as CDH2, SNAI2, VIM, ZEB1, and TWIST1. The overexpression of LMNA in HCC827/ER cells significantly inhibited EMT and cell proliferation, and this inhibitory effect of LMNA was enhanced in the presence of 2.5 μM erlotinib. Furthermore, a decrease in LMNA expression resulted in a higher nuclear deformability and cytoskeletal changes. In HCC827/ER cells, AKT, FGFR, ERK1/2, and c-fos phosphorylation levels were higher than those in HCC827 cells; Furthermore, overexpression of LMNA in HCC827/ER cells reduced the phosphorylation of AKT, ERK1/2, c-fos, and FGFR. In conclusion, our findings first demonstrated that downregulation of LMNA promotes acquired EGFR-TKI resistance in NSCLC with EGFR mutations by EMT. LMNA inhibits cell proliferation and migration of erlotinib-resistant cells via inhibition of the FGFR/MAPK/c-fos signaling pathway. These findings indicated LMNA as a driver of acquired resistance to erlotinib and provided important information about the development of resistance to erlotinib treatment in NSCLC patients with EGFR mutations.

Keywords: EGFR-TKI resistance; LMNA; epithelial–mesenchymal transition; non-small-cell lung cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Profound changes in the global transcriptional landscape between parental and erlotinib-resistant cells. (A) Heatmap of genes that were differentially expressed (DEGs) between HCC827 and HCC827/ER cells. The color represents the level of transformed gene expression of log10. (B) Volcano plot displaying genes detected by RNA-Seq. Red points represent up-regulated DEGs (FoldChange ≥ 2, q value ≤ 0.001). Blue points represent down-regulated DEGs (FoldChange ≤ −2, q value ≤ 0.001). Gray points represent non-DEGs. (C) KEGG pathway enrichment analysis of genes up-regulated or down-regulated in pairwise comparison of HCC827 and HCC827/ER cells. FDR ˂ 0.05 signified statistical significance. (D) Biological process, (E) cellular components, and (F) molecular function annotations of gene ontology were significantly enriched in genes that were differentially expressed between HCC827 and HCC827/ER cells. Top 10 terms of each class were performed. FDR ˂ 0.05 signified statistical significance.
Figure 2
Figure 2
Relationship between LMNA and the EMT-related gene between HCC827 and HCC827/ER cells. (A) Heatmap illustrating the expression of genes related to LMNA and EMT between HCC827 and HCC827/ER cells. (B) An interaction network using the STRING online database and Cytoscape software for 22 common gene signatures involved in resistance to erlotinib and EMT mechanisms. (C) A total of 10 genes were identified as hub genes from 22 candidate genes using the CytoHubba tool in Cytoscape: LMNA, EGFR, PTEN, CDH1, CDH2, VIM, SNAI2, TWIST1, AKT1, and AKT2. (D) Relative expression levels of genes related to LMNA and EMT in HCC827 and HCC827/ER cells were confirmed by qPCR. Data are presented as mean ± SD. ** p< 0.001, *** p < 0.001. (E) The expression of laminA/C, laminB1, E-cadherin, N-cadherin, vimentin, and snail was analyzed by Western blot in HCC827 and HCC827/ER cells. GAPDH was used as a loading control.
Figure 3
Figure 3
Effect of LMNA on cell proliferation and resistance to erlotinib in HCC827 and HCC827/ER cells. (A) The mRNA levels and (B) protein expression of LMNA were determined by qRT-PCR and Western blot after lentiviral infections of HCC827 cells with shRNA and HCC827/ER cells with LMNA, respectively. (C) Effect of LMNA knockdown or overexpression on erlotinib efficacy was determined by MTT assay. Data are presented mean ± SD from three independent experiments: ** p < 0.01 versus HCC827/ER cells, ## p < 0.01 versus HCC827 cells. (D, E) A colony-formation assay was performed to investigate the antiproliferation of LMNA in combination with indicated erlotinib in parental and resistant cells. Data are presented as mean ± SD from three independent experiments. Significant differences are indicated as follows: Student’s t-test, *** p < 0.001. (F) and (G) A BrdU incorporation assay was performed in HCC827 and HCC827/ER cells to assess the antiproliferative effect of LMNA combined with 5 nM or 2.5 μM erlotinib after 24 h of incubation, respectively. Data are presented as mean ± SD from three independent experiments. Significant differences are indicated as follows: Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4
Effect of LMNA on the migration and invasive capabilities of HCC827 and HCC827/ER cells. Representative images show the migration and invasion ability of HCC827, HCC827/shLMNA, HCC827/ER, and HCC827/ER-LMNA cells. Invasive cells were stained and counted under a microscope. Data are presented mean ± SD from five randomly selected visual fields. Significant differences are indicated as follows: Student’s t-test, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
LMNA regulation of EMT in erlotinib-acquired resistant lung adenocarcinoma cells; morphological changes from an epithelial phenotype to a spindle-shaped EMT-like morphology in HCC827 cells after knockdown of LMNA. EMT-like morphology in HCC827/ER cells was reversed by overexpression of LMNA. Levels of laminA/C (green), E-cadherin (green), and vimentin (red) were determined by immunofluorescence assay. Nuclei were visualized using DAPI staining (blue). Data are presented mean ± SD from three independent experiments. Significant differences are indicated as follows: Student’s t-test, * p < 0.05, *** p < 0.001.
Figure 6
Figure 6
LMNA regulation of cytoskeletal changes and nuclear deformability. Cytoskeletal F-actin was stained with rhodamine phalloidin (red). Nuclei were visualized using DAPI staining (blue). LaminA/C was determined by immunofluorescence assay (green). Representative pictures of fluorescent laminA/C, F-actin, and nuclei are shown. Scale bar = 100 μm.
Figure 7
Figure 7
LMNA reversal of EMT by regulation of the FGFR/MAPK/c-fos signaling pathway. (A) Western blot analysis was performed to detect the expression of laminA/C, P-FGFR, P-AKT, P-ERK1/2, and P-c-fos in HCC827 and HCC827/ER cells. (B) HCC827/ER cells were transfected with LMNA expression vectors and treated with or without 2.5 μM erlotinib for 24 h, then subjected to Western blot analysis using the indicated primary antibody. Data are presented mean ± SD from three independent experiments. Significant differences are indicated as follows: Student’s t-test, ** p < 0.01, *** p < 0.001.
Figure 8
Figure 8
Graphic abstract of molecular mechanisms of laminA/C-regulated EMT promoting acquired resistance to erlotinib in NSCLC. LaminA/C inhibited the phosphorylation of AKT and FGFR to prevent cell proliferation of erlotinib-resistant cells. Meanwhile, laminA/C decreased ERK1/2 and c-fos activation and decreased vimentin and ZEB1 expressions, resulting in reversing of the process of EMT and improving of erlotinib sensitivity in NSCLC with EGFR mutation. By Figdraw (www.figdraw.com).
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