Inhibition of LIMK by Cofilin-1 peptidomimetics enhances actin depolymerization and reduces metastasis of non-small cell lung cancer

Figures

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  Fig. 1. High expression of LIMK/CFL-1 correlates with poor prognosis in NSCLC patients. A Heatmap showing the expression of the CFL-1, CFL-2 and DSTN genes in non-cancerous and tumor tissues from patients with lung adenocarcinoma, based on TCGA RNA sequencing data. B Expression levels of the CFL-1, CFL-2, and DSTN gene were analysis in paired or unpaired tumor tissues and corresponding adjacent normal tissue from lung adenocarcinoma patients using TCGA RNA sequencing data. *p < 0.05. C Representative IHC staining of the CFL-1 protein in NSCLC tissues from clinical patients. The scale bar is 50 μm lengths. D The IHC scores of CFL-1 in normal lung tissues and lung cancer tissues at different stages (stages I, II, and III) were analyzed using a tissue array. *p < 0.05. E Comparison of overall survival rates in NSCLC patients. The expression of CFL-1 combined with LIMK gene expression was stratified by three groups: CFL-1 high with LIMK high, CFL-1 low plus LIMK low and others. F Comparison of progression-free survival rates in NSCLC patients. The expression of CFL-1 combined with LIMK gene expression was stratified by three groups: CFL-1 high with LIMK high, CFL-1 low plus LIMK low and…

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  Fig. 2. The serine-3 phosphorylation of CFL-1 is a crucial factor in driving alterations in cell morphology and cell motility. A Heatmap of microarray results (GSE42407) of the mRNA expression in CL1-0 (poorly invasive) and CL1-5 (highly invasive) lung cancer cells. B Comparison of LIMK2, pCFL-1, CFL-1 and destrin protein expression in CL1-0 and CL1-5 cells. GAPDH was used as an internal control. C Western blot analysis of the expression of the CFL-1, pCFL-1 and destrin proteins after CFL-1 overexpression with pCDH vector control or pCDH-CFL-1 in CL1-0 cells, or after CFL-1 knockdown with nontarget control (shLuc) in CL1-5 cells. GAPDH was used as an internal control. D Invasive abilities of CL1-0 cells following CFL-1 overexpression with pCDH vector control or pCDH-CFL-1, and CL1-5 cells following the elimination of CFL-1 with non-target control (shLuc). Scale bar: 50 μm. *p < 0.05. E Western blot analysis of the expression of CFL-1 and pCFL-1 (Ser3) in various lung cancer cells. GAPDH was used as an internal control for protein loading (Left). Densitometric quantification of total CFL-1 and pCFL-1 expression was performed using ImageJ software, and results are presented as mean ± SD.…

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  Fig. 3. CMP acted as a competitive decoy of endogenous CFL-1 in lung cancer cells. A Schematic representation of the CMP sequence and its reverse counterpart (RV-CMP), corresponding to amino acids 1–16 of the CFL-1 protein fused to a cell-penetrating sequence. B Western blot analysis of pCFL-1, CFL-1 and GAPDH expression following treatment with various concentrations of CMP or RV-CMP. Quantification of pCFL-1/CFL-1 ratio at different CMP or RV-CMP doses is shown. *p < 0.05. C Cellular uptake of 111In-DTPA-RV-CMP or 111In-DTPA-CMP at different incubation times in CL1-5 cells. *p < 0.05. D NanoSPECT/CT imaging of CL1-5 tumor-bearing mice injected with 99mTc-DTPA-CMP at 3 h post-injection (Sag. Sagittal plane, Cor. Coronal plane). E Representative images of the wound healing assay of CL1-5 cells treated with different doses of RV-CMP or CMP. The scale bar is 100 μm lengths. Quantification of migration abilities of CL1-5 cells was shown. *p < 0.05. F Representative images of the Matrigel transwell assay of CL1-5 cells treated with different dose of RV-CMP or CMP. The scale bar is 50 μm lengths. The quantification of the invasion abilities of CL1-5 cells were shown. *p < 0.05.

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  Fig. 4. CMP regulates actin dynamics to inhibit the ability to metastasis lung cancer cells. A Comparison of the actin polymerization rate and depolymerization rate between CL1-5 cells with CMP or solvent control treatment. *p < 0.05. B Comparison of cell viability in Beas2B, CL1-0, CL1-5, A549, and H1299 cells with different dose of CMP treatment using the MTT assay. C Immunofluorescence staining of CFL-1 and pCFL-1 with DAPI in CL1-5 cells treated with solvent or CMP. Scale bar: 20 μm. The fluorescence intensity was quantified. *p < 0.05. D Western blot analysis of fractionated lysates showing pCFL-1 and CFL-1 levels in the cytosol (α-tubulin as marker) and nucleus (TBP as marker). The pCFL-1/CFL-1 ratio for each fraction is quantified. *p < 0.05. E Western blot analysis for the expression of pCFL-1, CFL-1, E-cadherin, N-cadherin, Vimentin, MMP-9 and GAPDH with different doses of CMP treatment. F The corresponding densitometric quantification of pCFL-1/CFL-1 and EMT markers (E-cadherin, N-cadherin, Vimentin and MMP-9, each normalized to GAPDH) is shown. The data are represented as mean ± SD. *p < 0.05. G Western blot analysis of pCFL-1, total CFL-1, LIMK2, and GAPDH expression in CL1-0…

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  Fig. 5. CMP inhibits lung cancer metastasis in vivo. A The experimental scheme for orthotopic lung cancer model was treated by CMP intravenous injection. B Bioluminescence imaging of the response of lung cancer to CMP and solvent control after 20 days of treatment. Quantification of photon signaling by CMP treatment versus solvent control using the In Vivo Imaging System (n = 5). *p < 0.05. C Representation bioluminescence and microscopic images were evaluated using the IVIS system. Quantification of ex-vivo bioluminescence signal after CMP treatment versus solvent control using IVIS (n = 5). *p < 0.05. D Representative microscopic images of H&E staining and the number of metastatic tumors were counted (n = 5). *p < 0.05. E Representative microscopic images of IHC staining of lung tumor tissue after CMP treatment. The scale bar is 100 μm lengths. *p < 0.05. F Representative microscopic images of H&E staining of normal tissue organ after CMP treatment. G Body weight of the mice in CMP treatment group and the solvent control (saline) group during the experimental course (n = 5). ns not significant. H A Kaplan–Meier plot is displayed for solvent control (saline) or CMP treated mice,…