Cell death-inducing DFF45-like effector C intervenes the progression of nonsmall-cell lung cancer via modulating lipid metabolism

Abstract

Background: The cell death-inducing DFF45-like (CIDE) effector family, plays a crucial role in lipid droplet formation and stability. Its N-terminal and C-terminal domains are closely linked to cell apoptosis and lipid droplet growth. However, the expression and functional role of CIDEC in nonsmall-cell lung cancer (NSCLC) remain unexplored. This study aims to investigate the expression patterns and functional implications of CIDEC in NSCLC, with a particular focus on its role in lipid droplet metabolism and tumor progression.

Methods: We analyzed CIDEC expression levels in NSCLC tissues compared to normal lung tissues using immunohistochemistry and quantitative PCR. To elucidate the functional role of CIDEC, we performed in vitro and in vivo experiments involving overexpression and knockdown of CIDEC in NSCLC cell lines. Proliferation, invasion, and migration assays were conducted to assess the impact of CIDEC on tumor cell behavior. Additionally, lipid droplet morphology and triacylglycerol (TAG) content were evaluated using fluorescence microscopy and biochemical assays. The expression of ATGL, a downstream gene of CIDEC, was also measured to explore the mechanistic link between CIDEC and lipid droplet lipolysis.

Results: Our findings revealed that CIDEC expression was significantly downregulated in NSCLC tissues compared to normal tissues. Functionally, overexpression of CIDEC inhibited the proliferation, invasion, and migration of NSCLC cells both in vitro and in vivo . Furthermore, CIDEC overexpression led to an increase in lipid droplet diameter and TAG content, whereas CIDEC knockdown resulted in the reduction and fragmentation of large lipid droplets. Mechanistically, CIDEC deficiency was associated with increased ATGL expression and reduced lipid droplet content, while CIDEC overexpression correlated with elevated TAG levels and decreased ATGL protein levels. These results suggest that low CIDEC expression in NSCLC promotes tumor progression by enhancing ATGL-mediated lipolysis.

Conclusions: This study is the first to establish a link between CIDEC expression and lipid droplet lipolysis in NSCLC. Our findings indicate that CIDEC plays a critical role in regulating lipid metabolism and tumor progression in lung cancer. The downregulation of CIDEC in NSCLC promotes tumorigenesis by enhancing ATGL-induced lipolysis, highlighting CIDEC as a potential therapeutic target for lung cancer through metabolic reprogramming of lipid droplets. These insights provide a novel direction for targeted treatment strategies in NSCLC.

Keywords: apoptosis; cell death-inducing DFF45-like; lipid; non-small cell lung cancer.

Figure 1.

Expression profile of CIDEC in NSCLC and construction of stable NSCLC cell lines with CIDEC overexpression and knockdown. (A) Average expression levels of CIDEC gene in NSCLC tumor tissues (n = 1017) and adjacent/normal tissues (n = 686) in TCGA and GTEx databases. (B) Western Blot detection of CIDEC protein expression in lung cancer cell lines and normal lung epithelial cell lines, with (C) statistical results compared to the protein expression of lung epithelial cell BEAS-2B. (D) Green fluorescence observed in H1299 cells 72 h after virus infection. (E) Green fluorescence observed in A549 cells 72 h after virus infection. (F, H) Western blot detection of CIDEC protein expression level in H1299 and H1975 cells after overexpression of CIDEC-oe lentivirus and (G, I) gray value statistical graph. (J, L) Western blot detection of CIDEC protein expression level in A549 and PC9 cells after silencing of CIDEC-shRNA and (K, M) gray value statistical graph (*P < 0.01, **P < 0.05, ***P < 0.001).

Figure 2.

Effects of CIDEC expression intervention on apoptosis in NSCLC cells. (A) Flow cytometry detection of the effect of CIDEC expression intervention on apoptosis in H1299 cells and H1975 cells in the CIDEC-OE group and vector group, as well as (A’) the statistical chart of the total apoptotic cells; (B) A549 cells and PC9 cells in the shCIDEC#3 group and shNC group, as well as (B’) the statistical chart of the total apoptotic cells. (C–F) Western Blot detection of the influence of CIDEC expression intervention on apoptosis-related proteins in H1299 cell CIDEC-OE group and vector group and A549 cell shCIDEC#3 group and shNC group (*P < 0.01, **P < 0.05, ***P < 0.001).

Figure 3.

Effects of CIDEC overexpression and knockdown on the migratory and invasion capacity of NSCLC cells. (A, B) Scratch assay detecting the effect of CIDEC expression intervention on cell migration in H1299 and H1975 cells in the CIDEC-OE group and vector group; A549 and PC9 cells in the shCIDEC#3 group and shNC group. (C, D) The quantity of cells that passed through the Transwell chambers in the CIDEC-OE group and vector group of H1299 cells and H1975 cells. (E, F) The number of cells that passed through the Transwell chambers in the shCIDEC#3 group and shNC group of A549 cells and PC9 cells. (Scale bar: 200 µm; *P < 0.01, **P < 0.05, ***P < 0.001).

Figure 4.

Impacts of CIDEC expression on intracellular lipid droplet morphology and distribution in NSCLC cells. (A–F) Representative images of oil red O staining shows the morphology of lipid droplets within cells at 400×. (A, B) H1299 and H1975 cells transfected with either empty vector or CIDEC overexpression plasmid; (D, E) A549 and PC9 cells subjected to CIDEC knockdown and corresponding negative control. (C, F) Statistical analysis of largest LDs’ area per group. (G–I) Representative images of Bodipy 493/503 fluorescent staining illustrating lipid droplet morphology and intracellular distribution in CIDEC-overexpressing cells, imaged under a 400 × fluorescence microscope. (G, H) H1299 and H1975 cells with vector control and CIDEC overexpression; (I) Statistical analysis of largest LDs’ diameter in each group. Scale bar = 50 µm. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Lipidomics results of LC/MS detection after overexpression of CIDEC. Heatmap shows the relative content of TAGs in (A) H1299-Vector cells and H1299-CIDEC-OE cells; (B) H1975-Vector cells and H1975-CIDEC-OE cells, (n = 3). (C) Top 10 significant TAG relative quantification in H1299-Vector cells and H1299-CIDEC-OE cells. (D) Top 10 significant TAG relative quantification in H1975-Vector cells and H1975-CIDEC-OE cells. (E) Relative total quantification of TAG, DAG, and FFA in H1299-Vector cells and H1299-CIDEC-OE cells. (F) Relative total quantification of TAG, DAG, and FFA in H1975-Vector cells and H1975-CIDEC-OE cells. (G) ATGL protein level in the CIDEC-OE group and vector group of H1299 cells and H1975 cells. (H) Gray value histogram of ATGL protein level (*P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 6.

CIDEC reduces the malignant biological behavior of NSCLC by downregulating ATGL. (A) Proliferation capacity of H1299 cells overexpressing CIDEC and co-expressing ATGL. (B) Proliferation capacity of H1975 cells overexpressing CIDEC and co-expressing ATGL. (C, D) Apoptosis tendency of H1299 cells overexpressing CIDEC and co-expressing ATGL. (E, F) Apoptosis tendency of H1975 cells overexpressing CIDEC and co-expressing ATGL. (G, H) Invasive and migratory ability of H1299 cells overexpressing CIDEC and co-expressing ATGL. (I. J) Invasive and migratory ability of H1975 cells overexpressing CIDEC and co-expressing ATGL. (*P < 0.05, **P < 0.01, *** P < 0.001).

Figure 7.

Impacts of CIDEC expression on tumorigenicity in nude mouse Xenograft models. (A, D) Comparison of growth curves and final size, weight of subcutaneous xenograft tumors in each group on day 28. (B, E) Line graphs showing the volume-time growth curves of the tumors that were visible to the naked eye on day 10, measured every 3 days. (C, F) Statistical graphs of tumor weight in each group on day 28. (G, H) Immunohistochemical staining shows the expression of CIDEC and ATGL proteins in the tissue of nude mouse tumors. Tissues from subcutaneously transplanted tumors in nude mice were embedded in paraffin and stained with IHC to observe the expression of CIDEC and ATGL after overexpression or knockdown of CIDEC (Scale bar: 100 µm; NS P > 0.5, *P < 0.05, **P < 0.01, ***P < 0.001).