Introduction: Cancer stem cells (CSCs) play critical roles in lung adenocarcinoma (LUAD) progression, and fatty acid oxidation is key for CSC growth and survival. Therefore, investigating the molecular mechanisms regulating fatty acid β-oxidation in LUAD is important for its treatment. Methods: Bioinformatics analysis assessed CPT1B and MITF expression and their correlation in LUAD tissues, as well as the pathways enriched by CPT1B. qRT-PCR assessed expression of CPT1B and MITF, while CCK-8 and sphere-forming assays were used to measure cell viability and stemness, respectively. Dual staining detected lipid accumulation, while kits were used to measure fatty acid β-oxidation and glycerol content. qRT-PCR was used to assay expression of lipid oxidation genes. Western blot was used to examine expression of stem cell-related markers. Dual-luciferase assay and ChIP assay were used to verify the binding relationship between MITF and CPT1B. Results: CPT1B was found to be highly expressed in LUAD and enriched in linoleic acid metabolism pathway and α-linolenic acid metabolism pathway. Functional experiments showed that CPT1B could promote stemness in LUAD cells by regulating fatty acid β-oxidation. Additionally, CPT1B was found to be regulated by the upstream transcription factor MITF, which was lowly expressed in LUAD and could downregulate CPT1B expression. Rescue experiments revealed that CPT1B/MITF axis could affect stemness in LUAD cells by regulating fatty acid β-oxidation. Conclusion: Transcription factor MITF inhibited transcription of CPT1B to regulate fatty acid β-oxidation, thereby suppressing stemness in LUAD cells. MITF and CPT1B may become new targets for LUAD.

Lung adenocarcinoma (LUAD) is a highly heterogeneous malignant tumor [1]. The primary cause of its malignancy progression may be tumor cell proliferation and distant metastasis [2]. Previous studies have explored molecular targets and related mechanisms that affect the malignant progression of LUAD. For instance, Jiang et al. [3] found that lnc-REG3G-3-1/miR-215-3p facilitates brain metastasis of LUAD via manipulation of Lectin and SLC2A5. Liu et al. [4] discovered that circ_0004140/miR-1184/CCL22 axis promotes the progression of LUAD through circ_0004140. In addition, it has been found that sodium selenite weakens the malignant progression of LUAD by inhibiting stemness [5], indicating that inhibiting tumor cell stemness may be one of the strategic approaches to improve the treatment of LUAD. Therefore, finding a selective method to specifically inhibit tumor stem cells and their features could provide a strategy for LUAD therapy.

Cancer stem cells (CSCs) are cancer cells with self-renewal capability pivotal in tumor progression [6]. Previous studies have investigated factors that affect CSCs in LUAD, including epithelial-mesenchymal transition and tumor microenvironment. Feliciano et al. [7] disclosed that inhibiting transition of epithelial cells by regulating the epithelial-mesenchymal transition process can suppress stem cell characteristics and decrease the number of lung CSCs. Zhao et al. [8] discovered that hypoxia induces the stemness of CD166-positive LUAD stem cells and causes dismal prognosis in patients. In addition, tumor metabolic reprogramming also plays a crucial role in tumor stemness. Wang et al. [9] found that CPT1B promotes breast cancer stemness by manipulating fatty acid β-oxidation. However, no study has explored the molecular mechanism by which fatty acid β-oxidation affects tumor cell stemness in LUAD. Therefore, this study explored and delineated molecular mechanism by which CPT1B regulated fatty acid β-oxidation and affected stemness of LUAD cells, generating a new perspective for LUAD diagnosis and treatment.

CPT1B is the main homolog of the CPT family, and the CPT1B gene plays a crucial role in regulating fat breakdown and energy supply [10]. Previous studies have found that CPT1B acts as an oncogene and drives oncogenic cascade of various tumors, including breast cancer, bladder cancer, and so on [9, 11]. For example, in prostate cancer, the expression of CPT1B is significantly upregulated, and CPT1B overexpression can drive cell proliferation and resistance to enzalutamide [12]. However, the role of CPT1B in regulating fatty acid β-oxidation and stemness of LUAD cells has not been elucidated. Therefore, this study investigated the effect of CPT1B on fatty acid β-oxidation and cell stemness in LUAD, providing a reliable molecular therapeutic target for clinical treatment of LUAD.

In this study, we analyzed expression and biological function of CPT1B in LUAD and clarified molecular mechanism by which CPT1B affected the stemness of LUAD cells. CPT1B was significantly upregulated in LUAD tissues and cells, and high expression of CPT1B was enriched in the linoleic acid pathway and α-linolenic acid pathway. By treating with Bezafibrate, we found that overexpression of CPT1B enhanced stemness of LUAD cells via modulation of fatty acid β-oxidation. In addition, CPT1B had an upstream transcription factor. Rescue experiments presented that overexpression of CPT1B could reverse the suppressive effect of MITF overexpression on the stemness of LUAD cells. Our study demonstrated that transcription factor MITF inhibited the transcription of CPT1B, which regulated fatty acid β-oxidation and affected stemness in LUAD cells, generating new insight into the development of targeted drugs for LUAD.

Bioinformatics

LUAD mRNA expression data (normal: 59, tumor: 535) was downloaded from TCGA. The “EdgeR” package was used to analyze differences of mRNAs (|logFC| >2.0, padj <0.01). Gene of interest was determined through literature citation. The hTFtarget was utilized to predict potential transcription factors upstream of the target genes in LUAD, and transcription factors were determined using Pearson correlation. JASPAR was utilized to predict binding sites between target genes and transcription factors. GSEA was utilized for pathway enrichment analysis of target genes.

Cell Culture

Human LUAD cell lines PC-9, A549, Calu-3, normal bronchial epithelial cell line BEAS-2B, and human embryonic kidney cell line 293T were all purchased from BNCC (China). PC-9 and BEAS-2 cells were maintained in DMEM-H medium (Gibco, USA) +10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA). A549 cells were cultured in F-12K medium (Gibco, USA) +10% FBS and 1% penicillin-streptomycin. Calu-3 cells were cultured in EMEM medium (Gibco, USA) +10% FBS and 1% penicillin-streptomycin. 293T cells were cultured in DMEM-H medium +10% FBS, 1% penicillin-streptomycin, and 2 mM l-glutamine. All cells were cultured at 37°C with 5% CO2.

Cell Transfection

si-CPT1B, oe-CPT1B, oe-MITF, and corresponding negative controls were accessed from Ribobio (China), DMSO was from Sigma (USA), and Bezafibrate (100 mg tablets) was from MedChemExpress (USA). A549, Calu-3, and 293T cells were plated into 24-well plates and transfected with Lipofectamine 3000 (Invitrogen, USA). Functional assays were performed on the cells after 48 h of culture.

qRT-PCR

Total RNA was extracted from LUAD cells with TRIzol reagent (Invitrogen, USA). The isolated total RNA was reversely transcribed into cDNA with PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Japan). qRT-PCR was performed with TB Green® Premix DimerEraser (TaKaRa, Japan). β-Actin was utilized as an internal reference, and 2−∆∆CT was utilized for analysis. Primers used are shown in Table 1.

Table 1.

Primers used in qRT-PCR

GeneSequence
CPT1B Forward primer 5′-TTC​TGC​CTT​TAC​TTG​GTC​TCC-3′ 
Reverse primer 5′-ACA​TGC​GGA​TCT​GGG​ATT​G-3′ 
MITF Forward primer 5′-TTA​AGC​GTA​AGC​ATA​GCC​AT-3′ 
Reverse primer 5′-ACA​GCA​AGC​CCA​ACG​GCA​G-3′ 
Ppara Forward primer 5′-GCT​ATC​ATT​ACG​GAG​TCC​ACG-3′ 
Reverse primer 5′-TCG​CAC​TTG​TCA​TAC​ACC​AG-3′ 
Hmgcs2 Forward primer 5′-TCC​CTT​TAC​CTC​TCC​ACT​CAC-3′ 
Reverse primer 5′-CCA​TAA​GAG​AAG​GCA​CCA​ATC​C-3′ 
Cpt1a Forward primer 5′-GAA​TAA​CCC​AGA​GTA​CGT​GTC​C-3′ 
Reverse primer 5′-GAC​TTA​TAA​TCC​CCG​TCT​CAG​G-3′ 
Acox1 Forward primer 5′-TGC​CTA​TGC​CTT​CCA​GTT​TG-3′ 
Reverse primer 5′-AAA​GCC​TTC​AGT​CCA​GCG-3′ 
β-Actin Forward primer 5′-ACC​TTC​TAC​AAT​GAG​CTG​CG-3′ 
Reverse primer 5′-CCT​GGA​TAG​CAA​CGT​ACA​TGG-3′ 
GeneSequence
CPT1B Forward primer 5′-TTC​TGC​CTT​TAC​TTG​GTC​TCC-3′ 
Reverse primer 5′-ACA​TGC​GGA​TCT​GGG​ATT​G-3′ 
MITF Forward primer 5′-TTA​AGC​GTA​AGC​ATA​GCC​AT-3′ 
Reverse primer 5′-ACA​GCA​AGC​CCA​ACG​GCA​G-3′ 
Ppara Forward primer 5′-GCT​ATC​ATT​ACG​GAG​TCC​ACG-3′ 
Reverse primer 5′-TCG​CAC​TTG​TCA​TAC​ACC​AG-3′ 
Hmgcs2 Forward primer 5′-TCC​CTT​TAC​CTC​TCC​ACT​CAC-3′ 
Reverse primer 5′-CCA​TAA​GAG​AAG​GCA​CCA​ATC​C-3′ 
Cpt1a Forward primer 5′-GAA​TAA​CCC​AGA​GTA​CGT​GTC​C-3′ 
Reverse primer 5′-GAC​TTA​TAA​TCC​CCG​TCT​CAG​G-3′ 
Acox1 Forward primer 5′-TGC​CTA​TGC​CTT​CCA​GTT​TG-3′ 
Reverse primer 5′-AAA​GCC​TTC​AGT​CCA​GCG-3′ 
β-Actin Forward primer 5′-ACC​TTC​TAC​AAT​GAG​CTG​CG-3′ 
Reverse primer 5′-CCT​GGA​TAG​CAA​CGT​ACA​TGG-3′ 

CCK-8 Cell Viability Assay

Cell viability was assayed with CCK-8. Transfected cells were seeded in 96-well plates with three replicates per group. After 0, 24, 48, and 72 h, 10 μL of CCK-8 solution (MedChemExpress, USA) was added to each well, and cells were further cultured for 2 h. The absorbance at 450 nm was measured with a microplate reader (Bio-Rad, USA).

Lipid Droplet Fluorescence Staining

After digestion, cells were evenly plated in 6-well plates with coverslips and induced for differentiation and transfection as described above. Cells were fixed with ice-cold methanol to quench the green fluorescence. Following three washes with PBS, cells were stained with a 1:1,000 dilution of Bodipy 493/503 solution (GLPBIO, USA) for 30 min to stain the lipid droplets. Subsequently, cells were stained with a 1:1,000 dilution of Hoechst 33258 (Sigma, USA) for 30 min to stain the nuclei. After washing with PBS, cells were mounted with glycerol and imaged under a fluorescence microscope [9].

Measurement of Intracellular Fatty Acid β-Oxidation and Glycerol Content

Cells were seeded in 24-well plates with three replicates per group. Fatty acid β-oxidation was assessed with a β-hydroxybutyrate (ketone body) colorimetric assay kit (Millipore, USA), and glycerol content was assessed with a triglyceride assay kit (Abcam, UK) [13].

Stem Cell Sphere Formation Assay

Cells were harvested and washed to remove serum and dissociated into a single-cell suspension in serum-free RPMI-1640 with 20 ng/mL EGF, 20 ng/mL recombinant human bFGF, and 2% B27 supplement (Invitrogen, USA). Cells were then plated in ultra-low attachment 24-well plates (1,000 cells/well). Seven days later, A549 and Calu-3 cell spheres were counted (defined as >20 cells/sphere). All measurements were performed in triplicate [14].

Western Blot

Total protein was isolated from cells using RIPA lysis buffer (Thermo Fisher Scientific, USA). The total protein concentration was determined with a BCA assay kit (Vazyme, China). After separation by SDS-PAGE, proteins were transferred onto PVDF membranes and sealed with TBST buffer containing 5% nonfat milk for 2 h. Membranes were then incubated overnight at 4°C with primary rabbit monoclonal antibodies: anti-Naong, anti-CD3, anti-CD4, and anti-β-actin (Abcam, UK). The following day, membranes were incubated with a secondary antibody (HRP-conjugated goat anti-rabbit IgG) (Abcam, UK) for 2 h, and the chemiluminescent signal was detected using a gel imaging system.

Dual-Luciferase Reporter Assay

293T cells were plated in 24-well plates and transfected with pGL3-Basic-CPT1B-WT-Vector, pGL3-Basic-CPT1B-Vector, pGL4.70 [hRluc] luciferase reporter vectors (Promega, USA), oe-NC, and oe-MITF. Following 48 h of culture, luciferase activity was assessed on the Dual-Luciferase Reporter Gene Assay System (Promega, USA).

ChIP Assay

ChIP was performed with an EZ-Zyme chromatin preparation kit (Millipore, USA). Rabbit anti-MITF monoclonal antibody (Abcam, UK) was used to precipitate DNA crosslinked with MITF, and anti-IgG (Abcam, UK) was a negative control. qRT-PCR is performed to assess DNA fragments using primers in Table 2.

Table 2.

Primers used in ChIP qRT-PCR

PrimerSequence
Site Forward primer 5′-CTT​GGG​GAG​GAG​GAG​AGT​GA-3′ 
Reverse primer 5′-CCC​CAG​AGT​CTC​GTG​AGG​AT-3′ 
PrimerSequence
Site Forward primer 5′-CTT​GGG​GAG​GAG​GAG​AGT​GA-3′ 
Reverse primer 5′-CCC​CAG​AGT​CTC​GTG​AGG​AT-3′ 

Statistical Analysis

Data are presented as mean ± SEM. Statistical analysis was conducted on GraphPad 8.0. Mann-Whitney was used for comparisons between two groups, and Kruskal-Wallis was used for multiple comparisons. Dunnett’s test was used for post hoc analysis. A p value <0.05 represented statistically significant.

Upregulation of CPT1B Expression in LUAD Tissues and Cells

CPT1B plays a role in driving cancer progression [15]. Analysis of TCGA-LUAD indicated that CPT1B was significantly upregulated in LUAD tissues (Fig. 1a). Further qRT-PCR analysis of CPT1B expression revealed a significant increase in LUAD cells compared to normal bronchial epithelial cells BEAS-2B (Fig. 1b). These results indicated that CPT1B was significantly overexpressed in LUAD.

Fig. 1.

Upregulation of CPT1B expression in LUAD tissue and cells. a The expression of CPT1B in LUAD tissue from TCGA database. b qRT-PCR results of CPT1B expression. The asterisk (*) indicates p < 0.05.

Fig. 1.

Upregulation of CPT1B expression in LUAD tissue and cells. a The expression of CPT1B in LUAD tissue from TCGA database. b qRT-PCR results of CPT1B expression. The asterisk (*) indicates p < 0.05.

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CPT1B Promotes LUAD Stemness by Regulating Fatty Acid β-Oxidation

To investigate the pathway through which CPT1B affects the biological function of LUAD, we conducted KEGG pathway analysis of CPT1B in LUAD. The results revealed that CPT1B is related to fatty acid-related pathways such as KEGG_ALPHA_LINOLENIC_ACID_METABOLISM (NES = 1.83, p = 0.006) and KEGG_LINOLEIC_ACID_METABOLISM (NES = 1.55, p = 0.051) (Fig. 2a; online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000534547). To dissect the impact of CPT1B on fatty acid β-oxidation, si-CPT1B was transfected into A549 cells. qRT-PCR analysis reported that CPT1B level was reduced in A549 cells (Fig. 2b). CCK-8 disclosed that the knockdown of CPT1B reduced the viability of A549 cells (Fig. 2c). Further analysis using BODIPY 493/503 staining and Hoechst double staining revealed that knockdown of CPT1B significantly decreased fluorescence intensity of lipid staining, indicating that knockdown of CPT1B could inhibit lipid accumulation in cells (Fig. 2d). The determination of fatty acid β-oxidation and triglyceride content showed that knockdown of CPT1B significantly inhibited fatty acid β-oxidation and triglyceride synthesis in cells (Fig. 2e, f). qRT-PCR analysis of lipid oxidation genes Ppara, Hmgcs2, Cpt1a, and Acox1 showed that knockdown of CPT1B significantly downregulated levels of these genes (Fig. 2g). Thus, CPT1B promoted fatty acid β-oxidation in LUAD. Previous literature has reported that CPT1B can regulate fatty acid β-oxidation to affect stemness in cells [9, 16]. Treatment of A549 cells with the fatty acid oxidation agonist Bezafibrate and subsequent sphere-forming assays showed that knockdown of CPT1B significantly inhibited sphere-forming ability, while Bezafibrate treatment reversed this effect (Fig. 2h–i). Western blot of stem cell markers Naong, CD133, and Oct-4 presented that knockdown of CPT1B markedly reduced their expression, while Bezafibrate treatment reversed the suppressive impact of CPT1B knockdown on stem cell marker expression (Fig. 2j).

Fig. 2.

Silencing CPT1B represses the stemness of LUAD cells by regulating fatty acid β-oxidation. a KEGG pathway analysis of the CPT1B, enrichment score indicates the change in score of enrichment analysis on the ranked gene list. Hits plot shows the position of the genes in the gene set on the ranked gene list, and the ranked list metric is the distribution of the rank values of all the genes after sorting. b qRT-PCR results of transfection efficiency. c The results of the CCK-8 assay measuring cell viability. d BODIPY 493/503 staining and Hoechst double staining of lipid droplets in cells. e, f The determination of fatty acid β-oxidation and triglyceride content in cells, respectively. g The expression of lipid oxidation genes. h, i The results of the sphere-forming assay measuring cell stemness. j Western blot results of stem cell markers. The asterisk (*) indicates p < 0.05.

Fig. 2.

Silencing CPT1B represses the stemness of LUAD cells by regulating fatty acid β-oxidation. a KEGG pathway analysis of the CPT1B, enrichment score indicates the change in score of enrichment analysis on the ranked gene list. Hits plot shows the position of the genes in the gene set on the ranked gene list, and the ranked list metric is the distribution of the rank values of all the genes after sorting. b qRT-PCR results of transfection efficiency. c The results of the CCK-8 assay measuring cell viability. d BODIPY 493/503 staining and Hoechst double staining of lipid droplets in cells. e, f The determination of fatty acid β-oxidation and triglyceride content in cells, respectively. g The expression of lipid oxidation genes. h, i The results of the sphere-forming assay measuring cell stemness. j Western blot results of stem cell markers. The asterisk (*) indicates p < 0.05.

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At the same time, overexpression of CPT1B was performed on Calu-3 cells, and qRT-PCR results showed that compared with the NC group, overexpression of CPT1B significantly increased the expression level of CPT1B in Calu-3 cells (Fig. 3a). CCK-8 revealed that overexpression of CPT1B promoted the vitality of Calu-3 cells (Fig. 3b). Further analysis using BODIPY 493/503 staining and Hoechst double staining showed that overexpression of CPT1B significantly promoted the fluorescence intensity of lipid staining, indicating that overexpression of CPT1B can enhance lipid accumulation in cells (Fig. 3c). The determination of fatty acid β-oxidation and triglyceride content showed that overexpression of CPT1B significantly promoted the accumulation of fatty acids in cell β-oxidation and synthesis of triglycerides (Fig. 3d, e). qRT-PCR analysis of lipid oxidation genes Ppara, Hmgcs2, Cpt1a, and Acox1 showed that overexpression of CPT1B significantly upregulated the levels of these genes (Fig. 3f). The treatment of Calu-3 cells with the fatty acid oxidation agonist Bezafibrate and subsequent spheroidization showed that overexpression of CPT1B significantly promoted spheroidization, while the spheroidization ability was more significant after co-treatment with Bezafibrate (Fig. 3g, h). Western blot of stem cell biomarkers Naong, CD133, and Oct-4 showed that overexpression of CPT1B significantly increased its expression, while the expression ability of stem cell biomarkers was more significant after co-treatment with Bezafibrate (Fig. 3i). Taken together, our results confirmed that CPT1B could promote LUAD stemness by regulating fatty acid β-oxidation.

Fig. 3.

Overexpression of CPT1B promotes the stemness of LUAD cells by regulating fatty acid β-oxidation. a qRT-PCR results of transfection efficiency. b The results of the CCK-8 assay measuring cell viability. c BODIPY 493/503 staining and Hoechst double staining of lipid droplets in cells. d, e The determination of fatty acid β-oxidation and triglyceride content in cells, respectively. f The expression of lipid oxidation genes. g, h The results of the sphere-forming assay measuring cell stemness. i Western blot results of stem cell markers. The asterisk (*) indicates p < 0.05.

Fig. 3.

Overexpression of CPT1B promotes the stemness of LUAD cells by regulating fatty acid β-oxidation. a qRT-PCR results of transfection efficiency. b The results of the CCK-8 assay measuring cell viability. c BODIPY 493/503 staining and Hoechst double staining of lipid droplets in cells. d, e The determination of fatty acid β-oxidation and triglyceride content in cells, respectively. f The expression of lipid oxidation genes. g, h The results of the sphere-forming assay measuring cell stemness. i Western blot results of stem cell markers. The asterisk (*) indicates p < 0.05.

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MITF Is an Upstream Regulatory Molecule of CPT1B

To explore the potential molecular mechanism of how CPT1B affected stemness of LUAD cells through fatty acid β-oxidation, we predicted the possible potential transcription factors upstream of CPT1B by hTFtarget and then took the intersection with the differentially downregulated genes to get 29 potential transcription factors (Fig. 4a). Correlation analysis of these 29 potential transcription factors with CPT1B revealed that MITF was negatively correlated with CPT1B and had the highest correlation (cor = −0.284, p = 2.23E−11) (Fig. 4b; online suppl. Table 2). A JASPAR query on the transcription factor MITF revealed that it had binding sites with CPT1B (Fig. 4c). It has been reported that MITF can be used as a transcriptional suppressor in lung cancer [17]. Therefore, we chose MITF for further study. T test analysis determined that MITF was significantly underexpressed in tumor tissues (Fig. 4d). qRT-PCR detected a significant downregulation of MITF expression levels in LUAD cells (Fig. 4e). Subsequently, dual-luciferase assays and ChIP experiments verified the binding relationship between MITF and CPT1B (Fig. 4f, g). Luciferase activity of CPT1B-WT was significantly reduced in 293T cells overexpressing MITF, while the difference in luciferase activity was not significant in the CPT1B-MUT group. Anti-MITF significantly increased the enrichment of CPT1B. These results indicated that the transcription factor MITF could target and regulate CPT1B gene.

Fig. 4.

MITF is an upstream regulatory molecule of CPT1B. a UpSet plot of transcription factors predicted by the hTFtarget database. b Correlation scatter plot between the predicted MITF transcription factor and CPT1B; (c) binding site of MITF predicted by JASPAR. d The expression of MITF in LUAD tissue analyzed through bioinformatics. e The expression of MITF was tested by qRT-PCR. f, g Dual-luciferase reporter assay and ChIP experiments verifying the binding relationship between MITF and CPT1B. The asterisk (*) indicates p < 0.05.

Fig. 4.

MITF is an upstream regulatory molecule of CPT1B. a UpSet plot of transcription factors predicted by the hTFtarget database. b Correlation scatter plot between the predicted MITF transcription factor and CPT1B; (c) binding site of MITF predicted by JASPAR. d The expression of MITF in LUAD tissue analyzed through bioinformatics. e The expression of MITF was tested by qRT-PCR. f, g Dual-luciferase reporter assay and ChIP experiments verifying the binding relationship between MITF and CPT1B. The asterisk (*) indicates p < 0.05.

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The Transcription Factor MITF Inhibits the Transcription of CPT1B, Thereby Regulating Fatty Acid β-Oxidation to Affect the Stemness of LUAD Cells

Existing literature has reported that MITF is a transcriptional inhibitory factor [18] and exerts a repressive role in non-small cell lung cancer [17]. To dissect the molecular mechanism of MITF modulating CPT1B in LUAD, we constructed the following groups: oe-NC+oe-NC, oe-MITF+oe-NC, oe-NC+oe-CPT1B, and oe-MITF+oe-CPT1B. qRT-PCR presented that overexpression of MITF significantly inhibited expression of CPT1B, while overexpression of CPT1B significantly increased that of CPT1B. The rescue experiment reported that overexpression of CPT1B rescued the suppressive impact of MITF overexpression on CPT1B expression (Fig. 5a). CCK-8 assay results showed that overexpression of MITF significantly inhibited cell viability, while overexpression of CPT1B significantly increased cell viability. Overexpression of MITF rescued the promoting impact of CPT1B overexpression on cell viability (Fig. 5b). Lipid droplet staining results presented that overexpression of MITF reduced lipid accumulation, while overexpression of CPT1B promoted lipid accumulation. Overexpression of MITF rescued the stimulatory impact of CPT1B overexpression on lipid accumulation (Fig. 5c). Results of fatty acid β-oxidation and triglyceride content measurements showed that overexpression of MITF could inhibit fatty acid β-oxidation and triglyceride synthesis, while overexpression of CPT1B could promote fatty acid β-oxidation and triglyceride synthesis. Overexpression of MITF rescued the stimulatory impact of CPT1B overexpression on fatty acid β-oxidation and triglyceride synthesis (Fig. 5d, e). qRT-PCR results of lipid oxidation genes displayed that overexpression of MITF repressed expression of lipid oxidation genes, while overexpression of CPT1B increased expression of lipid oxidation genes. Overexpression of MITF could reverse the promoting effect of CPT1B overexpression on lipid oxidation gene expression (Fig. 5f). Results of cell sphere formation and Western blot manifested that overexpression of MITF repressed cell sphere formation ability and expression of stem cell markers. Overexpression of CPT1B could promote these effects, which could be rescued by overexpression of MITF (Fig. 5g–i).

Fig. 5.

Transcription factor MITF inhibits the transcription of CPT1B, regulating fatty acid β-oxidation and affecting the stemness of LUAD cells. a qRT-PCR results of transfection efficiency in oe-NC+oe-NC, oe-MITF+oe-NC, oe-NC+oe-CPT1B, and oe-MITF+oe-CPT1B groups. b The results of the CCK-8 assay measuring cell viability. c BODIPY 493/503 staining and Hoechst double staining of lipid droplets in cells. MFI means mean fluorescence intensity. d, e The determination of fatty acid β-oxidation and triglyceride content in cells, respectively. f The expression of lipid oxidation genes. g, h The results of the sphere-forming assay measuring cell stemness. i Western blot results of stem cell markers. The asterisk (*) indicates p < 0.05.

Fig. 5.

Transcription factor MITF inhibits the transcription of CPT1B, regulating fatty acid β-oxidation and affecting the stemness of LUAD cells. a qRT-PCR results of transfection efficiency in oe-NC+oe-NC, oe-MITF+oe-NC, oe-NC+oe-CPT1B, and oe-MITF+oe-CPT1B groups. b The results of the CCK-8 assay measuring cell viability. c BODIPY 493/503 staining and Hoechst double staining of lipid droplets in cells. MFI means mean fluorescence intensity. d, e The determination of fatty acid β-oxidation and triglyceride content in cells, respectively. f The expression of lipid oxidation genes. g, h The results of the sphere-forming assay measuring cell stemness. i Western blot results of stem cell markers. The asterisk (*) indicates p < 0.05.

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Meanwhile, we constructed the following groups: si-NC+si-NC, si-MITF+si-NC, si-NC+si-CPT1B, and si-MITF+si-CPT1B. qRT-PCR outcomes presented that silence of MITF significantly promoted expression of CPT1B, while silence of CPT1B remarkably decreased expression level of CPT1B. The rescue experiment reported that silence of CPT1B rescued promotive impact of MITF silence on CPT1B expression (Fig. 6a). CCK-8 assay results showed that silence of MITF significantly promoted cell viability, while silence of CPT1B significantly decreased cell viability. Silence of MITF rescued inhibiting the impact of CPT1B silence on cell viability (Fig. 6b). Lipid droplet staining results presented that silence of MITF promoted lipid accumulation, while silence of CPT1B inhibited lipid accumulation. The silence of MITF rescued the stimulatory impact of CPT1B silence on lipid accumulation (Fig. 6c). Results of fatty acid β-oxidation and triglyceride content measurements showed that silence of MITF could promote fatty acid β-oxidation and triglyceride synthesis, while silence of CPT1B could inhibit fatty acid β-oxidation and triglyceride synthesis. The silence of MITF rescued the stimulatory impact of CPT1B silence on fatty acid β-oxidation and triglyceride synthesis (Fig. 6d–e). qRT-PCR results of lipid oxidation genes displayed that silence of MITF promoted expression of lipid oxidation genes, while silence of CPT1B inhibited expression of lipid oxidation genes. The silence of MITF could reverse inhibiting effect of CPT1B silence on lipid oxidation gene expression (Fig. 6f). Results of cell sphere formation and Western blot manifested that silence of MITF promoted cell sphere formation ability and expression of stem cell markers. The silence of CPT1B could inhibit these effects, which could be rescued by the silence of MITF (Fig. 6g–i). In conclusion, the transcription factor MITF repressed stemness of LUAD cells by manipulating fatty acid β-oxidation through suppression of transcription of CPT1B.

Fig. 6.

Transcription factor MITF inhibits the transcription of CPT1B, regulating fatty acid β-oxidation and affecting the stemness of LUAD cells. a qRT-PCR results of transfection efficiency in si-NC+si-NC, si-MITF+si-NC, si-NC+si-CPT1B, and si-MITF+si-CPT1B groups. b The results of the CCK-8 assay measuring cell viability. c BODIPY 493/503 staining and Hoechst double staining of lipid droplets in cells. MFI means mean fluorescence intensity. d, e The determination of fatty acid β-oxidation and triglyceride content in cells, respectively. f The expression of lipid oxidation genes. g, h The results of the sphere-forming assay measuring cell stemness. i Western blot results of stem cell markers. The asterisk (*) indicates p < 0.05.

Fig. 6.

Transcription factor MITF inhibits the transcription of CPT1B, regulating fatty acid β-oxidation and affecting the stemness of LUAD cells. a qRT-PCR results of transfection efficiency in si-NC+si-NC, si-MITF+si-NC, si-NC+si-CPT1B, and si-MITF+si-CPT1B groups. b The results of the CCK-8 assay measuring cell viability. c BODIPY 493/503 staining and Hoechst double staining of lipid droplets in cells. MFI means mean fluorescence intensity. d, e The determination of fatty acid β-oxidation and triglyceride content in cells, respectively. f The expression of lipid oxidation genes. g, h The results of the sphere-forming assay measuring cell stemness. i Western blot results of stem cell markers. The asterisk (*) indicates p < 0.05.

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LUAD, with the highest mortality rate among lung cancers, takes up around 40% of all cases [19]. Despite the continuous improvement in treating LUAD in recent decades, survival quality of LUAD patients is still unfavorable. Therefore, it is urgently needed to understand molecular pathways facilitating LUAD progression and their related targets. CPT1B, an important isoform of CPT1 and a muscle-type carnitine palmitoyltransferase 1, plays a crucial role in regulating oxidation of fatty acids [20]. In this study, CPT1B was abnormally upregulated in LUAD and was enriched in the linoleic acid pathway and α-linolenic acid pathway, which can promote fatty acid oxidation. Previous studies have shown that fatty acid oxidation is necessary for cancer cell growth and survival [21], and inhibiting fatty acid oxidation can suppress tumor cell stemness and tumor growth. For example, lncRNA MACC1-AS1 regulated by mesenchymal stem cells facilitates stemness of gastric cancer cells through fatty acid oxidation [22]. Pike et al. [23] found that inhibiting fatty acid oxidation with etomoxir damages NADPH production and enhances reactive oxygen species, leading to ATP consumption and cell death in glioblastoma cells. In addition, studies have found that CPT1B can promote cancer progression by regulating fatty acid β-oxidation. For example, PITNC1 upregulates CPT1B expression and promotes gastric cancer metastasis by regulating fatty acid β-oxidation [24]. In this study, we used a fatty acid oxidation activator for rescue experiments and disclosed that CPT1B facilitated stemness of LUAD cells by regulating fatty acid β-oxidation. Our research results revealed the function of CPT1B in LUAD stem cells and its mechanism of action and suggested that targeting the fatty acid oxidation pathway may be an effective way to inhibit LUAD tumor stem cells.

To explore and delineate the potential mechanism of regulating CPT1B in LUAD, we found through bioinformatics analysis that there was an upstream transcription factor, MITF, that regulated CPT1B. Further molecular experiments validated that MITF could directly target and bind to CPT1B. MITF, a transcription factor associated with microphthalmia, recognizes E-Box sequence (CATGTG) in the promoter region of target genes via the BHLH-ZIP domain, regulating transcription of downstream genes and exerting its function [25]. For example, in renal angiomyolipoma, the same type of MITF (MITF-A) is highly expressed, promoting cell proliferation and invasion [26]. Hsiao et al. [17] found that silencing MITF drives cell malignant progression by upregulating FZD7 in non-small cell lung cancer. In this study, a significant negative correlation between MITF and CPT1B was revealed. It is worth noting that the correlation coefficient between MITF and CPT1B indicates a weak correlation, but the strength of the correlation may be influenced by multiple factors, including sample size and data variability. Furthermore, the biological relationship between these two genes may not necessarily lead to a strong correlation. Nevertheless, we believe that this correlation is still worth reporting as it provides a deeper understanding of the potential regulatory relationship between MITF and CPT1B in the context of our research. At the same time, MITF was downregulated in LUAD tissues and cells, and that overexpression of MITF could regulate fatty acid β-oxidation by inhibiting CPT1B transcription, thus suppressing stemness of LUAD cells. This suggested that MITF/CPT1B axis may be an actionable target for inhibiting LUAD tumor stem cells.

Viewed in total, we demonstrated that, for the first time, transcription factor MITF inhibited CPT1B transcription and regulated fatty acid β-oxidation, affecting the stemness of LUAD cells. Our data suggested that MITF/CPT1B axis may be a new potential target for LUAD. However, our study still had some limitations, such as the lack of verification of the impact of CPT1B on stemness of LUAD cells at animal and clinical levels. Therefore, the ongoing step is to collect clinical samples to investigate mechanism of MITF/CPT1B regulatory axis in modulating stemness of LUAD, laying a theoretical basis for LUAD treatment. In conclusion, this study deepens our understanding of molecular mechanisms of LUAD development and offers new insights for LUAD treatment.

The cell lines used in this study were obtained from BNCC (China). Ethical approval for the use of this is not required in accordance with local/national guidelines.

The authors declare that they have no competing interests.

The study was not funded by any organization.

W.J.T. and H.G.T. conceived and designed the study. Z.M.C. collected the data. S.H.X. analyzed and interpreted the data. W.J.T. and H.Y. contributed to the manuscript writing and editing. All authors read and approved the final manuscript.

Additional Information

Weijian Tang and Hongguang Tang contributed equally.

All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author.

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