TIMM8B promotes oxidative phosphorylation and glycolysis by inhibiting the mtROS/ASK1/JNK signaling pathway in ovarian cancer

Abstract

Background

Ovarian cancer is a complicated and heterogeneous disease. In this study, we investigated the functional significance of the gene TIMM8B, which is differentially expressed in ovarian cancer to better understand the molecular processes involved in the development of this disease.

Methods

RNA sequencing was performed on ovarian cancer tissues and adjacent noncancerous tissues. The mRNA expression profiles obtained from the sequencing data (transcripts), TCGA-OV, and GSE14407 were subsequently used to identify common DEGs. GO, KEGG pathway, and PPI network analyses of these common DEGs were conducted. The expression of TIMM8B was examined in ovarian cancer tissues and cell lines. The effects of TIMM8B on cellular behaviors, such as proliferation, apoptosis, migration, invasion, and energy metabolism, were assessed by conducting cell-based assays. Additionally, the regulation of these processes by TIMM8B through the mtROS/ASK1/JNK signaling pathway was investigated.

Results

A total of 233 common DEGs were identified in ovarian cancer. The results of the GO analysis revealed enrichment in extracellular matrix organization, collagen-containing extracellular matrix, and transmembrane transporter activity, among others. The results of the KEGG pathway analysis revealed the involvement of DEGs in pathways such as oxidative phosphorylation and glycolysis/gluconeogenesis. TIMM8B was upregulated in ovarian cancer tissues and cell lines. TIMM8B enhanced oxidative phosphorylation, glycolysis, proliferation, migration, and invasion and inhibited apoptosis in ovarian cancer cells. TIMM8B was found to exert its effects through the suppression of mtROS/ASK1/JNK signaling.

Conclusion

TIMM8B may regulate the mtROS/ASK1/JNK pathways, leading to an increase in oxidative phosphorylation and glycolysis. Targeting TIMM8B and its associated signaling pathway may help in the development of new treatment approaches for ovarian cancer.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13062-025-00663-6.

Introduction

In women, ovarian cancer is the deadliest of all gynecological tumors. Although the available standard treatments, such as tumor reduction surgery and taxane and platinum chemotherapy, initially yield positive responses, relapse and progression are highly likely [1]. Therefore, new targeted therapeutic techniques are urgently needed to improve patient outcomes.

Human cells contain six small TIM proteins, TIMM8A, TIMM8B, and TIMM9 [2]. TIMM8A and TIMM8B are homologous genes with significant differences in the presumed substrate-binding regions [3]. TIMM8B is expressed mainly in endocrine and skeletal muscle tissues [2]. TIMM8A has a greater effect on neuron-like cells (SH-SY5Y), whereas TIMM8B has a more prominent effect on HEK293 cells [4]. Mutations in the TIMM8B gene can result in a rare X-linked disorder known as Mohr-Tranebjaerg syndrome, which is characterized by hearing loss, neurological issues, and visual impairment [5, 6]. However, only a few bioinformatics analyses have focused on the role of TIMM8B in cancer [7, 8]. TIMM8B has been found to be highly expressed in breast and colon cancer [8, 9]. A study linked an increase in the expression of TIMM8B to poorer overall survival rates in lung adenocarcinoma (LUAD) patients [10]. Additionally, TIMM8B plays a role in shaping the immunosuppressive tumor microenvironment in LUAD, affecting immune cell infiltration and the response to immunotherapy. However, the function of TIMM8B in ovarian cancer remains unexplored.

Mitochondria are required for various cellular activities, including energy production, cell division, and programmed cell death [11]. TIMM8B functions as a chaperone in the mitochondrial intermembrane and is required for guiding and integrating hydrophobic transmembrane proteins into the inner mitochondrial membrane [12]. Therefore, TIMM8B may regulate ovarian cancer by affecting mitochondrial function. Metabolic reprogramming is a key marker in human tumors [13, 14]. In many solid tumors, glucose is oxidized from the mitochondria to the cytoplasm, and in the cytoplasm, glucose is rapidly metabolized into lactic acid [15, 16]. Some types of cancer depend solely on glycolysis for energy metabolism [17, 18]. Different subgroups of tumors exhibit different metabolic preferences, with some preferring aerobic glycolysis and others preferring oxidative phosphorylation (OXPHOS) [19–23]. The main source of energy for epithelial ovarian cancer cells is OXPHOS, not glycolysis [24]. Studies performed on cell cultures and mice indicated that compared to normal epithelial cells, ovarian cancer cells exhibit an increase in OXPHOS and glycolysis [25, 26]. However, the effects of TIMM8B on these metabolic pathways in ovarian cancer cells and its regulatory mechanism have not been investigated.

Mitochondria produce ATP using the electron transport chain (ETC) of OXPHOS, which is composed of several multisubunit complexes, including complexes I, II, III, and IV [27]. Studies have reported defects in ETC complexes in cells lacking TIMM8B [4]. In HEK293 cells lacking TIMM8B, glycolysis is disrupted [4]. TIMM8B can serve as an auxiliary assembly factor, contributing to the formation of the mitochondrial complex IV S3 subcomplex [28]. Therefore, TIMM8B may affect both OXPHOS and glycolysis. The ETC, which is mediated by OXPHOS complexes, acts as the origin of mitochondrial reactive oxygen species (mtROS). Under normal physiological conditions, about 0.2–2% of electrons deviate from the electron transport chain and interact with oxygen to form superoxides or hydrogen peroxide [29, 30]. During OXPHOS, a defective expression of the complex (CO I-V) induces a “leakage” transfer of electrons to molecular oxygen, which increases ROS production [31]. Dysfunction of complex IV is associated with an increase in mtROS and cytotoxicity [32]. Therefore, TIMM8B may regulate the production of mtROS by regulating OXPHOS.

The production of ROS can activate the ASK/JNK signaling pathway [33–35]. Therefore, TIMM8B may also play a role in this system. ASK1 is a 170-kDa protein that consists of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain [36]. ASK1 can be activated by various stimuli, including TNF, ROS, or serum starvation, which oxidize Trx1 to release it from ASK1 [37, 38]. The ROS/ASK1/JNK signaling axis is crucial in ovarian cancer. Inhibiting ROS can significantly decrease the activation of JNK and ASK1 [39]. The activation of the ROS/ASK1/JNK signaling pathway is essential for increasing cisplatin sensitivity in A2780/DDP cells treated with ABT-737. PUMA triggers ROS/ASK1/JNK activation to promote apoptosis in ovarian cancer cells [40].

In this study, we showed that TIMM8B promotes OXPHOS, reduces mtROS, and inhibits the ASK/JNK signaling pathway in ovarian cancer cells. Inhibition of this pathway leads to an increase in OXPHOS, glycolysis, cell proliferation, migration, and invasion, along with a decrease in cell apoptosis.

Materials and methods

Patients, sampling, and RNA sequencing

Patients with ovarian cancer admitted to The Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital, Peking University Cancer Hospital Yunnan) donated tissue samples of ovarian cancer and adjacent tissues for the study. The Ethics Committee of The Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital, Peking University Cancer Hospital Yunnan) reviewed and approved the study protocol according to the Declaration of Helsinki, and all participants provided informed consent.

RNA sequencing was conducted on three pairs of tissue samples provided by Novogene Co., Ltd. The purified total RNA was used for constructing a cDNA library, and the quality of the libraries was evaluated using an Agilent 2100 bioanalyzer. Then, the PCR product was sequenced using the Illumina platform, where the fluorescence signal from every cluster was captured and transformed into its corresponding base; using this method, the sequencing data were obtained.

Data collection and bioinformatics analysis

The mRNA expression profiles of patients with ovarian cancer (TCGA-OV) were obtained from The Cancer Genome Atlas (TCGA) database. The GSE14407 dataset was obtained from the Gene Expression Omnibus (GEO) database.

The mRNA expression patterns in three sets of tissue samples (Transcript), TCGA, and GSE14407 datasets were subsequently analyzed, and the differentially expressed genes (DEGs) were identified using the criteria of an adjusted p-value < 0.05 and a |log2FoldChange|> 0.585. Next, we used the Venn software online to identify the common DEGs across the three datasets. To investigate these DEGs further, we used the clusterProfiler package in R for gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis.

The online tool STRING (https://cn.string-db.org/) was used to evaluate the protein–protein interaction (PPI) network to investigate the relationships among these DEGs (with a maximum number of interactors = 0 and confidence score ≥ 0.4).

Cell culture

We purchased the human normal ovarian epithelial cell line IOSE80 (TCH-C402) from Haixing Biological Technology Co., Ltd. The ovarian cancer cell lines OVCAR3 (iCell-h168) and SKOV3 (FH0135) were purchased from iCell and Shanghai Fuheng Biological Co., Ltd., respectively. The cells were cultured at 37 °C with 5% CO₂ in RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum.

Cell transfection

To knock down TIMM8B, short hairpin RNAs (shRNAs) and nontargeting RNA (scrambled RNA) were designed and synthesized. The shRNA sequences used were as follows: 5′-CATCACTTCATGGAGTTATGT-3′ (sh-TIMM8B-1); 5′-GTGTAGACCGCTTCATTGACA-3′ (sh-TIMM8B-2); and 5′-CATCACTTCATGGAGTTATGT-3′ (sh-TIMM8B-3). The sequences of TIMM8B were also designed and synthesized. The TIMM8B sequence was inserted into the lentiviral vector JS043 containing the XhoI cloning site, resulting in the formation of the overexpressed recombinant plasmid JS043-TIMM8B. In contrast, the interference sequence was incorporated into the lentiviral vector JS039 containing the EcoRI and BamHI cloning sites, which led to the formation of the interference recombinant plasmid JS039-shTIMM8B. The recombinant plasmids were subsequently amplified and verified to confirm their integrity. The lentivirus particles were prepared by cotransfecting JS043-TIMM8B, JS039-shTIMM8B, or the respective control vectors with lentiviral packaging plasmids into 293 T cells. The concentration and viral titer of the lentivirus particles were determined. Finally, the lentivirus mixture containing JS043-TIMM8B and JS039-ShTIMM8B was used to infect SKOV3 and OVCAR3 cells, and the effectiveness of the infection was observed 72 h after infection.

RT-qPCR

RNA was extracted from the cells using TRIzol reagent, after which the resulting lysis products were treated with chloroform. The RNA obtained was used to measure the concentration of total RNA and synthesize cDNA using All-in-one qRT SuperMix (R333-01, Vazyme). Then, qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme). The primers used were as follows: TIMM8B-F: 5′-GAATCGCCTAGACTCTCGCA-3′; TIMM8B-R: 5′-GGTGATGGCAAGAGTGGTGT-3′; GAPDH-F: 5′-GTTCGTCATGGGTGTGAACC-3′; and GAPDH-R: 5′-CATCCACAGTCTTCTGGGTG-3′. GAPDH was used as an internal reference for quantifying mRNA levels, and relative expression levels were calculated using the 2^–ΔΔCt method. The expression was normalized to that of GAPDH.

Western blotting

Cell lysis solution (P0013, Beyotime) was used to extract total protein from the cells. After protein extraction, a BCA protein assay kit (P0010, Beyotime) was used for protein quantification, followed by the separation of the proteins via SDS-PAGE. The membranes were incubated with the following primary antibodies: TIMM8B (1:1000, abs152377, Absin), cyclin E1 (1:2000, AF0144, Affinity), p27 (1:1000, 3686S, Cell Signaling Technology), cyclin D1 (1:3000, ab134175, Abcam), p21 (1:1000, 14472S, Cell Signaling Technology), E-cadherin (1:1000, 14472S, Cell Signaling Technology), vimentin (1:1000, ab8978, Abcam), Snail (1:1000, ab216347, Abcam), N-cadherin (1:1000, 13116S, Cell Signaling Technology), BCL-XL (1:1000, AF6414, Affinity), BCL-2 (1:1000, AF6139, Affinity), BAX (1:2000, 29,552–1-AP, Proteintech), cleaved-Caspase 3 (1:2000, AF0722, Affinity), cleaved-Caspase 7 (1:2000, AF4023, Affinity), NDUFS1 (Complex I, 1:1000, DF7041, Affinity), SDHB (Complex II, 1:1000, DF12732, Affinity), CYTB polyclonal (Complex III, 1:2000, 55,090–1-AP, Proteintech); Complex IV (1:1000, 4850, Cell Signaling), ATP5A (Complex V, 1:1000, ab14748, Abcam), ASK1 (1:1000, 8662S, Cell Signaling Technology), p-JNK (1:1000, AF3318, Affinity), JNK (1:1000, AF6318, Affinity), and GAPDH (1:5000, AB0037, Abways) at 4 °C overnight. For incubation, the samples were exposed to either rabbit anti-mouse HRP (Beyotime, A0216) or goat anti-rabbit HRP (Beyotime, A0208).

Determination of cell viability

A cell proliferation detection kit (abs152377, Absin) was used to evaluate cell viability following the manufacturer’s guidelines. MTS solution (10 μL) was added to the culture and then incubated at 37 °C for 4 h. The absorbance was measured at 490 nm. The experiment was conducted in triplicate.

Colony formation

Cell suspensions were formed by initially digesting the cultured cells with 0.25% pancreatic enzymes. The cell suspensions were then counted using blood cell counting plates. Next, the cells were inoculated in six-well plates, with each well containing 300 cells. The plates were cultured at 37 °C and 5% CO₂. The size of the clones was regularly observed, and the culture was stopped when a certain number of visible clones appeared in the Petri dish. The cells were fixed by removing the medium and treating them with 4% paraformaldehyde. Next, the cells were stained with 0.2% crystal violet before being washed and dried, and the colonies were photographed.

Transwell assay

To measure cell migration, cells digested using pancreatic enzymes were mixed with a serum-free medium to form a cell suspension. The cell suspension (200 μL) was added to the upper chamber, and a medium containing 10% serum (500 μL) was added to the lower chamber. Then, the cells were cultured at 37 °C for 24 h. Next, the cells were fixed with 4% paraformaldehyde for 10 min and treated with formaldehyde containing 0.1% crystal violet (C02121, Beyotime) for 10 min. The cells were photographed after rinsing the extra dye with PBS.

To conduct the cell invasion assay, the wells were coated with Orgmatrix gel (#111,005, GENOM Bio, Hangzhou, China) and incubated at 37 °C for 4 h before adding the cell suspension. The remaining steps of the experimental process were the same as those for cell migration.

Cell apoptosis and the cell cycle

A cell apoptosis detection kit (40310ES50, Yeasen) was used to assess cell apoptosis. The cells (1 × 10⁵) were collected and stained with annexin V-FITC and PI staining solution. After 15 min of incubation in the dark, the samples were analyzed using a flow cytometer.

A cell cycle and apoptosis analysis kit (C1052, Beyotime) was used to measure cell proliferation. After the cells were fixed with 70% ethanol precooled in an ice bath at 4 °C for 2 h, they were stained with 0.5 mL of propidium iodide solution and incubated at 37 °C for 30 min in the dark. The samples were subsequently detected by conducting flow cytometry.

Tumor induction in nude mice

Four groups of healthy nude mice, aged 4–6 weeks, were randomly established, with each group containing five mice. Each mouse received a dose of 5 × 10⁶ cells in the armpit area. Throughout the feeding period, the mice had unlimited access to food and water, with the room temperature set at 22–25 °C. The mice were monitored every week, and their tumor volume was measured. After 30 days, the tumor tissue was collected to measure the tumor weight and volume. All animal experiments were approved by the Animal Experimental Ethics Review Committee of Kunming Medical University.

Measurement of hexokinase activity, lactate production, and ATP content

Glucose uptake (hexokinase activity) and lactate production in ovarian cancer cells were evaluated using a hexokinase activity assay kit (BC0740, Solarbio) and lactic acid content assay kit (BC0740, Solarbio), respectively, following the protocols provided by the manufacturers. The levels of ATP were measured using an ATP assay kit (S0026, Beyotime) following the instructions provided by the manufacturer.

Measurement of mtROS

Pancreatic enzymes were used to digest the cells, and the cell concentration was subsequently adjusted to 1 × 10⁶ cells/mL using a medium. After obtaining a 500 μL cell suspension, MitoSOX (M36005, Thermo Fisher) was added at a final concentration of 5 μM, and the mixture was thoroughly mixed. Following incubation for 20 min at 37 °C in an incubator in the dark, the cell suspension was centrifuged, and the supernatant was removed. The cells were then washed three times with PBS that had been preheated to 37 °C. Finally, 500 μL of DPBS (14,190,144, Gibco) was added, and the proteins were detected using a flow cytometer.

Seahorse cell energy metabolism experiment

The Seahorse XF Cell Mito Stress Test Kit (103,015–100, Agilent) was used for the determination of oxidative phosphorylation. Firstly, the probe plate was hydrated by adding 200 μL of sterile water to the wells and incubating overnight in a 37 °C incubator. After discarding the sterile water, 200 μL of XF calibration solution was added to each well, and the entire probe plate device was placed in an O₂-free cell incubator at 37 °C for 60 min of hydration. Agilent's dedicated DMEM phenol-red-free medium was used for the detection solution by adding 100 × pyruvic acid, glucose, and glutamine. The cultured cells were then removed, the cell culture medium was aspirated, leaving 20 μL, and the cells were washed twice with the detection solution before adding 160 μL of the detection solution. The cell culture plates were placed in a 37 °C incubator for 60 min, ready for testing by the machine. Oligomycin was added to well A, FCCP was added to well B, and Rot-AA was added to well C, followed by placing the probe plate on the cell wells and finally detecting the results on the cell energy metabolism analyzer (Seahorse XFe96, Agilent).

The Seahorse XF Cell Glycolysis Stress Test Kit (103,020–100, Agilent) is used for the determination of glycolysis. The first step was to hydrate the probe plate by adding 200 μL of sterile water to the wells and allowing it to incubate overnight at 37 °C. Next, the sterile water was removed, and 200 μL of XF calibration solution was added to each well. The entire probe plate device was then placed in an O₂-free cell incubator at 37 °C for 60 min for further hydration. Agilent's specialized DMEM phenol-free red medium was utilized, along with 100 × Glutamin added as the detection solution. Once the cultured cells were removed and the cell culture medium was aspirated, leaving 20 μL, the cells were washed twice with the detection solution. Subsequently, 160 μL of the detection solution was added before placing the cell culture plates in a 37 °C incubator for 60 min in preparation for testing by the machine. Oligomycin, FCCP, and Rot-AA were then added to wells A, B, and C respectively. The probe plate was placed on the cell wells, and the results were detected using the cell energy metabolism analyzer (Seahorse XFe96, Agilent).

Statistical analysis

The mean ± standard deviation (SD) was used to present all data in this study. The differences between groups were analyzed using GraphPad Prism 8.0 (GraphPad Software, USA). The differences between groups were determined by conducting Student’s t-tests, whereas the differences among multiple groups were determined by conducting a one-way analysis of variance followed by Tukey’s post hoc test. Survival analysis of ovarian cancer patients with available survival data was conducted using the Kaplan–Meier plotter. All results were considered to be statistically significant at P < 0.05.

Results

Identification of DEGs in ovarian cancer and functional enrichment analysis of DEGs

We conducted RNA sequencing on ovarian cancer tissues (OVs) and adjacent noncancerous tissues (NTs). We established certain criteria for differential gene analysis, specifically an adjusted p-value < 0.05 and a |log2FoldChange|> 0.585. The mRNA expression profiles obtained from the sequencing data (transcripts), TCGA-OV dataset, and GSE14407 dataset were analyzed to identify common DEGs. In ovarian cancer (Fig. 1A), 123 downregulated and 110 upregulated common DEGs were identified.

Fig. 1.

Identification of DEGs in ovarian cancer and functional enrichment analysis of DEGs. A Authentication of 231 common DEGs in the three datasets was performed using the Venn diagram software. B Gene ontology analysis of common DEGs in ovarian cancer was performed. C KEGG pathway analysis of common DEGs in ovarian cancer was performed. D The 30 core genes from the PPI network were constructed using the STRING online database

Next, the GO annotations of all 233 DEGs were analyzed (Fig. 1B). The biological processes were enriched primarily in extracellular matrix organization and extracellular structure organization. The cellular component annotations were primarily concentrated in the collagen-containing extracellular matrix and the mitochondrial inner membrane. Regarding molecular functions, primary active transmembrane transporter activity and protein kinase A binding were the main enriched terms.

A KEGG pathway analysis was conducted to identify the pathways associated with these DEGs. The DEGs were enriched in pathways such as human papillomavirus infection, diabetic cardiomyopathy, OXPHOS, and glycolysis/gluconeogenesis (Fig. 1C).

The 30 core genes in the PPI network were shown in Fig. 1D. Among the top 10 genes, TIMM8B was a DEG and had the largest |log2FC|(2.3936) (Table 1). Therefore, we focused on studying TIMM8B.

Table 1.

The top 10 core genes in the PPI network

Genes	log2FC	Adjusted p-value
GAPDH	1.105224598	0.317426959
BCL2	− 1.044996902	0.261360814
EZH2	0.989913355	0.351190106
H4C6	–	–
FBN1	− 0.781750242	0.379209366
COX5B	1.778409154	0.042480331
HNRNPA1	− 0.909386603	0.190518625
MRPL12	1.576279348	0.256983343
PDGFRA	− 1.313134062	0.027381317
TIMM8B	2.393696818	0.028903394

TIMM8B was upregulated in ovarian cancer

Compared to that in neighboring tissues, the expression of TIMM8B in ovarian cancer tissues was significantly elevated (Fig. 2A). Additionally, the TIMM8B mRNA and protein levels were higher compared to those in ISOE-80 cells (Figs. 2B, C). Patients who exhibited low expression of TIMM8B had a greater overall survival rate than those with high TIMM8B expression (Fig. 2D).

Fig. 2.

TIMM8B was highly expressed in ovarian cancer. A TIMM8B was highly expressed in the ovarian cancer tissues of six patients; adjacent noncancerous tissues (NTs). B RT-qPCR was performed to detect the expression of TIMM8B in ovarian cancer cells. C Western blotting analysis was conducted to measure the protein level of TIMM8B in ovarian cancer cells. D Kaplan–Meier plotter online tools were used to identify the prognostic information related to the core gene TIMM8B, and two Kaplan–Meier plots were generated from a single dataset, each using different probes to represent distinct transcripts; **P < 0.01, compared to the NT group; ^#P < 0.05, compared to the ISOE-80 group; ^##P < 0.01, compared to the ISOE-80 group

TIMM8B promoted the proliferation and inhibited the apoptosis of ovarian cancer cells

To investigate how TIMM8B affects the behavior of the SKOV3 and OVCAR3 cell lines, we created cells in which TIMM8B was either overexpressed or knocked down (Figs. 3A, B and 4A, B). The overexpression of TIMM8B increased cell viability and colony formation in SKOV3 and OVCAR3 cells (Fig. 3C and D). Conversely, knocking down TIMM8B reduced these two behaviors in these cells (Fig. 4C, D). The overexpression of TIMM8B also promoted cyclin D1 and cyclin E while decreasing the levels of P21 and P27 (Fig. 3E). Knocking down TIMM8B had the opposite effects (Fig. 4E). Additionally, the overexpression of TIMM8B caused a significant reduction in the G0/G1 phase ratio in SKOV3 and OVCAR3 cells (Fig. 3F). In contrast, the reduction in the expression of TIMM8B substantially delayed the G0/G1 phase (Fig. 4F).

Fig. 3.

TIMM8B overexpression enhanced the proliferation of SKOV3 and OVCAR3 cells. A The mRNA level of TIMM8B in TIMM8B-overexpressing cells was evaluated by RT-qPCR. B Western blotting assays were conducted to determine the protein levels of TIMM8B in TIMM8B-overexpressing cells. C The effect of TIMM8B overexpression on cell viability was determined by the MTS assay. D The effect of TIMM8B overexpression on cell proliferation was assessed by conducting a colony formation assay. E Western blotting assays were conducted to measure the levels of cyclin D1, cyclin E, P21, and P27 proteins in TIMM8B-overexpressing cells. F Flow cytometry assays were performed to determine the effect of TIMM8B overexpression on the progression of the cell cycle; **P < 0.01, compared to the Lv group

Fig. 4.

TIMM8B knockdown enhanced the proliferation of SKOV3 and OVCAR3 cells. A The mRNA level of TIMM8B in TIMM8B-knockdown cells was evaluated by RT-qPCR. B Western blotting assays were conducted to measure the protein levels of TIMM8B in TIMM8B-knockdown cells. C The effect of TIMM8B knockdown on cell viability was determined by the MTS assay. D The effect of TIMM8B knockdown on cell proliferation was assessed by conducting a colony formation assay. E Western blotting assays were performed to measure the levels of cyclin proteins in TIMM8B-knockdown cells. F Flow cytometry was conducted to determine the effect of TIMM8B knockdown on cell cycle progression; **P < 0.01, compared to the sh-NC group

The overexpression of TIMM8B in SKOV3 and OVCAR3 cells considerably decreased the degree of apoptosis (Fig. 5A). Additionally, it increased the levels of BCL 2 and BCL-XL while decreasing the levels of BAX, BAK, cleaved caspase 3, and cleaved caspase 7 (Fig. 5B). Knocking down TIMM8B had the opposite effect.

Fig. 5.

TIMM8B inhibited the apoptosis of SKOV3 and OVCAR3 cells. A The effect of TIMM8B on apoptosis was determined by flow cytometry. B Western blotting was conducted to detect the effects of TIMM8B on the levels of apoptosis-related proteins (BCL 2, BCL-XL, BAX, BAK, cleaved caspase 3, and cleaved caspase 7); **P < 0.01, compared to the Lv group; ^##P < 0.01, compared to the sh-NC group

TIMM8B enhanced the migration, invasion, and EMT of ovarian cancer cells

The overexpression of TIMM8B increased the migration and invasion of SKOV3 and OVCAR3 cells (Fig. 6A, B). In contrast, these cells exhibited a decrease in migration and invasion after TIMM8B was knocked down. The overexpression of TIMM8B led to elevated protein levels of N-cadherin, vimentin, and snail (Fig. 6C). Moreover, it decreased the level of E-cadherin. Knocking down TIMM8B had the opposite effect.

Fig. 6.

TIMM8B enhanced the migration, invasion, and EMT of SKOV3 and OVCAR3 cells. A and B The effects of TIMM8B on cell invasion and migration were determined by conducting Transwell assays. C Western blotting was performed to detect the effects of TIMM8B on the levels of EMT-related proteins (N-cadherin, vimentin, snail, and E-cadherin); **P < 0.01, compared to the Lv group; ^##P < 0.01, compared to the sh-NC group

TIMM8B promoted tumor growth in nude mice

The overexpression of TIMM8B promoted tumor formation, whereas knocking down TIMM8B inhibited tumor formation in mice (Fig. 7A). Additionally, the overexpression of TIMM8B increased the volume and weight of tumors, whereas knocking down TIMM8B decreased tumor suppressive volume and weight (Fig. 7B and C). Additionally, the overexpression of TIMM8B increased the levels of N-cadherin, vimentin, and snail proteins in tumors (Fig. 7D). Moreover, it decreased the level of E-cadherin. In contrast, knocking down TIMM8B had the opposite effect.

Fig. 7.

TIMM8B promoted tumor formation in nude mice. A Representative images of xenograft tumors. B The volume of the transplanted tumor was measured. C The weights of the transplanted tumors were recorded. D Western blotting was performed to detect the effects of TIMM8B on the levels of EMT-related proteins (N-cadherin, vimentin, snail, and E-cadherin); *P < 0.05, compared to the Lv group; **P < 0.01, compared to the Lv group; ^##P < 0.01, compared to the sh-NC group

TIMM8B increased mitochondrial oxidative phosphorylation and aerobic glycolysis in ovarian cancer cells

The overexpression of TIMM8B increased hexokinase activity and lactate production (Fig. 8A, B). However, silencing TIMM8B had the opposite outcome. Additionally, overexpressing TIMM8B also increased ATP concentration in SKOV3 and OVCAR3 cells, whereas knocking down TIMM8B significantly decreased the ATP concentration (Fig. 8C). Moreover, the overexpression of TIMM8B increased the levels of mitochondrial complex I, complex II, complex III, complex IV, and complex V in SKOV3 and OVCAR3 cells (Fig. 8D, E). Knocking down TIMM8B increased the protein levels of these molecules. Overexpressing TIMM8B reduced mtROS levels, whereas knocking down TIMM8B had the opposite effect (Fig. 8F).

Fig. 8.

TIMM8B enhanced mitochondrial OXPHOS and aerobic glycolysis in the SKOV3 and OVCAR3 cell lines. A The effects of TIMM8B on hexokinase activity were measured. B Effects of TIMM8B on lactic acid production. C Effects of TIMM8B on ATP concentration. D and E Effects of TIMM8B on the levels of mitochondrial complex subunits were determined by Western blotting assays. F Effects of TIMM8B on the level of mtROS; **P < 0.01, compared to the Lv group; ^##P < 0.01, compared to the sh-NC group

In the detection of glycolytic rate, TIMM8B knockdown reduced the glycolytic rate of SKOV3 and OVCAR3 cells, mainly manifested in the reduction of glycolysis, glycolytic capacity, and glycolytic reserve of the cells (Fig. 9). Conversely, in the detection of oxidative phosphorylation rate, TIMM8B knockdown also resulted in a decrease in the oxidative phosphorylation rate of SKOV3 and OVCAR3 cells, prominently affecting basal respiration, maximal respiration, ATP production, and spare respiratory capacity. On the other hand, the overexpression of TIMM8B led to an increase in both glycolysis rate and oxidative phosphorylation rate in SKOV3 and OVCAR3 cells.

Fig. 9.

TIMM8B increased glycolysis rate and oxidative phosphorylation rate in SKOV3 and OVCAR3 cells. ^*P < 0.05, compared to the sh-NC group; ^**P < 0.01, compared to the sh-NC group; ^#P < 0.05, compared to the Lv group; ^##P < 0.01, compared to the Lv group

TIMM8B inhibited mtROS/ASK1/JNK to regulate the behavior and energy metabolism of ovarian cancer cells

Overexpressing TIMM8B decreased the levels of ASK1 and p-JNK, whereas reducing TIMM8B led to an increase in these molecules in SKOV3 and OVCAR3 cells (Fig. 10A). However, the mtROS scavenger Mito-TEMPO (MT), which is a mitochondria-targeted superoxide dismutase mimetic, was found to reduce the impact of TIMM8B-knockdown in SKOV3 and OVCAR3 cells (Fig. 10B). These findings indicated that TIMM8B suppresses mtROS, resulting in the suppression of the ASK1/JNK pathway.

Fig. 10.

TIMM8B inhibited the activation of the mtROS/ASK1/JNK pathway. A The effects of TIMM8B on the ASK1/JNK signaling pathway were determined by Western blotting assays. B The mtROS scavenger Mito-TEMPO (MT), which is a mitochondria-targeted superoxide dismutase mimetic, reduced the effects of TIMM8B on the ASK1/JNK signaling pathway, as determined by Western blotting assays; ^**P < 0.01, compared to the sh-NC group; ^##P < 0.01, compared to the Lv group; ^$$P < 0.01, compared to the sh-TIMM8B group

Knocking down TIMM8B reduced cell viability and invasion, and increased the degree of apoptosis in SKOV3 and OVCAR3 cells (Figures S1A–C). Knocking down TIMM8B also significantly decreased lactate production and the concentration of ATP (Figure S1D, E). However, MT reduced the effect of TIMM8B-knockdown. The knockdown of TIMM8B decreased the glycolysis rate of SKOV3 and OVCAR3 cells, resulting in reduced glycolysis, glycolytic capacity, and glycolytic reserve in the cells (Figures S2). Treatment with MT reversed the effects of TIMM8B knockdown. In addition, the reduction in TIMM8B levels also led to a decrease in the oxidative phosphorylation rate of SKOV3 and OVCAR3 cells, specifically affecting basal respiration, maximal respiration, ATP production, and spare respiratory capacity. Once again, MT was able to reverse the impact of the decline in TIMM8B levels.

The JNK agonist anisomycin (AM) reduced the cloning of SKOV3 and OVCAR3 cells overexpressing TIMM8B and blocked the cell cycle (Fig. 11A, B). The cyclin-related proteins, including cyclin D1 and cyclin E, decreased, whereas the levels of P21 and P27 increased in SKOV3 and OVCAR3 cells overexpressing TIMM8B (Fig. 11C). AM also suppressed the migration and invasion of TIMM8B-overexpressing SKOV3 and OVCAR3 cells (Fig. 11D, E). AM increased the level of E-cadherin and decreased the levels of N-cadherin, vimentin, and snail in SKOV3 and OVCAR3 cells overexpressing TIMM8B (Fig. 11F). Moreover, the JNK agonist AM enhanced the apoptosis rate of SKOV3 and OVCAR3 cells overexpressing TIMM8B (Fig. 11G), decreasing the levels of BCL 2 and BCL-XL and increasing the levels of BAX, BAK, cleaved caspase 3, and cleaved caspase 7 (Fig. 11H, I).

Fig. 11.

TIMM8B inhibited mtROS/ASK1/JNK to regulate the behavior and energy metabolism in the SKOV3 and OVCAR3 cell lines. A A colony formation assay was performed in TIMM8B-overexpressing cells after treatment with AM (JNK agonist, 1 μg/mL). B Flow cytometry was conducted to determine cell cycle progression in TIMM8B-overexpressing cells after treatment with AM. C Western blotting assays were performed to measure the levels of cyclin proteins in TIMM8B-overexpressing cells after treatment with AM. D and E Cell invasion and migration were determined by conducting a Transwell assay in TIMM8B-overexpressing cells after treatment with AM. F Western blotting analysis was performed to measure the levels of EMT-related proteins (N-cadherin, vimentin, snail, and E-cadherin) in TIMM8B-overexpressing cells after treatment with AM. G Cell apoptosis was detected by flow cytometry in TIMM8B-overexpressing cells after treatment with AM. H and I Western blotting analysis was performed to measure the levels of apoptosis-related proteins (BCL 2, BCL-XL, BAX, BAK, cleaved caspase 3, and cleaved caspase 7) in TIMM8B-overexpressing cells after treatment with AM; ^**P < 0.01, compared to the sh-NC group; ^##P < 0.01, compared to the TIMM8B group

The JNK agonist AM decreased glucose consumption (hexokinase activity), lactate production, and ATP concentration in SKOV3 and OVCAR3 cells overexpressing TIMM8B (Fig. 12A–C). AM also decreased the levels of the mitochondrial complex in SKOV3 and OVCAR3 cells overexpressing TIMM8B (Fig. 12D, E).

Fig. 12.

TIMM8B inhibited mtROS/ASK1/JNK to regulate energy metabolism in the SKOV3 and OVCAR3 cell lines. A Hexokinase activity was measured in TIMM8B-overexpressing cells after AM treatment. B Lactic acid production was evaluated in TIMM8B-overexpressing cells after AM treatment. C ATP concentrations were assessed in TIMM8B-overexpressing cells after AM treatment. D and E Western blotting analysis was performed to measure the levels of mitochondrial complex subunits; ^**P < 0.01, compared to the sh-NC group; ^##P < 0.01, compared to the TIMM8B group

These findings suggested that TIMM8B inhibits apoptosis by suppressing mtROS/ASK1/JNK, thereby promoting glycolysis, mitochondrial OXPHOS, proliferation, migration, and invasion.

Discussion

Warburg et al. initially proposed that the main role of glucose in cells is to produce ATP. They argued that when respiration is irreversibly damaged, the efficiency of ATP acquisition through phosphorylation decreases significantly. However, subsequent studies reported that tumor cells still retain functional mitochondria and can undergo OXPHOS. The tumorigenicity of cancer cell lines decreases when the depletion of mitochondrial DNA is specifically targeted [41–43]. In genetically normal proliferating cells and virus-infected cells, glucose is preferentially converted to lactic acid [44–46]. This indicates that the “Warburg effect” may be a controlled metabolic condition that enables cells to adjust to elevated biosynthetic requirements. In this study, overexpressing TIMM8B in SKOV3 and OVCAR3 cells significantly increased aerobic glycolysis and OXPHOS. Compared to the demand for reductive equivalents such as precursor molecules and NADPH, cancer cells that are highly proliferative display a moderate increase in ATP consumption [47]. Moreover, the activity of the TCA cycle, which produces NADH and ATP, negatively regulates glucose metabolism. Therefore, converting surplus pyruvate into lactic acid can help sustain intracytoplasmic glucose metabolism without inhibiting excess mitochondrial ATP production [47]. These findings indicate that lactate production in cancer cells may facilitate a modest increase in OXPHOS. Analysis of the metabolic functions of lymphoma cells showed that lymphoma cells that utilize OXPHOS cell lines depend more on mitochondrial respiration than non-OXPHOS lymphoma cells [48]. In situ mouse models of human glioblastoma have shown that glucose can be metabolized to either lactic acid or oxidized by mitochondria [49]. By conducting single-cell RNA sequencing, researchers found that genes related to aerobic glycolysis and OXPHOS were upregulated simultaneously in high-grade serous ovarian cancer [50]. Similarly, a subpopulation of melanomas heavily relies on OXPHOS instead of glycolysis [20, 51]. These findings indicate that within a group of tumors with similar clinical characteristics, glucose metabolism can either be glycolytic or oxidative. However, we exclusively examined the effects of TIMM8B on OXPHOS and glycolysis in SKOV3 and OVCAR3 cells. Hence, future studies should examine how TIMM8B affects other ovarian cancer cell lines.

Cellular metabolic reprogramming in cancer cells is aimed at increasing their adaptability; however, as proliferating cells multiply, their electron transport flux can surpass that of ATP synthase. This leads to the production of harmful mtROS, decreasing cell viability, retarding growth, and ultimately leading to cell apoptosis [52–54]. Chronic oxidative stress occurs when the levels of ROS exceed the ability of mitochondria and cells to detoxify them, causing the mitochondrial permeability transition pore (mtPTP) to open [55]. The activated mtPTP results in the formation of an open channel in mitochondria. This subsequently leads to the release of matrix solute, causing mitochondrial swelling and significant damage to the mitochondria [56]. These findings suggest that excessive levels of ROS harm cell functions, such as OXPHOS, and do not favor tumor occurrence and development. In this study, the overexpression of TIMM8B significantly increased OXPHOS. A significant increase in OXPHOS may lead to the production of high quantities of mtROS. However, the overexpression of TIMM8B decreased mtROS levels. Therefore, in ovarian cancer, TIMM8B may promote OXPHOS and reduce mtROS produced by electron leakage. Besides oxidative stress, other mechanisms, such as decreased mitochondrial ΔΨ and ATP levels [57–59], can activate mtPTP. These findings suggest that the increase in ATP production by TIMM8B also contributes to the protection of mitochondria. Hence, TIMM8B can increase OXPHOS in ovarian cancer cells, simultaneously reducing the production of mtROS, which can improve mitochondrial protection.

We did not assess how TIMM8B increases OXPHOS and decreases mtROS production in ovarian cancer cells. Complexes I, III, and IV participate in multiple higher-order structures referred to as respiratory chain supercomplexes or respiratory bodies [60–62]. However, in HEK293 and SH-SY5Y cells, deleting TIMM8B substantially decreases the copper chaperone COX17 for complex IV [4]. A decrease in COX17 levels reduces properly assembled respiratory chain hypercomplexes [63]. Moreover, the lack of TIMM8B in HEK293 cells leads to a decrease in the level of the factor HIGD2A, which is responsible for assembling the mitochondrial respiratory chain [4]. HIGD2A deficiency can cause severe defects in mitochondrial respiration and also in the assembly and function of complex IV [64, 65]. HIGD2A is extremely important for incorporating COX3 into complex IV and incorporating complex IV into the hypercomplex [65]. Therefore, TIMM8B may regulate complex IV and the formation of respiratory chain supercomplexes. Respiratory chain supercomplexes have multiple functions. Individual respiratory chain complexes have higher stability, and their electron transport efficiency through the substrate channel is greater [66, 67]. The creation of respiratory chain supercomplexes occurs in response to an increase in energy requirements in the cell. These supercomplex structures also decrease the production of mtROS and increase the stability and distribution of the complexes in the inner mitochondrial membrane in a protein-dense environment [68, 69]. Thus, TIMM8B may enhance OXPHOS and decrease mtROS levels by regulating the assembly of respiratory chain supercomplexes. However, this hypothesis needs to be tested and validated in future studies.

Mitochondria play crucial roles not only in metabolism but also in programmed cell death [11]. A key protein family involved in mitochondria-mediated apoptosis is the BCL-2 family, which includes members that can either inhibit apoptosis, such as BCL-2 and BCL-XL, or facilitate apoptosis, such as BAX and BAK [70]. In this study, TIMM8B increased the levels of BCL-2 and BCL-XL while decreasing the levels of BAX, BAK, cleaved caspase 3, and cleaved caspase 7. These changes decreased cell apoptosis. Studies suggest that the entry of BAX/BAK into the mitochondrial outer membrane cytochrome c and DDP/TIMM8A is released, leading to the activation of DRP1-mediated fission. This results in mitochondrial fragmentation, and eventually, cell division during programmed cell death [71]. The DDP-TIMM8B chimera was constructed using amino acid residues 52–83 of TIMM8B to replace amino acid residues 59–97 of DDP. Although it still contains the conserved twin Cx3C motif, it does not interact with Drp1 as DDP/TIMM8A does. These findings suggest that TIMM8B may regulate apoptosis through other mechanisms. In this study, the antiapoptotic effect of TIMM8B was reversed when a JNK agonist was used. These findings suggested that TIMM8B may inhibit the activation of ASK1/JNK, thus inhibiting the apoptosis of ovarian cancer cells. However, further assessment is needed to understand how TIMM8B regulates JNK activation to induce the apoptosis of ovarian cancer cells. JNK primarily targets mitochondria via pro-apoptotic signaling [72]. When cytochrome c is injected directly into the cytoplasm of JNK-mutated murine embryonic fibroblasts, they do not exhibit defects in apoptosis [73]. The JNK signaling pathway has many targets that can affect mitochondria, including BCL-XL, BCL-2, BID, BIM, BAD, and BAX [74]. Therefore, when TIMM8B inhibits mtROS/ASK1, it may suppress JNK activation, thus decreasing the activity of apoptotic proteins and increasing the activity of antiapoptotic proteins. Consequently, apoptosis is inhibited in ovarian cancer cells.

Strong associations were found between cell cycle regulators, such as cyclins, and mitochondrial morphology and respiratory function. Cyclin D1 in the G1 phase can decrease mitochondrial activity through CDK4 kinase, thus increasing mitochondrial size and activity in hepatocytes lacking cyclin D1 [75]. Additionally, during the G1/S transition, hyperfused mitochondria can promote the accumulation of cyclin E [76]. In colon cancer cells, mitochondrial respiration is the major source of ATP production during the G2/M stage, with a greater proportion of mitochondrial OXPHOS [77]. In this study, TIMM8B increased cell cycle progression in SKOV3 and OVCAR3 cells. Consequently, TIMM8B may increase ATP production to provide the energy necessary for the cell cycle, especially during the G2/M phase. Additionally, mitochondrial OXPHOS may also affect migration and invasion. Rotenone restrains the TCA cycle and the aggressiveness of ovarian cancer cells by affecting oxygen consumption, ultimately inhibiting the mitochondrial respiratory chain [78]. In contrast to primary tumor cells, metastatic cells in humans and mice present higher levels of mitochondrial biogenesis and ATP production. PGC-1α induces OXPHOS in breast cancer cell metastasis [79]. The increased levels of TIMM8B examined in this study increased the movement and infiltration of SKOV3 and OVCAR3 cells, potentially through an increase in OXPHOS. Additionally, JNK agonists were used to study the effect of TIMM8B on the behavior of cells, indicating that TIMM8B might regulate these mechanisms via the JNK pathway.

The production of ATP relies on OXPHOS complexes. Defects in complexes I and III result in a decrease in ATP production through OXPHOS [80]. Mutations in the complex V gene are commonly associated with ATP synthase deficiency [80]. Certain inhibitors (molecules) can disrupt metabolism, cause oxidative damage, and promote cancer cell death by targeting the polymer complex of the OXPHOS pathway. Metformin is an OXPHOS inhibitor that functions by blocking mitochondrial complex I, thus decreasing mitochondrial ATP production [81, 82]. Rotenone and 2-thenoyltrifluoroacetone can independently induce autophagy and cell death by inhibiting complex I or complex II [83]. Atovaquone, an FDA-approved drug for malaria that hinders the activity of complex III, is used for treating solid tumors [84, 85]. TIMM8B inhibitors can act like these molecules to inhibit mitochondrial OXPHOS in ovarian cancer treatment.

Mitochondria exist in a variety of shapes and sizes, from short spheres to highly fused webs [86]. Shape itself affects the dynamics of biochemical reactions and the formation of protein complexes in the mitochondrial matrix [87]. It may also be related to exercise and the production of ROS or ATP. In general, short mitochondria are more mobile, but exhibit higher levels of mtROS [88]. Mito-TEMPO (MT, an mtROS scavenger) restored the expression level of translocase of the outer membrane 20 (TOM20) in HK2 cells exposed to tert-butyl hydroperoxide [89]. TOM20 is a critical protein responsible for maintaining the integrity of both the outer and inner mitochondrial membranes [90]. Therefore, TIMM8 may affect mitochondrial integrity by influencing mtROS. Shear-induced mtROS can lead to mitochondrial fission in ischemic/reperfusion exposed endothelial cells [91]. Interestingly, mitochondrial fission can also boost the production of mtROS and Cyt C [92]. Therefore, TIMM8B may inhibit mitochondrial fission by inhibiting mtROS. Additionally, there is a possibility of a direct impact on mitochondrial fusion/fission, as TIMM8 functions as a chaperone within the mitochondrial intermembrane [12]. A new project focusing on examining the effects of TIMM8B on mitochondrial fusion/fission could be initiated in the future.

Conclusion

In this study, we proposed that TIMM8B might promote OXPHOS and inhibit mtROS/ASK1/JNK, which enhances OXPHOS, glycolysis, proliferation, migration, and invasion and decreases the degree of apoptosis in ovarian cancer cells. We introduced a new approach to elucidate the regulatory mechanism of TIMM8B in ovarian cancer; our findings increased our knowledge regarding the pathogenesis of this disease.

Author contributions

YZ conceptualized and designed the present study and provided administrative support. YJ, JQL, XQY and HYH made contributions to the acquisition of data. WTZ, LFZ, CHA, YBX and SFT analyzed and interpreted the data. YJ and JQL were involved in drafting the manuscript. YZ critically revised the manuscript for significant intellectual content. The final manuscript was read and approved by all authors.

Funding

This work was supported by grants from the Major Science and Technology Projects in Yunnan Province (Grant No.: 202001AS070033), The Basic research in Yunnan Province (Kunming Medical University Joint Project) (Grant Nos.: 202201AY070001-138, 202201AY070001-162, 202301AY070001-250), The innovative research team of Yunnan Province (Grant No.: 202305AS350020), National Natural Science Foundation of China (Grant Nos.: 81860485, 82060425), the Applied Basic Research Foundation of Yunnan Province Science and Technology Department (Grant Nos.: 202201AT070044, 202301AT070246, 202401AY070001-152), Yunnan Fundamental Research Kunming Medical University Projects (Grant No.: 202501AY070001-036), The Basic Research Foundation of Yunnan Province (Grant No.: 202201AT070044), Yunnan Xingdian Talent Project "Young talents" to WT Zhao (Grant No.: CZ0127-910373), The Basic Research Foundation of Yunnan Province Local Universities (Grant Nos.: 202401BA070001-063, 202401BA070001-082), and Yunnan Provincial Department of Science and Technology-Kunming Medical University Applied Basic Research Joint Special General Project (Grant No.: 202501AY070001-110).

Data availability

Data will be made available on reasonable request.

Declarations

Ethics approval and consent to participate

The Ethics Committee of The Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital, Peking University Cancer Hospital Yunnan) reviewed and approved the study protocol in accordance with the Declaration of Helsinki. All patients provided informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Animal ethics

All animal experiments were approved by the Animal Experimental Ethics Review Committee of Kunming Medical University.