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. 2024 Feb 28;56(3):356–365. doi: 10.3724/abbs.2023246

HSPA8-mediated stability of the CLPP protein regulates mitochondrial autophagy in cisplatin-resistant ovarian cancer cells

Role of HSPA8/CLPP in cisplatin-resistant OC

Xinxin Kou 1, Xiaoxia Yang 1, Zheng Zhao 1, Lei Li 1,*
PMCID: PMC10984867  PMID: 38419499

Abstract

Currently, platinum agents remain the mainstay of chemotherapy for ovarian cancer (OC). However, cisplatin (DDP) resistance is a major reason for chemotherapy failure. Thus, it is extremely important to elucidate the mechanism of resistance to DDP. Here, we establish two DDP-resistant ovarian cancer cell lines and find that caseinolytic protease P (CLPP) level is significantly downregulated in DDP-resistant cell lines compared to wild-type ovarian cancer cell lines (SK-OV-3 and OVcar3). Next, we investigate the functions of CLPP in DDP-resistant and wild-type ovarian cancer cells using various assays, including cell counting kit-8 assay, western blot analysis, immunofluorescence staining, and detection of reactive oxygen species (ROS) and apoptosis. Our results show that CLPP knockdown significantly increases the half maximal inhibitory concentration (IC 50) and mitophagy of wild-type SK-OV-3 and OVcar3 cells, while CLPP overexpression reduces the IC 50 values and mitophagy of DDP-resistant SK-OV-3 and OVcar3 cells. Next, we perform database predictions and confirmation experiments, which show that heat shock protein family A member 8 (HSPA8) regulates CLPP protein stability. The dynamic effects of the HSPA8/CLPP axis in ovarian cancer cells are also examined. HSPA8 increases mitophagy and the IC 50 values of SK-OV-3 and OVcar3 cells but inhibits their ROS production and apoptosis. In addition, CLPP partly reverses the effects induced by HSPA8 in SK-OV-3 and OVcar3 cells. In conclusion, CLPP increases DDP resistance in ovarian cancer by inhibiting mitophagy and promoting cellular stress. Meanwhile, HSPA8 promotes the degradation of CLPP protein by regulating its stability.

Keywords: cisplatin resistance, ovarian cancer, caseinolytic protease P, heat shock protein family A member 8, mitophagy

Introduction

Ovarian cancer is the 3rd most common gynecologic malignancy worldwide and the most lethal gynecologic malignancy [1]. Currently, the incidence and mortality rates of ovarian cancer continue to increase [2]. The mainstream chemotherapy for epithelial ovarian cancer (EOC) is a platinum agent combined with taxane [3]. However, patients eventually develop tolerance to cisplatin (DDP)-based therapy after a few cycles of treatment, which further increases the mortality rate [4]. Thus, there is an urgent need to elucidate the underlying mechanism of DDP resistance in ovarian cancer.

DDP acts by binding to DNA and subsequently causing DNA damage, inhibition of DNA replication, and induction of cell apoptosis [5]. Based on DDP pharmacological studies and its biological activities, various mechanisms have been proposed to contribute to DDP resistance, including an increased cellular efflux of drugs [6], reduced drug influx [7], inhibition of apoptosis [8], dysregulation of DNA damage repair systems [9], enhanced activity of drug-metabolizing enzymes [ 10, 11], and an altered cellular microenvironment [ 12– 14]. The reasons for DDP resistance in DDP-resistant cancer cells include a reduction in drug accumulation, DDP inactivation resulted from its reaction with glutathione and metallothionein, and a rapid repair of DNA lesions [ 9, 15]. Copper efflux transporters, such as Cu-transporting P-type ATPases (ATP7A and ATP7B), have been shown to regulate the efflux of DDP [ 16, 17]. Recent studies have shown that changes in mitochondrial function and abnormal autophagy play key roles in DDP resistance in ovarian cancer [ 18– 20], which suggests that mitochondrial molecules are involved in DDP resistance.

Mitochondrial caseinolytic protease P (CLPP) is a serine protease located in the mitochondrial matrix and is involved in mitochondrial protein metabolism (proteostasis) and oxidative stress by facilitating the degradation of misfolded or damaged proteins and thus the maintenance of protein metabolism homeostasis [ 21, 22]. Due to the functions of CLPP in mitochondria, it has multiple effects on tumors [23]. In acute myeloid leukemia, the genetic and chemical activation of CLPP selectively kills cancer cells by degrading respiratory chain protein substrates and disrupting mitochondrial structure and function; however, CLPP has no effects on noncancerous cells [24]. In pancreatic ductal adenocarcinoma (PDAC), the chemical activation of CLPP increases the degradation of respiratory chain complexes, which causes an endoplasmic reticulum stress response that suppresses the growth of PDAC cells [25]. In glioblastoma, CLPP activators induce synthetic lethality by inhibiting oxidative energy metabolism and reducing cell viability [26]. In EOC, the mitochondrial deficits induced by CLPP inhibit the growth and metastasis of EOC cells [27]. However, the role of CLPP in DDP resistance in ovarian cancer remains unclear.

Heat shock protein family A member 8 (HSPA8) belongs to the heat shock protein 70 (HSP70) family [28]. It plays important physiological roles in protein metabolism and homeostasis, both of which depend on constant protein degradation and resynthesis [29]. Under normal or stressful conditions, eukaryotic cells remove misfolded proteins via autophagy. In chaperone-mediated autophagy, HSPA8 recognizes and targets cytosolic proteins with a signature exposed pentapeptide motif (KFERQ) [30]. After being recognized by HSPA8 and binding to lysosomal-associated membrane protein 2A, the target proteins are translocated into the lysosomal lumen for degradation [31].

In this study, we explored the effects of CLPP on mitophagy in wild-type or DDP-resistant ovarian cancer cells. Specifically, our database predictions and subsequent experiments indicated that the HSPA8 gene might regulate the stability of CLPP. Taken together, our findings suggest that HSPA8-mediated stability of the CLPP protein regulates mitochondrial autophagy in DDP-resistant ovarian cancer cells. Our results elucidate one of the mechanisms by which drug resistance occurs, provide new insights into cisplatin resistance in ovarian cancer, and will facilitate the development of new clinical therapies for ovarian cancer.

Materials and Methods

Cell lines and cell transfection

The human ovarian cancer cell lines SK-OV-3 (ATCC ® HTB-77 TM) and OVcar3 (ATCC ® HTB-161 TM) were purchased from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in RPMI 1640 medium (Gibco, Waltham, USA) supplemented with 10% fetal bovine serum (Gibco) at 37°C in a 5% CO 2 atmosphere. DDP-resistant SK-OV-3 and OVcar3 cells were developed from wild-type SK-OV-3 and OVcar3 cells by using a stepwise procedure in which the cells were exposed to increasing concentrations of DDP in culture medium for more than 6 months.

Real-time PCR analysis

TRIzol reagent (Invitrogen, Carlsbad, USA) was used to extract total RNA from cells and tissue samples. A reverse transcription kit (Takara, Tokyo, Japan) was used according to the kit’s instructions to perform reverse transcription. Quantitative polymerase chain reaction was performed by using SYBR Green Master Mix on a LightCycler 480 PCR system (Roche Diagnostics, Indianapolis, USA). The PCR reaction was carried out at 95°C for 10 min, followed by 40 cycles of denaturation (95°C for 15 s), annealing, and extension (60°C for 1 min). Relative gene expression levels were calculated using the 2 –ΔΔCt method, and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) served as an internal control. The sequences of primers used for RT-PCR are shown in Supplementary Table S1.

Western blot analysis

Total proteins were extracted from treated cells, and the protein concentration in each extract was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, USA). Next, a 40 μg aliquot of protein from each extract was separated by Tris-glycine SDS-PAGE (4% to 20%), and the protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, St Louis, USA), which were subsequently blocked with nonfat milk. The membranes were then incubated for 8–10 h with the following primary antibodies: anti-CLPP (ab124822; Abcam, Cambridge, UK), anti-PTEN-induced kinase 1 (PINK1) (ab300623; Abcam), anti-parkin (ab77924; Abcam), anti-HSPA8 (ab265664; Abcam) anti-microtubule-associated protein light chain 3 (LC3B) (ab48394; Abcam), and anti-β-actin antibody (ab8227; Abcam). The primary antibody was then detected after incubation with a secondary antibody conjugated with horseradish peroxidase. The protein signals were visualized with an enhanced chemiluminescence (ECL) kit (Pierce).

Cell counting kit-8 (CCK-8) assay

Cell viability was determined by CCK-8 assay on transfected and/or treated cells. After replacing the used media with fresh media, the cells were transferred into 96-well plates. Next, 20 μL of CCK-8 solution (Beyotime, Shanghai, China) was added to each well, and the cells were incubated for 3 h. Finally, the optical density (OD) of each well was measured at 450 nm with a microplate reader (Thermo Labsystems, Helsinki, Finland).

Immunofluorescence (IF) staining

Ovarian cancer cells were fixed for 30 min with 4% polyformaldehyde and then permeabilized for 10 min with 0.6% Triton X-100 before being blocked with goat serum. Next, following an overnight incubation at 4°C with anti-CLPP (ab124822; Abcam) or anti-LC3B (ab48394; Abcam) antibody, the cells were incubated for 1 h in the dark with a secondary antibody conjugated to fluorescein isothiocyanate (FITC; Invitrogen). Next, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) staining solution (ab104139; Abcam) at ambient temperature. Finally, the cells were imaged with a fluorescence laser microscope (Olympus, Tokyo, Japan).

ROS detection

The treated cells were harvested and incubated with 10 μM DCFH-DA (mlbio, Shanghai, China) for 30 min in the dark at 37°C. Following incubation, the cells were harvested with trypsin/ethylenediaminetetraacetic acid, and their intracellular levels of reactive oxygen species (ROS) were measured by flow cytometry (Beyotime, Shanghai, China) as previously described [32].

Cell apoptosis detection

Apoptotic cells were detected using an Annexin V-FITC Apoptosis Detection kit (Keygen, Guangzhou, China). In brief, the cancer cells were harvested with 0.25% trypsin, after which they were washed twice with phosphate buffered saline and resuspended in 500 μL of binding buffer. The cells were then incubated with 5 μL of antibody against Annexin V-FITC and 5 μL of propidium iodide (PI) for 15–20 min in the dark. After incubation, the apoptotic cells were detected with a BD Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, USA).

Statistical analysis

The results were analyzed using IBM SPSS Statistics for Windows (Version 20 software), and data are expressed as the mean± standard deviation. GraphPad 7.0 software (GraphPad, La Jolla, USA) and Biorender (Biorender-JavaShuo) were used to draw the graphs. Student’s t test was used to compare mean values between 2 groups, and one-way analysis of variance was used to compare differences among multiple groups. P<0.05 was considered statistically significant.

Results

CLPP expression was downregulated in DDP-resistant ovarian cancer cells

First, we established DDP‐resistant cell lines (SK-OV-3/DDP and OVcar3/DDP) by gradually increasing the cisplatin concentration in SK-OV-3 and OVcar3 cells. We then verified the viability of SK-OV-3 and OVcar3 cells under both normal conditions and DDP treatment conditions. As shown in Figure 1A, constant exposure to DDP significantly increased the survival rates of SK-OV-3 and OVcar3 cells. The IC 50 values of DDP-resistant SK-OV-3/DDP cells and OVcar3/DDP cells were significantly increased compared to those of untreated wild-type SK-OV-3 and OVcar3 cells, which indicated the successful establishment of DDP‐resistant ovarian cancer cell lines. After the establishment of the DDP‐resistant cell lines, the levels of CLPP messenger RNA (mRNA) and CLPP protein in the cisplatin-resistant groups were lower than those in the cisplatin-sensitive groups ( Figure 1B,C), as determined by qRT-PCR and western blot analysis. IF staining also showed reduced CLPP protein levels in SK-OV-3/DDP and OVcar3/DDP cells ( Figure 1D). These results demonstrated that CLPP expression was significantly decreased in DDP-resistant ovarian cancer cells compared to wild-type SK-OV-3 and OVcar3 cells.

Figure 1 .

Figure 1

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CLPP expression is upregulated in DDP-resistant ovarian cancer cells

(A) SK-OV-3 and OVcar3 cells were treated with DDP (0, 0.1, 1, 5, 10, 15, 20, 30, 40, 50, or 60 μg/mL) and examined for cell viability by the CCK-8 assay. DDP-resistant ovarian cancer cells were established. (B) The levels of CLPP mRNA in wild-type and DDP-resistant ovarian cancer cells were examined by real-time PCR. (C) The levels of CLPP protein in the cells were examined by western blot analysis. (D) The distribution of CLPP protein in the cells was examined by immunofluorescence staining (magnification 200× and 400×). ***P<0.001 compared to the control group. CLPP, caseinolytic protease P; DDP, cisplatin; CCK-8, cell counting kit-8; PCR, polymerase chain reaction. Control, wild-type SK-OV-3 and OVcar3 cell lines.

Effects of CLPP knockdown in wild-type ovarian cancer cells

As shown in Figure 2A, CLPP knockdown significantly increased the IC 50 values of the SK-OV-3 and OVcar3 cells. Studies have shown that the autophagy process of cancer cells is involved in resistance to chemotherapy [33]. Therefore, we next examined the levels of mitochondrial autophagy-related proteins ( i. e., PINK1 and Parkin) as well as the levels of autophagy-associated proteins (LC3II/I) in cells treated with DDP (IC 25). CLPP interference was successfully achieved in the wild-type OVcar3 and SK-OV-3 cell lines ( Figure 2B,C). SK-OV-3 and OVcar3 cells with CLPP knockdown were exposed to different concentrations of DDP (0–20 μg/mL), and IC 50 values were calculated based on cell viability. The western blot analysis results showed that CLPP knockdown increased the levels of PINK1, Parkin, and LC3-II proteins ( Figure 2C). The levels of LC3B protein in SK-OV-3 and OVcar3 cells were also increased by CLPP knockdown ( Figure 2D). These results indicated that CLPP knockdown can promote autophagy and mitochondrial autophagy in cancer cells. To further explore the oxidative stress and apoptosis of the cells, the ROS levels and apoptotic rates were detected by flow cytometry. As shown in Figure 2E,F, CLPP knockdown decreased the ROS levels and apoptosis rates of wild-type SK-OV-3 and OVcar3 cells treated with DDP (IC 25).

Figure 2 .

Figure 2

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The effects of CLPP knockdown on wild-type ovarian cancer cells

(A) Wild-type ovarian cancer cells were transfected with a CLPP interference sequence and then treated with different concentrations of DDP; the IC50 values were calculated based on cell viability. (B) The levels of CLPP mRNA in the cells were examined by real-time PCR. (C) The protein levels of CLPP, PINK1, Parkin, and LC3II/I were examined by western blot analysis. (D) The distribution of LC3B protein in the cells was examined by immunofluorescence staining (magnification 200× and 400×). (E) ROS levels were measured by flow cytometry. (F) Cell apoptosis was examined by flow cytometry. ***P<0.001 compared to the NC group. NC, negative control; CLPP, caseinolytic protease P; DDP, cisplatin; ROS, reactive oxygen species; si, small interfering; PCR, polymerase chain reaction.

Effects of CLPP overexpression in DDP-resistant ovarian cancer cells

After determining the effects of CLPP in wild-type ovarian cancer cells, we subsequently investigated the effects of CLPP in cisplatin-resistant ovarian cancer cells. As shown in Figure 3A, CLPP overexpression significantly decreased the IC 50 values of DDP-resistant SK-OV-3 and OVcar3 cells. Our data also showed that CLPP overexpression was successfully achieved in the DDP-resistant SK-OV-3 and OVcar3 cell lines ( Figure 3B,C). Next, DDP-resistant SK-OV-3 and OVcar3 cells overexpressing CLPP were exposed to different concentrations of DDP (0–60 μg/mL), and IC 50 values were calculated based on cell viability. Western blot analysis results showed that CLPP overexpression decreased the levels of PINK1, Parkin, and LC3II/I protein expression in cells treated with DDP (IC 25) ( Figure 3C). The levels of LC3B protein were also decreased in DDP-resistant SK-OV-3 and OVcar3 cells overexpressing CLPP ( Figure 3D). CLPP overexpression increased the ROS levels and apoptosis rates in DDP-resistant SK-OV-3 and OVcar3 cells undergoing DDP treatment (IC 25) ( Figure 3E,F). Furthermore, our data showed that overexpression of CLPP could reduce the levels of cellular and mitochondrial autophagy in cisplatin-resistant cells, increase the ROS levels and apoptosis rates of ovarian cancer cells, and thus improve the drug sensitivity of ovarian cancer cells.

Figure 3 .

Figure 3

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The effects of CLPP overexpression on DDP-resistant ovarian cancer cells

(A) DDP-resistant ovarian cancer cells were transfected with CLPP overexpression plasmids and subsequently treated with different concentrations of DDP; the IC50 values were calculated based on cell viability. (B) The levels of CLPP mRNA in the cells were examined by real-time PCR. (C) The protein levels of CLPP, PINK1, Parkin, and LC3II/I were examined by western blot analysis. (D) The distribution of LC3B protein in the cells was examined by immunofluorescence staining (magnification 200× and 400×). (E) ROS levels were measured by flow cytometry. (F) Cell apoptosis was examined by flow cytometry. **P<0.01, *** P<0.001 compared to the vector group. Vector, empty carrier; CLPP, caseinolytic protease P; DDP, cisplatin; ROS, reactive oxygen species; si, small interfering; PCR, polymerase chain reaction.

HSPA8 affects CLPP protein stability in ovarian cancer cells

After revealing that CLPP is involved in the DDP resistance of ovarian cancer, we investigated the underlying mechanism for its effect on DDP resistance. Our initial database predictions and experiments suggested that HSPA8 is involved in regulating CLPP protein stability. Thus, to explore the interaction between HSPA8 and CLPP, HSPA8 overexpression was achieved in the wild-type SK-OV-3 and OVcar3 cell lines ( Figure 4A). Our results showed that CLPP mRNA level remained unchanged after HSPA8 overexpression ( Figure 4B). Conversely, CLPP protein level was decreased by HSPA8 overexpression ( Figure 4C). Similarly, HSPA8 knockdown was performed in DDP-resistant SK-OV-3 and OVcar3 cell lines ( Figure 4D). The level of CLPP mRNA was not altered by HSPA8 knockdown ( Figure 4E), while the CLPP protein level was increased by HSPA8 knockdown ( Figure 4F). Next, cycloheximide (CHX) was used to inhibit total cellular protein synthesis. Under conditions of CHX treatment, HSPA8 overexpression promoted the degradation of CLPP protein in SK-OV-3 and OVcar3 cells ( Figure 4G). To further verify this process, a proteasome inhibitor (MG132) was used to treat HSPA8-overexpressing cells. The results showed that MG132 reversed the reductions in CLPP expression induced by HSPA8 ( Figure 4H). These results indicated that HSPA8 reduces CLPP protein levels by facilitating its stability.

Figure 4 .

Figure 4

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HSPA8 affects CLPP protein stability in ovarian cancer cells

(A) HSPA8 overexpression was established in SK-OV-3 and OVcar3 cells as confirmed by real-time PCR. (B) The levels of CLPP mRNA in the transfected SK-OV-3 and OVcar3 cells were determined by real-time PCR. (C) The levels of CLPP protein in the transfected SK-OV-3 and OVcar3 cells were determined by western blot analysis. (D) HSPA8 knockdown was established in DDP-resistant SK-OV-3 and OVcar3 cells as confirmed by real-time PCR. (E) The levels of CLPP mRNA in the transfected DDP-resistant SK-OV-3 and OVcar3 cells were determined by real-time PCR. (F) The levels of CLPP protein in the transfected DDP-resistant SK-OV-3 and OVcar3 cells were determined by western blot analysis. (G) The levels of CLPP protein in HSPA8-overexpressing SK-OV-3 and OVcar3 cells treated with CHX were determined by western blot analysis. (H) SK-OV-3 and OVcar3 cells with stable overexpression of HSPA8 were treated with a proteasome inhibitor (MG132, 20 μM). The levels of HSPA8 and CLPP proteins were detected by western blot analysis. ***P<0.001 compared to the vector or NC group. Vector, empty carrier; NC, negative control; CLPP, caseinolytic protease P; CHX, cycloheximide; DDP, cisplatin; HSPA8, heat shock protein family A member 8; M, marker; ns, not significant; PCR, polymerase chain reaction; si, small interfering.

HSPA8 affects wild-type ovarian cancer cell phenotypes by downregulating CLPP

As mentioned above, HSPA8 affects the stability of CLPP protein. To further investigate the dynamic effects of HSPA8 and CLPP on the DDP resistance of ovarian cancer, wild-type SK-OV-3 and OVcar3 cells were cotransfected with HSPA8 and CLPP-overexpression plasmids. Next, the transfected cells were exposed to different concentrations of DDP (0–20 μg/mL), and the IC 50 values were calculated based on cell viability. As shown in Figure 5A, HSPA8 overexpression significantly increased the IC 50 values of the SK-OV-3 and OVcar3 cells, while CLPP overexpression partially reversed the effects induced by HSPA8 overexpression. Next, the level of CLPP mRNA was confirmed by qRT-PCR ( Figure 5B). These results showed that CLPP protein level was decreased by HSPA8 overexpression and restored by CLPP overexpression, while the level of HSPA8 protein was not affected by CLPP overexpression ( Figure 5C). With regard to mitochondrial autophagy and autophagy-related proteins, HSPA8 overexpression increased the levels of PINK1, Parkin, and LC3II/I proteins, while CLPP overexpression restored the protein expression levels induced by HSPA8 overexpression ( Figure 5D). LC3B protein expression was increased by HSPA8 overexpression and restored by CLPP overexpression ( Figure 5E). Furthermore, cellular ROS levels and apoptosis rates were decreased by HSPA8 overexpression and restored by CLPP overexpression ( Figure 5F,G). These results indicated that HSPA8 could inhibit mitochondrial autophagy by downregulating CLPP expression and thereby promote the expressions of mitochondrial autophagy-related proteins and the interactions between LC3-II/I and mitochondrial autophagy-related proteins.

Figure 5 .

Figure 5

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HSPA8 affects wild-type ovarian cancer cell phenotypes by downregulating CLPP expression

(A) Wild-type ovarian cancer cells were cotransfected with HSPA8 and CLPP overexpression plasmids and then treated with different concentrations of DDP; the IC50 values were calculated based on cell viability. (B) The levels of CLPP mRNA in the cells were examined by real-time PCR. (C,D) The levels of CLPP, PINK1, Parkin, and LC3II/I proteins were examined by western blot analysis. (E) The distribution of LC3B protein in the cells was examined by immunofluorescence staining (magnification 200× and 400×). (F) ROS production was measured by flow cytometry. (G) Cell apoptosis was examined by flow cytometry. ### P<0.001 compared to the vector group; ***P<0.001 compared to the HSPA8 group. CLPP, caseinolytic protease P; DDP, cisplatin; HSPA8, heat shock protein family A member 8; M, marker; ns, not significant; PCR, polymerase chain reaction; ROS, reactive oxygen species.

HSPA8 knockdown upregulates CLPP and thus affects DDP-resistant ovarian cancer cell phenotypes

Next, DDP-resistant SK-OV-3 and OVcar3 cells were treated with small interfering RNA (specifically targeting HSPA8 and CLPP) to achieve the knockdown of HSPA8 and CLPP. The cells were exposed to different concentrations of DDP (0–60 μg/mL), and the IC 50 values were calculated based on cell viability. As shown in Figure 6A, HSPA8 knockdown significantly decreased the IC 50 values of DDP-resistant SK-OV-3 and OVcar3 cells, while CLPP knockdown partially restored the effects induced by HSPA8 knockdown. Next, the level of CLPP mRNA was confirmed by qRT-PCR ( Figure 6B). The level of CLPP protein was increased by HSPA8 knockdown and restored by CLPP knockdown; the level of HSPA8 protein was reduced by HSPA8 knockdown but not affected by CLPP knockdown ( Figure 6C). With regard to mitochondrial autophagy-related proteins, HSPA8 knockdown decreased the PINK1, Parkin, and LC3II/I protein levels, while CLPP knockdown restored the protein levels induced by HSPA8 knockdown ( Figure 6D). LC3B protein expression was decreased by HSPA8 knockdown and restored by CLPP knockdown ( Figure 6E). The cellular ROS levels and apoptotic rates were increased by HSPA8 knockdown and restored by CLPP knockdown ( Figures 6F,G). Taken together, these results suggest that HSPA8 mediates cisplatin-resistant ovarian cancer cell phenotypes via CLPP-dependent signaling.

Figure 6 .

Figure 6

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HSPA8 knockdown upregulates CLPP expression to affect DDP-resistant ovarian cancer cell phenotypes

(A) DDP-resistant ovarian cancer cells were transfected with HSPA8 and the CLPP interference sequence and then treated with different concentrations of DDP; the IC50 values were calculated based on cell viability. (B) The levels of CLPP mRNA in the cells were examined by real-time PCR. (C, D) The protein levels of CLPP, HSPA8, PINK1, Parkin, and LC3II/I were examined by western blot analysis. (E) The distribution of LC3B protein in cells was examined by immunofluorescence staining (magnification, 200× and 400×). (F) ROS production was measured by flow cytometry. (G) Cell apoptosis was examined by flow cytometry. ### P<0.001 compared to the si-NC group; ***P<0.001 compared to the si-HSPA8 group. si-NC, negative control; CLPP, caseinolytic protease P; DDP, cisplatin; HSPA8, heat shock protein family A member 8; M, marker; ns, not significant; PCR, polymerase chain reaction; ROS, reactive oxygen species; si, small interfering.

Discussion

Currently, platinum agents remain the main chemotherapy drugs for ovarian cancer. DDP chemotherapy combined with surgery is also the primary treatment for malignant ovarian cancer, and a majority of seriously ill patients benefit from this therapy [34]. A platinum agent plus a taxane is the main type of chemotherapy for EOC [3]. However, the occurrence of resistance is the main reason for treatment failure. Most patients with advanced EOC suffer from disease recurrence and develop platinum resistance, after which the choice of other therapeutic options and their effects are diminished [35]. Thus, elucidating the mechanisms of resistance occurrence and identifying drug-resistant biomarkers are vital for overcoming this dilemma and developing effective treatments.

In this study, 2 DDP-resistant ovarian cancer cell lines were established, and CLPP was found to be significantly downregulated in DDP-resistant cells. CLPP, which is a mitochondrial protease, participates in mitochondrial proteostasis and cellular stress. It is often reported to have a tumor-suppressive effect [23]. In ovarian cancer, the activation of CLPP by small molecules significantly inhibits cellular proliferation, adhesion, and metastasis and induces G1 phase arrest, cellular stress, and cell death [ 27, 36, 37]. Our results demonstrated that knockdown of CLPP significantly increased the IC 50 values of wild-type ovarian cancer cells, and the IC 50 values of DDP-resistant ovarian cancer cells were decreased by CLPP overexpression, which is consistent with the tumor-suppressive effects of CLPP. These results provide the first evidence for the involvement of CLPP in DDP resistance in ovarian cancer.

Mitochondria are the energy factories of cells and play a key role in the metabolic reprogramming of cancers [38]. In addition to regulating bioenergetic metabolism, mitochondria also regulate key signals involved in epigenetic processes, oxidative stress, protein metabolism, and the initiation and execution of apoptosis [39]. These multiple functions give mitochondria the ability to perceive different stresses and help cells adjust to challenges from their microenvironment [39]. Mitochondrial autophagy is a selective form of autophagy that involves the PINK1-Parkin and BNIP3/NIX pathways. These two pathways target damaged mitochondria for autophagosome degradation and reduce the levels of reactive oxygen species in mitochondria via LC3II, thereby promoting the survival of various types of cancer cells and tumors exposed to cytotoxic stress. LC3 occurs in two forms, i.e. LC3-I and LC3-II. Both forms are produced posttranslatively in various cells, and the ratio of LC3-II/I correlates with the degree to which autophagosomes are created [40]. The mammalian LC3 allotroph, LC3B, has been widely used as an autophagy membrane marker. Jin et al [41] showed that inhibition of LC3B could increase the chemical sensitivity of ovarian cancer cells. The protein kinase PINK1 and the E3 ubiquitin ligase Parkin control the specific elimination of dysfunctional or redundant mitochondria and can thereby fine-tune the mitochondrial network and maintain energy metabolism [42]. BNIP3 is a member of the Bcl-2 protein family, which belongs to the BH3 subfamily containing only the BH3 domain [41]. The removal of damaged mitochondria is achieved via both cell autophagy and mitochondrial autophagy. NIX, a protein on the outer membrane of mitochondria, recruits LC3 II/I to form autophagosomes that wrap damaged mitochondria. This autophagy process can help to maintain intracellular stability by allowing damaged mitochondria to be cleared [ 43, 44]. Thus, mitochondrial function is thought to be closely related to DDP resistance. Research has shown that mitochondrial alterations and abnormal mitophagy play a key role in DDP resistance [19], and inhibition of mitophagy is a promising intervention strategy for combating DDP resistance in ovarian cancer [20]. In this study, we examined the effect of CLPP on mitophagy in ovarian cancer cells undergoing DDP treatment. Our data showed that CLPP knockdown promoted the expressions of PINK1, Parkin, and LC3II/I proteins in wild-type ovarian cancer cells, while CLPP overexpression decreased these levels of expression. This suggests that CLPP can regulate mitochondrial autophagy in cisplatin-resistant ovarian cancer cells.

HSPA8 is a conserved molecular chaperone that plays an indispensable role in the cellular stress response [9]. High levels of HSPA8 expression have been detected in various cancer cells and are thought to promote cancer cell proliferation and autophagy [45]. HSPA8 is highly expressed in acute myeloid leukemia, and patients with high HSPA8 expression tend to have a poor prognosis [46]. HSPA8 ablation inhibits cell proliferation and enhances the chemosensitivity of imatinib-resistant chronic myeloid leukemia cells to imatinib [47]. HSPA8 overexpression promotes cell viability and autophagy in pancreatic cancer cells [48]. In this study, our database predictions and experimental analyses showed that HSPA8 is involved in regulating CLPP protein stability. HSPA8 did not affect CLPP mRNA expression, but HSPA8 overexpression promoted the degradation of CLPP protein. In addition, we further investigated the effects of HSPA8 on ovarian cancer cells. Our data showed that HSPA8 increased the IC 50 values and mitophagy and inhibited ROS production and cell death. Consistent with previous studies [ 49, 50], our findings also suggest that HSPA8 has oncogenic functions.

In summary, we successfully generated 2 DDP-resistant ovarian cancer cell lines and found that CLPP increased the DDP resistance of ovarian cancer cells by inhibiting mitophagy and promoting cellular stress. HSPA8 promotes the degradation of CLPP protein by regulating its stability. Our study reveals a novel regulatory axis of DDP resistance in ovarian cancer cells and suggests promising targets (i.e., HSPA8, CLPP, and mitochondrial protein homeostasis) for overcoming DDP resistance.

Supporting information

125TabS1
125TabS1.pdf (63.3KB, pdf)

Supplementary Data

Supplementary data is available at Acta Biochimica et Biphysica Sinica online.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grant from the construction Joint Project of Henan Province Medical Science and Technology Research Plan in 2022 (No. LHGJ20220200).

References

  • 1.Kuroki L, Guntupalli SR. Treatment of epithelial ovarian cancer. BMJ. . 2020;371:m3773. doi: 10.1136/bmj.m3773. [DOI] [PubMed] [Google Scholar]
  • 2.Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. . 2020;70:7–30. doi: 10.3322/caac.21590. [DOI] [PubMed] [Google Scholar]
  • 3.Tropé C, Kaern J. Adjuvant chemotherapy for early-stage ovarian cancer: review of the literature. J Clin Oncol. . 2007;25:2909–2920. doi: 10.1200/JCO.2007.11.1013. [DOI] [PubMed] [Google Scholar]
  • 4.Liu Y, Zhu K, Guan X, Xie S, Wang Y, Tong Y, Guo L, et al. TTK is a potential therapeutic target for cisplatin-resistant ovarian cancer. J Ovarian Res. . 2021;14:128. doi: 10.1186/s13048-021-00884-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Dasari S, Bernard Tchounwou P. Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol. . 2014;740:364–378. doi: 10.1016/j.ejphar.2014.07.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Zhao Y, Hong X, Chen X, Hu C, Lu W, Xie B, Zhong L, et al. Deregulation of exo70 facilitates innate and acquired cisplatin resistance in epithelial ovarian cancer by promoting cisplatin efflux. Cancers. . 2021;13:3467. doi: 10.3390/cancers13143467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Wu G, Peng H, Tang M, Yang M, Wang J, Hu Y, Li Z, et al. ZNF711 down-regulation promotes CISPLATIN resistance in epithelial ovarian cancer via interacting with JHDM2A and suppressing SLC31A1 expression. eBioMedicine. . 2021;71:103558. doi: 10.1016/j.ebiom.2021.103558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Kutuk O, Arisan ED, Tezil T, Shoshan MC, Basaga H. Cisplatin overcomes Bcl-2-mediated resistance to apoptosis via preferential engagement of Bak: critical role of Noxa-mediated lipid peroxidation. Carcinogenesis. . 2009;30:1517–1527. doi: 10.1093/carcin/bgp165. [DOI] [PubMed] [Google Scholar]
  • 9.Duan M, Ulibarri J, Liu KJ, Mao P. Role of nucleotide excision repair in cisplatin resistance. Int J Mol Sci. . 2020;21:9248. doi: 10.3390/ijms21239248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Low HB, Wong ZL, Wu B, Kong LR, Png CW, Cho YL, Li CW, et al. DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death. Nat Commun. . 2021;12:2284. doi: 10.1038/s41467-021-22638-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Jin L, Chun J, Pan C, Li D, Lin R, Alesi GN, Wang X, et al. MAST1 drives cisplatin resistance in human cancers by rewiring cRaf-Independent MEK activation. Cancer Cell. . 2018;34:315–330.e7. doi: 10.1016/j.ccell.2018.06.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Li H, Yang P, Wang JH, Zhang J, Ma Q, Jiang Y, Wu Y, et al. HLF regulates ferroptosis, development and chemoresistance of triple-negative breast cancer by activating tumor cell-macrophage crosstalk. J Hematol Oncol. . 2022;15:2. doi: 10.1186/s13045-021-01223-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wang D, Zhao C, Xu F, Zhang A, Jin M, Zhang K, Liu L, et al. Cisplatin-resistant NSCLC cells induced by hypoxia transmit resistance to sensitive cells through exosomal PKM2. Theranostics. . 2021;11:2860–2875. doi: 10.7150/thno.51797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Zhai J, Shen J, Xie G, Wu J, He M, Gao L, Zhang Y, et al. Cancer-associated fibroblasts-derived IL-8 mediates resistance to cisplatin in human gastric cancer. Cancer Lett. . 2019;454:37–43. doi: 10.1016/j.canlet.2019.04.002. [DOI] [PubMed] [Google Scholar]
  • 15.Ghosh S. Cisplatin: The first metal based anticancer drug. Bioorg Chem. . 2019;88:102925. doi: 10.1016/j.bioorg.2019.102925. [DOI] [PubMed] [Google Scholar]
  • 16.Komatsu M, Sumizawa T, Mutoh M, Chen ZS, Terada K, Furukawa T, Yang XL, et al. Copper-transporting P-type adenosine triphosphatase (ATP7B) is associated with cisplatin resistance. Cancer Res. 2000, 60: 1312–6 . [PubMed]
  • 17.Ohbu M, Ogawa K, Konno S, Kanzaki A, Terada K, Sugiyama T, Takebayashi Y. Copper-transporting P-type adenosine triphosphatase (ATP7B) is expressed in human gastric carcinoma. Cancer Lett. . 2003;189:33–38. doi: 10.1016/S0304-3835(02)00462-7. [DOI] [PubMed] [Google Scholar]
  • 18.Zampieri LX, Grasso D, Bouzin C, Brusa D, Rossignol R, Sonveaux P. Mitochondria participate in chemoresistance to cisplatin in human ovarian cancer cells. Mol Cancer Res. . 2020;18:1379–1391. doi: 10.1158/1541-7786.MCR-19-1145. [DOI] [PubMed] [Google Scholar]
  • 19.Ricci F, Corbelli A, Affatato R, Chilà R, Chiappa M, Brunelli L, Fruscio R, et al. Mitochondrial structural alterations in ovarian cancer patient-derived xenografts resistant to cisplatin. Am J Cancer Res. 2021, 11: 2303–11 . [PMC free article] [PubMed]
  • 20.Vianello C, Cocetta V, Catanzaro D, Dorn Ii GW, De Milito A, Rizzolio F, Canzonieri V, et al. Cisplatin resistance can be curtailed by blunting Bnip3-mediated mitochondrial autophagy. Cell Death Dis. . 2022;13:398. doi: 10.1038/s41419-022-04741-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Nouri K, Feng Y, Schimmer AD. Mitochondrial ClpP serine protease-biological function and emerging target for cancer therapy. Cell Death Dis. . 2020;11:841. doi: 10.1038/s41419-020-03062-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lee YG, Kim HW, Nam Y, Shin KJ, Lee YJ, Park DH, Rhee HW, et al. LONP1 and ClpP cooperatively regulate mitochondrial proteostasis for cancer cell survival. Oncogenesis. . 2021;10:18. doi: 10.1038/s41389-021-00306-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Cormio A, Sanguedolce F, Pesce V, Musicco C. Mitochondrial caseinolytic protease p: a possible novel prognostic marker and therapeutic target in cancer. Int J Mol Sci. . 2021;22:6228. doi: 10.3390/ijms22126228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ishizawa J, Zarabi SF, Davis RE, Halgas O, Nii T, Jitkova Y, Zhao R, et al. Mitochondrial ClpP-mediated proteolysis induces selective cancer cell lethality. Cancer Cell. . 2019;35:721–737.e9. doi: 10.1016/j.ccell.2019.03.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Wang P, Zhang T, Wang X, Xiao H, Li H, Zhou LL, Yang T, et al. Aberrant human ClpP activation disturbs mitochondrial proteome homeostasis to suppress pancreatic ductal adenocarcinoma. Cell Chem Biol. . 2022;29:1396–1408.e8. doi: 10.1016/j.chembiol.2022.07.002. [DOI] [PubMed] [Google Scholar]
  • 26.Nguyen TTT, Shang E, Schiffgens S, Torrini C, Shu C, Akman HO, Prabhu VV, et al. Induction of synthetic lethality by activation of mitochondrial ClpP and inhibition of HDAC1/2 in glioblastoma. Clin Cancer Res. . 2022;28:1881–1895. doi: 10.1158/1078-0432.CCR-21-2857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kou X, Ding H, Li L, Chao H. Caseinolytic protease P (CLPP) activated by ONC201 inhibits proliferation and promotes apoptosis in human epithelial ovarian cancer cells by inducing mitochondrial dysfunction. Ann Transl Med. . 2021;9:1463. doi: 10.21037/atm-21-4321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Arispe N, Doh M, De Maio A. Lipid interaction differentiates the constitutive and stress-induced heat shock proteins Hsc70 and Hsp70. Cell Stress Chaper. . 2002;7:330. doi: 10.1379/1466-1268(2002)007&#x0003c;0330:LIDTCA&#x0003e;2.0.CO;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Stricher F, Macri C, Ruff M, Muller S. HSPA8/HSC70 chaperone protein. Autophagy. . 2013;9:1937–1954. doi: 10.4161/auto.26448. [DOI] [PubMed] [Google Scholar]
  • 30.Chiang HL, Terlecky SR, Plant CP, Dice JF. A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science. . 1989;246:382–385. doi: 10.1126/science.2799391. [DOI] [PubMed] [Google Scholar]
  • 31.Kaushik S, Cuervo AM. Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol. . 2012;22:407–417. doi: 10.1016/j.tcb.2012.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lee DG, Kam MK, Kim KM, Kim HS, Kwon OS, Lee HS, Lee DS. Peroxiredoxin 5 prevents iron overload-induced neuronal death by inhibiting mitochondrial fragmentation and endoplasmic reticulum stress in mouse hippocampal HT-22 cells. Int J Biochem Cell Biol. . 2018;102:10–19. doi: 10.1016/j.biocel.2018.06.005. [DOI] [PubMed] [Google Scholar]
  • 33.Mowers EE, Sharifi MN, Macleod KF. Functions of autophagy in the tumor microenvironment and cancer metastasis. FEBS J. . 2018;285:1751–1766. doi: 10.1111/febs.14388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Fields EC, McGuire WP, Lin L, Temkin SM. Radiation treatment in women with ovarian cancer: past, present, and future. Front Oncol. . 2017;7:177. doi: 10.3389/fonc.2017.00177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Gupta S, Nag S, Aggarwal S, Rauthan A, Warrier N. Maintenance therapy for recurrent epithelial ovarian cancer: current therapies and future perspectives—a review. J Ovarian Res. . 2019;12:103. doi: 10.1186/s13048-019-0579-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Fan Y, Wang J, Fang Z, Pierce SR, West L, Staley A, Tucker K, et al. Anti-tumor and anti-invasive effects of ONC201 on ovarian cancer cells and a transgenic mouse model of serous ovarian cancer. Front Oncol. . 2022;12:789450. doi: 10.3389/fonc.2022.789450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Tucker K, Yin Y, Staley SA, Zhao Z, Fang Z, Fan Y, Zhang X, et al. ONC206 has anti-tumorigenic effects in human ovarian cancer cells and in a transgenic mouse model of high-grade serous ovarian cancer. Am J Cancer Res. 2022, 12: 521–36 . [PMC free article] [PubMed]
  • 38.Zong WX, Rabinowitz JD, White E. Mitochondria and cancer. Mol Cell. . 2016;61:667–676. doi: 10.1016/j.molcel.2016.02.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Vega-Naredo I, Loureiro R, Mesquita KA, Barbosa IA, Tavares LC, Branco AF, Erickson JR, et al. Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells. Cell Death Differ. . 2014;21:1560–1574. doi: 10.1038/cdd.2014.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Kabeya Y. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. . 2000;19:5720–5728. doi: 10.1093/emboj/19.21.5720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Chourasia AH, Tracy K, Frankenberger C, Boland ML, Sharifi MN, Drake LE, Sachleben JR, et al. Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis. EMBO Rep. . 2015;16:1145–1163. doi: 10.15252/embr.201540759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Quinn PMJ, Moreira PI, Ambrósio AF, Alves CH. PINK1/PARKIN signalling in neurodegeneration and neuroinflammation. Acta Neuropathol Commun. . 2020;8:189. doi: 10.1186/s40478-020-01062-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Yan C, Luo L, Guo CY, Goto S, Urata Y, Shao JH, Li TS. Doxorubicin-induced mitophagy contributes to drug resistance in cancer stem cells from HCT8 human colorectal cancer cells. Cancer Lett. . 2017;388:34–42. doi: 10.1016/j.canlet.2016.11.018. [DOI] [PubMed] [Google Scholar]
  • 44.Ke PY, Chang CW, Hsiao YC. Baicalein activates parkin-dependent mitophagy through NDP52 and OPTN. Cells. . 2022;11:1132. doi: 10.3390/cells11071132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Nirdé P, Derocq D, Maynadier M, Chambon M, Basile I, Gary-Bobo M, Garcia M. Heat shock cognate 70 protein secretion as a new growth arrest signal for cancer cells. Oncogene. . 2010;29:117–127. doi: 10.1038/onc.2009.311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Li J, Ge Z. High HSPA8 expression predicts adverse outcomes of acute myeloid leukemia. BMC Cancer. . 2021;21:475. doi: 10.1186/s12885-021-08193-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Liu Z, Zheng W, Liu Y, Zhou B, Zhang Y, Wang F. Targeting HSPA8 inhibits proliferation via downregulating BCR-ABL and enhances chemosensitivity in imatinib-resistant chronic myeloid leukemia cells. Exp Cell Res. . 2021;405:112708. doi: 10.1016/j.yexcr.2021.112708. [DOI] [PubMed] [Google Scholar]
  • 48.Tian Y, Xu H, Farooq AA, Nie B, Chen X, Su S, Yuan R, et al. Maslinic acid induces autophagy by down‐regulating HSPA8 in pancreatic cancer cells. PhytoTher Res. . 2018;32:1320–1331. doi: 10.1002/ptr.6064. [DOI] [PubMed] [Google Scholar]
  • 49.Zhang Y, Shan N, Zhou W, Zhang S. Identification of HSPA8 as a candidate biomarker for endometrial carcinoma by using iTRAQ-based proteomic analysis. Onco Targets Ther. . 2016;9:2169–2179. doi: 10.2147/OTT.S97983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Ying B, Xu W, Nie Y, Li Y. HSPA8 is a new biomarker of triple negative breast cancer related to prognosis and immune infiltration. Dis Markers. 2022, 2022: 8446857 . [DOI] [PMC free article] [PubMed]

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