Inhibition of PI3K/mTOR increased the sensitivity of hepatocellular carcinoma cells to cisplatin via interference with mitochondrial‐lysosomal crosstalk

Abstract Objectives The genotoxicity of cisplatin towards nuclear DNA is not sufficient to explain the cisplatin resistance of hepatocellular carcinoma (HCC) cells; cisplatin interacts with many organelles, which can influence the sensitivity. Here, we explored the role of mitochondrial‐lysosomal crosstalk in the cisplatin resistance of HCC cells. Materials and methods Huh7 and HepG2 cells were subjected to different treatments. Flow cytometry was conducted to detect mitochondrial reactive oxygen species, mitochondrial mass, lysosomal function, mitochondrial membrane potential and apoptosis. Western blotting was performed to evaluate protein levels. The oxygen consumption rate was measured to evaluate mitochondrial function. Results Cisplatin activated mitophagy and lysosomal biogenesis, resulting in crosstalk between mitochondria and lysosomes and cisplatin resistance in HCC cells. Furthermore, a combination of cisplatin with the phosphatidylinositol‐3‐kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor PKI‐402 induced lysosomal membrane permeabilization. This effect changed the role of the lysosome from a protective one to that of a cell death promoter, completely destroying the mitochondrial‐lysosomal crosstalk and significantly enhancing the sensitivity of HCC cells to cisplatin. Conclusions This is the first evidence of the importance of mitochondrial‐lysosomal crosstalk in the cisplatin resistance of HCC cells and of the destruction of this crosstalk by a PI3K/mTOR inhibitor to increase the sensitivity of HCC cells to cisplatin. This mechanism could be developed as a novel target for treatment of HCC in the future.


| INTRODUC TI ON
Cisplatin (cis-diamminedichloroplatinum(II), CDDP), as a representative platinum drug, has shown efficacy in hepatocellular carcinoma (HCC) treatment, [1][2][3][4] but most hepatobiliary cancer guidelines do not recommend cisplatin as a first-line treatment because of the low sensitivity of this drug to HCC. [5][6][7][8][9] Cisplatin has also been shown to induce DNA damage; however, recent studies have found that genotoxicity accounts for only a small portion of the cytotoxicity of this drug. 10 Cisplatin can also interact with mitochondria, lysosomes, the endoplasmic reticulum and other organelles, 11,12 influencing the sensitivity of tumour cells to cisplatin. Therefore, identification of the target of cisplatin in HCC cells is very important for elucidation of the resistance mechanism.
Several studies, including our own, have reported that cisplatin enhanced the reactive oxygen species (ROS) levels in HCC cells. [13][14][15] This finding provides support for mitochondria as targets of cisplatin in HCC because these organelles are the major sites of ROS formation in the cell, 16,17 and the production of mitochondrial ROS (mtROS) is an important indicator of damaged mitochondrial antioxidant defence function. 18 Furthermore, low mtROS levels can activate the mitophagy-lysosome pathway to degrade damaged mitochondria and mtROS. [19][20][21][22] This phenomenon plays a protective role in cells 23 but has not been reported in the context of HCC chemotherapy. However, high levels of mtROS accumulation were seen to be closely associated with mitochondrial apoptosis in tumour cells. [24][25][26] mtROS are also important for the maintenance of lysosomal homeostasis. To maintain autophagic flux, tumour cells require the quantity and function of lysosomes to be maintained via lysosomal biogenesis and autophagic lysosomal reformation. 27 Low levels of ROS can activate transcription factor EB (TFEB)-mediated lysosomal biogenesis, ensuring mitophagy, 28 while high levels of ROS contribute to lysosomal membrane permeabilization (LMP), leading to the lysosomal release of cathepsin and hydrolase into the cytoplasm, causing apoptosis. [29][30][31] Therefore, we speculate that the mtROS levels in HCC cells play a key role in the maintenance of the homeostasis of mitochondria and lysosomes in the mitophagy-lysosome pathway.
The highly activated phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway in HCC cells is an important connector pathway involved in mitochondrial-lysosomal crosstalk. This pathway is involved in the regulation of mitochondrial metabolism and in the resistance to mitochondrial pathway apoptosis. 32 Kirstein found that the PI3K inhibitor BKM120 impaired mitochondrial function, 33 while inhibition of PI3K/mTOR increased the ROS levels in tumour cells. [34][35][36][37] Additionally, Madge reported that PI3K signalling was involved in the control of lysosomal activity and stability, 38 and Seitz showed that the dual PI3K/mTOR inhibitor NVP-BEZ235 stimulated enlargement of the lysosomal compartment, generated ROS and cooperated with chloroquine (CQ) to trigger LMP in neuroblastoma cells. 39 However, it remains unclear whether chemotherapeutic drugs combined with PI3K/mTOR inhibitors cause LMP.
In this study, we found that mtROS induced the mitophagy-lysosome pathway and lysosome biogenesis in response to cisplatin in HCC cells. We also demonstrated that mitochondrial-lysosomal crosstalk was involved in the resistance to cisplatin-induced apoptosis in HCC cells and in the maintenance of the cells in a stable state. When cisplatin was combined with the PI3K/mTOR inhibitor PKI-402, the mtROS levels in HCC cells increased significantly, thereby destroying the stable state. High mtROS levels mediated LMP, resulting in mitochondrial injury, which produced additional mtROS and aggravated mitochondrial and lysosomal damage, thus forming a vicious cycle and eventually leading to HCC cell apoptosis. This study provides novel insights into potential comprehensive treatments of HCC.

| Cell culture
The human HCC cell lines Huh7 and HepG2 were purchased from the Chinese Academy of Medical Sciences (China). The two cell lines were incubated at 37°C in 5% CO 2 and cultured in DMEM supplemented with 10% foetal bovine serum (FBS), 100 IU/mL penicillin and 100 µg/mL streptomycin.

| Cell metabolic activity assays
Cells were seeded in 96-well plates. After exposure of the cells to different concentrations of cisplatin, 500 μg/mL MTT solution was added, and the cells were maintained for 4 hours at 37°C. Then, DMSO was added. The optical density was measured at 570 nm using a CLARIOstar microplate reader (BMG Labtech GmbH, Germany). The cell metabolic activity was calculated as follows: cell metabolic activity = absorbance of the experimental group/absorbance of the control group × 100%.

| Western blotting analysis
Cells subjected to the different desired treatments were harvested and incubated in RIPA for 40 minutes at 4°C to isolate the total protein content. Protein concentrations were analysed using the were blocked with 5% (w/v) skim milk for 2 hours and then incubated with the different desired antibodies at 4°C overnight. Then, the membranes were incubated with the corresponding secondary antibodies at room temperature for 1.5 hours. Immunodetection was performed using ECL reagent (Thermo Fisher Scientific, Inc, USA), and visualization was performed using a Syngene bioimaging system (Synoptics, UK). Protein levels were quantified using Quantity One software and normalized to β-actin.

| Immunofluorescence staining and confocal laser microscopy
Cells were seeded onto coverslips in 24-well plates overnight and exposed to different experimental conditions, and then, the cells

| Oxygen consumption rate
Cells were plated in 96-well plates at a density of 8 × 10 4 cells/ well overnight. Then, the medium in all the wells was replaced with a preheated liquid mixture containing reconstituted MitoXpress-Xtra reagent, fresh culture medium and the different treatments. The wells were then sealed using preheated mineral oil.
Fluorescence decay was measured using a CLARIOstar microplate reader.

| Reverse transcription-quantitative polymerase chain reaction analysis
TRIzol (Invitrogen) was used to extract the total cellular RNA, and reverse transcription was performed to generate cDNA, which was amplified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The primer sequences are listed in

| RT 2 profiler PCR array system
The RT 2 Profiler PCR array system for human mitochondrial energy metabolism (PAHS-008Z; Qiagen, Germany) profiles the expression of 84 key genes involved in human mitochondrial energy metabolism (Supporting Information Table S1). RT-qPCRs were performed using a CFX96 Touch Real-Time PCR detection system.
Fluorescence intensities were analysed using Qiagen online software, and relative quantification was performed using the 2 −ΔΔC t method. Changes in expression of the 84 genes were visualized as a heatmap.

| Statistical analysis
All the data are representative of three independent experiments, each performed in triplicate. Statistical significance was analysed using one-way ANOVA, followed by Tukey or Newman-Keuls post hoc analysis. The analyses were performed with GraphPad Prism 5.0 statistical software (USA). *P < 0.05 was considered to indicate statistical significance; **P < 0.01 indicated a highly significant difference; and ***P < 0.001 indicated an extremely significant difference.

| Mitochondria are important targets of cisplatin in HCC cells
The MTT assay was used to evaluate cell metabolic activity

| Cisplatin induced mitochondrial fission and the mitophagy-lysosomal pathway in HCC cells
Reactive oxygen species damage mitochondria, which initiates mitochondrial quality control for maintenance of mitochondrial homeostasis. 42 To determine whether HCC cells overcome cisplatin-induced mitochondrial damage in this manner, the cells were cultured with cisplatin for various durations, stained with MitoTracker Red CMXRos and observed by confocal microscopy (Figure 2A)

| Cisplatin induced lysosomal biogenesis in HCC cells, contributed to mitophagy and caused synergistic mitochondrial-lysosomal crosstalk
Mitophagy consumes large numbers of lysosomes. 43 To verify the enhancement of lysosome biogenesis in HCC cells induced by cisplatin, LysoTracker Green staining was used to quantify lysosome levels. Lysosome numbers were shown to increase in a time-dependent manner ( Figure 3A,B). DQ Red BSA, which is a red BODIPY dye con-   Table S1. Changes are presented as a heatmap; green indicates downregulation, and red indicates upregulation. Data were derived from three experiments. F, OCR was measured after Huh7 cells were treated with 8 μg/mL cisplatin and HepG2 cells were treated with 12 μg/mL cisplatin. Relative OCR values are expressed as the mean ± SD; n = 3, ***P < 0.001. G, Huh7 cells were treated with 8 μg/mL cisplatin and H, HepG2 cells were treated with 12 μg/mL cisplatin for varying durations. Then, the cells were stained with MitoTracker Green FM and detected using flow cytometry. The percentage of cells with high MitoTracker fluorescence is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01, ***P < 0.001

| Mitochondrial-lysosomal crosstalk was important for the resistance of HCC cells to cisplatin
was aggravated, in the group treated with CQ and cisplatin. We also evaluated the mitochondrial membrane potential using JC-1 and obtained similar results ( Figure 4G). Annexin V-FITC(+) staining showed that, compared with cisplatin alone, treatment with rapamycin reduced the apoptosis rate in HepG2 and Huh7 cells, while treatment with CQ enhanced cisplatin-induced apoptosis in HCC cells F I G U R E 2 Cisplatin induced mitochondrial fission and the mitophagy-lysosomal pathway in HCC cells. A, Huh7 cells were treated with 8 μg/mL cisplatin, and HepG2 cells were treated with 12 μg/mL cisplatin for 12 and 24 h. Then, the cells were stained with MitoTracker Red CMXRos and observed by confocal laser microscopy; scale bar = 10 μm. B, Western blot detection of mitochondrial fusion proteins and mitochondrial fission proteins in Huh7 cells treated with 8 μg/mL cisplatin for 6, 12 and 24 h. The protein/beta-actin ratio is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01. C, Western blot detection of mitophagy-lysosomal pathway-related proteins in Huh7 cells treated with 8 μg/mL cisplatin for 6, 12 and 24 h. The protein/beta-actin ratio is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 F I G U R E 3 Cisplatin induced lysosomal biogenesis in HCC cells. A, Huh7 cells were treated with 8 μg/mL cisplatin, and B, HepG2 cells were treated with 12 μg/mL cisplatin for varying durations. Then, the cells were stained with LysoTracker Green DND-26 and detected using flow cytometry. The percentage of cells with high LysoTracker fluorescence is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01. C, Huh7 cells were treated with 8 μg/mL cisplatin, and D, HepG2 cells were treated with 12 μg/mL cisplatin for varying durations. Then, the cells were stained with DQ Red BSA and detected using flow cytometry. The percentage of cells with high DQ Red BSA fluorescence is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01. E, Colocalization of TFEB and nuclei in Huh7 cells treated with 8 μg/mL cisplatin and HepG2 cells treated with 12 μg/mL cisplatin for 8 h; scale bar = 10 μm. The percentage of nuclear localization is analysed by ImageJ and expressed as the mean ± SD; n = 3, ***P < 0.001. F, The mRNA levels of TFEB and the CLEAR system in Huh7 cells treated with 8 μg/ mL cisplatin and G, HepG2 cells treated with 12 μg/mL cisplatin for 8 h. Relative mRNA expression is expressed as the mean ± SD; n = 3, **P < 0.01, ***P < 0.001 ( Figure 4H,I). Taken together, these results indicated that mitochondrial-lysosomal crosstalk plays a protective role in the resistance of HCC cells to cisplatin.

| D ISCUSS I ON
Mitochondria are responsible for regulating various forms of cell death, including apoptosis and necrosis. 50 Therefore, to resist chemotherapeutic drugs, cancer cells must maintain mitochondrial homeostasis. Although most previous studies have suggested that the main target of cisplatin is nuclear DNA, recent studies have found that mitochondria are critical targets of cisplatin, 11 and this finding was supported by our present findings in HCC cells. The underlying mechanism may be associated with the genotoxicity of cisplatin towards mtDNA 51 ; cisplatin binds to mtDNA with higher efficiency than to nuclear DNA, 52 but the efficiency of DNA repair in mitochondria is consistently low. However, cells initiate mitophagy to maintain organelle homeostasis. Mitochondrial fission is considered to be a sorting mechanism for mitophagy, as demonstrated by PINK1/parkin-mediated mitophagy. Loss of the mitochondrial membrane potential will stabilize PINK1 at the outer membrane, which can recruit parkin, leading to mitochondrial fragmentation and subsequent mitophagy. 23,43,53 However, it is unknown whether there exists a similar mechanism of cisplatin resistance in HCC cells. In the F I G U R E 4 Mitochondrial-lysosomal crosstalk was important for the resistance of HCC cells to cisplatin. A, Western blot detection of mitophagy-lysosomal pathway-related proteins in Huh7 cells treated with 8 μg/mL cisplatin and/or 20 μmol/L CQ for 24 h. The protein/ beta-actin ratio is expressed as the mean ± SD; n = 3, **P < 0.01, ***P < 0.001. B, Huh7 cells were treated with 8 μg/mL cisplatin combined with 20 μmol/L CQ or 5 μmol/L rapamycin for 24 h and then stained with MitoSOX Red and detected using flow cytometry. The percentage of cells with high MitoSOX fluorescence is expressed as the mean ± SD; n = 3, **P < 0.01, ***P < 0.001. C, Huh7 cells (same treatment as A) and D, HepG2 cells were treated with 12 μg/mL cisplatin combined with 20 μmol/L CQ or 5 μmol/L rapamycin for 24 h and then stained with MitoTracker Green FM and detected using flow cytometry. The percentage of cells with high MitoTracker fluorescence is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. E, OCR was measured after Huh7 cells were treated with 8 μg/mL cisplatin combined with 20 μmol/L CQ or 5 μmol/L rapamycin. Relative OCR values are expressed as the mean ± SD; n = 3, *P < 0.05. F, OCR was measured after HepG2 cells were treated with 12 μg/mL cisplatin combined with 20 μmol/L CQ or 5 μmol/L rapamycin. Relative OCR values are expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01. G, Huh7 cells were stained with JC-1 and analysed by FlowJo. The percentage of cells with low red fluorescence and high green fluorescence is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01. H, Huh7 cells and I, HepG2 cells were stained with Annexin V and PI and analysed by FlowJo. The percentage of cells with high Annexin V-FITC fluorescence is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 present study, we showed that cisplatin can induce mitochondrial fission and depolarization in HCC cells, which is a precondition of mitophagy. 43 Western blot analysis showed that cisplatin activated PINK1/parkin-mediated mitophagy in HCC cells, which selectively targeted impaired mitochondria for degradation. The autophagy inhibitor CQ can inhibit mitophagy effectively and increase the cell The percentage of cells with high MitoSOX fluorescence is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. B, Huh7 cells were treated with 8 μg/mL cisplatin and/or 5 μmol/L PKI-402 for 12 h and then stained with AO and detected using flow cytometry. The percentage of cells with low AO fluorescence is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. C, Huh7 cells (same treatment as A) were stained with JC-1 and analysed by FlowJo. The percentage of cells with low red fluorescence and high green fluorescence is expressed as the mean ± SD; n = 3, *P < 0.05, **P < 0.01. D, Cytoplasmic proteins were extracted, and the levels of cathepsin B, cathepsin D and cytochrome C were detected by Western blotting. The protein/ alpha-tubulin ratio is expressed as the mean ± SD; n = 3, **P < 0.01, ***P < 0.001 Recent studies have suggested that mtROS production is a major inducer of LMP. 30 We also found that PKI-402 elevated mtROS levels in HCC cells, which were further enhanced by cisplatin. Interestingly, we found that cisplatin caused a low degree of LMP in HCC cells.
This finding is consistent with other studies that reported that an interaction between cisplatin and lysosomes caused LMP accompanied by apoptosis, which may be associated with the accumulation of cisplatin in lysosomes, where this compound initiates LMP.
We showed that LMP induced by PKI-402 combined with cispla- We also found that mtROS levels were positively correlated with LMP, mitochondrial depolarization and apoptosis, suggesting that mtROS levels are associated with mitochondrial and lysosomal damage, which further aggravate the accumulation of mtROS. Therefore, F I G U R E 7 Cisplatin combined with PKI-402 changed the role of the lysosome from a protective one to that of a cell death promoter in HCC cells mtROS is an important positive feedback regulator that destroys mitochondrial-lysosomal crosstalk.
In summary, we provide evidence that mitochondria are important targets of cisplatin in HCC cells. Cisplatin-induced mitophagy and lysosomal biogenesis constitute mitochondrial-lysosomal crosstalk, which is a crucial mechanism by which HCC cells overcome the cytotoxicity of cisplatin. We also showed that a combination of cisplatin and the PI3K/mTOR inhibitor PKI-402 induced LMP in HCC cells and then destroyed cisplatin-induced mitochondrial-lysosomal crosstalk, which significantly increased the sensitivity of HCC cells to cisplatin (Figure 7). This study will provide new ideas and candidate targets for comprehensive treatment of HCC.

ACK N OWLED G EM ENTS
This work was supported by grants from the Project of Hepatobiliary and Pancreatic Disease Translational Medicine Platform Construction (2017F009) and Finance Department of Jilin Province.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.