SHARPIN promotes cell proliferation of cholangiocarcinoma and inhibits ferroptosis via p53/SLC7A11/GPX4 signaling

Abstract SHARPIN is a tumor‐associated gene involved in the growth and proliferation of many tumor types. A function of SHARPIN in cholangiocarcinoma (CCA) is so far unclear. Here, we studied the role and function of SHARPIN in CCA and revealed its relevant molecular mechanism. The expression of SHARPIN was analyzed in cholangiocarcinoma tissues from patients using immunohistochemistry, quantitative PCR, and western blot analysis. Expression of SHARPIN was suppressed/overexpressed by siRNA silencing or lentiviral overexpression vector, and the effect on cell proliferation was determined by the CCK‐8 assay and flow cytometry. Accumulation of reactive oxygen species was measured with MitoTracker, and JC‐1 staining showed mitochondrial fission/fusion and mitochondrial membrane potential changes as a result of the silencing or overexpression. The ferroptosis marker solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and the antioxidant enzymes superoxide dismutase 1 (SOD‐1) and SOD‐2 were analyzed by western blot. The results showed that SHARPIN expression was increased in CCA tissue, and this was involved in cell proliferation. SHARPIN silencing resulted in accumulated reactive oxygen species, reduced mitochondrial fission, and a reduced mitochondrial membrane potential. Silencing of SHARPIN inhibited the ubiquitination and degradation of p53, and downregulated levels of SLC7A11, GPX4, SOD‐1, and SOD‐2, all of which contributed to excessive oxidative stress that leads to ferroptosis. Overexpression of SHARPIN would reverse the above process. The collected data suggest that in CCA, SHARPIN‐mediated cell ferroptosis via the p53/SLC7A11/GPX4 signaling pathway is inhibited. Targeting SHARPIN might be a promising approach for the treatment of CCA.


| INTRODUC TI ON
Cholangiocarcinoma (CCA) is a rare malignant adenocarcinoma that arises from the epithelial cells of the intra-and extra-hepatic bile ducts. It is the second most common hepatobiliary malignancy after hepatocellular carcinoma. 1 The incidence and mortality of CCA has increased worldwide over the past 30  and activation of survival signaling pathways. 5,6 In addition, SHARPIN was found to mediate the cell fate through the action of mitochondria, 7 although the detailed mechanism for this is not fully elucidated.
We considered the possibility that SHARPIN might regulate cell ferroptosis via mitochondria and that this might be relevant to CAA. To test this hypothesis, we investigated the effects of SHARPIN in mediating CAA cells to enter ferroptosis by alteration of mitochondrial function.
Ferroptosis is a recently recognized type of regulated cell death that differs from other types of regulated cell death in morphology, genetics, and biochemistry. 8 Mitochondria are involved in ferroptosis, and cysteine deprivation also plays a crucial role. Recent studies have shown that ferroptosis is involved in several diseases, and it has become the focus of research on the treatment and prognosis improvement of cancer. 9,10 Mechanistically, ferroptosis is triggered by suppressing a central regulator, glutathione peroxidase 4 (GPX4), which is required for the clearance of reactive oxygen species (ROS).
Reduced GPX4 activity can result from direct inhibition of its enzyme activity or promoted degradation by inducers. Indirect inhibition of GPX4 can also occur through blocking a glutamate/cysteine antiporter that exports intracellular glutamate in exchange for extracellular cysteine to generate glutathione. 11,12 Inhibition of cysteine uptake by p53 can sensitize cells to ferroptosis through downregulating the expression of SLC7A11 and GPX4, reduction of antioxidant capacity and resultant ROS accumulation. [13][14][15] In addition, SHARPIN has been reported to facilitate p53 poly-ubiquitination and its degradation in breast cancer. 16 Whether SHARPIN functions in CCA was previously unclear, but here we confirm that SHARPIN expression is increased in CCA, promotes cell proliferation, and suppresses ferroptosis. The present study identified the mechanism by which SHARPIN promotes CCA proliferation via the p53/SLC7A11/GPX4 signaling pathway.

| Cell culture and clinical tumor tissue
The two human CCA cell lines, HuCCT1 and Hccc9810, were purchased from the American Type Culture Collection. Ferrostatin-1(#HY-100579) was purchased from MCE. Cells were maintained in exponential growth phase in RPMI-1640 medium (Life Technologies) containing 10% FBS (Invitrogen), 100 U/mL penicillin, and 0.1% (w/v) streptomycin at 37°C in a humidified atmosphere of 5% CO 2 . Cells were used within 6 months of resuscitation. CCA tissue samples were obtained by surgical resection. The tumor and adjacent tissues extracted from clinical cases were formalin-fixed, paraffin-embedded, and sectioned for immunohistochemistry and immunofluorescence assays or immediately frozen for western blot and RT-qPCR assays.

| Establishment of SHARPIN silenced and stable overexpression cell lines
The HuCCT1 and Hccc9810 cells were transfected using Lipofectamine

| Cell viability and proliferation assay
After transfection, the viability of the transfected SHARPIN siRNA and lentiviral vectors cells were determined using the CCK8 assay.
Cells were plated in 96-well plates with 5 × 10 3 cells per well in three replicate wells. CCK-8 reagent (Dojindo Molecular Technologies) was added to each well (10 μL) and incubated for 1 h. Absorbance at 450 nm was recorded using a microplate reader (Victor Nivo 3F, Perkin Elmer).
For the EdU proliferation assay, cells were cultured, fixed, blocked, and incubated with EdU dyes (Thermo Fisher Scientific). The nuclei were stained with Hoechst dye 33,342 (Solarbio, life sciences) for 30 min at room temperature. Images of five randomly selected areas were recorded with a fluorescence microscope (Leica, DMI8). For flow cytometry, cell proliferation was analyzed without Hoechst dye 33342 staining. Data were acquired using a flow cytometer (FACSAria, Becton Dickinson) and analyzed with FCS Expression software (3.0).

| Reactive oxygen species production assay
The cells were seeded in plates at 2 × 10 5 cells per well and cultured overnight, then incubated with the ROS-detecting fluorescence dye 2,7-dichlorofluorescein diacetate (DCF-DA, 10 μM) at 37°C for 30 min. Partial cells were washed with PBS, intracellular ROS content was measured by flow cytometry, and partial cells were microscopically examined. Data were analyzed with FCS Expression software and Image J software.

| Mitochondrial membrane potential assay
The mitochondrial membrane potential was quantified by Mito

| Western blot analysis
Western blot analysis was performed as previously described. 17 Briefly, total protein was isolated using RIPA lysis buffer with PMSF. Protein concentration was determined using the bicinchoninic acid assay. Following 12% SDS-PAGE electrophoresis, proteins were transferred to PVDF membranes that were blocked with 5% BSA at room temperature for 1 h. The blots were probed (1:1000, # 20886-1-AP, Proteintech) followed by the appropriate secondary antibody, which was conjugated to HRP (anti-mouse IgG/anti-rabbit IgG, CST, 1:5000). Bands were visualized by chemiluminescence (#35050, Thermo Fisher) and quantified by ChemiDoc Touch (Bio-Rad). Data were analyzed with Quantity One analysis software (Bio-Rad).

| Immunohistochemistry
The tumor specimens were fixed in 4% formalin and embedded in paraffin. Sections were cut into 5 μm slices, antigen retrieval was performed with citrate buffer, and endogenous peroxidase activity was blocked with 3% H 2 O 2 . Samples were incubated with anti-SHARPIN antibodies (1:4000, # ab197853, Abcam) overnight at 4°C. After washing with PBS, the slices were incubated with HRP-conjugated secondary antibodies at 37°C for 30 min. Signals were detected with DAB. The nuclei were counterstained with hematoxylin for 1 min, washed three times with PBS, and examined by microscopy. The positively stained area was quantified using Image J software.

| Immunofluorescence
Tissue slices were obtained as described above. Slices were treated as described previously. DAPI was used to stain cell nuclei. Immunostaining was then examined using a Leica microscope (DM4B). Cells were analyzed with image-Pro Plus 6.0 (IPP 6.0; Media Cybernetics).

| Statistical analysis
Statistical analysis of data was performed using GraphPad Prism 6.0 (GraphPad Software) as previously described. All data are presented as mean ± SD from triplicate experiments and statistical significance is shown as *p < 0.05, **p < 0.01, and ***p < 0.001.

| Expression of SHARPIN is elevated in cholangiocarcinoma
To investigate the role of SHARPIN in cholangiocarcinoma, we first analyzed SHARPIN expression in CCA cancer specimens and in normal controls based on the publicly available database Gene Expression Profiling Interactive Analysis (GEPIA). 18 This revealed that SHARPIN mRNA levels were elevated in cholangiocarcinoma tissue samples compared to normal tissue ( Figure 1A). The association between SHARPIN expression and prognosis of CCA patients through the publicly available database GEPIA was also shown ( Figure S1). This finding was confirmed by detection of SHARPIN expression by immunohistochemistry and immunofluorescence in paired samples from patients with CCA ( Figure 1B-D). Both qPCR and western blot results showed elevated SHARPIN expression in tumor tissue compared to adjacent healthy tissue ( Figure 1E-G).
These findings suggested that SHARPIN expression was increased in CCA.

| SHARPIN promotes growth of cholangiocarcinoma cells
Two CCA cell lines (Hccc9810 and HuCCT1) were transfected with SHARPIN siRNA and lentiviral overexpression vectors. The knockdown efficiency was confirmed by western blot and qPCR ( Figure S2). The CCK-8 assay was performed to demonstrate that siSHARPIN had significantly decreased CCA cell viability and overexpression of SHARPIN would increase CCA cells viability (Figure 2A

| Silencing SHARPIN induced cholangiocarcinoma cells to generate reactive oxygen species in vitro
The effect of SHARPIN expression on cell ROS production was evaluated. Intracellular ROS levels were measured using the ROS-

| Knockdown SHARPIN inhibits the function of mitochondria
Previous studies have shown that excessive ROS generation can alter the shape of mitochondria, and this contributes to cell apopto-

| SHARPIN inhibits cellular ferroptosis through a p53/SLC7A11/GPX4 cascade
To further determine the molecular mechanism by which SHARPIN regulates ROS production and inhibits cell proliferation, western blots were performed that were stained for SHARPIN, p53, GPX4, solute carrier family 7 member 11 (SLC7A11), apoptosis-inducing factor mitochondria-associated 2 (AIFM2, also called ferroptosis suppressor The ferroptosis hallmark SLC7A11 and GPX4 had no obvious change compared with scramble ( Figure S3). In this way, SHARPIN would protect cells from ferroptosis and promote cell proliferation. This is summarized in Figure 6.

| DISCUSS ION
Cholangiocarcinoma is a highly aggressive liver tumor with high mortality rates that can be molecularly heterogeneous, and its incidence is increasing. 20 In China, the incidence of CCA currently increases by more than 5% per year. 21 In the search for factors that F I G U R E 4 SHARPIN could alter the mitochondrial membrane potential and mediate fusion or fission in CCA cell lines. (A, B) After cells were silenced with siSHARPIN and overexpression with lentiviral vector, mitochondria were stained with Mito Tracker (red), and the mean fluorescence intensity was quantified. (C, D) Mitochondrial membrane potential (Δψm) was measured using the JC-1 probe. The distribution of JC-1 aggregates (PE channel) and monomers (FITC channel) was determined by flow cytometry. Values (mean ± SD) from quintuplicate experiments are shown. ***p < 0.001 vs Scr siRNA group, # p < 0.05 vs SHARPIN NC group.
contribute to the growth and proliferation of these tumor cells, we turned to SHARPIN. It has been well documented that SHARPIN is overexpressed in many human cancer types, including prostate cancer, melanoma, hepatocellular carcinoma, and breast cancer. 6,16,22,23 SHARPIN can increase cell proliferation and reduce apoptosis while enhancing metastasis via induction of cell migration, invasion, and angiogenesis. A recent study reported that knockdown of SHARPIN expression results in impaired growth and survival of tumor cells.
However, its expression and potential function in CCA has so far not been elucidated. Here, we report that SHARPIN was upregulated in human CCA and that high SHARPIN expression is associated with cell growth and proliferation by protecting cells from ferroptosis.
Using immunofluorescence, immunohistochemistry and molecular assays (western blots and qPCR), we demonstrate that SHARPIN expression is significantly increased in tumor tissue and that its silencing/overexpression results in a reduced/promoted ability for cell growth and proliferation. This finding is consistent with previously reported roles of SHARPIN in multiple tumor studies. 6,16,22 Our work contributes to a growing body of literature that identifies human CCA cells. We also noticed that the cell ferroptosis markers SLC7A11 and GPX4 were significantly reduced, as were the antioxidant enzymes SOD-1 and SOD-2. SLC7A11 is a plasma membrane cysteine/glutamate antiporter. 26 Inhibition of SLC7A11 contributes to ferroptotic cell death. 27 The gene for GPX4 is located downstream of SLC7A11, which functions as a guard against ferroptosis induced by lipid ROS. Overexpression of GPX4 would thus block ferroptosis, while depletion or repression of its expression would augment the levels of ROS. [28][29][30] Cysteine uptake is inhibited by p53, and this sensitizes the cells to ferroptosis by repression of SLC7A11 and promotion of ROS production. 13 A later study revealed that the p53/SLC7A11/GPX4 signaling pathway is involved in mediating ferroptosis in prostate cancer cells. 14 We also found that SLC7A11 and GPX4 had not obviously changed after SHARPIN siRNA transfected CCA cell line treatment with ferroptosis inhibitor. Furthermore, we found SHARPIN was not able to alter the expression of FSP1, which was a maker of the FSP1-CoQ10 (GSH/GPX4-independent) ferroptosis pathway.
These results suggest that SHARPIN mediated CCA cells ferroptosis through the GSH/GPX4-dependent rather than the FSP1-CoQ10 pathway. 19 ROS production can impair mitochondrial membrane potential. The cytoplasmic enzyme SOD-1 and the mitochondrial SOD-2 are important antioxidant enzymes. 31 Both SOD-1 and SOD-2 are responsible for protecting the cell from oxidative stress, and our data show that SHARPIN silencing contributes to reduced SOD-1 and SOD-2 activity, thus promoting cell ferroptosis in CCA.
Here we demonstrate that SHARPIN is overexpressed in CCA, which inhibits ferroptosis and promotes cell proliferation. Our results further reveal that silencing of SHARPIN reduces p53 ubiquitination and, as a consequence, the expression of SLC7A11 and GPX4 is decreased, resulting in elevated levels of intracellular and mitochondrial oxidative stress, which will eventually lead to ferroptosis. Taken together, these findings uncover a SHARPIN/p53/ SLC7A11/GPX4 axis in CCA cells, which may facilitate the targeting of SHARPIN as a novel therapeutic strategy for treatment of human CCA.

AUTH O R CO NTR I B UTI O N S
J.Y. and W.W. designed the research, and B.Q. analyzed the data. J.Y. and C.Z. wrote the paper; C.Z., J.L, and K.Z. performed the experiments; H.O., B. W., and X. Z. purchased the reagents and materials; L.Z., K.S, and X.D. provided guidance on experimental technology.
All authors read and approved the final manuscript.

D I SCLOS U R E
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. F I G U R E 6 Mechanism of SHARPIN regulated cell ferroptosis via ubiquitinmediated p53 degradation and the SLC7A11/GPX4 signaling cascade.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Approval of the research protocol by an Institutional Reviewer
Board: The study protocol was approved by the Investigation Ethical