p62 aggregates mediated Caspase 8 activation is responsible for progression of ovarian cancer

Abstract Increasing evidence suggests that p62/SQSTM1 functions as a signalling centre in cancer. However, the role of p62 in tumour development depends on the interacting factors it recruits and its precise regulatory mechanism remains unclear. In this study, we investigated the pro‐death signalling recruitment of p62 with the goal of improving anti‐tumour drug effects in ovarian cancer treatment. We found that p62 with Caspase 8 high expression is correlated with longer survival time compared with cases of low Caspase 8 expression in ovarian cancer. In vivo experiments suggested that insoluble p62 and ubiquitinated protein accumulation induced by autophagy impairment promoted the activation of Caspase 8 and increased cell sensitivity to cisplatin. Furthermore, p62 functional domain UBA and LIR mutants regulated autophagic flux and attenuated Caspase 8 activation, which indicates that autophagic degradation is involved in p62‐mediated activation of Caspase 8 in ovarian cancer cells. Collectively, our study demonstrates that p62 promotes Caspase 8 activation through autophagy flux blockage with cisplatin treatment. We have provided evidence that autophagy induction followed by its blockade increases cell sensitivity to chemotherapy which is dependent on p62‐Caspase 8 mediated apoptosis signalling. p62 exhibits pro‐death functions through its interaction with Caspase 8. p62 and Caspase 8 may become novel prognostic biomarkers and oncotargets for ovarian cancer treatment.

p62 is also a substrate of selective autophagy and its degradation is mainly regulated by autophagy. 7 It is known that autophagosomes undergo maturation by fusion with lysosomes for degradation after targeted substrates are sequestered in the autophagosome. During this process p62 binds ubiquitinated proteins through its ubiquitin-associated (UBA) domain and anchors to LC3 in the autophagosome membrane through its LC3-interacting region (LIR) domain. 8,9 Recent evidence suggests that the completion of autophagy relies on smooth autophagic flux and renewal of autophagosomes whereas dysfunction in the autophagy pathway leads to the accumulation of p62 and damaged proteins that possibly results in cell death. 10 Caspase 8 is a key protein in the extrinsic apoptotic pathway and its activation depends on oligomerization and self-cleavage. Upon activation, full-length Caspase 8 (p55/53) is cleaved into p43/41 and p10 fragments that are released into the cytoplasm to activate downstream caspases. The canonical Caspase 8 activation pathway is dependent on cell-surface death receptors through the deathinducing signalling complex (DISC) 11 Thus, a recent study showed that TRAIL can induce p62-dependent activation of Caspase 8 in H460 lung cancer cells. 12 Furthermore, p62 accumulation promoted Caspase 8 activation in HCT116 cells treated with ABT263, an inhibitor of Bcl-2. 13 Therefore, exploration of the association between p62 and Caspase 8 might help elucidate the crosstalk between autophagy and apoptosis during ovarian cancer treatment.
Cisplatin is the first-line agent used in the treatment of ovarian cancer. However, a high risk of tumour relapse and drug resistance are obstacles in its clinical use. Initial studies demonstrated that the anti-tumour effects of cisplatin involved its interference with the DNA structure. 14,15 However, later reports showed that only approximately 1% of the cisplatin that enters into cells actually binds to DNA. 16 The accumulated cisplatin in the cytoplasm may act as a stressor that affects the function of a variety proteins and signalling pathways including Caspase 8 and autophagy. 17,18 Here, we examined whether p62 exhibits pro-death functions through its interaction with Caspase 8. Our data suggest high p62 and Caspase8 expression is correlated with longer survival time.
Furthermore we provide evidence that p62 regulates autophagic flux through its autophagy-related domains and suppression of autophagic flux promotes Caspase 8 recruitment and activation caused by p62 accumulation, which increases the sensitivity of ovarian cancer cells to cisplatin. Our results demonstrate that p62 and Caspase 8 may serve as potential prognostic biomarkers and oncotargets for individualized treatment of ovarian cancer patients.

| Ovarian cancer tissues microarray and immunohistochemistry
A total of 160 ovarian cancer tissues were purchased from Shanghai Outdo Biotech Co.,Ltd., collected between 2009 and 2013 ( Table 1).
Immunohistochemistry of tissues was carried out using primary antibodies against p62 (Abcam) and Caspase 8 (Proteintech). Sections were fixed with 4% (v/v) formaldehyde in PBS and then dehydrated and embedded in paraffin. Tissues were sliced into 5 µm sections and antigen retrieval was performed by heating for 15 minutes in a microwave. Horse serum albumin (5%) was used to block non-specific interaction. Sections were incubated with primary antibodies and then HRP-conjugated secondary antibodies; DAPI was used to stain nuclei. Images were acquired using an Olympus microscope.
For image analysis, Image Pro Plus 6.0 was used. The areas of total ovarian tumours and interested protein-positive integrated option density (IOD) were quantified. After calculating the quotient of IOD and total area, the median value was used to divide patients into 'high expression' and 'low expression' groups.

| Cytotoxicity assays
Cells (8 × 10 3 cells per well) were plated in 96-well plates and treated with drugs for 24 hours. MTT reagents were added and cells were incubated for 4 hours. Absorbance values were then measured at 570 nm using a Vmax Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA).

| Flow cytometry analysis
Cell death was analysed using Annexin-V FITC/PI (BD Biosciences, Franklin Lakes, NJ) staining. Cells were seeded in 6-well plates and treated with drugs as indicated. The attached and detached cells were harvested and subjected to Annexin-V FITC/PI staining according to the manufacturer's instructions. Samples were analysed using an Accuri C6 Flow Cytometer (BD Biosciences).

| Caspase activity assay
Cells were plated in 96-well plates. Caspase activity was evaluated using the Caspase-Glo 8 Assay (Promega) according to the manufacturer's instructions. A microplate reader (FLUOstar Omega, BMG LABTECH, Germany) was used for detection.

| TUNEL assay
Cell apoptosis was determined using the TUNEL Apoptosis Detection Kit (Roche, Mannheim, Germany) following the manufacturer's protocol.

| Cell fractionation
Cells were lysed in 1% Triton X-100 PBS with protease inhibitors on ice for 30 minutes and then centrifuged for 30 minutes at 16,000 g.
The insoluble fraction was lysed in 2% SDS with protease inhibitors at 60°C for 1 hour. Lysates were centrifuged for 30 minutes at 16,000 g and examined using immunoblotting.

| Western blotting
Cells were lysed in RIPA buffer with protease inhibitors. Lysates were cleared by centrifugation at 1000 g for 15 minutes at 4°C, boiled in loading buffer and resolved using SDS-PAGE. Proteins were transferred to PVDF membranes and membranes were blocked with 5% milk, followed by incubation with primary antibodies overnight at 4°C. Membranes were then incubated with HRP-conjugated secondary antibodies (Proteintech). ECL reagent (Thermo Fisher Scientific, Rockford, IL) was used for immunodetection and visualization using Syngene Bio Imaging (Synoptics, Cambridge, UK).

| Co-immunoprecipitation
Cells were lysed in NP40 lysis buffer plus protease inhibitors. Lysates were incubated on ice for 30 minutes and cleared by centrifugation at 4500 rpm for 15 minutes at 4°C. Lysates were incubated with antibody overnight at 4°C, followed by incubation with 25 µL protein A and G agarose (Beyotime, China). Beads were washed three times with 1 ml PBS and bound complexes were analysed using immunoblotting.
After blocking with bovine serum albumin for 30 minutes, cells were incubated with primary antibody overnight at 4°C. Cells were then incubated with FITC/Texas Red-conjugated secondary antibodies (Proteintech) at room temperature for 1 hour. The images were acquired using an Olympus FV1000 confocal laser microscope.

| mCherry-GFP-LC3 for determining autophagic flux
SKOV3 cells stably expressing mCherry-GFP-LC3 were transfected with different p62 plasmids as indicated and then treated with cisplatin for 12 hours. Cells were fixed with 4% PFA, washed with PBS and analysed using fluorescence microscopy. Images were acquired randomly and analysed using Image J.

| DQ Red BSA for determining lysosomal degradation
Cells were incubated with 20 µg/ml DQ Red BSA (DQ-BSA; Life Technologies) for 30 minutes at 37°C followed by a 2 hour chase in full media with or without 25 µM chloroquine. Cells were then fixed with 4% PFA for 20 minutes at room temperature and analysed using fluorescence microscopy. Images were analysed by Image J.

| GST pull-down assay
Tumour tissues were lysed in RIPA buffer with protease and phosphatase inhibitors and then lysates were centrifuged at 4500 rpm at 4°C for 15 minutes. wt-p62 and mutant p62 genes were inserted into the pGEX-4T vector. The expression products were purified and coated with glutathione-agarose beads.
After incubating with lysates overnight at 4 °C, the beads were Mice were randomized into four groups of three mice per group and the mice were intraperitoneally administered 3 mg/kg cisplatin and 35 mg/kg chloroquine every 2 days. The bodyweights and tumour volumes were recorded every day. Mice were sacrificed and tumours were dissected, weighed and photographed.

| Immunohistochemistry
Tumour specimens from mouse xenografts were fixed in 4% (v/v) formaldehyde in PBS and then dehydrated and embedded in paraffin.
Tissues were sliced into 5 µm sections and antigen retrieval was performed by heating for 15 minutes in a microwave. Horse serum albumin (5%) was used to block non-specific interaction. Sections were incubated with primary antibodies and then HRP-conjugated secondary antibodies; DAPI was used to stain nuclei. Images were acquired using an Olympus microscope.

| Statistical analysis
Experimental data were presented as mean ± standard deviation (SD) and carried out using Student's t test. P < 0.05 was considered statistically significant. Statistical analysis was performed with GraphPad Prism 5 (La Jolla, CA).

| The expression of p62 and Caspase 8 is correlated with prognosis of human ovarian cancer
Previous studies have examined p62 expression in ovarian cancer tissues. 2,3 We speculate that p62 exhibits pro-survival or pro-death functions depending on its interacting partners and thus we evalu-  Figure 1C). Furthermore, we found that expression of Caspase 8 was negatively correlated with tumour-node-metastasis (TNM) stages in tissues with high p62 expression Figure 1D. In addition, high Caspase 8 expression was associated with less relapse risks in patients who overexpressed p62 ( Figure 1E). Together, these results indicated that p62 and Caspase 8 may be prognostic factors for survival in ovarian cancer.

| p62 accumulation and Caspase 8 activation induced by autophagy impairment increase the sensitivity of ovarian cancer cells to cisplatin
To

| p62 mediates Caspase 8 activation during cisplatin-induced apoptosis in ovarian cancer cells
The UBA domain in p62 is a dimerization domain as well as ubiquitin-binding domain and previous studies revealed that dimerization spatially occludes ubiquitin binding. 19 Another report showed that expression of a p62 mutant deleted for the UBA domain reduced the level of apoptosis induced by HAMLET. 20 To determine how p62 participates in Caspase 8 activation, we overexpressed either wildtype p62 or the UBA truncated p62 mutant in SKOV3 and A2780 cells and then treated the cells with cisplatin. MTT assay showed that expression of the UBA truncated p62 mutant in cells treated with cisplatin resulted in significantly increased cell viability in both cell lines compared with expression of wild-type p62 ( Figure 3A,B).
Moreover, Hoechst staining revealed that compared with wt-p62, the nuclear fragmentation induced by cisplatin treatment was significantly reduced in cells overexpressing the UBA mutant compared with wild-type p62 ( Figure 3C,D). Annexin-V FITC/PI staining, as well as evaluation of Caspase 3 activity, further validated that the UBA truncated p62 mutant that lacked the ability to bind ubiquitinated F I G U R E 3 Abolishing the ability of p62 binding to ubiquitinated proteins decreases the sensitivity of ovarian cancer cells to cisplatin. (A,B), SKOV3 and A2780 cells were transfected with wt-p62 or ΔUBA-p62. After 24 hours, the cells were treated with varying doses of cisplatin. The viability of cells was analysed using MTT assay. Data are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs wt-p62. (C) SKOV3 and A2780 cells transfected with wt-p62 or ΔUBA-p62 were treated with 4.5 µg/ml or 6 µg/ml cisplatin for 24 hours and stained with Hoechst 33342. Cell morphology was observed using confocal microscopy. Arrows indicate apoptotic cells. Scale bar, 50 µm. We found that the Caspase 8 activation was significantly inhibited in A2780 cells transfected with the mutant p62 construct treated with cisplatin ( Figure 4E,F), indicating that p62 binding to ubiquitinated proteins and to LC3 are required for Caspase 8 activation.

| Autophagy degradation is involved in p62-mediated activation of Caspase 8 in ovarian cancer cells
Previous studies reported that insoluble aggregated proteins are mainly degraded by autophagy. 25 Other reports showed that the major components of insoluble fractions are aggresomes with ubiquitin conjugates that are substrates for autophagic degradation. 26 We found that the overexpression of wild-type p62 led to an increased amount of insoluble ubiquitinated protein accumulation, which was similar to the accumulation observed with autophagy inhibition caused by chloroquine treatment (Figure 2K), In contrast, the main part of ubiquitinated proteins were in the soluble fractions with p62 mutants transfection ( Figure 5A,B).
Our findings indicated that p62 promotes Caspase 8 activation and requires domains that are closely associated with autophagic degradation. To examine the impact on autophagic flux, we used the tandem mCherry-GFP-LC3 reporter assay. GFP is quenched rapidly in acid lysosomes, but mCherry is not; thus mCherry-only puncta correspond with mature autolysosomes, whereas double-positive mCherry/GFP puncta correspond with early autosomes. 27 Our results revealed that compared with wild-type p62, there were higher numbers of mature autophagosomes in SKOV3 cells transfected with the p62 mutants treated with cisplatin for 12 hours and 24 hours ( Figure 5C-E, Figure S1). As completion of autophagy involves fusing with lysosomes, we examined lysosome degradation using DQ Red BSA, a fluorescent probe that tracks through the endosomal pathway and is ultimately de-quenched following proteolytic cleavage in the lysosome. 28 We found that expression of p62 mutants lacking the LIR and UBA domains resulted in increased lysosome degradation and this degradation was suppressed by chloroquine treatment ( Figure 5F). However, no obvious changes in the levels of the lysosomal protein Lamp1 were observed among the transfection groups, which suggested that the number of lysosomes may not be increased ( Figure 5G). These results suggest that p62 LIR and UBA functional domains are involved in the regulation of autophagic degradation.

| p62 functions as a bridge for the recruitment and activation of Caspase 8 on autophagosome membranes in ovarian cancer cells
Yong et al showed that the cleavage and activation of Caspase 8 requires the autophagosomal membrane. 29 To further dissect the role of p62 and autophagy in the activation of Caspase 8, we performed GST pull-down assays and detected the binding of GST-wild-type-p62 and GST-p62 protein domain mutants to Caspase 8 from cisplatin-treated tumour tissues from xenografts. We found that wild-type p62 specifically interacted with

| D ISCUSS I ON
In this study, our data indicated that high expression of p62 and  Autophagic flux is a process that starts with the stepwise engulfment of cytoplasmic material targeted for degradation by the isolation membrane. 36 Distinguished from 'autophagic death' was reported to take place under conditions of defective apoptotic machinery, the initiation of cell death is triggered by the blocking of protective autophagy in this study. 37 We showed that p62 accumu- Further studies should address these questions to fully clarify the function of p62 in ovarian cancer.
In summary, our study has characterized the role of p62 in ovarian cancer. We found that high expression of p62 and Caspase 8 is associated with favourable prognosis and progression of ovarian cancer. p62 promotes the recruitment and activation of Caspase 8 on autophagic membranes through the autophagy-related domains in p62, which establish a crosstalk between autophagy and apoptosis through p62 and Caspase 8. p62/Caspase 8 may become promising prognostic biomarkers and onctargets for ovarian cancer treatment.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural Science

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