SHP-2 restricts apoptosis induced by chemotherapeutic agents via Parkin-dependent autophagy in cervical cancer

Studies have shown that SHP-2 expression is increased following HPV16/18 infection in cervical cancer. To confirm this conclusion, we selected the Hela (HPV18 +) and C33A (HPV-) cell line to examine the basic level of expression of SHP-2. As shown in Fig. 1a, the expression of SHP-2 was high in Hela cells, and low in C33A cells. Therefore, we selected the Hela and C33A cell line as the study object to investigate the influence of SHP-2 on the resistance to chemotherapy. Firstly, we examined the silencing efficiency of shRNA in Hela cell. As shown in Fig. 1b, SHP-2 expression was suppressed after transfection of SHP-2 shRNA1 and SHP-2 shRNA2. As expected, Oxaliplatin (Fig. 1c) and 5-FU (Fig. 1d) induced Hela cell apoptosis. Silencing SHP-2 promoted Hela cell apoptosis induced by these chemotherapeutic agents. Next, to investigate the effect of SHP-2 on chemotherapeutic induced apoptosis in C33A cells, the transfection of pCMV-SHP2-HA plasmid prompted SHP-2 to be expressed in C33A cells (Fig. 1e). Overexpression of SHP-2 reduced C33A cell apoptosis induced by Oxaliplatin (Fig. 1f) and 5-FU (Fig. 1g). The above results show that SHP-2 suppresses cervical cancer apoptosis induced by Oxaliplatin and 5-FU.

In order to further study the molecular mechanism of SHP-2 inhibiting apoptosis, we investigated the expression of apoptosis related proteins. As shown in Fig. 2a, Oxaliplatin and 5-FU induced bax, cleaved caspase 9, cleaved caspase 3 and cleaved PARP expression and reduced bcl-2 expression in Hela cell. Then, silencing SHP-2 can further promote bax, cleaved caspase 9, cleaved caspase 3 and cleaved PARP expression and repressed bcl-2 expression. In C33A cell, overexpression of SHP-2 reduced bax, cleaved caspase 9, cleaved caspase 3 and cleaved PARP expression and induced bcl-2 expression after Oxaliplatin and 5-FU treatment (Fig. 2b). Thus, SHP-2 improved the mitochondrial apoptosis induced by Oxaliplatin and 5-FU. Furthermore, the changes of cell ROS level were further investigated, and it was found that the silencing SHP-2 could raise the ROS level in Hela cells, while overexpressing SHP-2 could reduce the ROS level in C33A cells (Fig. 2c, d). The changes of ROS are inseparable from mitochondrial involvement, while autophagy effectively protects the mitochondrial function. Then the changes in autophagy levels were studied. Silencing SHP-2 could down-regulated LC3-II expression in Hela cells, while overexpressing SHP-2 could up-regulated LC3-II expression in C33A cells (Fig. 2e, f). These results suggest that SHP-2 inhibits apoptosis induced by chemotherapeutic drugs by autophagy.

To investigate the mechanism by which SHP-2 inhibits apoptosis induced by Oxaliplatin and 5-FU, we investigated the effects of SHP-2 on mitochondrial function. Usually, the action of chemotherapeutic drugs can lead to the loss of mitochondrial membrane potential, resulting in the release of toxic reactive oxygen intermediates, leading to the release of cytochrome c and apoptosis. To test whether SHP-2 affects mitochondrial damage, we measured mitochondrial membrane potential in Hela cell. CCCP treatment induced loss of mitochondrial membrane potential, and this was exacerbated after diminishing SHP-2 expression (Fig. 3a). Similar findings that CCCP treatment increased cellular Ca²⁺ level and this was aggravated after diminishing SHP-2 expression (Fig. 3b). The release of toxic reactive oxygen species was further examined, and the results showed that CCCP could up-regulate the ROS level. Silencing SHP-2 increased the ROS level in Hela cell induced by CCCP (Fig. 3c, d). These results suggested that SHP-2 protects mitochondria from damage in Hela cell.

To further study the mechanism of SHP-2 prevented mitochondrial damage, we hypothesized that autophagy is involved in this process. The changes in LC3 expression under CCCP stimulation were examined by western blots. As expected, the expression of LC3-II was up-regulated in different times of CCCP stimulation. Silencing SHP-2 inhibited LC3-II expression induced by these CCCP in Hela cell (Fig. 4a, b). Additionally, diminishing SHP-2 expression decreased GFP-LC3 puncta accumulation after treated with CCCP (Fig. 4c, d). The data indicated that silencing SHP-2 suppressed autophagy activation induced by CCCP in Hela cell.

Next, we further investigated the effect of SHP-2 on autophagy activation in C33A (HPV-) cell. Overexpression of SHP-2 not only up-regulated the basal level of LC3-II, but also increased the expression of LC3-II induced by CCCP (Fig. 5a, b). To further assess the status of autophagic flux, we used the mCherry-GFP-LC3 construct. Overexpression of SHP-2 increased the numbers of GFP-mCherry + (red) puncta, indicating an increase in autolysosomes. In contrast, large-sized GFP + mCherry + (yellow) puncta and few GFP-mCherry + (red) puncta were observed in bafilomycin A1 treatment group (Fig. 5c, d). This demonstrated overexpression of SHP-2 promoted autophagy activation in C33A (HPV-) cells.

To further investigate the mechanism of SHP-2 in protecting mitochondrial damage, we examined the effect of SHP-2 on clearance of damaged mitochondria. We therefore examined whether SHP-2 affect the subcellular distribution of p62 and/or induce its mitochondrial translocation. In non-stimulated Hela cell, p62 displayed diffuse cytoplasmic distribution, but CCCP induced p62-containing aggregates that were colocalized with mitochondria. Silencing SHP-2 suppressed p62-containing aggregates and -translocating to mitochondria (Fig. 6a). Similar results showed that CCCP also induced GFP-LC3 colocalized with mitochondria, however, silencing SHP-2 suppressed GFP-LC3 translocation to mitochondria (Fig. 6b). Cell fractionation confirmed these results. In non-stimulated Hela cell, very little p62 and LC3-II co-sedimented with mitochondria, but after incubation with CCCP, much more p62 and LC3-II was present in the mitochondrial fraction, however, p62 and LC3-II was vanishing in the mitochondrial fraction after silencing SHP-2 (Fig. 6c, d). These results suggest that SHP-2 involved in translocation of p62 and LC3 to mitochondria.

The canonical pathway for the clearance of damaged mitochondria by autophagy involves Parkin recruitment to the mitochondrion. In Fig. 6c and d, Parkin was also translocated into mitochondria after incubation with CCCP. Silencing SHP-2 inhibited Parkin presented in the mitochondrial fraction. Further studies showed that CCCP induced Parkin colocalized with mitochondria. Silencing SHP-2 suppressed Parkin colocalized with mitochondria in Hela cell (Fig. 7a). Moreover, silencing Parkin reduced p62 and mitochondria co-localization induced by CCCP and SHP-2 overexpression in C33A cell (Fig. 7b). Induction of mitochondrial poly-ubiquitination is likely to depend on the E3 ligase Parkin [6]. Indeed, silencing Parkin reduced mitochondrial poly-ubiquitination induced by CCCP and SHP-2 overexpression in C33A cell (Fig. 7c). Thus, SHP-2 regulates the clearance of damaged mitochondria dependent on the ubiquitin ligase function of Parkin.

Oxaliplatin, 5-FU and CCCP were purchased from Sigma-Aldrich (St. Louis, USA). Dye DAPI was purchased from Invitrogen (Carlsbad, USA). Paraformaldehyde (PFA) was purchased from Yonghua Chemical Technology (Jiangsu) Co. Ltd. (Changshu, China). Triton X-100 was purchased from Shanghai Chao Rui Biotech. Co. Ltd. (Shanghai, China). BSA was purchased from Roche Diagnosis (Shanghai) Ltd. (Shanghai, China).

Primary antibodies against caspase 3, caspase 9 and β-actin were obtained from Santa Cruz Biotechnology (CA, USA); LC3, Bax and Bcl-2 was from Bioworld (OH, USA) and antibodies against PARP, SHP-2, VDAC1, Tubulin, Tom20, Parkin and Poly-Ub were purchased from Cell Signaling Technology (Danvers, MA); Antibodies to SQSTM1/p62 were obtained from Abcam (Cambridge, UK). IRDye™ 800 conjugated secondary antibodies were obtained from Rockland Inc. (Philadelphia, USA). Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 594 donkey anti-mouse IgG were obtained from Invitrogen (CA, USA).

Human cervical cancer cell lines Hela (HPV18-positive) and C33A (HPV-negative) were purchased from the American Type Culture Collection (ATCC, USA). All cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco, USA) containing 10% fetal bovine serum at 37 °C in 5% CO₂.

Cells were harvested, washed and resuspended in PBS, then stained with the Annexin V/PI Cell Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China) according to the manufacturer’s instructions. Data acquisition and analysis were performed with a Becton–Dickinson FACS Calibur flow cytometer using Cell-Quest software (BD Biosciences, Franklin Lakes, NJ). The cells in early stages of apoptosis were Annexin V positive and PI negative, whereas the cells in the late stages of apoptosis were both Annexin V and PI positive.

The electrical potential difference across inner ΔΨ _m was monitored using the ΔΨ _m -specific fluorescent probe JC-1 (Beyotime Institute of Biotechnology, China). The ΔΨ _m -specific fluorescent probe JC-1 (Beyotime Institute of Biotechnology, China) exists as a monomer with an emission at 530 nm (green fluorescence) at low membrane potential but forms J-aggregates with an emission at 590 nm (red fluorescence) at higher potentials. Cells were harvested and incubated with JC-1 for 30 min at 37 °C in the dark, then resuspended in washing buffer and photographed with a confocal laser scanning microscope (Fluoview FV1000, Olympus, Tokyo, Japan).

Cells were loaded with 10 mM Fluo-3 AM (Beyotime Institute of Biotechnology, China) which combined with Ca²⁺ and produced strong fluorescence. After 30 min incubation at 37 °C in the dark, the cells were washed with PBS twice and the fluorescence intensity was measured by FACSCalibur flow cytometry (Becton–Dickinson) at Ex./Em. − 488/525 nm.

The level of ROS was detected using fluorescent dye 2,7-dichlorofluorescein-diacetate (DCFHDA, Beyotime Institute of Biotechnology, China). Levels of ROS was detected using fluorescent dye 2,7-dichlorofluorescein-diacetate (DCFHDA, Beyotime Institute of Biotechnology, China). Cells were collected and incubated with DCFH-DA for 30 min at 37 °C in the dark. The fluorescence intensity was measured using flow cytometry (FACSCalibur, Becton–Dickinson).

Mitochondrial fractionation kit (KeyGen Biotech, China) was used to get mitochondrial according to the following protocol. Cells were incubated 3.5 × 10⁷ cells/1 mL ice-cold mitochondrial lyses Buffer, then suspended and ground the cells with tight pestle on ice. The homogenate was subjected to centrifuging at 800 g for 5 min at 4 °C to remove nuclei and unbroken cells, and then added 0.5 mL supernatant above the 0.5 mL Medium Buffer in the new 1.5 mL tube gently. After centrifugation at 15,000g for 10 min at 4 °C, the supernatant was carefully removed and collected as the cytosolic fraction and the remaining mitochondrial pellet was resuspended in the mitochondrial extraction buffer.

GFP-LC3, mCherry-GFP-LC3 plasmid and pCMV-SHP2-HA plasmid (Addgene, MA, USA) and the shRNA targeting human SHP-2 or human Parkin, or control shRNA with scrambled sequence (Santa Cruz, CA, USA) were transfected using Lipofectamine 2000™ reagent (Invitrogen, CA, USA), according to the manufacturer’s instructions 50.

Western blot analysis was prepared as described previously (25). Protein samples were separated by 10% SDS-PAGE and transferred to onto nitrocellulose membranes. The membranes were blocked with 1% BSA at 37 °C for 1 h and incubated with indicated antibodies overnight at 4 °C, followed by IRDye800 conjugated secondary antibody for 1 h at 37 °C. Immunoreactive protein was detected with an Odyssey Scanning System (LI-COR Inc., Lincoln, Nebraska).

Cells were washed with PBS, fixed with 4% paraformaldehyde and washed again with PBS. Nonspecific receptors on cells were blocked for 1 h with 3% BSA. Rabbit anti-p62 (Abcam, Cambridge, UK), Rabbit anti-Parkin (CST, MA, USA), Rabbit anti-Poly-Ub (CST, MA, USA), mouse anti-Tom20 (CST, MA, USA) were used for immunostaining. Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 594 donkey anti-mouse IgG were used as secondary antibodies (Invitrogen, CA, USA). Samples were observed and captured with a confocal laser scanning microscope (Olympus Corp., Tokyo, Japan).

The data shown in the study were obtained in at least three independent experiments and all results represent the mean ± S.E.M. Differences between the groups were assessed by one-way ANOVA test. Details of each statistical analysis used are provided in the figure legends. Differences with P values < 0.05 were considered statistically significant.