The lives of patients with ovarian cancer are threatened largely due to metastasis and drug resistance. Endogenous peptides attract increasing attention in oncologic therapeutic area, a few antitumor peptides have been approved by the FDA for clinical use over the past decades. However, only few peptides or peptide-derived drugs with antiovarian cancer effects have been identified. Here we focused on the biological roles and mechanism of a peptide named PDHPS1 in ovarian cancer development. Our results indicated that PDHPS1 reduced the proliferation ability of ovarian cancer cells in vitro and inhibited the ovarian cancer growth in vivo. Peptide pull down and following mass spectrometry, Western blot and qRT-PCR revealed that PDHPS1 could bind to protein phosphatase 2 phosphatase activator (PTPA), an essential activator of protein phosphatase 2A (PP2A), which resulted in increase of phosphorylated YAP, further inactivated YAP, and suppressed the expression of its downstream target genes. Flow cytometry, cell membrane permeability test, and IHC staining study demonstrated that there were no observable side effects of PDHPS1 on normal ovarian epithelium and hepatorenal function. Besides, modification of membrane penetration could improve the physicochemical properties and biological activity of PDHPS1. In conclusion, our study demonstrated that the endogenous peptide PDHPS1 serves as an antitumor peptide to inhibit YAP signaling pathway though interacting with PTPA in ovarian cancer.

Ovarian cancer is the most lethal gynecologic malignancy, and patient prognosis has not improved significantly over the last several decades. According to the latest report of American Cancer Society, ovarian cancer is expected to account for about 5% of female cancer deaths in 2020 (1). The main reason for the high mortality rate is that most patients are diagnosed at stage III (51%) or stage IV (29%), with extensive metastasis (2). Surgery and platinum-based chemotherapy are main treatments for ovarian cancer, however, about 70% of patients with ovarian cancer get therapeutic resistant. Hence there is an urgent need for developing novel therapeutics.

Peptides are small biological molecules composed of monomer amino acids, they are ubiquitous in nature and play important roles in vital activity, such as cell growth (3), energy metabolism (4), material transport (5), signal transmission (6) and immune regulation (7, 8). On this basis, a variety of peptide drugs have been developed and applied to treatment of various diseases (9, 10). Moreover, antitumor peptides are seen as the most promising directions in the field of antitumor drugs (11). In recent years, a variety of antitumor peptides have been approved by the FDA and showed promising effects in clinic (12). Antitumor peptides provide a viable and attractive approach for the treatment of chemotherapy-insensitive or recurrent tumors. However, there are only few reports on peptides with anti-ovarian cancer function.

We previously identified an endogenous peptide PDHPS1 (peptide deregulated in hypertrophic scar-1; sequence: IATTTASAATAAAIGATPRAK) from skin tissue, whose precursor protein is nuclear mitotic apparatus protein 1 (NUMA1). NUMA1 plays an essential role in the formation and maintenance of the spindle poles and the alignment and the segregation of chromosomes during mitotic cell division (13). Our previous studies showed that PDHPS1 inhibited the proliferation of dermal fibroblasts and reduced collagen secretion of myofibroblasts, and the results has granted a Chinese invention patent (14), but the detailed mechanism remains unclear. In recent years, there have been many reports on the association between fibroblasts and tumors, and it has been proved that fibroblasts are involved in liver cancer, breast cancer, and ovarian cancer (15–17). Considering that tumor cells have high proliferative activity similar to fibroblasts, and the initiation and progression of solid tumors are often accompanied by the activation of fibroblasts and excessive deposition of collagen or other extracellular matrix, we speculate that PDHPS1 may also have an influence on the development of malignant tumors. Therefore, we intend to verify the potential effect of PDHPS1 on ovarian cancer, and further explore the specific mechanism of its action.

Cell culture

The human ovarian cancer cell line SKOV3 (RRID:CVCL_0532) and A2780 (RRID:CVCL_0134) were obtained from Jiangsu KeyGEN BioTECH Co., Ltd. (Nanjing, China). The human normal ovarian epithelial cell line IOSE386 (RRID:CVCL_E230) was a kind gift from Prof. Zhu Jin (General Hospital of Eastern Theater Command). SKOV3 and A2780 were authenticated in May 2015, and IOSE386 in October 2019, by STR DNA Profiling Analysis. SKOV3 was grown in McCoy's 5A medium (KeyGEN), and A2780 and IOSE386 cells were grown in DMEM (KeyGEN). Cell lines were maintained in culture supplemented with 10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 mg/mL streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO2 in a humidified incubator (Thermo Fisher Scientific). All cell lines were immediately expanded on delivery, numerous vials of low passage cells were preserved in liquid nitrogen and no vial of cells was passaged for more than 2 months. All cell lines are tested for Mycoplasma, purity, and contaminants before experiments.

Animals

All animal studies in this article were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University and conformed to the legal mandates and guidelines for the care and maintenance of laboratory animals. The animal use approval ID is IACUC-1601070. Mice used in this study were from Model Animal Research Center of Nanjing University, including BALB/c nude mice (RRID: IMSR_JCL:mID-0001) aged 6 weeks (female) and C57BL/6J mice (RRID:IMSR_JAX:000664) aged 6 weeks (female). The treatment scheme of this experiment does not use blind method because different treatments are identified by tail tags. On the basis of our previous experience, we used 4–6 animals per group, as this number allows us to get statistically significant data while keeping the number of animals to a minimum. The mice were randomly allocated to groups. Tumor length and width are measured with calipers to determine the tumor size, tumor volume = length × width × width/2 (mm3).

Chemical synthesis of peptides

The following peptides were manually synthesized by carrying out standard Fmoc-based solid -phase peptide syntheses by Shanghai Science Peptide Biological Technology Co., Ltd: PDHPS1: IATTTASAATAAAIGATPRAK; Scramble peptide for PDHPS1: AAAATTAAGPITTAIKTAARS; S1: IATTTAS; S2: AATAAAI; S3: GATPRAK; S4: AATAAAIGATPRAK;TAT-PDHPS1:GRKKRRQRRRPPQQIATTTASAATAAAIGATPRAK. In vitro studies, all peptides were dissolved in water at the desired concentrations and directly added to the culture medium of ovarian cancer cells. In vivo studies, peptides were dissolved in saline at the desired concentrations and then injected intraperitoneally into mice.

Cell counting kit-8 assay

Cells were seeded into 96-well plates at the density of 2 × 103 cells per well, corresponding concentrations of peptides were added into the medium according to the set groups. Each group had six multiple wells, and was cultured in an incubator at 37°C with 5% CO2 for 0, 24, 48, and 72 hours. A total of 10 μL Cell Counting Kit-8 (CCK8) was added to each well and incubated in the dark for 2 hours and was measured at the optical density (OD) at 450 nm.

Plane clone formation assay

Cells (1,000 cells/well) were seeded into 6-well plates with medium supplemented with 10% FBS, and 50 μmol/L of peptides or solvent were added into the medium according to the set groups. After cultured for 14 days, colonies were fixed with 4% paraformaldehyde at room temperature for 15 minutes and stained with 0.1% crystal violet for 15 minutes (Biosharp), and the numbers of visible colonies were counted.

Transwell assays

Transwell assays were performed using transwell inserts (8.0 μm pore size, Costar, MA, USA). 5 × 104 cells were seeded in transwell inserts (with 250 μL FBS-free medium containing 50 μmol/L peptides or solvent) uncoated (migration assays) or coated (invasion assays) with 60 μL Matrigel (Corning), while 750 μL medium containing 20% FBS was added to the bottom chamber of 24-well plates. Twenty-four (migration assays) or 48 hours (invasion assays) later, all the transwell inserts were fixed in 4% paraformaldehyde for 20 minutes and then stained with crystal violet. The cells were lysed by RIPA, and OD at 562 nm was detected for quantitative analysis.

Wound healing assay

Cells were cultured in 6-well plates to confluency. A scratch is made with a 200 μL pipette tip in each well. Cells were washed with PBS three times, then serum-free medium with corresponding concentrations of peptides were added into the wells according to the indicated groups. Each group has two duplicate wells, and was cultured in an incubator at 37°C with 5% CO2 for 48 hours. Images were taken at 0, 24, and 48 hours, and scratch area was analyzed using Image J (RRID: SCR_003070).

In vivo xenograft tumor model

During the tumor formation assay, 1 × 107 SKOV3 cells were injected subcutaneously into the right forelimb axilla of BALB/c female nude mice. Once tumors were detected, the mice (n = 6) were intraperitoneally injected once every other day with PDHPS1 (10 mmol/L/kg) or equal volume of saline. After 2 weeks, the mice were sacrificed, and tumor volumes and tumor weight were measured.

In vivo toxicity analysis

C57BL/6J female mice were intraperitoneal injected with PDHPS1 (10 mmol/L/kg) or equal volume of saline once every other day (n = 9 in each group) in a two-week period. Then five mice in each group were sacrificed on day 14 for blood biochemical test. The remaining mice (n = 4 in each group) were sacrificed one week later after drug withdrawal to analyze the recovery of possible toxic and side effects.

IHC

The mouse tumor tissues were cut into pieces of about 5 mm in length, and washed with cold 0.9% (m/v) NaCl. The samples were then fixed with 4% PFA, and embedded with paraffin. Subsequently, these samples were sectioned to 4-μm thickness slices, and stained with hematoxylin and eosin to estimate tumorigenesis. For TUNEL assay, the slices were stained with One-Step TUNEL Apoptosis Kit (RiboBio, #C11026). Apoptotic cells were counted using Image J.

For Ki67 or p-YAP staining, tissue sections were blocked with normal goat serum at room temperature for 1 hour, followed by incubating with rabbit anti-Ki67 antibody (Proteintech, #27309-1-AP, RRID: AB_2756525) or rabbit anti-p-YAP(Ser127) antibody (Cell Signaling Technology, #13008, RRID: AB_2650553) overnight at 4°C. After rinsing for three times with TBST, goat anti-rabbit antibody were applied for 30 minutes at room temperature, then DAB reagents were incubated according to the manufacturer's protocol of the IHC kit (Proteintech, #PK10006).

Cell apoptosis analysis

Cells were seeded in 60-mm diameter plates, after attaching overnight, 50 μmol/L PDHPS1 was added to individual wells of test group, with ddH2O as a control. After 24 hours of this treatment, cells were harvested and double stained with Annexin V-PE and 7-AAD or Annexin V-APC and PI. The results were analyzed by flow cytometry (Beckman Coulter).

Cell membrane permeability test

The influence of PDHPS1 on permeability of normal ovarian epitheliums was determined using SYTOX green nucleic acid stain (Thermo Fisher Scientific). IOSE386 cells were grown in a sterile 96-well plate (5,000 cells/well) for 24 hours, and treated with relevant serum-free medium for 12 hours. Then, cells were incubated with SYTOX green stain (1 μmol/L) for 15 minutes and subsequently treated with gradient concentrations (0 μmol/L, 10 μmol/L, 20 μmol/L, 50 μmol/L, 100 μmol/L) of PDHPS1. The fluorescence was measured every 15 minutes for 75 minutes by a Biotek Synergy H4 plate reader (BioTek Instruments, Vermont, USA, RRID:SCR_019750) with excitation and emission wavelengths at 485 and 528 nm, respectively. Cells treated with 0.5% Triton X-100 was used as positive control.

Peptide pull down assay

About 200 μg biotinylated peptides immobilized on streptavidin dynabeads (Invitrogen, #11205D) were incubated with cell lysate at 4°C overnight. The beads were washed three times and placed on a magnet to discard the supernatant. The immobilized proteins were visualized with either silver stain or Western blot.

Immunofluorescence assay

The cells were plated in 20-mm dishes (NEST, 801001) and treated by 50 μmol/L peptides or solvent for 48 hours. After gently washed with PBS buffer, cells were fixed with 4% formaldehyde in PBS buffer for 15 minutes and permeabilized with 0.1% Triton X-100 in PBS buffer for 10 minutes, then cells were blocked with PBS buffer containing 5% BSA for 30 minutes and incubated with primary antibodies for 1 hour at 37°C. After being washed three times, the cells were incubated with secondary antibodies for 30 minutes at 37°C, and then stained with DAPI (1 μg/mL) for 2 minutes, followed by extensive washing. The primary antibodies used were anti-YAP (Santa Cruz Biotechnology, #101199, RRID: AB_1131430).

Western blot assay

Cells were cultured for 48 hours in medium containing 50 μmol/L peptides, and then were collected and lysed. Equal amounts of protein lysate were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% (w/v) nonfat milk or BSA (for phosphorylated proteins detection) in Tris-buffered saline with 0.1% Tween (TBST) for 1 hour at room temperature and incubated with a primary antibody overnight at 4°C. The primary antibody used in this study are as follows: anti-PTPA (Proteintech, #10321-1-AP, RRID:AB_2284352), anti-ARP3 (Abcam, #ab49671, RRID:AB_2257830), anti β-actin (Santa Cruz Biotechnology, #sc-81178, RRID:AB_2223230), anti–α-tubulin (Proteintech, #11224-1-AP, RRID:AB_2210206), anti-GAPDH (Proteintech, #60004-1-Ig, RRID:AB_2107436), anti-pMST1 (Thr183)/pMST2(Thr180) (Cell Signaling Technology, #49332, RRID:AB_2799355), anti-MST2 (Proteintech, #12097-1-AP, RRID:AB_2198801), anti-pYAP (Ser127)(Cell Signaling Technology, #13008, RRID:AB_2650553), anti-YAP (Santa Cruz Biotechnology, #101199, RRID: AB_1131430). anti-Wnt-4(B-6) (Santa Cruz Biotechnology, #sc-376279, RRID:AB_10986273), anti–β-catenin (E-5)(Santa Cruz Biotechnology, #sc-7963, RRID:AB_626807), anti-phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199)(Cell Signaling Technology, #4228, RRID:AB_659940), anti-PI3K p85 (19H8)(Cell Signaling Technology, #4257, RRID:AB_659889), anti-phospho-Akt (Ser473)(Signaling Technology, #4060, RRID:AB_2315049), anti-Akt (Cell Signaling Technology, #9272, RRID:AB_329827), anti-Phospho-mTOR (Ser2448)(Cell Signaling Technology, #5536, RRID:AB_10691552), anti-mTOR (Cell Signaling Technology, #2983, RRID:AB_2105622). After an incubation with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-Mouse IgG-HRP (Biosharp, BL001A, RRID:AB_2827665), goat anti-Rabbit IgG-HRP (Biosharp, BL003A, RRID:AB_2827666) at 37°C for 1 hour, the protein bands were visualized by the enhanced chemiluminescence Plus kit (Beyotime). Images were captured by automatic chemiluminescence image analysis system (Tanon).

RNA extraction and quantitative real-time PCR

Cells were cultured for 48 hours in medium containing 50 μmol/L peptides, and then were collected. According to the manufacturer's protocol, total RNA was isolated and purified using the RNA extraction and purification kit (Thermo Fisher Scientific) and reversely transcribed into cDNA by using a Reverse Transcription Kit (Vazyme). SYBR Green Real time PCR Master Mix (Vazyme) was used for PCR and reagents were subjected to 95°C for 30 s and followed by 40 cycles of 95°C for 10 seconds, 60°C for 15 seconds and 72°C for 45 seconds. All the results were normalized to the GAPDH mRNA levels. The primer sequences are listed in Supplementary Table S1. 2–ΔΔCt method was performed to analyze all the target genes of the relative fold changes.

Statistical analysis

Data are presented as mean ± S.D. for continuous variables and as frequencies and proportions for categorical variables. The proliferation, transwell, apoptosis, qRT-PCR, and Western blot analyses were performed at least in triplicate and repeated at least three times with similar results. Statistical differences were analyzed by SPSS 20.0 (SPSS Inc., RRID: SCR_002865) with Student t test (comparing two variables) or one-way ANOVA (comparing three or more variables). P < 0.05 was considered to indicate a significant difference.

Data availability statement

The data generated in this study are available within the article and its Supplementary Data.

PDHPS1 is an endogenous stable peptide that can enter the cell

PDHPS1 consists of 21 amino acids, which is derived from the C-terminal (2090–2110) of NUMA1. Structure analysis on Simple Modular Architecture Research Tool (SMART, http://smart.embl.de/) showed that NUMA1 was composed of 2,115 amino acids and PDHPS1 was from its tail low complexity regions which contains a threonine-phosphorylation site (Supplementary Fig. S1A).

The cellular localization of peptides often provides information about their probable function, so we investigated whether PDHPS1 could penetrate cell membrane and its main localization in cells. FITC-labeled PDHPS1 was added into the culture medium of SKOV3 or A2780 cells with a final concentration of 50 μmol/L, after 24-hour coculture, laser-scanning confocal microscopy was used to monitor the distribution of peptide, and results showed that PDHPS1 could get inside cells and widely distributed in cytoplasm and nucleus of cells (Supplementary Fig. S1B).

PDHPS1 inhibits the proliferation of ovarian cancer cells in vitro

To investigate the potential role of PDHPS1 in ovarian cancer cells, CCK8 assay was performed, and the results showed that comparing with cells cultured in scrambled peptide (composed of rearranged amino acids of PDHPS1) or solvent treated groups, cells treated by 20 μmol/L PDHPS1 slowed down the growth, and cells treated by 50 μmol/L and 100 μmol/L PDHPS1 had a more obvious performance (Fig. 1A and B). On the basis of this result, we set the concentration of PDHPS1 to 50 μmol/L for subsequent cell experiments. Then, to further verify the growth inhibition effect of PDHPS1 on ovarian cancer cells, we carried out plate clone formation assay. The results were consistent with that of CCK8 assay, the number of cell clones formed in the 50 μmol/L PDHPS1 treated group was obviously less than that in the 50 μmol/L scrambled peptide or solvent treated groups (Fig. 1C and D). These results indicated that PDHPS1 could inhibit the proliferation of ovarian cancer cells in vitro. Subsequently, transwell assay was performed to monitor the function of ovarian cancer cells in response to PDHPS1. Results showed that 50 μmol/L PDHPS1 suppressed the number of SKOV3 and A2780 cells that enter the lower layer of transwells. Quantitative analysis confirmed the conclusion (Fig. 1E and F). While wound healing in SKOV3 and A2780 cells was not significantly inhibited in response to PDHPS1 compared with the control groups (Supplementary Fig. S2A–S2D). Considering that cells are starved in wound healing experiments, PDHPS1 may be more effective against cells with adequate nutrition or high activity.

Figure 1.

PDHPS1 significantly inhibits the growth of ovarian cancer cells. A, Cell proliferation of SKOV3 cells treated with gradient concentration of PDHPS1 measured by CCK8 assay. B, Cell proliferation of A2780 cells treated with gradient concentration of PDHPS1 measured by CCK8 assay. C, Cell proliferation of SKOV3 and A2780 cells treated with 50 μmol/L PDHPS1 or scrambled peptide were analyzed by colony formation assay. D, Quantitative analysis of the colonies formed in C. E, Cell invasion and migration of SKOV3 and A2780 cells treated with 50 μmol/L PDHPS1, scrambled peptide or solvent analyzed by transwell. F, Quantitative analysis of the invaded and migrated cells in E. G, Representative cell apoptosis results of SKOV3 cells treated with 50 μmol/L PDHPS1, scrambled peptide or solvent analyzed by flow cytometry. H, Quantitative analysis of the early stage, late stage and total apoptosis in G.

Figure 1.

PDHPS1 significantly inhibits the growth of ovarian cancer cells. A, Cell proliferation of SKOV3 cells treated with gradient concentration of PDHPS1 measured by CCK8 assay. B, Cell proliferation of A2780 cells treated with gradient concentration of PDHPS1 measured by CCK8 assay. C, Cell proliferation of SKOV3 and A2780 cells treated with 50 μmol/L PDHPS1 or scrambled peptide were analyzed by colony formation assay. D, Quantitative analysis of the colonies formed in C. E, Cell invasion and migration of SKOV3 and A2780 cells treated with 50 μmol/L PDHPS1, scrambled peptide or solvent analyzed by transwell. F, Quantitative analysis of the invaded and migrated cells in E. G, Representative cell apoptosis results of SKOV3 cells treated with 50 μmol/L PDHPS1, scrambled peptide or solvent analyzed by flow cytometry. H, Quantitative analysis of the early stage, late stage and total apoptosis in G.

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PDHPS1 induced cell apoptosis in ovarian cancer cells

The cell viability is often used to assess healthy cells based on the integrity of the cell membrane. To determine whether PDHPS1 damaged cells or induced cell apoptosis, we conducted the flow cytometry after cells cultured with 50 μmol/L peptides for 48 hours. Annexin V and PI staining showed that PDHPS1 treatment slightly increased numbers of apoptotic cells. (Fig. 1G and H). These results indicate that the antitumor effect by PDHPS1 was partly due to the cytolysis or apoptosis.

PDHPS1 inhibits ovarian tumors growth in vivo

To further assess the antitumor effect of PDHPS1 in vivo, the subcutaneous xenograft mouse model was established and PDHPS1(10 mmol/L/kg) or saline was intraperitoneally injected every two days after the xenograft tumor was visible (Fig. 2A). We found that BALB/c female nude mice intraperitoneally injected with PDHPS1 had a smaller tumor volume and a lower tumor weight compared with that of saline treated mice (Fig. 2BD). In addition, IHC staining against Ki67 in tumors from the two groups showed that PDHPS1 treatment could significantly inhibit cell proliferation (Fig. 2E and F). And TUNEL assay indicated that the apoptosis was increased in the PDHPS1 treated xenograft tumor as compared with control (Fig. 2G and H). These results demonstrated an inhibitory effect of PDHPS1 on ovarian cancer growth.

Figure 2.

PDHPS1 inhibits ovarian tumor growth in vivo. A, Schematics of the experimental design for xenograft models. B, The image of the xenograft tumors dissected from the indicated group of mice treated with PDHPS1 or control. C, The quantitative analysis of weights of the xenograft tumors in B. D, The subcutaneous tumor growth monitored over time in two groups. E, Representative image of the IHC results of Ki67 in the xenograft tumors of the two indicated group. F, Quantitation of Ki67+ cells. G, Representative image of the TUNEL results in the xenograft tumors of the two indicated group. H, Quantitation of apoptotic cells indicated in G.

Figure 2.

PDHPS1 inhibits ovarian tumor growth in vivo. A, Schematics of the experimental design for xenograft models. B, The image of the xenograft tumors dissected from the indicated group of mice treated with PDHPS1 or control. C, The quantitative analysis of weights of the xenograft tumors in B. D, The subcutaneous tumor growth monitored over time in two groups. E, Representative image of the IHC results of Ki67 in the xenograft tumors of the two indicated group. F, Quantitation of Ki67+ cells. G, Representative image of the TUNEL results in the xenograft tumors of the two indicated group. H, Quantitation of apoptotic cells indicated in G.

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PDHPS1 interacts with PTPA

Encouraged by the antitumor effect of PDHPS1, we next focused on exploring the specific mechanism of PDHPS1 on ovarian cancer. We carried out a biotin pull-down assay followed by SDS-PAGE and sliver staining to identify proteins interacting with PDHPS1. A band with the molecular weight of about 45kD was specifically identified in the pull-down lysate of biotin labeled PDHPS1-treated SKOV3 and A2780 cells (Fig. 3A). Mass spectrometry analysis of the proteins enriched in this band showed that 90 and 178 proteins were specifically identified in the biotin-labeled PDHPS1-treated SKOV3 and A2780 cells, respectively (Fig. 3B). The intersection of these two cell types obtained 24 proteins that may interact with PDHPS1 both in SKOV3 cells and A2780 cells (Supplementary Table S2).

Figure 3.

PDHPS1 inhibits YAP signaling by interacting with PTPA. A, The representative silver staining result of pulled-down proteins in the indicated groups. B, A Venn diagram of proteins in the strip verified by mass spectrometry, and there are 24 proteins specifically captured by PDHPS1. C, WB verification of PTPA expression in the lysis pulled down from the indicated group. D, WB analysis of PTPA expression in 50 μmol/L PDHPS1, scrambled peptide treated and control ovarian cancer cells. E, 50 μmol/L PDHPS1 treatment positively regulates the abundance of phosphorylated YAP protein in SKOV3 and A2780 cells. F, 50 μmol/L PDHPS1 treatment negatively regulates the expression of target genes of YAP in SKOV3 and A2780 cells. G, Representative immunofluorescence analysis results of YAP localization in SKOV3 cells. H, Quantitative analysis of the nuclear localized YAP in SKOV3 and A2780 cells. I, IHC assessment of phosphorylated YAP expression in the control and PDHPS1 treated xenograft. J, Quantitative analysis of the results in I.

Figure 3.

PDHPS1 inhibits YAP signaling by interacting with PTPA. A, The representative silver staining result of pulled-down proteins in the indicated groups. B, A Venn diagram of proteins in the strip verified by mass spectrometry, and there are 24 proteins specifically captured by PDHPS1. C, WB verification of PTPA expression in the lysis pulled down from the indicated group. D, WB analysis of PTPA expression in 50 μmol/L PDHPS1, scrambled peptide treated and control ovarian cancer cells. E, 50 μmol/L PDHPS1 treatment positively regulates the abundance of phosphorylated YAP protein in SKOV3 and A2780 cells. F, 50 μmol/L PDHPS1 treatment negatively regulates the expression of target genes of YAP in SKOV3 and A2780 cells. G, Representative immunofluorescence analysis results of YAP localization in SKOV3 cells. H, Quantitative analysis of the nuclear localized YAP in SKOV3 and A2780 cells. I, IHC assessment of phosphorylated YAP expression in the control and PDHPS1 treated xenograft. J, Quantitative analysis of the results in I.

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Among the 24 proteins, PTPA, ACADM, BZW2, BCCIP, ARP3 are closely related to the occurrence and development of tumors (18–22). Notably, PTPA was the most abundant in the samples and caught our attention. A subsequent peptide pull down assay followed by Western blot confirmed that PDHPS1 could bind to PTPA (Fig. 3C). However, PTPA protein levels were similar between PDHPS1 treated or the scrambled peptide-treated cells, indicating that PDHPS1 did not change the protein expression level of PTPA (Fig. 3D).

PDHPS1 regulates ovarian cancer development through targeting YAP1

Given that PDHPS1 could interact with PTPA, but not regulate the protein expression of PTPA, we thus speculated that PDHPS1 inhibit the progression of ovarian cancer by affecting the function of PTPA after binding to it.

PTPA is an activator of serine/threonine protein phosphatase 2A (PP2A). PP2A accounts for the majority of serine/threonine phosphatase activities in human cells and participates in cell-cycle control (23), DNA damage repair (24), cell metabolism (25), immune activation (26), and so on. The heterotrimeric PP2A holoenzyme comprises three types of subunits: the scaffolding subunit (A subunit, PP2Aa), the catalytic subunit (C subunit, PP2Ac), and the regulatory subunit (B subunit)(27). During the holoenzyme assembly process, PTPA can bind to PP2Ac and up-regulate the activity of PP2A (28). Previous studies implicate PP2A in the regulation of the Hippo pathway, it promotes the dephosphorylation of p-YAP, raise the level of active YAP and the expression of downstream target genes (29). The Hippo pathway effector, YAP has been considered an important oncogene (30, 31) that promotes ovarian cancer progression through regulating mechanical transduction (32), ferroptosis (33), and so on. Considering the unique property of PTPA and PP2A described above, we proposed that PDHPS1 may potentially abrogate PP2A-PTPA–mediated activation of YAP to inhibit ovarian cancer progression.

To establish the mechanism by which PDHPS1 controls YAP activity, we analyzed the phosphorylation level of YAP in cells treated by 50 μmol/L PDHPS1 for 48 h. WB results showed that PDHPS1 treatment significantly increased the level of phosphorylated YAP (Fig. 3E). Meanwhile, the mRNA expression levels of CTGF and CYR61, the known YAP-target genes, were significantly down-regulated (Fig. 3F), which further confirmed the inhibitory effect of PDHPS1 on YAP activity. We also analyzed the localization of YAP in the nucleus and cytoplasm, and the results indicated that the nucleus localized YAP was decreased while the cytoplasmic YAP was increased in the PDHPS1 treated ovarian cancer cells (Fig. 3G and H). IHC of subcutaneous tumors in mice also showed that the p-YAP level of PDHPS1-injected group was significantly higher than that of the control group (Fig. 3I and J). In addition, we examined the expression of proteins in Wnt/β-catenin and PI3K/AKT signaling pathways, which are also known regulated by PP2A, and there are no significant differences between control and PDHPS1-treated ovarian cancer cells (Supplementary Fig. S3). Taken together, these results identified PDHPS1 as a regulatory subunit of YAP that regulate ovarian cancer.

PDHPS1 has no obvious adverse effects on ovarian epithelium and hepatorenal function

To test whether PDHPS1 has the potential for clinical application, we tested its potential toxicity. First, flow cytometric analysis was used to determine whether PDHPS1 has impact on cell apoptosis of normal ovarian epithelial cells, IOSE386, and the results indicated that PDHPS1 treatment did not significantly affect the cell apoptosis rates compared with vehicle-treated group (Fig. 4A and B). Also, the cell membrane permeability was tested after adding different concentrations of PDHPS1 into culture medium of IOSE386. The results showed that there was no significant difference in the cell membrane permeability between PDHPS1 treatment group and vehicle-treated group (Fig. 4C), indicating that PDHPS1 has almost no damage to the membrane integrity of normal ovarian epithelial cells.

Figure 4.

PDHPS1 has no obvious adverse effects on ovarian epithelium and hepatorenal function. A, Representative flow cytometry analysis of control and PDHPS1 treated IOSE80 cells. B, Quantitative analysis of the apoptosis of control and PDHPS1-treated IOSE80 cells. C, The membrane permeability rate of gradient concentration of PDHPS1-treated IOSE80 cells. D, Body weight growth curves of the two groups of mice. E, Representative HE staining result of the liver and kidney of control and PDHPS1 treated mice in the administration and recovery group. F, Quantitative analysis of blood biochemical test of hepatorenal function indicators. The units of measurement: U/L for ALT, AST and ALP, mg/dL for BUN, and μmol/L for Scr.

Figure 4.

PDHPS1 has no obvious adverse effects on ovarian epithelium and hepatorenal function. A, Representative flow cytometry analysis of control and PDHPS1 treated IOSE80 cells. B, Quantitative analysis of the apoptosis of control and PDHPS1-treated IOSE80 cells. C, The membrane permeability rate of gradient concentration of PDHPS1-treated IOSE80 cells. D, Body weight growth curves of the two groups of mice. E, Representative HE staining result of the liver and kidney of control and PDHPS1 treated mice in the administration and recovery group. F, Quantitative analysis of blood biochemical test of hepatorenal function indicators. The units of measurement: U/L for ALT, AST and ALP, mg/dL for BUN, and μmol/L for Scr.

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In addition, we assessed whether PDHPS1 treatment would result in biotoxicity in mice. 10 mmol/L/kg PDHPS1 or saline were intraperitoneal injected in C57BL/6J mice once every 2 days, after a 2-week period of treatment, 5 mice in each group were taken to examine the effect of PDHPS1 on the structure and function of main organs (Fig. 4D). The results showed that the liver and renal tissue of mice in the PDHPS1 intraperitoneal injection group showed no significant pathological changes compared with the control group (Fig. 4E). The hepatorenal function indicators including ALT, AST, ALP, BUN, and SCR were all in normal range, showing no statistical difference between two groups (Fig. 4F). One week after drug withdrawal, the structure and function of the organs of the remaining 4 mice in each group were detected and results also showed no obvious hepatoxicity or nephrotoxicity of PDHPS1 (Fig. 4E and F). In addition, during the experiment, there was no significant difference in the weight gain between the two groups of mice (Fig. 4D).

Rational optimization of PDHPS1

On the basis of the PDHPS1 peptide, we further explored the optimization method. First, to explore the main functional region of PDHPS1 and verify the influence of the structure of PDHPS1 on its function, we designed a series of truncated variants of the PDHPS1 peptide: S1 (IATTTAS), S2 (AATAAAI), S3 (GATPRAK), S4 (AATAAIGATPRAK), S5 (IATTTASAATAAAI) (Supplementary Fig. S4A). Considering S5 is too hydrophobic to be synthesized, S1, S2, S3 and S4 were synthesized and functionally measured. CCK8 assay and the plate clone formation assay showed that none of the truncated variants affected proliferation of ovarian cancer cells (Supplementary Fig. S4B–S4E). The results indicated that the complete sequence of PDHPS1 may be the basis of its function.

Previous studies have shown that membrane-penetrating modification is a commonly used modification method for peptides, which can improve the physical properties of peptides and enhance their cell penetration and functional activity (9). On the basis of this theory, the trans-activator of transcription (TAT) sequences (GRKKRRQRRPPQQ), a membrane permeability element, was linked to the N-terminal of PDHPS1 and an optimized peptide TAT-PDHPS1 was synthesized. Notably, TAT-PDHPS1 was more soluble than PDHPS1 (Fig. 5A), and it displayed longer half-life and superior efficiency of cell uptake compared to PDHPS1 (Fig. 5B). Beyond better physical and chemical properties, TAT-PDHPS1 was found to gain stronger biological activity. Both CCK8 assay and the plate clone formation assay showed that 50 μmol/L TAT-PDHPS1 could inhibit the proliferation activity of ovarian cancer cells, and the inhibition ability of 50 μmol/L TAT-PDHPS1 was stronger than 50 μmol/L PDHPS1 (Fig. 5CE). We also detected the function of TAT-PDHPS1 in vivo, after subcutaneous xenograft mouse model was established as noted earlier, PDHPS1 (10 mmol/L/kg) or TAT-PDHPS1 (5 mmol/L/kg) were injected peritoneally once every other day for 14 days, and it showed that the inhibitory effect of 5 mmol/L/kg TAT-PDHPS1 was found to be comparable with that of 10 mmol/L of the PDHPS1 peptide (Fig. 5F and G). And TAT-PDHPS1 treatment markedly increased phosphorylation level of YAP, and more strongly inhibited the transcription of YAP target genes than PDHPS1 in A2780 cells (Fig. 5H and I).

Figure 5.

Transmembrane modification optimizes the biological function of PDHPS1. A, Solubility of PDHPS1 and TAT-PDHPS1 at specified concentrations. B, Microscopic images of FITC labeled PDHPS1 and TAT-PDHPS1 in SKOV3 cells at concentrations of 50 μmol/L. C, Comparison of effects of 50 μmol/L PDHPS1 and 50 μmol/L TAT-PDHPS1 on SKOV3 and A2780 cell proliferation (OD450). D, Comparison of effects of 50 μmol/L PDHPS1 and 50 μmol/L TAT-PDHPS1 on colony formation. E, Quantitative analysis of number of colony formation after 50 μmol/L PDHPS1 or TAT-PDHPS1 treatment. F, The image of the xenograft tumors dissected from the indicated group of mice treated with control, 10 mmol/L/kg PDHPS1 or 5 mmol/L/kg TAT-PDHPS1. G, The quantitative analysis of weights of the xenograft tumors in F. H, YAP protein expression and phosphorylation level as well as the mRNA expression of YAP target genes after 50 μmol/L PDHPS1 or TAT-PDHPS1 treatment in SKOV3 cells. I, YAP protein expression and phosphorylation level as well as the mRNA expression of YAP target genes after 50 μmol/L PDHPS1 or TAT-PDHPS1 treatment in A2780 cells.

Figure 5.

Transmembrane modification optimizes the biological function of PDHPS1. A, Solubility of PDHPS1 and TAT-PDHPS1 at specified concentrations. B, Microscopic images of FITC labeled PDHPS1 and TAT-PDHPS1 in SKOV3 cells at concentrations of 50 μmol/L. C, Comparison of effects of 50 μmol/L PDHPS1 and 50 μmol/L TAT-PDHPS1 on SKOV3 and A2780 cell proliferation (OD450). D, Comparison of effects of 50 μmol/L PDHPS1 and 50 μmol/L TAT-PDHPS1 on colony formation. E, Quantitative analysis of number of colony formation after 50 μmol/L PDHPS1 or TAT-PDHPS1 treatment. F, The image of the xenograft tumors dissected from the indicated group of mice treated with control, 10 mmol/L/kg PDHPS1 or 5 mmol/L/kg TAT-PDHPS1. G, The quantitative analysis of weights of the xenograft tumors in F. H, YAP protein expression and phosphorylation level as well as the mRNA expression of YAP target genes after 50 μmol/L PDHPS1 or TAT-PDHPS1 treatment in SKOV3 cells. I, YAP protein expression and phosphorylation level as well as the mRNA expression of YAP target genes after 50 μmol/L PDHPS1 or TAT-PDHPS1 treatment in A2780 cells.

Close modal

Explore the antitumor peptide is one of the main approaches to improve the prognosis of patients suffering ovarian cancer. This study proved that endogenous peptide PDHPS1 could inhibit the progress of ovarian cancer with no obvious toxic effect. This is the first peptide with anti-tumor function found from human skin tissue, and it has the potential for clinical application.

Previous reports have shown that the main mechanisms of anti-tumor peptides are as follows: (i) competitively bind to target proteins to prevents protein–protein interaction; (ii) simulate the conformational regulatory domain of the target protein and inhibit its conformation-dependent activation; (iii) behave as a drug carrier to target tumor cell surface receptors to enhance drug efficacy; (iv) destroy the cell membrane of tumor cells; (v) activate antitumor immune (12). In this study, we conducted peptide pull-down assay and confirmed PDHPS1 binding to PTPA, thus abrogating PP2A-PTPA–mediated activation of YAP. However, the specific binding sites of PDHPS1 interacting with PTPA and the specific effects on the binding of PTPA-PP2A still need further experimental verification.

Protein phosphorylation is the most common type of posttranslational modification and can affect various cellular activities (34). Most studies of protein phosphorylation mainly focused on kinases, actually phosphatases can also be used as regulatory targets. PP2A is the most important serine/threonine protein phosphatase that regulates the dephosphorylation of most phosphorylated serine and phosphorylated threonine in the body, and plays an important role in the activity of plentiful tumor-related proteins (35). Our study confirmed that PDHPS1 can bind to PTPA, the activator of PP2A, and affect the phosphorylation level of YAP, an important regulator of tumor. The regulatory effect of PDHPS1 on the phosphorylation level of YAP confirms the important role of phosphatase in the regulation of phosphorylated proteins, and meanwhile suggests that PDHPS1 may also have a potential therapeutic effect in other YAP-related tumors.

Phosphatases usually form an integrated structure with different substrate specificity by combining the same core structural subunits with different regulatory subunits. Previous studies have reported that a peptide named STRN3 can bind to PP2Aa, the scaffolding subunit of PP2A, and enhance the dephosphorylation of PP2A on MST, thus slowing the progression of gastric cancer (36). In our study, PDHPS1 did not change the phosphorylation level of MST, which may account for PDHPS1 regulating a different subunit of PP2A and affecting the affinity or activity of PP2A to different substrates. The property of enzyme specificity to substrate could avoid the effect of PP2A on other substrates or pathways, thus avoiding drug side effects. In addition, because both MST and YAP are important regulatory factors in the Hippo pathway, and MST is the direct upstream of YAP, it is suggested that the combination of PDHPS1 and STRN3 may produce synergistic inhibition of the Hippo pathway and play a more powerful role in tumor inhibition.

Peptides generally exhibit higher specificity, lower immunogenicity and fewer side effects than common compounds. One of the major drawbacks of peptide drugs is that they have a relative short half-life due to natural degradation or enzymatic hydrolysis. Fortunately, some current peptide modification methods have been used to partially overcome this problem. In this study, we added a TAT transmembrane element to the N-terminal of PDHPS1, TAT-PDHPS1 was not only highly soluble in water, but also had a longer half-life than the original sequence. In addition, the optimized peptide had a stronger inhibitory effect on YAP activity. In future, we may continue to try some other peptide modification strategies to further improve clinical applicability of PDHPS1.

In summary, we demonstrated that PDHPS1 inhibited ovarian cancer progress with no obvious toxic effect. Furthermore, PDHPS1 was shown to interact with PTPA, an essential activator of protein phosphatase 2A, which resulted in increase of phosphorylated YAP, further inactivated YAP and suppressed the expression of its downstream target genes. Therefore, PDHPS1 may serve as an anti-tumor peptide to inhibit YAP signaling pathway though interacting with PTPA in ovarian cancer, and it may provide a new treatment for ovarian cancer.

No disclosures were reported.

X. Pan: Methodology, writing–original draft, project administration, writing–review and editing. Z. Geng: Data curation, methodology. J. Li: Data curation, formal analysis, funding acquisition. X. Li: Data curation, formal analysis. M. Zhang: Data curation, formal analysis, funding acquisition. X. Wang: Resources, formal analysis, funding acquisition. Y. Cong: Data curation, formal analysis. K. Huang: Data curation, formal analysis. J. Xu: Conceptualization, formal analysis, supervision, funding acquisition, writing–review and editing. X. Jia: Resources, supervision, funding acquisition, writing–review and editing.

This work was supported by the National Natural Science Foundation of China (grant nos: 81602285 and 81872126), Jiangsu provincial key research and development program (grant no: BE2019621 and BE2019619), Nanjing Medical Science and Technique Development Foundation (grant nos: JQX17009), Research Innovation Program for Graduates of Jiangsu Province (grant nos: JX10413664 and JX22013553).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data