Protein tyrosine phosphatase PTPN3 promotes drug resistance and stem cell-like characteristics in ovarian cancer

The current standard treatment for ovarian cancer is aggressive surgery followed by platinum-based combination chemotherapy. Recurrence and chemotherapeutic drug resistance are the two main factors that account for the high mortality of most ovarian cancers. Liposomal doxorubicin is primarily used for the treatment of ovarian cancer when the disease has progressed after platinum-based chemotherapy. However, relatively little is known about the genomic changes that contribute to both cisplatin and doxorubicin resistance in high-grade serous ovarian cancer (HGSC) under the selective pressure of chemotherapy. Here, we found that protein tyrosine phosphatase PTPN3 gene expression was substantially increased in both cisplatin and doxorubicin-resistant ovarian cancer cells. Silencing of PTPN3 restored sensitivity to cisplatin and doxorubicin in resistant ovarian cancer cells. Down-regulation of PTPN3 also inhibited cell cycle progression, migration, stemness in vitro and the tumorigenicity of resistant ovarian cancer cells in vivo. Meanwhile, the expression of PTPN3 was found to be regulated by miR-199 in resistant ovarian cancer cells. These findings suggest that PTPN3 promotes tumorigenicity, stemness and drug resistance in ovarian cancer, and thus is a potential therapeutic target for the treatment of ovarian cancer.


Real time quantitative reverse transcription PCR (qRT-PCR) analysis
The total RNA was isolated using the RNeasy Mini Kit (QIAGEN) and examined using the NanoDrop ND-1000 UV-Vis Spectrophotometer. For mRNA detection, the total RNA was reverse transcribed using the 5X All-In-One RT MasterMix (Applied Biological Materials Inc. Canada). The qPCR was performed using EvaGreen 2X qPCR MasterMix (Applied Biological Materials Inc. Canada). For miRNA detection, the total RNA samples were polyadenylated and reverse transcribed for a two-step quantitative RT-PCR reaction using the miRNA cDNA Synthesis Kit and miRNA qPCR MasterMixes (Applied Biological Materials Inc. Canada) according to the manufacturer's instructions. The HPRT1 or U6 gene was used as an endogenous control, and fold changes were calculated via relative quantification (2 -ΔCt ).

Western blotting
The cells were plated in the six well plate and were allowed to grow to 90% confluent within 3 days. Cell lysate was prepared in RIPA buffer with the addition of protease inhibitor cocktail, phosphatase inhibitor cocktail and DTT (Dithiothreitol) (Sigma Aldrich, St. Louis, MO, USA). Protein concentration was calculated by the BCA Protein assay kit (Thermo Scientific, UK). NuPAGE Novex 4-12% Bis-Tris protein gels (Novex, Life Technologies) were used for all Western blotting assays. The PTPN3 and GAPDH primary and secondary antibody were bought from Sigma. Bands were detected using super signal west pico chemiluminescent substrate (Thermo Scientific, USA) after incubation with primary antibody and HRP-cojugated secondary antibody (Sigma).

Cell cycle analysis
The A2780CIS and A2780ADR cells were seeded in the six well plates and transfected with esiRNA for 72 h. Cells were collected by tripsinization and washed twice in PBS, fixed in 70% chilled ethanol by adding dropwise into the samples while vortexing and stored at 4 °C until analyzed. Immediately before analysis, cells were washed twice in PBS, and incubated with 250 μg/ml RNase A (Invitrogen) for 30 min at room temperature, followed by staining with PI (Sigma) at 20 μg/ml final concentration for 30 min incubation in dark. The cell cycle distribution and percentage of apoptotic cells were analysed using a FACS analyser (BD LSRFortessa). Ten thousand cells were analysed for each sample. Appropriate gating was used to select the singlecell population. The percentage of cells in sub-2n phase was determined using software (BD FACSDiva v6.0).

In vitro transwell cell migration assay
The transwell cell migration assay was carried out using 24 well Transwell chambers (8 μm pore size, BD Biosciences, CA, USA). In brief, 600 μl complete medium was added to the bottom chamber. The A2780CIS and A2780ADR cells were seeded in the six well plates and transfected with esiRNA for 24 h. The transfected cells were suspended in serum-free medium, and 500 μl of the cell suspension (containing 5×10 4 cells) was placed in the upper chamber.
After 24 hours, any non-migrated cells on the upper surface of the membrane were removed using a cotton swab, and the cells on the bottom surface of the membrane were fixed in 95% ethanol and stained with a 0.1% crystal violet solution. The stained migrated cells adhering to the bottom surface of the membrane were photographed and counted in five randomly selected areas under a 40× microscope field. Each experiment was repeated three times.

Soft agar colony formation assay
The A2780CIS and A2780ADR cells was seeding in the six well plates and transfected with validated shRNA shPTPN3 or shScramble (Sigma). The stable transfected cell clones were selected using puromycin (2ug/ml). The validated shRNA stable transfected A2780CIS and A2780ADR cells were seeded in six well plates in growth medium containing 0.7% soft agar (1ml per well) on top of a layer of growth medium containing 1.4% agar (1ml per well). Growth medium (1ml) with 10% FBS was added on top of the agar. The cell suspension was plated and cultured in a 37 o C incubator for around 3 weeks. After that, the colonies were fixed with methanol and stained with 0.05% crystal violet in 25% methanol. The colonies were pictured and counted under an inverted microscope.

Flow cytometric analysis of ALDH+ and CD133+ cell population
The validated shRNA stable transfected A2780CIS and A2780ADR cell suspensions were counted and incubated with primary antibodies CD133 (Miltenyi Biotec). ALDH+ enzymatic activity was defined using the ALDEFLUOR kit per protocol (Stem Cell Technologies, Vancouver, BC, Canada). For each sample ½ of cell/substrate mixture was treated with 50 mmol/L diethylaminobenzaldehyde (DEAB). Cells were incubated for 45 min. Gating was established using Propidium Iodide (PI)-exclusion for viability and ALDEFLUOR/DEAB treated cells were used to define negative gates. FACS was performed with ≥1×105 cells using the BD FACSCanto II (Becton Dickinson, San Diego, USA) 1 .

Sphere formation
Sphere culture was performed as previously described 2 . Briefly, the validated shRNA stable transfected A2780CIS and A2780ADR cells were plated in triplicate in ultra-low attachment plates in serum-free DMEM/F12 medium supplemented supplemented with 5 μg/mL insulin (Sigma), 20 ng/mL human recombinant epidermal growth factor (EGF, Peprotech), 10 ng/mL basic fibroblastic growth factor (bFGF, Peprotech) and B27 Supplement (Gibco). Cells were plated at the indicated density and from 1,000-10,000 cells/ml in subsequent passage.
Sphere formation was assessed 2 weeks after seeding the cells.

Luciferase reporter assay
The 3'-UTR sequence of PTPN3 predicted to interact with miR-199 or a mutated 3'-UTR sequence within the predicted target sites was synthesized and inserted into the XbaI and FseI sites of the pGL3 control vector (Promega, Madison, WI). These constructs were named as pGL3-PTPN3-3'UTR or pGL3-PTPN3-3'UTR-mut, respectively. For the reporter assay, HEK293 cells were plated onto 24-well plates and transfected with the above constructs and miR-199 mimics or mimics control using Lipofectamine 3000 (Thermo Fisher Scientific, USA).
A Renilla luciferase vector pRL-SV50 (Promega, Madison, WI) was co-transfected to normalize the difference in the transfection efficiency. After 48h, the cells were harvested and assayed using the dual-luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer's instructions. Results were obtained from three independent experiments performed in duplicate.

Survival and statistical analysis
The experimental data are presented as the mean ± standard deviation (SD). All statistical analyses were performed using ANOVA or a two-tailed Student's t test by GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA). The survival curves were created using the Kaplan-Meier method and statistically compared using a log-rank test. Differences were considered statistically significant when the P-values were less than 0.05.