Cell culture

Human pancreatic cancer cell lines BxPC-3, Panc-1, Capan-1, MIAPaCa-2, and AsPC-1, as well as human normal epithelial cell line HPNE, were obtained from the American Type Culture Collection. KP-4 and SUIT-2 were purchased from the Japanese Collection of Research Bioresources. PaTu8988S and PaTu8988T cells were obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The human embryonic kidney cell line HEK293T was purchased from System Biosciences (SBI, Mountain View, CA, USA). BxPC-3, AsPC-1, KP-4, and SUIT-2 cells were cultured in RPMI medium supplemented with 10% FBS. Panc-1, MIAPaCa-2, PaTu8988S, PaTu8988T, and HEK293T cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Capan-1 cells were cultured in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with 20% FBS. All cells were incubated at 37 °C in an atmosphere of 20% O2 and 5% CO2.

Plasmid construction

shRNA oligonucleotides were annealed and cloned into pLKO.1–Puromycin and Tet-pLKO-Puromycin lentiviral vectors (Addgene, Watertown, MA, USA). The sequences used for the respective target genes were as follows:

shScramble: 5′-CCTAAGGTTAAGTCGCCCTCG-3′

shWWP1 #1:5′-ATTGCTTATGAACGCGGCTTT-3′

shWWP1 #2:5′-ACAACACACCTTCATCTCCGT-3′

Transfection and lentivirus transduction

A lentiviral packaging system (SBI) was used to generate stably expressing cells according to the manufacturer’s protocol. Briefly, the plasmid and pPACK1 packaging plasmid mix were transfected into HEK293T cells using Effectene Transfection Reagent (Qiagen). After 48 h, the viruses were concentrated in the culture medium using PEG-it Reagent (SBI). The centrifuged pellet was resuspended in 1 × PBS, and aliquots were stored at – 80 °C. Cells were infected with the virus using a polybrene reagent (Merck Millipore), followed by selection with 2 µg/mL puromycin for at least 3 days.

Western blotting

Western blotting was performed as previously described [27]. Briefly, total lysate samples were separated on 12.5% or 5–20% gradient polyacrylamide gels (Fujifilm Wako Pure Chemicals) and transferred onto Immobilon-P membranes (polyvinylidene fluoride; Merck). After blocking with 5% skimmed milk, the membranes were probed with the appropriate primary antibodies diluted in Immunoshot Reagent I (Cosmo Bio) for 16 h at 4 °C. The corresponding HRP-conjugated secondary antibodies (GE HealthCare) were then added. Bound antibodies were visualized using the ImmunoStar LD Reagent (Fujifilm Wako Pure Chemicals). Blot images were obtained using a WSE-6100H LuminoGraph I (ATTO Corporation, Tokyo, Japan). The antibodies used in this study are listed in Table S1.

Immunoprecipitation

The cells were lysed using RIPA buffer (Fujifilm, Wako Pure Chemicals). Total lysates were precleared using Pierce Protein A/G Magnetic Beads (#88802, Thermo Fisher Scientific) for 15 min at 4 °C, and then immunoprecipitated using anti-PTEN or anti-rabbit IgG antibody (Table S1) bound with Pierce Protein A/G Magnetic Beads overnight at 4 °C. The immunoprecipitates were washed three times using RIPA buffer and eluted with Pierce IgG Elution Buffer (#21004, Thermo Fisher Scientific). Samples were separated on 5–20% gradient polyacrylamide gels (Fujifilm Wako Pure Chemicals) and subjected to western blot analysis.

Subcellular fractionation

BxPC-3 cells expressing TetOn shWWP1 were treated with or without 1 µg/mL of Dox and cultured for 48 h. Membrane and cytosolic fractions were subsequently isolated using the Subcellular Protein Fraction Kit for Cultured Cells (Thermo Fisher Scientific), following the manufacturer’s protocol.

Cell proliferation assay

Cell proliferation was evaluated using a crystal violet assay. Cells at the 0, 24, 48, 72, and 96 h time points were fixed using 10% formalin and stained using 0.05% crystal violet. They were then solubilized using 10% acetic acid, and the absorbance was measured using a Multiskan FC Microplate Photometer (Thermo Fisher Scientific).

Cell cycle analyses

Cells harvested from a 6-well plate were fixed in 70% ice-cold ethanol and incubated for 24 h at − 20 °C. The fixed cells were washed twice using 1 × PBS and resuspended in 990 µL of 1 × PBS with 10 µL ribonuclease solution (Nippon Gene) to exclude RNA contamination, followed by the incubation for 30 min at 25 °C. Cellular DNA was stained by adding 50 µL of propidium iodide (1 µg/µL in PBS; Fujifilm Wako Pure Chemical) and incubated for 1 min at 25 °C in the dark. Flow cytometry analyses were performed using a Guava Easy Cyte Plus instrument (GE Healthcare) and FlowJo software (BD Biosciences).

Apoptosis assay

The apoptosis assay was performed using the Caspase-Glo 3/7 Assay System (Promega Corporation, Madison, Wisconsin, USA). Briefly, 2.0 × 104 cells were seeded into 96 well-white-walled plates with clear bottoms (Greiner Bio-One). After culturing for 24 h, 100 µL of Caspase-Glo Detection Solution was added to each well, and the plate was incubated for 1 h at 25 °C. The luminescence was measured using a Glomax 96 Microplate Luminometer (Promega).

RNA sequencing

RNA sequencing was performed by Rhelixa Co. (Tokyo, Japan). BxPC-3 cells expressing TetOn shWWP1 were treated with or without 1 µg/mL of Dox and cultured for 48 h. Total RNA was extracted from the cells using the RNeasy Mini kit (QIAGEN), and the quality of the extracted RNA was assessed using a NanoDrop One spectrophotometer (Thermo Scientific) and Bioanalyzer (Agilent), ensuring an RNA integrity number (RIN) greater than 7. RNA libraries were prepared using the NEBNext Poly (A) mRNA Magnetic Isolation Module (#E7490, New England Biolabs (NEB), Ipswich, MA, USA) and the NEBNext UltraTM II Directional RNA Library Prep Kit (#E7760, NEB) according to the manufacturer's instructions. The prepared complementary DNA libraries were sequenced using a NovaSeq 6000 system (Illumina) to generate 150 base paired-end reads. Each sample generated approximately 8 G bases of data, corresponding to 53.3 million reads (26.7 million pairs) per sample. Quality control of the raw sequencing reads was performed using the Trimmomatic software (ver. 0.38) with the following settings: ILLUMINACLIP 2:30:10, LEADING = 20, TRAILING = 20; SLIDING WINDOW, 4:15; and MINLEN, 36. Low-quality reads and adapter sequences were also removed. The trimmed reads were then aligned to the reference genome (hg38) using the HISAT2 software (ver. 2.1.0). The transcript abundance was quantified using featureCounts (ver. 1.6.3) to calculate the raw read counts mapped to known exon regions, which were further normalized to Transcripts Per Million (TPM) values. Gene enrichment analyses were performed using GSEA. A false discovery rate (FDR) of less than 0.25 was considered statistically significant in the GSEA.

Subcutaneous xenograft model

For xenograft transplantation, 2.0 × 105 BxPC-3 cells expressing TetOn shWWP1 in 75 µL of RPMI were mixed with 75 µL of Matrigel TM-Growth Factor Reduced (#356231, Corning Incorporated, Corning, NY, USA), and immediately injected subcutaneously into the backs of male BALB/cAjcl-Foxn1nu mice (CREA Japan Inc). The mice were maintained for 2 weeks to allow the transplanted cells to develop tumors and then divided into two groups with similar distribution of tumor sizes: one group received Dox at 1 mg/mL in a 5 mg/mL sucrose solution, and the other group did not receive Dox. The mice were then maintained for an additional 2 weeks, after which the resulting tumors were excised and weighed.

Immunohistochemistry

Immunohistochemistry (IHC) was performed as previously described [28]. Briefly, fixed paraffin-embedded tissue microarrays T141c and PA805c (US Biomax, Derwood, MD, USA) were deparaffinized and incubated in Target Retrieval Solution (Dako Corp., Carpinteria, CA, USA) for 60 min at 98 °C for antigen retrieval. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 10 min. After blocking in 10% normal goat serum (Dako) for 30 min, the sections were incubated overnight at 4 °C with a primary antibody diluted in 2% bovine serum albumin/PBS with 0.1% Tween-20. The sections were incubated for 30 min with a biotinylated secondary antibody (Vector Laboratories), washed, incubated for 30 min with an avidin–biotin–peroxidase complex (Vectarstain ABC Elite kit; Vector Laboratories), and developed by 3,3'-diaminobenzidine (DAB) substrate solution (DAKO Japan). Hematoxylin was used for nuclear staining. The primary antibodies, conditions, and dilutions used are listed in Supplementary Table S1.

Inhibitor drug screening

Drug screening was performed using SCADS inhibitor kits (Kit I ver. 4, kit IV, ver. 2.4) obtained from the Molecular Profiling Committee (Ministry of Education, Culture, Sports, Science, and Technology, Japan). In addition, Buparlisib, Alpelisib, Taselisib, and TGX-221 (Selleck Chemicals, Houston, TX, USA) were included in the screening. Cells were plated at a seeding density of 100 cells/well in a 96-well plate (Greiner Bio-One) and incubated for 16 h. Subsequently, 178 drugs were dissolved in DMSO at the concentration of 100 µM and applied to the wells at a final concentration of 1 µM. Equal amounts of DMSO were added to the control wells. After 120 h of incubation, the number of cells in each well was examined using the CellTiter-Glo Assay System (Promega) according to the manufacturer’s instructions. Absorbance was measured using SpectraMax iD3 (Molecular Devices).

Drug sensitivity assay

To determine the IC50 values of the drugs, 1.0 × 104 BxPC-3 cells expressing TetOn shWWP1 were treated with doxycycline and stepwise concentrations of drugs for 120 h. Linsitinib and Torkinib were purchased from Selleck, and Indole-3-carbinol (I3C) was purchased from Millipore Sigma. Cell numbers after treatment with each drug were evaluated using a crystal violet assay. The absorbance of each sample was normalized to that of the control well, and dose–response curves were generated to determine the IC50 values. Drug synergism assessments were performed using the Combenefit Software (Cancer Research UK Cambridge Institute).

Statistical analyses

The data are presented as mean ± standard deviation (SD) for each study group. For comparison between the two groups, the Student’s t-test or Mann–Whitney U test was used according to the data distribution. χ2 test or Fisher’s exact test was used to evaluate the immunohistochemistry of tissue microarrays. One-way analysis of variance (ANOVA) followed by the post-hoc Tukey–Kramer multiple comparison test was used for the apoptosis assay. Two-way ANOVA followed by the post-hoc Tukey–Kramer multiple comparison test was used for the cell proliferation assay and cell cycle analyses. Statistical significance was set at a p-value < 0.05. Data analysis was performed using GraphPad Prism software (version 10; Developer; San Diego, CA, USA).