Cell culture and chemical reagents. The human PC cell lines (Capan2, Mia-paca2, SW1990, Bxpc3, PANC1 and PL45) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) or Cell Culture Center at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. These cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco®, Life Technologies TM, Carlsbad, USA) supplied with 10% fetal bovine serum (FBS, Gibco), streptomycin (100 µg/ml) and penicillin (100 U/ml) at 37°C in a humidified atmosphere containing 5% CO2. Topotecan (TPT), Doxorubicin (ADM), Gemcitabine (GEM) and Paclitaxel (PTX) were obtained from Selleck (Selleck Chemicals, China). Anti-HSP90α neutralizing antibody was obtained from Enzo Life Sciences (Plymouth Meeting, PA, USA). E-cadherin, Vimentin, Snail, phospho-AKTser473, AKT, phospho-ERK1/2Thr202/Tyr204, ERK and β-actin were purchased from Cell Signaling Technologies. Antibody to HSP90α was obtained from Abcam.
Wound-healing assay. Capan2 and PL45 cells were seeded in 6-well plates and cultured at 37°C. After overnight, a wound was generated using a pipette tip to make a straight scratch. The cells were treated with or without anti-HSP90α antibody or human recombinant HSP90α (rHSP90α) protein for 24 h. The width of the wound was measured under a microscope.
Transwell assays. The invasive assay was performed as described using transwell cell culture chambers (8 µM pore size polycarbonate membrane; Costar, Cambridge, MA). The membrane was coated with Matrigel (BD Biosciences, Bedford, MA). Capan2 and PL45 cells suspension without FBS (200 µL, 1×106/mL cells) was placed in the upper chamber, while the bottom chamber was filled with 600 µL of culture medium containing 10% FBS. Anti-HSP90α antibody or rHSP90α protein was added to the FBS-free medium in the upper chamber. Cells that migrated to the lower chamber were fixed with 4% paraformaldehyde for 20 min followed by staining with 0.1% crystal violet (Solarbio, Beijing, China)) for 10 min. The cells were randomly photographed under a light microscope.
Cell survival assays. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was adapted to measure cell viability using Capan-2 and PL45 cells. Cells were treated with different concentrations of GEM (0.03~20 µM), TPT (0.03~20 µM), PTX (0.03~20 µM) or ADM (0.003~3 µM) for 72 h. Then, MTT (0.5 mg/mL) solution was incubated for 4 h. The formazan crystals were solubilized in DMSO and measured by at 570 nm using microplate reader (Biotek Instruments, Inc. USA). IC50 values were calculated using GraphPad Prism 8 software.
Quantitative RT-PCR (qPCR). Total RNA was extracted by the Easypure RNA kit (Tansgen Biotech, Beijing China). cDNA was obtained using the TransScript One-Step gDNA Remove and cDNA Synthesis SuperMix kit (Tansgen, Beijing China). PCR amplifications were performed using a SYBR Green PCR Master Mix kit (Cat. QPK-201, Toyobo, Japan) and an ABI PRISM 7900 Sequence Detection system (Applied Biosystems, Foster City, CA, USA). The primers used for qPCR were as follows: LRP1, F, 5'-GATGAGACACACGCCAACTG-3', R, 5'-CGGCACTGGAACTCATCA-3'; E-cadherin, F, 5'-CAATGCCGCCATCGCTTAC-3', R, 5'-ATGACTCCTGTGTTCCTGTTAATG-3'; N-cadherin, F, 5'-GACAATGCCCCTCAAGTGTT-3', R, 5'-CCATTAAGCCGAGTGATGGT-3'; Vimentin, F, 5'-TCCGCACATTCGAGCAAAGA-3', R, 5'-ATTCAAGTCTCAGCGGGCTC-3'; Snail, F, 5'-GCTCCACAAGCACCAAGAGT-3', R, 5'-ATTCCATGGCAGTGAGAAGG-3'; GAPDH, F, 5'-GAGTCAACGGATTTGGTCGT-3', R, 5'-TTGATTTTGGAGGGATCTCG-3'. GAPDH was used as an internal control. The indicated gene expression was calculated according to the 2−ΔΔCt method.
Western blot assays. Treated and untreated PC cell lysates (30 µg) were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, UK). Membranes were blocked and incubated with specific primary antibody (1:1000) and followed by applying with the corresponding HRP-conjugated secondary antibodies. Bands were visualized by ECL detection reagent using Image Quant LAS 4000 (GE Healthcare, Piscataway, NJ, USA). The relative protein levels were calculated based on β-actin as the loading control and were densitometrically analyzed by ImageJ software (NIH, MD).
Antibodies specific for the following factors were used for western blotting: HSP90α, E-cadherin, Vimentin, Snail, phospho-AKTser473, AKT, phospho-ERK1/2Thr202/Tyr204, ERK and β-actin.
RNA interference assay. Short hairpin RNAs for LRP1 gene were obtained from GenePharma Co., Ltd. (Shanghai, China). shLRP1 plasmid and control shRNA were transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. The targeting sequences are shown as follow. Transfected cells were selected with puromycin. The LRP1 expression was confirmed by qPCR or western blot with specific antibody.
Data mining and bioinformatic analyses. LRP1 mRNA expression data from the PC dataset of The Cancer Genome Atlas (TCGA; https://tcga-data.nci.nih.gov) were analyzed using cBioportal for Cancer Genomics (http://cbioportal.org) web resources. RNA sequencing data (FPKM values) of gene expression were downloaded from the Genomic Data Commons (GDC, https://portal.gdc.cancer.gov/) using the R package TCGAbiolinks and were transformed into transcripts per kilobase million (TPM) values, which are more similar to those resulting from microarrays and more comparable between samples.13 The overall survival was investigated using the Kaplan-Meier method with the log-rank test. We set the high and low gene expression level groups by the median value. The overall survival plot was obtained with the hazard ratios (HR) and the 95% confidence interval information. Pearson’s correlation analysis and Spearman’s rank correlation analysis were conducted for gene expression level score.
Statistical analysis. The results are expressed as the mean values ± SD. Statistical significance of obtained data was calculated using Student's t test. P < 0.05 was considered statistically significant. We used Kaplan-Meier estimation to analyze survival rates and the log-rank test to determine the significance of the differences between two survival curves. A Cox-proportional hazards model was used for the univariate and multivariate analyses, and hazard ratio was calculated using a 95% confidence interval (CI). Correlations coefficients between LRP1 mRNA expression and snail expression were computed by Spearman and distance correlation analyses.