Clinical sample material
This study was approved by the ethics committee of Fukushima Medical University. Two hundred seventy-eight patients with breast cancer whose expression levels of NEDD4 mRNA were measured in Fukushima Medical University between February 2007 and November 2017 were enrolled in the study. To evaluate the simple association between prognosis of hormone receptor-positive breast cancer and NEDD4, hormone receptor-positive HER2-negative breast cancer cases were selected. Of these cases, 145 patients with pathological stage 0–II for whom neoadjuvant/adjuvant hormone therapy for 5–10 years was recommended in the therapeutic guideline were included in the study[11]. We also included 51 hormone-receptor negative patients including HER2 positive and triple negative breast cancer patients. Clinical information was obtained from medical records in October 2019. Overall survival (OS) was defined as the time from the date of breast cancer diagnosis to the date of death from any cause. Disease-free survival (DFS) was defined as the time from the date of surgery to the date of recurrence or death, whichever occurred first. Patients in whom no event was observed were censored on the day of documentation of last medical records.
NEDD4 mRNA expression analysis
Breast cancer tissues were obtained from surgical or biopsy specimens. The specimens were frozen and processed for total RNA extraction using Isogen (Nippon Gene Co., Ltd., Tokyo, Japan) and for poly(A)+RNA purification using MicroPoly(A) Purist kit (Ambion, Austin, TX, USA) [12]. The DNA microarray that was used for the poly(A)+RNA was named system 1, in which a set of synthetic polynucleotides (80-mers) representing 31,797 species of human transcript sequences including NEDD4 was printed on a glass slide using a custom arrayer. The DNA microarray that was used for total RNA was named System 2, in which a set of synthetic polynucleotides (80-mers) representing 14,400 species of human transcript sequences including NEDD4 was printed on a glass slide using a custom arrayer. For the RNA of the samples, SuperScript II (Invitrogen Life Technologies, Carlsbad, CA, USA) and Cyanine 5-dUTP (Perkin-Elmer Inc., Boston, MA, USA) were used to synthesize labeled cDNA from 2 µg of poly(A)+RNA in System 1, and 5 µg of total RNA in System 2. Human common reference RNA was prepared by mixing equal amounts of total RNA and poly(A)+RNA, which were extracted from 22 human cancer cell lines (A431, A549, AkI, hBL-100, HeLa, hepG2, hL60, ImR-32, Jurket, k562, kP4, mkN7, Nk-92, Raji, Rd, Saos-2, Sk-N-mC, SW-13, T24, U251, U937, and Y79). Using a same method for the human common reference RNA, Cyanine 3-dUTP (Perkin-Elmer Inc.) was used to synthesize labeled cDNA from 2 µg of Human Universal Reference RNA Type I (MicroDiagnostic, Tokyo, Japan) in System 1, and 5 µg of Human Universal Reference RNA Type II (MicroDiagnostic) in System 2. Hybridization was performed with a Labeling and Hybridization kit (MicroDiagnostic). Signals were measured using a GenePix 4000B Scanner (Axon Instruments, Inc., Union City, CA, USA) and then converted into primary expression ratios of the cyanine 5 intensity of each specimen to the cyanine 3 intensity of the Human Universal Reference RNA. Each ratio was normalized using GenePix Pro 3.0 software (Axon Instruments, Inc.). The primary expression ratios were converted into log2 values, which were designated as converted values. The converted log2 values of NEDD4 mRNA was used in the present study.
Cell culture
ER-positive human breast cancer cell line, MCF-7, and human embryonic kidney cell line, 293T, were purchased from ATCC (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Fujifilm Wako, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biowest, Nuaille, France), 100 units/ml penicillin G and 100 µg/ml streptomycin in a humidified 5% CO2 incubator at 37℃. As pretreatment for evaluating the cells with E2 stimulation or hydroxytamoxifen (TAM) exposure, the MCF-7 cells were cultured for two days in phenol red-free DMEM (Fujifilm Wako) containing 10% heat-inactivated FBS stripped of steroids by absorption to dextran-coated charcoal FBS (Biological Industries, Beit HaEmek, Israel). The MCF-7 cells were stimulated by 1 nM E2 (17b-estradiol, Sigma-Aldrich, St. Louis, MO, USA) for 48 hours. When simulating clinical tamoxifen therapy, the MCF-7 cells were exposed to 1 nM E2 + 2 mM TAM (4-Hydroxytamoxifen, Sigma-Aldrich) for 48–72 hours.
Small hairpin RNA (shRNA) mediated knockdown
Lentivirus vector expressing shRNA for NEDD4 (sh-NEDD4) was constructed in pRSI12-U6-sh-HTS4-UbiC-TagRFP-2A-Puro plasmid (Cellecta, Mountain View, CA, USA). Lentivirus vector expressing shRNA for no target genes (sh-control) was also constructed in the plasmid. The sh-NEDD4 or the sh-control was transfected into 293T cells together with two packaging plasmids, pCMV-VSV-G/RSV-Rev and pCAG-HIVgp (RIKEN Bio-Resource Center, Tsukuba, Japan), using FuGENE HD (Promega, Madison, WI, USA). At 48 hours post-transfection, the supernatants were collected and filtered. The lentivirus encoding the shRNA were incubated with MCF-7 cells for 48 h. The transduced cells were selected for additional incubation for 72 h in the presence of 1 µg/ml puromycin (Fujifilm Wako).
Cell proliferation assay
The sh-control or sh-NEDD4 knockdown MCF-7 cells were seeded in 96-well culture plates (7 × 103 cells/well). After overnight incubation, the cells were replaced in a medium containing ethanol or 1 nM E2 or 1 nM E2 + 2 mM TAM. After incubation, a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) solution was added to the wells. After incubating the cells for 2 hours at 37℃, absorbance was measured using a plate reader (Thermo Fisher Scientific, Waltham, MA, USA). The absorbance was proportional to cell proliferation.
Western Blots
Western blot samples were prepared from the cultured cells that were lysed in RIPA buffer (40 mM Tris-HCl, pH7.5, 1% NP-40, 150 mM NaCl, 2 mM EDTA, 2 mM Na3VO4, 50 mM NaF) containing protease inhibitor cocktail (Roche, Basel, Swiss). The samples were boiled for 5 min at 95°C. Lysates were centrifuged at 13,000 g for 20 min at 4℃. Proteins in the samples were separated in 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), and transferred to polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA, USA). The proteins were blotted with primary and secondary antibodies. The antibodies used were as follows: NEDD4, HER3, PTEN, phospho-HER3 (pHER3[Y1289]), RAS, ERK1/2, phospho-ERK1/2 (pERK1/2[T202/Y204]), AKT, phospho-AKT (pAKT[S473]) (Cell Signaling Technology, Danvers, MA, USA), ER (Thermo Fisher Scientific) and β actin (Sigma-Aldrich). Expression levels of the proteins were visualized by SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific) and ChemiDoc XRS Plus (Bio-Rad).
Statistics analysis
All of the analyses, including receiver operating characteristic (ROC) curve analysis, Kaplan-Meier survival analysis log-rank test, and Student’s t-test, were conducted using R version 4.0.3. Kaplan-Meier survival analysis and the log-rank test were used to compare differences in OS or DFS for each clinical categorical variable. The cell proliferation assays were expressed as means ± standard deviation. Statistical analysis of the cell proliferation assay was performed using the Student’s t-test (two-tail). P-values < 0.05 were considered statistically significant.