Cell culture
The human breast cancer MCF-7 cells were purchased from Institute of Cell Research, Chinese Academy of Sciences (Beijing, China). Doxorubicin (DOX)-resistant MCF-7DOX cells were established by induction with gradient concentrations (0.1-2 μg/mL) of ADR in vitro. Cells were cultured in DMEM (Sigma) supplemented with 10% FBS (Gibco), 100 units/ml of penicillin, 100 μg/ml, streptomycin in a 5% CO2 at 37°C. MCF-7DOX cells were cultured at a final concentration of 2.0 μg/mL.
Patient samples
Breast cancer samples were obtained from Breast disease Center, the affiliated Hospital of Qingdao University. In all, 112 surgical resected tumors from Feb. 2012 to 2018. Pathological diagnosis was verified by two pathologists independently. All human samples were collected with informed consents from the donors according to the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS). The study was performed after approval by the institutional review board (IRB) of the affiliated Hospital of Qingdao University. Written informed consent was obtained from each individual or patient.
Immunohistochemistry of PEAK1
Formalin-fixed paraffin-embedded tissue sections from excised specimens were processed according to standard procedures. Specific primary antibodies against PEAK1 (1:50) was purchased from Sigma-Aldrich (Shanghai, China). IHC staining for PEAK1 was performed as the manufacture’s instruction. The expression of PEAK1 was positive when 10% of tumor cells showed PEAK1 immunopositivity, and negative when less than 10% of tumor cells showed PEAK1 immunopositivity.
PEAK1 shRNA vector construction and transfection
The short-hairpin RNA direct against human PEAK1 gene (PEAK1 shRNA) was synthesized and constructed into the pcDNA3.1 expression vector (Shanghai, China) as the manufacture’s instruction. The constructed vectors (PEAK1 shRNA or NC shRNA) were transfected into the MCF-7 cells using Lipofectamine 3000 reagent (Invitrogen, Shanghai, China) as the manufacturer's instruction. The PEAK1 shRNA or NC shRNA transfected MCF-7 cells were selected by puromycin (10 mg/ml) for 5 days. The puromycin-resistant colonies were then picked and expanded. The relative protein was detected by Western blot assay.
Plasmid constructs and transfection
The full-length human PEAK1 adenovirus was constructed as to the manufacturer’s instructions using the AdEasy Adenoviral Vector System. Viral particles were produced by GenScript Biotechnology, China. The virus particles containing PEAK1 or control vector were used to infect MCF-7 cells. Transfected cells were selected with G418 (600 μg/ml, Gibco) for 10-12 days. The expression of PEAK1 in stable PEAK1 transfected colonies was detected by western blot analysis.
In vitro doxorubicin sensitivity by MTT assay
MCF-7 cells after transfection with Lv-PEAK1 or PEAK1 shRNA or its control for 48 h were plated in a 96-well plate in triplicate for 24 h, and the cells (300 cells / well) were then exposed to a concentration of 2.0 μg/mL Doxorubicin for 72 h. Subsequently, 20 μL MTT (Sigma-Aldrich) was added to each well and then incubated for 4 hours at 37°C and 5% CO2 humidified atmosphere. The optical density (OD) at 450 nm was measured and considered an indirect index of relative cell viability.
In vitro doxorubicin sensitivity by colony formation assay
MCF-7 cells after transfection with Lv-PEAK1 or PEAK1 shRNA or its control for 48 h were plated in triplicate at 1000 cells per well in 6-well plates with or without 2.0 μg/mL Doxorubicin and cultured for 12 days, then methanol-fixed and Giemsa-stained (GS, Sigma-Aldrich), which was followed by colony counting.
Matrigel invasion assay.
Cell migration and invasion was determined using a Transwell Chamber assay (BD Biosciences) according to the manufacturer's instructions. MCF-7 cells (1 × 105 cells) after transfection with Lv-PEAK1 or PEAK1 shRNA or its control for 24 h were added to the upper compartment (in triplicate), and DMEM plus 10% FCS was added to the lower compartment. Bestatin (Sigma) was added to both compartments. The cells were incubated for 24 h, and the total number of invaded cells was calculated as the manufacture’s instruction.
Western Blotting
Cells and tissues were lysed and protein concentration was measured with BCA protein assay reagents (Pierce). The proteins were then resolved on SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore) and probed with the primary antibodies: PEAK1 and a-Tubulin. Band densitometry analysis was performed using ImageJ software (NCI).
In vivo metastasis assay
Female BALB/c nude mice (5-6 weeks of age, 16-18 g) were obtained from National Rodent Shanghai Experimental Branch Center, Chinese Academy of Sciences (Shanghai, China). All experimental procedures involving animals were conducted in accordance with the institutional guidelines by the Affiliated hospital of Qingdao University. The stable Lv-PEAK1 or PEAK1 shRNA or its control transfected MCF-7 cells were cultured to log phase. 2 × 106 cells (n = 6 per group) were injected into mice via tail veins. After 5 weeks, the whole lung tissues were removaled and the numbers of visible nodules on the lung surface were numbered. H&E staining was used to evaluate the tumor metastasis.
In vivo growth and doxorubicin sensitivity assay
A total of stable PEAK1 shRNA transfected MCF-7DOX or stable Lv-PEAK1 transfected MCF-7 cells (5 × 106 ) were injected s.c. to the left front flank of mice on day 0. From day 3 to day 9, DOX was administered to all groups of mice by i.v. injection (0.1 mL, 10 mg/kg) for a total of 4 times at 2 day intervals. Tumor dimensions were measured in 2 dimensions with microcalipers every other day and tumor volume was calculated by the following formula: tumor volume = (length × width2)/2. Non-retrospective ethical approval obtained for the animal experiments conducted in the study.
Statistical analysis
Data were shown as mean ± SEM; Data were analyzed using Student t test and pearson χ2 test. Value of p <0.05 were considered significant.