Bioinformatics analysis indicates that lysophosphatidylcholine acyltransferase 1 (LPCAT1) and forkhead box A1 (FOXA1) are highly expressed in breast cancer tissues and their expression levels are correlated. Therefore, the aim of the present study was to investigate their involvement in the malignant progression and drug resistance of breast cancer. The clinical significance of LPCAT1 was analyzed using The Cancer Genome Atlas data. The enrichment of LPCAT1 in breast cancer cells was determined and the effects of LPCAT1 knockdown on cell proliferation, colony formation, migration, invasion and paclitaxel (PTX) resistance were evaluated. The association between LPCAT1 and FOXA1 was verified using luciferase reporter and chromatin immunoprecipitation assays. Thereafter, the ability of FOXA1 overexpression to regulate LPCAT1 regulation was evaluated. The results revealed that a high LPCAT1 level was associated with poor overall survival in patients with breast cancer. Furthermore, LPCAT1 was found to be highly expressed in breast cancer cells, and its knockdown resulted in suppressed proliferation, colony formation, migration and invasion, and weakened PTX resistance. Furthermore, FOXA1 overexpression attenuated the effects of LPCAT1 knockdown on cells, indicating that FOXA1 transcriptionally regulates LPCAT1. In summary, the present study reveals that LPCAT1 is transcriptionally regulated by FOXA1, which influences breast cancer cell proliferation, metastatic potential and PTX resistance.
According to cancer statistics released in 2021 (
In a study of glioblastoma, lysophosphatidylcholine acyltransferase 1 (LPCAT1) was suggested to play a role in remodeling the structure of the plasma membrane by modifying the phospholipid composition of the cytoplasmic membrane, increasing the stabilization of epidermal growth factor receptor, and transmitting and amplifying growth signals (
The aim of the present study was to investigate the involvement of LPCAT1 in breast cancer and to explore its mechanism. The Human Transcription factor Database (HumanTFDB) (
The UALCAN database (ualcan.path.uab.edu) was used to compare LPCAT1 and FOXA1 expression levels between tumor and normal tissues using TCGA data. GEPIA (gepia.cancer-pku.cn) was used to compare overall and disease-free survival between patients with low and high LPCAT1 expression using TCGA data. The HumanTFDB database (bioinfo.life.hust.edu.cn) predicted the binding sites for FOXA1 and the LPCAT1 promoter.
MCF-10A mammary epithelial cells and MDA-MB-231, BT-549, HCC1937, SK-BR-3 and MCF-7 breast cancer cell lines were obtained from Shanghai EK-Bioscience Biotechnology Co., Ltd. PTX-resistant MDA-MB-231 (MDA-MB-231/PTX) cells were generated by 3 months of continuous exposure to a stepwise steadily increasing concentration of PTX (0–100 nM; MedChemExpress) at 37°C as previously described (
In order to reduce the expression of LPCAT1 and overexpress FOXA1, MDA-MB-231 cells were transfected with short hairpin (sh)RNAs targeting LPCAT1 (sh-LPCAT1-1 and −2) and FOXA1-overexpression plasmids (oe-FOXA1), respectively. Cells transfected with non-targeting shRNA and empty plasmid served as the negative controls (sh-NC and oe-NC, respectively). These pLVX-shRNAs and pcDNA3.1 plasmids were constructed by VectorBuilder, Inc. Briefly, cells (1×104/well) were seeded in 96-well plates 1 day before transfection, and transfection with a final concentration of 50 nM shRNA and/or 15 nM overexpression plasmids was then performed for 48 h at 37°C using FuGENE® transfection reagents (Promega Corporation). The interval between transfection and subsequent experiments was 48 h. The target sequences were as follows: sh-LPCAT1-1, 5′-GGAACTCTGATCCAGTATATA-3′; sh-LPCAT1-2, 5′-GGGAACTCTGATCCAGTATAT-3′; and sh-NC, 5′-GCACTACCAGAGCTAACTCAG-3′.
TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was added to the cells, mixed and allowed to stand at room temperature for 5 min. Chloroform was then added, the lysate was centrifuged at 12,000 × g for 12 min at 4°C, the upper aqueous phase was collected and the RNA was precipitated with isopropanol. The isolated RNA was reverse transcribed to generate cDNA using a PrimeScript™ RT Reagent Kit (Takara Bio, Inc.). The reaction conditions for reverse transcription were as follows: 30°C for 10 min, 42°C for 30 min and 70°C for 15 min. A QuantiTect SYBR Green PCR kit (Qiagen, Inc.) was used for qPCR according to the manufacturer's protocol. The qPCR was performed in a 20-µl reaction system containing 10 µl Master Mix, 10 ng DNA template and 500 nM specific forward and reverse primers. The thermocycling reaction conditions were as follows: Predenaturation at 95°C for 15 min and 40 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec and extension at 68°C for 30 sec. The relative mRNA levels were measured using the 2−∆∆Cq method (
Protein was isolated from cells following treatment with RIPA lysis buffer (Life-iLab Bio) and quantified using a Nano 300 protein detector (YPH-Bio). Protein separation (30 µg per lane) was achieved using 10% SDS-polyacrylamide gel electrophoresis and the separated proteins were transferred to PVDF membranes (Roche Diagnostics). The membranes were incubated with 5% skimmed milk for 2 h at room temperature, with primary antibodies overnight at 4°C and HRP-conjugated secondary antibodies for 2 h at room temperature in sequence. The primary antibodies against LPCAT1 (cat. no. ab214034; 1:2,000), FOXA1 (cat. no. ab170933; 1:1,000), Ki67 (cat. no. ab92742; 1:5,000), proliferating cell nuclear antigen (PCNA; cat. no. ab92552; 1:1,000), MMP2 (cat. no. ab92536; 1:1,000), MMP9 (cat. no. ab76003; 1:1,000), Bcl-2 (cat. no. ab32124; 1:2,000), Bax (cat. no. ab32503; 1:1,000), cleaved caspase 3 (cat. no. ab32042; 1:500) and GAPDH (cat. no. ab9485; 1:2,500), and the secondary antibodies (cat. no. ab6721; 1:4,000) were all from Abcam. Blots were visualized after treatment with Immobilon ECL Ultra Western HRP (Merck KGaA) and gray values were analyzed with ImageJ software (v1.8.0; National Institutes of Health).
CCK-8 assay was used to evaluate the proliferation of transfected cells and the viability of resistant cells. In brief, transfected cells (3×103/well) were seeded in 96-well plates and cultured for 24, 48 and 72 h. The resistant cells were treated with PTX (0–100 nM) for 72 h at 37°C. CCK-8 solution (Absin Bioscience, Inc.) was added to each well and incubation was continued for another 2 h. The optical density (OD) was then determined at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).
Control and transfected MDA-MB-231 cells were seeded into culture dishes at a density of 500 cells/dish. They were cultured for 2 weeks and the medium was changed every 3 days. Thereafter, cells were washed twice with PBS, fixed with 4% paraformaldehyde (Merck KGaA) for 20 min at room temperature and stained with 0.5% crystal violet (Shanghai Gefan Biotechnology Co., Ltd.) for 20 min at room temperature. The colonies were counted manually. A cluster of >50 cells was considered a colony.
The migration and invasion of the control and transfected MDA-MB-231 cells were separately assessed using wound healing and Transwell assays, respectively. In the wound healing assay, cells were cultured until a confluent monolayer formed and a sterile pipette tip was used to generate a wound in the middle of the cells that were cultured in serum-free Leibovitz's L-15 medium. Images were captured at 0 and 24 h. In the Transwell assay, cells (1×104 cells/well) were cultivated in serum-free Leibovitz's L-15 medium in the upper chamber, which was pre-coated with Matrigel (Corning, Inc.) at 37°C for 1 h. Leibovitz's L-15 containing 20% FBS was loaded into the lower chamber. Following 24 h of incubation at 37°C, the invasive cells were fixed and stained with 0.1% crystal violet solution at room temperature for 15 min. Results for both assays were observed under a light microscope (magnification ×100; Olympus Corporation).
The apoptosis of MDA-MB-231 and MDA-MB-231/PTX cells with or without transfection was analyzed using Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology) and flow cytometry. Briefly, cells (1×105) were washed twice with precooled PBS and suspended in 1 ml binding buffer. A 100-µl sample of the cell suspension was transferred in a culture tube and incubated with Annexin V-FITC and propidium iodide at room temperature in the absence of light for 15 min. Results were obtained using flow cytometry using a BD FACSCanto™ instrument (BD Biosciences) and FlowJo version 10 software (GlowJo LLC).
The promoter site of LPCAT1 and a mutated form (CGCCCAGGC) of this site were cloned into a dual-luciferase reporter vector (Promega Corporation). The reporter vector was co-transfected along with oe-FOXA1 or oe-NC into MDA-MB-231 cells using FuGENE® transfection reagents (Promega Corporation). At 48 h post-transfection, the luciferase activity was assessed using the Dual-luciferase Reporter Assay System (Promega Corporation), according to the manufacturer's protocol, and normalized to
The association between LPCAT1 and FOXA1 was evaluated using a ChIP Detection Kit (cat. no. 17–295; EZ-ChIP; MilliporeSigma). Briefly, the MDA-MB-231 cells were treated with 1% formaldehyde, followed by lysis buffer and then sonicated. The cells were subsequently incubated with an anti-FOXA1 (cat. no. ab170933; 1:50; Abcam) or anti-IgG antibody (cat. no. ab172730; 1:50; Abcam) overnight at 4°C. Following the incubation, 60 µl protein A agarose beads was added to harvest the protein-DNA complex. The complex was washed in low-salt and high-salt washing buffers at 4°C, for 5 min each time, 4 times in total. The liquid was removed by centrifugation at 1,000 × g for 1 min at 4°C, and 5 mmol/l NaCl was added to retrieve the DNA. The enrichment of LPCAT1 was determined using RT-qPCR.
For statistical analysis, GraphPad Prism 8.0 (GraphPad Software, Inc.) was utilized. All data are presented as the mean ± SD and all experiments were performed ≥3 times independently. To compare differences between two and multiple groups, the unpaired Student's t-test and one-way ANOVA followed by Tukey's post hoc test were used, respectively. P<0.05 was considered to indicate a statistically significant difference.
Analysis performed using the UALCAN database revealed that LPCAT1 is expressed at significantly higher levels in breast cancer tissues compared with normal tissues (
CCK-8 (
The viability of MDA-MB-231 and MDA-MB-231/PTX cells following treatment with PTX (0–100 nM) was detected by a CCK-8 assay. The results indicated that MDA-MB-231 cells were significantly more sensitive to PTX than were the MDA-MB-231/PTX cells (
Analysis performed using the UALCAN database suggested that FOXA1 was also highly expressed in breast cancer tissues compared with normal breast tissues (
To evaluate the effect of FOXA1 overexpression on the regulatory role of LPCAT, the proliferation and colony formation ability of MDA-MB-231 cells co-transfected with sh-LPCAT1 and oe-FOXA1 were assessed. The co-transfection increased cell proliferation (
PTX has been extensively known for its antitumor activity. It has a broad range of anticancer properties and can be employed in the chemotherapy of various solid tumors (
The findings of the present study indicate that LPCAT1 modulates breast cancer cell proliferation, metastatic potential and drug resistance. Given the previous findings of LPCAT1 in various tumors (
The role of FOXA1 in breast cancer has been reported in previous studies. For example, one study showed that microRNA (miR)-100 inhibits the proliferation, migration and invasion of breast cancer cells by targeting FOXA1 (
In summary, the present study reveals that the presence of LPCAT1 contributes to breast cancer cell proliferation, metastatic potential and PTX resistance. Moreover, LPCAT1 is transcriptionally regulated by FOXA1, and the identification of this signaling pathway in PTX resistance suggests a new potential target for the alleviation of chemotherapy resistance. Follow-up experiments using animals with gene overexpression or knockdown are planned to verify this discovery, and new therapies may become available to patients in terms of this potential novel target.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HZ and YZ contributed to the study concept, experiments and analysis. HZ drafted the manuscript. HZ and YZ confirm the authenticity of all the raw data. Both authors read and approved the final version of the manuscript
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
LPCAT1 levels and their clinical significance. (A) Data from the UALCAN database based on The Cancer Genome Atlas data suggest that LPCAT1 is upregulated in breast cancer tissues. ***P<0.001 vs. normal. (B) Gene Expression Profiling Interactive Analysis database analysis implicates an association of LPCAT1 expression with overall and disease-free survival in breast cancer. Expression levels of LPCAT1 in various cell lines were determined using (C) RT-qPCR and (D) western blotting. **P<0.01, ***P<0.001 vs. MCF-10A. Expression levels of LPCAT1 in transfected MDA-MB-231 cells were determined using (E) RT-qPCR and (F) western blotting. ***P<0.001 vs. sh-NC. LPCAT1, lysophosphatidylcholine acyltransferase 1; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin; NC, negative control; BRCA, breast invasive carcinoma.
Role of LPCAT1 in cell proliferation and metastasis. (A) Cell proliferation was examined using a Cell Counting Kit-8 assay. (B) Colony formation was examined using a colony formation assay. (C) Expression levels of Ki67 and PCNA were determined using western blotting. (D) Cell migration and (E) invasion potential were assessed using wound healing and Transwell assays, respectively. Scale bar, 100 µm. (F) Expression levels of MMP2 and MMP9 were determined using western blotting. ***P<0.001 vs. sh-NC. LPCAT1, lysophosphatidylcholine acyltransferase 1; PCNA, proliferating cell nuclear antigen; sh, short hairpin; NC, negative control; OD, optical density.
Role of LPCAT1 in PTX resistance. (A) Viability of MDA-MB-231 and MDA-MB-231/PTX cells after PTX intervention was detected by a Cell Counting Kit-8 assay. (B and C) Proportion of apoptotic cells following treatment with 4 nM PTX was assessed by flow cytometry. (B) Representative plots and (C) quantified results are shown. (D) Enrichment of apoptosis-associated proteins in the cells was determined using western blotting. ***P<0.001 vs. MDA-MB-231; ###P<0.001 vs. MDA-MB-231/PTX + sh-NC. LPCAT1, lysophosphatidylcholine acyltransferase 1; PTX, paclitaxel; sh, short hairpin; NC, negative control; PI, propidium iodide; FITC-A, fluorescein isothiocyanate-Annexin V.
Association between FOXA1 and LPCAT1. (A) UALCAN database analysis based on The Cancer Genome Atlas data indicates that FOXA1 is highly expressed in breast cancer tissues. ***P<0.001 vs. normal. Expression levels of FOXA1 in MCF-10A and MDA-MB-231 cells were assessed using (B) RT-qPCR and (C) western blotting. ***P<0.001 vs. MCF-10A. Expression of FOXA1 in the MDA-MB-231 cells transfected with oe-FOXA1 was confirmed using (D) RT-qPCR and (E) western blotting. Expression of LPCAT1 in the transfected MDA-MB-231 cells was confirmed using (F) RT-qPCR and (G) western blotting. ***P<0.001 vs. oe-NC. (H) Binding sites for transcription factors FOXA1 and LPCAT1 promoters predicted using the HumanTFDB website. (I) LPCAT1 promoter activity was determined with a luciferase reporter assay. ***P<0.001 vs. LPCAT1 + oe-NC (WT). (J) Binding between FOXA1 and LPCAT1 was evaluated with a chromatin immunoprecipitation assay. ***P<0.001 vs. IgG. FOXA1, forkhead box A1; LPCAT1, lysophosphatidylcholine acyltransferase 1; RT-qPCR, reverse transcription-quantitative PCR; HumanTFDB, Human Transcription factor Database; oe, overexpression; NC, negative control; BRCA, breast invasive carcinoma; WT, wild type; MUT, mutant.
FOXA1 regulates LPCAT1. (A) Proliferation and (B) colony formation of MDA-MB-231 cells co-transfected with sh-LPCAT1 and oe-FOXA1 was assessed. (C and D) Expression levels of Ki67 and PCNA were determined using western blotting. (C) Representative images and (D) densitometrically quantified results are presented. (E) Cell migration and (F) invasion potential were assessed using wound healing and Transwell assays, respectively. Scale bar, 100 µm. (G) Expression levels of MMP2 and MMP9 were determined using western blotting. ***P<0.001 vs. control; ##P<0.01 and ###P<0.001 vs. sh-LPCAT1 + oe-NC. (H) Apoptosis after 4 nM PTX treatment was assessed by flow cytometry. (I) Enrichment of apoptosis-associated proteins in the co-transfected cells was determined using western blotting. ***P<0.001 vs. MDA-MB-231; ###P<0.001 vs. MDA-MB-231/PTX + sh-NC; ΔΔΔP<0.001 vs. sh-LPCAT1 + oe-NC. FOXA1, forkhead box A1; LPCAT1, lysophosphatidylcholine acyltransferase 1; sh, short hairpin; oe, overexpression; NC, negative control; PCNA, proliferating cell nuclear antigen; OD, optical density; PI, propidium iodide; FITC-A, fluorescein isothiocyanate-Annexin V.