Among all types of women’s cancers, breast cancer has the most new cases of diagnosed cancer type and is the second cause of cancer-related mortality worldwide [1
]. Currently, dysregulation of metabolic pathways, including fatty acid metabolic pathways, is considered as a risk factor for promoting breast cancer progression [2
]. Fatty acid metabolism comprises multiple pathways including fatty acid transport, de novo synthesis, fatty acid oxidation, etc., and emerging evidence has indicated that some of the fatty acid metabolic enzymes are related to different subtypes of breast cancer [3
]. Fatty acid binding proteins (FABP) are a family of proteins that bind long-chain fatty acids and are involved in facilitating transport and uptake of lipids. Overexpression of FABP5 and FABP7 is associated with triple-negative breast cancer and basal-like breast cancer [4
]. Acyl-CoA synthetase long-chain family member 4 (ACSL4) activity leads to long-chain fatty acid transport and long-chain fatty-acid-coenzyme A ligase. High ACSL4 expression is inversely associated with estrogen receptor expression and high ACSL4 expression is a biomarker for an aggressive breast cancer phenotype [7
]. Thus, these enzymes might serve as therapeutic targets and diagnostic markers in different subtypes of breast cancer.
CD36 is a transmembrane protein and mediates fatty acid uptake. Recent studies indicate that high CD36 expression is detected in breast cancer and CD36 function is important for cell growth in breast cancer cells and metastasis in metastasis-initiating breast cancer cells [10
]. Therefore, the fatty acid transport pathway is important for breast cancer progression. Except for CD36, ACSL, and FABP, solute carrier family 27 (SLC27) is also involved in the process of long-chain fatty acid uptake. SLC27, also named fatty acid transport proteins (FATP) or very long-chain acyl-CoA synthetases (ACSVL), is a family of six members (SLC27A1 through SLC27A6) for uptake of long-chain fatty acids [12
]. Each protein has different specific preferred substrates and tissue distribution [13
]. In addition, SLC27 family proteins, especially SLC27A1 (FATP1) and SLC27A4 (FATP4), display acyl-CoA synthetase (ACS) activity, which links to fatty acid synthesis, β-oxidation, and phospholipid synthesis [12
]. This suggests that SLC27 family proteins are involved in regulation of fatty acid uptake and down-streaming of lipid metabolic processes.
Currently, the role of SLC27 family proteins is not fully understood in breast cancer although the role of ACSL, FABP, and CD36 has been investigated. The aim of this study is to investigate whether SLC27 family proteins are associated with progression of breast cancer, including cell growth, migration, invasion, and potential regulatory mechanism in breast cancer cells.
The bioinformatic analysis revealed that high SLC27A4 was associated with breast cancer tissue and poor prognosis in breast cancer patients. In addition, our results suggest that silencing SLC27A4 expression inhibited cell growth, migration and invasion capacity in breast cancer cell lines. Bioinformatic analyses of SLC27A4-interacting network are linked to several types of metabolic pathways and regulation of cell size. Phospholipids are essential components for all membranes. During growth and cell cycle progression, the regulation of DNA synthesis and phospholipids synthesis/turnover must be integrated. The intracellular fatty acids pool is contributed from de novo fatty acid synthesis and extracellular fatty acid transport in breast cancer cells [21
]. Therefore, we supposed that SLC27A4-silencing should affect cell growth and cell cycle in both cell lines. The present results showed that cell growth was inhibited after SLC27A4-silencing. G2/M cell cycle arrest was observed in in shSLC27A4#02 Hs578T and increased protein expression of cyclin A2 and cyclin B1 was detected. Cyclin A2 and cyclin B1 activated cyclin-dependent kinase 1 (CDK1), which regulates mitotic entry and progression [22
]. Cyclin A2 regulates nuclear-envelope breakdown and then the cyclin B1-CDK1 complex is activated [23
]. A previous study demonstrated that inhibition of fatty acid synthase activity arrested the cancer cells at G2/M [24
]. When cancer cells are treated with the inhibitor of fatty acid synthase, the increased protein expression of cyclin B1 was observed. The cyclin A2 expression was significantly affected by the inhibitor [24
Fatty acid synthase and acetyl-CoA carboxylase 1 are critical enzymes involved in de novo fatty acid synthesis [25
]. It suggests that interference of intracellular fatty acids pool arrests cells at late G2/mitosis before anaphase/telophase. The degradation pathways of cyclin A2 and cyclin B1 might be attenuated by SLC27A4-silencing, and subsequently contribute to G2/M arrest. Perhaps CDK1 is involved in this cell cycle regulation in Hs578T. Interestingly, the cell cycle of SLC27A4-silencing MDA-MB-231 was not significantly changed. Knockdown of acetyl-CoA carboxylase 1 or fatty acid synthase disrupts fatty acids synthesis, acetyl-CoA and CoA production, and then induces apoptosis in breast cancer cells [26
]. Therefore, SLC27A4-silencing might also lead to slight cell death but not affect cell cycle progression. It will be further investigated in the future.
The cell cycle results were inconsistent between MDA-MB-231 and Hs578T. Hs578T is a cell line derived from primary tumor and its pathology is distinguishing infiltrating ductal carcinoma [28
]. By contrast, MDA-MB-231 is derived from pleural effusion and its pathology is distinguishing adenocarcinoma [28
]. Both cells have mutant p53, and wild type BRCA1. Hs578T has a HRAS
mutation, and MDA-MB-231 has B-Raf Proto-Oncogene
), Cyclin Dependent Kinase Inhibitor 2A
), and KRAS
]. In mammary cells, p53 is a key regulator of cell cycle [30
]. Because Hs578T has a mutant p53, p53 should not play a role in SLC27A4-mediated cell cycle regulation in Hs578T. The other tumor suppressor, such as wild type p16, might be important for regulating cell cycle in SLC27A4-silencing Hs578T.
In this study, the capacity of migration and invasion was suppressed after silencing SLC27A4 (Figure 5
). Generally, mesenchymal phenotype is usually associated with tumor migration, invasion, and poor clinical outcomes [32
]. A previous study decreasing FASN and FABP1 cause inhibition of EMT in breast cancer cells [16
]. The cell morphology of SLC27A4-silencing Hs578T and MDA-MB-231 was not significantly affected when compared to control groups. The Western blot analyses showed that the expression of transcription factors that promoted EMT was suppressed in SLC27A4-silencing cells. Moreover, bioinformatic analyses suggest that SLC27A4 is involved in regulation of cell size in breast cancer cells. Transforming growth factor-β (TGF-β) induced EMT and increased cell size through mammalian target of rapamycin (mTOR) signaling pathways [33
]. This data might suggest interaction between SLC27A4-mediated cell size regulation and TGF-β signaling pathways. BRAF is involved in the processes of EMT, stemness or metastasis in breast [34
]; thus, different BRAF status in two breast cancer cell lines might affect the EMT signaling pathways after SLC27A4 silencing.
Palmitic acid (a common saturated fatty acid, C16:0) or a high-fat diet enhances the metastatic potential of CD36+
metastasis-initiating breast cancer cells [11
]. Interestingly, a previous study indicated that CD36 enhances fatty acid uptake but does not transport fatty acid across the plasma membrane in a mammalian cell line [35
]. It implies that uptake of palmitic acid or other types of fatty acid is not fully dependent on CD36 in breast cancer. The other families of transporters are necessary for utilization of fatty acid outside cells. ACSL4 has a fatty acid transporter activity and its preferred substrate is arachidonic acid (a unsaturated fatty acid, C20:4) [36
]. Silencing ACSL4 in breast cancer cells affects the components of cell membranes, especially arachidonic acid [37
]. Arachidonic acid is also known as a fatty acid that links to cancer metastasis [38
]; on the other hand, palmitic acid and lignoceric acid (C24:0) are known substrates of SLC27A4 [13
]. In Figure 2
j–k, low capacity of fatty acid uptake was detected in SLC27A4-silencing Hs578T and MDA-MB-231. We speculate that knockdown of SLC27A4 might alter uptake of specific fatty acids and then change the composition of intracellular fatty acids pool. When compared to the substrates among SLC27A4, SLC27A1 and SLC27A6, oleic acid (C18:1) is a preferred substrate of SLC27A1 and SLC27A6, but not SLC27A4 [13
]. Oleic acid has revealed antitumor effects in several types of cancers [39
]. Perhaps this is why SLC27A4 expression is opposite to other SLC27 family proteins expression. Because Hs578T and MDA-MB-231 were maintained at media with normal fetal bovine serum that contained various types of fatty acids in the present study, we could not evaluate the effect of each suspicious fatty acid in SLC27A4-silencing cells. Culture medium fatty-acid withdrawal via Bio-Beads methods might be a strategy to investigate whether fatty acids are important factors to regulate cell growth, migration, and invasion. These issues will be further investigated in the future.
Currently, the interaction of SLC27A4 is not well-known in cancer cells. In lung cancer cell lines, SLC27A4 directly interacts with autophagy-related 4B cysteine peptidase (ATG4B) [40
]. There are no related studies indicating the interacting networks of SLC27A4 in breast cancer. Because modulation of a metabolic enzyme expression might affect the entire metabolic flux, investigating the interacting networks is essential for further studies. Thus, the TCSBN database was used for predicting the possible interacting networks and DAVID Bioinformatics Resources was used for understanding the functions of these genes. In high SLC27A4-expressing breast cancer tissues and low SLC27A4-expressing normal breast tissues, different biological processes were observed. In addition, our analysis revealed six SLC27A4-correlated genes including CANT1, GDI1, LLGL2, LRSAM1, SLC26A11, and TCBD were also associated poor prognosis in clinical samples. An emerging study demonstrates that LLGL2 involves in Hippo-YAP pathway which regulates bone metastasis in breast cancer [41
]. The function of other genes has not been investigated in breast cancer. It is worth investigating the interactions among SLC27A4 and these genes in breast tumor cells and tumor environment in future studies. We believe that SLC27A4 is a potential diagnostic marker for breast cancer. Because SLC27A4 is a transmembrane protein, blockage of extracellular SLC27A4 via a SLC27A4 antibody might be a novel therapeutic strategy against breast cancer due to disruption of the SLC27A4/CD36-mediated fatty acids transportation pathway. This issue should be further evaluated in animal tumor models and clinical specimens in the future.
4. Materials and Methods
4.1. Cell Culture
Human mammary cancer cell lines MDA-MB-231 (HTB-26™), Hs578T (HTB-126™), T47D (HTB-133™), and MCF-7 (HTB-22™) were purchased from American Type Culture Collection (Manassas, VA, USA). MCF-7, Hs578T, and T47D was respectively cultured in Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle Medium (DMEM), and RPMI1640 supplied with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA), 100 units/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B in 5% CO2 air atmosphere at 37 °C. In addition, MDA-MB-231 was cultured in Leibovitz’s L-15 Medium with 10% FBS, 100 units/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B in a CO2-free air atmosphere at 37 °C. All culture media and supplements were purchased from Lonza (Walkersville, MD, USA).
4.2. Bioinformatic Analysis
The expression in SLC27 gene family (SLC27A1-6) in breast cancer samples and nontumor breast samples across available datasets was evaluated by Oncomine Research Edition (http://www.oncomine.org
, v4.5; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The expression of SLC27A4 was further evaluated in the TCGA breast dataset in Oncomine Research Edition, either. Moreover, the expression of SLC27A4 in different subtypes and races of breast cancer samples was evaluated by the UALCAN (http://ualcan.path.uab.edu
]. The association between gene expression and overall survival rate of breast cancer patients was obtained from the Human Protein Atlas (https://www.proteinatlas.org
]. In addition, the images of SLC27A4 protein expression in normal breast and breast cancer tissues, and results staining intensity of SLC27A4 were obtained from the Human Protein Atlas database (Antibody: HPA007293). The high and low expression groups were separated by “best separation” on the website of the Human Protein Atlas. Distant metastasis-free survival (DMFS) was evaluated by Kaplan–Meier (KM) plotter (http://kmplot.com
] and high- and low-expression groups were divided according to the “auto select best cutoff” in the website. The interacting networks were determined according to the TCSBN database (http://inetmodels.com
]. “Maximum number of nodes” was set at 25 and “Edge Pruning Parameter (-log10 P)” was set at 3 in the TCSBN database. The interacting networks were drawn by Cytoscape version 3.6.1 [46
]. Functional annotation (biological process) was determined by DAVID Bioinformatics Resources (https://david.ncifcrf.gov
4.3. Western Blot Assay
To collect protein, cells were cultured in a 6-cm dish for 48 h and then were lysed in radioimmunoprecipitation lysis buffer (Millipore, Billerica, MA, USA) with protease inhibitor cocktail (Millipore) at a 1000:1 ratio. Protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Billerica, MA, USA), then separated on 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene difluoride (PVDF) membranes (Millipore). After 1 h blocking with 5% dried skimmed milk in tris-buffered saline with Tween-20 (TBST) buffer, the membrane was hybridized with the primary antibodies including anti-E-cadherin (1:1000, Cat. No. #610182), anti-N-cadherin (1:1000, Cat. No. #610921), anti-Vimentin (1:3000, Cat. No. #550513) were purchased from BD Transduction Laboratories™; anti-GAPDH (1:5000, Cat. No. #MAB374) was purchased from Millipore (USA); anti-α-SMA (1:1000, Cat. No. #A5228) was purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-Slug (1:1000, Cat. No. #9585S), anti-cyclin A2 (1:1000, Cat. No. #4656) and anti-cyclin B1 (1:1000, Cat. No. #4135) were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-SLC27A4 (1:2000, Cat. No. #ab199719) was purchased from Abcam (UK) at 4 °C overnight. After TBST washing three times, the membrane was then hybridized with anti-rabbit IgG or anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, USA). The results were acquired on Alpha Innotech FluorChem FC2 imaging system (ProteinSimple; Bio-Techne, Minneapolis, MN, USA).
4.4. Knockdown of SLC27A4
Lentivirus shRNAs were purchased from RNAi Core Facility (Taipei, Taiwan). The lentivurus-shRNA clones included: Lenti-emptyT (clone ID, TRCN0000089107; a vector control); Lenti-shSLC27A4 #98 (clone ID, TRCN0000043398; targeting sequence: 5′-CTTCACAGATAAACTGTTCTA-3′); Lenti-shSLC27A4 #02 (clone ID, TRCN0000043402; targeting sequence: 5′-CCGGGTCTTCATCAAGACCAT-3′). To silencing the gene expression, the Hs578T and MDA-MB-231 cells lines were complete culture media containing 8 μg/mL polybrene (EMD Millipore, Billerica, MA, USA) in a 6-cm dish at 37 °C for 30 min. Lentiviruses for Hs578T and MDA-MB-231 were added for infection at multiplicity of infection (MOI) = 5 and MOI = 3, respectively. After 24 h of incubation, the culture medium was refreshed with fresh culture media, with 2 μg/mL puromycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), for 48 h. The infected cells were maintained in medium with 2 μg/mL puromycin, and subsequently used in assays.
4.5. Fatty Acid Uptake Assay
The capacity of fatty acid uptake was determined by using the Free Fatty Acid Uptake Assay Kit (Fluorometric) according to the manufacturer’s instructions (cat. no. ab176768; Abcam, Cambridge, UK). Before fatty acid uptake assay, 1 × 104 Hs578T and MDA-MB-231 were seeded on a 96-well plate overnight. The cells were preincubated in serum-free media for 1 h after phosphate-buffered saline washing. Subsequently, cells were incubated in a fluorescent fatty acid mixture for 30 min. The results were evaluated by using a microplate fluorescence reader at 485/528 nm (FL × 800; BioTek Instruments Inc., Winooski, VT, USA). The fluorescence signals from wells containing assay mix without cells were used as the background and fluorescence quantification in vector control groups was set to 100% for relative quantification.
4.6. Real-Time Quantitative PCR
Total RNA was isolated via TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription of cDNA was performed via the PrimeScript RT reagent kit (Clontech Laboratories, Inc., Kusatsu, Japan). The relative PCR levels were determined by Fast SYBR-Green Master Mix (Applied Biosystems, Foster City, CA, USA) with the specific primer targeting human Solute Carrier Family 27 Member 4 (SLC27A4), 5′-TCCTGTGGGCTTTTGGTTGT-3′ and 5′-TGGCACCCAACTCAACACAT-3′, and human Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GAGTCAACGGATTTGGT CGT-3′ and 5′-TTGATTTTGGAGGGATCTCG-3′, on a Real-Time PCR system (StepOnePlus Real-Time PCT system; Applied Biosystems, Foster City, CA, USA). The relative mRNA expression was normalized to the GAPDH expression and calculated using the 2−ΔΔCt
4.7. WST-1 Assay
The cell proliferation of Hs578T and MDA-MB-231 was evaluated by WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) (Clontech, Mountain View, CA, USA). Briefly, 3 × 103 cells were respectively seeded in 96-well plates overnight. The culture media were replaced with 100 μL mixture containing 95 μL of fresh culture media and 5 μL of WST-1 reagent. For 24- or 48-h incubation, the absorbance at 450 nm was determined on a microplate spectrophotometer (PowerWave X340; BioTek, Winooski, VT, USA).
4.8. Colony Formation Assay
To determine the long-term effect, 500 cells were seeded in a six-well plate. Cell culture media were replaced each 3 days until 14 days after seeding. Colonies were stained with crystal violet (0.4 g/L; Sigma-Aldrich, St. Louis, MO, USA) and the number of colonies was counted.
4.9. Cell Cycle Analysis
Hs578T and MDA-MB-231 cells were harvested at 48 h incubation after subculture. Harvested cells were fixed with 70% ethanol overnight at 4 °C and then were washed by phosphate-buffered saline. Subsequently, cells were incubated with 1 U/mL of DNase-free RNase A and 5 μg/mL of propidium iodide for 30 min at 4 °C in the dark (Sigma-Aldrich, St. Louis, MO, USA). The cell cycle distribution was determined on a flow cytometry (BD Accuri C6 flow cytometer, BD Biosciences San Jose, CA, USA). Amount of G0/G1, S and G2/M phase cells were determined as a percentage of the total number of cells.
4.10. Wound Healing Assay
1.5 × 105
breast cancer cells were seeded into 24-well plates. A scratch was made by a 200 μL pipette tip when cells reached a complete confluent monolayer. After scratching, the suspended debris was removed by phosphate-buffered saline (PBS) washing. Subsequently, the cells were cultured in serum-free culture media (MDA-MB-231) or culture media with 1% FBS (Hs578T) for 24 h. The images were captured via a Leica inverted microscope and quantification was performed by TScratch software (version 1.0. Available at http://www.cse-lab.ethz.ch
4.11. Transwell Migration and Invasion Assay
Before performing the transwell migration assay, 3 × 104 breast cancer cells were seeded into a 24-well insert (Millicell Cell Culture Inserts 24-well Hanging Inserts, 8-μm PET, Millipore, St. Charles, MO, USA) in 300-μL serum-free medium, while 500 μL medium with 10% FBS was placed in the lower chamber. After culturing for 24 h, the transwell membrane on the 24-well insert was fixed with 500 μL 4% formaldehyde solution followed by 1% crystal violet staining. After removal of the cells on the upper surface, four images of each bottom membrane were captured using a Leica inverted microscope at ×100 magnification via Leica Applications Suite version 4.5.0™ (LAS v4.5) software (Leica Microsystems, Wetzlar, Switzerland). Invasion assay was performed by QCM ECMatrix Cell Invasion Assay, 24-well (8 μm), fluorimetric (Millipore, Billerica, MA, USA) according to the manufacturer’s instruction. Briefly, 1.2 × 105 cells in 300 μL serum-free culture media was added in the insert and 500 μL media with 10% FBS was placed in the lower chamber for 48 h. The results were evaluated on a Bio-tek FLX-800 Fluorescence & Luminescence Reader at the excitation (Ex) and emission (Em) wavelengths = 485/528 nm.
All graphs and statistics were made by the GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA). To examine statistical differences among all groups, a one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used. p < 0.05 was considered to indicate a statistically significant difference.