Metastasis is the main cause of cancer-specific death in patients with prostate cancer (PCa). Acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) is involved in the metabolic reprogramming of multiple types of cancer cells, but its role in PCa metastasis remains largely unknown. Here, we determined the effect of overexpression or small interfering RNA-mediated depletion of ACSL3 on the migratory and invasive abilities of human PCa cell lines. We also conducted phospho-protein microarray analysis to identify signaling pathway components affected by ACSL3 modulation. Overexpression of ACSL3 promoted the migration and invasion of PCa cells, whereas ACSL3 downregulation had the opposite effects. Mechanistically, phospho-protein analysis showed that ACSL3 regulated the phosphorylation of AKT and the expression of matrix metalloproteinase9. Our results support a potential role for ACSL3 in promoting the metastatic behavior of PCa, possibly via AKT/matrix metalloproteinase9 pathways. Thus, ACSL3 could be a novel target for the development of treatments for PCa.
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Prostate cancer (PCa) is the most common non-cutaneous malignancy and the third leading cause of cancer mortality among men in developed countries.[1] In China, the incidence of PCa is increasing steadily, which is at least partially due to the rapid changes in dietary habits.[2] Although there have been major advances in the treatment of PCa in the past several decades, the majority of patients eventually experience disease progression, regardless of multidisciplinary treatments.[3,4] Metastasis is the main cause of cancer-specific death in patients with PCa[5]; however, the mechanisms underlying metastasis remains unclear. There is thus a pressing need to understand the pathways contributing to PCa metastasis and to identify innovative novel treatments for PCa.
Recent work suggests that rewiring of pathways controlling energy production, including lipid metabolism, is a critical mechanism by which cancer cells support their aggressive behavior.[6] Such reprogramming is usually caused by aberrant activation of metabolic pathways. Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3) is one of a family of enzymes that convert long-chain free fatty acids into fatty acyl-CoA esters, which are substrates for lipid synthesis and catabolism via β-oxidation.[7] ACSL3 is reportedly involved in the metabolic reprogramming of several types of cancer, including lung cancer.[8] In addition, ACSL3 has been shown to promote castration-resistant prostate cancer (CRPC) via activation of androgen receptor signaling.[9,10] Nevertheless, the role of ACSL3 in PCa metastasis remains largely unknown.
This study aimed to investigate the role of ACSL3 in the progression of PCa by exploring the effects of modulating its expression on the behavior of human PCa cell lines in vitro. We also investigated the underlying mechanisms of action of ACSL3 by analyzing changes in the expression of a large panel of proteins and phospho-proteins involving in intracellular signaling and growth-promoting pathways.
The normal human prostate epithelial cell line RWPE-1 (CRL-11609) and prostate cancer cell lines PC3 (CRL-1435), LNCaP (CRL-1740), and 22Rv1 (CRL-2505) were purchased from the American Type Culture Collection (Manassas, VA) and were maintained as previously described.[11]
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and the concentration and purity were measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). cDNA synthesis was performed using 2 μg total RNA and SYBR Premix Ex Taq II (Takara, Japan). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was carried out in triplicate using SYBR Green I Master Mix (Roche, Mannheim, Germany) on a LightCycler 480 System (Roche). Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified as an endogenous control for normalization of ACSL3 expression. The primers were as follows, ACSL3: forward, 5′-CAATTACAGAAGTGTGGGACT-3′ and reverse, 5′-CACCTTCCTCCCAGTTCTTT-3′. GAPDH: forward, 5′-TCCTCTGACTTCAACAGCGACACC-3′ and reverse, 5′-TCTCTCTTCCTCTTGTGCTCTTGG-3′. The results are presented as the ratio of ACSL3 mRNA to GAPDH mRNA levels.
The ACSL3 expression construct pcDNA3.1(+)-ACSL3 was generated as previously reported[11] and verified by sequencing (BGI Tech, Shenzhen, China). PCa cells were transfected with pcDNA3.1(+)-ACSL3 using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were transfected at the confluency of approximately 70% to 80%. Plasmid DNA and lipofectamine 2000 were premixed and then added to the cells. After incubation for 6 hours, culture media were replaced by complete culture media. For small interfering RNA (siRNA) transfection, cells were transfected with 100 nM siRNA against ACSL3 (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen), complete culture medium was added to the cells at 6 hours post-transfection. All cells were harvested for further experiments at 48 hours post-transfection.
Western blotting was performed as previously reported[12] using primary antibodies against ACSL3 (Abcam, 1:2000), total AKT (1:2000), phospho-AKTSer473 (1:1000), MMP9 (1:2000), and GAPDH (1:3000) (all from Cell Signaling Technology). Secondary antibodies (Cell Signaling Technology) were incubated at 1:5000 for 1 hour Protein bands were visualized using FluorChem M (ProteinSimple, CA). For quantification, protein levels were normalized to that of GAPDH.
PC3 cells were seeded in 6-well plates at 80% confluency and incubated overnight. A wound was created in the cell monolayer by scratching with a 10-μL pipette tip, and the detached cells were removed by 2 washes with phosphate-buffered saline. The plates were then incubated for 24 hours. The wound area was visualized and photographed at 0, 12, and 24 hours incubation using a Zeiss LSM 780 (Carl Zeiss Microscopy GmbH, Jena, Germany). Mock-transfected PC3 cells were included as controls in all experiments.
The invasive ability of PC3 cells was assessed using a Matrigel-coated Transwell invasion assay, as previously described.[13] Cells were plated into upper champers which were coated with Matrigel (BD Bioscience, San Diego, CA), and 600 μL of culture media containing 15% of fetal bovine serum were added into the lower chambers. After 24 hours, invaded cells in the bottom sides of the inserts were stained and then photographed using Zeiss LSM 780 (Carl Zeiss Microscopy GmbH).
Phosphorylated and total proteins were detected using a Phospho Explorer Antibody Array (PEX100; Full Moon Biosystems, Sunnyvale, CA) as previously described.[11] This array consists of 1318 well-characterized antibodies specific for the phosphorylated proteins involved in multiple intracellular signaling and growth-promoting pathways. The paired antibodies for the same, but unphosphospecific sites were also involve in this array to allow measurement of the relative phosphorylation ratio. Each antibody is printed in duplicate.
In brief, proteins were extracted from mock-transfected PC3 cells and cells transfected with an ACSL3 overexpression vector. The proteins were then biotinylated, hybridized to the array chip, and imaged according to the kit instructions using an Axon GenePix 4000B with GenePix software Pro 6. Background signals were decounted, before analysis, and results were compared using the average ratio of the phosphorylated to non-phosphorylated protein from quadruplicate samples.
Data were statistically analyzed using SPSS 22.0 software (SPSS, Chicago, IL) and expressed as the means ± standard deviations (SD). Comparisons between groups were performed using 1-way analysis of variance or two-tailed Student’s t test. A P < .05 was considered statistically significant.
To evaluate ACSL3 mRNA and protein expression, we performed qRT-PCR and western blotting analysis, respectively, in the normal human prostate epithelial cell line RWPE-1 and the PCa cell lines 22Rv1, LNCaP, and PC3. We found that ACSL3 expression at both the mRNA and protein levels was not significantly different between RWPE-1, the non-metastatic 22Rv1, and the androgen-sensitive LNCaP cell lines. However, ACSL3 was expressed at a significantly lower level in the metastatic CRPC cell line PC3 (P < .05, Fig. 1). Therefore, we selected PC3 for further investigation.
To investigate the potential role of ACSL3 in PCa migration and invasion, we transfected PC3 cells with an ACSL3 overexpression vector or with an ACSL3-targeting siRNA and performed wound-healing and Transwell invasion assays. In the wound-healing assays, the migration of PCa cells at 24 hours incubation was markedly increased compared with the mock-transfected negative control (NC) cells (Fig. 2A). Conversely, siRNA-mediated silencing of ACSL3 reduced PC3 mobility to levels comparable to the control cells (Fig. 2A). We observed similar results in the Transwell invasion assays. The invasive behavior of PCa cells was significantly promoted by ACSL3 overexpression and significantly reduced by ACSL3 knockdown (Fig. 2B and C). These results suggest that ACSL3 positively regulates the migratory and invasive behavior of PCa cells.
To explore the potential mechanisms whereby ACSL3 promoted migration and invasion, we performed a microarray analysis of protein phosphorylation in mock-transfected and ACSL3-over-expressing PC3 cells. The microarray was composed of 1318 antibodies specific for non-phosphorylated and phosphorylated forms of proteins associated with numerous signaling and growth pathways, such as AKT, MAPK, AMPK, ERK, p53, FGF, and others (Fig. 3). This analysis showed that ACSL3 markedly affected the phosphorylation of a variety of signaling proteins in the AKT pathway (Fig. 3).
To validate the microarray results, we performed additional western blot analyses on ACSL3-overexpressing or -depleted cells. As shown in Figure 4A, ACSL3 overexpression significantly elevated phosphorylated AKT (Ser473) levels without affecting total AKT levels. Since MMP9 is widely regarded as a biomarker of metastatic capabilities in cancer,[14] we also analyzed the expression of MMP9. Notably, MMP9 protein levels were also upregulated by ACSL3 overexpression. In the contrary, knock-down of ACSL3 reduced phospho-AKT Ser473 and MMP9 levels in PC3 cells but did not affect total AKT levels (Fig. 4B).
Collectively, these results indicate that ACSL3 regulates the migratory and invasive behavior of PCa cells, possibly via effects on AKT phosphorylation and MMP9 expression, suggesting that ACSL3 may contribute to PCa metastasis.
Metastasis is the main cause of death in PCa patients and represents a great challenge to urologists worldwide. In China, about 20% to 30% of newly diagnosed PCa patients already have metastatic disease.[2,15] This is a grave concern, because the overall survival rate of patients with metastatic PCa is only 28% compared with 100% for patients with localized disease.[16,17] Early diagnosis is thus one of the most important factors in improving patient survival. However, we must also find treatments that suppress the progression of metastatic PCa. Although androgen receptor signaling is the pivotal pathway driving PCa progression, the efficacy of current therapies targeting androgen receptor signaling is unsatisfactory.[18] Therefore, there is a need for novel biomarkers and targets to improve the treatment of metastatic PCa.
Metabolic alterations commonly occur in cancer cells to support their rapid proliferation and metastasis. Increasing evidence has identified a role for alterations in fatty acid (FA) metabolism in cancer, prompting a search for new methods to target this pathway as a novel approach to cancer therapy.[19] FAs are essential for biosynthesis of membranes and generation of signaling molecules, and cancer cells can obtain FAs from exogenous sources as well as synthesize them de novo.[19] Indeed, de novo FA synthesis is aberrantly activated in multiple human malignancies, such as prostate, breast, and lung.[20,21] FAs require covalent modification by CoA via fatty acyl-CoA synthetases before entering the bioactive pool. ACSL3, 1 of 5 members of the ACSL family (1, 3, 4, 5, and 6), participated in production of fatty acyl-CoA esters via covalent modification of long-chain free Fas.[7] As such, several studies have revealed a critical role for ACSL3 in multiple cancer types. ACSL3 is overexpressed in lung cancer and promotes the tumorigenesis and survival of lung cancer cells.[8] Moreover, in vivo experiments demonstrated that silencing of ACSL3 impairs the oncogenic effect of human lung carcinoma cells.[8] ACSL3 is also an androgen-responsive gene and is highly expressed in normal prostate tissues and PCa.[22,23] Previous studies proposed that ACSL3 may contribute to the growth of CRPC through intratumoral steroidogenesis via modulation of steroidogenic genes (ie, AKR1C3 and UGT2B).[10,24] In terms of therapeutic implications, several findings have suggested that ACSL3 could be an excellent target for the development of novel treatments.[8] First, the catalytic activity of ACSL3 is required to exert its oncogenic effect, and it is unlikely that cells can compensate for its inhibition because ACSL3 is distal to FA synthase. Second, even partial suppression of ACSL3 enzymatic activity sufficiently achieved critical therapeutic effect without generalized toxicity. Finally, the ACSL inhibitor triacsin C induces apoptosis in several types of cancer cells both in vitro and in vivo.[25]
The AKT pathway has diverse functions in cell growth, differentiation, proliferation, metabolism, metastasis, and drug resistance.[26] AKT is activated by phosphorylation on Ser473. MMP9 is regarded as a biomarker of metastatic capabilities in cancer.[14] Here we explored the underlying mechanisms potentially involved in the ACSL3-mediated promotion of behaviors associated with metastasis. We found that AKT phosphorylation and MMP9 expression were both upregulated by ACSL3 overexpression, suggesting a possible scenario whereby AKT activates MMP9 to increase the metastatic ability of PCa. Since ACSL3 does not have kinase activity, future studies will investigate which molecules link ACSL3 upregulation to AKT phosphorylation and MMP9 upregulation. Of note, ACSL3 has been shown to induce the expression of signaling lipoproteins and membrane synthesis.[27]
In summary, our study shows that ACSL3 overexpression promotes the migration and invasion of PCa cells, suggesting that ACSL3 upregulation in cancer cells may contribute to metastasis. Furthermore, we identified AKT phosphorylation and MMP9 expression as downstream effects of ACSL3 that may support metastasis. Our results thus provide evidence that ACSL3 could be a novel target for the development of treatments for PCa.
None.
KL participated in the study design and writing of the paper. YHM participated in the literature research and writing of the paper. WHQ, JWH, and DJW performed the study. CH and WTH contributed to the acquisition of data. JST contributed to the analysis and interpretation of data. JGQ was actively involved in the study design, manuscript drafting, and revision.
This work was supported by the Pearl River S&T Nova Program of Guangzhou, China (no. 201710010039), the Natural Science Foundation of Guangdong Province, China (no. 2015A030313031 and 2017A030313898), the Science and Technology Program of Guangdong Province, China (no. 2017A020215028), the Science and Technology Program of Guangzhou (no. 201707010113), and the Basic Service Charge Young Teachers Cultivation Project of Sun Yat-sen University, China (no. 17ykpy48).
The authors declare no conflicts of interest.
