Alteration in lipid composition is an important metabolic adaptation by cancer cells to support tumorigenesis and metastasis. Fatty acid 2-hydroxylase (FA2H) introduces a chiral hydroxyl group at the second carbon of fatty acid (FA) backbones and influences lipid structures and metabolic signaling. However, the underlying mechanisms through which FA 2-hydroxylation is coupled to metabolic adaptation and tumor growth remain elusive. Here, we show that FA2H regulates specific metabolic reprogramming and oncogenic signaling in the development of colorectal cancer. FA2H is highly expressed in normal colorectal tissues. Assessments through deciphering both published high-throughput data and curated human colorectal cancer samples revealed significant suppression of FA2H in tumors, which is correlated with unfavorable prognosis. Experiments with multiple models of genetic manipulation or treatment with an enzymatic product of FA2H, (R)-2-hydroxy palmitic acid, demonstrated that FA 2-hydroxylation inhibits colorectal cancer cell proliferation, migration, epithelial-to-mesenchymal transition progression, and tumor growth. Bioinformatics analysis suggested that FA2H functions through AMP-activated protein kinase/Yes-associated protein (AMPK/YAP) pathway, which was confirmed in colorectal cancer cells, as well as in tumors. Lipidomics analysis revealed an accumulation of polyunsaturated fatty acids in cells with FA2H overexpression, which may contribute to the observed nutrient deficiency and AMPK activation. Collectively, these data demonstrate that FA 2-hydroxylation initiates a metabolic signaling cascade to suppress colorectal tumor growth and metastasis via the YAP transcriptional axis and provides a strategy to improve colorectal cancer treatment.
These findings identify a novel metabolic mechanism regulating the tumor suppressor function of FA 2-hydroxylation in colorectal cancer.
Colorectal cancer is currently the third most commonly diagnosed cancer and ranks second in cancer mortality, with the incidence in young patients continuing to rise (1). Despite improvements in diagnosis and treatment for colorectal cancer over the last decade, approximately one-third of patients develop metastasis with a poor prognosis (2). Immunotherapy drugs are effective in less than half of the patients with metastatic colorectal cancer with mismatch repair deficiency/microsatellite instability high, which represent only 10%–20% of all colorectal cancers (3). Defining the molecular mechanisms underlying progression and metastasis in colorectal cancer will help identify novel biomarkers and provide efficient therapeutic strategies to improve colorectal cancer treatment in combination with available cancer care.
Alterations in lipid metabolism is an important metabolic adaptation by cancer cells to support proliferation, growth, and dissemination (4). The rapidly proliferating cancer cells require a constant supply of fatty acids (FA) for membrane biogenesis, energy production, and protein modification (5). Moreover, changes in FA composition and remodeling of membrane lipids may also be involved in tumorigenic signaling (6, 7). Hydroxy fatty acids (OHFA) are present in various mammalian tissues, which can be provided by food intake and sources of microorganisms (8, 9), or can be endogenously generated (7, 10). Fatty acid 2-hydroxylase (FA2H) specifically introduces a chiral hydroxyl group at the second carbon of long chain FAs, leading to the generation of (R)-2-OHFAs and sphingolipids containing (R)-2-OHFAs (11). In cultured cells, FA2H facilitates cAMP-induced cell-cycle exit (12), and (R)-2-OH-ceramide is more potent in inducing apoptosis than its stereoisomer or non-OH-ceramide (13). The in vivo antitumor activities of FA2H and (R)-2-OHFAs were demonstrated in gastric cancer (14). On the other hand, high expression of FA2H was observed in chemical-induced hepatocellular carcinoma in rats (15). Moreover, FA2H levels in lung adenocarcinomas were higher than in tumor-free tissues, while being suppressed in squamous and neuroendocrine lung carcinomas (16). These results suggest the presence of previously unexplored cancer type–dependent mechanisms through which FA 2-hydroxylation is coupled to metabolic adaptation and tumor growth.
The Hippo signaling pathway modulates key target genes to regulate a multitude of biological processes, including cellular proliferation, differentiation, fate determination, and regeneration (17). In mammalian systems, the central components of this pathway comprise serine/threonine kinases mammalian sterile 20-like protein kinase 1/2 (MST1/2) and the large tumor suppressor 1/2 (LATS1/2), which phosphorylate their downstream transcription coactivators, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Phosphorylation of YAP/TAZ by MST1/2 recruits 14-3-3 proteins and stimulates nucleus exclusion or proteolytic degradation. This process prevents YAP/TAZ from binding to TEA DNA-binding proteins (TEAD1–4) and other transcription factors, which enables the transcription of proliferative and prosurvival genes. Accumulating evidence has suggested that the perturbation of the Hippo pathway contributes to cancer development (18). YAP has a greater effect on cellular physiology, including proliferation and migration, than TAZ (19), and aberrant YAP expression or activation is involved in tumor cell proliferation and metastasis in several types of cancer, including colorectal cancer (20). YAP is also regulated by signaling pathways independent of the Hippo/LATS cascade, including ERK, JNK, and p38 MAPKs (21, 22), as well as mechanical cues (23). Furthermore, YAP activity is inhibited by cellular energy depletion through several mechanisms involving both LATS and AMP-activated protein kinase (AMPK; refs. 24, 25).
Our previous study demonstrated that FA2H contributes to metabolic regulation and energy sensing (14). FA2H is highly expressed in colorectal tissues (26), but its potential regulation of colorectal cancer development remains unknown. Bioinformatics analysis in this study suggested that FA2H may function through AMPK and the YAP transcriptional axis. Our findings identified FA2H as a novel metabolic regulator of colorectal cancer tumor growth and metastasis.
Materials and Methods
Collection of human colorectal cancer tissues
Eighteen pairs of primary colorectal cancer tissues and noncancerous mucosal tissues (for Western blotting) and 63 cases of colorectal cancer tissues (for IHC) were obtained from patients who underwent curative surgery from 2016 to 2018 at the Department of General Surgery, the First Affiliated Hospital of Soochow University (Suzhou, Jiangsu, P.R. China). Another 141 formalin-fixed, paraffin-embedded tissues of colorectal cancer and 41 adjacent normal tissues were collected immediately after surgical resection from 2009 to 2014 at the Second Affiliated Hospital of Soochow University (Suzhou, Jiangsu, P.R. China). The clinicopathologic information is summarized in Supplementary Table S1. The study was conducted in accordance with the ethical principles stated in the Declaration of Helsinki and approved by the Biomedical Research Ethics Committee of Soochow University (Suzhou, Jiangsu, P.R. China). All of the patients provided written informed consent.
Cell cultures and treatments
Colorectal cancer cell lines (HCT8, RRID: CVCL_2478; HCT116, RRID: CVCL_0291; LOVO, RRID: CVCL_0399; RKO, RRID: CVCL_0504; SW480, RRID: CVCL_0546; and SW620, RRID: CVCL_0547) were purchased from the Cell Bank of the Chinese Academy of Sciences, which performs routine cell line authentication testing with SNP and short tandem repeat analyses. Cells were cultured in RPMI1640 Medium (HyClone) or DMEM (HyClone) containing 10% FBS (Gibco), 100 U/mL penicillin G sodium, and 100 μg/mL Streptomycin Sulfate (Gibco) at 37°C in a humidified atmosphere containing 5% CO2. Cell lines stably expressing FA2H-specific short hairpin RNA (shRNA) with the target sequence of 5′-GACAGAUCCUGCUAUGGAACC-3′ or a plasmid encoding human FA2H were generated using a Lentivirus Technique (GeneChem) according to the manufacturer's protocols. Some cells were treated with 2-deoxyglucose (Sigma), docosahexaenoic acid (Sigma), D942 (BioVision), or aminoimidazole carboxamide ribonucleotide (AICAR, Beyotime) for the indicated time before analyses. All cell lines were passaged for less than 40 times and tested negative using GMyc-PCR Mycoplasma Test Kit (Yeasen).
Protein extraction and Western blot analysis
Whole-protein extracts were prepared with ice-cold RIPA lysis buffer supplemented with cocktails of Protease and Phosphatase Inhibitors (Sigma) as described previously (14). Total proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were blocked with 5% nonfat milk and incubated sequentially with the indicated primary antibodies and horseradish peroxidase–conjugated secondary antibodies. The proteins were visualized by chemiluminescence and signals were quantified by ImageJ software (version: 1.4.3, RRID: SCR_003070) as described previously (14). Antibodies used in this study are listed in Supplementary Table S2. The phos-tag gels containing 20–50 μmol/L phosbind acrylamide (APExBIO) and MnCl2 were prepared according to the manufacturer's instructions.
IHC and immunofluorescence staining
The paraffin-embedded tissues were cut into 5 μm sections and were incubated with the indicated antibodies. A Tissue Staining Kit (Zhongshan Biotechnology) was used to visualize the protein and the final staining score was determined by the color intensity and positive cell rate as described previously (14). Cells seeded on coverslips were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. After blocking in 2% goat serum for 1 hour, cells were incubated with indicated primary antibodies followed by Alexa Fluor 488 secondary antibodies as described previously (27). Slides were then washed three times and stained with DAPI (Invitrogen). The images were taken with a Nikon ECLIPSE Ni microscope with a color camera and were processed by ImageJ software.
RNA extraction and qRT-PCR
Total RNAs were isolated using TRizol Reagent (Thermo Fisher Scientific) and reverse transcribed as described previously (14). qRT-PCR was carried out using gene-specific primers for FA2H, CTGF, and CYR61 with SYBR Green RT-PCR Kits (TaKaRa) and ran on a 7500 Real-Time PCR System (Thermo Fisher Scientific). The human 18s was used as a loading control. Primers used in this study are listed in Supplementary Table S3.
Transfection of siRNA
The siRNAs against human FA2H (specific target sequence: 5′-GCUAUUACCUCAUCAUGCUTT-3′), human AMPK (specific target sequence: 5′-GCAGAAGUAUGUAGAGCAAUC-3′), and human YAP (specific target sequence: 5′-GGUCAGAGAUACUUCUUAAAU-3′) were synthesized by IBSBIO. A scrambled siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) was used as a negative control. Cell lines were transfected with siRNA at a final concentration of 20 nmol/L using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's protocols. Experiments were performed at 48–72 hours posttransfection.
Cell viability assay
Cell proliferation property was determined using an MTT Assay Kit (Amresco) or a Cell Counting Kit-8 (CCK-8) Assay (APExBIO) according to the protocols provided by the manufacturers in 96-well plates with 2,000 cells per well.
Cell migration assay
Cell migration ability was evaluated using 24-well Transwell Chambers (pore size: 8 μm; Corning Inc.). A total of 5 × 104 cells were resuspended in 200 μL serum-free medium and seeded into the top chambers, and 750 μL medium containing 10% FBS was placed in the bottom chambers. Twenty-four hours later, the cells remaining on the top surface of the membrane were removed using a cotton swab. The filters were then fixed with 4% Paraformaldehyde (Beyotime) at room temperature for 10 minutes, and then stained with 0.1% Crystal Violet (Beyotime) for 15 minutes at room temperature. The cells that had migrated from the top to the bottom side of the filter were imaged and counted in five randomly selected fields per sample using a Light Microscope (magnification, 200×; Nikon Corporation).
Relative ATP levels were measured using an enhanced ATP Assay Kit (Beyotime) according to the protocols provided by the manufacturer. Total ATP levels were calculated from the luminescence signals and normalized by the protein concentrations.
BALB/c nude mice (SPF grade, 16–18 g, 3–5 weeks old, male) were purchased from Shanghai SLRC Laboratory Animal Co., Ltd. and housed in a pathogen-free room with a 12-hour light/dark cycle. A total of 2 × 106 cells were inoculated into the left dorsal flank of nude mice by subcutaneous injection. The growth of the implanted tumors was monitored as described previously (14). All animal experimental procedures were approved by the Animal Ethics Committee of Soochow University (Suzhou, Jiangsu, P.R. China).
RNA sequencing experiment
The RNA sequencing (RNA-seq) experiments were performed by Genewiz Co., Ltd. Total RNA was extracted from cells using an RNeasy Kit (Qiagen) according to the manufacturer's instructed protocol. Libraries were constructed by using the VAHTSTM mRNA-Seq V2 Library Prep Kit for Illumina (Vazyme Biotech) following Illumina's recommendations. Subsequently, libraries for all samples were sequenced on the high-throughput sequencing platform (Illumina HiSeq X Ten platform) with the 150-bp paired-end strategy. Raw sequence data for all six samples in this study were deposited into Genome Sequence Archive database in BIG Data Center (http://bigd.big.ac.cn/) under the accession number CRA002391 (BioProject PRJCA002193).
Gene expression analysis, gene ontology (GO, RRID:SCR_002811) biological process (BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway (RRID: SCR_012773) enrichment analyses, and gene set enrichment analysis (GSEA, RRID:SCR_003199) were performed as described in the online Supplementary Materials and Methods.
LC/MS-MS–based lipidomics analysis
Lipid extraction and lipidomics analysis were performed as described in the online Supplementary Materials and Methods. Differential lipid selection was based on the statistics from Welch two sample t test with a subsequent P < 0.05 and a fold change > 2.
All experiments were repeated for at least three times and data are presented as mean ± SEM. Student t test (paired or unpaired, two-tailed) was used to compare means between two groups. Pearson χ2 test or Fisher exact test was used to analyze the relationships between FA2H expression and clinicopathologic factors. Kaplan–Meier method with the log-rank test was used to investigate the prognostic value of FA2H for patients with colorectal cancer. Spearman rank correlation analysis was used to calculate the correlations between protein expression levels. P < 0.05 was considered to indicate a statistically significant difference.
Regulation and prognostic value of FA2H expression in colorectal cancer
We compared FA2H mRNA expression between clinical colorectal tumor specimens and noncancerous tissues on the basis of published microarray datasets (GSE18105, GSE21510, and GSE44076), and found that the FA2H mRNA level was significantly lower in colorectal cancer tumors across different datasets (Fig. 1A; P < 0.001). A significantly reduced FA2H protein level in cancerous tissues was confirmed by Western blot (Fig. 1B) and IHC (Fig. 1C and D; P < 0.05) analyses. Furthermore, the colorectal cancer samples with lymph node metastasis (LNM) had a lower FA2H protein level than those specimens without LNM (Fig. 1C and D; P < 0.001).
We next evaluated the relationship between FA2H levels and the clinicopathologic status of patients with colorectal cancer. As shown in Supplementary Table S1, lower FA2H levels in colorectal cancer tissues were observed in subgroups with larger tumor size (P = 0.005), deeper tumor invasion (P = 0.014), LNM (P < 0.001), and later tumor–node–metastasis stage (P < 0.001), but with no significant differences among subgroups stratified according to age, gender, or tumor location (P > 0.05). The Kaplan–Meier curves revealed that patients with colorectal cancer with lower FA2H protein level appeared to have a lower probability of overall survival as compared with those with higher FA2H expression (Fig. 1E; P = 0.0042), which was further validated by published microarray dataset GSE39582 (Fig. 1F; P = 0.033). Moreover, its impact on metastasis-free survival was evident in public dataset GSE28722 (Fig. 1G; P = 0.038). Collectively, both retrospective and public data indicate that reduced FA2H expression is correlated with unfavorable prognosis in patients with colorectal cancer and may contribute to colorectal cancer development and metastasis.
FA2H suppresses tumor growth and metastasis in colorectal cancer
To select appropriate colorectal cancer cell lines for functional studies, FA2H levels in six human colorectal cancer cell lines were evaluated. FA2H protein levels in SW480, LOVO, and RKO cells were generally lower than in HCT8, HCT116, and SW620 cells, which could be partially explained by their different mRNA levels (Supplementary Fig. S1A). Accordingly, SW480 and LOVO cells with FA2H overexpression, and HCT8 and SW620 cells with FA2H stably depleted by shRNA were established (Supplementary Fig. S1B and S1C). Moreover, FA2H expression in HCT116 cells was efficiently knocked down by siRNA (Supplementary Fig. S2A). Cell viability assays showed that FA2H overexpression attenuated (Fig. 2A; P < 0.001), whereas its depletion significantly enhanced cellular proliferation (Fig. 2B; P < 0.001; Supplementary Fig. S2B; P < 0.01). Moreover, the proliferation ability of LOVO cells was markedly suppressed by a FA2H product, (R)-2-hydroxy palmitic acid [(R)-2-OHPA], at 50 μmol/L (Fig. 2C; P < 0.001) with no effects observed by nonhydroxylated palmitic acid (PA) at the same concentration (Fig. 2C). Importantly, the enhanced proliferation by FA2H knockdown was reversed by supplementation with (R)-2-OHPA (Fig. 2B; P < 0.001).
We next assessed the role of FA2H in colorectal cancer tumorigenesis in vivo with subcutaneous xenograft models. SW480 cells with or without FA2H overexpression were transplanted into nude mice (7/group) and the tumor growth was monitored (Fig. 2D). Compared with the control group, tumor growth from cells overexpressing FA2H was substantially inhibited as judged by the tumor weight (Fig. 2E; P < 0.05) and size (Fig. 2F; P = 0.06, not significant, but marginal). Conversely, tumor growth from SW620 cells with stable FA2H knockdown was promoted as compared with wild-type control (5/group; Fig. 2G and H; P < 0.01).
The transwell migration assay showed that FA2H overexpression potently suppressed cell migration (Fig. 2I; P < 0.01), while its depletion approximately doubled the migration rate (Fig. 2J; P < 0.01; Supplementary Fig. S2C; P < 0.01). Moreover, exposure to (R)-2-OHPA significantly attenuated cell migration as compared with treatments with the BSA, while PA had minimal effect (Fig. 2K; P < 0.01). Importantly, the increased migration ability induced by FA2H knockdown was reversed by treatment with (R)-2-OHPA (Fig. 2J; P < 0.01). Collectively, these results indicate that FA2H overexpression or treatment with (R)-2-OHPA inhibits tumorigenesis and metastasis in colorectal cancer.
FA2H alleviates the YAP transcriptional axis in colorectal cancer cells
Our previous study evidenced that FA2H regulation of cell growth and sensitivity to cisplatin in gastric cancer involved transcriptional activation of Gli1 (14). However, neither overexpression nor depletion of FA2H affected the Gli1 protein level in colorectal cancer cells (Supplementary Fig. S3A and S3B), suggesting that FA2H regulates specific oncogenic transcriptional axis in a cancer type–dependent manner. To explore the downstream transcriptional axis regulated by FA2H in colorectal cancer, RNA-seq assay was performed to globally profile the transcriptomes of LOVO cells in response to FA2H overexpression. GO and KEGG pathway enrichment analyses showed that the upregulation of FA2H downregulated cell-cycle progression and adhesion, and upregulated the biological processes and pathways contributing to cancer suppression, including the TNF signaling pathway, cell-cycle arrest, and negative regulation of cell proliferation (Fig. 3A). Further integrative bioinformatics analyses on the basis of published microarray data revealed that FA2H overexpression could induce a transcriptome change similar to that observed in YAP knockdown (GSE92335) and phosphomimic mutation (GSE41509) in human colorectal cancer cell lines (Fig. 3B and C; P < 0.05), implicating a FA2H regulation of YAP activity in colorectal cancer cells.
To further verify these findings from the in silico analysis, we asked whether FA2H could regulate YAP phosphorylation, which promotes its cytoplasmic sequestering by 14-3-3 binding, thereby preventing its translocation into the nucleus (24, 25). FA2H overexpression markedly enhanced YAP phosphorylation as evidenced by immunoblotting with a phospho-YAP antibody (Fig. 3D; P < 0.05) and slower migration of YAP on phospho-tag–containing gels (Fig. 3E), while its depletion had the opposite effects (Fig. 3F; P < 0.05). Consequently, immunofluorescence staining (Fig. 3G) and fractionation assays (Fig. 3H; P < 0.05) showed that FA2H overexpression enhanced cytoplasmic sequestering of YAP, while decreasing its nuclear localization. Moreover, a lower level of nuclear YAP in xenografted tumors derived from SW480 cells with FA2H overexpression was observed (Fig. 3I), suggesting the involvement of YAP activity in tumor growth regulated by FA2H. This was supported by FA2H regulation of YAP target genes CTGF and CYR61, which encode proteins to promote tumor cell growth and metastasis (24, 25). FA2H overexpression induced a significant decrease in CTGF and CYR61 mRNA levels (Fig. 3J; P < 0.001), while its depletion markedly increased the transcription of CTGF (Fig. 3K; P < 0.001) and CYR61 (Fig. 3K; P < 0.01). Together, these findings indicate that FA2H promotes YAP phosphorylation and cytoplasmic retention, leading to suppression of YAP target genes and colorectal cancer cell proliferation and migration.
We next examined whether (R)-2-OHPA regulates YAP activity. Treatment of SW480 and LOVO cells with 50 μmol/L (R)-2-OHPA for 24 hours led to enhanced phosphorylation of YAP (Fig. 4A; P < 0.05), with limited effects observed by PA or its enantiomer (S)-2-OHPA at the same concentration. Analysis with phospho-tag gels revealed slower migration of YAP protein after treatment with (R)-2-OHPA, indicating increased phosphorylation (Fig. 4B). Immunofluorescence staining (Fig. 4C) and fractionation assays (Fig. 4D; P < 0.01) showed that the treatment of LOVO cells with (R)-2-OHPA promoted cytoplasmic sequestering of YAP. Moreover, (R)-2-OHPA induced a significant decrease of CTGF (P < 0.01) and CYR61 mRNA (P < 0.01) levels (Fig. 4E). These results indicate that FA2H may function through (R)-2-OHFAs, which could be used as lipid surrogates to promote YAP phosphorylation and suppress its transcriptional activity in colorectal cancer cells.
FA2H regulates colorectal cancer cell proliferation and migration in a YAP-dependent manner
Epithelial-to-mesenchymal transition (EMT) is an early event in the cancer metastasis accompanied by the upregulation of mesenchymal-associated proteins, such as vimentin, and downregulation of epithelial-associated markers, such as E-cadherin (28). FA2H overexpression significantly enhanced the expression of E-cadherin (Fig. 5A; P < 0.05), while decreased vimentin level (Fig. 5A; P < 0.05). Conversely, silencing of FA2H had the opposite effects on their protein levels (Fig. 5B; P < 0.01), suggesting an inhibitory regulation of the EMT process by FA2H. Moreover, FA2H depletion was not able to regulate E-cadherin, vimentin (Fig. 5C), cell proliferation (Fig. 5D), or cell migration in the absence of YAP (Fig. 5E). Collectively, these results indicate that FA2H suppresses cell proliferation, migration, and EMT through a YAP-dependent pathway.
FA2H regulates YAP transcriptional axis in colorectal cancer cells via AMPK pathway
To understand the underlying regulatory mechanism by which FA2H modulates YAP activity, GSEA for a published colorectal cancer microarray dataset (GSE39582) according to FA2H expression level (top 20% vs. the bottom 20%) was conducted. Interestingly, colorectal cancer samples with high FA2H expression were enriched for the genes upregulated in response to AMPK activation by IL15 treatment (Supplementary Fig. S4A; P < 0.05; ref. 29), while samples with low FA2H expression showed significant enrichment of genes downregu; lated by this AMPK pathway activator (Supplementary Fig. S4B; P < 0.05), suggesting FA2H might function via AMPK, a potential upstream regulator of the YAP pathway (24, 25). Indeed, FA2H overexpression significantly increased AMPK and ACC phosphorylation (Supplementary Fig. S4C), while FA2H knockdown did the opposite (Supplementary Fig. S4D). Moreover, treatment with (R)-2-OHPA had a similar effect on AMPK phosphorylation as did FA2H overexpression (Supplementary Fig. S4E; P < 0.05), while PA or (S)-2-OHPA had minimal effects.
To rule out the possibilities that other known upstream regulatory kinases may contribute to the observed regulation of YAP phosphorylation upon FA2H manipulations, we evaluated the phosphorylation of ERK, p38, and JNK levels and found no regulatory effects by either FA2H knockdown or overexpression (Supplementary Fig. S3A and S3B). We next examined whether FA2H-regulated YAP phosphorylation may require AMPK activity and found that AMPK knockdown diminished FA2H-induced YAP phosphorylation (Fig. 6A). Conversely, AMPK activator, AICAR or D942, was able to enhance YAP phosphorylation when FA2H was depleted (Fig. 6B and C). We next evaluated YAP transcriptional activity by measuring the expression of its target gene CTGF. FA2H could not suppress CTGF expression in AMPK-deficient cells (Fig. 6D), supporting that AMPK activity is important for FA2H inhibition of YAP activity.
To confirm the correlation of FA2H expression and the AMPK/YAP signaling pathway, we examined FA2H, p-AMPK, and CTGF in 63 cases of colorectal cancer tissues by IHC (Fig. 6E). Low levels of FA2H were correlated with low levels of p-AMPK and high levels of CTGF, while high levels of FA2H were correlated with high levels of p-AMPK and low levels of CTGF (Fig. 6F). Consistently, the GSEA showed significant enrichment of the YAP conserved signature in the colorectal cancer clinical samples with lower FA2H level (Fig. 6G), suggesting that FA2H inhibits colorectal cancer tumorigenesis and metastasis through regulation of AMPK/YAP signaling pathway.
FA2H regulates glucose and FA metabolism in colorectal cancer cells
Previous studies identified a cross-talk between glucose metabolism and the Hippo pathway (25). Thus, we examined the FA2H regulation of glucose transporters (GLUT), GLUT1 and GLUT2, which are highly expressed in colorectal cancer tissues (30). FA2H overexpression or treatment with (R)-2-OHPA in SW480 cells suppressed protein levels of GLUT1 and GLUT2 (Fig. 7A and B), leading to a significant decrease of cellular ATP levels (Fig. 7C). Similar to FA2H overexpression, inhibition of glucose utilization by 2-deoxyglucose increased AMPK and YAP phosphorylation in SW480 cells and no obvious additive effects were observed (Fig. 7D). These results indicate that FA2H 2-hydroxylation may activate AMPK in colorectal cancer cells partially by limiting glucose utilization. To gain more insights into the potential mechanisms by which FA2H regulates energy homeostasis, we performed lipidomics profiling in SW480 cells. A total of 167 lipid species with significantly different levels (P < 0.05 and fold change > 2) in cells with or without FA2H overexpression were identified (Fig. 7E; Supplementary Table S4). As anticipated, FA2H overexpression dramatically increased levels of sphingolipid species containing OHFAs. Interestingly, significant increases of polyunsaturated fatty acids (PUFA) and glycerol lipids containing PUFAs were observed in FA2H-overexpressing cells (Fig. 7E; Supplementary Table S4), which have been reported to inhibit glycolysis (31) and YAP activity (32). Involvement of PUFAs in AMPK activation in colorectal cancer cells was supported by the dose-dependent activation of AMPK and YAP phosphorylation by docosahexaenoic acid (Fig. 7F). In summary, we reveal a novel metabolic mechanism by which FA2H and (R)-2-OHPA modulate the YAP transcriptional axis and mediate colorectal cancer cell proliferation and metastasis (Fig. 7G).
Metabolic reprogramming is considered as a hallmark of cancer cells to acquire nutrients necessary for new biomolecule synthesis and tumorigenesis (4) and provides unique opportunities to overcome drug resistance to classical therapy by interfering with the metabolic abnormality. FAs are key precursors to synthesize membrane components and signaling molecules for cancer cell proliferation. Functions of FAs in tumorigenesis are determined by their structural modifications, including desaturation and hydroxylation (7, 33). Therefore, pharmaceutical manipulation of enzymes involved in FA modification is increasingly being recognized as a promising approach to treat cancers. FA2H is highly regulated in cancer cells and may contribute to the development and progression of several cancer types (14–16). Because of the great heterogeneity of cancer cells, the complex molecular mechanisms underlying FA2H regulation of tumorigenesis and metastasis in different cancers are mainly unclear. FA2H is highly expressed in normal colon tissues and this study revealed that FA2H regulates metabolic reprogramming, which is linked to YAP activity in colorectal tumorigenesis and metastasis and shows a promising prognostic value in colorectal cancer.
Previous studies demonstrated that FA2H expression was suppressed in gastric cancer and functioned as a tumor suppressor (14), but was upregulated in adenocarcinomas of lung cancer (16). Conflicting results on FA2H regulation in tumors and its association with survival have been reported in triple-negative breast cancers (34, 35). Additional studies are required to clarify FA2H functions in regulating tumor aggressiveness and survival in breast cancer and to determine whether this regulation is enzymatic activity dependent. There are several possibilities regarding the distinct regulation and functions of FA2H in different cancer types. First, the FA2H expression level and its relative contribution to cellular metabolism vary in different tissues (26), and FA2H in tissues with high expression likely plays a more pivotal role in maintaining normal homeostasis to suppress tumor growth. Second, additional enzymes (36) or exogenous sources (9) may provide 2-OHFAs in some tissues/cell types, which bypass FA2H for FA 2-hydroxylation. Third, FA2H may function through its many binding partners, with distinct functions in different cancer types (37). Nevertheless, our results demonstrated that FA2H functions as a tumor suppressor in colorectal cancer. It has been well established that cellular membrane fluidity regulated by FA composition is an important determinant of glucose uptake and utilization, which further regulates downstream energy and nutrient sensors, AMPK and mTOR (14). GSEA revealed a significant association between FA2H overexpression and AMPK activation, and the energy-sensing through AMPK may regulate Hedgehog signaling and Hippo pathway in cancer development (38). In gastric cancer, FA2H overexpression activated AMPK, leading to noncanonical suppression of Gli1 activity and enhanced chemosensitivity (14). However, overexpressing FA2H in colorectal cancer cells had minimal effect on the Gli1 level. GSEA suggested a potential regulation of the Hippo–YAP pathway, which is likely because of suppressed glucose utilization and glycolysis and activation of AMPK through limiting 1,6-bisphosphate (39) and/or dihydroxyacetone phosphate (40). Collectively, these results demonstrated that FA 2-hydroxylation initiates complex metabolic signaling network and regulates tumor growth through cancer type–dependent mechanisms. The metabolic and signaling contexts determining specific activation of downstream transcriptional pathways by FA 2-hydroxylation require further investigation.
Lipidomics analysis revealed an accumulation of PUFAs in colorectal cancer cells overexpressing FA2H. PUFAs may influence the Hippo pathway by G protein-coupled receptors (GPCR), whose effect was determined by the specific coupled G protein (41). The activation of FFA receptor 1 (also known as GPR40) repressed YAP phosphorylation by coupling to Gq in HEK293A cells overexpressing GPCRs (41), arguing against the potential involvement of GPR40 in PUFA-mediated activation of YAP phosphorylation by FA2H. However, a recent study suggested that PUFA inhibition of colorectal cancer was mediated by GPR40 and GPR120, which subsequently activated protein kinase A via Gs and induced the Hippo pathway activation (32). These results revealed that YAP phosphorylation is regulated by upstream pathways in a cell type- and context-specific manner. It has been suggested that PUFAs contribute to the activation of AMPK in skeletal muscle and liver and influence transcriptional regulation of lipid metabolism (42). Although the mechanisms underlying PUFA-mediated AMPK activation remain unexplored, this study suggests that the observed activation is may be because of suppressed glucose utilization by PUFAs (31). AMPK could function as a tumor suppressor or a tumor promoter, depending on the genetic context, metabolic dependency of cancer cells, and the surrounding microenvironment (43). Our studies of FA 2-hydroxylation–mediated AMPK activation suggested that sensing of distinct metabolic cues and coupling to different downstream signaling pathways contribute to determining the dominant face of AMPK in different carcinogenic scenarios.
FA2H and FA 2-hydroxylation have diverse functions in maintaining membrane homeostasis and cell signaling (10). Studies of subjects with impaired FA2H activity and genetic knockout animal models revealed that FA2H is essential for the normal functioning of the nervous system. FA2H also regulates differentiation and cell signaling of various cell types (epidermal keratinocytes, schwannoma cells, and adipocytes) and cancer metabolism as revealed in this study and recent studies. Most of the known FA2H functions were carried out through its generation of 2-OHFAs and 2-OH sphingolipids, which confers the structural stability of the membrane. In addition to traditional cancer therapeutics targeting proteins and nucleic acids, lipid therapy is becoming a very effective alternative and is supported by this study (44). Treatment with c-AMP analogue or the cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) induced FA2H expression levels (45, 46), although the exact signaling pathways involved in this regulation are still not clear. Interestingly, cyclic phosphatidic acid–induced increase in intracellular cAMP levels was associated with the inhibition of growth in colon cancer cells (47), and Δ9-THC induced apoptosis in colorectal cancer cells probably by inhibition of RAS–MAPK and PI3K–AKT survival signaling (48). Collectively, FA2H is involved in a wide range of metabolic and tumorigenic signaling pathways, which makes FA 2-hydroxylation a promising approach of lipid therapy of colorectal cancer.
There are some limitations to our study. The molecular mechanisms through which FA2H increases PUFA levels and coordinates FA and glucose metabolism remain to be determined. FA2H may help generate LXR ligands to promote PUFA synthesis (49) and this possibility is supported by LXR inhibition of the proliferation of colorectal cancer (50). Although the altered membrane mobility by FA2H may influence trafficking and lysosomal degradation of GLUTs, the mechanism leading to the decreased GLUT1 and GLUT2 in FA2H-overexpressing colorectal cancer cells remains elusive. A recent study revealed an effect of TAZ on cell proliferation in non–small cell lung cancer (51) and its potential involvement in FA2H regulation of colorectal cancer metastasis needs additional investigation. Moreover, the inhibitory efficiencies of (R)-2-OHFAs and FA2H overexpression in colorectal cancer development under different pathologic conditions require further evaluation. Finally, The Cancer Genome Atlas RNA-seq datasets revealed that FA2H expression is differentially regulated in different cancer types as compared with their normal tissues, and mechanisms responsible for FA2H regulation of tumorigenesis and metastasis across tissues warrant future investigation. Nevertheless, our results revealed a novel mechanism linking FA 2-hydroxylation with the Hippo–YAP pathway in regulating the growth and migration of colorectal cancer cells and identified a metabolic target to improve colorectal cancer treatment.
No disclosures were reported.
L. Sun: Conceptualization, data curation, formal analysis, validation, investigation, methodology, writing-original draft. X. Yang: Conceptualization, data curation, software, formal analysis, validation, investigation, methodology, writing-original draft. X. Huang: Investigation, methodology. Y. Yao: Investigation. X. Wei: Software, investigation. S. Yang: Investigation, methodology. D. Zhou: Investigation. W. Zhang: Investigation, methodology. Z. Long: Investigation, methodology. X. Xu: Investigation, methodology. X. Zhu: Resources, supervision, project administration. S. He: Resources, supervision, funding acquisition, project administration. X. Su: Conceptualization, resources, supervision, funding acquisition, investigation, methodology, writing-original draft, project administration, writing-review and editing.
This work was supported by The National Natural Science Foundation of China grants 31620103906 (to X. Su) and 81672348 (to S. He), National Science Foundation of Jiangsu Province of China grants BK20150006 (to X. Su) and BK20191172 (to S. He), and the project funded by Priority Academic Program Development of Jiangsu Higher Education Institutions.