Non-esterified very long-chain fatty acids are accumulated in colorectal cancer tissues


 Colorectal cancer (CRC) is a major cancer, and its precise diagnosis is especially important for the development of effective therapeutics. In a series of metabolome analyses, the levels of very long chain fatty acids (VLCFA) was shown to be elevated in CRC tissues, although the endogenous form of VLCFA has not been fully elucidated. In this study we analyzed the amount of non-esterified fatty acids, phospholipids and acyl-CoA species by liquid-chromatography–mass spectrometry and showed that VLCFA is accumulated as the non-esterified form in CRC tissues. We also showed that the expression level of elongation of very long-chain fatty acids 1 (ELOVL1) is increased, whereas fatty acid transport protein 4 (FATP4) is decreased in CRC tissues. Finally, we showed that the amount of non-esterified VLCFA species was significantly up-regulated in cultured cells overexpressing ELOVL1. Our results suggest that the upregulation of ELOVL1 and the down-regulation of FATP4 cooperatively lead to the accumulation of non-esterified VLCFA in CRC tissues.

occurrence, whereas the ratio of n-3 PUFA/n-6 PUFA did not correlate with CRC occurrence [2], These results suggest that the pathological contribution of the amount of fatty acids and lipid metabolism in CRC remains to be elucidated.
Very long-chain fatty acids (VLCFA) with no less than 23 or 24 carbons are endogenously synthesized as very long-chain fatty acyl-CoA (VLCFA-CoA) through a fatty acid elongation process [3]. Then VLCFA-CoA are incorporated into complex lipids such as phospholipids (PL).
Recently, several analysis using gas chromatography-mass spectrometry (GC-MS) revealed that VLCFA levels are elevated in plasma and surgical specimens from CRC patients [4]. However, the endogenous form of VLCFA in CRC tissues has not been fully understood due to the limited number of intensive lipidomic analyses focusing on VLCFA. Notably, fatty acyl moieties are liberated from complex lipids during the derivatizing process of sample preparation for GC-MS, which leads to loss of information about the endogenous lipid species containing VLCFA.
Advances in liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) have enabled simultaneous quantification of a large number of underivatized lipid species in biological samples. In this study, we analyzed underivatized lipid fractions from surgical specimens, from patients with CRC by LC-ESI-MS, and revealed that VLCFA are not accumulated as esterified, but as non-esterified form in CRC tissues. To gain a mechanistic insight, we also conducted expression analysis of genes involved in lipid metabolism in CRC tissues.

The level of VLCFA-CoA is not elevated in CRC tissue
VLCFA are initially produced as VLCFA-CoA through de novo fatty acid elongation process [3].
Therefore, we first profiled the amount of VLCFA-CoA species in CRC tissues. Among 59 acyl-CoA species quantified in CRC and adjacent normal tissues, we found that 3% of the total acyl-CoA species consisted of the VLCFA-CoA species, with no less than 23 carbons in their fatty acyl moiety. Tetracosenoyl-CoA (24:1-CoA) was the most abundant VLCFA-CoA species in both CRC and adjacent normal tissues (Fig. 1A, and Supplementary Table S1 on line). We could not observe any VLCFA-CoA species that significantly accumulated in CRC tissues (Fig. 1A), but rather, we found that the amount of two VLCFA-CoA species (23:0-CoA and 24:0-CoA) was significantly smaller in CRC tissues, than in normal tissues (Fig. 1B). These results show that the intracellular pool of VLCFA-CoA is not significantly increased in CRC tissues.

The level of phospholipid species containing VLCFA is not altered in CRC tissues
Acyl-CoA species serve as metabolic intermediates of fatty acids involved in the synthesis of complex lipids, post-translational modification and b-oxidation. PLs that mostly contain two acylmoieties are abundantly present as membrane components in cells, and thus could serve as an intracellular reservoir for free fatty acids. Therefore, we next analyzed PL species to examine whether VLCFA species are accumulated in esterified form in CRC tissues. We analyzed 39 PL species (7 PC, 7 PE, 7 PS, 7 PI, 7 PG and 4 SM species), in which acyl moieties were determined by scanning product ions from parental ions of each PL species. The amounts of most PL species (17 out of 19 species) with long-chain fatty acyl moiety (C16:0, C18:0, C18:1, C20:4 and C22:0) were significantly lower in CRC tissues than in adjacent normal tissues, and the amounts of only 7 out of 20 PL species with VLCFA moiety (C24:0 and C26:0) were smaller in CRC tissue ( Fig.   2A, B and Supplementary figure S1 online). We could not observe PL species, which amount was significantly higher in CRC tissues. These results show that VLCFA are not accumulated as esterified form in PL species, and indicate that the PL metabolism could be different according to the length of fatty acyl moieties of PL species in CRC tissues.

The level of non-esterified VLCFA is elevated in CRC tissues
Non-esterified VLCFA could be produced through deactivating VLCFA-CoA by acyl-CoA thioesterases or liberation from complex lipids such as PL by lipases. To profile the FFA species as non-esterified fatty acids in CRC tissues, we analyzed the underivatized FFA in total lipid fractions by LC-MS method. We used a C8 reversed-phase column and mobile phases adjusted to pH 9.0 by ammonium hydroxide, which are almost identical with the methods for acyl-CoA analyses developed so far [5]. We validated our method using two isotopically-labeled palmitic acids, and confirmed that 8-2000 pmol/injection of FFA species can be quantified with the present quantitative method (Supplementary Table S2 online). We then profiled each FFA species in CRC tissues from 24 patients. The amount of FFA 16:0 in CRC was significantly lower than in adjacent normal tissues. By contrast, the amount of non-esterified VLCFA species (FFA 24:0, 24:1, 26:0, and FFA 26:1) and FFA 22:1 was significantly higher in CRC tissues than in normal tissues (Fig. 3A). The portion of four non-esterified VLCFA species with no less than 24 carbons (FFA 24:0, 24:1, 26:0, 26:1) was also significantly higher in CRC than in adjacent normal tissues (Fig. 3B). These results show that VLCFA species are accumulated as non-esterified form in CRC tissues.

The expression level of ELOVL1 is correlated with non-esterified VLCFA level in CRC tissue
To explore the machinery that explains how non-esterified VLCFA is accumulated in CRC tissues, we first focused on the process of fatty acyl elongation. ELOVL1 and ABCD1 (ATP-binding cassette transporter subfamily D1) play critical roles in the de novo synthesis and degradation of VLCFA, respectively [3,6]. We examined the mRNA levels of ELOVL1 and ABCD1 in samples from 40 CRC patients, and found that the expression level of ELOVL1 was higher in CRC tissues than in adjacent normal tissues (Fig 4A), a result which is consistent with a previous report [7]. In contrast, the expression level of ABCD1 was not significantly altered ( Fig 4A). We also tested the significance of correlation by Pearson's correlation coefficient between each variable, consisting of CRC/normal ratio of ELOVL1 mRNA and the 15 FFA species analyzed in this study.
We found that the CRC/normal ratio of ELOVL1 mRNA is positively and significantly correlated with the CRC/normal ratio of saturated (FFA 20:0 and FFA 22:0) or mono-unsaturated FFA (FFA 22:1, FFA 24:1 and FFA 26:1) (Fig. 4B). These results suggest that ELOVL1 is involved in the accumulation of saturated or mono-unsaturated non-esterified fatty acids with no less than 20 carbons in CRC tissues.

The expression level of FATP4 is down-regulated in CRC tissues
Our lipidomic study revealed that the expression level of ELOVL1 is significantly increased, while VLCFA is accumulated as non-esterified form, but not as VLCFA-CoA or VLCFA-PL in CRC tissues ( Fig. 1-3). This discrepancy led us to examine the re-activation process of VLCFA by acyl-CoA synthetases, which convert non-esterified fatty acids into acyl-CoA species. Long chain fatty acyl-CoA synthetases (ACSL), long chain fatty acid transport proteins (FATP) and bubblegum (ACSBG) are especially important for the activation and metabolism of long chain fatty acids [8]. We thus quantified the mRNA levels of ACSL (ACSL1, and 3-6), FATP (FATP1-6) and ACSBG (ASCBG1 and 2), and found that only the expression level of FATP4 was significantly decreased in CRC tissues, as compared with adjacent normal tissues (Fig. 5). We further examined another possibility that non-esterified VLCFA is accumulated due to increased rate of hydrolysis of ceramides by ceramidases, since the amount of SM species was grossly decreased in CRC tissues (Fig. 2). We analyzed two acid ceramidases (ASAH1 and 2) and three neutral ceramidases (ACER1, 2 and 3), although a significant change could not be observed in the amount of these five ceramidases (Fig. 5). FATP4 is the gene responsible for ichthyosis prematurity syndrome, and it has been shown that FATP4 prefers very long-chain free fatty acids with no less than 24 carbons as substrate [9][10][11]. Therefore, these results indicate that the accumulation of non-esterified VLCFA is caused by the combined effects of up-regulation of ELOVL1 and down-regulation of FATP4 in CRC tissues.  Table S3 online). Notably, the amount of 26:0-CoA was much smaller than that of 26:1-CoA, whereas the amount of FFA 26:0 was relatively close to that of FFA 26:1 in both mock and ELOVL1 transfected cells ( Fig. 6A and B). These results suggest that saturated VLCFA-CoA synthesized by ELOVL1 is efficiently metabolized in CRC tissues.

Discussion
In this study, we found that saturated and mono-unsaturated VLCFA are accumulated as non-esterified form but not as esterified form such as acyl-CoA or phospholipid species, in CRC tissues ( Fig. 1-3). This result is partially consistent with a previous report, in which FFA 24:0 and FFA 24:1 were shown to be accumulated in both CRC tissues with and without lymph node metastasis [12]. It will be necessary to examine whether esterified VLCFA is accumulated in other lipid species such as glycolipids and neutral lipids, including triacylglycerol and cholesterylester, in CRC tissues. It has also been shown that the amount of total VLCFA, including esterified and non-esterified forms, are elevated in the sera of colorectal cancer patients [13]. It needs to be further examined whether the accumulation of non-esterified VLCFA in CRC tissues directly leads to the changes in the composition of serum fatty acids by comparing the fatty acid composition in the sera of pre-and post-surgery patients.
It is still unclear that how FATP4 is involved in the cancer progression in CRC tissues through the regulation of VLCFA metabolism. Recent analysis showed that the expression level of FATP4 in cancer tissues is inversely correlated with survival rates in breast cancer [14]. In contrast, we showed that the expression level of FATP4 is decreased in CRC tissues (Fig. 5), suggesting that FATP4 expression could be different between different types of cancer. Further analysis should be undertaken to clarify So far, we inhibited FATP4 expression by siRNA in ABCD1-deficient HeLa cells, in which PL species and acyl-CoA species harboring VLCFA moiety are accumulated [5]. However, significant difference could not be observed in the amount of non-esterified VLCFA, as compared with control-siRNA treated cells (data not shown).
Considering the higher expression level of ELOVL1 in CRC tissues ( . It has been shown that the VLCFA moiety is located at the sn-1 position of the glycerol backbone of PL [5,[16][17][18]; thus it is also possible that phospholipase A1 contributes to the accumulation of non-esterified VLCFA in CRC tissues. Three intracellular phospholipase A1 identified so far in mammals (PA-PLA1a/DDHD1/iPLA1a, p125/Se23IP/iPLA1b and KIAA0725p/DDHD2/iPLA1g) prefer PA, PE and PI but not PC as substrates [19]. Therefore, intracellular phospholipase A1 and phospholipase D cooperatively contribute to the production of non-esterified VLCFA. Note that ELOVL1 overexpression in HEK293T cells resulted in not only the accumulation of non-esterified VLCFA, but also esterified VLCFA such as acyl-CoA and PL ( Fig. 6A-D). In contrast, VLCFA is accumulated as non-esterified but not as esterified form in CRC tissues ( Fig. 1-3). These differences in the VLCFA profile might be attributed to the difference in the activity of enzymes involved in VLCFA metabolism such as acyl-CoA thieoesterases and intracellular phospholipase A1, between CRC tissues and HEK293T cells.
The present study showed that the amount of SM species with VLCFA was significantly decreased in CRC tissues (Fig. 2). A recent study showed that SM species containing VLCFA and cholesterol cooperatively play crucial roles in maintaining tight junctions, which is essential for the barrier functions of enterocytes [20]. VLCFA-CoA is transferred into sphingosine by ceramide synthases, and then ceramide species with VLCFA are converted into SM by sphingomyelin synthases. Therefore, it might be possible that the down-regulation of FATP4 expression causes the decline in the amount of VLCFA-CoA and VLCFA-containing SM, which leads to aberrant tight junctions in colonic epithelial cells.

Ethics
This study abides by the Declarations of Helsinki Principles, and the research protocol was approved by the Ethics Committee of Teikyo University (#19-153). We enrolled 40 consecutive patients treated with curative resection at Teikyo University Hospital, Japan during the period 2019-2020. The surgery of all of the patients was elective. Informed consent was obtained from all participants and the reporting of our research is in accordance with the STROBE guidelines [21].

Sample preparation
All colorectal tissue specimen were dissected into small pieces, ~5 mm. a side, frozen in liquid nitrogen within 24 hours after surgery, and stored at -80°C until lipid extraction. Sample preparation for PL and free fatty acid (FFA) analysis was conducted as reported previously with slight modification [18,22]. Briefly, the tissue specimen were homogenized in 1 mL of methanol using a Micro Smash MS-100R instrument (TOMY, Tokyo, Japan) at 3000 rpm for 10 s at 4°C. Samples were stored at −20°C until further analysis. For acyl-CoA analysis, the tissue specimen were homogenized in 0.9 mL of acetonitrile/isopropanol (3:1 by volume) and the acyl-CoA fraction was extracted as previously described [5]. Samples were stored at −20°C until further analysis. Homogenate protein concentrations were determined with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). The samples for the validation of the LC-MS method for FFA were prepared as described in supplemental materials online.

LC-MS/MS analysis
Quantitation of each PL species was conducted with a TSQ Quantum Ultra (Thermo Fisher Scientific) linked to an Accela HPLC system (Thermo Fisher Scientific). An InertSustain C18 metal-free column (2.1 mm i.d. × 50 mm, particle size 3.0 µm; GL Sciences, Tokyo, Japan) was used at 50°C. The mobile phases were acetonitrile/methanol/water ( Corp., Kyoto, Japan) as described previously [18]. The fatty acyl moieties of each PL were assigned by the detection of the product ions from fatty acyl residues and lysophospholipids. In addition, fatty acyl residue positions were also determined by comparing the spectral intensity of two lysophospholipid ions produced from precursor ions, as reported previously [24].
Quantitation of each FFA species was conducted with a TSQ Quantum Ultra linked to an Accela

Quantitative Real-time RT-PCR
Total RNAs from colorectal tissues were extracted using the ISOGEN kit (Nippongene, Toyama, Japan) and cDNA libraries were synthesized with a high capacity cDNA RT kit (Thermo Fisher Scientific). The sequences of the oligonucleotides used in the PCR reaction are listed in Supplementary Table S5 online. human ELOVL1 plasmid was constructed as described previously [25]. Three days after transfection, cell layers were washed with phosphate buffered saline, scraped from the dishes, homogenized in methanol, and used for FFA and acyl-CoA extraction as described above.

HEK293T cells (Riken
Homogenate protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.).

Statistical analysis was performed with either Student's two-tailed t-test, two-tailed paired t-test
or Pearson correlation coefficient. Differences were considered to be significant if the p-value was < 0.05. All statistical analyses were conducted with IBM SPSS Statistics version 27 (IBM, Armonk, NY, USA).

Data availability statement
The datasets obtained in the current study are available from the corresponding author on reasonable request.     Data represent the mean ± SD. Statistical analysis was performed with two-tailed paired t-test.