Ceramide synthase CERS4 gene downregulation is associated with KRAS mutation in colorectal cancer

Ceramide, the central molecule in sphingolipid synthesis, is a bioactive lipid that serves as a regulatory molecule in the anti-inflammatory responses, apoptosis, programmed necrosis, autophagy, and cell motility of cancer cells. In particular, the authors have reported differences in sphingolipid content in colorectal cancer tissues. The associations among genetic mutations, clinicopathological factors, and sphingolipid metabolism in colorectal cancer (CRC) have not been investigated. The objective of this study is to investigate the association between genes associated with sphingolipid metabolism, genetic variations in colorectal cancer (CRC), and clinicopathological factors in CRC patients. We enrolled 82 consecutive patients with stage I–IV CRC who underwent tumor resection at a single institution in 2019–2021. We measured the expression levels of genes related to sphingolipid metabolism and examined the relationships between CRC gene mutations and the clinicopathological data of each individual patient. The relationship between CRC gene mutations and expression levels of ceramide synthase (CERS), N-acylsphingosine amidohydrolase (ASAH), and alkaline ceramidase (ACER) genes involved in sphingolipid metabolism was examined CRES4 expression was significantly lower in the CRC KRAS gene mutation group (p = 0.004); vascular invasion was more common in colorectal cancer patients with high CERS4 expression (p = 0.0057). By examining the correlation between sphingolipid gene expression and clinical factors, we were able to identify cancer types in which sphingolipid metabolism is particularly relevant. CERS4 expression was significantly reduced in KRAS mutant CRC. Moreover, CRC with decreased CERS4 showed significantly more frequent venous invasion.

In recent years, it has been reported that ceramide plays an important role in ovarian cancer and lung cancer, and therapeutic agents to address impairments of the ceramide synthetic pathway are being developed 1,2 .Ceramide, a cell membrane lipid, and sphingosine-1 phosphate (S1P) are bioactive lipids in the sphingolipid synthesis pathway and play important roles in cell signaling.In vivo, they play opposite roles in cellular metabolism 3 .S1P stimulates cell survival, proliferation, and tissue regeneration, whereas ceramide has been shown to be involved in stress-related cellular responses and apoptosis 4,5 .
Apoptosis is associated with morphological changes at the membrane level of plasma and organelles, and the involvement of specific lipids in apoptosis has been established [6][7][8] .Maintaining a balance between ceramide and S1P is very important for cells as these bioactive lipids substantially contribute to cell fate determination.Alterations in ceramide levels have been recognized in pathological conditions such as inflammatory bowel disease, sepsis, and colon cancer 9,10 .Ceramide also plays important roles in regulating tumor growth and apoptosis; however, the mechanisms through which ceramide induces apoptosis are still unclear.
RAS is a group of four proteins involved in signal transduction that promotes cell proliferation.There are three RAS genes: KRAS, NRAS, and HRAS.Mutations in any of these genes produce abnormal RAS proteins.Point mutations in the RAS gene, particularly in the G12, G13, and Q61 hotspots, lead to a loss of GTPase activity, impaired GAP binding, and accumulation of active RAS protein and lead to activation of different intracellular signaling pathways.This aberrant signaling enhances proliferation and survival pathways in cancer cells, contributing to tumor development and progression across various types of carcinomas.It is thought that cancer can develop as a consequence due to signal transduction pathways that constantly promote cell proliferation 11 .It was reported that KRAS mutation colorectal cancer has a poorer prognosis than wild-type KRAS colorectal cancer 12,13 .One reason is that colorectal cancer with a KRAS mutation does not respond to cetuximab, whereas patients with colorectal cancer bearing wild-type KRAS benefited from cetuximab 14 .Elucidation of the mechanism of RAS mutation is very important due to its prominence as a marker for predicting the development and prognosis of new therapeutic agents.Our research focuses on establishing connections between the expression levels of sphingolipid-related genes expression levels and both KRAS mutations and various clinicopathological characteristics in patients diagnosed with colorectal cancer (CRC).We have chosen to investigate specific subtypes of CRC that are influenced by sphingolipid metabolism.

Gene expression that contributes to ceramide in colorectal cancer
To investigate the role of CERS1-6 in colorectal cancer, we examined the mRNA expression levels of CERS1-6 in colorectal cancer tissue and paired normal colorectal mucosal tissue.The data generally showed significantly higher expression levels in CRC tissue than in normal tissue, with much higher mRNA relative levels of CERS2 and CERS6 (Fig. 1).Next, we investigated the gene expression of the enzymes that disassemble the fatty acid CoA, which is the substrate for CERS1-CERS6: namely, ASAH1-2 and ACER1-3, participating in the metabolic processes of sphingosine or ceramide.(Fig. 2).The results for mRNA expression levels of ASAH-1 and ACER3 in CRC tissue were significantly higher in cancer tissues than in normal paired tissue, respectively (p = 0.0088, 0.0001).

Relationship between KRAS mutation and acyl-CoA synthase genes
CERS, ASAH, and ACER gene expressions, which each contributes ceramide synthesis and degradation, were used to compare the characteristics of wild-type KRAS and KRAS mutant cancers.As a result, CERS4 showed significantly low expression in KRAS mutant CRC (p = 0.004) (Table 2, Fig. 3).

Cut-off value of CERS4 expression in KRAS mutation
We performed ROC analyses to define the optimal cut-off value for CERS4 expression in colorectal cancer tissue.ROC analyses showed that a CERS4 cut-off value of 0.800 produced the best results, with an AUC of 0.694 (sensitivity: 0.83, specificity: 0.35) (Fig. 4).

Comparison of low-and high-CERS4 groups
The target group was divided into two groups with the cut-off value of CERS4 set to 0.80.CERS4 < 0.8 was set as the low-CERS4 group, and CERS4 > 0.8 was set as the high-CERS4 group.The two groups were compared for variables of age, gender, tumor location, histology, depth of tumor invasion, lymph node metastasis, lymph invasion, venous invasion, distant metastasis, CEA level, and KRAS, NRAS, and HRAS mutations.Venous invasion showed a significant difference, and venous invasion was significantly more prevalent in the low-CERS4 group (p = 0.0057).There were significantly more cases of KRAS wild-type CRC in high CERS4 group than in low CERS4 group (p = 0.0015) (Table 3).

Discussion
Currently, there is no information about the clinical significance of CERS genes in CRC progression.High expression levels of ceramide have been reported in patients with ovarian, lung and colorectal cancer 1,15,16 .In the present study, we comprehensively analyzed genes that contribute to ceramide synthesis and metabolism using colorectal cancer tissue.This study revealed two things.First, significant differences in gene expression between normal tissue and colorectal cancer tissue were observed for ASAH1, ACER3, CERS2, and CERS6.Second, we compared the expression of genes involved in ceramide synthesis and metabolism for KRAS mutations   Several relationships between gene expression related to ceramide synthesis and metabolism and cancer sites have been reported.Zhang et al. reported that the expression level of CERS2 was decreased in highly metastatic ovarian cancer cells 1 .In ovarian cancer, Sheng et al. reported an association of high expression of CERS2 with an unfavorable prognosis in patients 17,18 .
Some reports have suggested that overexpressed CERS6 promoted cancer invasion in lung cancer.They showed that overexpressed CERS6 in lung cancer synthesizes a bioactive lipid called C16 ceramide, which activates the intracellular protein kinase, RAC 1 complex.As a result, morphogenesis called lamellipodia, which is essential for cell migration, occurs on the cell surface, and cancer cells metastasize 2,16 .
In the present study, expression of CERS4 was reduced in KRAS mutant colorectal cancer.There are two possible pathways for this result.The first is that the Wnt pathway signal is involved in the regulation of CERS4 or the Wnt pathway may be involved in the KRAS mutation in colorectal cancer.Peters et al. used mice to show that CERS4 is highly expressed in the epidermis of adult mice and is localized in defined populations within the interfollicular epidermis and hair follicle sebaceous unit.They reported that decreased bone morphogenetic protein signaling in CERS4−/− mice may promote Wnt/β-catenin signaling and strongly stimulate the activation of hair follicle stem cells 17 .In the present study, there are no data to positively demonstrate that the Wnt pathway is involved in the suppression of CERS4 in KRAS mutant CRCs, but this is a hypothesis.

False positive rate (1 -specificity)
True positive rate (sensitivity)   The other possibility is that there is a relationship between KRAS mutations and the NF-kB (NF kappa B) pathway.NF-κB (NF kappa B) is a protein complex that acts as a transcription factor.Five types of proteins (NF-κB family) are known in mammals: p50, p52, p65 (RelA), c-Rel, and RelB 18,19 .It has been reported that p65 activity, which is one of the NF-KB family, was higher in tumors with KRAS mutation (50.8%) than in tumors with the wild-type KRAS gene (30.6%)(P = 0.012) 20 .In addition, there is a report that NF-KB activation is involved in KRAS mutant colorectal cancer, with NF-kB activity being higher than it is in wild-type KRAS colorectal cancer 21 .High expression of CERS4 was observed in liver cancer tissues, and it has been reported that the nuclear factor (NF)-κB signaling pathway was affected after knockdown of CERS4 in liver cancer cells 22 .Thus, even in KRAS mutant colorectal cancer, CERS4 expression may be reduced due to the influence of the NF-kB pathway.
It has been reported that overexpression of CERS4 and CERS6 in colon cancer cells induced the production of short-chain ceramides (C16: 0, C18: 0 and C20: 0 ceramides), weakened cell proliferation and promoted apoptosis 23 .This is consistent with the poor prognosis of G12V and G12C mutation colorectal cancers we reported compared to wild-type KRAS colorectal cancers 12,13 .The present finding that CERS4 functions as an important regulator of KRAS mutation, may suggest its potential use as a marker for CRC and may also guide the development of new drugs for CRC.
To assess the clinical attributes of individuals exhibiting elevated ceramide expression, we conducted a comparison of clinicopathological factors among CRC patients.Notably, the high CERS4 group displayed a higher prevalence of KRAS wild-type status and an increased incidence of positive venous invasion.It has been previously documented that CERS4 and CERS5 play a role in apoptosis within CRC 24 .Sphingosine 1-phosphate (S1P) is a blood-borne lipid mediator implicated in the regulation of vascular and immune systems.Blood flow and circulating S1P activate endothelial S1P1 to stabilize blood vessels in development and homeostasis 25 .Ceramides are apoptosis-inducing sphingolipids and precursors of other bioactive sphingolipids such as S1P.When ceramide is downregulated, its downstream S1P is also downregulated.This implies that CERS4 expression was reduced in the KRAS mutation group, potentially resulting in a diminished proapoptotic effect, may be why low-CERS4 cancers had more venous invasion.This study's limitation was that it included patients from just a single institution.Our study findings need further review and validation in more CRC patients.

Conclusions
By examining the correlation between sphingolipids and clinical factors, we were able to identify cancer types in which sphingolipid metabolism is particularly relevant.Our study found that CERS4 expression was significantly reduced in KRAS mutant CRC.Among the CRCs in which CERS4 was decreased, there were significantly more cases with venous invasion than in the cases in which CERS4 was not decreased.

Patient selection
A total of 82 patients with stage I-IV CRC diagnosed based on the 8th edition of the United States Joint Commission on Cancer (AJCC) 26 staging system and undergoing CRC resection at Teikyo University Hospital from 2019 to 2021 were enrolled in the study.This study was approved by Teikyo University ethics committee (Registration Number; 19-153).All participants provided informed consent, and our research findings are reported in adherence to the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines 27 .The experimental procedures conducted adhered to the ethical guidelines outlined in the Declaration of Helsinki, which is internationally recognized as the Code of Ethics for World Medicine.

Sample preparation
All colorectal tissue specimens were dissected into small pieces, approximately 5 mm a side, frozen in liquid nitrogen within 24 h after resection, and stored at − 80 °C until lipid extraction.Normal tissues were obtained by lifting and dissecting the mucosa layer.The specimens mainly consisted of mucosa tissues but did not contain circular or longitudinal muscles.

RAS and BRAF mutations analysis
Testing for RAS/BRAF mutations was performed at Hoken Kagaku Laboratories (Kanagawa, Japan) using samples collected from tumor tissues.The selected area of formalin-fixed paraffin-embedded samples (FFPEs) was deparaffined, followed by DNA isolation from samples using a QIAamp DNA FFPE Tissue Kit (Qiagen, Manchester, UK) according to the manufacturer's instructions.DNA quantification was done on a NanoDrop 2000c (ThermoFisher Scientific, Waltham, MA, USA).The obtained DNA was amplified by a PCR reverse-sequencespecific oligonucleotide (PCR-rSSO) method on an Applied Biosystems VeritiTM 200 Thermal Cycler (Thermo Fisher Scientific) using MEBGENTM RASKET-B kit (MBL, Tokyo, Japan).Cycling began with 5 min at 40 °C and 2 min at 95 °C, followed by 10 cycles at 95 °C for 20 s and 62 °C for 30 s, then 45 cycles at 90 °C for 20 s, 60 °C for 30 s and 72 °C for 30 s. Finally, extension was performed for 1 min at 72 °C, followed by 1 min at 95 °C before the product was allowed to cool to 4 °C.The amplified PCR product was then hybridized with probes in Beads Mix in Hybridization Buffer provided with MEBGENTM RASKET-B kit.This reaction was done at 95 °C for 2 min followed by 55 °C for 20 min.The product was purified according to the manufacturer's instructions and reacted with fluorescent phycoerythrin-labelled streptavidin.Fluorescence was measured by flow cytometry on a Luminex 100/200 System (Luminex, Austin, TX, USA), and the data were analyzed with the associated UniMAG software (Luminex).

Quantitative real-time RT-PCR
Total RNA from colorectal tissues was extracted using an ISOGEN kit (Nippongene, Toyama, Japan), and cDNA libraries were synthesized using a shigh-capacity cDNA RT kit (Thermo Fisher Scientific).For cDNA synthesis, 2 µg of total RNA was used to yield 40 µL of reaction product.Then 1 µL of cDNA sample was used as a template per reaction for quantitative real-time PCR.We prepared cDNA samples from the 82 patients enrolled in this study and used them for quantitative real-time PCR.Due to shortages of cDNA samples for some patients, only 40 of the 82 patients were analyzed for SMS.The obtained results were normalized to the expression level of β-actin (ACTB) in each cDNA sample.The sequences of the oligonucleotides used in the PCR reaction were obtained from PrimerBank (https:// pga.mgh.harva rd.edu/ prime rbank/) or were originally designed and are listed as Table 4.
Table 4. List of oligonucleotide sequences used in the PCR reactions that were designed independently.

Figure 4 .
Figure 4. ROC for CERS4 as a KRAS mutation factor for colorectal cancer was plotted to verify the optimum cutoff value of CERS4. https://doi.org/10.1038/s41598-023-43557-1www.nature.com/scientificreports/ AAG ATC ATT GCT CCT C ACA TCT GCT GGA AGG TGG ACA CERS1 CCA TGA CCC ACC ATC TGT CT TTG GTG AAC TCA AGC TGC AC CERS2 TCT ATA TCA CGC TGC CCC TG CTT GCC ACT GGT CAG GTA GA CERS3 ACT GTT GCT GGA ATT GCG TT TGT TAC AGG TCT GCG TCC AT CERS4 TCT CTG GTG CTG CTG TTA CA AGA TGA GGA AGA GAG CGT CG CERS5 CAT CTT CTT CGT GAG GCT GC TGT CCT GAT TCC TCC GAT GG CERS6 ACC TGA AGA ACA CGG AGG AG CCA GCA ATG CCT CGT ATT CC ASAH1 TCA GGA CCA ACG TAC AGA GG AAA AGG GCC AGG AAA GTT GC ASAH2 TGC ACA GGA CAA GTA GCA GA CCA TGA TGA AGG CAC GAC TG ACER1 CCT ATC AGA GCT CCG AGG TG AGT GGC CCG AAG ATG AAG AA ACER2 TCT CTC TGA TGA CCC TGG GA GAG GCA GCA TCA AAG TAG GC ACER3 ACT GGT GCG AGG AGA ACT AC GAA GCA CCA GGA TCC CAT TC SMS1 GCA TAT TAC ATC ACC ACG AGA CTC TT GGA GGT TCA TCT GGG AAG CTT SMS2 TTC AGC GGT CAC ACG GTT AC ACG AGG CGA ATA TTC TTT GATGA

Table 2 .
Relationship between KRAS status and the expression levels of genes involved in sphingolipid metabolism in colorectal cancer patients.
Quantities of CERS4 in colorectal cancer tissue were compared for RAS gene status.

Table 3 .
Relationship between CERS4 status and clinicopathological factors in colorectal cancer patients.