Altered mRNA expression levels of the major components of sphingolipid metabolism, ceramide synthases and their clinical implication in colorectal cancer
- Authors:
- Published online on: September 18, 2018 https://doi.org/10.3892/or.2018.6712
- Pages: 3489-3500
Abstract
Introduction
Colorectal cancer (CRC) is a malignancy derived from the colorectal epithelium and is the third most commonly diagnosed cancer type worldwide (1). Although the mortality rates of CRC have been decreasing due to screening, reduced risk factor prevalence and/or improved therapies (2,3), CRC remains a global health burden in terms of morbidity and mortality, with ~700,000 estimated mortalities annually (1). It has been reported that the complicated and complex pathogenetic mechanisms of CRC involve genomic rearrangements, chromatin remodeling, genetic mutations and epigenetic changes (4,5).
The sphingolipid rheostat is a proposed concept that may regulate cell fate decisions (6). The two major components of the sphingolipid rheostat are ceramide and sphinogosine-1 phosphate, which are interconvertible sphingolipid metabolites that regulate cell growth and survival by modulating sphingolipid rheostat-related signaling (6,7). Ceramide has tumor suppressive anticancer properties, including potentiating signaling networks that drive apoptosis, autophagy and cell cycle arrest (8). Ceramide synthases (CerSs) are integral membrane proteins of the endoplasmic reticulum that synthesize ceramides of different acyl chain lengths. To date, six CerS families have been identified in mammals (9). Dysregulation of CerS activity has been reported to be associated with tumor cell invasion (10), proliferation (11), apoptosis (12) and epithelial-mesenchymal transition (13), as well as with the prognosis of patients with cancer (14). For example, in head and neck squamous cell carcinoma, downregulation of CERS1 leads to apoptotic resistance (15), while CERS1 overexpression enhances growth-inhibitory effects (16). Additionally, CERS2, CERS4 and CERS6 mRNA expression levels are increased in breast cancer (17), and the upregulation of CERS4 and CERS6 leads to reduced cell proliferation and the induction of apoptosis (18). Given these results and the association of altered CerS expression with malignant transformation, the present study aimed to characterize the mRNA expression of various CerS genes in CRC and non-neoplastic adjacent tissues (NST).
The present study investigated the mRNA expression levels of various CerS genes using mRNA expression data from six independent CRC cohorts and a Korean CRC dataset. Furthermore, the clinical significance of altered CerS genes expression was evaluated in the Korean CRC dataset.
Materials and methods
Gene expression databases and cluster analysis
Gene expression RNAseq dataset (Level 3) and clinical data for The Cancer Genome Atlas Colon and Rectal Cancer (TCGA-COADREAD) cohort (19) were downloaded from the UCSC Xena (https://xena.ucsc.edu). CRC gene expression microarray data used in this study were downloaded from the publicly available GEO databases (http://www.ncbi.nlm.nih.gov/geo/): GSE21815 (20), GSE44076 (21), GSE44861 (22), GSE41258 (23) and GSE33113 (24). The GEO datasets used in this study include 562 CRC tissues and 222 NST from respective same patient groups. The downloaded raw data of GEO databases were normalized at the transcript and gene level using the Robust Multichip Average method (25). Cluster analysis was performed using Cluster 3.0 to classify the samples into statistically similar groups, and the resulting heatmaps were visualized in TreeView 1.6 (www.eisenlab.org/eisen). The four CerS genes present in the TCGA COADREAD, GSE44076 and GSE44861 cohorts were LASS2, LASS4, LASS5 and LASS6. The present study meets the publication guidelines provided by TCGA.
Patients and tissues
A total of 59 patients (mean age, 64.83±9.48; age range, 38–83; 34 males and 25 females) diagnosed with CRC were included in the present study. CRC and NSTs were obtained from patients undergoing surgery in Keimyung University Dongsan Medical Center (Daegu, Korea) between April 2008 and January 2010. Enrolled patients with CRC were classified according to the AJCC Tumor-Node Metastasis (TNM) staging criteria (26). Tissue samples were immediately frozen in liquid nitrogen and stored at −196°C until RNA isolation. Tissue samples were provided by Keimyung Human Bio-Resource Bank (Daegu, Korea). Written informed consent was obtained from each study participant and the protocols were approved by the Institutional Review Board of Keimyung University Dongsan Medical Center (approval no. 2015-11-059-001).
RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total cellular RNA was extracted from tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNA was quantified using NanoDrop 1000 (Thermo Fisher Scientific, Inc.). Each cDNA was synthesized from 2 µg total RNA using MMLV reverse transcriptase (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. qPCR was performed on the LightCycler® 480 Real-Time PCR system (Roche Diagnostics GmbH, Mannheim, Germany) using the specific primer pairs presented in Table I and SYBR-Green Premix (Toyobo Life Science, Osaka, Japan). The qPCR was performed using the following thermocycling conditions: 95°C for 10 min; followed by 45 cycles of 95°C for 10 sec, 60°C for 10 sec, and 72°C for 12 sec. Melting curve was analyzed to determine primer specificity. b-actin was used as a housekeeping gene for normalization, and a no-template sample was used as a negative control. qPCR data were analyzed using the 2−∆∆Cq method (27). Each experiment was performed three times.
Statistical analysis
Statistical analysis was performed using SPSS 22.0 (IBM Corp., Armonk, NY, USA). The cell viability data were analyzed using one-way analysis of variance and the Student-Newman-Keuls post hoc test. Differences between the groups were analyzed statistically using Student's t-test or Mann Whitney U test. The co-expression of the mRNAs of various CerS genes in TCGA-COADREAD cohort were searched using cBioPortal (http://cbioportal.org) (28). The association between inter-individual mRNA expression levels of CerS genes in Korean patients with CRC was assessed using Pearson's correlation coefficient analysis for continuous variables. Clinicopathological associations with the mRNA expression levels of various CerS genes in Korean CRC were analyzed using the Linear by linear association, the Pearson's Chi-square test and the Fisher's exact test for categorical variables. The mean value was used as the cut-off value (low and high) for categorical variables. P<0.05 was considered to indicate a statistically significant difference.
Transient transfection
Various human colorectal adenocarcinoma cell lines, HCT116, HT29, SW403 and SW480 cells, were plated onto 6-well plates at a density 7×105 cells/well and cultured overnight. pcDNA3.1-empty vector was used for plasmid constructs, including HA-tagged form of CERS2 (HA-CERS2) and HA-tagged form of CERS6 (HA-CERS6) constructs. All plasmids, including pcDNA3.1-empty vector, HA-CERS2 and HA-CERS6 were provided by Professor Anthony H. Futerman (Weizmann Institute of Science, Rehovot, Israel). The CRC cells were transfected with pcDNA3.1-empty vector, 2 µg HA-CERS2 and HA-CERS6 plasmid in 6-well plates using Lipofectamine reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. At 24 h after plasmid transfection, the subsequent experiments were conducted.
Western blot analysis
The transient transfected CRC cells were collected and washed twice with cold PBS, and cell pellets were prepared by suspending in modified radioimmunoprecipitation assay buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM Na3VO4 and 1 mM NaF) containing protease inhibitors (100 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin and 2 mM EDTA). The lysates were centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant fractions were collected. The total protein concentration was measured using Micro BCA™ Protein assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturers protocol. Cellular proteins (60 mg) were mixed with protein 5X sample buffer (Elpis Biotech., Inc., Daejeon, Korea) and heated at 95°C for 5 min. The proteins were separated by 10% SDS-PAGE and then electrotransferred to Immobilon-P membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blocked at room temperature with 5% skimmed dried milk in PBS/0.1% Tween-20 for 1 h, and incubated overnight at 4°C with anti-HA (1:2,000; mouse monoclonal; cat. no. SAB1411737) and anti-β-actin (1:2,000; mouse monoclonal; cat. no. A5441; both Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The membranes were then washed six times with PBS/0.1% Tween-20 (30 min each) and incubated with the corresponding secondary antibodies (horseradish peroxidase-conjugated, horse antibodies to mouse IgG; 1:2,000; cat. no. 7076; Cell Signaling Technology, Inc.) for 1 h at room temperature. Following washing six times in PBS/0.1% Tween-20, the specific protein bands were detected using an enhanced chemiluminescence western blotting kit (EMD Millipore), according to the manufacturer's protocol.
Results
Altered expression levels of sphingolipid metabolism-related genes in six independent CRC cohorts
To investigate whether the sphingolipid metabolism-related genes (29) are dysregulated in CRC tissues, the present study re-analyzed the raw data of six independent CRC cohorts. To begin with, the cancer gene expression RNAseq datasets of 380 CRC patients were taken from the TCGA-COADREAD cohort through UCSC Xena. Next, CRC gene expression microarray data were downloaded from the publicly available Gene Expression Omnibus databases. The CRC gene expression microarrays, GSE21815, GSE33113, GSE41258, GSE44076 and GSE44861, were analyzed for potential transcriptome changes. Hierarchical clustering revealed that various sphingolipid metabolism-related genes were dysregulated in carcinomatous tissues compared with NST of patients with CRC (Fig. 1). To identify the significance of altered mRNA expression levels between CRC and NST, Student's t-test or Mann Whitney U test were performed (P<0.05). As demonstrated in Fig. 2, hierarchical clustering revealed that various sphingolipid metabolism-related genes were significantly dysregulated in CRC tissues compared with NST from the same patient groups. The list of analyzed sphingolipid metabolism-related genes is presented in Table II. Sphingosine kinase 1 (SPHK1) and UDP-glucose glycoprotein glucosyltransferase 2 (UGGT2) were significantly upregulated in the CRC tissues of all cohorts, while 15-hydroxyprostaglandin dehydrogenase (HPGD), lysophosphatidic acid receptor 1 (LPAR1), N-acylethanolamine acid amidase (NAAA), sphingomyelin phosphodiesterase 1 (SMPD1) and sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) were significantly downregulated in the CRC tissues of all cohorts (Fig. 2 and Table II).
 | Table II.List of the analyzed genes involved in sphingolipid metabolism (Student's t-test, Mann Whitney U test; P<0.05). |
Dysregulation of various CerSs in six independent CRC cohorts
Next, the present study evaluated whether the mRNA expression levels of the four CerS genes, which are abundant in colorectal tissues (30), are dysregulated in human CRC specimens with respect to NST. As demonstrated in Fig. 3, among six cohorts, CERS2 mRNA levels were significantly increased in five independent cohorts, while CERS5 and CERS6 were significantly upregulated in four independent cohorts. The specific platforms of each cohort and their associated studies are listed in Table III.
| Table III.mRNA expression levels of CerS gene in colorectal cancer tissues of patients from various datasets used in the present study. |
Altered CerS genes mRNA expression in Korean patients with CRC
To determine whether there is altered CERS2, CERS4, CERS5 and/or CERS6 mRNA expression in Korean patients with CRC, the expression levels of these four CerSs were measured using qPCR in 59 paired CRC and NST specimens from Korean patients. Following exclusion of unqualified results, the qPCR data were analyzed. The present study revealed that mRNA expression levels of all four CerS genes were significantly upregulated in CRC tissues compared with corresponding NSTs (CERS2, P<0.001; CERS4, P=0.006; CERS5, P<0.001; CERS6, P<0.001; Fig. 4; Table IV).
| Table IV.mRNA expression levels of CerS gene in CRC tissues as compared with NST of Korean patients with CRC. |
Exogenous CERS2 and CERS6 expression decreases the viability of human CRC cells
It has previously been observed that CERS6-overexpression reduces the proliferation of CRC cells and induces apoptosis, whereas CERS2-overexpression increases the proliferation of CRC cells (18). To confirm the effect of overexpressing CerSs in CRC cells, HCT116, HT29, SW403 and SW480 cells were transiently transfected with constructs to overexpress HA-CERS2 and HA-CERS6, respectively. After 48 and 72 h, the numbers of viable cells were counted using a hemocytometer. As demonstrated in Fig. 5, overexpression of CERS2 and CERS6 decreased the viability of this panel of CRC cell lines.
Inter-individual associations between mRNA expression levels of CerS genes in patients with CRC
Combinational patterns of CerS gene expression, including CerS hetero-complexes and co-expression of CerS genes, serve important roles in sphingolipid metabolism (31,32). Therefore, associations between the mRNA levels of each CerS gene were identified in the TCGA-COADREAD cohort and in the Korean CRC cohort. Using cBioPortal to analyze the TCGA-COADREAD cohort, co-expression analysis revealed that CERS4 and CERS5 had high correlation coefficients (Pearson's correlation=0.36; Spearman's correlation=0.48; Fig. 6A). Next, these correlations were assessed using Pearson's correlation coefficient analysis in the 59 Korean patients with CRC. There were significant correlations between CERS2 and CERS4, and also between CERS5 and CERS6, with a Pearson's correlation coefficient value of 0.532 (P<0.001; Fig. 6B) and 0.439 (P=0.003; Fig. 6C), respectively. Furthermore, significant correlations between the mRNA expression levels of CERS2 and CERS4 (P=0.009) and of CERS5 and CERS6 (P<0.001) were identified using Fisher's exact test (Table V).
| Table V.Association between mRNA expression levels of various CerS genes and clinicopathological parameters in Korean patients with colorectal cancer. |
Association between mRNA expression levels of CerS genes and clinicopathological parameters of Korean patients with CRC
To determine the clinicopathological implications of dysregulated expression of specific CerS genes in CRC, the association between CerS gene mRNA level and clinicopathological characteristics, which are used to represent progression and aggressiveness, were evaluated. Prior to the statistical analysis, the 44 patients, whose clinical data were available, were classified according to each clinicopathological characteristic (Table V). The results obtained from the statistical analysis of the Korean cohort revealed that altered mRNA expression levels of CerS genes were not significantly associated with any clinical parameters, including sex, age, Tumor-Node-Metastasis stage, body mass index or carcinoembryonic antigen titer.
Discussion
Sphingolipid metabolism serves a critical role in mammalian cell growth arrest and survival (33). Accumulating evidence have demonstrated that CerS, a major component in sphingolipid metabolism (7), regulates various biological phenomenon, including apoptosis (34), cancer (17,35), ER stress (36), hepatopathy (37), hypoxia/re-oxygenation injury (38), lipid metabolism (39), neurodegeneration (40), and sensitivity to chemotherapeutic drugs and radiation (30). Although aberrant CerS expression is correlated with cell death and proliferation (10–14) in various types of cancer, much uncertainty remains regarding the dysregulated mRNA levels of CerS gene in CRC and the clinical implications of this.
The aims of the present study were to investigate the mRNA expression levels and functions of CerS genes, which are primarily expressed in the intestine (30,41), and analyze their clinicopathological implications in patients with CRC. To begin with, significantly dysregulated sphingolipid metabolism-related genes were identified in the heat-maps of 6 independent CRC cohorts (Fig. 2). The hierarchical clustering results demonstrated considerable dysregulation of sphingolipid metabolism-related genes in CRC tissues compared with corresponding NST of independent CRC cohorts. Among the considerably altered genes, certain genes were overlapping over 6 independent cohorts. SPHK1 and UGGT2 were significantly upregulated in CRC tissues. This result is in accordance with those of recent studies that indicated that SPHK1 is overexpressed and serves an important role in tumorigenesis, proliferation, invasiveness and metastasis in CRC (42,43). On the other hand, HPGD, LPAR1, NAAA, SMPD1 and SMPDL3A were all significantly downregulated in CRC tissues. HPGD is a cytoplasmic enzyme responsible for degrading PGE2 in colorectal tissue (44), and functions as a tumor suppressor gene in various types of cancer (45–48). The present study observed that HPGD was downregulated in 6 independent CRC cohorts (Fig. 2). However, little is known regarding the cellular functions and clinicopathological implications of LPAR1, NAAA, SMPD1, SMPDL3A and UGGT2 in CRC. Therefore, further studies investigating the functional role of these genes in CRC are required as these transcripts may be diagnostic markers or promising therapeutic candidates.
Additionally, the differential mRNA levels of CERS2, CERS4, CERS5 and CERS6 in CRC and NST were analyzed in 1,001 patients with CRC from 6 independent publicly-available CRC cohorts and a cohort of Korean patients with CRC. The results of the present study should be interpreted with caution as qPCR, RNA-Seq and microarray are different experimental platforms with different sensitivities, principles and dynamic ranges. Nonetheless, the results revealed that CERS2 was significantly upregulated in the majority of cohorts (Fig. 3; Table III) and in the cohort of Korean patients with CRC (Fig. 4; Table IV). It was recently demonstrated that CERS2-overexpression had no effect on the viability of HCT116 cells, whereas overexpressing CERS2 plus the addition of very-long chain acyl-CoAs significantly enhanced colony formation in HCT116 cells (18). Unlike these previous results, the present study revealed that CERS2-overexpression reduced the viability of various human CRC cells, including HCT116 cells (Fig. 5). The primary technical differences between these two experiments are the culture time following transfection and the use of different expression plasmids. Additionally, knockdown experiments were performed using shRNAs against CERS2 mRNA and CERS6 mRNA, knockdown of CerS2 or CerS6 did not affect the proliferation of CRC SW403 and SW480 cells (data not shown). Although the exact mechanism that underlies the effect on cell viability was not elucidated in the present study, it is possible that sustained cell culture time following transfection may affect the synthesis of ceramides of various chain lengths.
It has been reported that increased expression of CerS6 and C16:0-Ceramide resulted in a sensitization of SW620 cells to TRAIL-induced apoptosis (49), and CERS6-overexpression significantly inhibited the colony formation capacity and increased the apoptosis of HCT116 cells (18). In accordance with these previous results, the present study demonstrated that CERS6 is significantly upregulated in CRC tissues, compared with NST (Figs. 3 and 4; Table IV) and CERS6-overexpression led to inhibition of cell viability in various human CRC cells (Fig. 5). Notably, controversial results have demonstrated that CerS6 and C16:0-Ceramide protected cells against ER-stress in human head and neck squamous cell carcinomas (36). Although oncogenes are usually upregulated in cancer tissues compared with non-neoplastic tissues, previous studies and the results presented in the present study indicated that the roles of CerSs and ceramides of specific chain lengths are complicated and cell type-dependent. Notably, it was demonstrated that CERS4 was significantly upregulated, but only in the cohort of Korean patients with CRC (Fig. 4), while it was downregulated in the TCGA-COADREAD cohort (Fig. 3). Future studies specifically focused on CERS4 in different CRC populations are required in order to understand this phenomena.
Additionally, the present study evaluated correlations between inter-individual mRNA expression levels of CerS genes and their clinicopathological implications in patients with CRC. A recent study revealed that inter-individual differences in the mRNA expression levels of CerS genes are significantly correlated with each other in cancer tissues (17). Furthermore, Combinational patterns of CerS expression are involved in sphingolipid metabolism (31,32). The results of analyzing correlations between inter-individual CerS genes mRNA expression levels revealed a correlation between CERS4 and CERS5 in TCGA-COADREAD, between CERS2 and CERS4, and between CERS5 and CERS6 in the cohort of Korean patients with CRC. However, combinational patterns of CerS expression may be associated with sphingolipid metabolism. Therefore, it will be important to determine which components serve critical roles in sphingolipid metabolism in different disease and tissue settings. To the best of our knowledge, the present study was the first to investigate the clinicopathological implications of dysregulated CerS genes mRNA expression in CRC. However, no correlation was observed between mRNA expression levels of specific CerS genes and the investigated clinicopathological parameters.
In conclusion, the present study revealed that the mRNA expression levels of CERS2, CERS4, CERS5 and CERS6 were significantly upregulated or downregulated in various independent CRC cohorts, suggesting that dysregulated CerS gene expression may serve a role in CRC development.
Acknowledgements
The authors would like to thank all members of their research group for providing enthusiastic participation in the present study. The biospecimens for the present study were provided by the Keimyung Human Bio-Resource Bank, a member of the National Biobank of Korea, which is supported by the Ministry of Health and Welfare. All samples derived from the National Biobank of Korea were obtained following receipt of written informed consent and Institutional Review Board approval.
Funding
The present study was supported by the National Research Foundation of Korea Grant funded by the Korean Government (Ministry of Science, ICT & Future Planning; grant nos. 2017R1C1B5016670 and 2014R1A5A2010008).
Availability of data and materials
The datasets used during the present study are available from the corresponding author upon reasonable request. The primary and processed data used to generate the analyses presented here can be downloaded by registered users from The Cancer Genome Atlas at http://tcga-data.nci.nih.gov/tcga/tcgaDownload.jsp.
Authors' contributions
SWJ, WJP and SK contributed to the conception and design of the study, analysis of the data, interpretation of results and the writing of the manuscript. SWJ, WJP, HM and SK contributed to the acquisition of data. SWJ, WJP, HM, SKB, IH and SK performed the experiments. TKK contributed to the conception and design of the study. JWP and IH contributed to the conception and design of the study and provided guidance regarding the clinical implications of the study. WJP and SK reviewed and edited the manuscript. All authors read and approved the manuscript, and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Ethics approval and consent to participate
The experimental study was approved by the Institutional Review Board of Keimyung University Dongsan Medical Center (approval no. 2015-11-059-001). Written informed consent was obtained from each study participant.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interest.
Glossary
Abbreviations
Abbreviations:
|
CRC |
colorectal cancer |
|
CerS |
ceramide synthase |
|
TCGA-COADREAD |
The Cancer Genome Atlas Colon and Rectal Cancer |
|
qPCR |
quantitative polymerase chain reaction |
|
NST |
non-neoplastic surrounding colon tissues |
|
LPAR1 |
lysophosphatidic acid receptor 1 |
|
NAAA |
N-acylethanolamine acid amidase |
|
SPHK1 |
sphingosine kinase 1 |
|
HPGD |
15-hydroxyprostaglandin dehydrogenase |
|
SMPD1 |
sphingomyelin phosphodiesterase 1 |
|
SMPDL3A |
sphingomyelin phosphodiesterase acid-like 3A |
|
UGGT2 |
UDP-glucose glycoprotein glucosyltransferase 2 |
|
HA-CERS2 |
HA-tagged form of CERS2 |
|
HA-CERS6 |
HA-tagged form of CERS6 |
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