Abstract
Background/Aim: Lysophosphatidylinositol (LPI) is a subspecies of the lysophospholipid mediators produced when phospholipase hydrolyzes membrane phosphatidylinositol. Previously, we used mass spectrometry-based lipidomics to demonstrate that LPI is selectively elevated in colorectal cancer (CRC) tissues. Here, we hypothesized that the expression levels of the LPI biosynthetic enzyme and LPI receptor - DDHD domain containing 1 (DDHD1) and G protein-coupled receptor 55 (GPR55), respectively - may be correlated with malignant potential, and we evaluated their roles in the context of CRC. Materials and Methods: Colorectal specimens from 92 CRC patients underwent DDHD1 and GPR55 immunolabeling. Correlation between protein expression levels and clinicopathological variables was examined. Results: Depth of tumor invasion was positively correlated with DDHD1 expression. Regardless of the degree of invasion depth, GPR55 was highly expressed in CRC tissues. Neither DDHD1 nor GPR55 expression levels were associated with disease-free survival. Conclusion: DDHD1 expression is associated with depth of tumor invasion in CRC tissues and may be involved in tumor progression.
Lysophospholipids (LPLs) are generated through membrane phospholipid hydrolysis by phospholipase A1 (PLA1) or A2 (PLA2). Recent studies have revealed that LPLs exhibit significant biological activity as lipid mediators, by signaling through a set of G protein-coupled receptors. In addition, PLA2 comprises a group of enzymes that hydrolyze phospholipids at the sn-2 position to liberate fatty acids and 1-acyl LPLs, which exert various physiological effects and are involved in tumor growth and invasion (1, 2).
Owing to technological improvements in mass spectrometry, this technique is now well-suited to quantify lysophospholipid species in clinical tissues. In a previous study, we used liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) to comprehensively profile the LPLs present in human colorectal cancer (CRC) tissues. That study demonstrated that lysophosphatidylserine (LPS) and lysophosphatidylinositol (LPI) levels were higher in CRC tissues compared to normal tissues (3). Specifically, levels of 1-acyl 18:0 LPI and 1-acyl 18:1 LPI (both produced by PLA2) as well as 2-acyl 20:4 LPI (produced by PLA1) were higher in CRC tissues (3). In recently, similar studies have been reported for the levels of LPI species, which performed mass spectrometry in tissues or bloods of the patients with CRC and inflammatory bowel disease (4, 5). While LPS is reportedly involved in various cellular functions, relatively little is known about the roles of LPI in the context of CRC.
Regarding the role of LPS in CRC, expression of PS-PLA1, (an enzyme that produces LPS) correlates with CRC depth of invasion and hematogenous metastasis (6). We have also previously reported that LPS promotes the migratory capacity of colorectal cancer cell lines via the LPS receptor and the PI3K/Akt pathway (7). In other cancers, LPS also impacts several cellular response functions, including mast cell degranulation (8, 9), lymphocyte proliferation (10), fibroblast migration (11), and neurite outgrowth (12).
The roles of LPLs such as lysophosphatidic acid and sphingosine-1-phosphate (13, 14) in solid cancers are well-established. Regarding the potential role of LPI in cancer, it binds to G protein-coupled receptor 55 (GPR55) (15); high levels of GPR55 can be detected in the spleen, thymus, testis, small intestine, brain, and other organs and the receptor has been suggested to play an important role in immune function (16, 17). Furthermore, analyses of breast cancer and skin squamous cell carcinoma have demonstrated an association between overexpression of GPR55 and cell proliferation (18, 19). However, the roles of LPI, its biosynthetic enzyme and its receptor have not been well evaluated in the context of CRC. Classification of LPI into 1-acyl or 2-acyl species depends on the position at which fatty acids are linked to the glycerol backbone: PLA1 produces 2-acyl LPI, whereas PLA2 produces 1-acyl LPI. Intracellular PLA1 is named DDHD domain containing 1 (DDHD1) and, accordingly, produces 2-acyl LPI. PLA2 is involved in the production of both LPI and other LPLs, and many studies report its association with cancer (1, 2, 20, 21). However, whether a correlation exists between PLA1 and CRC remains unclear.
Since no existing reports have evaluated expression of GPR55 and DDHD1 in human CRC, the present study aimed to clarify whether GPR55 and DDHD1 are involved in the pathophysiology of CRC. Human CRC tissues underwent polyclonal immunolabeling of GPR55 and DDHD1, and correlation between protein expression levels and clinicopathological variables was analyzed.
Materials and Methods
Participants and samples. Patients undergoing surgery for cancer of the colon or rectum (superior to the peritoneal reflection) at the University of Tokyo Hospital between January and December 2012 were recruited. Of a total of 150 enrolled patients, 120 underwent curative surgery (complete resection), including lymph node dissection. Patients diagnosed with ulcerative colitis, familial adenomatous polyposis, multiple CRCs, or another advanced cancer type, and those who underwent emergency surgery, neoadjuvant chemoradiotherapy, or pre-surgical endoscopic mucosal resection were excluded. Surgical specimens were preserved in 10% buffered formalin and embedded in paraffin blocks. Patient records were reviewed for clinicopathological data, and all lesions were staged according to tumor-node-metastasis (TNM) Classification of Malignant Tumor guidelines were provided by the Union for International Cancer Control (UICC, 7th edition) (22). The study protocol was approved by the Research Ethics Committee of the Graduate School of Medicine, University of Tokyo (Tokyo, Japan) (approval no. G3552-8). All participants provided written informed consent. This study was conducted in accordance with the principles laid out in the 1964 Declaration of Helsinki and its subsequent amendments.
Immunohistochemical evaluation. Consecutive 3-μm-thick formalin-fixed, paraffin-embedded sections underwent manual immunohistochemical labeling. Briefly, following deparaffinization and rehydration, endogenous peroxidase activity was blocked using 3% hydrogen peroxidase in methanol for 15 min. Autoclave-assisted heat-induced antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0). Following incubation in 5% bovine serum albumin for 30 min to block non-specific binding-sites, slides were incubated overnight at 4°C in the presence of primary antibodies against GPR55 (polyclonal rabbit anti-human; bs-7686R; 1:200 dilution; Bioss Antibodies Inc., Woburn, Massachusetts, USA) and DDHD1 (polyclonal rabbit anti-human; PA5-62070; 1:500 dilution; Thermo Fisher Scientific, Minato-ku, Tokyo, Japan). After rinsing with deionized water, slides were incubated in the presence of a labeled polymer (Histofine®, Simple Stain MAX-PO (Rat), Nichirei Bioscience Inc., Chuo-ku, Tokyo, Japan) for 30 min at room temperature, followed by addition of diaminobenzidine solution for 7 min. A cocktail of Mayer’s/Lillie-Mayer’s hematoxylin (Mayer’s hematoxylin Solution® and Lillie-Mayer’s hematoxylin Solution®, Muto Pure Chemicals Co., LTD, Bunkyo-ku, Tokyo, Japan) was used as a counterstain. Finally, sections were dehydrated by means of exposure to progressively increasing percentages of ethanol, and were then incubated in xylene prior to mounting.
Slides were independently evaluated at a final magnification of 200× and immunolabeling intensity of cancer cells was classified into four grades: negative, weak, moderate, and strong according to a previous report (6) (Figures 1 and 2). Sections consisting of >50% moderate- or strong-grade cells were classified as GPR55-high/DDHD1-high, while those consisting of ≤50% of such cells were classified as GPR55-low/DDHD1-low. Each section was assessed by two independent observers blinded to clinical findings.
Colorectal cancer sections immunostained by a polyclonal antibody against human G protein-coupled receptor 55 (GPR55). GPR55 staining was mainly observed in cancerous cell membranes (relative to normal epithelia and stromal cells). The immunostaining intensity was classified into four grades, as following: negative, weak, moderate, and strong. Scale bar=200 μm.
Colorectal cancer sections immunostained by a polyclonal antibody against human DDHD domain containing 1 (DDHD1). DDHD1 staining was mainly observed in the cytoplasm of cancerous cells, which generally exhibited more intense staining for this molecule (relative to normal epithelia and stromal cells). The immunostaining intensity was classified into four grades, as following: negative, weak, moderate, and strong. Scale bar=200 μm.
Statistical analysis. For correlation of GPR55 and DDHD1 expression levels with patient clinicopathological features, unpaired t-test was used for comparing continuous variables, and the chi-square test was employed for comparing categorical data.
To determine prognostic value of GPR55 and DDHD1 expression levels, association of section grade with disease-free survival (DFS) rates of 92 patients who had undergone curative surgery was analyzed using the Kaplan-Meier method and log-rank test. All statistical analyses were performed using JMP Pro version 15.0.0 (SAS Institute, Cary, NC, USA). Associations were considered significant at p<0.05.
Results
Patient and lesion characteristics. Clinicopathological features are shown in Table I. There were 53 male and 39 female patients, with a median age at surgery of 67.5 years (range=38-90). Thirty-eight patients (41%) had right-sided colon cancers, while 54 patients (59%) had left-sided colon and upper rectum cancers. Histopathological analysis identified the tumor types carried by 75 patients as differentiated adenocarcinoma. The values for T1-2, T3-T4a were 22% and 78%, respectively. The mean tumor diameter was 45.6±24.4 mm. Lymphatic invasion, venous invasion, and lymph node metastasis were apparent in 33%, 70%, and 38% of the patients, respectively.
Patient clinicopathological characteristics.
Immunohistochemistry and section grading. Staining for GPR55 was most intense in cancer cell membranes (relative to normal epithelia and stromal cells). Staining for DDHD1 was observed mainly in the cytoplasm of cancer cells, which generally exhibited more intense staining for this molecule (relative to normal epithelia and stromal cells). Among 92 cases, 77 were graded as GPR55-high, 15 were graded as GPR55-low, 69 were graded as DDHD1-high, and 23 were graded as DDHD1-low.
Correlation of GPR55 and DDHD1 expression with clinicopathological variables. Associations between GPR55 expression levels and clinicopathological variables are shown in Table II. There were no significant differences in patient characteristics, tumor location or pathological features between the two groups.
Univariate analysis of the association between clinicopathological variables and G protein-coupled receptor 55 (GPR55) immunostaining.
Results of univariate analyses of associations between DDHD1 expression levels and clinicopathological variables are shown in Table III. High levels of DDHD1 expression were observed in patients with advanced tumor depth (p=0.03). However, no significant correlation was observed between DDHD1 expression and any other clinicopathological variable.
Univariate analysis of the association between clinicopathological variables and DDHD domain containing 1 (DDHD1) immunostaining.
Prognostic value of GPR55 and DDHD1 expression levels in patients with CRC. The DFS rate following curative surgery was not associated with the expression levels of either GPR55 or DDHD1 (five-year DFS rates for GPR55-low and -high groups, respectively: 78.0% and 67.0%, p=0.89; five-year DFS rates for DDHD1-low and -high groups, respectively: 77.0% and 70.0%, p=0.29) (Figures 3 and 4). The commonest sites of recurrence were the liver and lymph nodes (five patients per group), followed by the peritoneum (three patients) and local recurrence (two patients).
The disease-free survival (DFS) rate for the 92 patients who underwent curative surgery, as evaluated by the log-rank test. The continuous line indicates the DFS curve of the GPR55-high group and the dashed line that of the GPR55-low group.
The disease-free survival (DFS) rate for the 92 patients who underwent curative surgery, as evaluated by the log-rank test. The continuous line indicates the DFS curve of the DDHD1-high group and the dashed line that of the DDHD1-low group.
Discussion
Our previous work examining human CRC tissue metabolism via LC-MS/MS demonstrated higher levels of LPI and LPS than those in normal tissues (3). Specifically, levels of 1-acyl 18:0 LPI and 1-acyl 18:1 LPI (both produced by PLA2) as well as 2-acyl 20:4 LPI (produced by PLA1) were higher in CRC tissues (3). While LPS is reportedly involved in various cellular functions, relatively little is known about the roles of LPI in the context of CRC. In addition, although PLA2 is known to promote CRC (23), knowledge regarding the role of PLA1 in the context of CRC is lacking. To explore whether CRC patient clinicopathological factors were associated with the expression of molecules involved in LPI signaling, correlations between DDHD1 and GPR55 expression patterns and CRC clinicopathological features were elucidated.
Results demonstrated a significant positive correlation between depth of tumor invasion and DDHD1 expression. Previously, DDHD1 (the enzyme which preferentially generates 2-acyl LPI species) was known as phosphatidic acid-preferring PLA1 (PA-PLA1) (24). This enzyme is believed to hydrolyze cell membrane phosphatidylinositols to generate arachidonic acid-containing 2-acyl LPI (25). It has been reported that DDHD1 enhances proliferation and survival of CRC cells: its down-regulation decreases colon cancer cell viability and increases the rate of apoptosis of these cells in vitro. Furthermore, silencing DDHD1 inhibits MERK/ERK and PI3K/Akt signaling, and DDHD1 over-expression supports in vitro and in vivo cancer cell growth by stimulating ERK1/2 signaling (26). In conjunction with results of the present study, these findings suggest that up-regulation of DDHD1 may be involved in CRC progression.
Similarly, GPR55 is reportedly involved in the pathogenesis of neoplasia (27). In a murine model of colitis-associated CRC, GPR55–/– mice exhibited fewer tumors and lower total tumor areas than their wild-type counterparts. In addition, GPR55 up-regulation in CRC patients is associated with lower relapse-free survival rates (28). In contrast, the present study does not demonstrate a significant correlation between GPR55 over-expression and clinicopathological factors or clinical outcome. Results must be validated in larger cohorts, using orthogonal methods.
However, preliminary data indicate that while GPR55 expression in adenoma tissues is nearly absent (data not shown) and that GPR55 is expressed in CRC tissue samples in 77 of 92 patients (83.7%, Table II). It remains possible that expression of GPR55 becomes up-regulated during progression from adenoma to carcinoma, whether or not this is causally linked to pathogenesis. Further studies are required to evaluate GPR55 and DDHD1 expression dynamics during adenoma to carcinoma progression. As the bioactivity of 2-acyl LPI is markedly higher than that of other LPI species, this species may be preferentially involved in GPR55-mediated signal transduction (29).
In conclusion, the present study demonstrated that depth of tumor invasion is positively correlated with DDHD1 expression, suggesting that the DDHD1-LPI-GPR55 axis may play a role in the progression of CRC.
Acknowledgements
This research is supported by Grants-in-Aid for Scientific Research (C: grant number; 18K07194, C: grant number; 19K09114, C: grant number; 19K09115, C: grant number; 20K09051, Challenging Research (Exploratory): grant number; 20K21626) from Japan Society for the promotion of Science. This research is supported by the Project for Cancer Research and Therapeutic Evolution (P-CREATE), grant number: JP 19cm0106502 from the Japan Agency for Medical Research and Development (AMED).
- Received February 18, 2021.
- Revision received March 23, 2021.
- Accepted March 26, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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