Pei Chen, Shiwen Wang, Bo Chen, Bing Lu, Xiaojing Zhang, Xiaohui Jiang, Yao Wang. High LIMP2 expression serves as a predictor for improved clinical outcomes in gastric cancer patients[J]. Blood&Genomics, 2020, 4(2): 142-150. DOI: 10.46701/BG.2020022020119
Citation:
Pei Chen, Shiwen Wang, Bo Chen, Bing Lu, Xiaojing Zhang, Xiaohui Jiang, Yao Wang. High LIMP2 expression serves as a predictor for improved clinical outcomes in gastric cancer patients[J]. Blood&Genomics, 2020, 4(2): 142-150. DOI: 10.46701/BG.2020022020119
Pei Chen, Shiwen Wang, Bo Chen, Bing Lu, Xiaojing Zhang, Xiaohui Jiang, Yao Wang. High LIMP2 expression serves as a predictor for improved clinical outcomes in gastric cancer patients[J]. Blood&Genomics, 2020, 4(2): 142-150. DOI: 10.46701/BG.2020022020119
Citation:
Pei Chen, Shiwen Wang, Bo Chen, Bing Lu, Xiaojing Zhang, Xiaohui Jiang, Yao Wang. High LIMP2 expression serves as a predictor for improved clinical outcomes in gastric cancer patients[J]. Blood&Genomics, 2020, 4(2): 142-150. DOI: 10.46701/BG.2020022020119
Xiaohui Jiang, Department of General Surgery, Nantong Tumor Hospital, 48 Xisi Road, Nantong, Jiangsu 226361, China. E-mail: jxhyjl@163.com
Yao Wang, Department of General Surgery, the Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, Jiangsu 226021, China. E-mail: wangyao@ntu.edu.cn
Lysosomal integral membrane protein-2 (LIMP2) is an important component of innate immunity. However, its role in the anti-tumor response remains unknown. Here, we found that the level of LIMP2 mRNA was frequently upregulated in gastric cancer (GC) tissues according to the TCGA, which was confirmed by immunohistochemistry in 329 cases. LIMP2 expression was significantly associated with Lauren classification (P=0.042), depth of invasion (P=0.016), lymph node metastasis (P=0.039) and TNM stage (P=0.027). LIMP2 expression (P < 0.001), differentiation (P < 0.001), TNM stage (P < 0.001) and preoperative CEA (P=0.018) can be used as independent prognostic factors. GC patients with higher levels of LIMP2 mRNA experienced improved clinical outcomes. Mechanically, the TGF-β signaling pathway, the ERBB signaling pathway, and Toll-like receptor signaling were enriched in proteins with higher LIMP2. Moreover, LIMP2 expression was positively related with M1 tumor-associated macrophages (TAMs) in TCGA data, which was verified by capturing LIMP2 and CD86 co-expression in GC samples. The study suggests that LIMP2 expression in GC would predict improved outcomes with M1 TAMs infiltration.
Gastric cancer (GC) is the fifth most frequently occurring malignancy and the third leading cause of cancer-related mortality worldwide[1]. Although rates of gastric cancer incidence have decreased in Western countries, they are increasing in China[1]. During the early stages of GC, most patients are asymptomatic, and consequently, diagnosis is frequently made when the disease is at an advanced stage[2]. The 5-year net survival rate of patients with GC in China is 30%-39% due to postsurgical recurrence and metastasis[3]. Thus, it is of paramount importance to develop methods for evaluating and improving patients' survival.
The Cancer Genome Atlas (TCGA) program collected over 11 000 primary cancer samples, which provided abundant information for researchers. When we analyzed the TCGA data, we found that LIMP2 expression positively associated with oncogenes and tumor suppressor genes in the stomach adenocarcinoma database and is upregulated in human GC compared with normal human gastric samples.
Scavenger receptor class B member (SCARB2) is the gene of LIMP2 protein. LIMP2 is an abundantly expressed type Ⅲ double-transmembrane glycoprotein (MIM: 602257, Locus: NM_005506) with N-as well as C-terminal cytoplasmic tails consisting of 20 amino acids, and LIMP2 is primarily expressed in lysosomes and late endosomes in diverse cell types[4-5]. Sequence analysis classified LIMP2 as a member of the class B scavenger receptor family that includes CD36 and SR-B1[6-7]. LIMP2 and glucocerebrosidase(GCase) interact in a highly specific and pH-dependent manner in the endoplasmic reticulum, to mediate transport to lysosomes and late endosomes[8].
Deficiency in LIMP2 decreases the levels and activity of lysosomal GCase, accompanied by the accumulation of glucosylceramide, which eventually causes Gaucher disease and contributes to Parkinson disease[9-11]. Furthermore, LIMP2 functions as an efficient plasma membrane-localized receptor that mediates the uptake of enterovirus 71 and coxsackievirus A16, which are major causative agents of hand, foot and mouth disease[12-13]. Moreover, LIMP2 is required for the maintenance of endosomes and lysosomes and lipid transport[14-15]. LIMP2 mutations in humans may represent the major cause of myoclonus-renal failure syndrome, and evidence indicates that LIMP2 deficiency in mice causes deafness and peripheral neuropathy[16-17]. The level of LIMP2 mRNA increases approximability 2-4 fold in prostate cancer cells vs. nonmalignant cells[18]. Upregulation of LIMP2 expression can predict lymph node metastases in oral squamous cell carcinoma[19]. However, the expression levels and effects of LIMP2 on GC have never been studied before and are worthy of exploration.
In this study, we showed that LIMP2 mRNA expression levels correlated with upregulated tumor-associated genes in corresponding tumors in the TCGA database. The levels of LIMP2 were elevated in human gastric cancer tissues compared with normal tissues. Further, univariate and multivariate analysis revealed the potential of LIMP2 expression for predicting metastasis and survival of patients with GC.
MATERIALS AND METHODS
Patients' clinicopathologic characteristics
Paraffin-embedded tumor tissues (n = 329) were collected from patients with gastric cancer who underwent curative primary tumor resection with or without lymph node dissection at the Affiliated Hospital of Nantong University (Jiangsu, China), between April 2004 and December 2010. Clinical data including name, sex, age, pathological diagnosis, histological type, differentiation grade, TNM stage, lymph node metastasis, and preoperative serum CEA level were retrospectively collected from medical records. Patients who met the criteria as follows were selected: ①histological and clinical diagnosis of GC; ②no preoperative chemotherapy, radiotherapy, immuno-therapy, or molecularly targeted therapy; ③no serious complications; ③clinical and follow-up information available.
Data for survival, death, and the causes of death after surgery were updated through a review of the patients' medical records and telephone calls. We calculated the OS from the date of surgery to the date of death or last follow-up. The Human Research Ethics Committee of the Affiliated Hospital of Nantong University approved the study protocol. The research conducted in this study complied with the terms of the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.
Immunohistochemistry(IHC)
As previously described[20], tissue microarrays were baked for 2 hours at 60℃, deparaffinized in xylenes, and rehydrated through an ethanol series (70%, 80%, 95% and 100%). Endogenous peroxidase activity was quenched using 3% H2O2 and methanol for 15 min. Next, the sections were boiled in citric acid (MVS-0101, MXB Biotechnologies, FuZhou, China) for 30 min in a pressure cooker. Tissues were then incubated with primary mouse anti-LIMP2 antibody (ab176317, Abcam, Cambridge, UK), diluted 1:400, overnight at 4℃ in a moist chamber. The primary antibody was removed using PBS (3 × 5 min) and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (kit-0015, MXB) for 30 min at room tem-perature. Subsequently, the sections were stained using 3, 3´-diaminobenzidine (kit-0015, MXB) and counterstained with hematoxylin. The sections were then dehydrated and mounted on a glass slide.
IHC image segmentation and automated scoring
The Vectra Multispectral Imaging System (Perkin Elmer, Hopkinton, MA, USA) was used to analyze the IHC slides. Briefly, entire stained images were scanned, and multispectral image cubes were acquired using the Phenochart Whole Slide Contextual Viewer (Perkin Elmer). InForm v2.2 software was trained to identify segment tissue and extraneous substances. The nucleus of each cell was visualized using hematoxylin staining, and a tissue size range 30–50 µm was used to identify the nucleus to further improve the accuracy of identification. The cytoplasmic signal was obtained by sampling the perinuclear area. With the software trained to identify tissue and cell segmentation, the InForm system can effectively identify the nucleus and cytoplasm of each cell, and the data can be automatically applied to the 329 samples. To evaluate the accuracy of our image analysis system for identifying cell segmentation, two pathologists evaluated the image segment results. The accuracy rate for identifying cells reached 95%.
Staining intensity scores included staining that was undetectable (score: 0), weak (score: 1), moderate staining (score: 2), and strong (score: 3)[21]. The frequency of each intensity category (0, 1+, 2+, and 3+) cell was automatically counted in each core. The H score of LIMP2 expression was equal to the sum of the percentages of cells (0%–100%) in each intensity category. The final score was assigned using a con-tinuous scale between 0 and 300. With the help of Xtile software, a score of 66 was used as a cutoff for classifying patients into either the LIMP2high or LIMP2low groups.
Public datasets analysis
Normalized gene expression data for normal and cancer tissues, profiled using the TCGA database were downloaded on April 19, 2019. Gene expression values are presented as X = log2(X+1), where X is the normalized value of the fragments per kilobase transcript per million mapped reads. RNAseq data for 407 unmatched GC samples were used to analyze the levels of LIMP2 expressed by GC and normal gastric tissue samples.
Pathway enrichment analysis
GSEA is a threshold-free method for analyzing genes according to their expression ranks without gene filtering[22]. The levels of LIMP2 mRNA expressed by TCGA samples of stomach adenocarcinomas were previously grouped as LIMP2high or LIMP2low according to the median value. GSEA was performed according to the correlations of a list of genes with LIMP2 expression. The pathway comprising genes enriched at the top or bottom of the ordered gene list was searched using GSEA. This process was quantized using an enrichment score that reflects pathway enrichment in the top or bottom of the ranked gene list. The Q value (Q < 0.05) of the false-discovery rate was used to select pathways, and a normalized enrichment score reflecting pathway enrichment in this list was used to rank the identified pathway.
TIMER analysis
TIMER is computational tool used to investigate the molecular characteristics of tumor-immune inter-actions[23]. The expression level of six types of tumor-infiltrating immune cells (macrophages, neutrophils, B cells, CD4+ T cells, CD8+ T cells and dendritic cells) were precalculated for 10 897 tumors in the TCGA database, which allows users to infer the abundance of tumor-infiltrating immune cells form specific gene expression profiles. Here, we calculated the correlation between LIMP2 expression and the abundance with the six types of tumor-infiltrating immune cells listed above. Further, we applied GEPIA to reconfirm this correlation[23]. The Pearson correlation coefficient was determined to assess the correlated LIMP2 levels with a set of gene markers for tumor-infiltrating immune cells.
Statistical analysis
Data are expressed as the mean±SD from at least three independent experiments and were analyzed using a two-tailed Student t test for binary measures or ANOVA for more than two measures by using GraphPad 7.0 (GraphPad Software Inc., San Diego, CA, USA). Pearson's chi-square test was applied to investigate the relationships between LIMP2 levels and clinicopathologic variables using Stata 15.0 (Stata-Corp, College Station, TX, USA). The 5-year OS curves were generated and analyzed using the Kaplan-Meier method and the log-rank test. The Cox multivariate proportional hazards regression model was used to identify independent factors that influenced survival and recurrence by using SPSS 23.0 (SPSS, Chicago, USA). P < 0.05 was considered statistically significant.
RESULTS
LIMP2 is abundantly expressed in gastric cancer
By analyzing the TCGA stomach adenocarcinoma database, we found that LIMP2 mRNA was highly expressed in GC compared with gastric mucosa tis-sues (P < 0.0001, Fig. 1A) and significantly correlated with tumor associated genes such as BRAF (P < 0.0001, r=0.63), PTEN (P < 0.0001, r=0.57), APC(P < 0.0001, r=0.61), and VHL(P < 0.0001, r=0.5) (Fig. 1B). To verify the expression of LIMP2 in GC, we performed IHC analysis of a tissue array representing 260 GC tissue samples, 33 normal tissue samples and 54 benign samples. The automatic analysis by InForm software helped score tissues. The LIMP2 positive staining rate was significantly higher in GC tissue samples (59.61%, 155/260) than normal and benign samples (36.78%, 32/87) (P < 0.0001, Fig. 1C, Fig. 1D). As shown in Fig. 2, LIMP2 was more frequently expressed in GC tissues, but was negatively expressed in normal tissues. Meanwhile, the localization of LIMP2 expression was mainly in the cytoplasm of tumor cells.
Figure
1.
LIMP2 expression is abundant in GC.
A: TCGA analysis of LIMP2 mRNA expression in GC relative to normal tissues; ****P < 0.0001. B: Pearson correlation analysis of SCARB2, BRAF, PTEN, APC, and VHL. C: IHC analysis of LIMP2 protein expression in GC relative to normal tissues; ***P < 0.001. D: The percentage of LIMP2 expression in benign samples and GC tissue samples.
A: Negative LIMP2 staining in normal gastric tissues (a ×40, A ×400). B: Weak staining of LIMP2 in the cytoplasm of well differentiation (b ×40, B ×400). C: Moderate staining of LIMP2 in the cytoplasm of middle differentiation (c ×40, C ×400). D: Strong staining of LIMP2 in the cytoplasm of poor differentiation (d ×40, D ×400).
LIMP2 expression is associated with patients' clinical characteristics
The association between LIMP2 protein expression and clinicopathologic parameters of patients with GC are presented in Table 1. LIMP2 levels significantly correlated with Laurén classification (P=0.042), suggesting that downregulation of LIMP2 was more frequently associated with a worse classification. Moreover, decreased LIMP2 levels were significantly associated with advanced TNM stage (P=0.027), particularly with advanced T stage (P=0.016) and lymph node metastasis (P=0.039). There was no significant difference between LIMP2 expression and sex (P=0.562), age(P=0.185), differentiation (P=0.073), preoperative CEA levels (> 5 ng/mL, P=0.054), and distant metastasis (P=0.310). This analysis reveals that LIMP2 levels might play a certain role in GC.
Table
1.
Relationship between the expression of LIMP2 and clinicopathologic characteristics in gastric cancer patients
Characteristics
n
LIMP2 low or no expression[n(%)]
LIMP2 high expression[n(%)]
Pearson χ2
P
Sex
0.3367
0.562
Male
183
76(41.53)
107(58.47)
Female
077
29(37.66)
048(62.34)
Age
1.7569
0.185
< 60
094
43(45.74)
051(54.26)
≥60
166
62(37.35)
104(62.65)
Laurén classification
6.3183
0.042
Intestinal type
080
26(32.50)
054(67.50)
Diffuse type
160
74(46.25)
086(53.75)
Mixed type
020
05(25.00)
015(75.00)
Differentiation
3.2253
0.073
Poor
151
68(45.03)
083(54.97)
Well-middle
082
27(32.93)
055(67.07)
othersa
027
10(37.04)
017(62.96)
pT status
8.2975
0.016
T1 and T2
077
25(32.47)
052(67.53)
T3
084
29(34.52)
055(65.48)
T4a and T4b
099
51(51.52)
048(48.48)
pN status
4.2601
0.039
N0
104
34(32.69)
070(67.31)
N1, N2 and N3
156
71(45.51)
085(54.49)
pM status
1.0300
0.310
M0
247
98(39.68)
149(60.32)
M1a and M1b
013
07(53.85)
006(46.15)
TNM stage
9.2203
0.027
0 and Ⅰa and Ⅰb
055
18(32.73)
037(67.27)
Ⅱa and Ⅱb
076
23(30.26)
053(69.74)
Ⅲa and Ⅲb and Ⅲc
110
54(49.09)
056(50.91)
IV
019
10(52.63)
009(47.37)
Preoperative CEA
3.7056
0.054
≤5 ng/mL
159
59(37.11)
100(62.89)
> 5 ng/mL
041
22(53.66)
019(46.34)
Unknown
060
24(40.00)
036(60.00)
Othersa : mucinous adenocarcinoma, signet ring cell carcinoma and undifferentiated carcinoma.
LIMP2 expression indicates improved clini-cal outcomes of patients with GC
Among the 260 patients, 124 (47.69%) were alive and 136 (52.31%) had passed away at the last follow-up (median observation period, 58 months; range, 2 months to 113 months). Univariate analyses revealed that differentiation (HR: 0.594, P < 0.001), T stage (HR: 1.623, P < 0.001), lymph node metastasis (HR: 1.904, P < 0.001), distant metastasis (HR: 2.470, P=0.006), preoperative CEA (HR: 1.707, P=0.018), and LIMP2 level (HR: 0.508, P < 0.001) were significantly related to 5-year OS (Table 2). Multivariate analysis of survival revealed that differentiation (HR: 0.603, P=0.001), TNM stage (HR: 2.326, P < 0.001), and LIMP2 level (HR: 0.559, P=0.001) were independent predictors of OS (Table 2). Moreover, patients with high-levels of LIMP2 had better clinical outcomes compared with those with low-levels (Fig. 3).
Table
2.
Univariate and multivariable analysis of prognostic factors for 5-year survival in gastric cancer
Factors
Univariate analysis
Multivariate analysis (adjusted for age)
HR
P > |z|
95% CI
HR
P > |z|
95% CI
LIMP2 expression
High vs. low and none
0.508
< 0.001
0.361
0.714
0.559
< 0.001
0.395
0.790
Age (years)
≤60 vs. > 60
0.945
0.755
0.664
1.346
Sex
Male vs. female
0.781
0.179
0.544
1.120
Laurén classification
Intestinal type vs. Diffuse type vs. mixed type
1.052
0.726
0.791
1.401
Differentiation
Well and middle vs. poor
0.594
< 0.001
0.448
0.788
0.603
< 0.001
0.451
0.806
TNM stage
0 and Ⅰa and Ⅰb vs.Ⅱa and Ⅱb vs.Ⅲa and Ⅲb and Ⅲc vs. IV
Bioinformatics predicts LIMP2 functions in immunity
We used GSEA to analyze expression data acquired from the TCGA. Eight pathways were significantly associated with the LIMP2high group, and five of eight pathways were associated with the phenotypes of cancer cells. The TGF-β signaling pathway, the ERBB signaling pathway, and Toll-like receptor signaling were significantly enriched in the LIMP2high group (Fig. 4).
Figure
4.
The TGF-β signaling pathway(A), the ERBB signaling pathway(B), and the Toll-like receptor signaling pathways(C) that are enriched in the LIMP2-upregulated group.
LIMP2 participates in innate immunity by inducing the production of proinflammatory cytokines and tumor-infiltrating lymphocyte cells, which are important factors that affect survival[25-26]. TIMER analysis revealed that SCARB2 levels were significantly associated with macrophages (P < 0.0001, r=0.345) (Fig. 5A). Spearman analysis confirmed the significance of the correlation between SCARB2 levels and immune cells markers determined by GEPIA. CD80 and CD86 are hallmarks of M1 TAMs, and CD163 and CD200R1 are markers of M2 TAMs[27-28]. We found that LIMP2 levels were significantly associated with CD80 (P < 0.0001, r=0.37) and CD86 (P < 0.0001, r=0.45), but not with CD163 (P=0.067, r=0.074) and CD200R1 (P=0.00025, r=0.16) (Fig. 5B). Next, we performed immunohistochemistry to evaluate CD86+ macrophages and LIMP2+ cells in five patients with gastric cancer. It turned out that elevated CD86+ macrophages correlated with abundant LIMP2 expression (Fig. 6). These results indicate that LIMP2 is significantly related to M1 TAMs.
Figure
5.
LIMP2 expression associated with M1 infiltration.
A: TIMER analysis of the correlation between LIMP2 expression and immune purity, B cells, CD4+ T cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells. B: Pearson correlation analysis of LIMP2 expression associated with the macrophage hallmarks M1 and M2.
In the present study, we showed that relatively high levels of LIMP2 were significantly associated with a longer OS in patients with GC, which demonstrates that the expression level of LIMP2 is an independent prognostic factor. This result is the first data, to our knowledge, indicating that the level of LIMP2 expression is significantly associated with the prognosis of patients with GC who undergo curative surgery. Furthermore, our IHC analyses revealed that LIMP2 levels were higher in GC tissues compared with those detected in noncancerous tissues. Moreover, LIMP2 levels were significantly associated with Laurén classification, depth of invasion, lymph node metastasis, and TNM stage, but not with distant metastasis, the preoperative CEA level, age, sex, or differentiation. Therefore, our data identified that LIMP2 levels were negatively associated with the progression of gastric adenocarcinoma.
Our univariate analysis of survival indicated that LIMP2 expression, tumor differentiation, depth of invasion, lymph node metastasis, distant metastasis, TNM stage, and preoperative CEA level were significantly associated with the 5-year OS of patients with GC. Moreover, multivariate COX regression analysis showed that LIMP2 expression, tumor differentiation, and TNM stage can serve as prognostic markers for 5-year OS and that LIMP2 was an independent prog-nostic factor of OS.
We gained new information on the biological functions of LIMP2 using TIMER analysis. Here, we found that SCARB2 levels were significantly associated with macrophages' infiltration of tumors, indicating that SCARB2 induced macrophage-mediated innate immunity. GSEA revealed that the ERBB signaling pathway had a high enrichment score. In a previous study, LIMP2 was recognized to induce immune cells to produce cytokines in response to bacterial infections and regulate macrophage activation in response to infection with Helicobacter pylori through EGFR signaling[25, 29]. Moreover, EGFR influences the polarization of macrophages in the tumor microenvironment. Specifically, upregulation of EGFR expression inhibits the expression of the M2-related marker[30]. SCARB2 expression was also discovered significantly associated with M1 but not M2 related hallmark. Therefore, we assumed that upregulated LIMP2 expression facilitated the infiltration of macrophages with M1-related markers while inhibiting EGFR activity in GC. Thus, M1 TAMs in the tumor environment produced proinflammatory cytokines that enhanced the antitumor response to prolong survival.
However, our study had certain limitations. Firstly, the effects of LIMP2 on cancer pathogenesis, angiogenesis, and metastasis were not investigated, and animal models of the effect of LIMP2 on tumor progression has not been established. Secondly, the association between LIMP2 and macrophages requires experimental confirmation. Thirdly, our data indicating the prognostic value of LIMP2 must be validated before translation to the clinic, particularly in view of the heterogeneity of GC and associated mechanisms.
In conclusion, the present study showed for the first time that LIMP2 was highly expressed in GC tissues, and that patients with high LIMP2 levels experienced longer overall survival. Moreover, LIMP2 can serve as an independent prognostic factor for OS of GC.
Acknowledgments
This work was supported by the grants of National Natural Science Foundation of China (81572390), the 13th Five Years Talent's Subsidy Project in Science and Education of Jiangsu Province (ZDRCA2016051).
Conflict of interests: All authors have no conflict of interests.
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