Original Article Overexpression of ANGPTL2 and LILRB2 As Predictive and Therapeutic Biomarkers for Metastasis and Prognosis in Colorectal Cancer

Int J Clin Exp Pathol 2018;11(5):2281-2294 www.ijcep.com /ISSN:1936-2625/IJCEP0074587

Original Article Overexpression of ANGPTL2 and LILRB2 as predictive and therapeutic biomarkers for metastasis and prognosis in colorectal cancer

Jian He1, Jie Xu2, Xiaoting Yu1, Hailong Zhu1, Yu Zeng1, Desheng Fan1, Xianghua Yi1

1Department of Pathology, Tongji Hospital, Tongji University School of Medicine, Shanghai, P. R. China; 2School of Public Health, Xinxiang Medical University, Xinxiang, Henan Province, P. R. China Received February 14, 2018; Accepted March 24, 2018; Epub May 1, 2018; Published May 15, 2018

Abstract: LILRB2 is an inhibitory receptor involved in immune cells. A variety of cancer cells have been observed to express LILRB2, which has been related to development of cancers. Recently, ANGPTL2 was found to be bound to LILRB2 as a high affinity ligand. Expression and function of LILRB2 and ANGPTL2 in colorectal cancer (CRC) remain unknown. To explore differential expression, 364 CRCs, 5 adenomas, and 205 normal samples for LILRB2 and 338 CRCs, 5 adenomas, and 232 normal samples for ANGPTL2 were studied in Oncomine and GEO databases. We noted that LILRB2 was significantly increased in CRC compared to adenoma and normal tissues. ANGPTL2 was higher in adenoma than normal tissues and further increased in CRC than adenoma. Copy number of LILRB2 and ANGPTL2 DNA was also more increased in CRC than in normal tissue. Furthermore, immunohistochemistry analysis of 155 pairs of primary CRC and normal tissues verified the positive rates of LILRB2 and ANGPTL2 were 87.10% (135/155) and 97.44% (151/155) in CRC, with almost no expression in normal tissues. LILRB2 and ANGPTL2 were significantly associated with tumor size, worse cell differentiation, lymph node metastasis, and advanced disease stage. Levels of ANGPTL2 were adversely related to survival of CRC patients, consistent with results in GEPIA (TCGA data) database. Moreover, a significant positive correlation was found between LILRB2 and ANGPTL2 in CRC. These findings suggest that ANGPTL2 and LILRB2 play an important role in CRC occurrence and progression. ANGPTL2 and LILRB2 could serve as novel biomarkers for treatment and prognosis of CRC.

Keywords: ANGPTL2, LILRB2, colorectal cancer

Introduction Leukocyte immunoglobulin-like receptor sub- family B member 2 (LILRB2) is a member of the Colorectal cancer (CRC) is the third most leukocyte immunoglobulin-like receptor (LILR) frequent cancer and fourth leading cause of family and is also known as ILT-4 or CD85d. A malignant tumor related deaths. According to cell-surface receptor, LILRB2 can negatively data from International Agency for Research regulate the immune response by recruiting on Cancer (IARC) in 2012, there are about tyrosine phosphatases, like SHP1 or SHP2. 1,360,000 new cases and 690,000 deaths LILRB2 is predominantly expressed in mono- annually, worldwide [1]. Fortunately, targeted cytes, macrophages, and myeloid cells and therapy has significantly prolonged survival and also be expressed by endothelial cells, placen- enhanced the quality of life of CRC patients. tal trophoblasts, and decidual macrophages However, drug resistance to targeted drugs [2-5], suggesting that LILRB2 may play an im- remains a challenge for treatment of CRC. portant role in various physiological functions. Moreover, targeted drugs can cause adverse Interestingly, LILRB2 and its mouse homolog, reactions to some normal tissues which paired immunoglobulin-like receptor (PIRB), express the target. Therefore, more specific tar- were found to be overexpressed in leukemic gets or novel biomarkers need to be identified stem cells (LSCs) and hematopoietic stem cells to improve the curative effect and prognosis of (HSCs), critical to the maintenance of stemness CRC. [2]. Furthermore, LILRB2, as well as LILRB1 and ANGPTL2 and LILRB2 in colorectal cancer

Table 1. Correlation between LILRB2/ANGPTL2 expression and clinicopathological parameters LILRB2 expression ANGPTL2 expression Characteristics N P value N P value Low High Low High All case (155) Age (years) ≤73 79 38 41 0.453 78 18 60 0.018a >73 76 32 44 77 7 70 Gender Male 92 40 52 0.611 91 20 71 0.018a Female 63 30 33 64 5 59 Tumor size (cm) ≤4.95 77 26 51 0.005b 81 14 67 0.683 >4.95 78 44 34 74 11 63 Differentiation Well 23 16 7 0.011a 22 7 15 0.031a Moderate/Poor 132 54 78 133 18 115 Dukes stage A/B 75 40 35 0.048a 70 16 54 0.039a C/D 80 30 50 85 9 76 T classification T1 0 0 0 0.029a 0 0 0 0.558 T2 9 6 3 10 2 8 T3 53 30 23 56 11 45 T4 93 34 59 89 12 77 N classification N0 74 40 34 0.033a 76 16 60 0.102 N1/N2 78 30 51 79 9 70 M classification M0 137 61 76 0.661 137 20 117 0.173 Ml 18 9 9 18 5 13 Tumor location Proximal 59 27 32 0.906 59 9 50 0.816 Distal 96 43 53 96 16 80 Tumor site Colon 101 44 57 0.585 101 14 87 0.294 Rectum 54 26 28 54 11 43 aP<0.05, bP<0.01.

LILRB4, is expressed in many solid tumors Angiopoietin-like proteins (ANGPTLs) are a fam- [6-10]. High LILRB2 expression was found in ily constituting seven secreted glycoproteins non-small cell lung cancer (NSCLC) and breast with the same domain structure as angiopoi- cancer and has been associated with lymph etin [11]. ANGPTLs are extensive expressed in node metastasis and less number of tumor many tissues such as hematopoietic system infiltrating lymphocytes [8, 9]. Higher LILRB2 and vascular system and liver and play an has been found in more aggressive pancreatic important role in inflammation, angiogenesis, ductal carcinomas (PDAC) [10]. These results and lipid metabolism [11, 12]. Several ANGPT- suggest that LILRB2 could be involved in pro- Ls, including ANGPTL2, can stimulate activities gression of malignant tumors by non-immuno- of HSCs in humans and mice [13-15]. ANGPTL2 regulation and immunoregulation. However, has been found to induce inflammatory carci- expression and function of LILRB2 in CRC nogenesis in skin squamous cell carcinoma remains poorly understood. (SCC) [16]. Significantly increased ANGPTL2

2282 Int J Clin Exp Pathol 2018;11(5):2281-2294 ANGPTL2 and LILRB2 in colorectal cancer expression also has been observed in various Patients and samples solid tumors, regulating proliferation and metastasis of cells such as NSCLC, ovarian CRC samples from 155 patients were obtained cancer, and sarcoma [17-19]. In addition, over- from CRC patients at Tongji Hospital affiliated expressed ANGPTL2 can induce epithelial-to- to Tongji University from 2008 to 2010. None mesenchymal transition (EMT) in PDAC [20]. of the patients had received chemotherapy or These reports suggest that ANGPTL2 may be a radiotherapy before surgery and no periopera- critical factor in carcinogenesis and cancer pro- tive mortality was observed. There were 95 gression. However, the role of ANGPTL2 in CRC males (59.375%) and 65 females (40.625%) remains largely undefined. with median age of 73 years (range: 33-98 years). Patients were classified according to cri- Recently, LILRB2 has been identified as a teria described in the seventh edition of AJCC/ receptor for ANGPTL2 [2]. Co-expression of UICC. Main clinicopathological variables of the LILRB2 and ANGPTL2 has been reported in patients are summarized (Table 1). Samples NSCLC to promote the proliferation of cancer were contacted by phone to confirm their health cells [21]. To our knowledge, expression and status. Median follow up duration was 68 function of LILRB2 and ANGPTL2 in CRC has months (range = 1-95 months). This study was not been reported. Compared with other ANG- approved by the Review Board and Ethics PTLs, ANGPTL2 presents the highest affinity for Committee of Tongji Hospital affiliated to Tongji LILRB2 [2]. Thus, we speculated that there may University and written informed consent was be interaction between LILRB2 and ANGPTL2 obtained from each patient. and that this interaction may be involved in molecular mechanisms of CRC progression. Tissue microarray and immunohistochemistry Our present work was designed to evaluate the expression and role of both LILRB2 and CRC and adjacent normal tissues were con- ANGPTL2 in CRC. structed into tissue microarray (TAMs) blocks and cut into 4 μm serial sections. After being Materials and methods deparaffinized and rehydrated, the sections were heated in an autoclave for antigen re- Bioinformatics analysis trieval. Endogenous peroxidases were blocked with 0.3% H O solution and nonspecific back- We made a differential expression analysis 2 2 ground staining was blocked with serum. for mRNA expression and DNA copy number Immunohistochemical staining for LILRB2 and variation (CNV) of LILRB2 and ANGPTL2, in ANGPTL2 was performed with primary antibod- the Oncomine Cancer Microarray Database ies: anti-LILRB2 (1:150, LSBio) and anti-ANG- (https://www.oncomine.org), to assess the ex- PTL2 (1:200, Proteintech). Then, sections were pression pattern of LILRB2 and ANGPTL2 in incubated with a HRP-conjugated anti-mouse/ CRC. Meta-analysis of LILRB2 and ANGPTL2 expression was performed in datasets. Co- rabbit secondary antibody for 45 minutes and expression gene analysis of LILRB2 and visualized with 3,3’-diaminobenzidine solution ANGPTL2 was performed and presented in and counterstained with hematoxylin. Sections heatmap. Datasets in the database were used incubated with rabbit IgG substitute primary in which CRC type, sample sizes, folds change, antibodies were used as negative controls. All t-test values, and p values were obtained, specimens were evaluated by two independent showing a significant difference between the pathologists unaware of the clinical records of two groups. The filter criteria were P<0.05, fold patients. Scores were obtained as the sum of change: all, gene rank: all. Original data of the intensity score (0 = negative, 1 = weak, 2 = microarray was downloaded from the Gene moderate, 3 = strong) multiplied by the pro- Expression Omnibus (GEO) database (https:// portion score representing the percentage of www.ncbi.nlm.nih.gov/geo/). Survival analysis stained cells (1 = 10-24%, 2 = 25-49%, 3 = of LILRB2 and ANGPTL2 using TCGA data was 50 -74%, 4 = 75-100%). Brown staining of tumor performed in Gene Expression Profiling Inter- cells >10% was considered as positive. High active Analysis (GEPIA) Database (http://gepia. expression of LILRB2 or ANGPTL2 was defined cancer-pku.cn/index.html). Oncomine, GEO, as score ≥6. Otherwise, scores represented low and GEPIA are all free public databases. expression.

2283 Int J Clin Exp Pathol 2018;11(5):2281-2294 ANGPTL2 and LILRB2 in colorectal cancer

Table 2. Expression of LILRB2 and ANGPTL2 in various malig- 134 significant unique analyses, res- nant tumors pectively. In CRC, LILRB2 was upreg- LILRB2 ANGPTL2 ulated in 12 datasets which com- Type of cancer (No. of analyses) (No. of analyses) plied with inclusion criteria, including (Oncomine database) Cancer vs. Normal Cancer vs. Normal 10 mRNA datasets and 2 DNA datas- Up-regulated Up-regulated ets. For ANGPTL2, 15 datasets con- Bladder cancer 4 0 sistent with inclusion criteria were upregulated, including 13 mRNA Brain and CNS cancer 11 18 datasets and 2 DNA datasets. More- Breast cancer 25 4 over, LILRB2 and ANGPTL2 also were Cervical cancer 5 1 found to be upregulated in many Colorectal cancer 12 15 datasets of digestive system tumors, Esophageal cancer 3 8 such as esophageal cancer, gastric Gastric cancer 11 12 cancer, liver cancer, and pancreatic Head and neck cancer 9 7 cancer (Table 2). Ten mRNA datasets Kidney cancer 14 10 from 5 microarrays, including 274 Leukemia 7 16 CRC and 111 normal samples, were Liver cancer 4 3 analyzed for mRNA differential expression of LILRB2. The fold change Lung cancer 2 2 of LILRB2 mRNA in CRC was 1.156 to Lymphoma 22 18 2.25, compared with normal tissues Melanoma 2 2 (Figure 1A; Table 3). Twelve mRNA Myeloma 1 1 datasets from 6 microarrays, includ- Other cancers 11 7 ing 296 CRC and 138 normal sam- Ovarian cancer 0 0 ples, were implicated in the differen- Pancreatic cancer 4 4 tial expression analysis of ANGPTL2. Prostate cancer 3 3 The fold change of ANGPTL2 mRNA Sarcoma 4 5 in CRC was 1.213 to 2.557 (Figure Significant unique analyses 152 134 1D; Table 3). Ten LILRB2 mRNA data- CNS: central nervous system. sets and 12 ANGPTL2 mRNA datasets were analyzed by meta-analysis, with results showing that the median Statistical analysis gene rank of LILRB2 and ANGPTL2 in overexpressed genes was 5132.5 and 4020.0, res- SPSS software version 20.0 was used for pectively (P = 0.004, P = 0.005). Furthermore, statistical analyses. Data are represented as expression of LILRB2 mRNA and ANGPTL2 mean ± standard deviation (SD). Student’s mRNA was increased in cecum adenocarcino- t-test was used to compare data between two ma, colon adenocarcinoma, rectal adenocarci- groups. Association between expression of noma, rectosigmoid adenocarcinoma, colon LILRB2 or ANGPTL2 and clinicopathological mucinous adenocarcinoma, and rectal muci- 2 variables was analyzed using χ test. Overall nous adenocarcinoma. Fold change of LILRB2 survival (OS) curves were constructed using and ANGPTL2 mRNA in subtypes of CRC was Kaplan-Meier method and analyzed using log- 1.276 to 1.786 and 1.213 to 1.733, compared rank test. Spearman’s correlation analysis was with normal colon tissues, respectively (Figures used to analyze correlation between LLILRB2 2, 3). and ANGPTL2 expression. P value <0.05 was considered to indicate a statistically significant Expression of LILRB2 and ANGPTL2 in normal difference. colons, colon adenomas, and colon cancer tissues Results

Expression of LILRB2 and ANGPTL2 in CRC The original data of the Skrzypczak Colorectal 2 and other digestive system cancers microarray were downloaded from GEO database (Table 4). It was noted that there was no In Oncomine database, we found that LILRB2 significant difference in LILRB2 mRNA expres- and ANGPTL2 were upregulated in 152 and sion levels between colon adenoma and nor-

2284 Int J Clin Exp Pathol 2018;11(5):2281-2294 ANGPTL2 and LILRB2 in colorectal cancer

Figure 1. LILRB2 and ANGPTL2 expression in CRC and normal tissues at mRNA and DNA (copy number variation) level. A. LILRB2 mRNA (Hong Colorectal microarray). B, C. LILRB2 DNA (Kurashina Colon microarray). D. ANGPTL2 mRNA (Hong Colorectal microarray). E, F. ANGPTL2 DNA (Kurashina Colon microarray).

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Table 3. LILRB2 and ANGPTL2 overexpression datasets in CRC Gene Microarray (mRNA) Dataset t-Test Fold change P value LILRB2 Kaiser Colon Rectal Mucinous Adenocarcinoma (n = 4) vs. Normal (n = 5) 4.279 1.426 0.002b Rectosigmoid Adenocarcinoma (n = 10) vs. Normal (n = 5) 3.109 1.311 0.006b Colon Mucinous Adenocarcinoma (n = 13) vs. Normal (n = 5) 3.106 1.729 0.004b Cecum Adenocarcinoma (n = 17) vs. Normal (n = 5) 2.620 1.310 0.010a Rectal Adenocarcinoma (n = 8) vs. Normal (n = 5) 2.262 1.786 0.027a Colon Adenocarcinoma (n = 41) vs. Normal (n = 5) 2.785 1.276 0.009b Skrzypczak Colorectal Colon Carcinoma Epithelia (n = 10) vs. Normal (n = 5) 3.061 1.156 0.005b Hong Colorectal Colorectal Carcinoma (n = 70) vs. Normal (n = 12) 3.081 2.250 0.003b Gaedcke Colorectal Rectal Adenocarcinoma (n = 65) vs. Normal (n = 65) 3.604 1.376 2.30E-4c Skrzypczak Colorectal Colorectal Carcinoma (n = 36) vs. Normal (n = 24) 1.693 1.183 0.049a ANGPTL2 Gaedcke Colorectal Rectal Adenocarcinoma (n = 65) vs. Normal (n = 65) 10.903 2.374 5.70E-20c Skrzypczak Colorectal 2 Colon Carcinoma (n = 5) vs. Normal (n = 10) 11.289 2.557 3.58E-7c Colon Carcinoma (n = 5) vs. Normal (n = 10) 8.360 2.252 4.11E-5c Skrzypczak Colorectal Colorectal Carcinoma (n = 36) vs. Normal (n = 24) 4.906 2.371 6.54E-6c Hong Colorectal Colorectal Carcinoma (n = 70) vs. Normal (n = 12) 6.688 2.348 2.22E-7c Kaiser Colon Colon Mucinous Adenocarcinoma (n = 13) vs. Normal (n = 5) 3.963 1.733 0.001b Rectal Adenocarcinoma (n = 8) vs. Normal (n = 5) 2.808 1.607 0.009b Rectal Mucinous Adenocarcinoma (n = 4) vs. Normal (n = 5) 2.546 1.284 0.019a Colon Adenocarcinoma (n = 41) vs. Normal (n = 5) 3.196 1.432 0.009b Rectosigmoid Adenocarcinoma (n = 10) vs. Normal (n = 5) 1.878 1.247 0.042a Cecum Adenocarcinoma (n = 17) vs. Normal (n = 5) 2.143 1.213 0.033a TCGA Colorectal Colon Mucinous Adenocarcinoma (n = 22) vs. Normal (n = 22) 1.964 1.305 0.028a aP<0.05, bP<0.01, cP<0.001. mal colon tissues (P = 0.2368). While com- in overexpressed genes, respectively (P = pared with colon adenoma, LILRB2 mRNA 0.002, P = 0.021). These results indicate that was significantly increased in colon cancer and DNA copy number of LILRB2 and ANGPTL2 was fold change was 2.218 (P = 0.011). Similarly, more increased in CRC than in normal tissues. expression of ANGPTL2 mRNA showed a significant difference between normal colons and Co-expression analyses of LILRB2 and colon adenoma tissues, with fold change of ANGPTL2 in CRC 1.213 (P = 0.017). Compared with colon adenomas, ANGPTL2 mRNA was further increased Three expression profile microarrays were in colon cancer, with fold change of 1.71 found containing co-expression data of LILRB2 (P<0.001). and ANGPTL2. Results revealed that LILRB2 mRNA and ANGPTL2 mRNA had a similar DNA copy number variations of LILRB2 and expression patterns and the correlation coeffi- ANGPTL2 cients in 3 microarrays were 0.368, 0.293, and 0.238, respectively (Table 5; Figure 4). In addi- CNV of LILRB2 and ANGPTL2 was analyzed in tion, correlation between LILRB2 and ANGPTL2, Kurashina Colon microarray, including 188 at mRNA level, was examined in GEPIA data- samples. Compare to normal tissues, DNA copy base using The Cancer Genome Atlas (TCGA) number of LILRB2 and ANGPTL2 increased in data. These results showed that there was a CRC. Compared with normal tissues, the fold significant positive correlation between mRNA change of LILRB2 in CRC was 1.082, 1.056 (P expression of LILRB2 and ANGPTL2 in colon = 2.07E-6, P = 0.005) (Figure 1B, 1C), and adenocarcinoma (COAD) and rectal adeno- 1.11, 1.045 (P = 0.041, P = 0.001) (Figure 1E, carcinoma (READ). Correlation coefficient was 1F) for ANGPTL2 in datasets, respectively. 0.73 and 0.65, respectively (P<0.01, P<0.01) Meta-analysis results of LILRB2 and ANGPTL2 (Figure 5A, 5C). However, there was no correla- copy number showed the median gene rank of tion between LILRB2 and ANGPTL2 in normal LILRB2 and ANGPTL2 was 3187.0 and 2415.5 colons and rectum tissues (P = 0.23, P = 0.62)

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Figure 2. Differential expression analysis of LILRB2 in subtype of CRC. A. Colon (n = 5) vs. Cecum Adenocarcinoma (n = 17), fold change = 1.310, P = 0.010. B. Colon (n = 5) vs. Colon Adenocarcinoma (n = 41), fold change = 1.276, P = 0.009. C. Colon (n = 5) vs. Rectal Adenocarcinoma (n = 8), fold change = 1.786, P = 0.027. D. Colon (n = 5) vs. Rectosigmoid Adenocarcinoma (n = 10), fold change = 1.311, P = 0.006. E. Colon (n = 5) vs. Colon Mucinous Adenocarcinoma (n = 13), fold change = 1.729, P = 0.004. F. Colon (n = 5) vs. Rectal Mucinous Adenocarcinoma (n=4), fold change = 1.426, P = 0.002.

(Figure 5B, 5D). In conclusion, LILRB2 and in the cytoplasm of CRC cells. In contrast, ANGPTL2 were significantly positive correlated LILRB2 and ANGPTL2 were scarcely stained in in CRC tissues. adjacent normal colorectal tissue cells (Figure 6). Positive rates of LILRB2 and ANGPTL2 in LILRB2 and ANGPTL2 protein expression in CRC samples were 87.10% (135/155) and primary CRC tissues 97.44% (151/155), respectively. High expression of LILRB2 in CRC tissues was associated Expression of ANGPTL2 and LILRB2 in 155 pri- with tumor size (P = 0.005), worse cell differen- mary CRC tissues was examined by immunohis- tiation (P = 0.011), advanced Dukes stage (P = tochemistry. LILRB2 was found in membranes, 0.048), T stage (P = 0.029) and lymph node cytoplasm, or both, and ANGPTL2 was observed metastasis (P = 0.033) (Table 1). Similarly, high

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Figure 3. Differential expression analysis of ANGPTL2 in subtype of CRC. A. Colon (n = 5) vs. Cecum Adenocarci- noma (n = 17), fold change = 1.213, P = 0.033. B. Colon (n = 5) vs. Colon Adenocarcinoma (n = 41), fold change = 1.432, P = 0.009. C. Colon (n = 5) vs. Rectal Adenocarcinoma (n = 8), fold change = 1.607, P = 0.009. D. Colon (n = 5) vs. Rectosigmoid Adenocarcinoma (n = 10), fold change = 1.247, P = 0.042. E. Colon (n = 5) vs. Colon Mucinous Adenocarcinoma (n = 13), fold change = 1.733, P = 0.001. F. Colon (n = 5) vs. Rectal Mucinous Adenocarcinoma (n = 4), fold change = 1.284, P = 0.019. expression of ANGPTL2 in CRC tissues was Correlation between LILRB2, ANGPTL2 protein associated with ages of more than 73 years (P expression, and OS of CRC patients = 0.018), female (P = 0.018), worse cell differentiation (P = 0.031), and advanced Dukes An important question is whether expression of stage (P = 0.039) (Table 1). No significant cor- LILRB2 and ANGPTL2 could be used to predict relation was observed between LILRB2 and prognosis of CRC patients. In survival analysis age, gender, and tumor location. ANGPTL2 in GEPIA database by TCGA data, significant dif- expression had no relation to tumor size, TNM ferences were observed in the OS and disease- stage, and tumor location (Table 1). Further- free survival (DFS) at mRNA level between more, expression of LILRB2 and ANGPTL2 ANGPTL2 high expression group and low ex- showed significant correlation at the protein pression group of CRC patients (P = 0.0042, P level (r = 0.505, P<0.001). = 0.0013) (Figure 7A, 7B). No difference of OS

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Table 4. Fluorescence values of LILRB2 and ANGPTL2 probe in Skrzypczak Colorectal 2 microarray LILRB2 (Reporter: 207697_x_at) ANGPTL2 (Reporter: 213004_at) Tissue Subtype GEO ID ID_REF Tissue Subtype GEO ID ID_REF Colon (normal) GSM523242 3.66491 Colon Adenoma GSM523244 3.7952 Colon (normal) GSM523243 3.60744 Colon Adenoma GSM523247 4.19941 Colon (normal) GSM523246 3.70634 Colon Adenoma GSM523251 4.1066 Colon (normal) GSM523268 3.60522 Colon Adenoma GSM523252 3.87153 Colon (normal) GSM523269 3.68977 Colon Adenoma GSM523281 3.83996 Colon (normal) GSM523270 3.689 Colon Carcinoma GSM523248 4.80783 Colon (normal) GSM523271 4.42752 Colon Carcinoma GSM523249 5.16897 Colon (normal) GSM523273 4.60052 Colon Carcinoma GSM523250 5.96317 Colon (normal) GSM523274 3.85642 Colon Carcinoma GSM523253 5.40926 Colon (normal) GSM523280 4.02841 Colon Carcinoma GSM523275 4.77843 Colon Adenoma GSM523244 3.59502 Colon Adenoma GSM523247 3.81785 Colon Adenoma GSM523251 3.79166 Colon Adenoma GSM523252 3.60936 Colon Adenoma GSM523281 3.59599

Table 5. LILRB2 and ANGPTL2 co-expression microarrays LILRB2 ANGPTL2 Microarray Cancer Type Sample (n) Correlation Reporter Reporter Smith Colorectal 2 Colorectal Adenocarcinoma 210146_x_at 219514_at 55 0.368 213004_at 213001_at Jorissen Colorectal Colorectal Adenocarcinoma 210146_x_at 211148_s_at 74 0.293 Ayers Colorectal Colon Carcinoma 207697_x_at 219514_at 59 0.238

Figure 4. Co-expression analysis of LILRB2 and ANGPTL2 in CRC. A. Smith Colorectal 2 microarray. B. Jorissen Colorectal microarray. C. Ayers Colorectal microarray. and DFS was found in LILRB2 (P = 0.37, P = expression group (P = 0.0303) (Figure 7C). 0.57) (Figure 7D, 7E). In addition, log-rank test Levels of ANGPTL2 negatively correlated with analysis showed that OS of high expression of OS of CRC patients. However, no significant dif- ANGPTL2 protein group was lower than the low ference in OS between LILRB2 high and low

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Figure 5. Correlation analysis of LILRB2 and ANGPTL2 expression using TCGA data in GEPIA database. A. Correla- tion of LILRB2 and ANGPTL2 in colon adenocarcinoma. B. Correlation of LILRB2 and ANGPTL2 in normal colon. C. Correlation of LILRB2 and ANGPTL2 in rectum adenocarcinoma. D. Correlation of LILRB2 and ANGPTL2 in normal rectum. TPM: transcripts per million.

Figure 6. Expression of LILRB2 and ANGPTL2 in primary CRC and normal tissues. A, E. Normal control tissues, - (×200). B, F. Well differentiation CRC tissues, + (×200). C, G. Middle differentiation CRC tissues, ++ (×200). D, H. Poor differentiation CRC tissues, +++ (×200).

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Figure 7. Relationship between LILRB2 or ANGPTL2 expression and patient survival. A, D. OS/mRNA level, low (n) = 181, high (n) = 181. B, E. DFS/mRNA level, low (n) = 181, high (n) = 181. C, F. OS/protein level, low ANGPTL2 (n) = 134, high ANGPTL2 (n) = 21, low LILRB2 (n) = 88, high LILRB2 (n) = 67. protein expression groups was found (P = 0.44) These results, likewise, supported the increase (Figure 7F). of LILRB2 and ANGPTL2 in CRC. In addition, we found that mRNA expression of LILRB2 and Discussion ANGPTL2, in each subtype of CRC, was higher than normal tissues. This further suggests that Past studies have shown that expression of LILRB2 and ANGPTL2 may play a role as onco- LILRB2 is increased in multiple tumor tissues gene in development of CRC. including acute myeloid leukemia cells, breast cancer, and NSCLC [8, 9, 22]. Overexpression Occurrence of CRC is a multistep biological pro- of LILRB2 can promote occurrence and devel- cess. About 85% of CRCs originate from adeno- opment of tumors and is closely related to prog- matous polyps. It is essential to understand the nosis [21, 23, 24]. We used public microarray roles of LILRB2 and ANGPTL2 in the evolution databases to evaluate expression of LILRB2 of CRC. Therefore, we examined expression of and ANGPTL2 in CRC. Our results showed LILRB2 and ANGPTL2 in normal tissues, adeno- that both LILRB2 and ANGPTL2 were overex- mas, and CRC tissues. Our results showed that pressed, not only in CRC but also in other expression of LILRB2 in colon adenomas was digestive system cancers. Expression of LILRB2 not significantly different from that in normal and ANGPTL2 was significantly upregulated at tissues but expression of ANGPTL2 in colon mRNA and DNA levels in CRC, indicating that adenomas was higher than in normal tissues. It increased mRNA of LILRB2 and ANGPTL2 may can be speculated that ANGPTL2 may play a be caused by change of DNA copy number and role in the transition from normal tissue to ade- increased LILRB2 and ANGPTL2 may play a noma by binding to LILRB2. In addition, expres- role. In order to reduce error and expand the sion of LILRB2 and ANGPTL2 in cancer tissues sample size, we performed meta-analysis of was significantly higher than in adenomas. High LILRB2 and ANGPTL2 at mRNA and DNA levels. expression of LILRB2 and ANGPTL2 in CRC indi-

2291 Int J Clin Exp Pathol 2018;11(5):2281-2294 ANGPTL2 and LILRB2 in colorectal cancer cates that LILRB2 and ANGPTL2 interaction tor LILRB2 as a ligand in CRC? No synergistic plays a role in promoting the transformation of expression of LILRB2 and ANGPTL2 in CRC adenoma into carcinoma. In particular, high has been reported. Therefore, expression of expression of LILRB2 was not observed in ade- ANGPTL2 in CRC was examined in this study. It nomas, proving that LILRB2 is closely related to was noted that expression of ANGPTL2 in CRC occurrence and development of cancer. Levels tissues increased significantly. The positive of LILRB2 protein in CRC tissues was further rate of ANGPTL2 in CRC tissues was 97.44%. In confirmed by immunohistochemistry and was addition, the level of ANGPTL2 had a significant significantly higher than normal tissues. In this correlation with clinicopathological parame- study, the positive expression rate of LILRB2 in ters, suggesting that ANGPTL2 was involved in CRC was 87.10%. development of CRC. Analysis of the correlation between ANGPTL2 protein and OS showed that Currently, there are no reports on expression of survival time of patients with high ANGPTL2 LILRB2 in CRC tissues. This study takes the expression was shorter than those with low lead in verifying the high expression of LILRB2 expression of ANGPTL2. A consistent OS and in CRC. In addition, LILRB2 was found to be DFS result also was obtained at the mRNA level correlated with tumor size, worse cell differen- using TCGA data in GEPIA database. tiation, and advanced disease stage, suggesting that LILRB2 may be involved in progression The relationship between LILRB2 and ANGPTL2 of CRC. Moreover, we analyzed correlation was comprehended by co-expression analysis, between LILRB2 and OS of CRC patients. providing evidence for the binding of ANGPTL2 Unexpectedly, we were unable to find a signifi- to LILRB2 in CRC. It was found that there was cant correlation between LILRB2 and OS. We a significant positive correlation between LIL- explored mRNA expression levels of LILRB2 RB2 and ANGPTL2 in many microarrays, in using TCGA data in GEPIA database and found Oncomine database. Furthermore, LILRB2 and that LILRB2 mRNA in CRC was upregulated, ANGPTL2 also presented a highly positive cor- compared with corresponding normal tissues. relation by TCGA data. These results suggest While in survival analysis, there was no signifi- that ANGPTL2 plays a biological role acts as a cant difference between LILRB2 mRNA high ligand binding to LILRB2 receptors in CRC. In expression group and low expression group. addition, some stromal cells also expressed Results of survival analysis at mRNA level in LILRB2 and ANGPTL2, suggesting that the inhi- GEPIA database were consistent with the bition function of LILRB2 for immune cells may results at protein level. There may be some rea- play a role in the microenvironment around can- sons that significant correlation was found cer cells. ANGPTL2 may also be secreted between expression of LILRB2 and OS. First, by cancer cells and be involved as a soluble the results may have been interfered by sam- ligand of LILRB2. LILRB2 and ANGPTL2 may pling error for patients from a single area. Second, LILRB2 might not be an independent play immunosuppression and tumor sustaining prognostic factor. As ligand of LILRB2, ANGPTL2 roles. Application of bioinformatics analysis negative correlation with OS and DFS may laid a foundation for our hypothesis. Large sam- explain this phenomenon, in part. Therefore, ple sizes avoid errors produced by small sam- the prognosis significance of LILRB2 still ple sizes in a single study. requires much research. In conclusion, LILRB2 and ANGPTL2 play impor- Previously, ANGPTL2 has been considered as tant roles in CRC, especially in the transforma- an “orphan ligand”, but a recent study has tion from normal and precancerous lesions to found that ANGPTL2 is a high affinity ligand CRC. They are also correlated with prognosis. for LILRB2 and supports development of a vari- This study presents a perspective for occur- ety of malignant tumors. It was reported that rence and progression of CRC and provides a ANGPTL2 binds LILRB2 and supports develop- basis for exploring LILRB2 and ANGPTL2 in ment of LSCs and leukemia [2]. The combina- CRC. It also has a guiding significance for clini- tion of ANGPTL2 and LILRB2 has also been cal treatment and prognosis of CRC. The molec- found to promote growth of tumor cells in ular mechanisms still require further study in NSCLC [24]. Does ANGPTL2 also bind to recep- the future.

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