CHITINASE-LIKE PROTEINS AS PROMISING MARKERS IN CANCER PATIENTS

In the present review we collected the main studies regarding the role of chitinase-like proteins (CLps), belonging to the family of Glyco_18 domain-containing proteins, in different cancers. In humans, 3 chitinaselike proteins have been identified: YKL-40 (CHI3L1), YKL-39 (CHI3L2) and stabilin-1-interacting chitinase-like protein (SI-CLp). CLps are produced by several types of cells and combine the properties of cytokines and growth factors. The high levels of CLps were identified in the circulation of the patients with inflammatory diseases and various types of tumors. We highlighted the main known functions of CLps in normal and pathological conditions, their contribution to metastasis development, angiogenesis, invasion and other processes in cancer, the correlation of the levels of CLps with tumour progression. our data also contribute to the understanding of question how CLp could be useful for cancer patient benefit.


Common features of CLPs family
Chitinase-like proteins (CLPs) are structurally resemble chitinases that belong to a group of proteins, which are widely expressed in nature, and distributed in a wide range of organisms, including mammals, bacteria, plants, insects, viruses.Proteins with chitinase activity represent evolutionary ancient enzymes responsible for degradation of chitin, which is the second most abundant natural compound [1].
Mammalian chitinases and CLPs belong to glycosyl hydrolase family 18 (GH18) [2] due to presence of highly conserved Glyco_18 domain, responsible for sugar-binding, and catalytic site, which is essential for hydrolysis of chitin.The prehistoric purpose of GH18 existence is the degradation of complex sugar compounds, such as cellulose or chitin, via disruption of strong covalent or glycosylic bonds in polysaccharidic chains that compose polymer molecules.There are only two mammalian chitinases identified as functionally active enzymes, which are known as Acidic Mammalian Chitinase (AMCase) and Chitotriosidase (CHIT1) and they are both expressed in human [3,4].AMCase was firstly revealed in macrophages from patients with Gaucher disease [5].The source of secreted chitotriosidase is abnormal lipid-laden macrophages that can be classified as a variation of alternatively activated macrophages, expressing CD68, CD14, HLA class II, CD163, CCL18 and IL-1-receptor antagonist, but not CD11b, CD40 and pro-inflammatory cytokines [6].
YKL-39 is known to bind to chitooligosaccharides (GlcNac)5 and (GlcNac)6 [11,12], that was demonstrated by glycan array screening, intrinsic tryptophan fluorescence and isothermal titration calorimetry (ITC).There are more binding targets known for YKL-40; it can bind to type I collagen that was revealed by affinity chromatography and surface plasmon resonance [13], to chitooligosaccharides, that shown in protein X-ray crystallography assay [14]; (GlcNac)5 and (GlcNac)4 that revealed by the Western blot [15] and heparin demonstrated by heparin affinity and HPLC chromatography [16].ITC analysis showed that SI-CLP can bind to galactosamine, glucosamine, chitooligosacharide, (GlcNac)4, ribose and mannose [17].It was demonstrated by surface plasmon resonance analysis that YM1 can bind to glucosamine, galactosamine and glucosamine polymers [18].

The main sources and functions of CLPs
The secretion of chitinase-like proteins was found in macrophages, neutrophils, epithelial СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ.2018; 17(4): 99-105 оБзоры cells, chondrocytes and synovial cells, vascular smooth muscle cells as well as tumor cells (including breast, colon, kidney, lung, ovarian, prostate, uterine, osteosarcoma, glioblastoma) and their expression was regulated by various cytokines and hormones [1,12].YKL-39 is predominantly secreted by chondrocytes and synoviocytes and is recognized as a biochemical marker for the activation of chondrocytes and progression of osteoarthritis in humans [19].
YKL-40 is secreted by chondrocytes, synoviocytes, differentiated vascular smooth muscle cells, fibroblastlike synovial cells, by macrophages in the atherosclerotic plaque, tumor cells in many cancers [1,20,21].In vivo, YKL-40 expression was found in places with intensive tissue remodeling [1].YKL-40 regulates cell proliferation, migration, adhesion, macrophage differentiation, as well as extracellular matrix assembly and correlates with an elevated level of YKL-40 in chronic inflammation and connective tissue turnover [1,22].YKL-40 promotes the proliferation of chondrocytes and fibroblasts, migration and reorganization of vascular endothelial cells as well as inflammation and remodeling of extracellular matrix [1,16].It induces the migration of vascular smooth muscle cells (VSMC) [16] and promotes the growth of human synovial cells, skin and fibroblasts.High YKL-40 level was detected in serum of patients with rheumatoid arthritis (RA) and in patients with type 2 diabetes [1].SI-CLP expression was found in various tumor cell lines, Raji cells, Jurkat cells, as well as in CD3+ T-cells, in the synovial fluid of patients with osteoarthritis or rheumatoid arthritis [17].
Expression of YKL-40 mRNA in human monocyte was strongly upregulated by IFN-gamma, and inhibited by IL-4 and dexamethasone [9].There are also evidences that YKL-40 is secreted by macrophages during the late stages of differentiation.YKL-40 gene expression was up-regulated in monocytes stimulated with granulocyte-macrophage colonystimulating factor, in colony-stimulating factor stimulated monocytes and in lipopolysaccharide stimulated monocytes [23,24].
Human macrophages produce also SI-CLP and its expression is induced by Th2 cytokine IL-4 and glucocorticoid dexamethasone [9].In vivo, high amounts of SI-CLP were detected in macrophages from bronchoalveolar lavage of patients with chronic airway inflammation [17].
In macrophages, SI-CLP is primarily localized in the secretory lysosomes.Kzhyshkowska et al. demonstrated that the intracellular sorting of SI-CLP in alternatively activated macrophages was mediated by the scavenger receptor stabilin-1, which was specifically expressed on subpopulations of tissue macrophages and sinusoidal endothelial cells in liver, spleen, lymph node and bone marrow.Stabilin-1 recognized SI-CLP in trans-Golgi network and delivered it to the late endosomes and consequently into Lamp1-positive and CD63-positive lysosomes [9].The pattern of intracellular YKL-39 distribution was similar to the pattern demonstrated for SI-CLP suggesting that YKL-39 can be secreted by the lysosomal secretory pathway.Endogenous YKL-39 was found in the trans-Golgi network, where it was partially co-localized with stabilin-1.Using GST pulldown assay we showed that stabilin-1 can act as an intracellular sorting receptor for YKL-39 [25].

The role of CLPs in angiogenesis and chemotaxis
Among all chitinases and chitinase-like proteins, the pro-angiogenic activity and function of YKL-40 in various types of cancer progression were well studied.Angiogenic properties of YKL-40 in cancer development were demonstrated in breast and brain cancers where the expression level of YKL-40 was associated with tumor vascular formation [26,27].Immunohistochemical analysis of human breast cancer demonstrated a correlation between blood vessel density and YKL-40 protein expression [26].In mouse model of breast cancer, YKL-40 was demonstrated to promote tumor growth by supporting angiogenesis.In mouse model of melanoma and glioblastoma, the inhibiting effect of anti-YKL-40 monoclonal antibody on tumor growth was shown [28,29].It was revealed that YKL-40 can facilitate tumor angiogenesis by interacting with syndecan-1 on endothelial cells and metastasis by stimulating production of MMP-9, CCL2 and CXCL2 [30].
Several studies on in vitro tube formation and endothelial cell migration have demonstrated that YKL-40 has stimulating effect on the endothelial cells that is similar to the effect of endothelial growth factor (VEGF) [27].YKL-40-heparin interaction promotes the interaction with syndecan-1 and αvβ3 integrin, leading to activation of the ERK1/2 pathway and stimulation of VEGF [26,27].In glioblastoma, transient suppression of VEGF substantially increased YKL-40 expression and promoted tumor angiogenesis [31].Anti-VEGF neutralizing antibody did not improve HMVECs tube formation and migration induced by YKL-40, thus confirming that pro-angiogenic effects of YKL-40 on HMVECs were not affected by VEGF [26].
Chitinase-like proteins can influence chemotactic activity of various cells directly or indirectly.Using an in vitro microchemotaxis transwell system model, Nio et al. demonstrated that YM1 acted as a chemotactic factor for eosinophils, T-lymphocytes and bone marrow cells [32].YKL-40 was shown to affect chemotaxis of VSMC [16], THP-1 cells [33] and bronchial smooth muscle cells [34].For THP-1 and VSMC cells, purified YKL-40 induced chemotaxis directly.In contrary, for REVIEWS bronchial smooth muscle cells and SW480, YKL-40 enhanced secreted levels of IL-8, thus providing a chemotactic effect.
YKL-40 significantly increased the migration and invasion ability of CL1-1 NSCLC (non-small cell lung cancer) cell lines by regulating EMT (Epithelial Mesenchimal Transition) genes.In YKL-40 overexpressed cell line, the expression of E-cadherin, a marker of epithelial cells, was significantly lower; and the expression of markers of mesenchymal cells (N-cadherin, Vimentin) was significantly higher as well as other EMT regulators (Snail, Slug, and Twist) [35].Moreover, inhibition of YKL-40 reduced the tube formation in vitro and suppressed tumor growth, angiogenesis, and progression of brain tumors [28].
Analysis of biological functions of YKL-39 demonstrated that it is unique that CLP combines monocyte attracting and pro-angiogenic activities, which essential for tumor progression [25].The angiogenesis assay showed that recombinant YKL-39 induced tube formation of HUVEC cells 6 times higher than that observed in the negative control group, and this induction was more than 60% of positive control.The chemotactic effect of YKL-39 on primary monocytes was approximately 2 times higher after 1 h and more than 5 times higher after 3 h compared to control, and this effect was comparable with the effect of MCP-1/CCL2 chemokines [25].

CLPs in cancer
YKL-40 is expressed by several types of solid tumors including breast, colon, lung, kidney, head and neck, liver, bladder, prostate, stomach, ovary, pancreas, osteosarcoma, thyroid, glioblastoma and endometrial cancers.Microarray analysis identified YKL-40 gene as one of the most overexpressed genes in glioblastoma, papillary thyroid carcinoma, and chondrosarcoma [36].YKL-40 protein expression was found in biopsies of glioblastomas, breast cancer and colon cancer.In vitro YKL-40 was secreted by the following human cancer cell lines: osteosarcoma, glioblastoma, colon cancer, ovarian cancer, prostate cancer and malignant melanoma [37].YKL-40 protein expression was found in tumor associated macrophages (TAM) in patients with melanoma [37].YKL-40 protein was not expressed in small cell lung cancer cells, but YKL-40 mRNA expression was elevated in TAM [36].
In tumors, YKL-40 may contribute to the proliferation and differentiation of malignant cells, protect the cancer cells from apoptosis, stimulate angiogenesis, and regulate extracellular tissue remodeling [23].In non-small cell lung cancer, YKL-40 may also regulate (PI3K)/AKT/mTOR pathway, which is related with cell transformation, tumor survival, invasion and metastasis, and is a central feature of EMT [23].In breast cancer, YKL-40 levels were inversely correlated with expression of GATA3 and E-cadherin, which regulate cell-cell contacts and act as tumor inhibitors [37].The high risk of tumor progression may be explained either by the fact that cancer cells and TAM produce YKL-40, or that chronic inflammation causes both elevated plasma YKL-40 and cancer.
In our study we showed that the elevated levels of YKL-39 expression in tumors after neoadjuvant chemotherapy (NAC) were associated with increased risk of distant metastasis and poor response to NAC in patients with nonspecific invasive breast carcinoma [25].Moreover, in the study of gene expression of M2 macrophage markers (YKL-39 and CCL18) we found that in breast cancer patients, who received anthracycline-containing NAC, the absence of clinical response was associated with the presence of M2+ macrophage phenotype (YKL-39-CCL18 + or YKL-39 + CCL18-) [38].Kavsan et al. reported the increased expression of CHI3L2 gene in glioblastoma [39].However, there is still insufficient data on the association of both YKL-39 gene and protein level with tumor progression, and no data on SI-CLP in tumor progression are available.

YKL-40 is a marker of late stages of cancer
Elevated plasma YKL-40 was found in patients with metastatic pancreatic and ovarian adenocarcinoma [36].In patients with gastric cancer, serum levels of YKL-40 were significantly higher compared to those observed in healthy population, and the increased YKL-40 level indicated more aggressive phenotype of tumor [40].Plasma YKL-40 level was elevated in approximately 80% of patients with metastatic renal cell carcinoma [37].Dupont et al. showed that serum YKL-40 was upregulated in 65% of patients with stage I and II ovarian cancer in contrast to 74-91% of patients with stage III and IV cancer [41].In patients with small cell lung cancer, the highest percentage of the patients who had elevated serum YKL-40 level was associated with advanced disease compared to local one.More than 80% of patients with metastatic renal cell cancer and more that 40% of patients with metastatic malignant melanoma and metastatic prostate cancer had also elevated serum YKL-40.In patients with glioblastoma, the serum YKL-40 level was higher in patients with glioblastoma multiforme compared to patients with lower grade gliomas [23].In breast cancer, increased serum levels of YKL-40 were found more frequently in patients with metastatic cancer compared to patients with early cancer [23].YKL-40 is associated with cancer aggressiveness.It was reported that not serum but urine YKL-40 level can be helpful in the diagnosis of bladder cancer in the assistance to BTA protein.Urine YKL-40 level was significantly higher in all invasive subgroups (T1, T2-T4, and T1-T4) compared to low stage (Ta) and can help determine treatment regimen in early invasive stages [42].
СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ.2018; 17(4): 99-105 оБзоры YKL-40 as an independent marker of tumor progression Serum YKL-40 as a prognostic marker was independent of serum carcinoembryonic antigen in patients with colorectal cancer, of serum CA-125 and CA15-3 in patients with ovarian cancer, of estrogen receptor status, KRAS mutation status, of serum HER2 in patients with metastatic breast cancer, of serum prostate-specific antigen in patients with metastatic prostate cancer, and of serum lactate dehydrogenase in patients with small cell lung cancer or metastatic malignant melanoma and of clinical parameters (i.e., age, performance status, tumor stage, histology), indicating that serum YKL-40 reflects other pathogenic aspects of tumor progression than these tumor markers [23].Plasma YKL-40 in pre-treatment patients was shown to be an independent prognostic biomarker of short overall survival both at time of first cancer diagnosis and at time of relapse in patients with different types of adenocarcinoma (breast, colorectal, endometrial, non-small cell lung, ovary, cervix and prostate), in patients with head and neck and cervix squamous cell carcinoma [36].
In gastric cancer, high YKL-40 protein level was an independent biomarker of short survival and was associated with tumor invasion, lymph node metastasis [43].In patients with localized or advanced small cell lung carcinoma, high plasma YKL-40 levels before chemotherapy independently predicted short survival [44].Pre-treatment plasma and serum level of YKL-40 was an independent prognostic biomarker in patients with metastatic prostate cancer [36].Serum level of YKL-40 is also an independent marker for the aggressiveness of metastatic breast cancer [1].High plasma YKL-40 in patients with metastatic colorectal cancer before treatment was associated with short progression free survival and short overall survival, independently of KRAS status [45].However, serum concentrations of YKL-40 do not show high sensitivity for early diagnostics of cancer and YKL-40 cannot be used as a single screening marker for diagnosis of cancer [1,23].

Elevated YKL-40 level may serve as a useful potential prognostic biomarker for cancer patients
Serum levels of YKL-40 are indicative for the poor prognosis of metastatic process.Increased plasma concentration of YKL-40 is related to poor prognosis and shorter survival in patients with breast cancer, gastric cancer, ovarian cancer, colorectal carcinoma, metastatic prostate carcinoma, melanoma and many other cancers.High plasma YKL-40 levels predicted an absolute 10-year risk of gastrointestinal cancer in patients who were >70 years and smokers [46].
For advanced pancreatic cancer it was shown that the combination of plasma YKL-40, CA-19-9 and osteopontin was more sensitive compared to CA-19-9 alone [47].High plasma YKL-40 was a predictor of chemoresistance in patients with ovarian cancer and breast cancer during treatment [36].In recurrent breast cancer, high serum YKL-40 was associated with metastasis and high tumor grade and was elevated mostly in case of visceral and bone metastasis and less in case of lymph nodes metastasis.Moreover, the highest serum YKL-40 levels were found in patients with more than two different metastatic sites [23].
It was found that colorectal cancer patients with elevated serum YKL-40 after surgery had significantly shorter recurrence-free period and overall survival than patients with normal serum YKL-40, indicating that YKL-40 may be useful for the monitoring of cancer patients [23].

Conclusion remarks
In the present review we shortly highlighted the main features of CLPs, their key function and their ability to contribute to tumor progression.Nowadays we have clear evidences about the correlation with survival, invasion, metastasis etc.only for YKL-40 protein.There are a lot of studies related to the YKL-40 serum levels with cancer aggressiveness and disease progression.However, many fundamental aspects regarding the function, mechanisms of action and regulation of YKL-40 as well as YKL-39 and SI-CLP in cancer remain unclear.Problems regarding the direct or indirect contribution of YKL-39 and SI-CLP to tumor progression remain to be solved.
Future translational researches combining basic and clinical basis are needed and should give the answers on the main questions: "Are CLPs useful clinical biomarker for patients with cancer?" and "Can CLPs potentially become new targets for cancer therapy?".