I. Overview of liver metastasis of colorectal cancer
The liver is the most common and metastatic organ for colorectal tumors. It is reported that about 150,000 new colorectal cancers are discovered in the United States every year, and 1/4-1/3 of them belong to advanced stage, i.e. stage IV of TNM stage. In 1990, according to the American Cancer Society, 61,000 people died of colorectal cancer in that year, of which nearly 40,000 died of liver metastases.
At the time of initial consultation, about 10% were Duke’s stage A, 20%-30% were stage B, 30%-40% were stage C, and the rest were metastatic patients. The most predominant metastatic organ is the liver. About 60% of patients have metastases at the time of first diagnosis of colorectal cancer. Within 5 years after surgical resection of the primary lesion, liver metastases can occur in 50% of patients. It has been reported that liver metastases can occur in 36%-81% of colorectal cancer cases at autopsy after death [3]. This high incidence may be an important factor affecting the overall level of treatment of colorectal tumors.
Regarding the natural survival of liver metastases from colorectal cancer without any treatment, it is generally short, with a mean survival of 6-10 months and a median survival of 4.5 or 6.6 months, and even a six-month survival rate of 0 has been reported in some cases [1]. The above situation fully illustrates that liver metastasis from colorectal cancer has a poor prognosis if not treated actively, and is one of the main causes of death from colorectal cancer.
Unfortunately, only about 20% of patients with liver metastases are suitable for surgical treatment at the time of diagnosis due to the excessive size of liver metastases, their close relationship with large blood vessels and multiple metastases in the liver [4]. According to the current status, it seems difficult to improve the surgical resection rate simply by improving surgical techniques. Only through early detection of the existence of liver metastases is it possible to treat them as early as possible, thus increasing the survival rate and improving the prognosis.
Mechanisms of liver metastasis formation of colorectal cancer
The formation of liver metastasis from colorectal cancer is a complex process involving the role of multiple factors between tumor cells and the host. The main processes of its metastasis include.
1. Invasion of extracellular matrix At the site of primary foci, the adhesion between cancer cells is reduced, the charge density on the surface of cancer cells is increased, the mutual repulsive force is increased, the cells are easy to move, and various enzymes secreted by tumor cells (such as serine proteases and matrix metalloproteinases) digest the extracellular matrix and are easy to adhere and connect with the basement membrane, resulting in tumor cells breaking through the barrier formed by connective tissue.
2. Entering portal system Cancer cells adhere to basement membrane and vascular endothelial cells, the vascular endothelial cytoskeleton is affected and the cell gap is increased. Under the action of matrix metalloproteinase, heparinase, cytokinin, elastase and urokinase, cancer cells cross the vascular endothelium and basement membrane and enter the portal system.
3.Cancer cells survive in circulation Most of the cancer cells that enter the blood are killed by the host, some are dormant, and a small subpopulation of cells with high metastatic potential can adhere to the vascular endothelium and penetrate the vessel wall to form metastatic foci.
4.Proliferation of cancer cells in the liver Whether cancer cells can grow in a new microenvironment after penetrating the blood vessels is the key to the final formation of metastatic foci. Cancer cells can form liver metastases only when they proliferate microvascularly under suitable intrahepatic microenvironmental conditions [7].
The formation of liver metastases from colorectal cancer is related to the biological characteristics of cancer cells, the immune status of the body and the microenvironment of the liver. The formation of CRC liver metastases can be simply expressed as follows: primary colorectal cancer – portal vein tract blood – liver – hepatic vein into the lung circulation to the whole body (or into the bile).
III. Molecular markers related to liver metastasis of colorectal cancer
Infiltration and metastasis benefit from the involvement of some proteins, such as stimulation of adhesion between tumor cells and host cells or extracellular matrix; protein hydrolysis of tumor cells to host barriers such as basement membrane, directional movement of tumor cells, and clonal growth within the target organ.
(I) Alteration of cell adhesion-related receptors
1. calmodulin-ligand protein system: β-cadherin (β-catenin) is a cytoskeletal protein that binds to the C-terminus of calmodulin (E-cadherin) and the N-terminal end binds to α-cadherin (α-cat), forming the E-cad/β-cat/α-cat complex, which is attached to actin filaments by α-cat [8] . the E-calmodulin The E-cad/β-cat/α-cat complex is the main adhesion molecule that maintains the polarity, morphology and histological integrity of epithelial cells. With the loss of complex function, inter-tumor cell adhesion decreases and easily detaches, invading the surrounding tissue. It has been shown that deletion of β-catenin is associated with liver metastasis in colorectal cancer [9].
Carcinoembryonic antigen (CEA): CEA is a highly glycosylated cell surface glycoprotein, a member of the immunoglobulin superfamily, which is an adhesion molecule of tumor cells and plays an important role in the recurrence and metastasis of colorectal cancer. It is most widely used in the early detection of liver metastasis from colorectal cancer [10]. CEA receptors are present on hepatic Kupffer cells and induce cytokines (IL-1α, IL-1β, IL-6, TNFα) secretion from Kupffer cells. It induces the expression of intercellular adhesion molecules by hepatic sinusoidal endothelial cells, thus increasing tumor adhesion and retention in the liver [11].
3. CD44: The adhesion factor CD44 on the surface of tumor cells is a transmembrane hyaluronan receptor that mediates adhesion to endothelial cells. The high expression of its variants CD44v6 and CD44v8-10 is thought to be closely related to liver metastasis of colorectal cancer [12,13].CD44 splice variants may affect the migration and motility of tumor cells by promoting adhesion of tumor cells to vascular endothelial cells and extracellular matrix, promoting tumor cell invasion into the stroma, and affecting the aggregation and distribution of tumor cytoskeletal proteins The combination of several aspects thus affects the formation of tumor metastasis [14]. Tumor cells expressing CD44 variants can evade recognition by the host immune system during the metastasis process and be spared from clearance.
Integrins: These molecules mainly mediate the adhesion of cells to the extracellular matrix (ECM), allowing the integration of the intracellular skeleton with the extracellular matrix to form a whole, and are also involved in cell-to-cell adhesion. It mediates adhesion mainly through recognition of arginine-glycine-aspartate (RGD) tripeptide sites.Takamura H et al. showed positive staining for integrin αVLA3 in 58% (11/19) of colorectal cancer tissues with liver metastases using immunohistochemical assays, which was significantly higher compared to non-metastatic tissues (0%). The increased integrin α3β1 suggests that it may play an important role in liver metastasis of colorectal cancer.
5, Sialylated Lewis oligosaccharide-X (sLex) antigen: SLX is also a ligand for endothelial leucocyte adhesion molecule-1 (ELAM-1), as a receptor for E-selectin on the surface of hepatic vascular endothelial cells. ligand of ELAM-1, which plays an important role in the liver metastasis of colorectal cancer. Tumor cells with high expression of sLex antigen shed from the primary foci, enter the vasculature, adhere to the hepatic vascular endothelium and grow and form liver metastases, which are more likely to infiltrate the basement membrane, adhere to activated human vascular endothelial cells and form liver metastases. Studies have confirmed that sLex antigen expression in metastatic foci of colorectal cancer is stronger than that of sLex antigen in the primary foci [18,19].
The tumor-associated antigens CA 19-9 and sLex are expressed throughout the colon. These glycoantigens are elevated as colon cancer evolves from non-metastatic to metastatic tumors, and sLex is a common feature of colon cancer cells that tend to metastasize. Certain antigens associated with serum antigens are also associated with tumor progression; CA19-9 antigen levels are significantly higher in adenoma and colon cancer than in normal mucosa. Other lectins such as lactose-binding lectin or galactose-binding lectin-3 (galectin-3) have also been associated with tumorigenic transformation and metastatic evolution [20, 21].
(ii) Protease alterations
Tumor cells detach from the basement membrane or extracellular matrix, and cellular proteases degrade the basement membrane or extracellular matrix to infiltrate into lymphatic vessels or blood vessels to constitute infiltration and metastasis. The extracellular matrix (ECM) degrading proteases include serine proteases, metalloproteases, elastases and cysteine proteases. Among them, metalloproteinase family (MMPs) is considered as the most important group of proteases and an important target for anti-tumor metastasis. The most studied in colorectal cancer are matrix metalloproteinases and urokinase, etc.
1. Matrix metalloproteases (MMP) MMP is a metal-dependent endopeptidase with protease activity against various components of ECM. It is regulated by tissue inhibitors of metalloproteinases (TIMP) and membrane-type matrix metalloproteinase (MT-MMP).
MMP can be divided into the following groups: collagenases (or gelatinases), stromelysin, matrilysin, and MT-MMT. the most important collagenases are MMP-2 and -9, which hydrolyze collagen from the ECM. MMP in normal colon tissue is present only in neutrophils in blood vessels and in macrophages in collecting lymph nodes. The amount of MMP-2 and -9 is higher in carcinomas and correlates with Dukes stage.
2. plasminogen activator DeBruin et al. demonstrated that urokinase-type plasminogen activator (uPA) activity was low in normal colonic mucosa, whereas tissue-type plasminogen activator (TTPA) activity was low. type plasminogen activator (tPA) activity was high, but the opposite was true in colon cancer, and the activity in colon adenoma was in between.
The level of uPA expression was negatively correlated with prognosis, and it has been shown that uPA protein levels significantly correlate with tumor angiogenesis. Urokinase-type fibrinolytic activator receptor (uPA receptor, uPAR) is highly expressed in human gastrointestinal tumors including gastric, pancreatic, and colorectal cancers. uPAR expression is still not completely elucidated in relation to colon tumor formation. The level of PAI-1 in urokinase-type fibrinolytic enzyme activator (uPA inhibitor, PAI) tumors is negatively correlated with prognosis.
3, Type II serine protease ST14/SNC19 was screened in 1993 from a meiotic hybridization library of colorectal cancer with the ability to degrade extracellular matrix, which was associated with metastasis. It was also confirmed that its action substrates are uPA and HGF/SF precursors (BC-uPA and Pro-HGF/SF), which are associated with tumor adhesion, migration, infiltration and vascular growth. In colorectal cancer studies, lymph node metastasis was high in those with high ST14/SNC19 expression in the paracancerous mucosa.
Applying Microarray technology, comparing ST14 before and after transfection of RKO cells, 26 metastasis-related genes were found to be differentially expressed. Among them, 15 were metastasis-promoting genes and 11 inhibited tumor cell metastasis. The expression of metastasis-promoting genes was up-regulated, such as growth factors, cell adhesion factors, metastasis-related protease classes, signal transduction molecules and oncogenes.
The expression of metastasis-inhibiting genes was down-regulated, such as Maspin and TIMP2. Changes in all of these genes may lead to enhanced cell metastasis. However, five of the genes whose expression was down-regulated were pro-metastatic genes and seven of the genes whose expression was up-regulated were metastasis suppressor genes, while it was possible that the metastatic ability was diminished. The effect of these on metastasis needs to be confirmed by further studies. High expression of ST14 increased cell migration ability, invasion ability, promoted ECM degradation, and depolarized the skeletal protein F-actin by confocal microscopy. This type of change was also confirmed by high through gene microarray assay.
Genes whose expression was significantly up-regulated after SNC19 transfection expression were: BRMS1, CSAP8, caveolin-1, CSF1, C-ETS2, ETV4, HPSE, IGF2, KISS1, MGAT5,, CSAP9, H-Ras, ICAM5, KAI1, MMP15, MMP16, MUC1, PIK3. PAI2, TGFA.
The genes whose expression was significantly down-regulated after SNC19 transfection expression were MMP3, MMP7, Maspin, BCSG1, Osteopontin, and TIMP-2.
4. Maspin, a member of the serine protease inhibitor superfamily, was obtained by meiotic hybridization and differential display techniques between normal breast epithelium and breast cancer by Zou et al. in 1994. Applying the mRNA differential display analysis technique, the Institute of Oncology of Zhejiang University found that maspin expression was increased in paired specimens of gastric, and pancreatic and colorectal cancers. In the application of gene microarray study, maspin expression was also found to be significantly elevated in 16 cases of colorectal cancer by testing 21 pairs of paired specimens of colorectal cancer and normal mucosal tissues. Antisense maspin transfection of colorectal cancer COLO205 cell line showed morphological changes of increased aggregation, enhanced adhesion and irregular morphology of colorectal cancer cells. The study showed that Maspin gene is associated with colorectal cancer metastasis, but the specific mechanism of its action still needs further study to elucidate.
5.Altered expression of other proteases and protease inhibitors The expression of histone protease D (cathepsin D) affects the infiltration of cancer cells. Secreted histone proteases are mainly derived from stromal cells such as fibroblasts. In colorectal cancer, histone D is also expressed in malignant cells, but the relationship with prognosis is unclear because of the great variability in its expression.
(iii) Vascular growth factor
There is a synergistic effect on this. These factors can also stimulate tumor cell growth through autocrine must stimulation and paracrine secretion. The production of various vascular growth factors is obviously important for the rapid proliferation and infiltrative growth of tumors, because an abundant blood supply is necessary for tumor growth. b) They stimulate the proliferation of vascular endothelial cells and the production of other capillary-forming factors. b-FGF and TGF-b, a (TGF-b and a colorectal cancer cells can produce angiogenin [25]/) basic fibroblast growth factor (b-FGF) [25], transforming growth factor
About 100 years ago, it was found that the blood supply in tumors was much richer than in surrounding normal tissues, whether induced by tumor cells or not. 1971 Folkman proposed that tumor growth is dependent on blood supply and that without blood flow, oxygen and nutrients tumors would become resting, remain 2-3 mm3 in size and then apoptotic [26]. There are a series of family members of vascular growth factors, and there is the presence of vascular growth factor-related receptor (VEGFR) on the surface of tumor cells. The binding of this transmembrane receptor to the ligand VEGF activates tyrosine kinase (TK), which in turn leads to the proliferation of vascular endothelium or/and lymphatic vascular endothelium.
Among the VEGF family members, VEGF A is the main mediator of vascular proliferation, and is associated with VEGF B, C, D and E, and each has associated VEGFR1, 2 and 3 and placental growth factor (PIGF). lymphatic vascular endothelial cells.
In addition to promoting endothelial cell proliferation, VEGF can regulate endothelial cell wandering, increase vascular permeability, and modulate immune responses to regulate vascular lymphatic vessel proliferation. Apparently vascular endothelial cells are involved in tumor development, infiltration and metastasis.
(IV) Other metastasis-related molecules
The nm23 gene was isolated by Steeg from a murine melanoma cell line with different metastatic potential. nm23 may regulate cell proliferation through the process of serine phosphorylation. Among them, the nm23-H1 isoform is more closely related to tumor metastasis [27]. The human homologous gene nm23-HI is also localized on chromosome 17q21, and this band is frequently lost heterozygously in colorectal cancer. It has been observed in human colorectal cancer that the nm23-H1 gene LOH may be a late event in the malignant progression of colorectal cancer and is closely associated with severe aggressive behavior of colorectal cancer, and thus suggests that the nm23-H1 gene may be involved in the regulation of the malignant progression and metastatic process of human colorectal cancer [28]. However, as studies have progressed, opposite conclusions have been made [29].
2, PRL3 gene Vogelstein and colleagues used serial analysis of gene expression (SAGE.) to identify a phosphatase of regenerating liver metastases that was highly expressed in all 12 colon cancers, PRL-3 (phosphatase of regenerating liver-3, also known as PTP4A3Protein tyrosine phosphatase type IVA3), which was not expressed in its matched primary tumor of the same individual. It belongs to a new class of isoprenylated phosphatases [30].
3. bone bridge protein (OPN) OPN is a secreted glycosylated phosphoprotein, which is not only closely related to the mineralization of bone tissue, but also to various physiological and pathological phenomena such as Ca2+, NO and other cellular messengers, macrophage chemotactic response, smooth muscle cell proliferation, metastasis and infection of tumors, and is considered as a new cell signaling molecule [31]. It has been shown that the expression of OPN is significantly higher in liver metastatic tissues of colorectal cancer compared with the primary tumor [32], and its ligand-receptor interaction may play an important role in promoting liver metastasis of colorectal cancer. Ding Ling et al. demonstrated that tumor cells expressing Osteopontin had reduced homogeneous adhesion and easily detached from the original tumor masses. In contrast, the heterogeneous adhesion ability was increased, such as the increased adhesion between vascular endothelial cells, which could easily adhere to the metastatic site. From the immunohistochemical observation, the tumor tissue and its surrounding liver tissues were positively colored, which may be related to the presence of its receptor CD44 and integrin in hepatocytes.
Ras protein is mainly involved in cell proliferation and signal transduction; Rab protein is involved in regulating intracellular membrane traffic; Rho protein (30% homology with Ras) plays a regulatory role in the composition of cytoskeletal network. Rho protein (30% homology with Ras) plays a regulatory role in the composition of cytoskeletal network, thus affecting cell morphology [33]. It was shown that Rho protein overexpression was detected in liver metastases from colorectal cancer [34]. It was found that defects in Ki-ras codon 12 and liver metastatic potential of colorectal cancer are closely related [35].
Although more genes related to liver metastasis of colorectal cancer and related molecular events are being revealed through the persistent efforts of researchers. Vogelstein et al. suggested that hundreds of tumor-associated genes are linked to each other in small “nodes” and their networks, and the nodes are linked to each other as a group of networks [45]. The occurrence of liver metastasis must involve the synergistic or antagonistic effects of several genes and proteins, and the inconsistent results of some studies have caused confusion in the research work.
V. Molecular biology and bioinformatics of liver metastasis in colorectal cancer
More and more scientists apply the viewpoint of systems biology to study the genome level, transcription level and protein expression level as a whole system to understand the whole life system from a larger and deeper level. For liver metastasis of colorectal cancer, the Institute of Oncology of Zhejiang University has integrated chromosome copy number variation data to analyze the differences of gene expression profiles among different phenotypes, and studied the expression of genes contained in a specific segment of chromosomes. And the following studies were conducted.
(1) Seven chromosomal amplification regions and six chromosomal deletion regions were identified in the CGH analysis of 50 human primary colorectal cancers, among which a high frequency deletion at 16q was not reported in the same way. The 16q20-21 region was analyzed by bioinformatics to find genes associated with liver metastasis, and further verified by biotechnological methods one by one to obtain relatively holistic information.
(2) The HG-U95A microarray from AFFYMETRIX was applied to analyze the whole genome expression profiles of 13 cases of colorectal cancer tissues and 12 cases of liver metastases from colon cancer, and 9 cases of corresponding normal colorectal mucosal tissues were used as controls. When the same screening statistical algorithm and the same classification method were used, the integration of chromosome copy number variation data with gene microarray data could substantially improve the tumor classification accuracy from 36% to 95% and obtain a set of liver metastasis-associated genes of colorectal cancer.