Molecular Mechanism of Hepatic Metastasis of Colorectal Cancer

Fig. 3.1

Expression of OPN in colorectal cancer and hepatic metastatic tissue

By combining the related functional detection results of colorectal cancer cells after OPN transfection, a hypothesis is proposed that OPN influences occurrence and development of hepatic metastasis of colorectal cancer: for the colorectal cancer cells with high expression of OPN, homotypic adhesion capability between cells is reduced so that tumor cells are easy to detach from primary focus and complete the first step of metastasis; GJIC function between cancer cells is inhibited and intercellular communication is weakened; OPN also can enhance heterotypic adhesion between colorectal cancer cell and blood vessel endothelial cell, and expression of metastasis-related gene CD44 is strengthened, and expression of E-cadherin is inhibited, which urges tumor cell to adhere to extracellular matrix. During the course of reflux through portal vein, OPN provides possibility for colorectal cancer cell to invade peripheral vessel and to easily remain in the liver. The combined ligand-receptor action between OPN and its chemotactic receptor CD44 and another receptor integrin makes colorectal cancer easy to form metastatic focus in the liver, which demonstrates that OPN is one of the important genes involved that can influence occurrence and development of hepatic metastasis of colorectal cancer.

Through the experimental study on expression of OPN and metastasis-related mechanism, and combining their related literature, we deduce the possible action mechanism of OPN that promotes hepatic metastasis of colorectal cancer. OPN is secreted from colorectal cancer cell, which accounts for decreased expression of adhesion molecules such as E-cadherin, reduced homotypic adhesion and adhesion force between cancer cells, inhibited GJIC function, and enhanced invasive movement capability of cells. Thus cancer cells can be detached from the primary focus to enter peripheral circulation so as to initiate metastasis of colorectal cancer. At the same time, due to the expression of OPN, expression of another metastasis-related factor CD44 also increases, and heterotypic adhesion action between colorectal cancer cell and ECM and blood vessel endothelial cell is enhanced. In addition, chemotactic receptor CD44 of OPN and another receptor integrin exist in the liver. Ligand-receptor action between them makes colorectal cancer easy to form metastatic focus in the liver.

3.4.2 Secreted Protein, Acidic and Rich in Cysteine-Like 1 (SPARCL1)

Based on the results of Affymetrix GeneChip system and by adoption of mathematical analysis method singular value decomposition (SVD), the Tumor Research Institute of Zhejiang University conducted an integrated analysis on cytobands and genes expression profile data, of which cytoband 4q22 with the most significant difference was further analyzed, and discovered the main contributing gene osteopontin (OPN) of high expression and the main contributing gene secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) of low expression.

Both SPARCL1 and osteopontin belong to adhesion molecule that mediates cell matrix mutual action. SPARCL1 was discovered in the study on non-small cell lung cancer conducted by Schraml et al. for the first time in 1994, named as MAST9 [30]. SPARCL1 protein belongs to secreted protein, acidic and rich in cysteine (SPARC) family, with 62 % of homologous nature as secreted protein, acidic and rich in cysteine (SPARC) sequence. Both of them have cysteine-rich follistatin-like (FS) structure field structural domain and extracellular calcium binding (EC) structural domain. But N end of SPARCL1 is far longer than that of SPARC, as shown in Fig. 3.2. It is also named as SPARC-like 1 because its structure is highly homologous as SPARC. In recent years, the study discovered that downregulation of the expression of SPARCL1 in CD133+/CD34+ cell indicates the possible regulation related to malignant tumors and other diseases [31].

Fig. 3.2

Schematic diagram of action mechanism of OPN in hepatic metastasis of colorectal cancer. RGD arg-gly-asp (arginine-glycine-aspartic acid), GJIC gap junction intercellular communication (cell gap junction)

Immunohistochemical detection discovered that expression of SPARCL1 in cytoplasm and cell membrane of non-metastatic colorectal cancer is significantly higher than that in primary focus tissue of hepatic metastatic colorectal cancer, but its expression in hepatic metastatic focus is significantly lower than the paired primary focus tissue of colorectal cancer. Through real-time quantitative PCR validation, the difference in the abovementioned SPARCL1 is also significant in mRNA level.

Western blot and RT-PCR method verified that there is no expression of SPARCL1 protein and mRNA in the three kinds of colorectal cancer cell lines, i.e., RKO, SW480, and SW620. In in vitro experiment, by adoption of MTT method and through cell scratch test, it was discovered that SPARCL1 recombinant protein did not significantly change the proliferation capability of the three kinds of colorectal cancer cells, but migration capability of RKO cells was weakened significantly. Such inhibitory action was not observed in SW480 and SW620.

The present studies on SPARCL1 are summed up as follows: (1) Expression level of SPARCL1 is high in non-metastatic colorectal cancer tissue while low in hepatic metastatic focus, which indicates that SPARCL1 perhaps is an early event in the process of hepatic metastasis of colorectal cancer and may serve as the marker for early prediction of hepatic metastasis of colorectal cancer. (2) In in vitro experiment, it was observed that SPARCL1 recombinant protein mainly has the ability of inhibiting the migration of colorectal cancer cells. SPARCL1 may become a candidate target in hepatic metastatic treatment.

3.4.3 Mammary Serine Protease Inhibitor (Maspin)

Maspin was obtained through suppression subtractive hybridization and differential display between the normal mammary epithelial cells and breast cancer cells by Zhou et al. in 1994. Encoded protein maspin has relatively high homology with other members of serine proteinase inhibitor superfamily, of which homology with equine and human neutral, monocyte elastase inhibitor is 43 % and 39 % respectively. Expression of maspin has been found in mammary epithelial cells, mammary myoepithelial cells, and prostatic epithelial cells.

By applying mRNA differential display analysis technology, the Cancer Research Institute of Zhejiang University screened out ten pairs of paired specimens of cancer tissue of solid tumor (two pairs of cardia cancer, esophageal cancer, gastric cancer, colorectal cancer, and ovarian cancer, respectively) and normal tissue through 32 primers from ten pairs of clinical tumor tissue samples. One hundred twenty-seven differential display fragments were obtained. A human solid tumor-related gene EST pool was preliminarily established. Among these differential display fragments, we found that expression of maspin increased in four pairs of specimens of gastric cancer, cardia cancer, and colorectal cancer. In the study applying gene chip of more than 12,000 genes and EST, we also found that expression of maspin significantly increased in 16 pairs of colorectal cancer through the detection for 21 pairs of specimens of colorectal cancer and normal mucosa tissue.

Through the experimental study relating to the expression of maspin in colorectal cancer and metastasis, it was found that:

1.

When importing exogenous antisense maspin (AsCOLO205) into COLO205 colorectal cancer cell line, it was discovered that CD44 was positively related to maspin and negatively related to CD62, while there is no significant change in CD54.
2.

When applying gene chip and bioinformatical analysis on the change in expression of AsCOLO205, change was discovered in adhesion-related genes, such as cadherin genes; movement-related genes, such as actin-gene; and cell information transmission-related genes, such as cell linker protein gene.
3.

When applying laser confocal technology to detect functional change in cell linker protein of AsCOLO205, it was discovered that expression level of gap junction protein of AsCOLO205 increased, and the function was somewhat restored, but its function is still poor compared with normal fibroblast.
4.

Antisense maspin transfected COLO205 cell line of colorectal cancer. Morphological changes such as aggregation and proliferation of colorectal cancer cells and enhancement of adhesion occurred.

Therefore, we infer that possible action mechanism of maspin in colorectal cancer metastasis is as follows: At the early stage of metastasis of colorectal cancer, expression of maspin will increase; adhesion function of colorectal cancer cells is reduced through downregulation of the expression of some adhesion molecules, such as CD44 and cadherin, which is favorable for detaching cancer cells from primary tumor so as to initiate metastasis. Upregulation of P-selectin makes cancer cells easy to become oncogenic at the transportation stage so as to improve survival capability of cancer cells. At the late stage of colorectal cancer metastasis, i.e., the stage where secondary tumor is formed, downregulation of the expression of maspin enhances the adhesion capability of colorectal cancer cells so that oncogenesis of cancer cell increases, which is favorable for forming secondary tumor in target organ; growth of cancer cell is also accelerated, which is favorable for growth of colorectal cancer. Intercellular communication of cancer cells is restored to a certain degree, which is favorable for cancer cells to quickly obtain the characteristics of a certain tumor. Perhaps this is one of the reasons for the difference in phenotype between secondary tumor and primary one in most cases.

3.4.4 ST14 Gene

ST14 gene was discovered by the study group of Prof. Zheng Shu of the Cancer Research Institute of Zhejiang University for the first time. The encoded ST14 protein belongs to type II serine proteinase. It is deemed as one of the embers of protein hydrolase family that involves tumor-invasive metastasis [32]. Besides self-activation, possessing activity of collagenase, and degradation of extracellular matrix, this protein can identify and activate such proteins as proteinase-activated receptor 2 (PAR2), precursor of hepatocyte growth factor/scatter factor (HGF/SF), and precursor of urokinase-type plasminogen activation factor [33, 34]. These proteins are closely associated with the growth and metastasis of tumor [35].

The Cancer Research Institute of Zhejiang University applied GeneChip (microarray) technology to detect the change in gene expression profile before and after colorectal cancer cell line RKO transfected ST14 gene and discovered that high expression of ST14 gene may cause upregulation of the metastasis-related integrin β1 (ITGB1), matrix metalloproteinase 1 (MMP1), MALAT1, AP1S3 genes, and oxidative phosphorylation pathway (as shown in Table 3.1).

Table 3.1

Differential expression genes after RKO cell line transfected ST14

Top 10 upregulation probe sets			Top 10 downregulation probe sets
	Probe set ID	Gene symbol		Probe set ID	Gene symbol
1	1553538_s_at	N/A^a	1	1554237_at	SDCCAG8
2	1553551_s_at	MTND2	2	1555623_at	N/A^a
3	1553575_at	N/A^a	3	1555801_s_at	ZNF533
4	1555461_at	N/A^a	4	1558048_x_at	N/A^a
5	1555653_at	MTND5	5	1558105_a_at	N/A^a
6	1555731_a_at	AP1S3	6	1561775_at	N/A^a
7	1558250_s_at	N/A^a	7	1564220_a_at	N/A^a
8	1558678_s_at	MALAT1	8	1569110_x_at	PDCD6
9	1560514_at	LOC285205	9	200600_at	MSN
10	1561042_at	ITGB1	10	200916_at	TAGLN2

^a N/A not available

According to the present study results, encoded protein of ST14 may participate in signal transduction, jointly influence matrix degradation and epithelium migration, and enhance tumor infiltration and metastasis through direct degradation of extracellular matrix and activation of other membrane proteins or matrix source proteins in certain condition, such as growth factor, protein hydrolase, G protein-linked receptor on cell surface, etc. It can be summed up as ST14-centered network relation, where ECM, scHGF/SF, and Pro-UPA are its stroma, F-action, and HAI-1 are binding proteins (as shown in Figs. 3.3, 3.4, and 3.5).