Up-regulation of Thy-1 Promotes Invasion and Metastasis of Hepatocarcinomas

Hepaticarcinoma is the fifth most common cancer and one of the leading causes of cancer death worldwide. Especially with quick development of liver disease due to hepatitis B virus infection, incidence of hepaticarcinoma has been on the rise and earlier recurrence after operation or liver transplant is common. The underlying root cause is the existence of cancer stem cells (CSCs) (Shupe et al., 2005; Sell et al., 2008; Alison et al., 2009; Chiba et al., 2009; Lee et al., 2009), which, despite their small number, play a major role in tumor origination, development, invasion and earlier metastasis (Reya et al., 2001; Couzin et al., 2003). Oval cells are the stem cells of liver (Bird et al., 2008; Strick-Marchand et al., 2008; Okabe et al., 2009; Shupe et al., 2009). Recently a great deal of research suggested a positive relationship between its proliferation and the incidence of hepaticarcinoma (Alison et al., 2005; Alison et al., 2006; Wu et al., 2006). Thy-1, as a marker protein of oval cells, has recently been suggested to play a role in potential liver cancer stem cells (Dahlke et al., 2003; Ceafalan et al., 2005; Masson et al., 2006; Yang et al., 2008; Yang et al., 2008; Xu et al., 2009). As a gene expressed during embronic period or under pathologic state, AFP (alpha fetoprotein) participates in


Introduction
Hepaticarcinoma is the fifth most common cancer and one of the leading causes of cancer death worldwide.Especially with quick development of liver disease due to hepatitis B virus infection, incidence of hepaticarcinoma has been on the rise and earlier recurrence after operation or liver transplant is common.The underlying root cause is the existence of cancer stem cells (CSCs) (Shupe et al., 2005;Sell et al., 2008;Alison et al., 2009;Chiba et al., 2009;Lee et al., 2009), which, despite their small number, play a major role in tumor origination, development, invasion and earlier metastasis (Reya et al., 2001;Couzin et al., 2003).Oval cells are the stem cells of liver (Bird et al., 2008;Strick-Marchand et al., 2008;Okabe et al., 2009;Shupe et al., 2009).Recently a great deal of research suggested a positive relationship between its proliferation and the incidence of hepaticarcinoma (Alison et al., 2005;Alison et al., 2006;Wu et al., 2006).Thy-1, as a marker protein of oval cells, has recently been suggested to play a role in potential liver cancer stem cells (Dahlke et al., 2003;Ceafalan et al., 2005;Masson et al., 2006;Yang et al., 2008;Yang et al., 2008;Xu et al., 2009).As a gene expressed during embronic period or under pathologic state, AFP (alpha fetoprotein) participates in

Up-regulation of Thy-1 Promotes Invasion and Metastasis of Hepatocarcinomas
Bian-Qiao Cheng, Yi Jiang*, Dong-Liang Li, Jing-Jing Fan, Ming Ma embryonic development and cell division process (Kang et al., 2006).Recent research suggested (Kuhlmann et al., 2006) AFP possessed stem cell characteristics and could be regarded as an ideal marker predicting earlier liver cancer recurrence after operation.However, the shortcoming of AFP loss expression in some samples required a more accurate and specific marker to predict liver cancer occurrence and recurrence independent of or in combination with AFP marker.E-cadherin, a member of cadherin family, mediates cell-cell adhesions and its loss of function could cause breakdown of cell-cell conjunctions.It is therefore commonly used to predict invasion and metastasis of various kinds of malignant tumor.Based on findings above, this article was aimed to investigate the relationship between thy-1, AFP and E-cadherin and to unravel the function of thy-1 in hepaticarcinoma.

Patients and sample collection
Fifty paraffin-embedded tissues with liver cancer who had undergone laparotomy (46/50 had curative hepatic resection and 4/50 had liver transplation; 18/50 well-differentiated and 32/50 poorly differentiated) were included in the study.Pathology diagnosis was made according to the histology of tumor specimens and examinations were performed by experienced pathologists.Fifty corresponding peri-tumorous healthy tissues were used as controls which were confirmed according to pathological diagnosis.The majority of HCC (53 cases) and cirrhosis or hepatitis patients (36 cases) were positive for serum hepatitis B surface antigen.All the blood sample studies were approved by the Institutional Review Board of the fuzhou general hospital.

Immunohistochemistry and Immunocytochemistry
Immunohistochemical evaluation on 4mm-thin tissue sections from formalin-fixed and paraffinembedded samples was performed for thy-1 (sc-53456, dilution1:200; Santa Cruz Biotechnology Inc., Santa Cruz CA), AFP(sc-51506, dilution1:200;Santa Cruz Biotechnology Inc., Santa Cruz CA;), E-cadherin(sc-8426, dilution1:200;Santa Cruz Biotechnology Inc., Santa Cruz CA).The staining reactions were interpreted in the presence of tissue mast cells in portal fields of the liver as internal controls, while AFP and E-cadherin as external controls for embryonic tissues and breast duct carcinoma respectively.

Immunocytochemistry
HepG2 cells were plated at 100,000 cells per well one night before the experiment.Breast carcinoma cell line DBA-DA231 and un-transfected hepg2 cells were used as positive and negative controls respectively.Hepg2cells were transfected with pReceiver-M29/ thy-1 expression vector and treated with Aspirin in the test group according to the manufacturer's instructions of vltrasensitiveTMS-P (mouse/rabbit) kit (Maixin, Fujian China).Briefly, cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.3% Triton-100 for 10 min.The following steps were performed in the same way as immunohistochemistry.

Immunosorbent assay
Thy-1, AFP and E-cadherin ELISA.wereperformed using ELISA kit (R&D) as per manufacturer's instructions.Briefly, 50 µl standard or 50 µl of 1:5 diluted samples was dispensed to standard or sample wells, respectively.Thrity minutes after the addition of 50 µl enzyme label reagent at 37 °C, chromogenic agent A and B were added to react for 10 min at 37 °C with protection from light, following which stop buffer was added.Thereafter, the absorbance at 450 nm was measured with an enzyme-labeling instrument (ELX-800 type).

Statistical analysis
Descriptive statistics, t-test, χ 2 test and One-way ANOVA with LSD multiple comparison were performed.Fisher precise probabilistic method was used to analyze expression intensity, Spearman for correlation analysis.SPSS software (version 11.5 for Windows; spss Inc., Chicago, IL) was used for all statistical analyses.P value less than 0.05 was considered significant.

Results
Altered expression pattern of thy-1, AFP and E-cadherin in liver cancers.Immunohistochemical staining showed reactivities of thy-1, AFP and E-cadherin in 40%, 20%, and 30% of peritumoral normal tissues, whereas 72%, 76% and 64% of neopastic liver tissues respectively.Staining intensities for thy-1 and AFP in normal peritumoral samples were significantly weaker than in tumor samples, but E-cadherin staining showed the opposite results (Figure 1).In addition, we detected a significant correlation between the degree of differentiation of hepatocarcinomas and the expression of   thy-1, AFP and E-cadherin, whereby poorly differentiated hepatocarcinomas were more likely to express thy-1 and AFP but less likely to express E-cadherin than welldifferentiated ones (poorly differentiated: thy-1, AFP and E-cadherin expressed in 81.2%, 68.8% and 53.1% of samples respectively; well-differentiated: thy-1, AFP and E-cadherin expressed in 38.8%, 38% and 83.3% of samples respectively) (Table1).
Lack of correlation between thy-1 and AFP, but negative correlation between thy-1 and E-cadherin.
Both positive expression samples or negative for thy-1 and AFP was 27or 5, Positive expression samples for thy-1of 7 cases but negative 1for AFP and negative 11 for thy-1 but positive for AFP was also observed, there was no significant difference between them (rs=0.116,p=0.421).Both positive expression samples for thy-1 and E-cadherin was 18, both negative for them was 2, positive for thy-1 was 16 but negative for E-cadherin was 14, negative correlation was detected between them (rs=-0.336,p=0.017) (Table 2).In addition, from 53 liver cancer serum samples, levels of thy-1, AFP and E-cadherin detected were 3.694 2.961, 3.121 1.965, and 1.591 0.696 respectively.No correlation was found between AFP and thy-1 (rs=0.046,p=0.746), but there was a negative correlation between thy-1 and E-cadherin (rs=-0.334,p=0.027) (Table 3).
Enhanced Thy-1 expression reduces E-cadherin level.In order to further understand the relationship between Thy-1 and E-cadherin, we transfected hepg2 cells pReceiver-M29/thy-1 expression vector and looked for changes in expression of E-cadherin and thy-1 using RT-PCR.Immunocytochemistry revealed higher expression of thy-1 but lower expression E-catherin in pReceiver-M29/thy-1 transfected hepg2 cells as compared to non-transfected hepg2 cells and differences in expression levels were statistically significant (p<0.05).
when 0.5µmol/ml Aspirin was used to inhibit the wnt/b-catenin signaling pathway (Dihlmann et al., 2003); E-cadherin level was slightly elevated but thy-1 expression was dramatically reduced in hepg2 cells transfected with pReceiver-M29/thy-1 (Figure 2).In addition, E-cadherin staining was obviously weaker in transfected cells, but slightly higher than in non-transfected hepg2 cells treated with Aspirin (Figure 3).Trasnfected hepg2 cells also frequently showed double or multiple nuclei, but this phenotype did not change after Aspirin treatment.

Discussion
Thy-1 is a 25-37KD glycosylphosphatidylinositolanchored glycoprotein expressed mainly in leukocytes and involved in cell-cell and cell -matrix interactions (Rege et al., 2006;Rege et al., 2006).Recently research showed its expression in thymocytes, mesenchyme stem cells, embryo haemopoietic stem cells, smooth muscle cells, and cd34+myeloma cells.Moreover, thy-1 is regarded as a tumor stem cell marker in some cancers (Chen et al., 2005;Yamazaki et al., 2009;Mercati et al., 2009;True et al., 2010).Yang et al findings suggested thy-1+/CD45+ (Donnenberg et al., 2010, Yang et al., 2008) hepatocarcinoma compared to thy-1-/CD45-in liver cancer possessed the ability of conspicuous neoplasia, and thy-1+/CD44+ hepatocarcinoma cells showed enhanced tendency of invasion and metastasis.Subsequent research also supported thy-1 as liver cancer stem cells.In this study we found positive expression of thy-1 in 72% (36 /50 cases) of liver cancer samples with hepatitis B surface antigen positive compared with 40% (20/50 cases) of peritumoral liver tissues including cirrhosis or hepatitis, and the difference was significant (p=0.000?).We further investigated the relationship between thy-1 expression and differentiation degree of liver cancers.Higher expression of thy-1 was found in poorly differentiated hapatocarcinoma than in well-differentiated ones with staining intensity correlating to degree of differentiation and these findings are similar to a former smaller-scale study (Rege et al., 2006).They found decreasing number of CD117+/CD90+ cells from the neoplastic (2.48 +/-0.67) and peritumoral region (0.88 +/-0.12) to the area of para-tumoral (normal) parenchyma (0.13 +/-0.04).This suggested thy-1 could be a tumor stem cell marker of liver cancer.
Tumorigenesis is a multi-step genetic event with activation of oncogenes and surpression of anti-oncogene.We aimed to determine whether thy-1 could interact with other proteins or genes to influence hepatocarcinoma development.Our study deteced a correlation between AFP and thy-1, as well as between E-cadherin and thy-1.A protein expressed during embryo period or pathologic state, AFP participates in embryonic development and cell division.Some recent studies consider (Kuhlmann et al., 2006) AFP as a super marker of oval cells, showing conspicuous clonal expansion in cells of liver cancers.Form tumor and self-renewal capability, AFP possess stem cell characteristic, play an important role in carcinogenesis and recurrence and metastasis of liver cancer.E-cadherin is a calcium-dependent cellular adhesion molecule existing in normal endothelial cells which maintains the structural integrity of tissues.Down-regulation of E-cadherin caused invasion and metastasis of tumor.Here we investigated the expression of AFP and E-cadherin in all liver cancer samples and interestingly their expression levels correlated to thy-1.AFP expresson is higher in poorly differentiated hepaticarcinomas than in well-differentiated ones, but the reverse is true for E-cadherin.In addition, a negative correlation was detected between thy-1 and E-cadherin but there is no relationship between AFP and thy-1.The same expression patterns were observed in 53 blood samples collected from liver cancer patients with blood analytes quantified using ELISA.From findings above we speculate that high expression of thy-1 but low expression of E-cadherin in hepatocarcinoma could be regarded as markers to predict invasion and early metastasis.
In order to further prove that thy-1 possibly participates in invasion and metastasis, we transfected pReceiver-M29/ Thy-1 expression vector (this part was not presented in this article) into hepg2 cells.Immunocytochemistry and RT-PCR results indicated obvious down-regulated expression of E-cadherin but up-regulated expression of thy-1 in transfected hepg2 cells.Under normal condition, E-cadherin is connected to actin cystoskeleton thereby regulates intercellular adhesion by forming a E-cadherin/ catenins Complex with β-catenin and α-catenin (Nieset et al., 1997).Previous study showed that down-regulation or mutation of any member of the complex would result in loss of adhesion ability that is related to tumor invasion and metastasis and therefore the prognosis of the disease (Czyzewska et al., 2010).β-catenin/TCF dimer could also down -regulate E-cadherin transcription and attenuate cell adhesion.On the other hand, Aspirin was found to attenuate β-Catenin expression, suppress cell proliferation, while induce apoptosis (DiMmann et al., 2001).Cording to findings of a previous study (Fromowitz et al., 1987), 0.5µmol/ml Aspirin was used to treat hepg2 cells transfected with thy-1.After 48h treatment, significantly lower expression of β-catenin was detected (this part result was not presented in this article data no shown).In addition, E-cadherin expression slightly increased but thy-1 level decreased compared to hepg2 cells which were transfected but not treated with Aspirin, suggesting that thy-1 and E-cadherin participate in invasion and metastasis of liver cancer.
Taken together, our study demonstrated for the first time that there is no correlation between thy-1 and AFP in liver cancer, but up-regulated expression of thy-1 plays critical roles in the invasion and metastasis of hepatocarcinoma.Furthermore, we also presented the mechanism of thy-1-induced invasion and metastasis of liver cancer with down-regulation of E-cadherin and upregulation of β-catenin.However, high recurrence and DOI:http://dx.doi.org/10.7314/APJCP.2012doi.org/10.7314/APJCP. .13.4.1349Up-regulation of Thy-1 Promotes Invasion and Metastasis of Hepatocarcinomas early invasion/metastasis of cancer are complex processes.Therefore, understanding detailed actions of thy-1 in hepatocarcinoma and designing therapeutic strategies such as RNAi or gene knock-out directed towards thy-1 would hold promise for liver cancer therapy.