Abstract:
The present invention relates to a biomarker associated with a cancer and a method using the biomarker to evaluate a risk of proliferation, invasion, or metastasis of a cancer. The method of the present invention comprises the following steps: (A) providing a tissue sample to evaluate for risk of proliferation, invasion, or metastasis of a cancer, wherein the tissue sample comprises a non-cancer region, and a suspected cancer region; (B) detecting expression levels of a biomarker and a predetermined standard in the non-cancer region and the suspected cancer region respectively, wherein the biomarker is T-cell lymphoma invasion and metastasis 2 (TIAM2); (C) comparing the expression levels of the biomarker and the predetermined standard in the non-cancer region to the expression levels of the biomarker and the predetermined standard in the suspected cancer region.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The present invention relates to a biomarker associated with a cancer and a method using the biomarker to evaluate the risk of proliferation, invasion, or metastasis of a cancer and, more particularly, to a biomarker and a method using the biomarker which can be used to evaluate the risk of proliferation, invasion, or metastasis of cancer in an early stage. 
         [0003]    2. Description of Related Art 
         [0004]    In recent years, it is found that foods or food additives, and environmental pollutions are blamed for directly causing cancer. It is also found that not only in Taiwan, but also in the developed countries around the whole world, the incidence rates of cancers are quite high. Additionally, according to the data published by the American Cancer Society, cancer is perhaps the most significant threat to public health. 
         [0005]    If the proliferation, invasion, or metastasis of cancers can be detected in the early stage, the survival rate of patients with cancers can be greatly increased. Hence, many studies focus on developing methods for analyzing or predicting cancers in the early stage. Currently, as the developments of gene profiling and proteomics, biomarkers for analyzing or predicting cancers are well developed, such as α-fetoprotein (AFP), T-cell lymphoma invasion and metastasis 1 (TIAM1), and N-cadherin. 
         [0006]    However, all of them are applied with limitations. For example, AFP is a useful serological marker for diagnosis of hepatocellular carcinoma (HCC), but it showed 40% false-negative. Furthermore, up-regulation of N-cadherin is only correlated with recurrence after surgery, and TIAM1 overexpression was observed in 63.8% poor prognosis of HCC patients. Besides, since these proteins also expresses in normal liver tissue, the determination of pathological overexpression of given protein may not be easy. Hence, it is desirable to provide a biomarker and a method using the biomarker, which can improve the accuracy of predicting the risk of proliferation, invasion, or metastasis of a cancer. 
         [0007]    In addition, hepatocellular carcinoma (HCC), i.e. liver cancer, is the fifth-most common cancer worldwide and shows high prevalence in Asia and Africa. Despite the heterogeneous etiology, one unique feature of HCC is its aggressiveness with early vascular invasion and metastasis. If the proliferation and the invasion/metastasis in hepatocellular carcinoma can be predicted in the early stage, the incidence rate and the mortality of the liver cancer can be greatly decreased. Therefore, it is also desirable to provide a biomarker and a method using the biomarker, which can predict the risk of proliferation, invasion, or metastasis of a liver cancer precisely. 
       SUMMARY OF THE INVENTION 
       [0008]    The object of the present invention is to provide a method for evaluating a risk of proliferation, invasion, or metastasis of a cancer, so the risk of proliferation, invasion, or metastasis of a cancer can be predicted precisely through the method of the present invention. 
         [0009]    Another object of the present invention is to provide a biomarker for evaluating a risk of proliferation, invasion, or metastasis of a cancer, which can be used to evaluate whether a tissue in a suspected cancer region is a cancer tissue or not. 
         [0010]    To achieve the object, the method for evaluating risk of proliferation, invasion, or metastasis of a cancer of the present invention comprises the following steps: (A) providing a tissue sample to evaluate for risk of proliferation, invasion, or metastasis of a cancer, wherein the tissue sample comprises a non-cancer region, and a suspected cancer region, and the tissue sample is preferably selected from the group consisting of liver tissue, breast tissue, pancreas tissue, brain tissue, thymus tissue, prostate tissue, colon tissue, or other solid tissues; (B) detecting expression levels of a biomarker and a predetermined standard in the non-cancer region and the suspected cancer region respectively, wherein the biomarker is T-cell lymphoma invasion and metastasis 2 (TIAM2); (C) comparing the expression levels of the biomarker and the predetermined standard in the non-cancer region to the expression levels of the biomarker and the predetermined standard in the suspected cancer region through the following equation (I): 
         [0000]      Value=(the expression level of the biomarker in the suspected cancer region/the expression level of the predetermined standard in the suspected cancer region)−(the expression level of the biomarker in the non-cancer region/the expression level of the predetermined standard in the non-cancer region)  [Equation (I)]
 
         [0000]    wherein when the value is positive, this indicates high risk of proliferation, invasion, or metastasis of liver cancer, breast cancer, thymus cancer, prostate cancer, colon cancer, pancreas cancer, or other solid cancers; and when the value is negative, this indicates high risk of proliferation, invasion, or metastasis of brain cancer. 
         [0011]    In addition, the present invention also provides a biomarker for evaluating risk of proliferation, invasion, or metastasis of a cancer, which is selected from the group consisting of nucleotides, a complementary of the nucleotides, a derivative of the nucleotides, a protein, a derivative of the protein, a peptide of the protein, a mutation of the protein of TIAM2. 
         [0012]    Preferably, the biomarker of TIAM2 protein was used in the method of the present invention. 
         [0013]    When the method of the present invention is used to evaluate whether a person is at a risk of proliferation, invasion, or metastasis of a cancer or not, the prediction accuracy can be greatly improved. In addition, the present invention also provides a novel biomarker, TIAM2, which expresses specifically in tumor cells. Hence, when the biomarker of the present invention is used to evaluate whether a person is at a risk of proliferation, invasion, or metastasis of a cancer or not, the prediction accuracy can be further improved. More especially, the method and the biomarker of the present invention can be used to evaluate the risk of the invasion or metastasis of a solid cancer, which cannot be accomplished by the conventional method and biomarker. Hence, the method and the biomarker of the present invention can predict the severity of the solid cancer in an early stage, and thereby doctors can provide effective treatment to patients to delay the invasion or metastasis of tumor cells into vessels. 
         [0014]    According to the method of the present invention, the non-cancer region means a region of normal tissue. Preferably, the non-cancer region is surrounded around the suspected cancer region. In addition, the risk means a possibility that the cells in the suspected cancer region are tumor cells, or a possibility that a person to be evaluated suffers from a cancer. In addition, when the value is positive, this indicates that the suspected cancer region is a cancer region, i.e. a region of tumor tissue, and the person is at risk of proliferation, invasion, or metastasis of a cancer. Furthermore, the method of the present invention can be used to predict not only the proliferation of a cancer, but also the invasion or metastasis of a cancer. When the value is positive/negative, this indicates that the tumor cells may invade or metastasize into blood vessels. The invasion or metastasis of the tumor cells may cause the cancer become severe, and metastatic cancers may be developed. 
         [0015]    In addition, according to the method and the biomarker of the present invention, TIAM2 is T-cell lymphoma invasion and metastasis 2 short fowl (TIAM2S), or T-cell lymphoma invasion and metastasis 2 long form (TIAM2L). Preferably, TIAM2 is TIAM2S. In addition, according to the method and the biomarker of the present invention, the sequence of the nucleotides of TIAM2 is SEQ ID NO: 1, the sequence (i.e. the expressed sequence) of the nucleotides of TIAM2S is SEQ ID NO: 2, and the sequence of the protein of TIAM2S is SEQ ID NO: 3. 
         [0016]    According to the biomarker and the method of the present invention, cancer may be liver cancer, breast cancer, pancreas cancer, brain cancer, thymus cancer, prostate cancer, colon cancer, or other solid cancers. Preferably, the cancer is liver cancer. 
         [0017]    In addition, according to the method of the present invention, the tissue sample may be any tissue nodule to be detected. For example, the tissue sample can be nodules of liver, breast, pancreas, brain, thymus, prostate, colon, or other tissues. Preferably, the tissue sample is liver nodules. 
         [0018]    Furthermore, according to the method of the present invention, the expression level may be a protein expression level, or an mRNA expression level. 
         [0019]    When the expression level is a protein expression level, it can be detected through any conventional analysis for protein expression. Preferably, the protein expression level is detected through Western blot analysis, Gel electrophoresis, Enzyme-linked immunosorbent assay (ELISA), Immunohistochemistry (IHC), Immunoprecipitation (IP), or Mass spectrum analysis (MS). More preferably, the protein expression level is detected through Western blot analysis. In addition, the predetermined standard used in the analysis for protein expression can be α-tubulin, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), or β-actin. Preferably, the predetermined standard is α-tubulin, or β-actin. More preferably, the predetermined standard is α-tubulin. 
         [0020]    When the expression level is an mRNA expression level, it can be detected through any conventional analysis for RNA expression. Preferably, the mRNA expression level is detected through quantitative real-time reverse transcription PCR, or reverse transcription PCR. More preferably, the mRNA expression level is detected through quantitative real-time reverse transcription PCR. In addition, the predetermined standard used in the analysis for mRNA expression can be 18S ribosomal RNA , α-tubulin mRNA, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, or β-actin mRNA. More preferably, the predetermined standard is 18S ribosomal RNA. 
         [0021]    Other objects, advantages, and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0022]      FIG. 1  is a diagram showing the results of the expression of TIAM2S mRNA in normal and tumor tissues of brain, liver, thymus, breast, prostate, pancreas, and colon; 
           [0023]      FIG. 2  is a diagram showing the results of the expression of TIAM2S mRNA in HCC cells; 
           [0024]      FIG. 3  is a diagram showing the quantified results from Western blot analysis; 
           [0025]      FIG. 4  is a diagram showing the correlation of TIAM2S protein expression in HCC samples with different pathology stages; 
           [0026]      FIG. 5  is a diagram showing the results of a cell proliferation assay; 
           [0027]      FIG. 6  is a diagram showing the results of a cell invasion assay; and 
           [0028]      FIGS. 7A-7C  are diagrams showing the results of in vivo tumorigenicity assay. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0029]    The present invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described. 
         [0000]    Identification of Endogenous TIAM2S Expressions in Normal Tissues and Tumor Tissues with Semi-quantitative RT-PCR and Western Blot 
         [0030]    The cDNA panels for different human tissues including normal brain tissue, brain tumor tissue, normal liver tissue, liver tumor tissue, normal thymus tissue, thymus tumor tissue, normal breast tissue, breast tumor tissue, normal prostate tissue, prostate tumor tissue, normal pancreas tissue, pancreas tumor tissue, normal colon tissue, and colon tumor tissue were purchased from Clontech (Palo Alto, Calif.) and (Biochain, Hayward, Calif.). TIAM2S-specific PCR primers were used to amplify TIAM2S. The PCR parameters were 95° C. for 5 minutes followed by 30 cycles of amplification at 95° C. for 30 seconds, 64° C. for 20 seconds, and 72° C. for 20 seconds, with a final extension at 72° C. for 10 minutes. The PCR products were separated on 8% acrylamide gel. The relative expression levels of TIAM2S in different tissues were measured from each PCR product by spot density function using Alphaimage 1200 (Alpha Innotech Corporation, San Leandro, Calif.), and normalized by the density of 200 bp band of the 1 Kb plus DNA marker (Invitrogen). The results of the expression of mRNA in each tissue are shown in  FIG. 1 . In addition, normal human tissues including normal brain tissue, normal liver tissue, normal thymus tissue, normal breast tissue, normal prostate tissue, normal pancreas tissue, and normal colon tissue also were purchased from (Biochain, Hayward, Calif.). Antibodies used for Western blot were purchased as follows: rabbit anti-α-tubulin, from Cell Signaling Technology (MA, USA); goat anti-TIAM2 antibodies from Santa Cruz Biotech (CA, USA). 
         [0031]    The Western blot analysis was performed as follow. First, 30 μg of protein was injected into 6% SDS-PAGE in Tris-glycine-SDS buffer (10 mM Tris, 50 mM glycine, 0.1% SDS, pH 8.0), and separated with electrophoresis. Then, the proteins on the SDS-PAGE were transferred onto PVDF membranes, and the membranes were blocked in TBST (10 mM Tris-HCl, pH7.5, 150 mM NaCl, and 0.05% Tween 20) containing 5% non-fat milk for at least 1 hour and incubated overnight with primary TIAM2S antibodies. The membranes were washed four times in TBST and incubated with horseradish peroxidase-conjugated secondary antibody for 2 hour. After washing and staining the membranes, the signals on the membranes were detected with an enhanced chemiluminescence (ECL) system (PerkinElmer Life Science, Waltham, Mass.). In the experiment of the Western blot analysis, α-tubulin was used as a control for analyzing the total amount of the TIAM2S proteins. 
         [0032]    As shown in the results of  FIG. 1 , the expression of TIAM2S mRNA was detected in liver tumor tissue, breast tumor tissue, and pancreas tumor tissue. However, the expression of TIAM2S mRNA was not detected in normal liver tissue, normal breast tissue, and normal pancreas tissue. On the contrary, the expression of TIAM2S mRNA was detected in normal brain tissue, normal thymus tissue, normal prostate tissue, and normal colon tissue, but not detected in brain tumor tissue, thymus tumor tissue, prostate tumor tissue, and colon tumor tissue. Furthermore, only normal human brain was detected with abundant TIAM2S protein expression which indicated the expression of TIAM2S protein may be controlled by special translational regulation. Taken together, these results indicate that the aberrant mRNA expression of TIAM2S is observed in many solid tumors which indicated TIAM2S is a potential tumor associated gene and the final gene product of TIAM2S protein is more suitable to be a marker than TIAM2S mRNA. 
       Collection of HCC Specimens 
       [0033]    A total of 88 paired (tumor part and matched non-tumor part) and 3 metastatic HCC samples were collected from patients with liver cancers (mean age 57.9±15.6, ranging from 13 to 85 years) undergoing surgical operation. Among them, 28 and 59 pairs were collected from the Department of Surgery, National Cheng Kung University Hospital and the Taiwan Liver Cancer Network (TLCN), respectively. Diagnosis of liver cancer was confirmed by histological examination. Sample collection protocols were approved by the IRB Committees and informed consents were obtained from patients. Tissues were taken separately from cancerous and surrounding normal parts of the resected specimens and were immediately frozen until further processed for nucleic acid and protein extractions. An additional seven metastatic HCC specimens from four individual patients were collected from the Tumor Tissue Bank in the Department of Pathology, National Cheng Kung University Hospital and 30 benign hemangiomas (8 males and 22 females) were from TLCN as negative controls. Among the 91 paired HCC samples, 32 paired samples (35%) were under pathologic stage I, 26 paired samples (29%) were under pathologic stage II, and 25 paired samples (27%) were under pathologic stage III. 
         [0000]    Identification of TIAM2S mRNA Expression in HCC Cells with Quantitative Real-Time RT-PCR 
         [0034]    Total RNA from frozen specimens of HCCs were isolated using Rezol C&amp;T (Protech Technology, Taipei, Taiwan) according to the manufacturer&#39;s protocol. qRT-PCR of TIAM2S and 18S ribosomal RNA (18S rRNA) was performed with TaqMan assays (Applied Biosystems, Foster City, Calif.) according to the manufacturer&#39;s protocol. The levels of TIAM2S mRNA expression in each of the HCC tumor samples were measured (i.e., the 2 −ΔΔCt  method) and normalized to the expression level of the normal part from each specimen. All measurements were performed in triplicate and the experiments were repeated at least twice. Twenty pairs of HCC samples were used to examine the ectopic expression of TIAM2S mRNA in HCC cells, and the mRNA expression level of TIAM2S in each tumor cell was normalized to its normal counterpart, 18S rRNA. The results of the TIAM2S mRNA expression in HCC cells are shown in  FIG. 2 . 
         [0035]    As shown in the results of  FIG. 2 , the TIAM2S mRNA expression in HCC cells is higher than that in normal tissue, and especially the results from paired-t test demonstrated that TIAM2S mRNA is significantly expressed in tumor cells (**: P=0.0043). In addition, examination of TIAM2S mRNA in 20 paired HCC cells revealed that 65% (13/20) of HCC cells showed an increase (from 2- to 9-fold) of TIAM2S mRNA in tumor. 
         [0000]    Identification of TIAM2S Protein Expression in HCC Cells with Western Blot Analysis 
         [0036]    Approximately 150 mg of the aforementioned HCC samples was dissected and snap-frozen in liquid nitrogen. The frozen samples were added to 1 mL RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF and the cocktail of protease inhibitors) and homogenized by using tissue grinder immediately on ice. The lysate was rinsed twice with 1 mL RIPA buffer and constantly agitated at 4° C. for 20 min to maintain homogenization, followed by centrifugation for 20 minutes at 19,600×g at 4° C. The supernatants were collected and subjected to western blot analysis. 
         [0037]    The results of the Western blot analysis show that TIAM2S protein only express in tumor cells, but does not express in normal cells. In addition, none or trace amounts of TIAM2S protein were detected in the hemangiomas. These results indicate that TIAM2S protein specifically express in HCC cells. 
         [0038]    In addition, the Western blot density of TIAM2S protein in HCC cells and normal cells from each patient was normalized to that of α-tubulin in HCC cells and normal cells respectively. Herein, the Western-blot density of TIAM2S and α-tubulin in HCC cells and normal cells has to be from the same patient. The normalization was performed by the following equation (II), and the density and the value are summarized in the following Table 1. 
         [0000]      Value=[(the density of TIAM2S protein in HCC cells/the density of α-tubulin in HCC cells)−(the density of TIAM2S protein in normal liver cells/the density of α-tubulin in the normal liver cells)]  [Equation (II)]
 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
             
             
               
                   
                   
               
               
                   
                 Normal liver cells 
                 HCC cells 
                   
               
             
          
           
               
                 Patient No. 
                 TIAM2S 
                 α-tubulin 
                 TIAM2S 
                 α-tubulin 
                 Value 
               
               
                   
               
             
          
           
               
                 1 
                 0.2 
                 4. 
                 5.9 
                 5.5 
                 1.02 
               
               
                 2 
                 0.3 
                 4.1 
                 8.2 
                 5.9 
                 1.32 
               
               
                 3 
                 0.3 
                 3.1 
                 0.4 
                 3.6 
                 0.01 
               
               
                 4 
                 0.2 
                 4.8 
                 2.7 
                 5.2 
                 0.48 
               
               
                 5 
                 0.2 
                 3 
                 5.8 
                 4.6 
                 1.19 
               
               
                 6 
                 0.3 
                 4.7 
                 1.7 
                 6.8 
                 1.19 
               
               
                   
               
               
                 Value: the density of ectopic expression of TIAM2S protein 
               
             
          
         
       
     
         [0039]    As shown in Table 1, the values of each paired sample are shown in positive, which means that the TIAM2S protein expression is positively related to the formation of liver cancer. 
         [0040]    Furthermore,  FIG. 3  is a plot showing the results from Western blot analysis of 69-paired HCCs, which were quantified with spot density function using alpha-imager system. After 69 HCC samples were detected with Western blot analysis, the results indicate that 60 (87%) HCC samples showed aberrant TIAM2S protein expression in tumor cell, and the paired-t test shows that the ectopic expression of TIAM2S is significant in HCC cells (***: P&lt;0.0001). 
         [0041]    In addition, the correlations between TIAM2S expression levels and various clinical features were also analyzed. If the HCC samples are under stages II and/or III, it indicates that the probability of tumor invasion into vessels is high. Therefore, the risk of invasion/metastasis of tumor cells is increased. As shown in  FIG. 4 , the expression of TIAM2S protein was increased in HCC samples with the invasive phenotype (stage II and III), but not in those with the non-invasive phenotype (stage I, *: P&lt;0.05). According to the results shown in  FIG. 4 , the TIAM2S indeed can be used as a biomarker to evaluate the risk of invasion or metastasis of tumor, especially liver cancer. 
         [0042]    According to the aforementioned results, the method of the present invention, which uses TIAM2S as a biomarker to evaluate a risk of proliferation of a liver cancer, has high accuracy (87%). However, the method using AFP as a biomarker has an accuracy of about 50%. Hence, the method of the present invention has higher accuracy than that using AFP as a biomarker. In addition, the biomarker TIAM2S of the present invention can also be used to evaluate a risk of invasion/metastasis of a liver cancer, which cannot be accomplished by the conventional biomarker AFP. 
       Cell Proliferation Assay 
       [0043]    The human hepatoma cell line, HepG2, was used to generate stable cloned cell lines for overexpression of recombinant TIAM2S. One control clone of HepG2/pcDNA3.1A+ (V1A3) and two independent clones of HepG2/pcDNA3.1A+_TIAM2S (T1A1 and T2C1) were obtained after selection for 30 days using G418. Cells were maintained at 37° C. in a humidified atmosphere of 5% CO 2  in α-MEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 700 μg/mL G418, and 10% FBS for further applications. 
         [0044]    Cell proliferation was measured using a colorimetric assay according to the manufacturer&#39;s protocol (CellTiter 96 Aqueous One Solution cell proliferation assay; Promega, Madison, Wis.). In brief, stable clones were seeded at 5×10 3  cells/well in 96-well culture plates (each group had four wells). The effect of overexpression of TIAM2S on cell growth was determined at various time points. The intensity of the color was measured at 490 nm using a 96-well microplate reader (Labsystems, Multiskan EX, Helsinki, Finland). The corrected absorbance (after subtracting the control blanks) was used to determine the proliferative response. All experiments were independently repeated at least three times. The relative proliferation rates of stable clones carrying control vector (V1A3) and recombinant TIAM2S (T1A1 and T2C1) are shown in  FIG. 5 . 
         [0045]    According to the results shown in  FIG. 5 , HepG2-TIAM2S cells (T1A1 and T2C1) continued to grow while HepG2-control cells (V1A3) stopped growing after day 4 (*: P&lt;0.05, **: P&lt;0.01, ***: P&lt;0.001). It means that stable expression of recombinant protein, HepG2-TIAM2S cells, promotes the growth ability in HepG2 cells (a low dedifferentiated HCC cell line). 
       Cell Invasion Assay 
       [0046]    The aforementioned control clone of HepG2/pcDNA3.1A+ (V1A3) and independent clones of HepG2/pcDNA3.1A+_TIAM2S (T1A1 and T2C1) were used in the present experiment. A transwell with an 8-μm pore size polycarbonate membrane filter (Millipore, Temecula, Calif.) was coated with a uniform layer of 15.15 μg/cm 2  Matrigel basement membrane matrix. The coated transwell was air-dried completely. Before carrying out the experiment, the Matrigel was reconstituted by adding 40 μL α-MEM with 0.1% FBS and incubated at 37° C. for 1 hour. Trypsinized cells (1.5×10 5 ) were suspended in 100 μL α-MEM with 0.1% FBS and added into the upper camber of the transwell. α-MEM (700 μL) containing 10% FBS was added to the lower camber. After 24 hours of incubation, the non-invading cells and the upper side of the pore membrane were removed with a cotton swab. The cells on the lower surface of the membrane were fixed for 15 minutes with methanol and stained with 0.2% crystal violet for 30 minutes. The invading cells were counted by dividing the membrane area into five fields and visualizing the fields at 200× magnification. 
         [0047]    The sum number of the violated stained cells from five randomly selected fields on the transwell is shown in  FIG. 6  (*: P&lt;0.05, **: P&lt;0.01, ***: P&lt;0.001). As shown in  FIG. 6 , the number of invaded cells was greater in T1A1 (P&lt;0.001) and T2C1 (P&lt;0.05) cells than in control V1A3 cells. Thus, the data revealed that TIAM2S increased the invasive ability of HepG2 cells, and it suggests that TIAM2S is involved in promoting growth and increasing invasiveness in TIAM2S-expressing cells. 
       In Vivo Tumorigenicity Assay 
       [0048]    The control (V1A3) and TIAM2S stable clones (T1A 1 and T2C1) were subcutaneously injected into 8-week-old ICR nu/nu mice to examine the tumorigenicity. About 5×10 6  cells from individual cloned cell lines were injected (in 0.2 μL PBS) subcutaneously into the dorsal region using a 27-gauge needle. Nine mice were used for each cell line and the tumors were palpated weekly after inoculation. Tumor volumes were measured after 3 weeks using a caliper (calculated as length×width×height×0.52) until the tumor volume reached 1,000 mm 3  or after 8 weeks. The mice were then euthanized; tumors were excised and embedded in OCT. HE-stained sections from various excised tumors were evaluated for invasion ability. The results are shown in  FIGS. 7A-7C . 
         [0049]      FIG. 7A  shows the Xenograft tumor volume in mice injected with control clones (V1A3),  FIG. 7B  shows the Xenograft tumor volume in mice injected with TIAM2S stable clones (T1A1), and  FIG. 7C  shows the Xenograft tumor volume in mice injected with TIAM2S stable clones (T2C1). 
         [0050]    As shown in  FIGS. 7A-7C , the total number of tumor-growing mice was slightly higher in the TIAM2S group (9/18; 50%) than in the control group (3/9; 33%). Compared to the TIAM2S-expressing group, tumors grown from the control group were round and smooth and they were more easily stripped and dissociated from the adjacent tissues. In contrast, tumors grown from the TIAM2S-expressing group were multi-module with rough edges and were hard to separate from the adjacent tissues. The ratio of tumors showing the invasive phenotype was 0% (0/3) and 55.6% (5/9), respectively, in the control and TIAM2S groups. Recombinant TIAM2S expression was detected in tumors grown from TIAM2S-expressing cells but not from control cells. 
         [0051]    In addition, results of histological analysis demonstrated that TIAM2S-expressing tumors tightly contacted or even penetrated into the surrounding skeletal muscle layer and showed an unclear boundary. In contrast, tumors grown from the control group showed clear boundaries between tumor and stroma cells and showed no connection with neighboring skeletal muscle. These data are in agreement with the cellular tumorigenic assays and further support the role of TIAM2S expression in promoting cell growth and invasion. 
         [0052]    According to the results of the cell proliferation assay, the cell invasion assay and the in vivo tumorigenicity assay, the expression of TIAM2S may increase the invasive ability, so it may play a role in cancer metastasis. Therefore, the biomarker of the present invention, TIAM2S, can be used not only to evaluate a risk of proliferating a cancer, but also to predict the invasive and metastasis ability of a cancer. 
         [0053]    Although the present invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed.