Clinical Significance of TROP2 Expression in Colorectal Cancer

Colorectal cancer is one of the most prevalent cancers in the world. In Japan, the disease rate of colorectal cancer patients has doubled over the past 20 years, with ∼75 of 100,000 people suffering from the disease today. Additionally, colorectal cancer has been the second cause of death in neoplastic disease (1). Recently, molecular target therapy and cancer immunotherapy for solid cancers have been introduced to the clinic (2–5). However, indications for these therapies are limited due to the low frequency of target gene expression, unstable effectiveness, and/or severe side effects (5, 6). Thus, there is a pressing need to explore novel cancer-specific genes to serve as molecular targets for therapy and cancer specific immunotherapy.

In this study, the gene expression profiles were compared between cancer cells and normal epithelial cells using the combined techniques of laser microdissection and cDNA microarray analysis to explore cancer-related or cancer-specific genes. From this analysis, several cancer-related genes were identified. Among them, TROP2 showed markedly different expression between cancer cells and normal epithelial cells.

Fornaro et al. cloned the TROP2 gene, which encodes a 35,709-Da type 1 transmembrane protein with a single transmembrane domain and is homologous to TROP1/KSA/GA733-2. Moreover, TROP2, a cell surface receptor, has been shown to play a role in regulating the growth of carcinoma cells (7). TROP2 is also responsible for gelatinous drop-like corneal dystrophy (8). TROP2 is expressed at high levels in human trophoblast cells and it has been reported as one of the highly specific genes to the tumors, such as ovarian and bladder cancers (9–12). On the other hand, our institute developed tumor-specific immunotherapy using MAGE peptide-pulsed dendritic cells (13). We are planning to expand the therapy using other tumor-specific genes. Depending on the outcome of this analysis, TROP2 may become a candidate gene. In addition, the clinical significance of the gene has not been studied in colorectal cancer. Thus, we investigated TROP2 expression in clinical samples of colorectal cancer to determine its clinicopathologic significance.

Tissue sampling, laser microdissection, and cDNA microarray analysis. The samples of cancer tissues and noncancerous tissues were obtained from 16 patients with colorectal cancer who underwent surgical resection in Kyushu University Hospital (Beppu, Japan). Written informed consent was obtained from all patients. Tumors and adjacent normal tissues were immediately embedded in Tissue-Tek OCT compound medium (Sakura, Tokyo, Japan) and were kept frozen at −80°C until laser microdissection was done. Serial 8-μm frozen sections were generated by a cryostat. Sections were mounted onto a foil-coated glass slide, 90 FOIL-SL25 (Leica Microsystems, Wetzlar, Germany) for laser microdissection. Slides were stained with H&E at room temperature and dehydrated with ethanol. The Application Solutions Laser Microdissection System (Leica Microsystems) was introduced for laser microdissection to obtain the cancer cells and normal epithelial cells and to discard the mesenchymal tissues. Laser microdissection was done for several sequential sections and total RNA was extracted from each section (14). As the extracted total RNA was insufficient for hybridization to the cDNA microarray, the RNA was subjected to T7-based RNA amplification (15). The purity and concentration of the amplified RNA were determined by an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) as described previously (14). In brief, high-quality amplified RNA run on a bioanalyzer typically has the shape of a hump peak and one marker peak, indicating no contamination of rRNA. Of 16 cancer and 16 normal microdissected and T7-based amplified RNA samples, 8 samples from the cancer sections and 10 samples from the normal sections were determined to be of sufficient quality. Each 8 RNA samples from cancer sections and the mixture of 10 RNA samples from normal epithelium were hybridized competitively to a cDNA microarray containing 12,814 genes. A list of genes on this cDNA microarray is available from http://www.agilent.com/chem/genelists.

Microarray analysis. After subtracting the local and global background signals, the expression values were calculated as the intensity of the dye-normalized red (Cy5) and green (Cy3) channel signals. The data flagged as poor quality according to the Agilent data extraction software were removed from the analysis. All data calculated by data extraction software were imported to the Rosetta Luminator system version 2.0 (Rosetta Biosoftware, Kirkland, WA). Candidate genes were selected that fulfilled the following criteria: the control (Cy3) intensity was <700, the fold changes were >2.5, and the P was <0.01 (14). Moreover, within the selected genes that met these criteria, genes that were up-regulated in three or more RNA samples (eight total samples) were further analyzed.

Semiquantitative real-time PCR. The semiquantitative real-time reverse transcription-PCR assay used 74 operatively resected paired cancer and normal samples that were not used for microarray analysis. Total RNA was extracted from each bulk sample and cDNA was synthesized from 8.0 μg total RNA as described previously (16). The purity and concentration of total RNA were determined using an Agilent 2100 Bioanalyzer. The following primers were used to amplify the TROP2 gene: sense primer 5′-GCCTACTACTTCGAGAGGGACA-3′ and antisense primer 5′-CAGTTCCTTGATCTCCACCTTC-3′. The glyceraldehyde-3-phosphate dehydrogenase (sense primer 5′-TTGGTATCGTGGAAGGACTCA-3′ and antisense primer 5′-TGTCATCATATTTGGCAGGTTT-3′) gene was used as an internal control. The reaction was done in a LightCycler system (Roche Applied Science, Indianapolis, IN) using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Mannheim, Germany). Details of each reaction are described elsewhere (17). Briefly, thermal cycling for all genes was initiated with a denaturation step of 95°C for 10 minutes and then consisted of 40 cycles at 95°C for 10 seconds, 65°C (60°C for glyceraldehyde-3-phosphate dehydrogenase) for 10 seconds, and 72°C for each optimal length (1 second/25 bp). All calculated concentrations of target genes were divided by the amount of the endogenous reference (glyceraldehyde-3-phosphate dehydrogenase) to obtain the normalized TROP2 expression values. Each assay was done in triplicate.

Immunohistochemistry. Of 74 cases, immunohistochemical staining was done in 34 selected cases. Immunohistochemical studies of TROP2 were done on formalin-fixed, paraffin-embedded surgical sections. After deparaffinization and blocking, the antigen-antibody reaction was incubated overnight at 4°C. The LSAB2 kit (DAKO, Kyoto, Japan) was applied to detect the signal of the TROP2 antigen-antibody reaction. All sections were counterstained with hematoxylin. The purified goat polyclonal antibody against the purified recombinant human TROP2 extracellular domain (R&D Systems, Inc., Minneapolis, MN) was used at 5 μg/mL.

Statistical analysis. Quantitative real-time reverse transcription-PCR data were calculated with JMP 5 for Windows software (SAS Institute, Inc., Cary, NC). Differences between groups were estimated using the Student's t test and the χ² test. The survival curves were estimated by the Kaplan-Meier method and the comparison between the curves was made by the log-rank test. The relative risk (RR) was calculated using the Cox proportional hazards model. A probability level of 0.05 was chosen for statistical significance.

Microarray analysis. In the microarray analysis, 84 genes were identified that had a higher expression level in cancer cells than in normal epithelial cells. Among these genes, some are involved with signal transduction (27.4%), transcription (16.7%), transport (13.1%), metabolism (4.8%), cell adhesion (4.8%), secretion (3.6%), apoptosis (3.6%), and an unknown function (16.7%; Table 1). Of these genes, some were already reported as associated with colorectal cancer aggressiveness, such as MMP7 (18, 19), MMP11 (20, 21), CD44 (22–24), CD61 (25, 26), CRIPTO (27, 28), ENC1 (29), SLC7A5 (30), DPEP1 (31), and activin A (32, 33).

TROP2 expression in colorectal cancer samples and corresponding normal tissues. Quantitative real-time reverse transcription-PCR was done on 74 paired samples to show TROP2 mRNA expression in clinical samples. Quantitative real-time reverse transcription-PCR showed that TROP2 expression in cancer samples was significantly higher (averaged expression values of cancer were 8.34-fold higher; P < 0.0001) than those in normal samples (Fig. 1). Figure 2 shows the results of immunohistochemical studies of TROP2 expression in representative clinical samples of well-differentiated adenocarcinoma (Fig. 2A), moderately differentiated adenocarcinoma (Fig. 2B), poorly differentiated adenocarcinoma (Fig. 2C), and mucinous adenocarcinoma (Fig. 2D). The majority of the TROP2 expression was observed in the cancer cells, the minority in stromal cells, and none in the normal colonic epithelium. Immunohistochemical studies revealed that the staining was strong (n = 7), moderate (n = 9), or weak (n = 18) in the tumor cells, whereas very weak or none in the normal cells in all 34 cases. There was a significant difference in immuohistochemical staining between the tumor and the normal samples (P < 0.01), and the data are similar to those obtained from mRNA expression analysis. All 16 tumors with strong or moderate immunohistochemical expression showed higher mRNA expression values (>0.2). The expression of TROP2 mRNA relatively associated with protein expression. Among the undifferentiated cell types, several regions of the cancer had strong immunohistochemical staining for TROP2 (Fig. 2C and D).

High TROP2 expression correlates with clinicopathologic variables. The experimental samples were divided into two groups [the high expression group with TROP2 expression values (>0.2; n = 26) and the remaining samples in the low expression group (n = 48)] to investigate TROP2 expression in association with clinicopathologic variables (Table 2). The border of the two groups was defined by an upper limit, including 95% of the expression values of the normal samples. The incidence in liver metastasis was significantly higher (P = 0.005) in the high expression group (9 of 26, 34.6%) than in the low expression group (4 of 48, 8.3%), and the incidence of cancer death was significantly higher (P = 0.046) in the high expression group (9 of 26, 34.6%) than in the low expression group (7 of 48, 14.6%). The high expression group also had inclinations of deeper invasion (P = 0.064) and more lymph node metastasis (P = 0.125) than the low expression group.

Survival analysis. The 5-year actuarial overall survival rates in patients with high TROP2 mRNA expression levels and those with low levels were 48.5% and 83.3%, respectively. Moreover, the high expression group also showed poorer prognosis in the Kaplan-Meier survival curve (P = 0.0036; Fig. 3). Table 3 revealed the result of RR and the 95% confidence interval (95% CI) by the Cox proportional hazards model. Table 3A shows the univariate analysis for all clinicopathologic variables. Regarding these variables, lymph node metastasis (RR, 2.711; 95% CI, 1.526-5.681; P = 0.0004), liver metastasis (RR, 2.409; 95% CI, 1.381-4.059; P = 0.003), depth of invasion (RR, 2.127; 95% CI, 1.276-3.675; P = 0.0041), histologic grade (RR, 2.097; 95% CI, 1.098-5.313; P = 0.0225), and high TROP2 expression (RR, 2.026; 95% CI, 1.216-3.501; P = 0.0071) were statistically significant. Table 3B shows the result of multivariate analysis in the final model, which included lymph node metastasis, depth of invasion, histologic grade, tumor site, and lymphatic permeation. In this model, the variable of high TROP2 expression was an independent prognostic predictor for the patients with colorectal cancer (RR, 2.38; 95% CI, 1.29-4.74; P = 0.005; Table 3B). Of the variables that were entered in the multivariate analysis, the variable of liver metastasis was excluded because there was a significant correlation with the variable of high TROP2 expression.

For further understanding of cancer biology, it is important to identify cancer-related genes. To find such genes, a combination of techniques, laser microdissection and cDNA microarray analysis, was applied to clinical samples. Laser microdissection can isolate cancer cells from tumor tissue that consists of mixed populations of carcinoma cells and stromal cells, such as fibroblasts, macrophages, and lymphocytes (16). RNA extracted from these tissues was subjected to rigorous procedures for quality to ensure its utility in cDNA microarray analysis. Almost 50% to 60% of the frozen surgical samples passed the RNA quality check according to our experience. Through using both laser microdissection and microarray analysis, 84 cancer-related genes were identified that were overexpressed in cancer cells compared with normal cells in the colon.

This study showed that TROP2 was one of the most highly expressed genes in colorectal cancer cells compared with normal colon tissue. TROP2 is reported to be highly expressed in several types of cancers. For example, Nakajima et al. revealed that TROP2 is overexpressed in esophageal cancer, and the titer of serum TROP2 antibody correlated with tumor size (34). With respect to colon cancer, Kanai et al. studied the expression of mRNA of genes altered by 5-azacytidine treatment in cancer cell lines and found that TROP2 was overexpressed in clinical colorectal cancers (35). However, they did not investigate the correlation between the TROP2 expression status and the clinicopathologic factors. Our study of a comparison of both groups showed a significant difference in liver metastasis, and this was associated with cancer-related death. Although there were no significant differences in the other pathologic factors examined, there was a tendency in serosal invasion between the groups. Therefore, these results suggest that high TROP2 expression has an association with biological aggressiveness of colorectal cancer.

The reason why the cancers with high TROP2 expression show aggressive behavior is unclear. There are some studies suggesting the biological mechanism of high TROP2 expression associated with the aggressiveness of cancer. Fornaro et al. suggested that TROP2 not only acts as a calcium signal transducer but also has a receptor function activity. TROP2 has homology to serum insulin-like growth factor-II–binding proteins and has a conservative structure for potential cytoplasmic phosphorylation sites (7). Basu et al. revealed that the phosphorylation occurred on Ser³⁰³ by protein kinase C (36).

We are investigating the predicted function of TROP2 gene and taking notice of its intracellular domain of phosphatidylinositol 4,5-bisphosphate–binding sequence. Phosphatidylinositol 4,5-bisphosphate is the most important lipid in the cytoplasmic leaflet of the plasma membrane and is responsible for a wide range of membrane-related phenomena that consist of the functions of endocytosis, exocytosis, cytoskeletal attachment, enzyme activation, actin-binding protein, production of three second messengers, ion-channel activation, and binding site for PH and other domains (37). EI Sewedy et al. investigated that the phosphatidylinositol 4,5-bisphosphate–binding site of TROP2 has an ability of being phosphorylated by protein kinase C (38); thus, the signal transduction through TROP2 has a possibility of causing the aggressiveness of cancer cells. Consequently, we assume that the TROP2 gene has a receptor activity and influences the aggressive behavior of colorectal cancer cells during tumor progression. To investigate its biological function, we are planning the following experiments: (a) Establishment of the transfectant of the TROP2 gene to clarify the dominant proliferation and progression of invasiveness. (b) Interference of the TROP2 gene expression by small interfering RNA (or short hairpin RNA) to examine the alteration of its proliferation and invasiveness. (c) Verification of the binding activity between TROP2 and phosphatidylinositol 4,5-bisphosphate and exploration of the alteration of the binding activity by protein kinase C. (d) Utilization of the transfectant with mutant TROP2 gene, which lacks the phosphatidylinositol 4,5-bisphosphate–binding site to compare the result of in vitro analysis with that of experiment 1. Thus, high TROP2 expression would contribute to biological aggressiveness of cancer cells through paracrine and autocrine signaling pathway. Nonetheless, more precise understanding of the mechanism that is responsible for the aggressiveness is necessary.

Because TROP2 is overexpressed in colon cancer cells and not expressed in normal cells, it is a novel target for treatment. In fact, Mangino G et al. showed that TROP2 is one of the target molecules recognized by human CTLs (39). We investigated the effectiveness of cancer-specific immunotherapy using MAGE antigen as a dendritic cell therapy (13, 40–42). However, the patients that can be treated with this therapy are limited because the patients must meet conditions, such as adequate HLA typing and positive MAGE gene expression in the tumor. The percentage of such patients is ∼10% to 40% in several types of cancers. If TROP2 can become an immunotherapeutic molecular target, the combined specific immunotherapy with MAGE and TROP2 genes would be a powerful new treatment modality for colorectal cancer patients.

In conclusion, our results indicated that TROP2 mRNA and protein were overexpressed in colorectal cancer cells and high TROP2 expression status correlated with liver metastasis and poor prognosis. These findings suggest that TROP2 is one of the cancer-related genes that are associated with cancer aggressiveness and is one of the predictors of survival in patients with colorectal cancer. The overexpression of this gene has a possible role for assessment of the postoperative adjuvant therapy of colorectal cancer patients. Furthermore, this gene will be useful not only for diagnostics but also molecular targeted therapy.

We thank Dr. S. Tanaka for helpful discussion and T. Shimooka, K. Ogata, M. Oda, M. Kasagi, and Y. Nakagawa for excellent technical assistance.