Trop2 Protein Research Articles

The trophoblast cell surface antigen 2 and miR-125b axis in urothelial bladder cancer.

Human trophoblast cell surface antigen 2 (Trop-2) is a 40-kDa transmembrane glycoprotein that was first identified as a marker of human trophoblast cells. Trop-2 acts on cell proliferation, adhesion, and migration by activating a number of intracellular signalling pathways. Elevated Trop-2 expression has been demonstrated in several types of cancer and correlated with aggressiveness and poor prognosis. Since no data are available on Trop-2 in bladder cancer (BC), the purpose of the study was to determine its levels in tissue specimens from normal individuals and patients with BC at different stages. Moreover, since according to recent evidence Trop-2 is a miR-125b target, miR-125b expression was also assessed in tissue specimens. Finally, the effect of the Trop-2/miR-125b axis on the proliferation and migration of BC cells was evaluated in vitro.The Trop-2/miR-125b axis was seen to be differentially expressed in normal urothelium, non-invasive BC and invasive BC tissue. Significant miR-125b down-regulation was associated with a significant increase in Trop-2 protein levels in BC tissue and correlated with disease severity. In vitro analysis confirmed the role of miR-125b in down-modulation of Trop-2 protein levels and showed that Trop-2/miR-125b axis affects cellular proliferation in bladder tissue.In conclusion, our findings highlight a role for the Trop-2/miR-125b axis in BC progression and suggest Trop-2 and miR-125b as diagnostic/prognostic marker candidates as well as druggable targets for innovative therapeutic approaches.

Abstract 463: Immunohistochemical study of TROP-2 protein expression in thyroid lesions

Abstract Introduction Tumor-associated calcium signal transducer 2 (TROP-2) is a transmembrane glycoprotein overexpressed in various types of epithelial carcinomas from lung, breast, stomach, ovary, cervix, colon and pancreas. A recent immunohistochemical (IHC) study indicated that TROP-2 is also overexpressed in thyroid papillary carcinoma, but the sensitivity is only 82% in formalin-fixed and paraffin-embedded thyroid (FFPE) tissues when a polyclonal anti-TROP-2 antibody was used. A high quality of TROP-2 antibody for IHC applications is needed to improve the sensitivity of the detection of thyroid papillary carcinoma in FFPE tissues. Methodology A rabbit monoclonal antibody (clone SP295) against human TROP-2 protein was developed. Western blot was performed using SK-BR-3 cell lysates to verify the specificity of the TROP-2 antibody. An IHC study of TROP-2 was performed on normal tissue and tumor arrays from the thyroid and other organs. TROP-2 staining intensity was semi-quantitatively scored using a 0-3 scale. The sensitivity and specificity of the TROP-2 IHC assay for detecting papillary carcinoma were determined using 65 cases of thyroid papillary carcinoma, 29 cases of follicular carcinoma, and 31 cases of other thyroid conditions (normal, adenoma, Grave's disease, thyroiditis, Goiter, medullary carcinoma, clear cell carcinoma, squamous cell carcinoma, and undifferentiated carcinoma). Results A single band at 33 kDa was detected by SP295 on a western blot. In normal tissues, TROP-2 is detectable by IHC mostly in squamous epithelia of many normal organs (skin, pharynx, esophagus, and cervix) and cuboidal and columnar epithelium (breast, kidney, liver, uterus, pancreas, prostate and transitional epithelium of the bladder). In thyroid lesions, the TROP-2 positive rate is 100% (65/65) in papillary adenocarcinoma and it is negative in all other thyroid conditions (normal, adenoma, Grave's disease, thyroiditis, Goiter, medullary carcinoma, clear cell carcinoma, squamous cell carcinoma, and undifferentiated carcinoma). The sensitivity and specificity of TROP-2 IHC for detecting papillary carcinoma were both 100% in this study. Conclusions Rabbit monoclonal TROP-2 antibody (clone SP295) is highly sensitive and specific for detecting TROP-2 in FFPE tissues and it can be used to identify thyroid papillary carcinoma. Citation Format: Zhiming Liao. Immunohistochemical study of TROP-2 protein expression in thyroid lesions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 463.

Trop2 is overexpressed in gastric cancer and predicts poor prognosis.

The cell surface protein Trop2 is overexpressed in a variety of human cancers. Trop2 expression increases tumor development and metastasis and reduces patient survival. However, little is known about the role of Trop2 expression and its prognostic value in gastric cancer (GC), particularly in Chinese populations. We analyzed Trop2 expression in GC tissues collected from Chinese GC patients. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry on tissue microarrays were performed to assess levels of Trop2 mRNA and protein in GC, and correlations between Trop2 expression and clinical characteristics and prognosis were analyzed. Trop2 expression was higher in GC tissues than in neighboring non-tumor tissues. Increased Trop2 protein levels in GC were associated with increased differentiation, tumor node metastasis stage, tumor size, lymph node metastasis, distant metastasis, and H. pylori infection. GC patients with high Trop2 expression also had poor overall survival rates. These data suggest Trop2 is a useful prognostic biomarker for GC.

Association of lung adenocarcinoma expression of Trop-2 protein with tumor infiltrating CD8+ T lymphocytes.

e18509 Background: Trop-2, a component of tight junctions, is highly expressed in many epithelial cancers, and its intracellular expression has been associated with prolonged survival in breast ade...

Trop2 inhibition suppresses the proliferation and invasion of laryngeal carcinoma cells via the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway.

The cell surface glycoprotein Trop2 is overexpressed in various types of epithelial cancer. Laryngeal carcinoma is one of the most common types of head and neck cancer and in a previous study, the expression of Trop2 in laryngeal squamous cell carcinoma (LSCC) was identified as an independent prognostic factor. However, the biological significance of Trop2 in LSCC development remains unclear. In the current study, Trop2 protein expression in fresh LSCC tissue and paracancerous tissue was investigated using western blotting. Trop2 in the Hep2 laryngeal cell line was subsequently suppressed by transfection with small interfering RNA (siRNA). The effects of knockdown of Trop2 on cell viability, migration, invasiveness and ERK/MAPK pathway activity were investigated in the current study. The expression of Trop2 in fresh LSCC tissue was demonstrated to be significantly greater than that in paracancerous tissue. Trop2 expression was also identified to be required for proliferation, migration and invasiveness of Hep2 laryngeal carcinoma cells, as all were blocked by siRNA-mediated Trop2 inhibition. Notably, the ERK/MAPK signaling pathway and cell cycle factor, cyclin D1, were identified to be suppressed following the knockdown of Trop2 in Hep2 cells. These observations suggest that Trop2 serves an oncogenic role in LSCC and has potential as a therapeutic target.

Expression of Trop2 in bladder cancer is modulated by miR125b: in vivo and in vitro analyses

Human trophoblastic cell surface antigen 2 (Trop-2) is a 40-kDa transmembrane glycoprotein, first identified as a cell surface marker for human trophoblast cells (1). Elevated expression of Trop-2 has been shown in several types of epithelial cancers and correlated with tumour aggressive and poor prognosis (2-3). The first aim of this study was to evaluate the variation of the Trop-2 expression in normal urothelium and urothelial bladder cancer. The immunohistochemical results showed an increase of Trop-2 levels in bladder cancer tissues with the increase of the severity of the pathology. Recent data identified Trop-2 as a target for miR-125b suggesting a pos sible role of miR-125b in the modulation of Trop-2 protein expression (4). The second aim was to verify if Trop-2 could be a target for miR-125b in bladder cells and to evaluate the possible role of miR-125b in the modulation of Trop-2 protein expression in normal bladder as well as in urothelial bladder cancer. In vitro we showed a contribution of miR-125b in deregulation of Trop-2 protein expression in a bladder cell line and we found that the expression of miR-125b was inversely correlated with the expression of Trop-2 protein on a cohort of bladder cancer tissues. We concluded to investigate in a larger population the use of Trop-2 and/or miR-125b as potential diagnostic markers in urothelial bladder cancer.

Increased expression of Trop2 correlates with poor survival in extranodal NK/T cell lymphoma, nasal type

Human trophoblastic cell surface antigen 2 (Trop2) has been suggested to play an important role in the development of solid tumors. However, the expression of Trop2 in extranodal NK/T cell lymphoma, nasal type (ENKTL) and the relationship with the clinical characteristics of this disease remain poorly understood. In this study, one-step quantitative PCR reverse transcription-polymerase chain reaction and immunohistochemical staining with tissue sections were employed to evaluate the expression of Trop2 in ENKTL. Furthermore, the relationship between Trop2 expression and prognosis of ENKTL was investigated. Expression of Trop2 mRNA and protein was significantly higher in ENKTL tissue than in corresponding non-lymphomatous tissue (p = 0.04 and p < 0.001, respectively). Expression of Trop2 protein in ENKTL was associated with lymph node involvement and poor overall survival (p = 0.045 and p = 0.018, respectively). Kaplan-Meier analysis and the logrank test indicated that lymph node involvement (p = 0.0481), single therapy strategy (p = 0.0037), and high expression of Trop2 (p = 0.0042) are significantly correlated with poor prognosis of ENKTL patients. The data suggest that Trop2 expression reflects a more malignant phenotype and may serve as an unfavorable prognostic factor for ENKTL.

Trop2 regulates motility and lamellipodia formation in cultured fetal lung fibroblasts

Proliferation and migration of fibroblasts are vital for fetal lung development. However, the regulatory mechanisms are poorly understood. We have previously shown that TROP2 gene expression is closely associated with fetal lung cell proliferation in vivo and that TROP2 knockdown decreases proliferation of fetal lung fibroblasts in culture. We hypothesized that the Trop2 protein also regulates the morphology and motility of fetal lung fibroblasts. Fibroblasts isolated from fetal rat lungs (gestational age embryonic day 19) adopted a myofibroblast-like morphology in culture. Trop2 protein was localized to lamellipodia. TROP2 siRNA significantly decreased: TROP2 mRNA levels by 77%, the proportion of cells containing Trop2 protein by 70%, and cell proliferation by 50%. TROP2 siRNA also decreased the degree of motility as determined by the number of gridlines that cells moved across (2.2 ± 0.2 vs. 3.2 ± 0.2; P < 0.001). TROP2 knockdown altered cell morphology, causing a notable absence of lamellipodia and abnormal localization of components of the cell migration apparatus, and it reduced phosphorylated ERK1 and ERK2 levels. In contrast, TROP2 overexpression significantly increased: TROP2 mRNA levels by 40-fold, cell proliferation by 40%, the proportion of cells that were motile by 20%, and the number of gridlines that cells moved across (2.1 ± 0.2 vs. 1.6 ± 0.1; P < 0.001). Our data suggest that Trop2 regulates cell proliferation and motility and that it does so by regulating the ERK pathway and several critical components of the cell migration apparatus.

Trop-2 protein overexpression is an independent marker for predicting disease recurrence in endometrioid endometrial carcinoma

BackgroundEndometrial cancer is the most common gynecologic malignancy in developed countries. Trop-2 is a glycoprotein involved in cellular signal transduction and is differentially overexpressed relative to normal tissue in a variety of human adenocarcinomas, including endometrioid endometrial carcinomas (EEC). Trop-2 overexpression has been proposed as a marker for biologically aggressive tumor phenotypes.MethodsTrop-2 protein expression was quantified using tissue microarrays consisting of formalin-fixed paraffin-embedded specimens from 118 patients who underwent surgical staging from 2001–9 by laparotomy for EEC. Clinicopathologic characteristics including age, stage, grade, lymphovascular space invasion, and medical comorbidities were correlated with immunostaining score. Univariate and multivariate analyses were performed for overall survival, disease-free survival, and progression-free survival in relation to clinical parameters and Trop-2 protein expression.ResultsClinical outcome data were available for 103 patients. Strong Trop-2 immunostaining was significantly associated with higher tumor grade (p=0.02) and cervical involvement (p<0.01). Univariate analyses showed a significant association with reduced disease-free survival (DFS) (p=0.01), and a trend towards significance for overall and progression-free survival (p=0.06 and p=0.05, respectively). Multivariate analyses revealed Trop-2 overexpression and advanced FIGO stage to be independent prognostic factors for poor DFS (p=0.04 and p <0.001, respectively).ConclusionsTrop-2 protein overexpression is significantly associated with higher tumor grade and serves as an independent prognostic factor for DFS in endometrioid endometrial cancer.

Significance of EpCAM and TROP2 expression in non-small cell lung cancer

BackgroundThe tumor-associated calcium signal transducer (TACSTD) genes, originally designated epithelial cell adhesion molecule (EpCAM) and TROP2, represent true oncogenes. Little is known about EpCAM and TROP2 gene expression in non-small cell lung carcinoma (NSCLC). This study evaluated EpCAM and TROP2 protein expression and clinicopathologic significance in cases of NSCLC.MethodsTissue microarray blocks acquired from 164 cases of NSCLC, including 100 cases of adenocarcinoma (AdC) and 64 of squamous cell carcinoma (SCC), were examined by immunohistochemical staining for EpCAM, and TROP2. The results were correlated with clinicopathologic data.ResultsEpCAM and TROP2 were significantly overexpressed in SCC than in AdC (P < 0.01). In AdC, EpCAM overexpression was closely related to sex, histologic grade, pathologic T stage, pathologic N stage, and TNM stage, and TROP2 overexpression was only related to histologic grade (P < 0.05, respectively). In SCC, correlations were evident between EpCAM overexpression and TNM stage (P = 0.01), and between TROP2 overexpression and pathologic T stage (P = 0.02). EpCAM overexpression showed no significance with overall survival in AdC and SCC patients. However, TROP2 overexpression in AdC had a positive influence on overall survival (P = 0.02) and disease-free survival (P = 0.03). In particular, AdC patients with stage II or III showed better overall survival (P = 0.05) and disease-free survival (P = 0.04).ConclusionsWhile EpCAM and TROP2 show weak and non-complete membranous staining in normal bronchial epithelium and pneumocyte, their complete membranous expression in carcinoma suggests their role in carcinogenesis. EpCAM and TROP2 were more frequently overexpressed in SCC. EpCAM overexpression had no prognostic value in this study, but TROP2 overexpression showed better survival in AdC patients and might be a better prognostic marker in advanced stage AdC.

TROP2 is epigenetically inactivated and modulates IGF‐1R signalling in lung adenocarcinoma

Trop-2, a cell surface glycoprotein, contains both extracellular epidermal growth factor-like and thyroglobulin type-1 repeat domains. Low TROP2 expression was observed in lung adenocarcinoma tissues as compared with their normal counterparts. The lack of expression could be due to either the loss of heterozygosity (LOH) or hypermethylation of the CpG island DNA of TROP2 upstream promoter region as confirmed by bisulphite sequencing and methylation-specific (MS) polymerase chain reaction (PCR). 5-Aza-2′-deoxycytidine treatment on lung cancer cell (CL) lines, CL1-5 and A549, reversed the hypermethylation status and elevated both TROP2 mRNA and protein expression levels. Enforced expression of TROP2 in the lung CL line H1299 reduced AKT as well as ERK activation and suppressed cell proliferation and colony formation. Conversely, silencing TROP2 with shRNA transfection in the less efficiently tumour-forming cell line H322M enhanced AKT activation and increased tumour growth. Trop-2 could attenuate IGF-1R signalling-mediated AKT/β-catenin and ERK activation through a direct binding of IGF1. In conclusion, inactivation of TROP2 due to LOH or by DNA methylation may play an important role in lung cancer tumourigenicity through losing its suppressive effect on IGF-1R signalling and tumour growth.

Upregulation of Trop-2 quantitatively stimulates human cancer growth

Trop-2 is a calcium signal transducer that is associated with transformed cell growth in experimental systems. However, its role in human cancer remains essentially unknown. In this study, we profiled Trop-2 expression in normal human tissues at the mRNA and protein levels. We then systematically compared Trop-2 mRNA and protein levels in tumours with their tissues of origin. We find that Trop-2 expression is invariably upregulated in tumours, regardless of baseline expression in normal tissues, which suggests a corresponding selective advantage. Thus, we investigated the outcome of Trop-2 upregulation on tumour growth. Overexpression of wild-type Trop-2 was shown to be necessary and sufficient to drive cancer growth in a widely invariant manner across cell type and species. Upregulation of Trop-2 was shown to quantitatively stimulate tumour growth, as proportional to expression levels in vivo, and tumour cell growth was abrogated by somatic knockdown of Trop-2 expression. On the other hand, we found no evidence of tumour-associated TROP2 mutations, nor of TROP2 induction of oncogenic transformation per se. Our data support a model where above-baseline expression of wild-type Trop-2 is a key driver of human cancer growth.

Loss of Trop2 Promotes Carcinogenesis and Features of Epithelial to Mesenchymal Transition in Squamous Cell Carcinoma

Trop2, an oncogenic cell surface protein under investigation as a therapeutic target, is commonly overexpressed in several epithelial tumor types yet its function in tumor biology remains relatively unexplored. To investigate the role of Trop2 in epithelial carcinogenesis, we generated Trop2(-/-) mice, which are viable and possess a normal lifespan. Contrary to expectations, Trop2 loss fails to suppress keratinocyte transformation. Instead, ras-transformed Trop2(-/-) keratinocytes preferentially pass through an epithelial to mesenchymal transition (EMT) and form tumors with spindle cell histology. Furthermore, Trop2 loss renders Arf-null mice susceptible to the formation of biphasic sarcomatoid carcinomas containing both squamous and spindle cell components upon carcinogen exposure in an otherwise skin cancer-resistant strain (C57BL/6). Immortalized keratinocytes derived from Trop2(-/-)Arf(-/-) mice exhibit enhanced proliferative and migratory capacity as well as increased activation of mitogen-activated protein kinase and Src prior to transformation. The clinical relevance of these findings was supported by studying the molecular epidemiology of Trop2 in primary head and neck squamous cell carcinomas. This analysis revealed that Trop2 mRNA levels are decreased in a subset of tumors with features of EMT, and total loss of Trop2 protein expression is observed in the spindle cell component of sarcomatoid carcinomas. Therefore, while previous studies have emphasized the potential importance of Trop2 gain of function, these results uncover a role for Trop2 loss in tumorigenesis and the mesenchymal transdifferentiation observed in a subset of squamous cell carcinomas.

Influence of TROP-2 gene on the ability of human breast cancer cell metastasis

Objective To study the effects of tumor-associated calcium signal transducer-2 (TROP-2) gene small interfering RNA(siRNA) on adhesion and invasion of human breast cancer cell. Methods Real time PCR was used to evaluate the TROP-2 mRNA of seven human breast cancer cell lines Bcap-37 ,LCC1 ,MCF-7 ,MDA-MB-231,MDA-MB-435, MDA-MB-468 ,and ZR75-1. The cell line of TROP-2 highest expression was transfected with different dose of TROP-2 siRNA. The expression of TROP-2 mRNA and protein were determined by Real-time quantitative PCR and immumoflurescence method. The cell adhesion was evaluated by MTT assay,and invasion was exmined by hoyden chamber,respectively. Results Cell line MCF-7 showed the highest elevation of TROP-2 mRNA in seven breast cancer cell lines. The results from real-time quantitative PCR and immumoflurescence method showed that TROP-2 mRNA and protein reduced in time-and dose-dependent manners( P ＜ 0.01 ;P ＜ 0.01 ). The adhesive rate of siRNA groups(5 nM,10 nM,and 20 nM)was(52.9 +2.5)％ ,(25.6 ±2.3)％, ( 12.8 +2.2)％ (P ＜0.01 ) ,respectively.The transwell results showed that the invasion cells was(78 ± 17), (39 ± 15), ( 19 ± 16), ( 136 +25 ) and( 139 ±21 )in different groups(5,10,20 nM siRNA,and controls) ,respectively(P ＜0.01). Conclusion TROP-2 gene might play an important role in adhesion and invasion of human breast cancer cell. siRNA targeted TROP-2 could effectively inhibit adhesion and invasion of human breast cancer cell. Key words: Breast Neoplasms; Tumor-associated calcium signal transducer-2

The oncogeneTrop2regulates fetal lung cell proliferation

The factors regulating growth of the developing lung are poorly understood, although the degree of fetal lung expansion is critical. The oncogene Trop2 (trophoblast antigen 2) is upregulated during accelerated fetal lung growth, and we hypothesized that it may regulate normal fetal lung growth. We investigated Trop2 expression in the fetal and neonatal sheep lung during accelerated and delayed lung growth induced by alterations in fetal lung expansion, as well as in response to glucocorticoids. Trop2 expression was measured using real-time PCR and localized spatially using in situ hybridization and immunofluorescence. During normal lung development, Trop2 expression was higher at 90 days gestational age (GA; 4.0 ± 0.8) than at 128 days GA (1.0 ± 0.1), decreased to 0.5 ± 0.1 at 142 days GA (full term ∼147 days GA), and was positively correlated to lung cell proliferation rates (r = 0.953, P < 0.005). Trop2 expression was regulated by fetal lung expansion, but not by glucocorticoids. It was increased nearly threefold by 36 h of increased fetal lung expansion (P < 0.05) and was reduced to ∼55% of control levels by reduced fetal lung expansion (P < 0.05). Trop2 expression was associated with lung cell proliferation during normal and altered lung growth, and the TROP2 protein colocalized with Ki-67-positive cells in the fetal lung. TROP2 was predominantly localized to fibroblasts and type II alveolar epithelial cells. Trop2 small interfering RNA decreased Trop2 expression by ∼75% in cultured fetal rat lung fibroblasts and decreased their proliferation by ∼50%. Cell viability was not affected. This study demonstrates that TROP2 regulates lung cell proliferation during development.

Expression and clinical significance of Trop-2 in human pancreatic cancer

To investigate the expression and its clinical significance of Trop-2 in human pancreatic cancer. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence assay were used to characterize the expression of Trop-2 in human pancreatic cancer. The expression of Trop-2 protein in 31 tumor tissue samples, including peritumoral tissue from patients with pancreatic cancer, was detected with immunohistochemistry by tissue microarray. The clinicopathological characteristics and the tissue expression of Trop-2 protein were analyzed statistically. RT-PCR, immunofluorescence assay and immunohistochemistry by tissue microarray showed a high expression of Trop-2 in pancreatic cancer. The expression rate of Trop-2 was much higher in pancreatic cancer than that in peritumoral tissues (87.1% vs 9.7%). And a high expression of Trop-2 in pancreatic cancer was correlated with the low-differentiated changes. It had statistical significance (P < 0.05). In contrast, no statistically significant correlation was found between the expression of Trop-2 and gender or age. The expression of Trop-2 is correlated with the development and malignancy of pancreatic cancer. As a clinical prognostic marker, Trop-2 may be a potential therapeutic target for pancreatic cancer.

Effects of down-regulation of tmnor-associated calcium signal transducer-2 gene expression by RNA interference on adhesion, migration and invasion of gastric cancer cells

Objective To study the effects of tumor-associated calcium signal transducer-2 (TROP-2) gene small interfering RNA (siRNA) on adhesion, migration, and invasion of human gastric cancer cells.Methods Real time polymerase chain reaction (PCR) was used to detect the TROP-2 mRNA expression of human gastric cancer cell lines MGC-803, HC, C-27 and BGC-823. The cells with highest expression of TROP-2 were transfected with different doses of TROP-2 siRNA. The expression of TROP-2 mRNA and protein was detected by real-time quantitative PCR and immumoflureseence method. Cell adhesion, migration,and invasion were exmined by hoyden chamber, respectively. Results Cell line BGC-823 showed the highest elevation of TROP-2 mRNA among three gastric cancer cell lines. Real-time quantitative PCR and immumoflurescence method revealed that the expression of TROP-2 mRNA and protein was reduced in a time- and dose-dependent manner ( r = 0. 935 ; r = 0. 922). The ability of adhesion, migration and invasion of BGC-823 cells treated with TROP-2 siRNA was decreased as compared with control group (P <0. 01 ). Conclusion TROP-2 gene might play an important role in adhesion, migration, and invasion of human gastric cancer cells, siRNA targeted TROP-2 could effectively inhibit adhesion, migration, and invasion of human gastric cancer cells. Key words: Gastric carcinoma; TROP-2; RNA interference; Invasion

Trop-2 overexpression as an independent marker for poor overall survival in ovarian carcinoma patients

Background Prognostic factors currently available are insufficient to predict the clinical course of epithelial ovarian cancer (EOC). In a previous microarray study we identified the human trophoblast cell surface antigen Trop-2 as one of the top differentially expressed genes in serous papillary EOCs compared to normal human ovarian surface epithelial (HOSE) short-term cultures. The aim of the present investigation was to analyse Trop-2 expression at mRNA and protein level and to assess its prognostic significance in EOC. Methods Using quantitative real-time PCR we tested a total of 104 fresh-frozen EOC tissues and 24 HOSE for Trop-2 mRNA expression. Trop-2 protein expression was then examined by immunohistochemistry in matched formalin-fixed paraffin-embedded EOC samples and in 13 normal ovaries. Finally, we correlated Trop-2 expression to EOC conventional clinicopathological features and patient outcomes. Results We found a significant Trop-2 mRNA and protein upregulation in EOCs compared to normal controls ( p < 0.001). Trop-2 protein overexpression was significantly associated with the presence of ascites ( p = 0.04) and lymph node metastases ( p = 0.04). By univariate survival analysis, Trop-2 protein overexpression was significantly associated with decreased progression-free ( p = 0.02) and overall survival ( p = 0.01). Importantly, Trop-2 protein overexpression was an independent prognostic marker for shortened survival time in multivariate Cox regression analysis ( p = 0.04, HR = 2.35, CI 95% = 1.03–5.34). Conclusions Our results indicate, for the first time, that Trop-2 protein overexpression correlates with an aggressive malignant phenotype and may constitute a novel prognostic factor for EOC. The targeting of Trop-2 overexpression by immunotherapeutic strategies may represent an attractive and potentially effective approach in patients harbouring EOC.

TROP2 is a major determinant of growth and metastatic spreading of human cancer

10510 Background: The TROP2 gene encodes a transmembrane calcium signal transducer, involved in the regulation of cell- cell adhesion. Methods: To identify the role of Trop-2 in transformed cells, we studied its expression pattern and function by DNA array and SAGE analysis, Northern and Western blotting, flow cytometry, confocal microscopy and IHC in experimental systems and in man. Overexpression or down-regulation of Trop-2 and directed mutagenesis were used to identify its role in tumor growth and metastasis in vivo. Results: DNA microarray, EST, SAGE, RT-PCR and Northern blot analysis of human tumors revealed expression of the TROP2 gene in most cancers. IHC analysis of human tumors (1755 cases) revealed a corresponding overexpression of Trop-2 protein. Trop-2 potently stimulated the growth of tumor cells, whereas TROP2 siRNA inhibited it. Deletion of the cytoplasmic region of Trop-2 abolished the growth stimulatory capacity, as did mutagenesis of the S303 PKC phosphorylation site. Proteomic analysis showed that multiple PKC isoforms partecipate to the Trop-2 signaling network. In vivo imaging showed dynamic colocalization of PKCs and Trop-2 in vivo in membrane ruffles and podosomes. DN PKCs and siRNA abolished Trop-2-induced growth. Strikingly, comparative global gene expression analysis revealed that TROP2 was the only gene up-regulated across different metastatic models, tumor types and animal species. IHC analysis revealed a dramatic up-regulation in metastases from colon, stomach, breast and ovary tumors in man. To assess if Trop-2 may play a causal role in metastatic spreading, TROP2-transfected KM12SM colon cancer cells were orthotopically injected in nude mice. TROP2-overexpressing transfectants demonstrated increased metastatic potential to the liver. Deletion of the HIKE domain of Trop-2 severely diminished, whereas that of the whole cytoplasmic region vastly increased metastatic diffusion, indicating the existence of both metastatic enhancers and silencers in the Trop-2 cytoplasmic tail. Conclusions: Our findings demonstrate that Trop-2 is a novel, widespread, stimulator of human cancer growth and a unique marker and causal factor of metastatic cancer, and candidate Trop-2 as a target of novel diagnostic and therapeutic procedures. No significant financial relationships to disclose.

Serological identification of TROP2 by recombinant cDNA expression cloning using sera of patients with esophageal squamous cell carcinoma.

We applied serological analysis of recombinant cDNA expression libraries (SEREX) to cases of esophageal squamous cell carcinoma (SCC) to identify tumor antigens. One of the clones identified was TROP2, which is known as calcium signal transducer. To evaluate the clinical significance of serum anti-TROP2 antibodies (s-TROP2-Abs) in patients with esophageal SCC, the presence of s-TROP2-Abs was analyzed by Western blotting using bacterially expressed TROP2 protein. We found that 23 of 75 (31%) patients were positive for s-TROP2-Abs. Positivity in terms of s-TROP2-Abs showed a significant association with tumor size but not with other clinicopathological features. The protein expression levels of TROP2 were much higher in esophageal SCC cell lines as compared to those in normal esophageal mucosa and its immortalized cells although the mRNA expression levels were not necessarily elevated in malignant cell lines and tissues. Immunohistochemical studies showed that the expression of TROP2 protein in esophageal SCC specimens was noticeably higher than that found in mild hyperplasia of esophageal mucosae. Thus, s-TROP2-Abs seemed useful in the diagnosis of SCC and may be a candidate for serum tumor markers.