Overexpression Of EpCAM Correlated With Castration-resistant Prostate Cancer

Castration-resistant prostate cancer is the major cause of the death of prostate cancer.The mechanism of androgen-resistant prostate cancer incidence has now become one ofthe hotspots of the prostate cancer research. Epithelial cell adhesion molecule is a class Itransmembrane glycoprotein wich restrictedly expressed in the basement membrane ofthe normal epithelial cells but highly expressed in rapidly proliferating carcinomas.Overexpression of EpCAM are the promising target for the diagnosis andimmunotherapy of the malignancies as its expression closely correlated with theoccurrence and development of the malignancies. So far, there has no study about therelationship of EpCAM overexpression and the occurrence of castration-resistant prostatecancer.In this study, we used the methods of immunohistochemistry and tissue microarrayto detect the expression of EpCAM in6normal prostate,24benign prostatic hyperplasiaand210prostate cancer tissues. The results showed that the expression of EpCAM innormal prostate was low and was restrictedly expressed in the basement membrane andcell junction of the normal epithelial cells. The the expression level of EpCAM in benign prostatic hyperplasia cells was increased both in the basement membrane and the celljunction. In the prostate cancer tissues, the expression level of EpCAM was significantlyincreased. The expression pattern of EpCAM was aberrant which did not only expressein the basement membrane and the cell junction but also in the top of the cancer cells.Western-blot detected the expression of EpCAM in prostate cancer tissues and adjacentnormal tissues and the results confirmed that the expression of EpCAM was significantlyhigher in the prostate cancer tissuse than which in the adjacent normal tissues. Thecorrelation of EpCAM overexpression with the clinicopathological parameters of theprostate cancer was statistically analyzed. The results showed that the overexpression ofEpCAM significantly correlated with the clinical pathological grade, lymph nodemetastasis, distant metastasis as well as pre-operative serum PSA levels of the prostatecancer. Moreover, the metastasis-free survival and the overall survival of the patientswith EpCAM high expression were significantly short than those with EpCAM lowexpression.Western-blot detected the expression of EpCAM in four prostate cancer cell lines ofDU-145，PC-3，LNCaP and VCaP. The result showed that the amount of EpCAMexpressed in the LNCaP cells was the highest followed by the PC-3, DU-145and VCAPcells. Using the the EpCAM-shRNA Lentiviral particles and the appropriate controlshRNA Lentiviral vector infected the prostate cancer cells of LNCaP and established thestably transfected cell lines of EpCAM-shRNA5and control cell lines of Vector3. Theexpression levels of AR and PSA were detected in the cell lines of EpCAM-shRNA5and Vector3, the results showed that the expression levels of AR and PSA inEpCAM-shRNA5cells was significantly lower than which in Vector3cells. Thesandwich ELISA assay confirmed the PSA levels in the EpCAM-shRNA5cell culturesupernatant was lower than in the supernatant of Vector3cells. MTT assay, TUNELmethod, Annexin V/PI double staining, cell scratch assay and Transwell experimentswere used to detect the proliferation, anti-apoptosis, migration and invasive ability of thecells of EpCAM-shRNA5and Vector3in vitro. The results showed that the abilities ofproliferation, anti-apoptosis, migration, and invasion of EpCAM-shRNA5cells were significantly lower than the Vector3cells. Using the method of mice tumor cultivationmodel to detect the ability of tumor forming of both cell in vivo, the results showed thatthe tumorigenicity of EpCAM-shRNA5cells in vivo was significantly lower than Vector3cells.Our findings suggest that EpCAM overexpression in prostate cancer may promotethe ability of proliferation, anti-apoptosis, migration and invasion of prostate cancer cellsby promoting the expression of AR and made the prostate cancer cells to adapt to thecastration levels of androgen.The morphology of the Vector3cells was similar to LNCaP cells withspindle-shaped, thin filopodia and almost non-existent connections between cells.However， the morphology of EpCAM-siRNA5cell changed from elongatedspindle-shaped into a more regular pebble-like cell, the thin filopodia disappeared, andthe connection appeared between the relatively sparse of the cells. These morphologicalchanges suggested that the correlation of EpCAM expression and the occurrence ofEpithelial-mesenchymal transition (EMT). EMT associated protein marker was detectedin the cells of EpCAM-siRNA and Vector3. The result showed that the expression levelof epithelial cell-specific protein of E-cadherin and Cytokeratin was increased andinterstitial cell-specific protein of Vimentin and fibronectin expression was decreased inthe EpCAM-shRNA5cells compared to the Vector3cells. Immunofluorescence stainingfurther confirmed the closed relation of EpCAM expression with EMT. Due to theoccurrence of EMT cells would confer cells some of the characteristics of stem cells, andthe expression of EpCAM in some tumor tissues is also regarded as a stem cellassociated markers. Therefore, we further verify the stem cells characteristics inEpCAM-shRNA5and Vector3cells. Using colony formation assay to detect the abilityof colony formation of two cell lines. The result showed that the ability of colonyformation of EpCAM-shRNA5cells was significantly lower than Vector3cells. Furtherdetection the expression of stem cell related transcription factors OCT4, Sox2, NANOG.The expression levels of three stem cell-related transcription factors were significantlylow in EpCAM-shRNA5cells than Vector3cell.Suggesting that EpCAM can promotethe survival of prostate cancer cell in the castration levels of androgens not only through increasing the level of AR expression but also promoting the occurrence of EMT andconferring tumor cells on stem cell properties.In summary, our study firstly illustrated the correlation of EpCAM overexpressionwith the castration-resistant prostate cancer and confirmed that EpCAM overexpressioncan promote the survival of prostate cancer cell in the castration levels of androgens notonly through increasing the level of AR expression but also promoting the occurrence ofEMT and conferring tumor cells on stem cell properties. The result of our study canprovide an promising candidate of the target molecules for the immunotherapy of theCastration-resistant prostate cancer.