The Mechanism Of UBE2T Promoting Epithelial Mesenchymal Transformation And Metastasis In Prostate Cancer

Prostate cancer (PC) is one common carcinoma in men worldwide and it is one of the main causes of death. In China, the incidence of prostate cancer is lower than that in the developed countries, but increasing incidence of PC at annual 12.1% is found recently because of the increasing economic level, human ideological emancipation and changes of life-style. Big cities are the main areas of PC happening. Recently, surgery combined with radioactive therapy and chemotherapy is the main treatment of PC. If PC was diagnosed earlier, the patients with PC could get an effective surgery and a good prognosis. But when most PC patients are diagnosed, metastasis foci has been found in other parts of the bodies due to less symptoms happen in early stage of prostate cancer. Most PC patients die from complication of PC.Epithelial mesenchymal transformation (EMT) is considered as the key procedure in the initial period of metastasis of carcinomas, and mesenchymal epithelial transformation (MET) is considered as the key procedure in the terminal phase of metastasis of carcinomas. After EMT, cancer cells change from epithelial phenotype to mesenchymal phenotype and move freely due to the decreasing adhesion ability and increasing motility and as a result, cancer cells obtain stronger abilities of move, invasion and metastasis that cancer cells could invade through the basement membrane and go into the circulatory system. After MET, cancer cells grow at the metastasis foci and form tumors. The steps above are the key of PC metastasis and finding new genes regulating EMT or MET is the focus of PC research.The ubiquitination pathway is the main way for protein degradation in which ubiquitin (polypeptide with 76 amino acids), powered by ATP, is transferred from ubiquitin-activating enzyme (E1) to ubiquitin-conjugating enzyme (E2), then ubiquitin ligase (E3) bind both E2 and substrate and transfer ubiquitin to substrate. The substrate with one or more ubiquitin could be recognized by proteasome and degraded into polypeptides. E2 plays a critical role in ubiquitination pathway. Ubiquitin-conjugating enzyme 2T (UBE2T) is a putative member of E2 family and the research about UBE2T focus on its role in DNA damage repair pathways, mainly in Fanconi anemia pathway (FA pathway). In FA pathway, UBE2T participates FANCD2 ubiquitination and activates DNA damage repair pathway. Of note, in another research, UBE2T acts as an oncogene inhibiting cancer suppressor gene BRCA1/BRCA2 in breast cancer that promotes breast cancer cells proliferation and metastasis. But few papers about UBE2T are reported and mechanism of UBE2T is not clear. Therefore it is important to uncover the role and mechanism of UBE2T.In this research, we mainly studied the mechanistic roles of UBE2T in promoting cell proliferation and metastasis in prostate cancer which improved our understanding of the function of UBE2T as well as implied UBE2T as a potrential target in prostate cancer treatment.Part I:Correlation analysis of UBE2T expression level and prostate cancer and breast cancer metastasis[Objective]To detect the expression levels of UBE2T in prostate cancer tissues and breast cancer tissues with different clinical pathologic stages and analyze the relationship between UBE2T levels and tumor metastasis.[Methods]1. Tissue chip of prostate cancer (PR483a) from Alenabio company in Xi’an was used for immunohistochemical stainging of UBE2T in normal prostate tissue, prostate cancer in situ and prostate cancer with distant metastasis.2. Tissue chip of breast cancer (BR1321) from Alenabio company in Xi’an was used for immunohistochemical detection of UBE2T expression in normal breast tissue, breast cancer in situ and breast cancer with distant metastasis.[Results]1. Through immunohistochemical stain for UBE2T, UBE2T expressed lowest in normal prostate tissue and higher in prostate cancer in situ and highest in prostate cancer with distant metastasis.2. Through immunohistochemical stain for UBE2T, UBE2T expressed lowest in normal breast tissue and higher in breast cancer in situ and highest in breast cancer with distant metastasis.[Conclusions]1. UBE2T expressed more significantly in metastatic tumor tissue than that in tumor tissue in situ and expressed lowest in normal tissue predicating that UBE2T closely related to metastasis of carcinomas.Part Ⅱ:UBE2T promotes prostate cancer cells proliferation, EMT and invasion[Objective]Base on the conclusion that UBE2T maybe related with prostate cancer metastasis in tissue array, we try to analyze if UBE2T could promote proliferation and metastasis of prostate cancer cells through in vitro and in vivo tests.[Methods]1. UBE2T overexpressing plasmid pBabe-UBE2T and control vector are retrovirally transfected into prostate cancer cells Du145 and PC3 to establish cell lines: Du145-pBabe-UBE2T, Du145-pBabe, PC3-pBabe-UBE2T, PC3-pBabe.2ng/ml puromycin is used as the selection agent. RT-PCR and western blot are used to detect the UBE2T expression in indicated cells.2. UBE2T silencing plasmid pBabe-UBE2T and control are retrovirally transfected into prostate cancer cells Du145 and PC3 to establish cell lines: Du145-pSuper-shUBE2T.A, Du145-pSuper-shUBE2T.D, Du145-pSuper, PC3-pSuper-shUBE2T.D, PC3-pSuper.2ng/ml puromycin is used as the selection agent. RT-PCR and western blot are used to detect the UBE2T expression in indicated cells.3. Detect the influence of UBE2T on prostate cancer cell proliferation through MTT and colony formation in established cell lines above.4. Observe the morphology of established cell lines under microscope and detect the expressions of EMT markers (E-cadherin, Vimentin, Fibronectin, alpha-smooth muscle acting or α-SMA) through western blot, immunofluorescence and immunohistochemical stain.5. Using the established cell lines above, detect the influence of UBE2T on motility of prostate cancer cells through scratch assay and traswell assay; detect the influence of UBE2T on invasion of prostate cancer cells through matrigel assay.6. Establish nude mice model of tumor formation through subcutaneously injecting UBE2T overexpressing cells and corresponding control cells to show the influence of overexpressing UBE2T on tumor growth; establish nude mice model of metastasis through tail vein injecting UBE2T overexpressing cells and corresponding control cells to show the influence of overexpressing UBE2T on invasion and metastasis.7. Establish nude mice model of tumor formation through subcutaneously injecting UBE2T silencing cells and control showing the influence of silencing UBE2T on tumor growth; establish nude mice model of metastasis through tail vein injecting UBE2T silencing cells and control showing the influence of silencing UBE2T on invasion and metastasis.8. Detect UBE2T expressions in tumors and metastatic foci through immunohistochemical stain.[Results]1. Successfully established UBE2T overexpressing cell lines and control: Du145-pBabe-UBE2T, Du145-pBabe, PC3-pBabe-UBE2T, PC3-pBabe.2. Successfully established UBE2T silencing cell lines and control: Du145-pSuper-shUBE2T.A, Du145-pSuper-shUBE2T.D, Du145-pSuper PC3-pSuper-shUBE2T.D, PC3-pSuper.3. Overexpressing UBE2T enhanced prostate cancer cell proliferation, silencing UBE2T inhibited growth of prostate cancer cells.4. UBE2T changed prostate cancer cells from epithelial morphology to mesenchymal phenotype.5. UBE2T increased the expression of mesenchymal markers (Vimentin, Fibronectin, a-SMA) and decreased the expression of epithelial marker (E-cadherin).6. UBE2T promoted motility and metastasis of prostate cancer cells.7. UBE2T promoted tumor growth of prostate cancer in tumor formation assay in vivo.8. UBE2T promoted metastasis of prostate cancer cells in metastatic assay in vivo.[Conclusions]1. UBE2T promotes proliferation of prostate cancer cells.2. UBE2T regulates EMT, motility, invasion and metastasis of prostate cancer cells.3. UBE2T promotes tumor growth and metastasis of prostate cancer cells in vivo.Part Ⅲ:The mechanism of UBE2T promoting EMT and metastasis of prostate cancer cells[Objective]In tests in vitro, UBE2T could promote proliferation, migration and metastasis of prostate cancer cells, but the mechanism is not clear. In this part, we try to find the possible mechanism.[Methods]1. Tandem affinity purification and Mass Spectrometry (TAP-MS) analyzes the possible targets of UBE2T.2. Confirm the relationship between UBE2T and Vimentin in tissue array3. Half-life assay for Vimentin by western blot with UBE2T overexpression or FBXW7 overexpression.4. In binding assay in vitro, confirm the combination between UBE2T and Vimentin, FBXW7 and Vimentin.5. Western blot for Vimentin expression with UBE2T or FBXW7 or both UBE2T and FBXW7.6. Co-TP for the combination between FBXW7 and Vimentin with different expressions of UBE2T.7. Confirm whether 95th amino acid is the binding site of UBE2T to Vimentin.8. Mutant 95th amino acid and detect proliferation, migration and invasion of prostate cancer cells.[Results]1. In Tap-MS, Vimentin was one target of UBE2T.2. In tissue array, UBE2T promoted Vimentin expression in prostate cancer.3. UBE2T delayed the degradation of Vimentin but FBXW7 promoted this progression.4. UBE2T restrained the degradation of Vimentin by FBXW7.5. UBE2T interrupted the combination between FBXW7 and Vimentin.6. The 95th amino acid is the binding site of UBE2T to Vimentin.9. Mutant of 95th amino acid could inhibit proliferation, migration and invasion of prostate cancer cells.[Conclusion]1. Vimentin is the target of UBE2T and UBE2T delays the degradation of Vimentin.2. UBE2T interrupts the combination between FBXW7 and Vimentin further affecting Vimentin expression.3. The 95th amino acid is the binding site of UBE2T to Vimentin.