The Role And Mechanism Of UBE2T Promoting The Stemness Of Breast Cancer Cells

As is reported, breast cancer (BC) is the most frequently diagnosed female cancer and its incidence shows an incresing trend in China. Nowadays, the relapse and metastasis of BC can’t be completely controlled for lack of effective therapies. This circumstance causes a high mortality in intermediate and advanced BC cancer patients.According to the reports, the breast cancer stem cells (BCSCs) show a stronger self-healing capability after radiotherapy or chemotherapy compared with the common BC cells. The minor amount of remaining BCSCs are able to proliferate again then finally cause the relapse and metastasis of BC. Thus, BCSCs have been considered to play a crucial role in cancer growth, metastasis, prognosis and the resistance to tumor chemotherapy. Therefore, the BCSCs targeted therapy may provide a possibility to permanently cure BC and significantly increase the survival rate.The ubiquitin-conjugating enzyme 2T (UBE2T) is a member of ubiquitin-conjugating enzyme and its elevated expression has been considered to associate with the occurence of bladder, prostate and lung cancers. Besides, our previous study has confirmed that UBE2T plays an oncogenic role in prostate cancer through promoting epithelial-mesenchymal transition (EMT) of prostate cancer cells. Moreover, UBE2T has been reported to play a positive role in promoting the proliferation and metastasis of BC cells via suppressing the expression of BRCA1. However, there is still no study to explore the biological function and molecular mechansim about UBE2T in mediating the stemness of BC cells. Here, our study about the mechanism of UBE2T promoting the sternness of BC cells may provide a novel therapeutic method for BC.ObjectiveTo explore the biological function and molecular mechanism of UBE2T mediating the sternness of BC cells.Methods1. Western blotting and qRT-PCR were carried out to detect the expression levels of UBE2T in different BC cell lines (BT549, HCC1428, MCF-7 and MDA-MB-231) and the normal breast epithelial cell line(MCF10A).2. Western blotting and qRT-PCR were carried out to analyze the expression levels of UBE2T in BC tissues and their corresponding adjacent non-cancerous tissues.3. To explore the biological function and potentially molecular mechanism of UBE2T mediating the sternness of BC cells, UBE2T overexpressing, UBE2T silencing plasmids and their corresponding controls were respectively transfected into BC cells.4. Sphere formation assay and soft agar assay were carried out to detect the potential role of UBE2T regulating the temness of BC cells.5. Flow cytometry was carried out to detect the role of UBE2T in altering the proportion of CD44+CD24-/low subpopulations in BC cells.6. Western blotting was performed to detect the potential role of UBE2T in regulating the experssion of cancer stem cell transcription factors (SOX2, OCT4 and NANOG).7. To analyze the potential molecular mechanism of UBE2T mediating the sternness of BC cells, the expression of mTOR and its phosphorylation (p-mTOR) were detected by Western blotting.8. To explore whether mTOR acted as an mediator in UBE2T-induced upregulation of SOX2, OCT4 and NANOG, Western blotting was performed to analyze the expression levels of SOX2, OCT4 and NANOG in BC cells treated with rapamycin.9. To further explore the role of mTOR in UBE2T promoting sternness of BC cell, sphere formation assay was performed on the BC cells treated with rapamycin, an mTOR inhibitor.Results1. The results of Western blotting and qRT-PCR showed the expression of UBE2T in BC cell lines (BT549, HCC1428, MCF-7 and MDA-MB-231) were much higher than it in the normal breast epithelial cell line (MCF10A). Besides, the expression of UBE2T in MDA-MB-231 and BT549 is higher than it in MCF-7 and HCC1428.2. The results of Western blotting and qRT-PCR showed the expression levels of UBE2T in BC tissues were much higher than their corresponding adjacent non-cancerous tissues.3. The UBE2T expression levels in established breast cancer lines were detected by Western blotting and qRT-PCR:(1). We successfully established UBE2T overexpressing and controlled BC cell lines: MCF-7-pBabe-UBE2T, MCF-7-pBabe-Vector, HCC1428-pBabe-UBE2T and HCC1428-pBabe-Vector.(2). We successfully established UBE2T silencing and controlled BC cell lines: BT549-pSuper-shUBE2T.A, BT549-pSuper-shUBE2T.D, BT549-pSuper-Vector, MDA-MB-231-pSuper-shUBE2T.A, MDA-MB-231-pSuper-shUBE2T.D and MDA-MB-231-pSuper-Vector.4. The results of sphere formation assay and soft agar assay indicated overexpression of UBE2T could promote the sternness of BC cells.5. The flow cytometry showed overexpression of UBE2T was able to increase the proportion of CD44+CD24-/low subpopulations while silencing expression of UBE2 T decrease the proportion in BC cells.6. The results of Western blotting showed that overexpression of UBE2T could significantly increase the expression levels of CSC transcription factors (SOX2, OCT4 and NANOG) while silencing expression of UBE2T decrease the expression of these transcription factors.7. The results of Western blotting showed overexpression of UBE2T could increase the expression of mTOR and its phosphorylation level while silencing expression of UBE2T could decrease the expression of mTOR and its phosphorylation.8. The result of Western blotting showed mTOR inhibitor was able to decrease the elevated expression of CSC transcription factors (SOX2, OCT4 and NANOG) induced by overexpression of UBE2T.9. The result of sphere formation assay showed mTOR inhibitor was able to decrease the the number of spheres in UBE2T overexpressing BC cell lines, which indicated mTOR inhibitor could weaken the sternness induced by overexpression of UBE2T.ConclusionOur study reveals the positive role of UBE2T regulating the sternness of BC cell. Furthermore, we preliminarily confirm that UBE2T can regulate the expression of SOX2, OCT4 and NANOG mediated by mTOR and then plays its crucial role in promoting the sternness of BC cells. As far as we know, this is the first time to reveal the biological function and potential molecular mechanism of UBE2T in promoting the sternness of BC cells. These findings suggest a potential genie target of BC treatment.