Expression and function of the tumor antigen MAGE-A3 in bladder cancer cell lines

MAGE-A3 is a member of the type I Melanoma Antigen Gene family and is expressed in various cancers including bladder cancer. MAGE-A3 represents a candidate antigen as a possible target for cancer immunotherapy. In particular, MAGE-A3 vaccination protocols are investigated in clinical trials in...

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Bibliographic Details
Main Author: Zhou, Gaofeng
Contributors: Hänze, J. (PD Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2020
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Summary:MAGE-A3 is a member of the type I Melanoma Antigen Gene family and is expressed in various cancers including bladder cancer. MAGE-A3 represents a candidate antigen as a possible target for cancer immunotherapy. In particular, MAGE-A3 vaccination protocols are investigated in clinical trials in bladder cancer. So far, functional studies on MAGE were mainly performed in melanoma cells, myeloma cells, lung cancer and breast cancer cells whereas the function of MAGE-A3 in bladder cancer cells is largely unknown. Here, we analyzed the expression of MAGE-A3 in a panel of human bladder cancer cell lines and selected appropriate cells for functional studies. Furthermore, we established potent knockdown of MAGE-A3 by RNA interference and harnessed this technique for functional analysis of MAGE-A3. MAGE-A3 mRNA levels where highest in UMUC-3, 5637 and T24 cells whereas no detectable levels were observed in EJ-28 cells. BFTC-905 and HT-1376 cells exhibited intermediate MAGE-A3 mRNA levels. For further experiments, T24, UMUC-3 and EJ-28 cells were selected and MAGE-A3 expression was confirmed at protein level by immunocytochemistry. Potent siRNA knockdown of MAGE-A3 mRNA was validated by RT-PCR exhibiting down-regulation of MAGE-A3 mRNA by approximately 80 % in T24 and UMUC-3 cells. Evidence of MAGE-A3 knockdown could also be confirmed in a non-quantitative way by immunocytochemistry. EJ-28 cells that displayed no detectable MAGE-A3 mRNA abundance were included in these experiments as control cells. At the functional level, silencing of MAGE-A3 resulted in significant increased proliferation, cell count and colony formation in T24 und UMUC-3 cells, whereas EJ-28 cells were unaffected. Apoptosis was reduced after silencing of MAGE-A3 in Summary 46 T24 cells. In order to get some mechanistic clue for this observation, proteomic array analysis of cell cycle regulatory and apoptotic proteins was performed in T24 cells demonstrating increased level of livin and decreased levels of cyclin-dependent kinase inhibitor p21 and tumor suppressor protein phospho-p53 forms after silencing of MAGE-A3. Thus, at the functional level we could demonstrate anti-proliferative and pro-apoptotic effects of MAGE-A3 indicating an anti-oncogenic characteristic. The anti-proliferative and pro-apoptotic effects of MAGE-A3 were accompanied by down-regulation of livin and up-regulation of p21 and phospho-p53 forms likely contributing to or mediating the observed effects. In sum, we selected suitable bladder cancer cell lines for analysis of MAGE-A3 and established efficient silencing of MAGE-A3. Interestingly, we revealed an important so far non-described anti-oncogenic function of MAGE-A3 in bladder cancer cells. This aspect should be considered when employing immunotherapeutic strategies targeting MAGE-A3 tumor antigen by antibodies.
Physical Description:59 Pages
DOI:https://doi.org/10.17192/z2020.0369