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
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
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.