Splice variants of the forkhead box protein AFX exhibit dominant negative activity and inhibit AFXα-mediated tumor cell apoptosis

Background: Loss-of-function in the apoptosis-inducing genes is known to facilitate tumorigenesis. AFX (FOXO4), a member of forkhead transcription factors functions as a tumor suppressor and has 2 isoforms, AFXα (505 a.a.) and AFXζ (450 a.a.). In human cancer cells, we identified an N-terminally deleted form of AFXα (α198-505), translated from a downstream start and 2 short N-terminal AFX proteins (90, and 101 a.a.) produced by aberrant splicing. Methods and Findings: We investigated the expression and role of these AFX variants. Cell transduction study revealed that short N-terminal AFX proteins were not stable. Though α(198-505) protein expression was detected in the cytoplasm and nucleus, α(198-505) expressing cells did not show a nucleocytoplasmic shuttling mediated by PI3 kinase signaling. Whereas, we observed this shuttling in cells expressing, either AFXα or AFXζ protein. AFXζ and α(198-505) lost the ability to transactivate BCL6 or suppress cyclin D2 gene expression. These variants did not induce cancer cell death whereas AFXα resulted in apoptosis. We found that AFXζ and α(198-505) suppress the AFXα stimulation of BCL6 promoter in a dose dependent manner, indicating dominant negative activity. These variants also inhibited AFXα induction of apoptosis. Conclusions: Loss of function by aberrant splicing and the dominant negative activity of AFX variants may provide a mechanism for enhanced survival of neoplastic cells.