Splice variants of the forkhead box protein AFX exhibit dominant negative activity and inhibit AFXalpha-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, AFXalpha (505 a.a.) and AFXzeta (450 a.a.). In human cancer cells, we identified an N-terminally deleted form of AFXalpha (alpha198-505), translated from a downstream start and 2 short N-terminal AFX proteins (90, and 101 a.a.) produced by aberrant splicing.


RESULTS
We investigated the expression and role of these AFX variants. Cell transduction study revealed that short N-terminal AFX proteins were not stable. Though alpha(198-505) protein expression was detected in the cytoplasm and nucleus, alpha(198-505) expressing cells did not show a nucleocytoplasmic shuttling mediated by PI3 kinase signaling. Whereas, we observed this shuttling in cells expressing either AFXalpha or AFXzeta protein. AFXzeta and alpha(198-505) lost the ability to transactivate BCL6 or suppress cyclin D2 gene expression. These variants did not induce cancer cell death whereas AFXalpha resulted in apoptosis. We found that AFXzeta and alpha(198-505) suppress the AFXalpha stimulation of BCL6 promoter in a dose dependent manner, indicating dominant negative activity. These variants also inhibited AFXalpha 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.
Lee
Eun Jig
EJ
Endocrinology, Yonsei University College of Medicine, Seoul, Korea.
Kim
Jeong Mo
JM
Lee
Mi Kyung
MK
Jameson
J Larry
JL
D017398:Alternative Splicing; D017209:Apoptosis; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D056742:Cyclin D2; D016213:Cyclins / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D005799:Genes, Dominant; D016147:Genes, Tumor Suppressor; D006367:HeLa Cells; D006801:Humans; D009369:Neoplasms / Q000378:metabolism*; D020033:Protein Isoforms; D051560:Proto-Oncogene Proteins c-bcl-6; D014157:Transcription Factors / Q000378:metabolism*
C084103:BCL6 protein, human; C080336:CCND2 protein, human; D018797:Cell Cycle Proteins; D056742:Cyclin D2; D016213:Cyclins; D004268:DNA-Binding Proteins; C108803:FOXO4 protein, human; D051858:Forkhead Transcription Factors; D020033:Protein Isoforms; D051560:Proto-Oncogene Proteins c-bcl-6; D014157:Transcription Factors