A functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS-mediated cell growth.

SP/KLF (Specificity protein/Krüppel-like factor) transcription factors comprise an emerging group of proteins that may behave as tumour suppressors. Incidentally, many cancers that display alterations in certain KLF proteins are also associated with a high incidence of KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue) mutations. Therefore in the present paper we investigate whether SP/KLF proteins suppress KRAS-mediated cell growth, and more importantly, the potential mechanisms underlying these effects. Using a comprehensive family-wide screening of the 24 SP/KLF members, we discovered that SP5, SP8, KLF2, KLF3, KLF4, KLF11, KLF13, KLF14, KLF15 and KLF16 inhibit cellular growth and suppress transformation mediated by oncogenic KRAS. Each protein in this subset of SP/KLF members individually inhibits BrdU (5-bromo-2-deoxyuridine) incorporation in KRAS oncogenic-mutant cancer cells. SP5, KLF3, KLF11, KLF13, KLF14 and KLF16 also increase apoptosis in these cells. Using KLF11 as a representative model for mechanistic studies, we demonstrate that this protein inhibits the ability of cancer cells to form both colonies in soft agar and tumour growth in vivo. Molecular studies demonstrate that these effects of KLF11 are mediated, at least in part, through silencing cyclin A via binding to its promoter and leading to cell-cycle arrest in S-phase. Interestingly, similar to KLF11, KLF14 and KLF16 mechanistically share the ability to modulate the expression of cyclin A. Collectively, the present study stringently defines a distinct subset of SP/KLF proteins that impairs KRAS-mediated cell growth, and that mechanistically some members of this subset accomplish this, at least in part, through regulation of the cyclin A promoter.

Sp5l is a mediator of Fgf signals in anteroposterior patterning of the neuroectoderm in zebrafish embryo.

The neuroectoderm is patterned along the anterior-posterior axis in vertebrate embryos. Fgf signals are required to induce the posterior neuroectodermal fates, but they repress the anterior fate. Sp5l/Spr2, an Sp1-like transcription factor family member, has been shown to be required for development of mesoderm and posterior neuroectoderm. We demonstrate here that repression of the anterior neuroectodermal markers fez and otx1 by fgf17b or fgf3 coincides with induction of sp5l in the anterior neuroectoderm, and that this repression is efficiently rescued by simultaneous sp5l knockdown. On the other hand, sp5l knockdown is able to inhibit inductive activity of ectopic Fgf signals on the expression of the posterior neuroectodermal markers gbx2, hoxb1b, and krox20. Furthermore, effect of overexpression of a dominant negative Fgf receptor on anteroposterior patterning of the neuroectoderm is rescued by sp5l overexpression. Taken together, these data suggest that sp5l mediates the functions of Fgf signals in anteroposterior patterning of the neuroectoderm during zebrafish embryogenesis.

Sp2 localizes to subnuclear foci associated with the nuclear matrix.

We have reported that extracts prepared from many human and mouse cell lines show little or no Sp2 DNA-binding activity and that Sp2 has little or no capacity to stimulate transcription of promoters that are activated by Sp1, Sp3, and Sp4. Using an array of chimeric Sp1/Sp2 proteins we showed further that Sp2 DNA-binding activity and trans-activation are each negatively regulated in mammalian cells. As part of an ongoing effort to study Sp2 function and regulation we characterized its subcellular localization in comparison with other Sp-family members in fixed and live cells. We report that 1) Sp2 localizes largely within subnuclear foci associated with the nuclear matrix, and 2) these foci are distinct from promyelocytic oncogenic domains and appear to be stable during an 18-h time course of observation. Deletion analyses identified a 37 amino acid sequence spanning the first zinc-"finger" that is sufficient to direct nuclear matrix association, and this region also encodes a bipartite nuclear localization sequence. A second nuclear matrix targeting sequence is encoded within the Sp2 trans-activation domain. We conclude that Sp2 preferentially associates with the nuclear matrix and speculate that this subcellular localization plays an important role in the regulation of Sp2 function.