Here, we identify SF2 as a dose-dependent effector of cyclin D1b production, and provide evidence of a tumor-associated mechanism that alters the influence of the G/A870 polymorphism. While minigene analyses of individual alleles () and evaluation of non-neoplastic human tissue support a role for the A870 allele in promoting transcript-b
and resultant cyclin D1b production, the influence of the A-allele is thought to be relieved in PCa (11
). The present data demonstrate that the RNA binding protein SF2, which is induced as a function of PCa progression, predicts for cyclin D1b (not cyclin D1a) elevation in human disease (). By contrast, cyclin D1b production was attenuated in model systems of SF2 depletion (). Functional studies in PCa cells heterozygous for the polymorphism unexpectedly demonstrated that SF2 predominantly associates with and induces transcript-b
derived from the G870-allele, however, association with and production of transcript-b
from the A-allele can still occur ( and ). Together, these studies provide a novel mechanism by which cyclin D1b oncogene production induced in human disease, and identify tumor-associated SF2 as an allele selective effector of cyclin D1b.
Despite the potent oncogenic activity of cyclin D1b, knowledge of the factor(s) that regulate the CCND1
alternative splicing event are poorly defined. The present identification of SF2 as an effector of transcript-b
and cyclin D1b production using both in vitro
models and analyses of human tumors provides strong evidence linking splicing factor deregulation to oncogene activation. It is tempting to speculate as to whether SF2 can cooperate with few effectors of cyclin D1b production that were identified in other tissue types. For example, in colorectal cells it was observed that knockdown of the SWI/SNF chromatin remodeling complex subunit Brahma (BRM) increased transcript-b
production without altering transcript-a
). These findings are of note, as BRM was recently shown to be significantly downregulated in human PCa (35
), and it has yet to be determined whether BRM loss might also impact SF2 levels. In a separate study, a chromosomal translocation-derived transcription factor known to be upregulated in Ewing’s sarcoma (EWS-FLI1) was found to enhance cyclin D1b production by diminishing the rate of transcriptional elongation (10
). More recently, it was demonstrated that Sam68 promotes cyclin D1b production through a splicing repressive mechanism by blocking U1-70k association, a constitutive spliceosome accessory factor of the U1 snRNP that is necessary for 5’ splice site recognition (36
). It is well established that chromatin remodeling complexes can alter RNA Pol II accessibility by alteration of the native chromatin structure (38
); moreover, pre-mRNA splicing occurs co-transcriptionally, and is aided by the function of SR proteins (including SF2) which can bind to Pol II and selected SWI/SNF subunits (BAF155 and BAF53A) (39
). Therefore, an attractive hypothesis is that SF2 could act in concert with either BRM or EWS-FLI1 to modulate the CCND1
splicing event. Given the marked pro-tumorigenic activity of cyclin D1b, these collective observations further underscore the importance of delineating the mechanisms that regulate or influence the SF2-mediated alternative splicing event in models of disease relevance.
With regard to clinical relevance, it is notable that in PCa cells, SF2 exhibited an allele selective effect on the alternative splicing event. Previous studies showed that in non-neoplastic tissue, the A870 allele is associated with higher transcript-b
production but that the influence of the A-allele was lost in PCa specimens (11
); these findings suggested tumor-associated factors may either bypass the impact of the polymorphism or bolster production of transcript-b
from the G-allele. The present data support the hypothesis that SF2 may serve as such a factor, since SF2 induction (such as occurs in human disease) predominantly binds to transcript-b
derived from the G-allele and promotes accumulation of this transcript. Production of transcript
-b still occurs from the A-allele (about 40%), however, appears to remain unaltered following modulation of SF2 expression, thus it will be of interest to examine the functional relationship between SF2 and the A-allele. Examination of how SF2 influences allele-specific cyclin D1b production in other tumor types in which cyclin D1b levels are elevated as a function of tumorigenesis (e.g. colon, bladder or breast carcinoma) will be critical (6
). Given the propensity of SF2 to preferentially bind to and induce cyclin D1b from the G-allele, the present data indicate that SF2 may promote intron inclusion at the CCND1
exon4-intron4 boundary, and precedence for a splicing repressor function of SF2 was previously established for the MNK2 kinase (42
). It cannot be presently ruled out that the impact of SF2 could be manifest through other means, given the ability of SF2 to affect mRNA metabolism, mRNA transport and/or stability, and mTOR-mediated translation (43
). Future analyses will be directed at defining the action of SF2 at the exon4-intron4 boundary.
Finally, the present findings provide new insight into a potential means through which SF2 promotes cellular transformation. It is noteworthy that SF2 can independently induce transformation and induce tumor growth in vivo
), thus demonstrating phenotypes similar to those observed with cyclin D1b (3
). As SF2 levels correlated with cyclin D1b in human PCa, these data implicate cyclin D1b as a possible downstream effector of SF2-mediated cellular transformation in the prostate. Additional mechanisms are predicted to contribute to this event, as perturbations in spliceosome function and RNA processing proteins have been recently identified as major contributors to genomic instability (45
). Importantly, dysregulation of splicing factors has also been shown to accelerate PCa progression and metastases, such as observed by deregulated expression of SRp40, or in the presence of specific, cancer-risk associated polymorphisms in the intronic region of the KLF6 tumor suppressor that create novel SRp40 binding sites (46
). Given the disease relevance of this event, it will be imperative to determine not only the consequence of SR protein dysregulation, but whether these events act in concert to promote tumor progression. Ongoing studies suggest that tumor-associated SF2 induction may induce and enhanced migration and invasion phenotype in prostate cancer cells (data not shown), providing the impetus to discern the impact of SF2 on development of tumor metastases.
In summary, our study identifies SF2 as a novel, clinically relevant effector of CCND1 alternative splicing, capable of promoting allele-specific induction of cyclin D1b in prostate cancer. The findings presented are among the first to determine how the cyclin D1b oncogene is enhanced in human disease, and provide the foundation for future studies directed at developing mechanisms to target oncogene induction.