Our results demonstrate that Sf1 levels modulate the incidence of TGCT in two different mouse models of TGCTs. Haploinsufficiency of Sf1 correlated with decreases in tumor incidences in Sf1+/-;Ter/Ter and Sf1+/-;M19/+ mice. This argues for a common role of SF1 in germ cell transformation in both strains considering that different genetic defects are responsible for germ cell tumorigenesis in the two strains. Reduction of Sf1 levels likely reduces germ cell transformation rates and results in an overall reduced number of testicular tumors. Another possibility is that lower Sf1 levels in germ cells may result in reduced viability of germ cells. Thus fewer germ cells survive to transform. However, PN1 testes of Sf1+/- mice did not appear to have fewer germ cells compared to wild-type mice and Sf1+/- mice are fertile.
Sf1 levels in both MOLF and M19 strains were found to be significantly lower than in the 129 strain. Thus, different mouse inbred strains inherently express varying Sf1 levels in their tissues and the Sf1 levels could likely influence the tumorigenic potential of cells from these strains. Because the 129 strain has higher Sf1 levels and is permissive for TGCT development, we propose that Sf1 is an ‘oncogenic’ genetic susceptibility factor from 129 that promotes TGCT development.
Interestingly, Sf1 levels are observed to inherently vary in tissues of different inbred strains (NCBI Geo Profiles). This inherent variation in Sf1 levels could likely influence normal and disease phenotypes in mice.
We note that no significant tumor incidences have been observed for the MOLF inbred strain, which has inherently low Sf1
levels. Of the reported studies, TGCT development is found in the M19 (129.MOLF Chr 19) consomic strain (21
) and suppression of mammary tumorigenesis has been reported in FVB/N-Tg (MMTV-PyMT) and MOLF F1 hybrid mice (36
The difference in SF1 levels between the 129 and MOLF strains may be due to a number of factors such as differences in the sequence and activity of the promoters between strains, differences in the nature of alternate spliced Sf1 variants or SNPs (single nucleotide polymorphisms) in the transcripts that affect stability.
Contrary to other observations, we found that Sf1 levels are low in M19 but TGCT incidence is high in this strain. In this case, it appears that the protective effect of low Sf1 levels does not overcome the effects of multiple tumor promoting loci present in M19. However, when we reduce the dosage of tumor promoting loci in M19 to M19/+, then we clearly observe the protective effect of lower Sf1 levels in M19/+;Sf1+/-. TGCT incidences correlate with Sf1 levels in M19/+ and M19/+;Sf1+/- mice. Thus, the protective effect of Sf1 deficiency can be overshadowed by the presence of multiple tumor promoting loci in the genome.
An earlier study reported a gene trap inactivated Sf1
mouse line in which the gene trap was inserted in the promoter region of Sf1
). Treatment of Sf1
+/- mice with an organotropic carcinogen resulted in higher number of colon tumors (32
). This indicated that lower Sf1
levels are oncogenic and this is contradictory to our data. One explanation for this discrepancy could be that Sf1
functions as an oncogene or tumor-suppressor depending on the cell type. In colon cells, Sf1
may be responsible for generating a majority of splice variants with tumor-suppressor function whereas in the testes, Sf1
may generate mostly oncogenic splice variants. Thus lowering Sf1
levels in the colon enhances tumorigenesis whereas lowering Sf1
levels in the testes attenuates tumorigenesis. Another possibility is that application of DNA damaging carcinogen likely induces additional oncogenic mutations in colon cells, and the effects of multiple oncogenes outweigh the protective effect of Sf1
deficiency. A third possibility is that because the Sf1
+/- mouse backgrounds in the two studies are different, this influences the differences in results.
SF1 has been shown to be critical for HeLa cell viability (31
). We detected SF1 expression in developmentally important organs, such as the heart and brain (Supplementary Fig.5
). Presumably, there are cell-type specific pre-mRNA targets of SF1 that are critical for cell and embryonic viability.
In the testes, SF1 is found in both the germ cells and supporting cells of the seminiferous tubules. It is possible that SF1 function in the supporting Sertoli cells of the testes may also be important for germ cell tumorigenesis. Overall, alternative splicing events are most prevalent in the testis and brain (38
), implying that splicing regulation is especially important in these tissues.
Transformation of cells frequently occurs in conjunction with dysregulation in alternative splicing (39
). Because splicing factors usually regulate production of a variety of different mRNAs, changes in either the level or function of splicing factors can cause global changes in RNA levels and splicing variants. Specific splicing factors have been found to be overexpressed or downregulated in cancer tissues (42
). Studies on human testicular tumors have found novel testicular cancer associated splice isoforms (44
) which could serve as potential diagnostic or prognostic markers.
Interestingly, in humans, SF1 has also been implicated in immune responses (46
). SF1 interacts with the branchpoint sequence within the intron of the histocompatibility leukocyte antigen, HLA-DQBβ. Naturally occurring and disease associated branchpoint mutations in DQβ1 intron 3 show impaired SF1 binding and altered splicing which influences their gene expression (46
). A recent global analysis of the human transcriptosome revealed that many genes have subtle genetic changes, such as SNPs in splice site usage (47
). Splicing differences due to SNPs were found to be frequent in human populations, affect disease-causing genes and likely contribute to phenotypic diversity and susceptibility to complex diseases.
In another study, oncogenic alternate splice variants were generated when SF1 was transiently transfected into human colon cancer cell lines and this correlated with cell transformation (37
). This again indicates a proto-oncogenic capability of SF1 in human cells.
In summary, we provide genetic evidence showing that deficiency of SF1 suppresses testicular tumorigenesis. SF1, an RNA binding protein (48
) necessary for spliceosome assembly of specific pre-mRNAs (24
) likely regulates alternative splicing of pre-mRNAs in the testes. Thus, higher Sf1
levels are oncogenic in the testes. Our data leads us to propose that SF1 is a tumor susceptibility factor for germ cell tumorigenesis.