The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

The RNA recognition motif protein RBM11 is a novel tissue-specific splicing regulator.

Mammalian tissues display a remarkable complexity of splicing patterns. Nevertheless, only few examples of tissue-specific splicing regulators are known. Herein, we characterize a novel splicing regulator named RBM11, which contains an RNA Recognition Motif (RRM) at the amino terminus and a region lacking known homology at the carboxyl terminus. RBM11 is selectively expressed in brain, cerebellum and testis, and to a lower extent in kidney. RBM11 mRNA levels fluctuate in a developmentally regulated manner, peaking perinatally in brain and cerebellum, and at puberty in testis, in concomitance with differentiation events occurring in neurons and germ cells. Deletion analysis indicated that the RRM of RBM11 is required for RNA binding, whereas the carboxyl terminal region permits nuclear localization and homodimerization. RBM11 is localized in the nucleoplasm and enriched in SRSF2-containing splicing speckles. Transcription inhibition/release experiments and exposure of cells to stress revealed a dynamic movement of RBM11 between nucleoplasm and speckles, suggesting that its localization is affected by the transcriptional status of the cell. Splicing assays revealed a role for RBM11 in the modulation of alternative splicing. In particular, RBM11 affected the choice of alternative 5' splice sites in BCL-X by binding to specific sequences in exon 2 and antagonizing the SR protein SRSF1. Thus, our findings identify RBM11 as a novel tissue-specific splicing factor with potential implication in the regulation of alternative splicing during neuron and germ cell differentiation.

TLR stimulation of prostate tumor cells induces chemokine-mediated recruitment of specific immune cell types.

TLRs boost antimicrobial response mechanisms by epithelial cells and represent the first line of defense at mucosal sites. In view of these immunomodulatory properties, TLR stimulation may represent a novel means to activate anticancer immune responses. In the present study, the ability of TLR ligands to affect the recruitment of different immune cell populations by human prostate cancer cell lines and the underlying mechanisms were investigated. We showed that LNCaP and DU-145 cells express functionally active TLR3 and TLR5. Treatment with their respective agonists, polyinosinic:polycytidylic acid and flagellin, rapidly triggered NF-kappaB-dependent upregulation of different inflammatory molecules, as assayed by microarray and ELISA. Furthermore, we demonstrated that conditioned media from polyinosinic:polycytidylic acid- and flagellin-treated LNCaP and DU-145 cells induced the recruitment of different leukocyte subpopulations, suggesting that TLR stimulation is able to activate the earliest step of immune response mediated by soluble factors. Interestingly, the more aggressive cancer cell line PC3 expressed TLR3 and TLR5 but failed to respond to TLR agonists in terms of NF-kappaB activation and the ability to attract immune effectors. Overall, these data show for the first time that TLR3 and TLR5 stimulation of human prostate cancer cells triggers the production of chemokines, which, in turn, favor the attraction of immune effectors, thereby representing a tool to enhance the efficacy of conventional therapies by stimulating anticancer immune responses.

Genotoxic stress causes the accumulation of the splicing regulator Sam68 in nuclear foci of transcriptionally active chromatin.

DNA-damaging agents cause a multifaceted cellular stress response. Cells set in motion either repair mechanisms or programmed cell death pathways, depending on the extent of the damage and on their ability to withstand it. The RNA-binding protein (RBP) Sam68, which is up-regulated in prostate carcinoma, promotes prostate cancer cell survival to genotoxic stress. Herein, we have investigated the function of Sam68 in this cellular response. Mitoxantrone (MTX), a topoisomerase II inhibitor, induced relocalization of Sam68 from the nucleoplasm to nuclear granules, together with several other RBPs involved in alternative splicing, such as TIA-1, hnRNP A1 and the SR proteins SC35 and ASF/SF2. Sam68 accumulation in nuclear stress granules was independent of signal transduction pathways activated by DNA damage. Using BrU labelling and immunofluorescence, we demonstrate that MTX-induced nuclear stress granules are transcriptionally active foci where Sam68 and the phosphorylated form of RNA polymerase II accumulate. Finally, we show that MTX-induced relocalization of Sam68 correlates with changes in alternative splicing of its mRNA target CD44, and that MTX-induced CD44 splicing depends on Sam68 expression. These results strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell that modulates alternative splicing in response to DNA damage.

An emerging role for nuclear RNA-mediated responses to genotoxic stress.

Defects in the regulation of alternative splicing have strong relevance in the onset and progression of several types of human cancer. Modulation of alternative splicing allows cancer cells to adapt to hostile environments through production of specific mRNA variants. In particular, genotoxic stress exerted by chemotherapeutic drugs or irradiation strongly affects splicing of many genes. A key role in this aberrant regulation is played by the unbalanced expression of several splicing factors in cancer cells. Among them, the RNA-binding protein Sam68, which is overexpressed in various tumors, was shown to accumulate in nuclear foci of active transcription, together with other splicing regulators, and to affect splicing of target mRNAs in response to genotoxic stress. We suggest that subcellular redistribution of splicing factors is guided by changes in chromatin conformation elicited by DNA-damaging drugs. This event might represent an escape mechanism used by cancer cells to survive to genotoxic insults through expression of pro-survival, cancer-specific gene products.

Phosphorylation of eIF4E by MNKs supports protein synthesis, cell cycle progression and proliferation in prostate cancer cells.

Deregulation of the phosphatidyl inositol trisphosphate kinase/AKT/mammalian target of rapamycin (mTOR) and RAS/mitogen-activated protein kinase (MAPK)/MNK pathways frequently occurs in human prostate carcinomas (PCas) and leads to aberrant modulation of messenger RNA (mRNA) translation. We have investigated the relative contribution of these pathways to translational regulation and proliferation of PCa cells. MNK-dependent phosphorylation of eIF4E is elevated in DU145 cells, which have low basal levels of AKT/mTOR activity due to the expression of the tumor suppressor PTEN. In contrast, eIF4E phosphorylation is low in PC3 and LNCaP cells with mutated PTEN and constitutively active AKT/mTOR pathway, but it can be strongly induced through inhibition of mTOR activity by rapamycin or serum depletion. Remarkably, we found that inhibition of MNKs strongly reduced the polysomal recruitment of terminal oligopyrimidine messenger RNAs (TOP mRNAs), which are known targets of mTOR-dependent translational control. Pull-down assays of the eIF4F complex indicated that translation initiation was differently affected by inhibition of MNKs and mTOR. In addition, concomitant treatment with MNK inhibitor and rapamycin exerted additive effects on polysomal recruitment of TOP mRNAs and protein synthesis. The MNK inhibitor was more effective than rapamycin in blocking proliferation of PTEN-expressing cells, whereas combination of the two inhibitors suppressed cell cycle progression in both cell lines. Microarray analysis showed that MNK affected translation of mRNAs involved in cell cycle progression. Thus, our results indicate that a balance between the activity of the AKT/mTOR and the MAPK/MNK pathway in PCa cells maintains a defined translational level of specific mRNAs required for ribosome biogenesis, cell proliferation and stress response and might confer to these cells the ability to overcome negative insults.

The RNA-binding protein Sam68 is a multifunctional player in human cancer.

Src associated in mitosis, of 68 kDa (Sam68) is a KH domain RNA-binding protein that belongs to the signal transduction and activation of RNA family. Although ubiquitously expressed, Sam68 plays very specialized roles in different cellular environments. In most cells, Sam68 resides in the nucleus and is involved in several steps of mRNA processing, from transcription, to alternative splicing, to nuclear export. In addition, Sam68 translocates to the cytoplasm upon cell stimulation, cell cycle transitions or viral infections, where it takes part to signaling complexes and associates with the mRNA translation machinery. Recent evidence has linked Sam68 function to the onset and progression of endocrine tumors, such as prostate and breast carcinomas. Notably, all the biochemical activities reported for Sam68 seem to be implicated in carcinogenesis. Herein, we review the recent advancement in the knowledge of Sam68 function and regulation and discuss it in the frame of its participation to neoplastic transformation and tumor progression.

Alternative splicing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68.

Human cyclin D1 is expressed as two isoforms derived by alternate RNA splicing, termed D1a and D1b, which differ for the inclusion of intron 4 in the D1b mRNA. Both isoforms are frequently upregulated in human cancers, but cyclin D1b displays relatively higher oncogenic potential. The splicing factors that regulate alternative splicing of cyclin D1b remain unknown despite the likelihood that they contribute to cyclin D1 oncogenicity. In this study, we report that Sam68, an RNA-binding protein frequently overexpressed in prostate cancer cells, enhances splicing of cyclin D1b and supports its expression in prostate cancer cells. Chromatin immunoprecipitation and RNA coimmunoprecipitation experiments showed that Sam68 is recruited to the human CCND1 gene encoding cyclin D1 and that it binds to cyclin D1 mRNA. Transient overexpression and RNAi knockdown experiments indicated that Sam68 acts to enhance endogenous expression of cyclin D1b. Minigene reporter assays showed that Sam68 directly affected alternative splicing of CCND1 message, with a preference for the A870 allele that is known to favor cyclin D1b splicing. Sam68 interacted with the proximal region of intron 4, and its binding correlated inversely with recruitment of the spliceosomal component U1-70K. Sam68-mediated splicing was modulated by signal transduction pathways that elicit phosphorylation of Sam68 and regulate its affinity for CCND1 intron 4. Notably, Sam68 expression positively correlates with levels of cyclin D1b, but not D1a, in human prostate carcinomas. Our results identify Sam68 as the first splicing factor to affect CCND1 alternative splicing in prostate cancer cells, and suggest that increased levels of Sam68 may stimulate cyclin D1b expression in human prostate cancers.