Splicing targeting drugs highlight intron retention as an actionable vulnerability in advanced prostate cancer.

BACKGROUND: Advanced prostate cancer (PC) is characterized by insensitivity to androgen deprivation therapy and chemotherapy, resulting in poor outcome for most patients. Thus, advanced PC urgently needs novel therapeutic strategies. Mounting evidence points to splicing dysregulation as a hallmark of advanced PC. Moreover, pharmacologic inhibition of the splicing process is emerging as a promising option for this disease. METHOD: By using a representative androgen-insensitive PC cell line (22Rv1), we have investigated the genome-wide transcriptomic effects underlying the cytotoxic effects exerted by three splicing-targeting drugs: Pladienolide B, indisulam and THZ531. Bioinformatic analyses were performed to uncover the gene structural features underlying sensitivity to transcriptional and splicing regulation by these treatments. Biological pathways altered by these treatments were annotated by gene ontology analyses and validated by functional experiments in cell models. RESULTS: Although eliciting similar cytotoxic effects on advanced PC cells, Pladienolide B, indisulam and THZ531 modulate specific transcriptional and splicing signatures. Drug sensitivity is associated with distinct gene structural features, expression levels and cis-acting sequence elements in the regulated exons and introns. Importantly, we identified PC-relevant genes (i.e. EZH2, MDM4) whose drug-induced splicing alteration exerts an impact on cell survival. Moreover, computational analyses uncovered a widespread impact of splicing-targeting drugs on intron retention, with enrichment in genes implicated in pre-mRNA 3'-end processing (i.e. CSTF3, PCF11). Coherently, advanced PC cells displayed high sensitivity to a specific inhibitor of the cleavage and polyadenylation complex, which enhances the effects of chemotherapeutic drugs that are already in use for this cancer. CONCLUSIONS: Our study uncovers intron retention as an actionable vulnerability for advanced PC, which may be exploited to improve therapeutic management of this currently incurable disease.

The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer.

Alternative polyadenylation (APA) yields transcripts differing in their 3'-end, and its regulation is altered in cancer, including prostate cancer. Here we have uncovered a mechanism of APA regulation impinging on the interaction between the exonuclease XRN2 and the RNA-binding protein Sam68, whose increased expression in prostate cancer is promoted by the transcription factor MYC. Genome-wide transcriptome profiling revealed a widespread impact of the Sam68/XRN2 complex on APA. XRN2 promotes recruitment of Sam68 to its target transcripts, where it competes with the cleavage and polyadenylation specificity factor for binding to strong polyadenylation signals at distal ends of genes, thus promoting usage of suboptimal proximal polyadenylation signals. This mechanism leads to 3' untranslated region shortening and translation of transcripts encoding proteins involved in G1/S progression and proliferation. Thus, our findings indicate that the APA program driven by Sam68/XRN2 promotes cell cycle progression and may represent an actionable target for therapeutic intervention.

The androgen receptor couples promoter recruitment of RNA processing factors to regulation of alternative polyadenylation at the 3' end of transcripts.

Prostate cancer (PC) relies on androgen receptor (AR) signaling. While hormonal therapy (HT) is efficacious, most patients evolve to an incurable castration-resistant stage (CRPC). To date, most proposed mechanisms of acquired resistance to HT have focused on AR transcriptional activity. Herein, we uncover a new role for the AR in alternative cleavage and polyadenylation (APA). Inhibition of the AR by Enzalutamide globally regulates APA in PC cells, with specific enrichment in genes related to transcription and DNA topology, suggesting their involvement in transcriptome reprogramming. AR inhibition selects promoter-distal polyadenylation sites (pAs) enriched in cis-elements recognized by the cleavage and polyadenylation specificity factor (CPSF) complex. Conversely, promoter-proximal intronic pAs relying on the cleavage stimulation factor (CSTF) complex are repressed. Mechanistically, Enzalutamide induces rearrangement of APA subcomplexes and impairs the interaction between CPSF and CSTF. AR inhibition also induces co-transcriptional CPSF recruitment to gene promoters, predisposing the selection of pAs depending on this complex. Importantly, the scaffold CPSF160 protein is up-regulated in CRPC cells and its depletion represses HT-induced APA patterns. These findings uncover an unexpected role for the AR in APA regulation and suggest that APA-mediated transcriptome reprogramming represents an adaptive response of PC cells to HT.

DNA Damage Regulates the Functions of the RNA Binding Protein Sam68 through ATM-Dependent Phosphorylation.

Cancer cells frequently exhibit dysregulation of the DNA damage response (DDR), genomic instability, and altered RNA metabolism. Recent genome-wide studies have strongly suggested an interaction between the pathways involved in the cellular response to DDR and in the regulation of RNA metabolism, but the molecular mechanism(s) involved in this crosstalk are largely unknown. Herein, we found that activation of the DDR kinase ATM promotes its interaction with Sam68, leading to phosphorylation of this multifunctional RNA binding protein (RBP) on three residues: threonine 61, serine 388 and serine 390. Moreover, we demonstrate that ATM-dependent phosphorylation of threonine 61 promotes the function of Sam68 in the DDR pathway and enhances its RNA processing activity. Importantly, ATM-mediated phosphorylation of Sam68 in prostate cancer cells modulates alternative polyadenylation of transcripts that are targets of Sam68, supporting the notion that the ATM-Sam68 axis exerts a multifaceted role in the response to DNA damage. Thus, our work validates Sam68 as an ATM kinase substrate and uncovers an unexpected bidirectional interplay between ATM and Sam68, which couples the DDR pathway to modulation of RNA metabolism in response to genotoxic stress.

Non-Coding RNAs and Splicing Activity in Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) are the most common tumors in adolescent and young men. Recently, genome-wide studies have made it possible to progress in understanding the molecular mechanisms underlying the development of tumors. It is becoming increasingly clear that aberrant regulation of RNA metabolism can drive tumorigenesis and influence chemotherapeutic response. Notably, the expression of non-coding RNAs as well as specific splice variants is deeply deregulated in human cancers. Since these cancer-related RNA species are considered promising diagnostic, prognostic and therapeutic targets, understanding their function in cancer development is becoming a major challenge. Here, we summarize how the different expression of RNA species repertoire, including non-coding RNAs and protein-coding splicing variants, impacts on TGCTs' onset and progression and sustains therapeutic resistance. Finally, the role of transcription-associated R-loop misregulation in the maintenance of genomic stability in TGCTs is also discussed.

Oncogenic dysregulation of pre-mRNA processing by protein kinases: challenges and therapeutic opportunities.

Alternative splicing and polyadenylation represent two major steps in pre-mRNA-processing, which ensure proper gene expression and diversification of human transcriptomes. Deregulation of these processes contributes to oncogenic programmes involved in the onset, progression and evolution of human cancers, which often result in the acquisition of resistance to existing therapies. On the other hand, cancer cells frequently increase their transcriptional rate and develop a transcriptional addiction, which imposes a high stress on the pre-mRNA-processing machinery and establishes a therapeutically exploitable vulnerability. A prominent role in fine-tuning pre-mRNA-processing mechanisms is played by three main families of protein kinases: serine arginine protein kinase (SRPK), CDC-like kinase (CLK) and cyclin-dependent kinase (CDK). These kinases phosphorylate the RNA polymerase, splicing factors and regulatory proteins involved in cleavage and polyadenylation of the nascent transcripts. The activity of SRPKs, CLKs and CDKs can be altered in cancer cells, and their inhibition was shown to exert anticancer effects. In this review, we describe key findings that have been reported on these topics and discuss challenges and opportunities of developing therapeutic approaches targeting splicing factor kinases.

Sempervirine inhibits RNA polymerase I transcription independently from p53 in tumor cells.

In the search of small molecules that can target MDM2/p53 pathway in testicular germ cell tumors (TGCTs), we identified sempervirine (2,3,4,13-tetrahydro-1H-benz[g]indolo[2,3-a]quinolizin-6-ium), an alkaloid of Gelsemium sempervirens, that has been previously proposed as an inhibitor of MDM2 that targets p53-wildtype (wt) tumor cells. We found that sempervirine not only affects cell growth of p53-wt cancer cells, but it is also active in p53-mutated and p53-null cells by triggering p53-dependent and independent pathways without affecting non-transformed cells. To understand which mechanism/s could be activated both in p53-wt and -null cells, we found that sempervirine induced nucleolar remodeling and nucleolar stress by reducing protein stability of RPA194, the catalytic subunit of RNA polymerase I, that led to rRNA synthesis inhibition and to MDM2 block. As shown for other cancer cell models, MDM2 inhibition by nucleolar stress downregulated E2F1 protein levels both in p53-wt and p53-null TGCT cells with the concomitant upregulation of unphosphorylated pRb. Finally, we show that sempervirine is able to enter the nucleus and accumulates within the nucleolus where it binds rRNA without causing DNA damage. Our results identify semperivirine as a novel rRNA synthesis inhibitor and indicate this drug as a non-genotoxic anticancer small molecule.

Splicing Dysregulation as Oncogenic Driver and Passenger Factor in Brain Tumors.

Brain tumors are a heterogeneous group of neoplasms ranging from almost benign to highly aggressive phenotypes. The malignancy of these tumors mostly relies on gene expression reprogramming, which is frequently accompanied by the aberrant regulation of RNA processing mechanisms. In brain tumors, defects in alternative splicing result either from the dysregulation of expression and activity of splicing factors, or from mutations in the genes encoding splicing machinery components. Aberrant splicing regulation can generate dysfunctional proteins that lead to modification of fundamental physiological cellular processes, thus contributing to the development or progression of brain tumors. Herein, we summarize the current knowledge on splicing abnormalities in brain tumors and how these alterations contribute to the disease by sustaining proliferative signaling, escaping growth suppressors, or establishing a tumor microenvironment that fosters angiogenesis and intercellular communications. Lastly, we review recent efforts aimed at developing novel splicing-targeted cancer therapies, which employ oligonucleotide-based approaches or chemical modulators of alternative splicing that elicit an impact on brain tumor biology.

c-MYC empowers transcription and productive splicing of the oncogenic splicing factor Sam68 in cancer.

The splicing factor Sam68 is upregulated in many human cancers, including prostate cancer (PCa) where it promotes cell proliferation and survival. Nevertheless, in spite of its frequent upregulation in cancer, the mechanism(s) underlying its expression are largely unknown. Herein, bioinformatics analyses identified the promoter region of the Sam68 gene (KHDRBS1) and the proto-oncogenic transcription factor c-MYC as a key regulator of Sam68 expression. Upregulation of Sam68 and c-MYC correlate in PCa patients. c-MYC directly binds to and activates the Sam68 promoter. Furthermore, c-MYC affects productive splicing of the nascent Sam68 transcript by modulating the transcriptional elongation rate within the gene. Importantly, c-MYC-dependent expression of Sam68 is under the tight control of external cues, such as androgens and/or mitogens. These findings uncover an unexpected coordination of transcription and splicing of Sam68 by c-MYC, which may represent a key step in PCa tumorigenesis.

The Splicing Factor PTBP1 Promotes Expression of Oncogenic Splice Variants and Predicts Poor Prognosis in Patients with Non-muscle-Invasive Bladder Cancer.

Purpose: Non-muscle-invasive bladder cancer (NMIBC) is a malignant disease characterized by high heterogeneity, which corresponds to dysregulated gene expression and alternative splicing (AS) profiles. Bioinformatics analyses of splicing factors potentially linked to bladder cancer progression identified the heterogeneous nuclear ribonucleoprotein I (i.e., PTBP1) as candidate. This study aimed at investigating whether PTBP1 expression associates with clinical outcome in patients with NMIBC.Experimental Design: A cohort of 152 patients presenting with primary NMIBC (pTa-pT1) was enrolled. Primary NMIBCs were assessed for PTBP1 expression by IHC, and the results were correlated with clinical data using Kaplan-Meier curves and Cox regression analyses. Cell proliferation and survival assays were performed to assess the function of PTBP1. Furthermore, the impact of PTBP1 on the AS pattern of specific bladder cancer-related genes was investigated in cancer cell lines and in patients' specimens.Results: Public datasets querying highlighted a positive correlation between PTBP1 expression and NMIBC progression, which was then confirmed by IHC analysis. High PTBP1 expression was associated with worse clinical outcome in terms of incidence of tumor relapse and survival in patients with NMIBC. Interestingly, downregulation of PTBP1 in bladder cancer cell lines affected prosurvival features. Accordingly, PTBP1 modulated AS of bladder cancer-related genes in cell lines and patient's specimens.Conclusions: PTBP1 expression correlates with disease progression, poor prognosis, and worse survival in patients with NMIBC. Downregulation of PTBP1 expression affects prosurvival features of bladder cancer cells and modulates AS of genes with relevance for bladder cancer, suggesting its role as an outcome-predictor in this disease. Clin Cancer Res; 24(21); 5422-32. ©2018 AACR.

Alternative polyadenylation of ZEB1 promotes its translation during genotoxic stress in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extremely poor prognosis. The standard chemotherapeutic drug, gemcitabine, does not offer significant improvements for PDAC management due to the rapid acquisition of drug resistance by patients. Recent evidence indicates that epithelial-to-mesenchymal transition (EMT) of PDAC cells is strictly associated to early metastasization and resistance to chemotherapy. However, it is not exactly clear how EMT is related to drug resistance or how chemotherapy influences EMT. Herein, we found that ZEB1 is the only EMT-related transcription factor that clearly segregates mesenchymal and epithelial PDAC cell lines. Gemcitabine treatment caused upregulation of ZEB1 protein through post-transcriptional mechanisms in mesenchymal PDAC cells within a context of global inhibition of protein synthesis. The increase in ZEB1 protein correlates with alternative polyadenylation of the transcript, leading to shortening of the 3' untranslated region (UTR) and deletion of binding sites for repressive microRNAs. Polysome profiling indicated that shorter ZEB1 transcripts are specifically retained on the polysomes of PDAC cells during genotoxic stress, while most mRNAs, including longer ZEB1 transcripts, are depleted. Thus, our findings uncover a novel layer of ZEB1 regulation through 3'-end shortening of its transcript and selective association with polysomes under genotoxic stress, strongly suggesting that PDAC cells rely on upregulation of ZEB1 protein expression to withstand hostile environments.

Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method.

RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of a RBP to specific regions of its target RNAs in adherent human cell lines.

Regulation of BCL-X splicing reveals a role for the polypyrimidine tract binding protein (PTBP1/hnRNP I) in alternative 5' splice site selection.

Alternative splicing (AS) modulates many physiological and pathological processes. For instance, AS of the BCL-X gene balances cell survival and apoptosis in development and cancer. Herein, we identified the polypyrimidine tract binding protein (PTBP1) as a direct regulator of BCL-X AS. Overexpression of PTBP1 promotes selection of the distal 5' splice site in BCL-X exon 2, generating the pro-apoptotic BCL-Xs splice variant. Conversely, depletion of PTBP1 enhanced splicing of the anti-apoptotic BCL-XL variant. In vivo cross-linking experiments and site-directed mutagenesis restricted the PTBP1 binding site to a polypyrimidine tract located between the two alternative 5' splice sites. Binding of PTBP1 to this site was required for its effect on splicing. Notably, a similar function of PTBP1 in the selection of alternative 5' splice sites was confirmed using the USP5 gene as additional model. Mechanistically, PTBP1 displaces SRSF1 binding from the proximal 5' splice site, thus repressing its selection. Our study provides a novel mechanism of alternative 5' splice site selection by PTBP1 and indicates that the presence of a PTBP1 binding site between two alternative 5' splice sites promotes selection of the distal one, while repressing the proximal site by competing for binding of a positive regulator.

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-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.

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.