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Transcription-associated cyclin-dependent kinases (CDKs) regulate the transcription cycle through sequential phosphorylation of RNA polymerase II (RNAPII). Herein, we report that dual inhibition of the highly homologous CDK12 and CDK13 impairs splicing of a subset of promoter-proximal introns characterized by weak 3′ splice sites located at larger distance from the branchpoint. Nascent transcript analysis indicated that these introns are selectively retained upon pharmacological inhibition of CDK12/13 with respect to downstream introns of the same pre-mRNAs. Retention of these introns was also triggered by pladienolide B (PdB), an inhibitor of the U2 small nucelar ribonucleoprotein (snRNP) factor SF3B1 that recognizes the branchpoint. CDK12/13 activity promotes the interaction of SF3B1 with RNAPII phosphorylated on Ser2, and disruption of this interaction by treatment with the CDK12/13 inhibitor THZ531 impairs the association of SF3B1 with chromatin and its recruitment to the 3′ splice site of these introns. Furthermore, by using suboptimal doses of THZ531 and PdB, we describe a synergic effect of these inhibitors on intron retention, cell cycle progression and cancer cell survival. These findings uncover a mechanism by which CDK12/13 couple RNA transcription and processing, and suggest that combined inhibition of these kinases and the spliceosome represents an exploitable anticancer approach.

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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.

PDF file - 146K, Effect of TAT-SNAG peptide on migration, invasion and proliferation of p53-null HCT116 cells

PDF file - 76K

PDF file - 153K, Effect of LSD1 silencing on migration and invasion of HCT116 cells

PDF file - 5MB, Effect of Slug silencing on invasion and EMT markers expression of HTLA230 cells

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.

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.

Homologous recombination and chromosome segregation in meiosis rely on the timely expression of two splice variants of the endonuclease SPO11, named α and β, which respectively skip or include exon 2. However, in spite of its physiological importance, the mechanism underlying Spo11 alternative splicing in meiosis is still unknown. By screening the activity of factors that are predicted to bind the alternatively spliced region of Spo11, we identified hnRNPH as a key regulator of SPO11α splicing in mouse spermatocytes. Although hnRNPH was not upregulated in meiosis concomitantly with the switch in splicing, its recruitment to Spo11 pre-mRNA was favored by selective modulation of RNA polymerase II (RNAPII) phosphorylation and processivity in proximity of exon 2. The hnRNPH binding sites were localized near those of splicing factors that promote SPO11β splicing, suggesting that hnRNPH favors exon 2 skipping by competing out positive regulators. Indeed, hnRNPH binds proximal to a consensus motif for Sam68, a positive regulator of SPO11β splicing in vitro and in vivo, and it interferes with Sam68 binding to the Spo11 pre-mRNA. Thus, our work reveals that modulation of RNAPII dynamics in concert with hnRNPH recruitment exerts a combinatorial control of the timely regulated Spo11 splicing during meiosis.

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.

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.

Significance High-risk neuroblastomas (NBs) show undifferentiated/poorly differentiated morphology as a distinctive feature. We have identified the transcription factor ZNF281 as a factor that can counteract the neuronal differentiation of primary neurons in culture and NB cells. The expression of ZNF281 is inhibited by TAp73 and promoted by MYCN. In turn, ZNF281 inhibits the expression of GDNF and NRP2, two proteins associated with neuronal differentiation. In patients with NB, the expression of ZNF281 is higher in high-risk patients and is associated with worse prognosis. Understanding the molecular mechanisms that regulate neuronal differentiation is relevant for the identification of defects in this process that underlie the development of tumors such as NB, in which an aberrant differentiation arrest has occurred., Derangement of cellular differentiation because of mutation or inappropriate expression of specific genes is a common feature in tumors. Here, we show that the expression of ZNF281, a zinc finger factor involved in several cellular processes, decreases during terminal differentiation of murine cortical neurons and in retinoic acid-induced differentiation of neuroblastoma (NB) cells. The ectopic expression of ZNF281 inhibits the neuronal differentiation of murine cortical neurons and NB cells, whereas its silencing causes the opposite effect. Furthermore, TAp73 inhibits the expression of ZNF281 through miR34a. Conversely, MYCN promotes the expression of ZNF281 at least in part by inhibiting miR34a. These findings imply a functional network that includes p73, MYCN, and ZNF281 in NB cells, where ZNF281 acts by negatively affecting neuronal differentiation. Array analysis of NB cells silenced for ZNF281 expression identified GDNF and NRP2 as two transcriptional targets inhibited by ZNF281. Binding of ZNF281 to the promoters of these genes suggests a direct mechanism of repression. Bioinformatic analysis of NB datasets indicates that ZNF281 expression is higher in aggressive, undifferentiated stage 4 than in localized stage 1 tumors supporting a central role of ZNF281 in affecting the differentiation of NB. Furthermore, patients with NB with high expression of ZNF281 have a poor clinical outcome compared with low-expressors. These observations suggest that ZNF281 is a controller of neuronal differentiation that should be evaluated as a prognostic marker in NB.

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.

The use of Ginkgo biloba leaf extract as nutraceutical is becoming increasingly common. As a consequence, the definition of a reliable toxicological profile is a priority for its safe utilization. Recently, contrasting data have been reported on the carcinogenic potential of Ginkgo biloba extract in rodent liver. We measured viability, Reactive Oxygen Species (ROS), apoptosis, colony-forming efficiency, genotoxicity by comet assay, and gene expression changes associated with hepato-carcinogenicity in human cells of hepatic origin (HepG2 and THLE-2) treated with different concentrations (0.0005–1.2 mg/mL) of Ginkgoselect®Plus. Our analyses highlighted a decrease of cell viability, not due to apoptosis, after treatment with high doses of the extract, which was likely due to ROS generation by a chemical reaction between extract polyphenols and some components of the culture medium. Comet assay did not detect genotoxic effect at any extract concentration. Finally, the array analysis detected a slight decrease in the expression of only one gene (IGFBP3) in Ginkgo-treated THLE-2 cells as opposed to changes in 28 genes in Aflatoxin B1 treated-cells. In conclusion, our results did not detect any significant genotoxic or biologically relevant cytotoxic effects and gross changes in gene expression using the Ginkgo extract in the hepatic cells tested. © 2017 The Authors

ZNF281 is a zinc-finger factor involved in the control of cellular stemness and epithelial-mesenchymal transition (EMT). Here, we report that ZNF281 expression increased after genotoxic stress caused by DNA-damaging drugs. Comet assays demonstrated that DNA repair was delayed in cells silenced for the expression of ZNF281 and treated with etoposide. Furthermore, the expression of 10 DNA damage response genes was downregulated in cells treated with etoposide and silenced for ZNF281. In line with this finding, XRCC2 and XRCC4, two genes that take part in homologous recombination and non-homologous end joining, respectively, were transcriptionally activated by ZNF281 through a DNA-binding-dependent mechanism, as demonstrated by luciferase assays and Chromatin crosslinking ImmunoPrecipitation experiments. c-Myc, which also binds to the promoters of XRCC2 and XRCC4, was unable to promote their transcription or to modify ZNF281 activity. Of interest, bioinformatic analysis of 1971 breast cancer patients disclosed a significant correlation between the expression of ZNF281 and that of XRCC2. In summary, our data highlight, for the first time, the involvement of ZNF281 in the cellular response to genotoxic stress through the control exercised on the expression of genes that act in different repair mechanisms.

Neuroblastoma (NB) is the most common extracranial childhood tumor classified in five stages (1, 2, 3, 4 and 4S), two of which (3 and 4) identify chemotherapy-resistant, highly aggressive disease. High-risk NB frequently displays MYCN amplification, mutations in ALK and ATRX, and genomic rearrangements in TERT genes. These NB subtypes are also characterized by reduced susceptibility to programmed cell death induced by chemotherapeutic drugs. The latter feature is a major cause of failure in the treatment of advanced NB patients. Thus, proper reactivation of apoptosis or of other types of programmed cell death pathways in response to treatment is relevant for the clinical management of aggressive forms of NB. In this short review, we will discuss the most relevant genomic rearrangements that define high-risk NB and the role that destabilization of p53 and p73 can have in NB aggressiveness. In addition, we will propose a strategy to stabilize p53 and p73 by using specific inhibitors of their ubiquitin-dependent degradation. Finally, we will introduce necroptosis as an alternative strategy to kill NB cells and increase tumor immunogenicity.

Tumor progression to metastasis is a complex, sequential process that requires proliferation, resistance to apoptosis, motility and invasion to colonize at distant sites. The acquisition of these features implies a phenotypic plasticity by tumor cells that must adapt to different conditions by modulating several signaling pathways1 during the journey to the final site of metastasis. Several transcription factors and microRNA play a role in tumor progression, but less is known about the control of their expression during this process. Here, we demonstrate by ectopic expression and gene silencing that the proto-oncogene c-Myb activates the expression of the 5 members of miR200 family (miR200b, miR200a, miR429, miR200c and miR141) that are involved in the control of epithelial-mesenchymal transition (EMT) and metastasis in many types of cancers. Transcriptional activation of miR200 by c-Myb occurs through binding to myb binding sites located in the promoter regions of miR200 genes on human chromosomes 1 and 12. Furthermore, when c-Myb and the transcriptional repressor ZEB1 are co-expressed, as at the onset EMT, the repression by ZEB1 prevails over the activation by c-Myb, and the expression of miR200 is inhibited. We also demonstrate that during EMT induced by TGF-β, the promoters of miR200 genes are methylated, and their transcription is repressed regardless of the presence of repressors such as ZEB1 and activators such as c-Myb. Finally, we find a correlation between the expression of c-Myb and that of four out of 5 miR200 in a data set of 207 breast cancer patients. © 2013 Landes Bioscience.

The process of epithelial–mesenchymal transition (EMT) which is required for cancer cell invasion is regulated by a family of E-box–binding transcription repressors, which include Snail (SNAIL1) and Slug (SNAI2). Snail appears to repress the expression of the EMT marker E-cadherin by epigenetic mechanisms dependent on the interaction of its N-terminal SNAG domain with chromatin-modifying proteins including lysine-specific demethylase 1 (LSD1/KDM1A). We assessed whether blocking Snail/Slug-LSD1 interaction by treatment with Parnate, an enzymatic inhibitor of LSD1, or TAT-SNAG, a cell-permeable peptide corresponding to the SNAG domain of Slug, suppresses the motility and invasiveness of cancer cells of different origin and genetic background. We show here that either treatment blocked Slug-dependent repression of the E-cadherin promoter and inhibited the motility and invasion of tumor cell lines without any effect on their proliferation. These effects correlated with induction of epithelial and repression of mesenchymal markers and were phenocopied by LSD1 or Slug downregulation. Parnate treatment also inhibited bone marrow homing/engraftment of Slug-expressing K562 cells. Together, these studies support the concept that targeting Snail/Slug-dependent transcription repression complexes may lead to the development of novel drugs selectively inhibiting the invasive potential of cancer cells. Cancer Res; 73(1); 235–45. ©2012 AACR.