Histone Marks-Dependent Effect on Alternative Splicing: New Perspectives for Targeted Splicing Modulation in Cancer?

Alternative splicing (AS) is a tightly regulated mechanism that generates the complex human proteome from a small number of genes. Cis-regulatory RNA motifs in exons and introns control AS, recruiting positive and negative trans-acting splicing regulators. At a higher level, chromatin affects splicing events. Growing evidence indicates that the popular histone code hypothesis can be extended to RNA-level processes, such as AS. In addition to nucleosome positioning, which can generate transcriptional barriers to shape the final splicing outcome, histone post-translational modifications can contribute to the detailed regulation of single exon inclusion/exclusion. A histone-based system can identify alternatively spliced chromatin stretches, affecting RNAPII elongation locally or recruiting splicing components via adaptor complexes. In tumor cells, several mechanisms trigger misregulated AS events and produce cancer-associated transcripts. On a genome-wide level, aberrant AS can be the consequence of dysfunctional epigenetic splicing code, including altered enrichment in histone post-translational modifications. This review describes the main findings related to the effect of histone modifications and variants on splicing outcome and how a dysfunctional epigenetic splicing code triggers aberrant AS in cancer. In addition, it highlights recent advances in programmable DNA-targeting technologies and their possible application for AS targeted epigenetic modulation

Transcription Factors in Cancer: When Alternative Splicing Determines Opposite Cell Fates

Alternative splicing (AS) is a finely regulated mechanism for transcriptome and proteome diversification in eukaryotic cells. Correct balance between AS isoforms takes part in molecular mechanisms that properly define spatiotemporal and tissue specific transcriptional programs in physiological conditions. However, several diseases are associated to or even caused by AS alterations. In particular, multiple AS changes occur in cancer cells and sustain the oncogenic transcriptional program. Transcription factors (TFs) represent a key class of proteins that control gene expression by direct binding to DNA regulatory elements. AS events can generate cancer-associated TF isoforms with altered activity, leading to sustained proliferative signaling, differentiation block and apoptosis resistance, all well-known hallmarks of cancer. In this review, we focus on how AS can produce TFs isoforms with opposite transcriptional activities or antagonistic functions that severely impact on cancer biology. This summary points the attention to the relevance of the analysis of TFs splice variants in cancer, which can allow patients stratification despite the presence of interindividual genetic heterogeneity. Recurrent TFs variants that give advantage to specific cancer types not only open the opportunity to use AS transcripts as clinical biomarkers but also guide the development of new anti-cancer strategies in personalized medicine

Tackling Alzheimer's Disease with Existing Drugs:A Promising Strategy for Bypassing Obstacles

The unmet need for the development of effective drugs to treat Alzheimer's disease has been steadily growing, representing a major challenge in drug discovery. In this con-text, drug repurposing, namely the identification of novel therapeutic indications for approved or investigational compounds, can be seen as an attractive attempt to obtain new medications reducing both the time and the economic burden usually required for research and development programs. In the last years, several classes of drugs have evidenced promising beneficial effects in neurodegenerative diseases, and for some of them, preliminary clinical trials have been started. This review aims to illustrate some of the most recent examples of drugs reprofiled for Alzheimer’s disease, considering not only the finding of new uses for existing drugs but also the new hypotheses on disease pathogenesis that could promote previ-ously unconsidered therapeutic regimens. Moreover, some examples of structural modifica-tions performed on existing drugs in order to obtain multifunctional compounds will also be described

NF-Y loss triggers p53 stabilization and apoptosis in HPV18-positive cells by affecting E6 transcription

The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NFYA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response

Concurrent inhibition of enzymatic activity and NF-Y-mediated transcription of Topoisomerase-IIα by bis-DemethoxyCurcumin in cancer cells

Topoisomerase-IIa (TOP2A) enzyme is essential for cell viability due to its fundamental role in DNA metabolism and in chromatin organization during interphase and mitosis. TOP2A expression is finely regulated at the transcriptional level through the binding of the CCAAT-transcription factor NF-Y to its promoter. Overexpression and/or amplification of TOP2A have been observed in many types of cancers. For this reason, TOP2A is the target of the most widely successful drugs in cancer chemotherapy, such as TOP2A poisons, which stabilize TOP2A-DNA cleavage complexes and create DSBs, leading to chromosome damage and cell death. We previously reported that the Curcumin-derivative bis-DemethoxyCurcumin (bDMC) is an anti-proliferative agent that inhibits cell growth by concomitant G1/S and G2/M arrest. Here we showed that bDMC irreversibly induces DSBs in cancer cells, but not in normal cells, by targeting TOP2A activity and expression. TOP2A ablation by siRNA corroborates its contribution to apoptosis induced by bDMC. Short-term exposure to bDMC induces retention of TOP2A-DNA intermediates, while longer exposure inhibits TOP2A transcription by affecting expression and sub-cellular localization of NF-Y subunits. ChIP analysis highlighted reduced recruitment of NF-Y to TOP2A regulatory regions, concomitantly to histone deacetylation and decreased gene transcription. Our findings suggest that the dual activity of bDMC on TOP2A represents a novel therapeutic strategy to induce persistent apoptosis in cancer cells and identify NF-Y regulation as a promising approach in anti-cancer therapy

Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications

Potent Anti-Cancer Properties of Phthalimide-Based Curcumin Derivatives on Prostate Tumor Cells

Metastatic castration-resistant prostate cancer is commonly treated with chemotherapy, whose effect is less than satisfactory. This raised the need for novel agents for the treatment of prostate cancer. In the present study, five phthalimide-based curcumin derivatives were synthesized and completely characterized to assess improved stability, pharmacodynamics, and radical scavenging ability. To investigate the potential application in anti-cancer therapy, the anti-proliferative activity of the synthesized molecules was determined on aggressive prostate tumor cells. We demonstrated that the K3F21 derivative has increased potency compared to curcumin, in terms of GI50, anti-proliferative and anti-migrating activities. K3F21 inhibits anchorage-dependent and -independent growth of prostate cancer cells by altering the expression of key genes controlling cell proliferation, such as Cylins D1, B1 and B2, and apoptosis, among which Puma, Noxa, and Bcl-2 family members. Finally, the anti-cancer activity of K3F21 was demonstrated by the analysis of cancer-associated PI3K/AKT, ERK, and p38 signaling pathways

An autoregulatory loop controls the expression of the transcription factor NF-Y

The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer. Here we demonstrate that NF-Y subunits expression is finely regulated through transcriptional and post-translational mechanisms thus allowing control over basal expression levels. NF-Y negatively regulates the transcription of the genes encoding for its subunits. DNA pull-down/affinity purification assay coupled with Mass Spectrometry identified putative co-regulators, such as Lamin A, involved in NF-YA gene transcription level. We also evidentiate how the stability of the complex is severely affected by the absence of one subunit. Our results identified for the first time one of the mechanisms responsible for NF-Y expression, which may be involved in the aberrant expression and activity observed in tumor cells and other pathological conditions

NF-YA splice variants have different roles on muscle differentiation

The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors