Mutant p53 Protein and the Hippo Transducers YAP and TAZ: A Critical Oncogenic Node in Human Cancers.

p53 protein is a well-known tumor suppressor factor that regulates cellular homeostasis. As it has several and key functions exerted, p53 is known as "the guardian of the genome" and either loss of function or gain of function mutations in the TP53 coding protein sequence are involved in cancer onset and progression. The Hippo pathway is a key regulator of developmental and regenerative physiological processes but if deregulated can induce cell transformation and cancer progression. The p53 and Hippo pathways exert a plethora of fine-tuned functions that can apparently be in contrast with each other. In this review, we propose that the p53 status can affect the Hippo pathway function by switching its outputs from tumor suppressor to oncogenic activities. In detail, we discuss: (a) the oncogenic role of the protein complex mutant p53/YAP; (b) TAZ oncogenic activation mediated by mutant p53;

MicroRNA (miRNA)-mediated interaction between leukemia/lymphoma-related factor (LRF) and alternative splicing factor/splicing factor 2 (ASF/SF2) affects mouse embryonic fibroblast senescence and apoptosis.

Leukemia/lymphoma-related factor (LRF) is a transcriptional repressor, which by recruiting histone deacetylases specifically represses p19/ARF expression, thus behaving as an oncogene. Conversely, in mouse embryonic fibroblasts (MEF), LRF inhibition causes aberrant p19ARF up-regulation resulting in proliferative defects and premature senescence. We have recently shown that LRF is controlled by microRNAs. Here we show that LRF acts on MEF proliferation and senescence/apoptosis by repressing miR-28 and miR-505, revealing a regulatory circuit where microRNAs (miRNAs) work both upstream and downstream of LRF. By analyzing miRNA expression profiles of MEF transfected with LRF-specific short interfering RNAs, we found that miR-28 and miR-505 are modulated by LRF. Both miRNAs are predicted to target alternative splicing factor/splicing factor 2 (ASF/SF2), a serine/arginine protein essential for cell viability. In vertebrates, loss or inactivation of ASF/SF2 may result in genomic instability and induce G(2) cell cycle arrest and apoptosis. We showed that miR-28 and miR-505 modulate ASF/SF2 by directly binding ASF/SF2 3'-UTR. Decrease in LRF causes a decrease in ASF/SF2, which depends on up-regulation of miR-28 and miR-505. Alteration of each of the members of the LRF/miR-28/miR-505/ASF/SF2 axis affects MEF proliferation and the number of senescent and apoptotic cells. Consistently, the axis is coordinately modulated as cell senescence increases with passages in MEF culture. In conclusion, we show that LRF-dependent miRNAs miR-28 and miR-505 control MEF proliferation and survival by targeting ASF/SF2 and suggest a central role of LRF-related miRNAs, in addition to the role of LRF-dependent p53 control, in cellular homeostasis.

CircRNAs: role in human diseases and potential use as biomarkers.

Circular RNAs (circRNAs) are a class of endogenous RNAs characterized by a covalent loop structure. In comparison to other types of RNAs, the abundance of circRNAs is relatively low but due to the circular configuration, their stability is very high. In addition, circRNAs display high degree of tissue specificity. The sponging activity of circRNAs toward microRNAs is the best-described mode of action of circRNAs. However, the ability of circRNAs to bind with specific proteins, as well as to encode short proteins, propose alternative functions. This review introduces the biogenesis of circRNAs and summarizes the roles played by circRNAs in human diseases. These include examples of their functional roles in several organ-specific cancers, such as head and neck and breast and lung cancers. In addition, we review potential functions of circRNAs in diabetes, cardiovascular, and neurodegenerative diseases. Recently, a growing number of studies have demonstrated involvement of circRNAs in a wide spectrum of signaling molecular pathways, but at the same time many different and controversial views on circRNAs role and function are emerging. We conclude by offering cellular homeostasis generated by networks comprising circular RNAs, other non-coding RNAs and RNA-binding proteins. Accordingly, it is predictable that circRNAs, due to their highly stable nature and remarkable tissue specificity, will emerge as reliable biomarkers of disease course and treatment efficacy.

Circulating microRNAs as biomarkers to assist the management of the malignant germ-cell-tumour subtype choriocarcinoma.

Germ-cell-tumours (GCTs) are heterogeneous and management is complex. The current conventional biomarkers, alpha-fetoprotein and human-chorionic-gonadotropin (HCG), have limited utility for diagnosis/follow-up as secretion is restricted to specific malignant-GCT subtypes and long half-life can make interpretation and clinical decision-making challenging. We sought to identify circulating microRNAs that reflected choriocarcinoma disease activity more accurately than HCG in a metastatic primary mediastinal nonseminomatous-GCT (PMNSGCT) case with elevated diagnostic serum HCG (>250,000 U/L), consistent with pure choriocarcinoma. We undertook comprehensive microRNA profiling (n = 754 microRNAs) using two 384-well TaqMan Low-Density-Array cards in 16 serum samples; 10 from PMNSGCT diagnosis/follow-up and six controls. Key findings underwent confirmatory qRT-PCR. We identified a serum panel of choriocarcinoma-specific 'chromosome-19-microRNA-cluster' (C19MC) microRNAs that were highly elevated at diagnosis but fell rapidly on treatment and normalised before the second full chemotherapy course. We also re-confirmed serum elevation of the previously identified malignant-GCT marker miR-371a-3p at diagnosis. These circulating microRNA markers reflected choriocarcinoma disease activity more accurately than serum HCG and real-time knowledge would have assisted clinical decision-making. With further study, these microRNA markers will facilitate future management of such patients and are likely to result in improved outcomes.

The circRNA-microRNA code: emerging implications for cancer diagnosis and treatment.

Circular RNAs (circRNAs) comprise an emerging new class of endogenous RNAs expressed abundantly by the eukaryotic transcriptome. They are characterized by a covalently closed loop structure, resulting in RNA molecules that are more stable than linear RNAs. A growing number of studies indicate that circRNAs play critical roles in human diseases and show great potential as biomarkers and therapeutic targets. The molecular events determined by circRNA activity, the circRNA code, involve other types of noncoding RNA molecules, primarily microRNAs, long noncoding RNAs, and RNA-binding proteins. Herein, we mainly focus on the circRNA-microRNA code, showing how this relationship impacts the regulation of gene expression in cancer. The emerging roles for circRNAs in oncogenic pathways highlight new perspectives for the detailed molecular dissection of cancer pathogenesis and, at the same time, offer new opportunities to design innovative therapeutic strategies. Here, we review recent research advancements in understanding the biogenesis, molecular functions, and significance of circRNAs in cancer diagnosis and treatment.

A structure-guided molecular chaperone approach for restoring the transcriptional activity of the p53 cancer mutant Y220C.

Aim: The p53 cancer mutation Y220C creates a conformationally unstable protein with a unique elongated surface crevice that can be targeted by molecular chaperones. We report the structure-guided optimization of the carbazole-based stabilizer PK083. Materials & methods: Biophysical, cellular and x-ray crystallographic techniques have been employed to elucidate the mode of action of the carbazole scaffolds. Results: Targeting an unoccupied subsite of the surface crevice with heterocycle-substituted PK083 analogs resulted in a 70-fold affinity increase to single-digit micromolar levels, increased thermal stability and decreased rate of aggregation of the mutant protein. PK9318, one of the most potent binders, restored p53 signaling in the liver cancer cell line HUH-7 with homozygous Y220C mutation. Conclusion: The p53-Y220C mutant is an excellent paradigm for the development of mutant p53 rescue drugs via protein stabilization. Similar rescue strategies may be applicable to other cavity-creating p53 cancer mutations.

Aminobenzothiazole derivatives stabilize the thermolabile p53 cancer mutant Y220C and show anticancer activity in p53-Y220C cell lines.

Many cancers have the tumor suppressor p53 inactivated by mutation, making reactivation of mutant p53 with small molecules a promising strategy for the development of novel anticancer therapeutics. The oncogenic p53 mutation Y220C, which accounts for approximately 100,000 cancer cases per year, creates an extended surface crevice in the DNA-binding domain, which destabilizes p53 and causes denaturation and aggregation. Here, we describe the structure-guided design of a novel class of small-molecule Y220C stabilizers and the challenging synthetic routes developed in the process. The synthesized chemical probe MB710, an aminobenzothiazole derivative, binds tightly to the Y220C pocket and stabilizes p53-Y220C in vitro. MB725, an ethylamide analogue of MB710, induced selective viability reduction in several p53-Y220C cancer cell lines while being well tolerated in control cell lines. Reduction of viability correlated with increased and selective transcription of p53 target genes such as BTG2, p21, PUMA, FAS, TNF, and TNFRSF10B, which promote apoptosis and cell cycle arrest, suggesting compound-mediated transcriptional activation of the Y220C mutant. Our data provide a framework for the development of a class of potent, non-toxic compounds for reactivating the Y220C mutant in anticancer therapy.

The oncogenic role of circPVT1 in head and neck squamous cell carcinoma is mediated through the mutant p53/YAP/TEAD transcription-competent complex.

BACKGROUND: Circular RNAs are a class of endogenous RNAs with various functions in eukaryotic cells. Worthy of note, circular RNAs play a critical role in cancer. Currently, nothing is known about their role in head and neck squamous cell carcinoma (HNSCC). The identification of circular RNAs in HNSCC might become useful for diagnostic and therapeutic strategies in HNSCC. RESULTS: Using samples from 115 HNSCC patients, we find that circPVT1 is over-expressed in tumors compared to matched non-tumoral tissues, with particular enrichment in patients with TP53 mutations. circPVT1 up- and down-regulation determine, respectively, an increase and a reduction of the malignant phenotype in HNSCC cell lines. We show that circPVT1 expression is transcriptionally enhanced by the mut-p53/YAP/TEAD complex. circPVT1 acts as an oncogene modulating the expression of miR-497-5p and genes involved in the control of cell proliferation. CONCLUSIONS: This study shows the oncogenic role of circPVT1 in HNSCC, extending current knowledge about the role of circular RNAs in cancer.

Microarray dataset of Jurkat cells following miR-93 over-expression.

The dataset presented here represents a microarray experiment of Jurkat cell line over-expressing miR-93 after lentiviral transgenic construct transduction. Three biological replicates have been performed. We further provide normalized and processed data, log2 Fold Change based ranked list and GOterms resulting table. The raw microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number ArrayExpress: E-MTAB-4588.

microRNA-181a enhances cell proliferation in acute lymphoblastic leukemia by targeting EGR1.

Acute lymphoblastic leukemia (ALL) is an aggressive cancer that occurs in both children and adults. Starting from an integrated analysis of miRNA/mRNA expression profiles in 20 ALL patients, we identify a negative correlation between miR-181a and EGR1. Coherently, miR-181a over-expression in Jurkat T-ALL cells decreases EGR1 expression, increasing cell proliferation and enhancing the cell-cycle progression from G1 to S phase. We show that EGR1 is a new direct target of miR-181a. Our findings suggest that miR-181a behaves as an onco-miRNA in ALL by down-regulating EGR1.

Hypoxia/reoxygenation cardiac injury and regeneration in zebrafish adult heart.

AIMS: the adult zebrafish heart regenerates spontaneously after injury and has been used to study the mechanisms of cardiac repair. However, no zebrafish model is available that mimics ischemic injury in mammalian heart. We developed and characterized zebrafish cardiac injury induced by hypoxia/reoxygenation (H/R) and the regeneration that followed it. METHODS AND RESULTS: adult zebrafish were kept either in hypoxic (H) or normoxic control (C) water for 15 min; thereafter fishes were returned to C water. Within 2-6 hours (h) after reoxygenation there was evidence of cardiac oxidative stress by dihydroethidium fluorescence and protein nitrosylation, as well as of inflammation. We used Tg(cmlc2:nucDsRed) transgenic zebrafish to identify myocardial cell nuclei. Cardiomyocyte apoptosis and necrosis were evidenced by TUNEL and Acridine Orange (AO) staining, respectively; 18 h after H/R, 9.9±2.6% of myocardial cell nuclei were TUNEL(+) and 15.0±2.5% were AO(+). At the 30-day (d) time point myocardial cell death was back to baseline (n = 3 at each time point). We evaluated cardiomyocyte proliferation by Phospho Histone H3 (pHH3) or Proliferating Cell Nuclear Antigen (PCNA) expression. Cardiomyocyte proliferation was apparent 18-24 h after H/R, it achieved its peak 3-7d later, and was back to baseline at 30d. 7d after H/R 17.4±2.3% of all cardiomyocytes were pHH3(+) and 7.4±0.6% were PCNA(+) (n = 3 at each time point). Cardiac function was assessed by 2D-echocardiography and Ventricular Diastolic and Systolic Areas were used to compute Fractional Area Change (FAC). FAC decreased from 29.3±2.0% in normoxia to 16.4±1.8% at 18 h after H/R; one month later ventricular function was back to baseline (n = 12 at each time point). CONCLUSIONS: zebrafish exposed to H/R exhibit evidence of cardiac oxidative stress and inflammation, myocardial cell death and proliferation. The initial decrease in ventricular function is followed by full recovery. This model more closely mimics reperfusion injury in mammals than other cardiac injury models.

MicroRNA 218 mediates the effects of Tbx5a over-expression on zebrafish heart development.

tbx5, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart development. Tbx5 function in heart development appears to be exquisitely sensitive to gene dosage, since both haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt-Oram syndrome (HOS), a highly penetrant autosomal dominant disease characterized by congenital heart defects of varying severity and upper limb malformation. It is suggested that tight integration of microRNAs and transcription factors into the cardiac genetic circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Based on these considerations, we performed an in silico screening to identify microRNAs embedded in genes highly sensitive to Tbx5 dosage. Among the identified microRNAs, we focused our attention on miR-218-1 that, together with its host gene, slit2, is involved in heart development. We found correlated expression of tbx5 and miR-218 during cardiomyocyte differentiation of mouse P19CL6 cells. In zebrafish embryos, we show that both Tbx5 and miR-218 dysregulation have a severe impact on heart development, affecting early heart morphogenesis. Interestingly, down-regulation of miR-218 is able to rescue the heart defects generated by tbx5 over-expression supporting the notion that miR-218 is a crucial mediator of Tbx5 in heart development and suggesting its possible involvement in the onset of heart malformations.