P63 belongs to a family of transcription factors, which, while demonstrating striking conservation of functional domains, regulate distinct biological functions. Its principal role is in the regulation of epithelial commitment, differentiation and maintenance programs, during embryogenesis and in adult tissues. The p63 gene has a complex transcriptional pattern, producing two subclasses of N-terminal isoforms (TA and ΔN) which are alternatively spliced at the C-terminus. Here, we report the identification of two new C-terminus p63 variants, we named p63 δ and ε, that increase from 6 to 10 the number of the p63 isoforms. Expression analysis of all p63 variants demonstrates a tissue/cell-type-specific nature of p63 alternative transcript expression, probably related to their different cellular functions. We demonstrate that the new p63 variants as ΔN isoforms are active as transcription factors as they have nuclear localization and can modulate the expression of p63 target genes. Moreover, we report that, like ΔNp63α, ΔNp63δ and ε sustain cellular proliferation and that their expression decreases during keratinocyte differentiation, suggesting their involvement in this process. Taken together, our results demonstrate the existence of novel p63 proteins whose expression should be considered in future studies on the roles of p63 in the regulation of cellular functions.

Alzheimer’s Disease (AD) is the most common cause of dementia affecting the elderly population worldwide. We have performed a comprehensive transcriptome profiling of Late-Onset AD (LOAD) patients using second generation sequencing technologies, identifying 2,064 genes, 47 lncRNAs and 4 miRNAs whose expression is specifically deregulated in the hippocampal region of LOAD patients. Moreover, analyzing the hippocampal, temporal and frontal regions from the same LOAD patients, we identify specific sets of deregulated miRNAs for each region, and we confirm that the miR-132/212 cluster is deregulated in each of these regions in LOAD patients, consistent with these miRNAs playing a role in AD pathogenesis. Notably, a luciferase assay indicates that miR-184 is able to target the 3’UTR NR4A2 - which is known to be involved in cognitive functions and long-term memory and whose expression levels are inversely correlated with those of miR-184 in the hippocampus. Finally, RNA editing analysis reveals a general RNA editing decrease in LOAD hippocampus, with 14 recoding sites significantly and differentially edited in 11 genes. Our data underline specific transcriptional changes in LOAD brain and provide an important source of information for understanding the molecular changes characterizing LOAD progression.

A central role for mitochondrial dysfunction has been proposed in the pathogenesis of DS (Down's syndrome), a multifactorial disorder caused by trisomy of human chromosome 21. To explore whether and how abnormalities in mitochondrial energy metabolism are involved in DS pathogenesis, we investigated the catalytic properties, gene expression and protein levels of certain proteins involved in mitochondrial ATP synthesis, such as F1F0-ATPase, ANT (adenine nucleotide translocator) and AK (adenylate kinase), in DS-HSF (human skin fibroblasts with trisomic karyotype), comparing them with euploid fibroblasts. In DS-HSF, we found a strong impairment of mitochondrial ATP synthesis due to a reduction in the catalytic efficiency of each of the investigated proteins. This impairment occurred in spite of unchanged gene expression and an increase in ANT and AK protein content, whereas the amount of ATPase subunits was selectively reduced. Interestingly, exposure of DS-HSF to dibutyryl-cAMP, a permanent derivative of cAMP, stimulated ANT, AK and ATPase activities, whereas H89, a specific PKA (protein kinase A) inhibitor, suppressed this cAMPdependent activation, indicating an involvement of the cAMP/PKA-mediated signalling pathway in the ATPase, ANT and AK deficit. Consistently, DS-HSF showed decreased basal levels of cAMP and reduced PKA activity. Despite the impairment of mitochondrial energy apparatus, no changes in cellular energy status, but increased basal levels of L-lactate, were found in DS-HSF, which partially offset for the mitochondrial energy deficit by increasing glycolysis and mitochondrial mass.These results provide new insight into the molecular basis for mitochondrial dysfunction in DS and might provide a molecular explanation for some clinical features of the syndrome.

TRIM8 plays a key role in controlling the p53 molecular switch that sustains the transcriptional activation of cell cycle arrest genes and response to chemotherapeutic drugs. The mechanisms that regulate TRIM8, especially in cancers like clear cell Renal Cell Carcinoma (ccRCC) and colorectal cancer (CRC) where it is low expressed, are still unknown. However, recent studies suggest the potential involvement of some microRNAs belonging to miR-17-92 and its paralogous clusters, which could include TRIM8 in a more complex pathway. We used RCC and CRC cell models for in-vitro experiments, and ccRCC patients and xenograft transplanted mice for in vivo assessments. To measure microRNAs levels we performed RT-qPCR, while steady-states of TRIM8, p53, p21 and N-MYC were quantified at protein level by Western Blotting as well as at transcript level by RT-qPCR. Luciferase reporter assays were performed to assess the interaction between TRIM8 and specific miRNAs, and the potential effects of this interaction on TRIM8 expression. Moreover, we treated our cell models with conventional chemotherapeutic drugs or tyrosine kinase inhibitors, and measured their response in terms of cell proliferation by MTT and colony suppression assays. We showed that TRIM8 is a target of miR-17-5p and miR-106b-5p, whose expression is promoted by N-MYC, and that alterations of their levels affect cell proliferation, acting on the TRIM8 transcripts stability, as confirmed in ccRCC patients and cell lines. In addition, reducing the levels of miR-17-5p/miR-106b-5p, we increased the chemo-sensitivity of RCC/CRC-derived cells to anti-tumour drugs used in the clinic. Intriguingly, this occurs, on one hand, by recovering the p53 tumour suppressor activity in a TRIM8-dependent fashion and, on the other hand, by promoting the transcription of miR-34a that turns off the oncogenic action of N-MYC. This ultimately leads to cell proliferation reduction or block, observed also in colon cancer xenografts overexpressing TRIM8. In this paper we provided evidence that TRIM8 and its regulators miR-17-5p and miR-106b-5 participate to a feedback loop controlling cell proliferation through the reciprocal modulation of p53, miR-34a and N-MYC. Our experiments pointed out that this axis is pivotal in defining drug responsiveness of cancers such ccRCC and CRC.

Background: Aerobic glycolysis, namely the Warburg effect, is the main hallmark of cancer cells. Mitochondrial respiratory dysfunction has been proposed to be one of the major causes for such glycolytic shift. This hypothesis has been revisited as tumors appear to undergo waves of gene regulation during progression, some of which rely on functional mitochondria. In this framework, the role of mitochondrial complex I is still debated, in particular with respect to the effect of mitochondrial DNA mutations in cancer metabolism. The aim of this work is to provide the proof of concept that functional complex I is necessary to sustain tumor progression. Methods: Complex I-null osteosarcoma cells were complemented with allotopically expressed complex I subunit 1 (MT-ND1). Complex I re-assembly and function recovery, also in terms of NADH consumption, were assessed. Clones were tested for their ability to grow in soft agar and to generate tumor masses in nude mice. Hypoxia levels were evaluated via pimonidazole staining and hypoxia-inducible factor-1α (HIF-1α) immunoblotting and histochemical staining. 454-pyrosequencing was implemented to obtain global transcriptomic profiling of allotopic and non-allotopic xenografts. Results: Complementation of a truncative mutation in the gene encoding MT-ND1, showed that a functional enzyme was required to perform the glycolytic shift during the hypoxia response and to induce a Warburg profile in vitro and in vivo, fostering cancer progression. Such trigger was mediated by HIF-1α, whose stabilization was regulated after recovery of the balance between α-ketoglutarate and succinate due to a recuperation of NADH consumption that followed complex I rescue. Conclusion: Respiratory complex I is essential for the induction of Warburg effect and adaptation to hypoxia of cancer cells, allowing them to sustain tumor growth. Differently from other mitochondrial tumor suppressor genes, therefore, a complex I severe mutation such as the one here reported may confer anti-tumorigenic properties, highlighting the prognostic values of such genetic markers in cancer.

P53 is a central hub in controlling cell proliferation. To maintain genome integrity in response to cellular stress, p53 directly regulates the transcription of genes involved in cell cycle arrest, DNA repair, apoptosis and/or senescence. An array of post-translational modifications and protein-protein interactions modulates its stability and activities in order to avoid malignant transformation. However, to date it is still not clear how cells decide their own fate in response to different types of stress. We described here that the human TRIM8 protein, a member of the TRIM family, is a new modulator of the p53-mediated tumor suppression mechanism. We showed that under stress conditions, such as UV exposure, p53 induced the expression of TRIM8, which in turn stabilized p53 leading to cell cycle arrest and reduction of cell proliferation through enhancement of CDKN1A (p21) and GADD45 expression. TRIM8 silencing reduced the capacity of p53 to activate genes involved in cell cycle arrest and DNA repair, in response to cellular stress. Concurrently, TRIM8 overexpression induced the degradation of the MDM2 protein, the principal regulator of p53 stability. Co-immunoprecipitation experiments showed that TRIM8 physically interacted with p53, impairing its interaction with MDM2. Altogether, our results reveal a previously unknown regulatory pathway controlling p53 activity and suggest TRIM8 as a novel therapeutic target to enhance p53 tumor suppressor activity.