Retinoic acid and arsenic trioxide sensitize acute promyelocytic leukemia cells to ER stress.

Retinoic acid (RA) in association with chemotherapy or with arsenic trioxide (ATO) results in high cure rates of acute promyelocytic leukemia (APL). We show that RA-induced differentiation of human leukemic cell lines and primary blasts dramatically increases their sensitivity to endoplasmic reticulum (ER) stress-inducing drugs at doses that are not toxic in the absence of RA. In addition, we demonstrate that the PERK pathway, triggered in response to ER stress, has a major protective role. Moreover, low amounts of pharmacologically induced ER stress are sufficient to strongly increase ATO toxicity. Indeed, in the presence of ER stress, ATO efficiently induced apoptosis in RA-sensitive and RA-resistant APL cell lines, at doses ineffective in the absence of ER stress. Our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML.

Gain-of-function mutant p53 downregulates miR-223 contributing to chemoresistance of cultured tumor cells.

Mutant p53 proteins are expressed at high frequency in human tumors and are associated with poor clinical prognosis and resistance to chemotherapeutic treatments. Here we show that mutant p53 proteins downregulate micro-RNA (miR)-223 expression in breast and colon cancer cell lines. Mutant p53 binds the miR-223 promoter and reduces its transcriptional activity. This requires the transcriptional repressor ZEB-1. We found that miR-223 exogenous expression sensitizes breast and colon cancer cell lines expressing mutant p53 to treatment with DNA-damaging drugs. Among the putative miR-223 targets, we focused on stathmin-1 (STMN-1), an oncoprotein known to confer resistance to chemotherapeutic drugs associated with poor clinical prognosis. Mutant p53 silencing or miR-223 exogenous expression lowers the levels of STMN-1 and knockdown of STMN-1 by small interfering RNA increases cell death of mutant p53-expressing cell lines. On the basis of these findings, we propose that one of the pathways affected by mutant p53 to increase cellular resistance to chemotherapeutic agents involves miR-223 downregulation and the consequent upregulation of STMN-1.

Argonaute 2 sustains the gene expression program driving human monocytic differentiation of acute myeloid leukemia cells.

MicroRNAs are key regulators of many biological processes, including cell differentiation. These small RNAs exert their function assembled in the RNA-induced silencing complexes (RISCs), where members of Argonaute (Ago) family of proteins provide a unique platform for target recognition and gene silencing. Here, by using myeloid cell lines and primary blasts, we show that Ago2 has a key role in human monocytic cell fate determination and in LPS-induced inflammatory response of 1,25-dihydroxyvitamin D3 (D3)-treated myeloid cells. The silencing of Ago2 impairs the D3-dependent miR-17-5p/20a/106a, miR-125b and miR-155 downregulation, the accumulation of their translational targets AML1, VDR and C/EBPß and monocytic cell differentiation. Moreover, we show that Ago2 is recruited on miR-155 host gene promoter and on the upstream region of an overlapping antisense lncRNA, determining their epigenetic silencing, and miR-155 downregulation. These findings highlight Ago2 as a new factor in myeloid cell fate determination in acute myeloid leukemia cells.

Expression of TP53 mutation-associated microRNAs predicts clinical outcome in head and neck squamous cell carcinoma patients.

BACKGROUND: TP53 mutation is associated with decreased survival rate in head and neck squamous cell carcinoma (HNSCC) patients. We set out to identify microRNAs (miRNAs) whose expression associates with TP53 mutation and survival in HNSCC. PATIENTS AND METHODS: We analyzed TP53 status by direct sequencing of exons 2 through 11 of a prospective series of 121 HNSCC samples and assessed its association with outcome in 109 followed-up patients. We carried out miRNA expression profiling on 121 HNSCC samples and 66 normal counterparts. miRNA associations with TP53 mutations and outcome were evaluated. RESULTS: A TP53 mutation was present in 58% of the tumors and TP53 mutations were significantly associated with a shorter recurrence-free survival. This association was stronger in the clinical subgroup of patients subjected to adjuvant therapy after surgery. The expression of 49 miRNAs was significantly associated with TP53 status. Among these 49, we identified a group of 12 miRNAs whose expression correlates with recurrence-free survival and a group of 4 miRNAs that correlates with cancer-specific survival. The two groups share three miRNAs. Importantly, miRNAs that correlate with survival are independent prognostic factors either when considered individually or as signatures. CONCLUSIONS: miRNAs expression associates with TP53 status and with reduced survival after surgical treatment of squamous cell carcinoma of the head and neck.

MicroRNA-128-2 targets the transcriptional repressor E2F5 enhancing mutant p53 gain of function.

p53 mutations have profound effects on non-small-cell lung cancer (NSCLC) resistance to chemotherapeutic treatments. Mutant p53 proteins are usually expressed at high levels in tumors, where they exert oncogenic functions. Here we show that p53R175H, a hotspot p53 mutant, induces microRNA (miRNA)-128-2 expression. Mutant p53 binds to the putative promoter of miR128-2 host gene, ARPP21, determining a concomitant induction of ARPP21 mRNA and miR-128-2. miR-128-2 expression in lung cancer cells inhibits apoptosis and confers increased resistance to cisplatin, doxorubicin and 5-fluorouracyl treatments. At the molecular level, miR-128-2 post-transcriptionally targets E2F5 and leads to the abrogation of its repressive activity on p21(waf1) transcription. p21(waf1) protein localizes to the cytoplasmic compartment, where it exerts an anti-apoptotic effect by preventing pro-caspase-3 cleavage. This study emphasizes miRNA-128-2 role as a master regulator in NSCLC chemoresistance.

Mutant p53: an oncogenic transcription factor.

Inactivation of tumor-suppressor genes is one of the key hallmarks of a tumor. Unlike other tumor-suppressor genes, p53 is inactivated by missense mutations in half of all human cancers. It has become increasingly clear that the resulting mutant p53 proteins do not represent only the mere loss of wild-type p53 tumor suppressor activity, but gain new oncogenic properties favoring the insurgence, the maintenance, the spreading and the chemoresistance of malignant tumors. The actual challenge is the fine deciphering of the molecular mechanisms underlying the gain of function of mutant p53 proteins. In this review, we will focus mainly on the transcriptional activity of mutant p53 proteins as one of the potential molecular mechanisms. To date, the related knowledge is still quite scarce and many of the raised questions of this review are yet unanswered.

S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation.

The p53 paralogues p73, p63 and their respective truncated isoforms have been shown to be critical regulators of developmental and differentiation processes. Indeed, both p73- and p63-deficient mice exhibit severe developmental defects. Here, we show that S100A2 gene, whose transcript and protein are induced during keratinocyte differentiation of HaCaT cells, is a direct transcriptional target of p73beta and DeltaNp63alpha and is required for proper keratinocyte differentiation. Transactivation assays reveal that p73beta and DeltaNp63alpha exert opposite transcriptional effects on S100A2 gene. While DeltaNp63alpha is found in vivo onto S100A2 regulatory regions predominantly in proliferating cells, p73beta is recruited in differentiating cells. Silencing of p73 impairs the induction of S100A2 during the differentiation of HaCaT cells. Moreover, silencing of p73 or S100A2 impairs the proper expression of keratinocyte differentiation markers. Of note, p53 family members do not trigger S100A2 gene expression in response to apoptotic doses of cisplatin and doxorubicin.

The transcriptional repressor ZEB regulates p73 expression at the crossroad between proliferation and differentiation.

The newly discovered p73 gene encodes a nuclear protein that has high homology with p53. Furthermore, ectopic expression of p73 in p53(+/+) and p53(-/-) cancer cells recapitulates some of the biological activities of p53 such as growth arrest, apoptosis, and differentiation. p73(-/-)-deficient mice exhibit severe defects in proper development of the central nervous system and pheromone sensory pathway. They also suffer from inflammation and infections. Here we studied the transcriptional regulation of p73 at the crossroad between proliferation and differentiation. p73 mRNA is undetectable in proliferating C2C12 cells and is expressed at very low levels in undifferentiated P19 and HL60 cells. Conversely, it is upregulated during muscle and neuronal differentiation as well as in response to tetradecanoyl phorbol acetate-induced monocytic differentiation of HL60 cells. We identified a 1-kb regulatory fragment located within the first intron of p73, which is positioned immediately upstream to the ATG codon of the second exon. This fragment exerts silencer activity on p73 as well as on heterologous promoters. The p73 intronic fragment contains six consensus binding sites for transcriptional repressor ZEB, which binds these sites in vitro and in vivo. Ectopic expression of dominant-negative ZEB (ZEB-DB) restores p73 expression in proliferating C2C12 and P19 cells. Thus, transcriptional repression of p73 expression by ZEB binding may contribute to the modulation of p73 expression during differentiation.

The cyclin B1 gene is actively transcribed during mitosis in HeLa cells.

In mammalian cells, the expression level of the cyclin B1 gene plays a critical role in the progression through mitosis. Here we demonstrate that the transcriptional activity of the human cyclin B1 promoter, as well as the rate of gene transcription, is high during mitosis. Indeed, the cyclin B1 promoter maintains an open chromatin configuration at the mitotic stage. Consistent with this, we show that the cyclin B1 promoter is occupied and bound to NF-Y during mitosis in vivo. Our results provide the first example of RNA polymerase II-dependent transcription during mitosis in mammalian cells.

Absence of in vivo DNA-protein interactions in the DQA2 and DQB2 promoter regions.

The DQ subregion of the human major histocompatibility complex (HLA) contains two pairs of loci: the DQA1/B1 genes (hereafter called DQ1), coding for the DQ molecules, and the DQA2/B2 pseudogenes (hereafter called DQ2). These pseudogenes are highly homologous to the functional DQ1 genes and they have no apparent abnormal features in their sequences that could prevent their activity. Only recently a low expression of the DQA2 gene has been observed whereas the DQB2 transcript was never found. The comparison between the DQ1 and DQ2 regulatory sequences revealed several differences in their W, X, and Y cis-acting elements. To examine the DNA/protein interactions in the DQ promoter regions, we performed in vivo footprinting experiments. Whereas the functional DQ1 loci showed a series of DNA-protein contact points in the X and Y boxes, the promoters of the DQ2 pseudogenes displayed an unoccupied phenotype. These findings suggest that the very low level of DQA2 expression and the apparent lack of DQB2 activity are caused by the reduced binding affinity of specific transcription factors.

From p63 to p53 across p73.

Most genes are members of a family. It is generally believed that a gene family derives from an ancestral gene by duplication and divergence. The tumor suppressor p53 was a striking exception to this established rule. However, two new p53 homologs, p63 and p73, have recently been described [1-6]. At the sequence level, p63 and p73 are more similar to each other than each is to p53, suggesting the possibility that the ancestral gene is a gene resembling p63/p73, while p53 is phylogenetically younger [1,2].The complexity of the family has also been enriched by the alternatively spliced forms of p63 and p73, which give rise to a complex network of proteins involved in the control of cell proliferation, apoptosis and development [1,2,4,7-9]. In this review we will mainly focus on similarities and differences as well as relationships among p63, p73 and p53.

p53 regulates myogenesis by triggering the differentiation activity of pRb.

The p53 oncosuppressor protein regulates cell cycle checkpoints and apoptosis, but increasing evidence also indicates its involvement in differentiation and development. We had previously demonstrated that in the presence of differentiation-promoting stimuli, p53-defective myoblasts exit from the cell cycle but do not differentiate into myocytes and myotubes. To identify the pathways through which p53 contributes to skeletal muscle differentiation, we have analyzed the expression of a series of genes regulated during myogenesis in parental and dominant-negative p53 (dnp53)-expressing C2C12 myoblasts. We found that in dnp53-expressing C2C12 cells, as well as in p53(-/-) primary myoblasts, pRb is hypophosphorylated and proliferation stops. However, these cells do not upregulate pRb and have reduced MyoD activity. The transduction of exogenous TP53 or Rb genes in p53-defective myoblasts rescues MyoD activity and differentiation potential. Additionally, in vivo studies on the Rb promoter demonstrate that p53 regulates the Rb gene expression at transcriptional level through a p53-binding site. Therefore, here we show that p53 regulates myoblast differentiation by means of pRb without affecting its cell cycle-related functions.

Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells.

In mammals, molecular mechanisms and factors involved in the tight regulation of telomerase expression and activity are still largely undefined. In this study, we provide evidence for a role of estrogens and their receptors in the transcriptional regulation of hTERT, the catalytic subunit of human telomerase and, consequently, in the activation of the enzyme. Through a computer analysis of the hTERT 5'-flanking sequences, we identified a putative estrogen response element (ERE) which was capable of binding in vitro human estrogen receptor alpha (ERalpha). In vivo DNA footprinting revealed specific modifications of the ERE region in ERalpha-positive but not ERalpha-negative cells upon treatment with 17beta-estradiol (E2), indicative of estrogen-dependent chromatin remodelling. In the presence of E2, transient expression of ERalpha but not ERbeta remarkably increased hTERT promoter activity, and mutation of the ERE significantly reduced this effect. No telomerase activity was detected in human ovary epithelial cells grown in the absence of E2, but the addition of the hormone induced the enzyme within 3 h of treatment. The expression of hTERT mRNA and protein was induced in parallel with enzymatic activity. This prompt estrogen modulation of telomerase activity substantiates estrogen-dependent transcriptional regulation of the hTERT gene. The identification of hTERT as a target of estrogens represents a novel finding which advances the understanding of telomerase regulation in hormone-dependent cells and has implications for a potential role of hormones in their senescence and malignant conversion.

Down-regulation of cyclin B1 gene transcription in terminally differentiated skeletal muscle cells is associated with loss of functional CCAAT-binding NF-Y complex.

The observation that cyclin B1 protein and mRNAs are down-regulated in terminally differentiated (TD) C2C12 cells, suggested us to investigate the transcriptional regulation of the cyclin B1 gene in these cells. Transfections of cyclin B1 promoter constructs indicate that two CCAAT boxes support cyclin B1 promoter activity in proliferating cells. EMSAs demonstrate that both CCAAT boxes are recognized by the trimeric NF-Y complex in proliferating but not in TD cells. Transfecting a dominant-negative mutant of NF-YA we provide evidence that NF-Y is required for maximal promoter activity. Addition of recombinant NF-YA to TD C2C12 nuclear extracts restores binding activity in vitro, thus indicating that the loss of NF-YA in TD cells is responsible for the lack of the NF-Y binding to the CCAAT boxes. Consistent with this, we found that the NF-YA protein is absent in TD C2C12 cells. In conclusion, our data demonstrate that NF-Y is required for cyclin B1 promoter activity. We also demonstrate that cyclin B1 expression is regulated at the transcriptional level in TD C2C12 cells and that the switch-off of cyclin B1 promoter activity in differentiated cells depends upon the loss of a functional NF-Y complex. In particular the loss of NF-YA protein is most likely responsible for its inactivation.