Radiotherapy-induced miR-223 prevents relapse of breast cancer by targeting the EGF pathway.

In breast cancer (BC) patients, local recurrences often arise in proximity of the surgical scar, suggesting that response to surgery may have a causative role. Radiotherapy (RT) after lumpectomy significantly reduces the risk of recurrence. We investigated the direct effects of surgery and of RT delivered intraoperatively (IORT), by collecting irradiated and non-irradiated breast tissues from BC patients, after tumor removal. These breast tissue specimens have been profiled for their microRNA (miR) expression, in search of differentially expressed miR among patients treated or not with IORT. Our results demonstrate that IORT elicits effects that go beyond the direct killing of residual tumor cells. IORT altered the wound response, inducing the expression of miR-223 in the peri-tumoral breast tissue. miR-223 downregulated the local expression of epidermal growth factor (EGF), leading to decreased activation of EGF receptor (EGFR) on target cells and, eventually, dampening a positive EGF-EGFR autocrine/paracrine stimulation loop induced by the post-surgical wound-healing response. Accordingly, both RT-induced miR-223 and peri-operative inhibition of EGFR efficiently prevented BC cell growth and reduced recurrence formation in mouse models of BC. Our study uncovers unknown effects of RT delivered on a wounded tissue and prompts to the use of anti-EGFR treatments, in a peri-operative treatment schedule, aimed to timely treat BC patients and restrain recurrence formation.

HINCUTs in cancer: hypoxia-induced noncoding ultraconserved transcripts.

Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts is currently unknown. Starting from a genome-wide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named 'hypoxia-induced noncoding ultraconserved transcripts' (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category.

miR-145 participates with TP53 in a death-promoting regulatory loop and targets estrogen receptor-alpha in human breast cancer cells.

Understanding the consequences of miR-145 reintroduction in human breast cancer (BC) could reveal its tumor-suppressive functions and may disclose new aspects of BC biology. Therefore, we characterized the effects of miR-145 re-expression in BC cell lines by using proliferation and apoptosis assays. As a result, we found that miR-145 exhibited a pro-apoptotic effect, which is dependent on TP53 activation, and that TP53 activation can, in turn, stimulate miR-145 expression, thus establishing a death-promoting loop between miR-145 and TP53. We also found that miR-145 can downregulate estrogen receptor-alpha (ER-alpha) protein expression through direct interaction with two complementary sites within its coding sequence. In conclusion, we described a tumor suppression function of miR-145 in BC cell lines, and we linked miR-145 to TP53 and ER-alpha. Moreover, our findings support a view that miR-145 re-expression therapy could be mainly envisioned in the specific group of patients with ER-alpha-positive and/or TP53 wild-type tumors.

Prostaglandin E2 inhibits proliferation and migration of HTR-8/SVneo cells, a human trophoblast-derived cell line.

Normal placentation requires a highly coordinated control of proliferation, migration and invasiveness of extravillous trophoblast cells. Since prostaglandin E2 is a major prostanoid synthesized by intrauterine tissues and highly involved in pregnancy homeostasis, we examined the possibility that it modulates extravillous trophoblast cell functions. Here, we report the presence of mRNAs for prostaglandin E2 EP2 and EP4 receptor isoforms and of proteins in both first-trimester human chorionic villi and in the human trophoblast-derived HTR-8/SVneo cells. Moreover we found that: (i) this cell line releases prostaglandin E2 and the output is enhanced by interleukin-1beta; (ii) the prostanoid consistently inhibits serum- or epidermal growth factor-induced cell proliferation and also migration. An involvement of cAMP in the prostaglandin E2 antiproliferative action is suggested by the observation that the prostanoid greatly enhances cAMP level in HTR-8/SVneo cells and that forskolin inhibits cell proliferation; moreover the administration of prostaglandin E2 plus forskolin, a condition which evokes a synergistic enhancement of cAMP, induces a major impairment of cell growth. Provided that our data are applicable to the trophoblast tissue in vivo, we suggest that prostaglandin E2 exerts an important control on extravillous trophoblast cell functions, preventing an excessive proliferation and migration.

p27(kip1) functional regulation in human cancer: a potential target for therapeutic designs.

The mitotic cell cycle is a tightly regulated process that ensures the correct division of one cell into two daughter cells. Progress along the different phases of the cell cycle is positively regulated by the sequential activation of a family of serine-threonine kinases called CDKs (Cyclin Dependent Kinases). Their activity is counteracted by small proteins known as CDK inhibitors (CKI) that ensure the correct timing of CDK activation in the different phases of the cell cycle. The present review will deal with the role of one of this CKI, p27(kip1), in human cancer, focusing in particular on the mechanisms underlying its functional inactivation in tumor cells. p27(kip1) protein downregulation is usually achieved by proteasomal degradation and is often correlated to a worse prognosis in several types of human cancers, resulting in the reduction of disease free and overall survival. More recently, it has been proposed that p27(kip1) protein, rather than degraded, can be functionally inactivated. The mechanisms and the implications of these two types of p27(kip1) deregulation will be discussed and some potential therapeutic approaches targeting p27(kip1) functions will be proposed.

Targeted ribose methylation of RNA in vivo directed by tailored antisense RNA guides.

Eukaryotic ribosomal RNAs are post-transcriptionally modified by methylation at the ribose sugar of specific nucleotides. This takes place in the nucleolus and involves a family of small nucleolar RNAs (snoRNAs) with long regions (10-21 nucleotides) complementary to rRNA sequences spanning the methylation site--a complementary snoRNA is required for methylation at a specific site. Here we show that altering the sequence of the snoRNA is sufficient to change the specificity of methylation. Mammalian cells transfected with a snoRNA engineered to be complementary to an arbitrary rRNA sequence direct the methylation of the predicted nucleotide in that sequence. We have further identified structural features, both of the guide and substrate RNA, required for methylation and have used these to design an exogenous transcript, devoid of rRNA sequence, that is site-specifically methylated when coexpressed with an appropriate guide snoRNA. Endogenous non-ribosomal RNA can thus be targeted, possibly providing a highly selective tool for the alteration of gene expression at the post-transcriptional level.

Intron-encoded, antisense small nucleolar RNAs: the characterization of nine novel species points to their direct role as guides for the 2'-O-ribose methylation of rRNAs.

A growing number of small nucleolar RNAs (snoRNAs) are intron-encoded, contain the characteristic box C (UGAUGA) and box D (CUGA) motifs and exhibit long complementarities to conserved sequences in mature rRNAs. We have identified nine additional members of this family, U32 to U40. All but one are encoded in introns of ribosomal protein genes in vertebrates: U32 to U35 in rpL13a, U36 in rpL7a and U38 to U40 in rpS8. By contrast, U37 is encoded in elongation factor 2 gene. Interestingly, U32 and U36 each contain two complementarities (one to 18 S and the other to 28 S rRNA). U32 to U40 are fibrillarin-associated, devoid of a 5'-trimethyl-cap and display an exclusively nucleolar localization. They are all metabolically stable and roughly as abundant as previously reported members of this family. Characterization of their homologs in distant species shows that their 10 to 14 nt long rRNA complementarities are conserved. A clue on the function of this snoRNA family is provided by the comparative analysis of the largely expanded collection of their conserved duplexes with rRNA. Not only does each duplex span at least one site of 2'-O-ribose methylation in the rRNA but the modification site is always at the same position in the duplex, paired to the fifth nucleotide upstream from a box D motif in the snoRNA. Consistent with the notion that each snoRNA of this family guides one particular methylation along the rRNA sequence, we have detected several pairs of snoRNAs with overlapping complementarities to rRNA tracts with vicinal sites of ribose methylations. In each case, the two overlapping complementarities are shifted from each other by a distance equal to the spacing between the methylated sites which are thus found at the same position within each of the mutually exclusive duplexes. Finally, we have also identified, within three previously known snoRNAs, novel antisense elements able to form a canonical duplex around ribose-methylated sites in rRNA, which further supports the conclusion that the duplex structure provides the 2'-O-methyltransferase with the appropriate site-specificity on the substrate.

SnoRNA U21 is also intron-encoded in Drosophila melanogaster but in a different host-gene as compared to warm-blooded vertebrates.

U21 is an intron-encoded snoRNA in vertebrates which contains a 13-nt tract of complementarity to an invariant sequence in eukaryotic 28S rRNA. Here, we report the characterization of its Drosophila melanogaster homolog which is the first case of an intron-encoded snoRNA in an invertebrate metazoan. In D. melanogaster, U21 is encoded within the ARF-1 (ADP ribosylation factor 1) gene, whereas in chicken and mammals it is found in the ribosomal protein L5 gene. In D. melanogaster, like in vertebrates, U21 is devoid of a 5' trimethylguanosine cap, thus, likely resulting from processing of intronic RNA. The only portion of U21 sequence preserved between D. melanogaster and vertebrates, in addition to the hallmark box C and box D motifs, corresponds precisely to the 13-nt complementary to rRNA, pointing to an important role of the pairing of U21 to pre-rRNA.

Novel intron-encoded small nucleolar RNAs with long sequence complementarities to mature rRNAs involved in ribosome biogenesis.

Recently, several new snoRNAs encoded in introns of genes coding for ribosomal, ribosome-associated, or nucleolar proteins have been discovered. We are presently studying four of these intronic snoRNAs. Three of them, U20, U21, and U24, are closely related to each other on a structural basis. They are included in genes encoding nucleolin and ribosomal proteins L5 and L7a, respectively, in warm-blooded vertebrates. These three metabolically stable snoRNAs interact with nucleolar protein fibrillarin. In addition, they display common features that make them strikingly related to snoRNA U14. U14 contains two tracts of complementarity to 18S rRNA, which are required for the production of 18S rRNA. U20 displays a 21 nucleotide (nt) long complementarity to 18S rRNA. U21 contains a 13 nt complementarity to an invariant sequence in eukaryotic 28S rRNA. U24 has two separate 12 nt long complementarities to a highly conserved tract of 28S rRNA. Phylogenetic evidences support the fundamental importance of the pairings of these three snoRNAs to pre-rRNA, which could be involved in a control of pre-rRNA folding during preribosome assembly. By transfection of mouse cells, we have also analyzed the processing of U20 and found that the -cis acting signals for its processing from intronic RNA are restricted to the mature snoRNA sequence. Finally, we have documented changes of host genes for these three intronic snoRNAs during the evolution of eukaryotes.

U24, a novel intron-encoded small nucleolar RNA with two 12 nt long, phylogenetically conserved complementarities to 28S rRNA.

Following computer searches of sequence banks, we have positively identified a novel intronic snoRNA, U24, encoded in the ribosomal protein L7a gene in humans and chicken. Like previously reported intronic snoRNAs, U24 is devoid of a 5'-trimethyl-cap. U24 is immunoprecipitated by an antifibrillarin antibody and displays an exclusively nucleolar localization by fluorescence microscopy after in situ hybridization with antisense oligonucleotides. In vertebrates, U24 is a 76 nt long conserved RNA which is metabolically stable, present at approximately 14,000 molecules per human HeLa cell. U24 exhibits a 5'-3' terminal stem-box C-box D structure, typical for several snoRNAs, and contains two 12 nt long conserved sequences complementary to 28S rRNA. It is, therefore, strikingly related to U14, U20 and U21 snoRNAs which also possess long sequences complementary to conserved sequences of mature 18S or 28S rRNAs. In 28S rRNA the two tracts complementary to U24 are adjacent to each other, they involve several methylated nucleotides and are surprisingly close, within the rRNA secondary structure, to complementarities to snoRNAs U18 and U21. Identification of the yeast Saccharomyces cerevisiae U24 gene directly confirms the outstanding conservation of the complementarity to 28S rRNA during evolution, suggesting a key role of U24 pairing to pre-rRNA during ribosome biogenesis, possible in the control of pre-rRNA folding. Yeast S.cerevisiae U24 is also intron-encoded but not in the same host-gene as in humans or chicken.

Antisense snoRNAs: a family of nucleolar RNAs with long complementarities to rRNA.

A growing subset of small nucleolar RNAs (snoRNAs) contains long stretches of sequence complementarity to conserved sequences in mature ribosomal RNA (rRNA). This article reviews current knowledge about these complementarities and proposes that these antisense snoRNAs might function in pre-rRNA folding, base modification and ribosomal ribonucleoprotein assembly, in some cases acting as RNA chaperones.

U21, a novel small nucleolar RNA with a 13 nt. complementarity to 28S rRNA, is encoded in an intron of ribosomal protein L5 gene in chicken and mammals.

Following a search of sequence data bases for intronic sequences exhibiting structural features typical of snoRNAs, we have positively identified by Northern assays and sequence analysis another intron-encoded snoRNA, termed U21. U21 RNA is a 93 nt. long, metabolically stable RNA, present at about 10(4) molecules per HeLa cell. It is encoded in intron 5 of the ribosomal protein L5 gene, both in chicken and in the two mammals studied so far, human and mouse. U21 RNA is devoid of a 5'-trimethyl-cap and is likely to result from processing of intronic RNA. The nucleolar localization of U21 has been established by fluorescence microscopy after in situ hybridization with digoxigenin-labeled oligonucleotide probes. Like most other snoRNAs U21 contains the box C and box D motifs and is precipitated by anti-fibrillarin antibodies. By the presence of a typical 5'-3' terminal stem, U21 appears more particularly related to U14, U15, U16 and U20 intron-encoded snoRNAs. Remarkably, U21 contains a long stretch (13 nt.) of complementarity to a highly conserved sequence in 28S rRNA. Sequence comparisons between chicken and mammals, together with Northern hybridizations with antisense oligonucleotides on cellular RNAs from more distant vertebrates, point to the preferential preservation of this segment of U21 sequence during evolution. Accordingly, this complementarity, which overlaps the complementarity of 28S rRNA to another snoRNA, U18, could reflect an important role of U21 snoRNA in the biogenesis of large ribosomal subunit.

U20, a novel small nucleolar RNA, is encoded in an intron of the nucleolin gene in mammals.

We have found that intron 11 of the nucleolin gene in humans and rodents encodes a previously unidentified small nucleolar RNA, termed U20. The single-copy U20 sequence is located on the same DNA strand as the nucleolin mRNA. U20 RNA, which does not possess a trimethyl cap, appears to result from intronic RNA processing and not from transcription of an independent gene. In mammals, U20 RNA is an 80-nucleotide-long, metabolically stable species, present at about 7 x 10(3) molecules per exponentially growing HeLa cell. It has a nucleolar localization, as indicated by fluorescence microscopy following in situ hybridization with digoxigenin-labeled oligonucleotides. U20 RNA contains the box C and box D sequence motifs, hallmarks of most small nucleolar RNAs reported to date, and is immunoprecipitated by antifibrillarin antibodies. It also exhibits a 5'-3' terminal stem bracketing the box C-box D motifs like U14, U15, U16, or Y RNA. A U20 homolog of similar size has been detected in all vertebrate classes by Northern (RNA) hybridization with mammalian oligonucleotide probes. U20 RNA contains an extended region (21 nucleotides) of perfect complementarity with a phylogenetically conserved sequence in 18S rRNA. This complementarity is strongly preserved among distant vertebrates, suggesting that U20 RNA may be involved in the formation of the small ribosomal subunit like nucleolin, the product of its host gene.

Alternative pre-rRNA processing pathways in human cells and their alteration by cycloheximide inhibition of protein synthesis.

rRNA processing pathways in humans have been reinvestigated through systematic Northern-blot hybridizations of HeLa cell nuclear RNA with a collection of digoxigenin-labeled rDNA probes from different regions of the human rDNA transcriptional unit. In addition to the known 45S, 41S, 32S and 21S pre-rRNA, two major pre-rRNA fractions were identified; a '30S' (about 5800 nucleotides) precursor to 18S rRNA containing an external transcribed spacer at the 5' end (ETS) and internal transcribed spacer (ITS) 1 sequences and a '12S' (about 950 nucleotides) precursor to 5.8S rRNA containing ITS 2 sequences. These pre-rRNA species do not react with probes located near the 3'-terminal segments of ITS 1 or ITS 2, thus suggesting that processive endonuclease cuts occur within ITS spacer sequences. The simultaneous occurrence of at least two alternative 45S pre-rRNA processing pathways is deduced, which correspond to a different temporal order of endonuclease attack at the sites located near the 5' end of 18S rRNA and within ITS 1. In-vivo labeling experiments with [14C]uridine revealed that inhibition of protein synthesis with cycloheximide abolishes the endonuclease cut at the 5' end of 18S rRNA and the formation of 41S pre-rRNA, while the cut within ITS 1 and the processing to 32S and '30S' pre-rRNA remains relatively unaltered.

Alterations of nucleolar ultrastructure and ribosome biogenesis by actinomycin D. Implications for U3 snRNP function.

We have studied, at the electron microscope level, the reorganizations of nucleolar ultrastructure induced by actinomycin D (AMD) in different conditions of drug treatment associated with an inhibition of rRNA synthesis. We have analyzed in parallel the localizations of ribosomal genes, of their transcripts, of various pre-rRNA intermediates, as well as of U3 RNA and fibrillarin by in situ hybridization with nucleic acid probes and immunocytological detection on thin sections of human and mouse cells. Consistent with previous observations, dense fibrillar component (DFC) and granular component (GC) appear to contain distinct pre-rRNA species at different stages of their processing. DFC appears as a major site of U3 RNA accumulation, but a very substantial fraction of nucleolar U3 RNA is also found in GC, colocalizing with partially processed pre-rRNAs. Remarkably, the major nucleolar components retain their ultrastructural appearance when extensively depleted of their pre-rRNA moiety, and ribosomal genes are always detected over fibrillar center (FC), even after extended AMD treatments which result in the characteristic segregation of nucleolar components. Moreover, while for GC the U3 RNA and pre-rRNA contents evolve in parallel following the cessation of rRNA synthesis, a dramatic uncoupling is observed for DFC. The persistent presence of U3 RNA and fibrillarin after pre-rRNA depletion suggests that DFC could represent an anchorage site for U3 snRNPs, before their entering another cycle of pre-rRNA processing reactions.

Localization of U3 RNA molecules in nucleoli of HeLa and mouse 3T3 cells by high resolution in situ hybridization.

We have examined the ultrastructural localization of U3 RNA in the nucleoli of HeLa and mouse 3T3 cells by in situ hybridization with a biotinylated U3 DNA probe and subsequent detection of hybrids with electron microscopy by direct immunogold labeling. The highest levels of signal density for U3 RNA are detected over the dense fibrillar component (DFC) of the nucleolus, including the interfaces between DFC and the enclosed fibrillar center (FC) on the one hand and DFC and the granular component (GC) on the other hand. Lower but significant signals also are observed over GC, which indicate, taking into account the high relative volume of GC in a nucleolus, that a substantial fraction of U3 RNA is present in this compartment where the more mature forms of pre-rRNA accumulate. In parallel, the localization of fibrillarin was analyzed by immunogold detection, demonstrating that fibrillarin and U3 RNA have a roughly similar distribution, although quantitative measurements reveal that the signal ratio for both molecules exhibit significant differences among the major ultrastructural components of the nucleolus.

A sequence dimorphism in a conserved domain of human 28S rRNA. Uneven distribution of variant genes among individuals. Differential expression in HeLa cells.

In humans, cellular 28S rRNA displays a sequence dimorphism within an evolutionarily conserved motif, with the presence, at position +60, of either a A (like the metazoan consensus) or a G. The relative abundance of the two forms of variant genes in the genome exhibit large differences among individuals. The two variant forms are generally represented in cellular 28S rRNA in proportion of their relative abundance in the genome, at least for leucocytes. However, in some cases, one form of variant may be markedly underexpressed as compared to the other. Thus, in HeLa cells, A-form genes contribute to only 1% of the cellular content in mature 28S rRNA although amounting to 15% of the ribosomal genes. The differential expression seems to result from different transcriptional activities rather than from differences in pre-rRNA processing efficiency or in stabilities of mature rRNAs. G-form ribosomal genes were not detected in other mammals, including chimpanzee, which suggests that the fixation of this variant type is a rather recent event in primate evolution.

Presence of a differentially expressed U3A RNA variant in mouse. Structure and evolutive implications.

A U3 RNA variant has been identified in mouse, the abundance of which relative to the previously characterized major form (U3B) appears to vary to a large extent depending upon the cell origin. Its partial sequence analysis shows that it is clearly related to the U3A form previously described in rat. Sequence comparisons suggest that the separation of the two forms of U3 genes now found in rat and mouse represent a relatively ancient event in rodent evolution. While mouse U3B RNA is encoded by four clustered genes, the U3A variant is encoded by a unique gene. Both mouse U3 RNAs differ substantially in primary structure (more than 10% divergence). Although rodent U3 RNAs exhibit a largely similar secondary structure, a specific difference between the A and B form can nevertheless be observed.

Titration of variant DNA sequences differing by a single point-mutation by selective dot-blot hybridization with synthetic oligonucleotides.

A dot-blot hybridization procedure with synthetic oligonucleotide probes is reported, which allows the quantitative titration in genomic DNA of variant forms of repeated genes differing by a single nucleotide change. It involves the utilization of a pair of 22-base long oligonucleotides matching the two variant sequences and the choice of an hybridization temperature very close to the Td of the oligonucleotide/DNA duplexes. The selectivity is achieved through a competition between the cognate labeled and the non-cognate unlabeled probes in the hybridization mixture.

Phylogenetic calibration of the 5' terminal domain of large rRNA achieved by determining twenty eucaryotic sequences.

Due to their high information content and their particular mode of variation, large rRNA molecules potentially represent powerful indicators of phylogenetic relationships. Even partial sequences may suffice to generate reliable estimations, provided they correspond to well-chosen portions of the molecule. We have systematically analyzed a specific portion of the large rRNA (the region extending over nearly 400 nucleotides from the 5' end) as a general index of eucaryotic phylogeny. By means of fast and direct rRNA sequencing, we have determined the sequence of this region for 20 additional eucaryotes, including several representatives of each vertebrate class, an invertebrate metazoan (mussel), a fungus (Schizosaccharomyces pombe), and three higher plants. Comparative treatment of these new data and previously reported rRNA sequences shows that this region can serve as an indicator of eucaryotic phylogeny for evaluating both long-range and short-range relationships. Its conservative domains appear to possess a rather uniform rate of nucleotide changes in all the eucaryotic lineages analyzed and the phylogenetic tree we derived agrees with classical views.