Targeting ribosome biogenesis reinforces ERK-dependent senescence in pancreatic cancer.

Pancreatic adenocarcinomas (PDAC) often possess mutations in K-Ras that stimulate the ERK pathway. Aberrantly high ERK activation triggers oncogene-induced senescence, which halts tumor progression. Here we report that low-grade pancreatic intraepithelial neoplasia displays very high levels of phospho-ERK consistent with a senescence response. However, advanced lesions that have circumvented the senescence barrier exhibit lower phospho-ERK levels. Restoring ERK hyperactivation in PDAC using activated RAF leads to ERK-dependent growth arrest with senescence biomarkers. ERK-dependent senescence in PDAC was characterized by a nucleolar stress response including a selective depletion of nucleolar phosphoproteins and intranucleolar foci containing RNA polymerase I designated as senescence-associated nucleolar foci (SANF). Accordingly, combining ribosome biogenesis inhibitors with ERK hyperactivation reinforced the senescence response in PDAC cells. Notably, comparable mechanisms were observed upon treatment with the platinum-based chemotherapy regimen FOLFIRINOX, currently a first-line treatment option for PDAC. We thus suggest that drugs targeting ribosome biogenesis can improve the senescence anticancer response in pancreatic cancer.

SOCS1 inhibits migration and invasion of prostate cancer cells, attenuates tumor growth and modulates the tumor stroma.

BACKGROUND: The suppressor of cytokine signaling 1 (SOCS1) gene is repressed in prostate cancer (PCa) by epigenetic silencing and microRNA miR30d. Increased expression of the SOCS1-targeting miR30d correlates with higher biochemical recurrence, suggesting a tumor suppressor role of SOCS1 in PCa, but the underlying mechanisms are unclear. We have shown that SOCS1 inhibits MET receptor kinase signaling, a key oncogenic pathway in cancer progression. Here we evaluated the role of SOCS1 in attenuating MET signaling in PCa cells and tumor growth in vivo. METHODS: MET-overexpressing human DU145 and PC3 PCa cell lines were stably transduced with SOCS1, and their growth, migration and invasion of collagen matrix were evaluated in vitro. Cells expressing SOCS1 or the control vector were evaluated for tumor growth in NOD.scid.gamma mice as xenograft or orthotopic tumors. RESULTS: HGF-induced MET signaling was attenuated in SOCS1-expressing DU145 and PC3 cells. Compared with vector control cells, SOCS1-expressing cells showed reduced proliferation and impaired migration following HGF stimulation. DU145 and PC3 cells showed marked ability to invade the collagen matrix following HGF stimulation and this was attenuated by SOCS1. As xenografts, SOCS1-expressing PCa cells showed significantly reduced tumor growth compared with vector control cells. In the orthotopic tumor model, SOCS1 reduced the growth of primary tumors and metastatic spread. Intriguingly, the SOCS1-expressing DU145 and PC3 tumors showed increased collagen deposition, associated with increased frequency of myofibroblasts. CONCLUSIONS: Our findings support the tumor suppressor role of SOCS1 in PCa and suggest that attenuation of MET signaling is one of the underlying mechanisms. SOCS1 in PCa cells also appears to prevent the tumor-promoting functions of cancer-associated fibroblasts.

Suppressor of cytokine signaling 1-dependent regulation of the expression and oncogenic functions of p21(CIP1/WAF1) in the liver.

The SOCS1 gene coding for suppressor of cytokine signaling 1 is frequently repressed in hepatocellular carcinoma (HCC), and hence SOCS1 is considered a tumor suppressor in the liver. However, the tumor-suppressor mechanisms of SOCS1 are not yet well understood. SOCS1 is known to inhibit pro-inflammatory cytokine production and signaling and to promote activation of the p53 tumor suppressor. However, we observed that SOCS1-deficient mice developed numerous and large liver tumor nodules following treatment with the hepatocarcinogen diethylnitrosamine (DEN) without showing increased interleukin-6 production or activation of p53. On the other hand, the livers of DEN-treated Socs1-null mice showed elevated levels of p21(CIP1/WAF1) protein (p21). Even though p21 generally functions as a tumor suppressor, paradoxically many cancers, including HCC, are known to express elevated levels of p21 that correlate with poor prognosis. We observed elevated p21 expression also in the regenerating livers of SOCS1-deficient mice and in cisplatin-treated Socs1-null hepatocytes, wherein the p21 protein showed increased stability. We show that SOCS1 interacts with p21 and promotes its ubiquitination and proteasomal degradation. Besides, the DEN-treated livers of Socs1-null mice showed increased nuclear and cytosolic p21 staining, and the latter was associated with growth factor-induced, phosphatidylinositol 3-kinase-dependent phosphorylation of p21 in SOCS1-deficient hepatocytes. Cytosolic p21 is often associated with malignancy and chemo-resistance in many cancers. Accordingly, SOCS1-deficient hepatocytes showed increased resistance to apoptosis that was reversed by shRNA-mediated p21 knockdown. In the regenerating livers of Socs1-null mice, increased p21 expression coincided with elevated cyclinD levels. Correspondingly, SOCS1-deficient hepatocytes showed increased proliferation to growth factor stimulation that was reversed by p21 knockdown. Overall, our findings indicate that the tumor-suppressor functions of SOCS1 in the liver could be mediated, at least partly, via regulation of the expression, stability and subcellular distribution of p21 and its paradoxical oncogenic functions, namely, resistance to apoptosis and increased proliferation.

ARF1 controls proliferation of breast cancer cells by regulating the retinoblastoma protein.

The ADP-ribosylation factors (ARFs) 1 and 6 are small GTP-binding proteins, highly expressed and activated in several breast cancer cell lines and are associated with enhanced migration and invasiveness. In this study, we report that ARF1 has a critical role in cell proliferation. Depletion of this GTPase or expression of a dominant negative form, which both resulted in diminished ARF1 activity, led to sustained cell-growth arrest. This cellular response was associated with the induction of senescent markers in highly invasive breast cancer cells as well as in control mammary epithelial cells by a mechanism regulating retinoblastoma protein (pRB) function. When examining the role of ARF1, we found that this GTPase was highly activated in normal proliferative conditions, and that a limited amount could be found in the nucleus, associated with the chromatin of MDA-MB-231 cells. However, when cells were arrested in the G(0)/G(1) phase or transfected with a dominant negative form of ARF1, the total level of activated ARF1 was markedly reduced and the GTPase significantly enriched in the chromatin. Using biochemical approaches, we demonstrated that the GDP-bound form of ARF1 directly interacted with pRB, but not other members of this family of proteins. In addition, depletion of ARF1 or expression of ARF1T(31)N resulted in the constitutive association of pRB and E2F1, thereby stabilizing the interaction of E2F1 as well as pRB at endogenous sites of target gene promoters, preventing expression of E2F target genes, such as cyclin D1, Mcm6 and E2F1, important for cell-cycle progression. These novel findings provide direct physiological and molecular evidence for the role of ARF1 in controlling cell proliferation, dependent on its ability to regulate pRB/E2F1 activity and gene expression for enhanced proliferation and breast cancer progression.

The role of Stat5 transcription factors as tumor suppressors or oncogenes.

Stat5 is constitutively activated in many human cancers affecting the expression of cell proliferation and cell survival controlling genes. These oncogenic functions of Stat5 have been elegantly reproduced in mouse models. Aberrant Stat5 activity induces also mitochondrial dysfunction and reactive oxygen species leading to DNA damage. Although DNA damage can stimulate tumorigenesis, it can also prevent it. Stat5 can inhibit tumor progression like in the liver and it is a tumor suppressor in fibroblasts. Stat5 proteins are able to regulate cell differentiation and senescence activating the tumor suppressors SOCS1, p53 and PML. Understanding the context dependent regulation of tumorigenesis through Stat5 function will be central to understand proliferation, survival, differentiation or senescence of cancer cells.

PML a target of translocations in APL is a regulator of cellular senescence.

PML is the most frequent fusion partner of the RARalpha in the specific translocations associated with acute promyelocytic leukemia (APL). Models to explain the origin of this leukemia propose a block in cell differentiation due to aberrant repression of retinoic acid responsive genes and/or disruption of the function of the PML-containing nuclear bodies. Recently, PML has been identified as a regulator of replicative senescence and the premature senescence that occurs in response to oncogenic ras. This review discusses the idea that senescence is a general tumor suppressor mechanism related to terminal differentiation and disrupted during the establishment of APL and other cancers. According to this idea the PML-RARalpha fusion protein promotes leukemogenesis not only through repression of retinoic acid responsive genes, but also by way of interfering with several tumor suppressor proteins that cooperate to establish senescence. Retinoids and other drugs effective against APL do so by re-establishment of the senescence program, which also includes features of cell differentiation.

Hammerhead-mediated processing of satellite pDo500 family transcripts from Dolichopoda cave crickets.

This work reports the discovery and functional characterization of catalytically active hammerhead motifs within satellite DNA of the pDo500 family from several DOLICHOPODA: cave cricket species. We show that in vitro transcribed RNA of some members of this satellite DNA family do self-cleave in vitro. This self-cleavage activity is correlated with the efficient in vivo processing of long primary transcripts into monomer-sized RNA. The high sequence conservation of the satellite pDo500 DNA family among genetically isolated DOLICHOPODA: schiavazzii populations, as well as other DOLICHOPODA: species, along with the fact that satellite members are actively transcribed in vivo suggests that the hammerhead-encoding satellite transcripts are under selective pressure, perhaps because they fulfil an important physiological role or function. Remarkably, this is the third example of hammerhead ribozyme structures associated with transcribed repetitive DNA sequences from animals. The possibility that such an association may not be purely coincidental is discussed.

PML is induced by oncogenic ras and promotes premature senescence.

Oncogenic ras provokes a senescent-like arrest in human diploid fibroblasts involving the Rb and p53 tumor suppressor pathways. To further characterize this response, we compared gene expression patterns between ras-arrested and quiescent IMR90 fibroblasts. One of the genes up-regulated during ras-induced arrest was promyelocytic leukemia (PML) protein, a potential tumor suppressor that encodes a component of nuclear structures known as promyelocytic oncogenic domains (PODs). PML levels increased during both ras-induced arrest and replicative senescence, leading to a dramatic increase in the size and number of PODs. Forced PML expression was sufficient to promote premature senescence. Like oncogenic ras, PML increased the levels of p16, hypophosphorylated Rb, phosphoserine-15 p53, and expression of p53 transcriptional targets. The fraction of Rb and p53 that colocalized with PML markedly increased during ras-induced arrest, and expression of PML alone forced p53 to the PODs. E1A abolished PML-induced arrest and prevented PML induction and p53 phosphorylation in response to oncogenic ras. These results imply that PML acts with Rb and p53 to promote ras-induced senescence and provide new insights into PML regulation and activity.

Distribution of hammerhead and hammerhead-like RNA motifs through the GenBank.

Hammerhead ribozymes previously were found in satellite RNAs from plant viroids and in repetitive DNA from certain species of newts and schistosomes. To determine if this catalytic RNA motif has a wider distribution, we decided to scrutinize the GenBank database for RNAs that contain hammerhead or hammerhead-like motifs. The search shows a widespread distribution of this kind of RNA motif in different sequences suggesting that they might have a more general role in RNA biology. The frequency of the hammerhead motif is half of that expected from a random distribution, but this fact comes from the low CpG representation in vertebrate sequences and the bias of the GenBank for those sequences. Intriguing motifs include those found in several families of repetitive sequences, in the satellite RNA from the carrot red leaf luteovirus, in plant viruses like the spinach latent virus and the elm mottle virus, in animal viruses like the hepatitis E virus and the caprine encephalitis virus, and in mRNAs such as those coding for cytochrome P450 oxidoreductase in the rat and the hamster.

Expressing active ribozymes in cells.

Artificially engineered ribozymes can be used to specifically regulate expression of target genes. Such ribozymes can be synthesized chemically and delivered into the cell exogeneously. Alternatively, ribozymes can be produced by the cell endogenously, after introduction of the artificial gene into the cellular genome. In the latter case, the design of the artificial gene defines the ribozyme properties, such as: expression level, intracellular localization, folding and association with proteins. Generally speaking, design of the expression vector is critical to obtain active ribozyme molecules. This paper first describes factors that are known or predicted to affect ribozyme activity in the cell, then reviews various expression systems that have been specifically developed for ribozymes. Lastly, a recently developed ribozyme system termed snorbozymes (small nucleolar RNA:ribozyme hybrids) will be discussed. This powerful test system has generated several important observations that are likely to affect the future development of ribozyme technology.

The distribution of RNA motifs in natural sequences.

Functional analysis of genome sequences has largely ignored RNA genes and their structures. We introduce here the notion of 'ribonomics' to describe the search for the distribution of and eventually the determination of the physiological roles of these RNA structures found in the sequence databases. The utility of this approach is illustrated here by the identification in the GenBank database of RNA motifs having known binding or chemical activity. The frequency of these motifs indicates that most have originated from evolutionary drift and are selectively neutral. On the other hand, their distribution among species and their location within genes suggest that the destiny of these motifs may be more elaborate. For example, the hammerhead motif has a skewed organismal presence, is phylogenetically stable and recent work on a schistosome version confirms its in vivo biological activity. The under-representation of the valine-binding motif and the Rev-binding element in GenBank hints at a detrimental effect on cell growth or viability. Data on the presence and the location of these motifs may provide critical guidance in the design of experiments directed towards the understanding and the manipulation of RNA complexes and activities in vivo.

A small nucleolar RNA:ribozyme hybrid cleaves a nucleolar RNA target in vivo with near-perfect efficiency.

A hammerhead ribozyme has been localized to the yeast nucleolus by using the U3 small nucleolar RNA as a carrier. The hybrid small nucleolar RNA:ribozyme, designated a "snorbozyme," is metabolically stable and cleaves a target U3 RNA with nearly 100% efficiency in vivo. This is the most efficient in vivo cleavage reported for a trans-acting ribozyme. A key advantage of the model substrate featured is that a stable, trimmed cleavage product accumulates. This property allows accurate kinetic measurements of authentic cleavage in vivo. The system offers new avenues for developing effective ribozymes for research and therapeutic applications.

E1A signaling to p53 involves the p19(ARF) tumor suppressor.

The adenovirus E1A oncogene activates p53 through a signaling pathway involving the retinoblastoma protein and the tumor suppressor p19(ARF). The ability of E1A to induce p53 and its transcriptional targets is severely compromised in ARF-null cells, which remain resistant to apoptosis following serum depletion or adriamycin treatment. Reintroduction of p19(ARF) restores p53 accumulation and resensitizes ARF-null cells to apoptotic signals. Therefore, p19(ARF) functions as part of a p53-dependent failsafe mechanism to counter uncontrolled proliferation. Synergistic effects between the p19(ARF) and DNA damage pathways in inducing p53 may contribute to E1A's ability to enhance radio- and chemosensitivity.

Schistosome satellite DNA encodes active hammerhead ribozymes.

Using a computer program designed to search for RNA structural motifs in sequence databases, we have found a hammerhead ribozyme domain encoded in the Smalpha repetitive DNA of Schistosoma mansoni. Transcripts of these repeats are expressed as long multimeric precursor RNAs that cleave in vitro and in vivo into unit-length fragments. This RNA domain is able to engage in both cis and trans cleavage typical of the hammerhead ribozyme. Further computer analysis of S. mansoni DNA identified a potential trans cleavage site in the gene coding for a synaptobrevin-like protein, and RNA transcribed from this gene was efficiently cleaved by the Smalpha ribozyme in vitro. Similar families of repeats containing the hammerhead domain were found in the closely related Schistosoma haematobium and Schistosomatium douthitti species but were not present in Schistosoma japonicum or Heterobilharzia americana, suggesting that the hammerhead domain was not acquired from a common schistosome ancestor.

Amber suppression in Escherichia coli by unusual mitochondria-like transfer RNAs.

The "cloverleaf" base-pairing pattern was established as the structural paradigm of active tRNA species some 30 years ago. Nevertheless, this pattern does not accommodate the folding of certain mitochondrial tRNAs. For these recalcitrant tRNAs, we have proposed structures having from 5 to 10 base pairs in the anticodon stem rather than the canonical 6. The absence of these types of tRNAs in cytoplasmic translation systems, however, raises the possibility that they may not be bona fide alternate folding patterns for active tRNA molecules. For this reason, we have designed new tRNA genes based on our model of unusual mitochondrial tRNAs, having 7, 8, 9, and 10 base pairs in the anticodon stem with other modifications to the D-stem and connector regions. We show here that these synthetic genes produce tRNAs that actively suppress amber codons in vivo.

PolI-driven integrative expression vectors for yeast.

A novel expression vector for yeast has been constructed from the regulatory elements present in the polI promoter and the enhancer/termination region (E/T) of rDNA. Under some conditions, this promoter/vector combination produces small RNAs such as the hammerhead RNA sequence at levels comparable to polII- and polIII-dependent systems. No stable transcription product can be demonstrated with this vector when the enhancer/termination sequence is less than 100 nucleotides downstream from the promoter. On the other hand, high expression of a stable, hammerhead RNA molecule can be obtained from this vector by inserting a 400-bp fragment containing the ADH1 transcription termination region upstream of the E/T. RNAs produced by this vector are polyadenylated and multiple copies of this plasmid can be stably integrated into the yeast chromosome.

Efficient hammerhead ribozyme and antisense RNA targeting in a slow ribosome Escherichia coli mutant.

We have evaluated inhibition of the plasmid-born chloramphenicol acetyl transferase gene (CAT) by the hammerhead ribozyme and antisense RNA in Escherichia coli where the translation and transcription rates have been modified. Whereas neither antisense nor the hammerhead had an inhibitory effect on CAT activity in wild-type E. coli, both reduced the level of the messenger RNA and the activity of the CAT gene by almost 60% in a slow ribosome mutant. Streptomycin, which increases the speed of translation in this mutant strain, restored full CAT activity. The level of CAT activity expressed from a T7 RNA polymerase promoter was not affected by the presence of either antisense RNA or the hammerhead ribozyme. When the target gene was expressed from a chromosomal locus in wild-type E. coli, both antisense RNA and the hammerhead ribozyme showed some inhibitory activity, but the level of inhibition was significantly increased in the slow ribosome strain. This bacterial system offers a unique entry to the study of cellular factors which mediate the activity of ribozymes in vivo.

SnoRNAs as tools for RNA cleavage and modification.

Eukaryotic small nucleolar RNAs (snoRNAs) influence rRNA synthesis at two stages: nucleolytic processing and selection of nucleotides to be ribose methylated (Nm) or converted to pseudouridine (psi). The two modification functions and some processing activities involve direct base pairing of snoRNA with rRNA. In addition to rRNA-targeting sequences, snoRNA function depends on the presence of conserved box elements involved in snoRNA synthesis and localization. The present investigation is directed at using snoRNAs as tools for two purposes: 1) introducing nucleotide modifications into novel sites in rRNA and other snoRNAs, and: 2) targeting nucleolar RNAs for destruction using snoRNA:ribozyme chimers ('snorbozymes'). Early results demonstrate that snoRNAs can be used for both applications.

Stimulation of mitotic recombination upon transcription from the yeast GAL1 promoter but not from other RNA polymerase I, II and III promoters.

Homologous recombination in Saccharomyces cerevisiae and other organisms can be stimulated by transcription. Consistent with this, we find that recombination of a chromosomal ade1 allele with a plasmid-borne ADE1 ORF under the control of the GAL1 promoter increased from 6.1x10(-6) to 1.7x10(-4) when transcription of the plasmid locus was induced by growing the cells in the presence of galactose. Recombination could also be stimulated by over-expressing the Gal4 transcription factor in the presence of the GAL1-ADE1 plasmid, while culturing the cells in dextrose medium. However, when transcription of the same ORF was driven from the highly active promoters of the rDNA (RNA polymerase I), and ADH1 (RNA polymerase II) genes, only background levels of recombination (5-10x10(-6)) were observed, irrespective of the carbon source. Recombination was found to involve integration of the whole plasmid and to depend on RAD51, RAD52 and RAD54. The results indicate that increased accessibility of transcriptionally active chromatin is not sufficient to cause increased rates of this kind of reciprocal exchange.