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Previous studies from our laboratory showed that p21Cip1/WAF1 can be phosphorylated by Pim-1 kinase in vitro, implying that part of the function of Pim-1 might involve influencing the cell cycle. In the present study, site-directed mutagenesis and phosphorylated-specific antibodies were used as tools to identify the sites phosphorylated by Pim-1 and the consequences of this phosphorylation. What , it localizes primarily in the cytoplasm and the effect of phosphorylation on stability is minimal. Cotransfection of wild-type Pim-1 with p21 increases the rate of proliferation compared with cotransfection of p21 with kinase-dead Pim-1. Knocking down Pim-1 expression greatly decreases the rate of proliferation of H1299 cells and their ability to grow in soft agar. These data suggest that Pim-1 overexpression may contribute to tumorigenesis in part by influencing the cellular localization and stability of p21 and by promoting cell proliferation. (Mol Cancer Res 2007;5(9):909 -22)

In this report, we employed a lentiviral RNA interference screen to discover nucleolar DEAD/DEAH-box helicases involved in RNA polymerase I (Pol I)-mediated transcriptional activity. Our screen identified DHX33 as an important modulator of 47S rRNA transcription. We show that DHX33 is a cell cycle-regulated nucleolar protein that associates with ribosomal DNA (rDNA) loci, where it interacts with the RNA Pol I transcription factor upstream binding factor (UBF). DHX33 knockdown decreased the association of Pol I with rDNA and caused a dramatic decrease in levels of rRNA synthesis. Wild-type DHX33 overexpression, but not a DNA binding-defective mutant, enhanced 47S rRNA synthesis by promoting the association of RNA polymerase I with rDNA loci. In addition, an NTPase-defective DHX33 mutant (K94R) acted as a dominant negative mutant, inhibiting endogenous rRNA synthesis. Moreover, DHX33 deficiency in primary human fibroblasts triggered a nucleolar p53 stress response, resulting in an attenuation of proliferation. Thus, we show the mechanistic importance of DHX33 in rRNA transcription and proliferation.RNA is a highly structured macromolecule whose secondary and tertiary conformations facilitate an array of specific interactions with proteins. The DEAD/DEAH-box family of RNA helicases (here referred to as DDX/DHX) (3) is one such classification of RNA binding proteins that are capable of modifying the higher-ordered structures of RNA through the hydrolysis of ATP/nucleoside triphosphate (NTP) (41). DDX/ DHX proteins often form large multiprotein complexes that participate in fundamental biological activities such as RNA transcription, RNA editing, pre-mRNA splicing, ribosome biogenesis, and RNA decay (3).DDX/DHX helicases are named and characterized by the conserved DEAD/DEAH motif common among all family members. Through site-directed mutagenesis analysis, DEAD/ DEAH along with seven conserved peptide motifs have been found to participate in ATP/NTP binding, hydrolysis, and substrate binding (28). Despite the conservation of these peptide motifs, the remaining sequences within each RNA helicase family member vary widely. Specifically, differences exist between the two categories of DDX and DHX proteins. DDX proteins contain a unique Q motif at their N termini that distinguishes them from DHX proteins. It was proposed previously that the Q motif might sense the state of ATP in vivo (40), given that DHX-box proteins are promiscuous in their ability to utilize NTP (16).Ribosome biogenesis is a complex multistep process, the majority of which occurs in the nucleolus of the cell (24, 43). The transcription of ribosomal DNA (rDNA) is the initial and rate-limiting step in ribosome biogenesis, and as such, it is influenced by multiple levels of regulation (25). One of the key regulators of rDNA transcription is the upstream binding factor (UBF), a transcriptional transactivator that binds to the upstream core element of rDNA and subsequently bends rDNA (37). This change in the rDNA structure favors the binding of SL.1 as wel...

IntroductionThe DDX21 RNA helicase has been shown to be a nucleolar and nuclear protein involved in ribosome RNA processing and AP-1 transcription. DDX21 is highly expressed in colon cancer, lymphomas, and some breast cancers, but little is known about how DDX21 might promote tumorigenesis.MethodsImmunohistochemistry was performed on a breast cancer tissue array of 187 patients. In order to study the subcellular localization of DDX21 in both tumor tissue and tumor cell lines, indirect immunofluorescence was applied. The effect of DDX21 knockdown was measured by cellular apoptosis, rRNA processing assays, soft agar growth and mouse xenograft imaging. AP-1 transcriptional activity was analyzed with a luciferase reporter and bioluminescence imaging, as well as qRT-PCR analysis of downstream target, cyclin D1, to determine the mechanism of action for DDX21 in breast tumorigenesis.ResultsHerein, we show that DDX21 is highly expressed in breast cancer tissues and established cell lines. A significant number of mammary tumor tissues and established breast cancer cell lines exhibit nuclear but not nucleolar localization of DDX21. The protein expression level of DDX21 correlates with cell proliferation rate and is markedly induced by EGF signaling. Mechanistically, DDX21 is required for the phosphorylation of c-Jun on Ser73 and DDX21 deficiency markedly reduces the transcriptional activity of AP-1. Additionally, DDX21 promotes rRNA processing in multiple breast cancer cell lines. Tumor cells expressing high levels of endogenous DDX21 undergo apoptosis after acute DDX21 knockdown, resulting in significant reduction of tumorigenicity in vitro and in vivo.ConclusionsOur findings indicate that DDX21 expression in breast cancer cells can promote AP-1 activity and rRNA processing, and thus, promote tumorigenesis by two independent mechanisms. DDX21 could serve as a marker for a subset of breast cancer patients with higher proliferation potential and may be used as a therapeutic target for a subset of breast cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0449-z) contains supplementary material, which is available to authorized users.

Pim-2 kinase is one of three highly conserved Pim family members which are known to be involved in cell survival and cell proliferation. Here we demonstrate that like Pim-1, Pim-2 also phosphorylates the cell cycle inhibitor p21 Cip1/WAF1 (p21) on Thr145 in vitro and in vivo. Overexpression of Pim-2 in HCT116 cells leads to the increased stability of p21, and results in enhanced levels of both exogenous and endogenous p21 proteins. Knock-down of Pim-2 expression via siRNA results in reduced level of endogenous p21, indicating that like Pim-1, Pim-2 is another legitimate p21 kinase. However, Pim-2 has no influence on the nuclear localization of p21 in HCT116 cells. In addition, Pim-2 is able to arrest the cell cycle at G1/S phase and inhibit cell proliferation through phosphorylation of p21 in HCT116 cells. These data suggest that Pim-2 phosphorylation of p21 enhances p21's stability and inhibits cell proliferation in HCT116 cells.

The RNA helicase DHX33 has been shown to be a critical regulator of cell proliferation and growth. However, the underlying mechanisms behind DHX33 function remain incompletely understood. We present original evidence in multiple cell lines that DHX33 transcriptionally controls the expression of genes involved in the cell cycle, notably cyclin, E2F1, cell division cycle (CDC), and minichromosome maintenance (MCM) genes. DHX33 physically associates with the promoters of these genes and controls the loading of active RNA polymerase II onto these promoters. DHX33 deficiency abrogates cell cycle progression and DNA replication and leads to cell apoptosis. In zebrafish, CRISPR-mediated knockout of DHX33 results in downregulation of cyclin A2, cyclin B2, cyclin D1, cyclin E2, cdc6, cdc20, E2F1, and MCM complexes in DHX33 knockout embryos. Additionally, we found the overexpression of DHX33 in a subset of non-small-cell lung cancers and in Ras-mutated human lung cancer cell lines. Forced reduction of DHX33 in these cancer cells abolished tumor formation in vivo. Our study demonstrates for the first time that DHX33 acts as a direct transcriptional regulator to promote cell cycle progression and plays an important role in driving cell proliferation during both embryo development and tumorigenesis.

b DEAD/DEAH box RNA helicases play essential roles in numerous RNA metabolic processes, such as mRNA translation, premRNA splicing, ribosome biogenesis, and double-stranded RNA sensing. Herein we show that a recently characterized DEAD/ DEAH box RNA helicase, DHX33, promotes mRNA translation initiation. We isolated intact DHX33 protein/RNA complexes in cells and identified several ribosomal proteins, translation factors, and mRNAs. Reduction of DHX33 protein levels markedly reduced polyribosome formation and caused the global inhibition of mRNA translation that was rescued with wild-type DHX33 but not helicase-defective DHX33. Moreover, we observed an accumulation of mRNA complexes with the 80S ribosome in the absence of functional DHX33, consistent with a stalling in initiation, and DHX33 more preferentially promoted structured mRNA translation. We conclude that DHX33 functions to promote elongation-competent 80S ribosome assembly at the late stage of mRNA translation initiation. Our results reveal a newly recognized function of DHX33 in mRNA translation initiation, further solidifying its central role in promoting cell growth and proliferation. Mammalian cells maintain tight control of global mRNA translation through the production of ribosomes (1, 2); deregulation in mRNA translation is frequently found in human diseases (3-6) and is regarded as one of the many factors contributing to cancer development (7-9).Most eukaryotic protein translation initiation occurs by an ordered assembly of a preinitiation complex on the 5= cap of mRNA (10). After mature mRNA is transported into the cytosol, the distinct 5= cap of mRNA is recognized and bound by a large protein complex comprising eukaryotic initiation factor 4E (eIF4E), eIF4A, and eIF4G as well as poly(A)-binding protein (PABP) (1,11,12). These factors coordinately prevent mRNA degradation while priming mRNAs for translation initiation.The initial step in mRNA translation involves formation of a ternary complex between eIF2-GTP, Met-tRNA interference, and small 40S ribosomal subunits. This process is stimulated by the translation initiation factors eIF1, eIF3, eIF4F, and eIF5 (13). This large complex, termed the 43S preinitiation complex, attaches to the activated 5= cap of mRNA. Bound RNA helicases are responsible for unwinding various secondary structures in mRNA as the complex scans along the mRNA from the 5= end to the 3= end until it finds the initiation codon. The 60S large ribosome subunit then joins with the 40S subunit to form an 80S ribosome under guidance from eIF5B-GTP (2, 13). eIF2-GTP and eIF5B-GTP are then hydrolyzed into their GDP forms to promote the assembly of the functional initiation complex (14). The detailed mechanism of how elongation-competent 80S ribosomes are assembled prior to initiation or what triggers initiation is not well understood.Mammalian mRNAs often contain highly structured untranslated regions (UTRs) at the 5= ends of their open reading frame sequences that must be unwound to allow ribosome recruitment and scanning. Not su...

Oncogene c-Myc is frequently amplified and activated in human cancers. Deregulation of c-Myc protein has been shown to occur in 30% of all human cancers, especially in hematopoietic malignancies. As a transcription factor, c-Myc has been shown to regulate up to 15% of all human genome genes, controlling diverse cellular activities including cell cycle, ribosome biogenesis, protein synthesis, metabolism, apoptosis and angiogenesis. In this report, we provide evidence that the RNA helicase DHX33 is a critical downstream target of c-Myc. Myc binds to DHX33 upstream promoter region and stimulates its transcription. Elevated DHX33 protein is pivotal for c-Myc to drive tumor formation. Knockdown of DHX33 to basal levels in c-Myc overexpressing cells significantly reduced cell proliferation, cell migration and anchorage-independent cell growth in vitro and in vivo. Additionally, we found that DHX33 promotes MMP9, MMP14 and urokinase-type plasminogen activator (PLAU) transcription by directly binding to their promoters, thus promoting cancer cell migration. DHX33 protein was overexpressed in a certain subset of human non-Hodgkin's lymphoma tissues. Finally, knockdown of DHX33 significantly inhibits the development of Myc-induced acute myeloid leukemia. Overall, our results implicate the important role for DHX33 in Myc-induced cancer and point toward its potential therapeutic value in Myc driven cancers.