We analyzed 28 thymic epithelial tumors (TETs) using next-generation sequencing and identified a missense mutation (chromosome 7 c.74146970T>A) in GTF2I at high frequency in type A thymomas, a relatively indolent subtype. In a series of 274 TETs, we detected the GTF2I mutation in 82% of type A and 74% of type AB thymomas but rarely in the aggressive subtypes, where recurrent mutations of known cancer genes have been identified. Therefore, GTF2I mutation correlated with better survival. GTF2I β and δ isoforms were expressed in TETs, and both mutant isoforms were able to stimulate cell proliferation in vitro. Thymic carcinomas carried a higher number of mutations than thymomas (average of 43.5 and 18.4, respectively). Notably, we identified recurrent mutations of known cancer genes, including TP53, CYLD, CDKN2A, BAP1 and PBRM1, in thymic carcinomas. These findings will complement the diagnostic assessment of these tumors and also facilitate development of a molecular classification and assessment of prognosis and treatment strategies.

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

Previous studies from this laboratory have shown that human TS mRNA translation is regulated by its protein product in a negative autoregulatory manner. In this paper, we identify an RNA binding site for TS protein located within the first 188 nt of TS RNA. A 36-nt RNA sequence contained within this 188-nt fragment, corresponding to nt 75-110 and including the translational initiation site, binds TS protein with an affinity similar to that of both the full-length and the 188-nt TS RNA sequences. Variant RNAs with either a deletion or a mutation at the translational initiation region are unable to compete for TS protein binding. UV crosslinking studies reveal that an RNA fragment of "36 nt is protected from RNase Ti digestion by TS protein binding. A second TS protein-binding site is localized within the protein-coding region corresponding to nt 434-634. These findings demonstrate a specific interaction between human TS protein and its TS RNA and identify an RNA binding site that includes the translational initiation site.Thymidylate synthase (TS; EC 2.1.1.45) catalyzes the conversion of deoxyuridine monophosphate (dUMP) and 5,10-methylenetetrahydrofolate to thymidine monophosphate (dTMP) and dihydrofolate (1). Because this enzymatic reaction provides the sole de novo intracellular source of dTMP, TS is a critical therapeutic target enzyme in cancer chemotherapy (2, 3).In mammalian cells, the study of TS gene regulation has mainly focused on cell cycle-directed events. The increase in TS enzyme expression that accompanies entry into the S phase of the cell cycle appears to be regulated at both the transcriptional and the posttranscriptional level (4-9). More recently, Jolliff et al. (10) protein end product, TS, in an autoregulatory manner (13). Using an in vitro rabbit reticulocyte lysate system, we observed that recombinant human TS inhibited translation of TS mRNA. Preliminary studies using a gel mobility-shift assay confirmed binding of recombinant human TS to its corresponding mRNA.Here we provide evidence that TS protein binds specifically to TS RNA sequences in vitro. Two binding sites for TS have been identified, one located within the initial 188 nt of the TS RNA and the other between nt 434 and 634. Further studies reveal that a 35-nt RNA oligomer containing the TS translational initiation sequence retains the ability to specifically bind TS. MATERIALS AND METHODS Preparation of Plasmid Constructs and in Vitro mRNATranscripts. Full-length TS RNA transcript was synthesized with SP6 RNA polymerase (13). Full-length antisense TS RNA transcript was synthesized with T7 RNA polymerase after linearization of pcEHTS with Sst I. The pcEHTS-PST RNA (PST) transcript was synthesized after linearization of pcEHTS with Pst I. The 113-bp Pst I-EcoRI fragment from pcEHTS was cloned into the Pst I and EcoRI sites of pGEM-4Z (Promega), and pcEHTS-PST/EcoRI (PSTEcoRI) RNA was synthesized with SP6 RNA polymerase after linearization with HindIII. The 133-bp EcoRI-Ava I fragment from pcEHTS was cloned into the EcoRI...

Lung cancer is the number one cancer killer, and metastasis is the main cause of high mortality in lung cancer patients. However, mechanisms underlying the development of lung cancer metastasis remain unknown. Using genome-wide transcriptional analysis in an experimental metastasis model, we identified laminin γ2 (LAMC2), an epithelial basement membrane protein, to be significantly upregulated in lung adenocarcinoma metastatic cells. Elevated LAMC2 increased traction force, migration, and invasion of lung adenocarcinoma cells accompanied by the induction of epithelial-mesenchymal transition (EMT). LAMC2 knockdown decreased traction force, migration, and invasion accompanied by EMT reduction in vitro, and attenuated metastasis in mice. LAMC2 promoted migration and invasion via EMT that was integrin β1-and ZEB1-dependent. High LAMC2 was significantly correlated with the mesenchymal marker vimentin expression in lung adenocarcinomas, and with higher risk of recurrence or death in patients with lung adenocarcinoma. We suggest that LAMC2 promotes metastasis in lung adenocarcinoma via EMT and may be a potential therapeutic target. Cell Death and Differentiation (2015) 22, 1341-1352; doi:10.1038/cdd.2014.228; published online 16 January 2015Lung cancer is the leading cause of cancer-related death. 1 Non-small-cell lung cancer (NSCLC) accounts for~80-85% of lung cancers. 2 Only 20-30% of NSCLC are radically resectable and the majority of lung cancer patients succumb to the disease. The high mortality of NSCLC is largely attributable to late diagnosis, when metastases are present. 2 The molecular mechanisms governing metastasis of NSCLC remain poorly understood.Metastasis is a complex, multistep process, involving migration and invasion of malignant cells from the primary tumor to blood vessels, intravasation, and survival in the circulation, and ultimately extravasation, colonization, and formation of secondary tumor at distant target organs. 3 Each of these events is classically driven by the acquisition of genetic and/or epigenetic traits in tumor cells and the cooperation of nonneoplastic stromal cells. Increased or decreased expression of several genes in tumors has been found to be correlated with metastasis. 3 However, specific gene alterations that drive NSCLC metastasis remain largely elusive.Laminin γ2 (LAMC2) is a subunit of the heterotrimeric glycoprotein laminin-332 (LAM-332, formerly laminin-5), consisting of the α3, β3, and γ2 chains. Although LAMC2 is an important structural component of the epithelial basement membrane (BM) in various normal tissues, 4 there is an emerging evidence for a pathological role of LAMC2 monomer in cancer. It has been demonstrated that LAMC2 protein expression correlates with clinical outcome of stage I lung adenocarcinoma (ADC) patients. 5,6 In addition, noncontinuous expression pattern of LAMC2 predicts the prognosis of esophageal squamous cell carcinoma (SCC), 7 and secreted LAMC2 in the serum is associated with the aggressiveness of pancreatic cancer. 8 Besides, LAMC2 monom...

Dihydrofolate reductase (DHFR) is a critical enzyme in de novo purine and thymidylate biosynthesis. An RNA gel mobility shift assay was used to demonstrate a specific interaction between human recombinant DHFR protein and its corresponding DHFR mRNA. Incubation of DHFR protein with either its substrates, dihydrofolate or NADPH, or with an inhibitor, methotrexate, repressed its ability to interact with DHFR mRNA. An in vitro rabbit reticulocyte lysate translation system was used to show that the addition of exogenous human recombinant DHFR protein to in vitro translation reactions specifically inhibited DHFR mRNA translation. These studies suggest that the direct interaction between DHFR protein and its mRNA may be a mechanism for regulation of DHFR synthesis.

Thymidylate synthase (TS) catalyzes the reductive methylation of dUMP by 5,10-methylenetetrahydrofolate to generate thymidylate and dihydrofolate. This enzymatic reaction provides for the sole intracellular de novo source of thymidylate, an essential precursor for DNA biosynthesis. As a result, TS remains a critical target enzyme in cancer chemotherapy (25, 60a).In addition to its role in enzyme catalysis, there is evidence that TS also functions as an RNA binding protein (5-7). Studies from this laboratory have demonstrated that the translation of human TS mRNA is regulated by its own protein product via a negative autoregulatory mechanism (5). The repression of TS mRNA translation by TS is mediated by specific binding of the protein to at least two distinct cis-acting sequences on its own mRNA (6). The first site corresponds to a 188-nucleotide (nt) sequence that includes the translational start site, while the second site is contained within a 100-nt sequence in the protein-coding region. However, in the presence of the nucleotide substrate dUMP or 5-fluoro-2Ј-deoxyuridine-5Ј-monophosphate (FdUMP), TS is unable to directly interact with its own mRNA, thus allowing for the synthesis of new protein to proceed (5, 7). Several in vitro studies have shown that shortterm exposure of human colon and breast cancer cells to TSinhibitory compounds, such as 5-fluorouracil (5-FU) or the antifolate analog ZD1694, is associated with an increased level of TS protein expression but no corresponding change in the levels of TS mRNA (8,9,35). These findings are consistent with earlier observations that both the RNA binding and the translational inhibition activities of TS are impaired in the presence of either nucleotide and/or folate substrates. Thus, the ability to regulate the expression of TS at the translational level in the setting of acute cytotoxic stress suggests that this regulatory event has biological relevance. Moreover, this process may represent an important mechanism by which normal cellular synthetic function can be controlled and a mechanism for the rapid development of cellular resistance in response to exposure to nucleotide inhibitors of TS, such as 5-FU, and antifolate inhibitors of TS, such as ZD1694, LY231514, and AG331.An immunoprecipitation-reverse transcription (RT)-PCR technique was recently developed to isolate from an intact cultured human colon cancer cell line a TS-ribonucleoprotein (RNP) complex made up of TS protein and TS mRNA (10). In addition to complexing with its own mRNA, TS formed an RNP complex with the mRNA of the c-myc transcription factor. Subsequent studies with RNA electrophoretic gel mobility shift assays (EMSAs) confirmed that the interaction between TS and c-myc mRNA was specific and identified the C-terminal coding region as being an important cis-acting regulatory element (11). Furthermore, in vitro translation experiments demonstrated that TS protein specifically repressed the translation of c-myc mRNA (11). Recent work has shown that TS is * Corresponding author. Mailing ad...

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.Recently, there has been an increased interest in the characterization of translational regulatory mechanisms. There are a number of eukaryotic mRNAs whose expression is controlled at the level of translation (18). The regulated synthesis of the iron storage protein ferritin by iron represents one of the best-studied examples of this form of regulation (25). A stem-loop structure located within the 5' untranslated region (UTR) of ferritin mRNA, termed the iron-responsive element (IRE), represents the cis-acting element to which the IRE-binding protein binds (16,23). Recent studies have demonstrated that the redox state in the cell is an important determinant of the binding affinity of this protein to the IRE (15,17).Using an in vitro translation system, we showed that translation of human thymidylate synthase (TS) mRNA is regulated by its own protein product, TS, in a negative autoregulatory manner (7). Although translational autoregulation has been described in prokaryotic systems (2, 5), TS mRNA represents the first eukaryotic mRNA whose regulation is controlled in such a fashion. Furthermore, we demonstrated that incubation of TS protein with either the nucleotide substrate dUMP or the inhibitor 5-fluoro-dUMP repressed its inhibitory effect on TS mRNA translation. during the cell cycle is primarily regulated at the transcriptional level (1,19,30), there is now recent evidence suggesting control at the level of translation (22). These findings, taken together, offer supportive evidence for the model of TS translational autoregulation.The purpose of the present study was to identify a TS ribonucleoprotein (RNP) complex in a cultured cell system. With the use of specific antisera to TS, we show that TS protein is complexed with its corresponding TS RNA in human colon cancer cells. In addition, we present evidence demonstrating a specific interaction between the mRNA of the nuclear oncogene c-myc and TS protein. MATERIALS AND METHODSCell culture. The characteristics of the human colon cancer cell line H630 have been previously described (32). The resistant H630-R1O subline was selected in vitro for resistance to 5-FU by exposure of the parent H630 cell line to stepwise increases in 5-FU and was maintained in medium containing 10 p,. Cell lines were grown in 75-cm2 plastic tissue culture flasks (Falcon Labware, Oxnard, Calif.) in growth medium containing RPMI 1640 with 1...

The role of microRNAs in small-cell lung carcinoma (SCLC) is largely unknown. miR-34a is known as a p53 regulated tumor suppressor microRNA in many cancer types. However, its therapeutic implication has never been studied in SCLC, a cancer type with frequent dysfunction of p53. We investigated the expression of a panel of 7 microRNAs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in 31 SCLC tumors, 14 SCLC cell lines, and 26 NSCLC cell lines. We observed significantly lower miR-21, miR-29b, and miR-34a expression in SCLC cell lines than in NSCLC cell lines. The expression of the 7 microRNAs was unrelated to SCLC patients' clinical characteristics and was neither prognostic in term of overall survival or progression-free survival nor predictive of treatment response. Overexpression or downregulation of miR-34a did not influence SCLC cell viability. The expression of these 7 microRNAs also did not predict in vitro sensitivity to cisplatin or etoposide in SCLC cell lines. Overexpression or downregulation of miR-34a did not influence sensitivity to cisplatin or etoposide in SCLC cell lines. In contrast to downregulation of the miR-34a target genes cMET and Axl by overexpression of miR-34a in NSCLC cell lines, the intrinsic expression of cMET and Axl was low in SCLC cell lines and was not influenced by overexpression of miR-34a. Our results suggest that the expression of the 7 selected microRNAs are not prognostic in SCLC patients, and miR-34a is unrelated to the malignant behavior of SCLC cells and is unlikely to be a therapeutic target.