Stc1: a critical link between RNAi and chromatin modification required for heterochromatin integrity.

In fission yeast, RNAi directs heterochromatin formation at centromeres, telomeres, and the mating type locus. Noncoding RNAs transcribed from repeat elements generate siRNAs that are incorporated into the Argonaute-containing RITS complex and direct it to nascent homologous transcripts. This leads to recruitment of the CLRC complex, including the histone methyltransferase Clr4, promoting H3K9 methylation and heterochromatin formation. A key question is what mediates the recruitment of Clr4/CLRC to transcript-bound RITS. We have identified a LIM domain protein, Stc1, that is required for centromeric heterochromatin integrity. Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex. Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi. We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification.

Complete genome sequence of cnidium virus 2, a novel cytorhabdovirus isolated from Cnidium officinale in South Korea.

High-throughput sequencing identified a cytorhabdovirus, tentatively named "cnidium virus 2" (CnV2), in Cnidium officinale, and Sanger sequencing confirmed the genome sequence. CnV2 is 13,527 nucleotides in length and contains seven open reading frames in the order 3'-N-P-3-4-M-G-L-5', separated by intergenic regions. The full-length nucleotide sequence of CnV2 shares 19.4-53.8% identity with other known cytorhabdovirus genome sequences. The N, P, P3, M, G, and L proteins share 15.8-66.7%, 11-64.3%, 11.1-80.5%, 10.8-75.3%, 12.3-72.1%, and 20-72.7% amino acid sequence identity, respectively, with the cognate deduced protein sequences from known cytorhabdoviruses. CnV2 is related to other members of the genus Cytorhabdovirus, with sambucus virus 1 being the closest relative. Thus, CnV2 should be classified as a new member in the genus Cytorhabdovirus of the family Rhabdoviridae.

Complete genome sequence of a novel member of the genus Polerovirus from Cnidium officinale in South Korea.

The complete genome sequence of a novel virus found infecting Cnidium officinale, which we have named "cnidium polerovirus 1" (CnPV1), is 6,090 nucleotides in length, similar to those of other poleroviruses. Seven open reading frames (ORF0-5 and ORF3a) were predicted in this genome. CnPV1 shares 32.4%-38.9% full-length nucleotide sequence identity with other known polerovirus genome sequences. The putative P0, P1-2, P3-5, P3, and P4 proteins share 11.3%-19.5%, 37.1%-49.8%, 26.7%-39.5%, 40.8%-49.7%, and 40.8%-49.7% amino acid sequence identity, respectively, with homologous inferred protein sequences from known poleroviruses. Phylogenetic analysis of P1-2 and P3 sequences places CnPV1 with other members of the genus Polerovirus, indicating that it should be classified in a new distinct species.

Bacteria-derived metabolite, methylglyoxal, modulates the longevity of C. elegans through TORC2/SGK-1/DAF-16 signaling.

Gut microbes play diverse roles in modulating host fitness, including longevity; however, the molecular mechanisms underlying their mediation of longevity remain poorly understood. We performed genome-wide screens using 3,792 Escherichia coli mutants and identified 44 E. coli mutants that modulated Caenorhabditis elegans longevity. Three of these mutants modulated C. elegans longevity via the bacterial metabolite methylglyoxal (MG). Importantly, we found that low MG-producing E. coli mutants, Δhns E. coli, extended the lifespan of C. elegans through activation of the DAF-16/FOXO family transcription factor and the mitochondrial unfolded protein response (UPRmt). Interestingly, the lifespan modulation by Δhns did not require insulin/insulin-like growth factor 1 signaling (IIS) but did require TORC2/SGK-1 signaling. Transcriptome analysis revealed that Δhns E. coli activated novel class 3 DAF-16 target genes that were distinct from those regulated by IIS. Taken together, our data suggest that bacteria-derived MG modulates host longevity through regulation of the host signaling pathways rather than through nonspecific damage on biomolecules known as advanced glycation end products. Finally, we demonstrate that MG enhances the phosphorylation of hSGK1 and accelerates cellular senescence in human dermal fibroblasts, suggesting the conserved role of MG in controlling longevity across species. Together, our studies demonstrate that bacteria-derived MG is a novel therapeutic target for aging and aging-associated pathophysiology.

Co-treatment of birinapant with TRAIL synergistically induces apoptosis by downregulating cFLIP(L) in MDA-MB-453 cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received much attention owing to its ability to specifically induce cell death in cancer. However, several types of cancer, including some forms of breast cancer, are resistant to TRAIL. Various chemotherapeutic agents, phytochemicals, and TRAIL combination therapies have been proposed to resolve TRAIL resistance. Here, we explored the sensitization effect of birinapant on TRAIL-induced apoptosis in the MDA-MB-453 cell line. Although neither birinapant nor TRAIL showed any cytotoxic effect when used alone, apoptosis was induced when birinapant and TRAIL were used together. Our data suggest that the combination of birinapant and TRAIL induces downregulation of FLICE-like inhibitory protein (cFLIP) (L) protein expression. Interestingly, cFLIP(L) overexpression reversed apoptosis caused by co-treatment with TRAIL. Taken together, our results indicate that a combination of birinapant and TRAIL may be a promising treatment for TRAIL-resistant breast cancer.

A randomized, open-label, crossover study to compare the safety and pharmacokinetics of two tablet formulations of tenofovir (tenofovir disoproxil and tenofovir disoproxil fumarate) in healthy subjects.

PURPOSE: This study was performed to compare the pharmacokinetic properties and assess bioequivalence for the test formulation (HUG116 tablet; tenofovir disoproxil) and reference formulation (Viread tablet; tenofovir disoproxil fumarate). MATERIALS AND METHODS: A randomized, open-label, single-dosing, two-treatment, two-period, two-sequence cross-over study was conducted in 50 healthy subjects. All subjects were randomly assigned to one of the two sequences, and they received a single dose of test or reference formulation in the first period and the alternative formulation during the next period under fasting conditions. Serial blood samples for pharmacokinetic evaluation were collected up to 72 hours post dose, and the pharmacokinetic parameters were estimated by noncompartmental methods. Throughout the study, tolerability was assessed based on adverse events, vital signs, and clinical laboratory tests. RESULTS: The test formulation showed similar pharmacokinetic profiles to those of the reference formulation. The geometric mean ratio and 90% confidence interval (CI) of the test formulation to the reference formulation for maximum plasma concentration (Cmax) was 0.93 (0.87 - 0.99), and the corresponding value for the area under the concentration-time curve from time zero to time of last quantifiable concentration (AUCt) was 0.94 (0.89 - 0.99). Both CIs were within the conventional bioequivalence range of 0.8 - 1.25. The tolerability profile was not significantly different between the test and reference formulations. CONCLUSION: This study found that the PKs of the test formulation (HUG116 tablet; tenofovir disoproxil) and reference formulation (Viread tablet; tenofovir disoproxil fumarate) were similar, and the test formulation met the regulatory criteria for assuming bioequivalence with the reference formulation.

Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex.

Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast.

Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed the DNA damage sensitivity and the reduced HR efficiency associated with loss of ddb1(+) or cdt2(+). Furthermore, we demonstrate a role for nucleotide synthesis in postsynaptic gap filling of resected ssDNA ends during HR repair. Finally, we define a role for Rad3 (ATR) in nucleotide synthesis and HR through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which break-induced Rad3 and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.

Genome-wide functional analysis using the barcode sequence alignment and statistical analysis (Barcas) tool.

BACKGROUND: Pooled library screen analysis using shRNAs or CRISPR-Cas9 hold great promise to genome-wide functional studies. While pooled library screens are effective tools, erroneous barcodes can potentially be generated during the production of many barcodes. However, no current tools can distinguish erroneous barcodes from PCR or sequencing errors in a data preprocessing step. RESULTS: We developed the Barcas program, a specialized program for the mapping and analysis of multiplexed barcode sequencing (barcode-seq) data. For fast and efficient mapping, Barcas uses a trie data structure based imperfect matching algorithm which generates precise mapping results containing mismatches, shifts, insertions and deletions (indel) in a flexible manner. Barcas provides three functions for quality control (QC) of a barcode library and distinguishes erroneous barcodes from PCR or sequencing errors. It also provides useful functions for data analysis and visualization. CONCLUSIONS: Barcas is an all-in-one package providing useful functions including mapping, data QC, library QC, statistical analysis and visualization in genome-wide pooled screens.

Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast.

Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs.

A Novel Arginase Inhibitor Derived from Scutellavia indica Restored Endothelial Function in ApoE-Null Mice Fed a High-Cholesterol Diet.

Elevated endothelial arginase activity decreases nitric oxide (NO) production by competing with the substrate l-arginine, previously reported, and reciprocally regulating endothelial nitric oxide synthase (eNOS) activity. Thus, arginase inhibitors may help treat vascular diseases associated with endothelial dysfunction. A screening of metabolites from medicinal plants revealed that (2S)-5,2',5'-trihydroxy-7,8-dimethoxy flavanone (TDF) was a noncompetitive inhibitor of arginase. We investigated whether TDF reciprocally regulated endothelial NO production and its possible mechanism. TDF noncompetitively inhibited arginase I and II activity in a dose-dependent manner. TDF incubation decreased arginase activity and increased NO production in human umbilical vein endothelial cells and isolated mouse aortic vessels and reduced reactive oxygen species (ROS) generation in the endothelium of the latter. These TDF-mediated effects were associated with increased eNOS phosphorylation and dimerization but not with changes in protein content. Endothelium-dependent vasorelaxant responses to acetylcholine (Ach) were significantly increased in TDF-incubated aortic rings and attenuated by incubation with soluble guanylyl cyclase inhibitor. Phenylephrine-induced vasoconstrictor responses were markedly attenuated in TDF-treated vessels from wild-type mice. In atherogenic-prone ApoE(-/-) mice, TDF attenuated the high-cholesterol diet (HCD)-induced increase in arginase activity, which was accompanied by restoration of NO production and reduction of ROS generation. TDF incubation induced eNOS dimerization and phosphorylation at Ser1177. In addition, TDF improved Ach-dependent vasorelaxation responses and attenuated U46619-dependent contractile responses but did not change sodium nitroprusside-induced vasorelaxation or N-NAME-induced vasoconstriction. The findings suggest that TDF may help treat cardiovascular diseases by reducing pathophysiology derived from HCD-mediated endothelial dysfunction.

The CCR4-NOT complex is implicated in the viability of aneuploid yeasts.

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability.

Construction of the first compendium of chemical-genetic profiles in the fission yeast Schizosaccharomyces pombe and comparative compendium approach.

Genome-wide chemical genetic profiles in Saccharomyces cerevisiae since the budding yeast deletion library construction have been successfully used to reveal unknown mode-of-actions of drugs. Here, we introduce comparative approach to infer drug target proteins more accurately using two compendiums of chemical-genetic profiles from the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe. For the first time, we established DNA-chip based growth defect measurement of genome-wide deletion strains of S. pombe, and then applied 47 drugs to the pooled heterozygous deletion strains to generate chemical-genetic profiles in S. pombe. In our approach, putative drug targets were inferred from strains hypersensitive to given drugs by analyzing S. pombe and S. cerevisiae compendiums. Notably, many evidences in the literature revealed that the inferred target genes of fungicide and bactericide identified by such comparative approach are in fact the direct targets. Furthermore, by filtering out the genes with no essentiality, the multi-drug sensitivity genes, and the genes with less eukaryotic conservation, we created a set of drug target gene candidates that are expected to be directly affected by a given drug in human cells. Our study demonstrated that it is highly beneficial to construct the multiple compendiums of chemical genetic profiles using many different species. The fission yeast chemical-genetic compendium is available at http://pombe.kaist.ac.kr/compendium.

8-Methoxypsoralen Induces Apoptosis by Upregulating p53 and Inhibits Metastasis by Downregulating MMP-2 and MMP-9 in Human Gastric Cancer Cells.

Furanocoumarin 8-methoxypsoralen (8-MOP) is the parent compound that naturally occurs in traditional medicinal plants used historically. 8-MOP has been employed as a photochemotherapeutic component of Psoralen + Ultraviolet A (PUVA) therapy for the treatment of vitiligo and psoriasis. Although the role of 8-MOP in PUVA therapy has been studied, little is known about the effects of 8-MOP alone on human gastric cancer cells. In this study, we observed anti-proliferative effect of 8-MOP in several human cancer cell lines. Among these, the human gastric cancer cell line SNU1 is the most sensitive to 8-MOP. 8-MOP treated SNU1 cells showed G1-arrest by upregulating p53 and apoptosis by activating caspase-3 in a dose-dependent manner, which was confirmed by loss-of-function analysis through the knockdown of p53-siRNA and inhibition of apoptosis by Z-VAD-FMK. Moreover, 8-MOPinduced apoptosis is not associated with autophagy or necrosis. The signaling pathway responsible for the effect of 8-MOP on SNU1 cells was confirmed to be related to phosphorylated PI3K, ERK2, and STAT3. In contrast, 8-MOP treatment decreased the expression of the typical metastasis-related proteins MMP-2, MMP-9, and Snail in a p53-independent manner. In accordance with the serendipitous findings, treatment with 8-MOP decreased the wound healing, migration, and invasion ability of cells in a dose-dependent manner. In addition, combination treatment with 8-MOP and gemcitabine was effective at the lowest concentrations. Overall, our findings indicate that oral 8-MOP has the potential to treat early human gastric cancer, with fewer side effects.

Soluble CD93 induces differentiation of monocytes and enhances TLR responses.

The cell surface protein CD93 is known to be involved in the regulation of phagocytosis and cell adhesion. Although typically membrane-bound, a soluble form of CD93 (sCD93) has recently been identified. Currently, however, the role of sCD93 in monocyte function is unknown. In the current study, we analyzed the functional effects of sCD93 on THP-1 monocytic cells and human primary monocytes. Various forms of recombinant human sCD93 were used to investigate the effects of this molecule on both human primary monocytes and a monocytic cell line, THP-1. We found that sCD93 induced differentiation of monocytes to macrophage-like cells, as evidenced by activated cell adhesion and increased phagocytic activities. In addition, this differentiation resulted in an enhanced response to TLR stimulation in terms of differentiation marker expression and proinflammatory cytokine production. Furthermore, sCD93 enhanced LPS-stimulated TNF-α production even prior to monocyte differentiation. To investigate a possible role for sCD93 in the pathogenesis of chronic inflammatory diseases, we assessed the concentration of sCD93 in synovial fluid from patients with rheumatoid arthritis and found it to be significantly increased compared with synovial fluid from patients with osteoarthritis. Together, these data revealed a function for sCD93 that may have implications in inflammation and inflammatory diseases including rheumatoid arthritis.

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4.

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H(2)O(2)-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.

Significant conservation of synthetic lethal genetic interaction networks between distantly related eukaryotes.

Synthetic lethal genetic interaction networks define genes that work together to control essential functions and have been studied extensively in Saccharomyces cerevisiae using the synthetic genetic array (SGA) analysis technique (ScSGA). The extent to which synthetic lethal or other genetic interaction networks are conserved between species remains uncertain. To address this question, we compared literature-curated and experimentally derived genetic interaction networks for two distantly related yeasts, Schizosaccharomyces pombe and S. cerevisiae. We find that 23% of interactions in a novel, high-quality S. pombe literature-curated network are conserved in the existing S. cerevisiae network. Next, we developed a method, called S. pombe SGA analysis (SpSGA), enabling rapid, high-throughput isolation of genetic interactions in this species. Direct comparison by SpSGA and ScSGA of approximately 220 genes involved in DNA replication, the DNA damage response, chromatin remodeling, intracellular transport, and other processes revealed that approximately 29% of genetic interactions are common to both species, with the remainder exhibiting unique, species-specific patterns of genetic connectivity. We define a conserved yeast network (CYN) composed of 106 genes and 144 interactions and suggest that this network may help understand the shared biology of diverse eukaryotic species.

CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells.

Tamoxifen (TAM) is a selective estrogen receptor modulator used for breast cancer patients. Prolonged use of tamoxifen is not recommended for some patients. In this study, we aimed to identify molecular targets sensitive to TAM using a genome-wide gene deletion library screening of fission yeast heterozygous mutants. From the screening, casein kinase 1 gamma 2 (CSNK1G2), a serine-/threonine protein kinase, was the most sensitive target to TAM with a significant cytotoxicity in estrogen receptor-positive (ER+) breast cancer cells but with only a slight toxicity in the case of ER- cells. In addition, tumor sphere formation and expression of breast stem cell marker genes such as CD44/CD2 were greatly inhibited by CSNK1G2 knockdown in ER+ breast cancer cells. Consistently, CSNK1G2 altered ERα activity via phosphorylation, specifically at serine (Ser)167, as well as the regulation of estrogen-responsive element (ERE) of estrogen-responsive genes such as CTSD and GREB1. However, ERα silencing almost completely blocked CSNK1G2-induced TAM sensitivity. In ER+ breast cancer cells, combined treatment with TAM and CSNK1G2 knockdown further enhanced the TAM-mediated decrease in phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K) signaling but not extracellular signal-regulated kinase (ERK) signaling. Inversely, in ER- cells treated with TAM, only ERK and PI3K signaling was altered by CSNK1G2 knockdown. The CK1 inhibitor, D4476, partly mimicked the CSNK1G2 knockdown effect in ER+ breast cancer cells, but with a broader repression ranging from PI3K/AKT/mTOR/S6K to ERK signaling. Collectively, these results suggest that CSNK1G2 plays a key role in sensitizing TAM toxicity in ER+ and ER- breast cancer cells via differently regulating PI3K/AKT/mTOR/S6K and ERK signaling.

G-749 Promotes Receptor Tyrosine Kinase TYRO3 Degradation and Induces Apoptosis in Both Colon Cancer Cell Lines and Xenograft Mouse Models.

G-749 is an FLT3 kinase inhibitor that was originally developed as a treatment for acute myeloid leukemia. Some FLT3 kinase inhibitors are dual kinase inhibitors that inhibit the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase family and are used to treat solid cancers such as non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). AXL promotes metastasis, suppression of immune response, and drug resistance in NSCLC and TNBC. G-749, a potential TAM receptor tyrosine kinase inhibitor, and its derivative SKI-G-801, effectively inhibits the phosphorylation of AXL at nanomolar concentration (IC50 = 20 nM). This study aimed to investigate the anticancer effects of G-749 targeting the TAM receptor tyrosine kinase in colon cancer. Here, we demonstrate the potential of G-749 to effectively inhibit tumorigenesis by degrading TYRO3 via regulated intramembrane proteolysis both in vitro and in vivo. In addition, we demonstrated that G-749 inhibits the signaling pathway associated with cell proliferation in colon cancer cell lines HCT15 and SW620, as well as tumor xenograft mouse models. We propose G-749 as a new therapeutic agent for the treatment of colon cancer caused by abnormal TYRO3 expression or activity.

Knockdown of vps54 aggravates tamoxifen-induced cytotoxicity in fission yeast.

Tamoxifen (TAM) is an anticancer drug used to treat estrogen receptor (ER)‒positive breast cancer. However, its ER-independent cytotoxic and antifungal activities have prompted debates on its mechanism of action. To achieve a better understanding of the ER-independent antifungal action mechanisms of TAM, we systematically identified TAM-sensitive genes through microarray screening of the heterozygous gene deletion library in fission yeast (Schizosaccharomyces pombe). Secondary confirmation was followed by a spotting assay, finally yielding 13 TAM-sensitive genes under the drug-induced haploinsufficient condition. For these 13 TAM-sensitive genes, we conducted a comparative analysis of their Gene Ontology (GO) 'biological process' terms identified from other genome-wide screenings of the budding yeast deletion library and the MCF7 breast cancer cell line. Several TAM-sensitive genes overlapped between the yeast strains and MCF7 in GO terms including 'cell cycle' (cdc2, rik1, pas1, and leo1), 'signaling' (sck2, oga1, and cki3), and 'vesicle-mediated transport' (SPCC126.08c, vps54, sec72, and tvp15), suggesting their roles in the ER-independent cytotoxic effects of TAM. We recently reported that the cki3 gene with the 'signaling' GO term was related to the ER-independent antifungal action mechanisms of TAM in yeast. In this study, we report that haploinsufficiency of the essential vps54 gene, which encodes the GARP complex subunit, significantly aggravated TAM sensitivity and led to an enlarged vesicle structure in comparison with the SP286 control strain. These results strongly suggest that the vesicle-mediated transport process might be another action mechanism of the ER-independent antifungal or cytotoxic effects of TAM.