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Kaposi's sarcoma-associated herpesvirus (KSHV) is responsible for the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. The expression of immunosuppressive genes, such as IL-10 and CD274/PD-L1 is observed during KSHV-associated pathogenesis, and the modulation of the host immune system by KSHV contributes to establish viral persistence in the host. Understanding the mechanism that allow the virus to evade host cell immunity would be helpful to develop therapeutic strategy for KSHV malignancy. In this study, we show that KSHV replication and transcriptional activator (K-RTA), essential activator of the viral lytic cycle transactivates the CD274/PD-L1 gene promoter. Mechanistically, we demonstrate that the binding of K-RTA to the cellular specificity protein 1 (SP1) is critical for K-RTA-mediated CD274/PD-L1 promoter activation. These findings suggest that K-RTA cooperates with intracellular SP1 to activate the expression of CD274/PD-L1, which helps the virus regulate immune checkpoints to escape and survive.

In most colorectal cancers, Wnt/β-catenin signaling is activated by loss-of-function mutations in the adenomatous polyposis coli (APC) gene and plays a critical role in tumorigenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize Axins, a negative regulator of β-catenin, and upregulate β-catenin signaling. Tankyrase inhibitors downregulate β-catenin and are expected to be promising therapeutics for colorectal cancer. However, colorectal cancer cells are not always sensitive to tankyrase inhibitors, and predictive biomarkers for the drug sensitivity remain elusive. Here we demonstrate that the short-form APC mutations predict the sensitivity of colorectal cancer cells to tankyrase inhibitors. By using well-established colorectal cancer cell lines, we found that tankyrase inhibitors downregulated β-catenin in the drug-sensitive, but not resistant, colorectal cancer cells. The drug-sensitive cells showed higher Tcf/LEF transcriptional activity than the resistant cells and possessed “short” truncated APCs lacking all seven β-catenin-binding 20-amino acid repeats (20-AARs). In contrast, the drug-resistant cells possessed “long” APC retaining two or more 20-AARs. Knockdown of the long APCs with two 20-AARs increased β-catenin, Tcf/LEF transcriptional activity and its target gene AXIN2 expression. Under these conditions, tankyrase inhibitors were able to downregulate β-catenin in the resistant cells. These results indicate that the long APCs are hypomorphic mutants, whereas they exert a dominant-negative effect on Axin-dependent β-catenin degradation caused by tankyrase inhibitors. Finally, we established 16 patient-derived colorectal cancer cells and confirmed that the tankyrase inhibitor–responsive cells harbor the short-form APC mutations. These observations exemplify the predictive importance of APC mutations, the most common genetic alteration in colorectal cancers, for molecular targeted therapeutics. Mol Cancer Ther; 16(4); 752–62. ©2017 AACR.

Supplementary Figure S1: Full scans of the immunoblots shown in Figs. 1C, 2A and 4A. Supplementary Figure S2: Basal expression of tankyrase, Axins, and β-catenin and intracellular localization of β-catenin in CRC cell lines used in this study. Supplementary Figure S3: Response of colorectal cancer SW480 cells to tankyrase inhibitors. Supplementary Figure S4: Depletion of long APC enhances β-catenin signaling and restores tankyrase inhibitor-mediated downregulation of the signal. Supplementary Figure S5: Long but not short APC represses Wnt/β-catenin signal and its knockdown sensitizes β-catenin to tankyrase inhibitor-mediated downregulation. Supplementary Figure S6: Schematic models for the differential effects of the "short" and "long" APC mutants on tankyrase inhibitor (TNKSi)-mediated downregulation of β-catenin. Supplementary Figure S7: APC mutations and sensitivity to tankyrase inhibitors of the patient-derived CRC cells. Supplementary Figure S8: Effects of a tankyrase inhibitor G007-LK on colony formation and β-catenin knockdown on proliferation of patient-derived CRC cells (PDCs).

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Multiple regression analysis of tankyrase inhibitor sensitivities and genetic mutations in colorectal cancer cell lines

Purpose: Ulcerative colitis (UC) is associated with a high risk of colorectal cancer. To identify genes that could predict the development of cancer in UC, we conducted a DNA microarray analysis using nonneoplastic rectal mucosa of UC patients.Experimental Design: Gene expression in nonneoplastic mucosa of 53 UC patients were examined. Gene expression profiles were examined using human Genome U133 Plus 2.0 gene chip array (Affymetrix). Among 53 UC patients, 10 had UC-associated cancer (UC-Ca group) whereas 43 did not (UC-NonCa group).Results: By comparing gene expression profiles of nonneoplastic rectal mucosae between the UC-Ca and UC-NonCa groups, we could identify 40 genes that were differentially expressed between two groups. The list of discriminating genes included low-density lipoprotein receptor–related protein (LRP5 and LRP6). Previous studies suggested that LRP5 and LRP6 expression promotes cancer cell proliferation and tumorigenesis and are considered as candidate oncogenes. In the present study, both LRP5 and LRP6 showed significantly higher expression in the UC-Ca group, which suggests the importance of these genes in the development of UC-associated colorectal cancers. With the 40 selected discriminating genes, we did class prediction of the development of colorectal neoplasms in UC patients. Using the k-nearest neighbor method and the support vector machine, we could predict the development of UC-associated neoplasms with an accuracy of 86.8% and 98.1%, respectively.Conclusions: These findings have important implications for the early detection of malignant lesions in UC and may provide directions for future research into the molecular mechanisms of UC-associated cancer.

Supplementary Figure 1 from Gene Expression Signature and the Prediction of Ulcerative Colitis–Associated Colorectal Cancer by DNA Microarray

Supplementary Table S1 from Distal Colorectal Cancers with Microsatellite Instability (MSI) Display Distinct Gene Expression Profiles that Are Different from Proximal MSI Cancers

Supplementary Figure S1 from Distal Colorectal Cancers with Microsatellite Instability (MSI) Display Distinct Gene Expression Profiles that Are Different from Proximal MSI Cancers

Omic analyses of economically important animals, including Japanese Black cattle, are currently underway worldwide. In particular, tissue and developmental stage-specific transcriptome characterization is essential for understanding the molecular mechanisms underlying the phenotypic expression of genetic disorders and economic traits. Here, we conducted a comprehensive analysis of 124 transcriptomes across 31 major tissues from fetuses, juvenile calves, and adult Japanese Black cattle using short-read sequencing. We found that genes exhibiting high tissue-specific expression tended to increase after 60 days from fertilization and significantly reflected tissue-relevant biology. Based on gene expression variation and inflection points during development, we categorized gene expression patterns as stable, increased, decreased, temporary, or complex in each tissue. We also analysed the expression profiles of causative genes (e.g. SLC12A1, ANXA10, and MYH6) for genetic disorders in cattle, revealing disease-relevant expression patterns. In addition, to directly analyse the structure of full-length transcripts without transcript reconstruction, we performed RNA sequencing analysis of 22 tissues using long-read sequencing and identified 232 novel non-RefSeq isoforms. Collectively, our comprehensive transcriptomic analysis can serve as an important resource for the biological and functional interpretation of gene expression and enable the mechanistic interpretation of genetic disorders and economic traits in Japanese Black cattle.

Organic anion-transporting polypeptides (OATPs) 1A2 and OATP2B1 are expressed in the small intestine and are involved in drug absorption. We identified narirutin, which is present in grapefruit juice, as a novel OATP inhibitor. The citrus fruit jabara also contains high levels of narirutin; therefore, we investigated the inhibitory potency of jabara juice against OATPs. The inhibitory effects of various related compounds on the transport activity of OATPs were evaluated using OATP-expressing HEK293 cells. The IC50 values of narirutin for OATP1A2- and OATP2B1-mediated transport were 22.6 and 18.2 μM, respectively. Other flavanone derivatives from grapefruit juice also inhibited OATP1A2/OATP2B1-mediated transport (order of inhibitory potency: naringenin > narirutin > naringin). Five percent jabara juice significantly inhibited OATP1A2- and OATP2B1-mediated transport by 67 ± 11 and 81 ± 5.5%, respectively (p < 0.05). Based on their inhibitory potency and levels in grapefruit juice, the inhibition of OATPs by grapefruit juice is attributable to both naringin and narirutin. Citrus × jabara, which contains narirutin, potently inhibits OATP-mediated transport.

Eukaryotic initiation factor 2α (eIF2α) kinase general control nonderepressible 2 (GCN2) drives cellular adaptation to amino acid limitation by activating the integrated stress response that induces activating transcription factor 4 (ATF4). Here, we found that a multikinase inhibitor, GZD824, which we identified using a cell-based assay with ATF4 immunostaining, inhibited the GCN2 pathway in cancer cells. Indeed, GZD824 suppressed GCN2 activation, eIF2α phosphorylation, and ATF4 induction during amino acid starvation stress. However, at lower nonsuppressive concentrations, GZD824 paradoxically stimulated eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under unstressed conditions. Such dual properties conceivably arose from a direct effect on GCN2, as also observed in a cell-free GCN2 kinase assay and shared by a selective GCN2 inhibitor. Consistent with the GCN2 pathway inhibition, GZD824 sensitized certain cancer cells to amino acid starvation stress similarly to ATF4 knockdown. These results establish GZD824 as a multikinase GCN2 inhibitor and may enhance its utility as a drug under development. SIGNIFICANCE STATEMENT: GZD824, as a direct general control nonderepressible 2 (GCN2) inhibitor, suppresses activation of the integrated stress response during amino acid limitation, whereas it paradoxically stimulates this stress-signaling pathway at lower nonsuppressive concentrations. The pharmacological activity we identify herein will provide the basis for the use of GZD824 to elucidate the regulatory mechanisms of GCN2 and to evaluate the potential of the GCN2-activating transcription factor 4 pathway as a target for cancer therapy.

JQ1 disrupts the binding of bromodomain and extra-terminal (BET) family of proteins to acetylated histones, modulates the expression of various genes, and inhibits the proliferation of cancer cells. We established two JQ1-resistant sublines from human colorectal cancer HCT116 cells. These resistant cells showed an 8- to 9-fold higher resistance to JQ1, and a 2- to 4-fold higher resistance to various anti-cancer agents, such as doxorubicin, etoposide, mitoxantrone, SN-38, cisplatin, and methotrexate than the parental HCT116 cells. The JQ1-resistant cells expressed higher levels of TRAF2 and NCK-interacting protein kinase (TNIK), cyclin D1 (CCND1), cyclin E1 (CCNE1), and their corresponding mRNAs than the parental cells. TNIK is a regulator of Wnt/β-catenin signaling and is known to transactivate CCND1. Transient transfection of HCT116 cells with a TNIK expression plasmid resulted in the upregulation of cyclin D1, cyclin E1, and their corresponding mRNAs, as well as an increase in CCNE1 promoter activity. Furthermore, luciferase assay revealed that the JQ1-resistant cells showed high CCNE1 promoter activity. These results suggest that TNIK also transactivates CCNE1. Three stable TNIK transfectant clones of HEK293 cells expressed 1.5- to 2-fold higher levels of TNIK, cyclin D1, and cyclin E1 than the parental cells. The 293/TNIK-6 cells, which expressed the highest level of TNIK among the transfectants, showed a 2.3-fold higher resistance to JQ1 than the parental cells. These results suggest the possible involvement of TNIK in cellular resistance to JQ1.

TSPO2 (translocator protein 2) is a transmembrane protein specifically expressed in late erythroblasts and has been postulated to mediate intracellular redistribution of cholesterol. We identified TSPO2 as the causative gene for the HK (high-K+) trait with immature red cell phenotypes in dogs and investigated the effects of the TSPO2 defects on erythropoiesis in HK dogs with the TSPO2 mutation and Tspo2 knockout (Tspo2(?/?)) mouse models. Bone marrow?derived erythroblasts from HK dogs showed increased binucleated and apoptotic cells at various stages of maturation and shed large nuclei with incomplete condensation when cultured in the presence of erythropoietin, indicating impaired maturation and cytokinesis. The canine TSPO2 induces cholesterol accumulation in the endoplasmic reticulum and could thereby regulate cholesterol availability by changing intracellular cholesterol distribution in erythroblasts. Tspo2(?/?) mice consistently showed impaired cytokinesis with increased binucleated erythroblasts, resulting in compensated anemia, and their red cell membranes had increased Na,K-ATPase, resembling the HK phenotype in dogs. Tspo2-deficient mouse embryonic stem cell?derived erythroid progenitor (MEDEP) cells exhibited similar morphological defects associated with a cell-cycle arrest at the G(2)/M phase, resulting in decreased cell proliferation and had a depletion in intracellular unesterified and esterified cholesterol. When the terminal maturation was induced, Tspo2(?/?) MEDEP cells showed delays in hemoglobinization; maturation-associated phenotypic changes in CD44, CD71, and TER119 expression; and cell-cycle progression. Taken together, these findings imply that TSPO2 is essential for coordination of maturation and proliferation of erythroblasts during normal erythropoiesis.

We attempted to estimate the additive genetic variance explained by each autosome, using genotype data of 33,657 single nucleotide polymorphism (SNP) markers in 2271 Japanese Black fattened steers. Traits were cold carcass weight, ribeye area, rib thickness, subcutaneous fat thickness, estimated yield percentage, and marbling score. Two mixed linear models were used: One is that (model 1) incorporating a genomic relationship matrix (G matrix) constructed by using all available SNPs, and another (model 2), incorporating two G matrices constructed by using the SNPs on one autosome and using those on the remaining autosomes. Genomic heritabilities estimated using model 1 were moderate to high. The sums of the proportions of the additive genetic variance explained by each autosome to the total genetic variance estimated by using model 2 were90%. For carcass weight, the proportions explained by Bos taurus autosomes 6, 8, and 14 were higher than those explained by the remaining autosomes. In some cases, the estimated proportion was close to 0. The results obtained from model 2 could provide a novel insight into the genetic architecture, such as heritability per chromosome, of carcass traits in Japanese Black cattle, although further careful investigation would be required.

Some oncoproteins along with stress kinase general control non-derepressible 2 (GCN2) can ensure the induction of activating transcription factor 4 (ATF4) to counteract amino acid deprivation; however, little is known regarding the role of the oncogenic EGFR-PI3K pathway. In this study, we demonstrate that both mutated EGFR and PIK3CA contribute to ATF4 induction following GCN2 activation in NSCLC cells. The inhibition of EGFR or PI3K mutant proteins, pharmacologically or through genetic knockdown, inhibited ATF4 induction without affecting GCN2 activation. A downstream analysis revealed that the oncogenic EGFR-PI3K pathway may utilize mTOR-mediated translation control mechanisms for ATF4 induction. Furthermore, in NSCLC cells harboring co-mutations in EGFR and PIK3CA, the combined inhibition of these oncoproteins markedly suppressed ATF4 induction and the subsequent gene expression program as well as cell viability during amino acid deprivation. Our findings establish a role for the oncogenic EGFR-PI3K pathway in the adaptive stress response and provide a strategy to improve EGFR-targeted NSCLC therapy.

Tyrosine kinase inhibitors (TKIs) are established drugs in the therapy of FLT3-ITD mutated acute myeloid leukemia (AML). However, acquired mutations, such as D835 in the tyrosine kinase domain (FLT3-ITD/D835), can induce resistance to TKIs. A cap analysis gene expression (CAGE) technology revealed that the gene expression of BCL2A1 transcription start sites was increased in primary AML cells bearing FLT3-ITD/D835 compared to FLT3-ITD. Overexpression of BCL2A1 attenuated the sensitivity to quizartinib, a type II TKI, and venetoclax, a selective BCL2 inhibitor, in AML cell lines. However, a type I TKI, gilteritinib, inhibited the expression of BCL2A1 through inactivation of STAT5 and alleviated TKI resistance of FLT3-ITD/D835. The combination of gilteritinib and venetoclax showed synergistic effects in the FLT3-ITD/D835 positive AML cells. The promoter region of BCL2A1 contains a BRD4 binding site. Thus, the blockade of BRD4 with a BET inhibitor (CPI-0610) downregulated BCL2A1 in FLT3-mutated AML cells and extended profound suppression of FLT3-ITD/D835 mutant cells. Therefore, we propose that BCL2A1 has the potential to be a novel therapeutic target in treating FLT3-ITD/D835 mutated AML.

Nobiletin is a polymethoxylated flavone from citrus fruit peels. Among other bioactivities, it acts antioxidative, anti-inflammatory, neuroprotective, and cardiovascular-protective. Nobiletin exerts profound anticancer activity in vitro and in vivo but the underlying mechanisms are not well understood.The aim was to unravel the multiple modes of action against cancer cells by bioinformatic and transcriptomic techniques and their verification by molecular pharmacological methods.The in silico methods used were COMPARE analysis of transcriptomic data, signaling pathway analysis, transcription factor binding motif analysis in promoter sequences of target genes, and molecular docking. The in vitro methods used were resazurin assay, isobologram analysis, generation of stably SOX5-tranfected cells, and Western blotting.Nobiletin was cytotoxic against a wide range of cell lines from different tumor types, including diverse phenotypes to established anticancer drugs (e.g., P-glycoprotein, ABCB5, p53, EGFR). Cross-resistance profiling with 83 standard anticancer drugs revealed a correlation to antihormonal anticancer drugs, which can be explained by the phytoestrogenic features of nobiletin. Transcriptomic analysis showed that the responsiveness of tumor cells was predictable by their specific mRNA expression profile. Nobiletin bound to the transcription factor SOX5 in silico. SOX5 conferred resistance to the control drug doxorubicin but collateral sensitivity to nobiletin in HEK293 cells transfected with a lentiviral GFP-tagged pLOCORF-SOX5 vector. The combination of nobiletin and doxorubicin synergistically killed HEK293-SOX5 cells in isobologram analyses, implying attractive new treatment options.Nobiletin represents an interesting candidate for cancer therapy with broad-spectrum activity and multiple modes of action. The identification of novel targets (i.e., SOX5) may allow its use for targeted tumor therapy in individualized treatment protocols.

The nitrogen structure in coals has been focused on as a key factor for reducing NOx. Solid-state nuclear magnetic resonance (NMR) with high spectral resolution can be an effective tool for analyzi...

Intensive use of a few elite sires has increased the risk of the manifestation of deleterious recessive traits in cattle. Substantial genotyping data gathered using single-nucleotide polymorphism (SNP) arrays have identified the haplotypes with homozygous deficiency, which may compromise survival. We developed Japanese Black cattle haplotypes (JBHs) using SNP array data (4843 individuals) and identified deleterious recessive haplotypes using exome sequencing of 517 sires. We identified seven JBHs with homozygous deficiency. JBH_10 and JBH_17 were associated with the resuming of estrus after artificial insemination, indicating that these haplotypes carried deleterious mutations affecting embryonic survival. The exome data of 517 Japanese Black sires revealed that AC_000165.1:g.85341291C>G of IARS in JBH_8_2, AC_000174.1:g.74743512G>T of CDC45 in JBH_17, and a copy variation region (CNVR_27) of CLDN16 in JBH_1_1 and JBH_1_2 were the candidate mutations. A novel variant AC_000174.1:g.74743512G>T of CDC45 in JBH_17 was located in a splicing donor site at a distance of 5 bp, affecting pre-mRNA splicing. Mating between heterozygotes of JBH_17 indicated that homozygotes carrying the risk allele died around the blastocyst stage. Analysis of frequency of the CDC45 risk allele revealed that its carriers were widespread throughout the tested Japanese Black cattle population. Our approach can effectively manage the inheritance of recessive risk alleles in a breeding population.

Background Calf mortality generally occurs in calves prior to weaning, which is a serious problem in cattle breeding. Several causative variants of monogenic Mendelian disorders in calf mortality have been identified, whereas genetic factors affecting the susceptibility of calves to death are not well known. To identify variants associated with calf mortality in Japanese Black cattle, we evaluated calf mortality as a categorical trait with a threshold model and performed a genome-wide copy number variation (CNV) association study on calf mortality. Results We identified a 44-kb deleted-type CNV ranging from 103,317,687 to 103,361,802 bp on chromosome 5, which was associated with the mortality of 1–180-day-old calves. The CNV harbored C1RL, a pseudogene, and an IncRNA localized in the C1R and C1S gene cluster, which is a component of the classical complement activation pathway for immune complexes for infectious pathogens. The average complement activity in CNVR_221 homozygotes at postnatal day 7 was significantly lower than that of wild-type animals and heterozygotes. The frequency of the risk allele in dead calves suffering from diarrhea and pneumonia and in healthy cows was 0.35 and 0.28, respectively (odds ratio = 2.2, P = 0.016), suggesting that CNVR_221 was associated with the mortality of Japanese Black calves suffering from an infectious disease. Conclusions This study identified a deleted-type CNV associated with the mortality of 1–180-day-old calves. The complement activity in CNVR_221 homozygotes was significantly lower than that in heterozygotes and wild type animals. The frequency of the risk allele was higher in dead calves suffering from an infectious disease than in healthy cows. These results suggest that the existence of CNVR_221 in calves could be attributed to a reduction in complement activity, which in turn leads to susceptibility to infections. Thus, the risk allele could serve as a useful marker to reduce the mortality of infected Japanese Black calves.

Background Sesquiterpene lactones having α-methylene-γ-lactone moiety are promising natural metabolites showing various biological activity. One of the major metabolites isolated from Pulicaria undulata, 2α-hydroxyalantolactone (PU-1), has not been investigated in detail yet. Multidrug resistance (MDR) represents a major obstacle for cancer chemotherapy and the capability of novel natural products to overcoming MDR is of great interest. Purpose Exploring the molecular modes of action for potent natural product metabolites. Methods The resazurin reduction assay was employed to evaluate the cytotoxicity of PU-1 on sensitive and their corresponding drug-resistant cell lines (overexpressing P-glycoprotein, BCRP, ABCB5, ΔEGFR, or TP53 knockout). Gene expression profiling was performed by transcriptome-wide mRNA microarray in the human CCRF-CEM leukemic cells after treatment with PU-1. The top significantly up- or down-regulated genes were identified by Chipster program and analyzed using Ingenuity Pathway Analysis (IPA) software. Finally, flow cytometry and Western blotting were performed for cell cycle analyses and apoptosis detection. Results The sesquiterpene lactone, PU-1, showed potent cytotoxicity towards the drug-sensitive and -resistant cell lines. Transcriptome-wide mRNA expression profiling and pathway analysis pointed to genes involved in DNA damage response and G2/M cell cycle arrest. G2/M arrest was verified by flow cytometry and further confirmed by the upregulation of p21 and downregulation of p-CDC25C expression in Western blotting. Moreover, the suggested DNA damage checkpoint regulation was confirmed by immunofluorescence and Western blotting by upregulation of pS345 Chk1, p-H3 and γ-H2AX. Furthermore, PU-1 inhibited PI3K/AKT pathway, which is involved in signaling DNA damage and G2/M arrest. Cells ultimately induced apoptosis upon PU-1 treatment. Conclusions PU-1 is a potent natural product inhibiting otherwise drug-resistant human tumor cell growth through DNA damage, G2/M cell cycle arrest and apoptosis.

Background Carnosic acid (CA) is one of the main constituents in rosemary extract. It possesses valuable pharmacological properties, including anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer activities. Numerous in vitro and in vivo studies investigated the anticancer profile of CA and emphasized its potentiality for cancer treatment. Nevertheless, the role of multidrug-resistance (MDR) related mechanisms for CA's anticancer effect is not yet known. Purpose We investigated the cytotoxicity of CA against known mechanisms of anticancer drug resistance (P-gp, ABCB5, BCRP, EGFR and p53) and determined novel putative molecular factors associated with cellular response towards CA. Study design Cytotoxicity assays, bioinformatic analysis, flow cytometry and western blotting were performed to identify the mode of action of CA towards cancer cells. Methods The cytotoxicity to CA was assessed using the resazurin assays in cell lines expressing the mentioned resistance mechanisms. A pharmacogenomic characterization of the NCI 60 cell line panel was applied via COMPARE, hierarchical cluster and network analyses. Flow cytometry was used to detect cellular mode of death and ROS generation. Changes in proteins-related to apoptosis were determined by Western blotting. Results Cell lines expressing ABC transporters (P-gp, BCRP or ABCB5), mutant EGFR or p53 were not cross-resistant to CA compared to their parental counterparts. By pharmacogenomic approaches, we identified genes that belong to different functional groups (e.g. signal transduction, regulation of cytoskeleton and developmental regulatory system). These genes were predicted as molecular determinants that mediate CA tumor cellular responses. The top affected biofunctions included cellular development, cellular proliferation and cellular death and survival. The effect of CA-mediated apoptosis in leukemia cells, which were recognized as the most sensitive tumor type, was confirmed via flow cytometry and western blot analysis. Conclusion CA may provide a novel treatment option to target refractory tumors and to effectively cooperate with established chemotherapy. Using pharmacogenomic approaches and network pharmacology, the relationship between cancer complexity and multi-target potentials of CA was analyzed and many putative molecular determinants were identified. They could serve as novel targets for CA and further studies are needed to translate the possible implications to clinical cancer treatment.

3',4',7-Trimethoxyflavone (TMF) has been reported to show a potent reversal effect on drug resistance mediated by breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2). In this study, we designed and synthesized five derivatives with either a hydroxy group or a fluorine atom at C-5 and several kinds of capping moiety at the C-7 hydroxy group, on the same 3',4'-dimethoxy-substituted flavone skeleton. We subsequently evaluated the efficacies of these compounds against BCRP-expressing human leukaemia K562/BCRP cells. Reversal of drug resistance was expressed as the concentration of compound causing a twofold reduction in drug sensitivity (RI50 ). Of the synthesized compounds, the reversal effect of 5-hydroxy-3',4',7-trimethoxyflavone (HTMF, RI50 7.2 nm) towards 7-ethyl-10-hydroxycamptothecin (SN-38) was stronger than that of TMF (RI50 18 nm). Fluoro-substituted 5-fluoro-3',4',7-trimethoxyflavone (FTMF, RI50 25 nm) and monoglycosylated 7-(β-glucosyloxy)-5-hydroxy-3',4'-dimethoxyflavone (GOHDMF, 91 nm) also exhibited reversal effects, whereas the di- and triglycoside derivatives did not. TMF, HTMF and FTMF at 0.01-10 μm upregulated the K562/BCRP cellular accumulation of Hoechst 33342 nuclear staining dye. In addition, western blotting revealed that treatment of K562/BCRP cells with 0.1 μm TMF, HTMF or FTMT suppressed the expression of BCRP. HTMF showed the strongest inhibition of BCRP-mediated efflux and suppression of BCRP expression of the three effective synthesized flavones.

Telomere maintenance by telomerase activity supports the infinite growth of cancer cells. MST-312, a synthetic telomerase inhibitor, gradually shortens telomeres at non-acute lethal doses and eventually induces senescence and apoptosis of telomerase-positive cancer cells. Here we report that MST-312 at higher doses works as a dual inhibitor of telomerase and DNA topoisomerase II and exhibits acute anti-proliferative effects on cancer cells and xenografted tumours in vivo. Our cell-based chemical fingerprinting approach revealed that cancer cells with shorter telomeres and lower expression of lamin A, a nuclear architectural protein, exhibited higher sensitivity to the acute deleterious effects of MST-312, accompanied by formation of telomere dysfunction-induced foci and DNA double-strand breaks. Telomere elongation and lamin A overexpression attenuated telomeric and non-telomeric DNA damage, respectively, and both conferred resistance to apoptosis induced by MST-312 and other DNA damaging anticancer agents. These observations suggest that sufficient pools of telomeres and a nuclear lamina component contribute to the cellular robustness against DNA damage induced by therapeutic treatment in human cancer cells.

Internal tandem duplication (ITD) in Fms-like tyrosine kinase 3 (FLT3) is frequently observed in acute myeloid leukemia (AML). Quizartinib, gilteritinib, and midostaurin are inhibitors against FLT3-ITD that have good efficacy for FLT3-ITD-positive AML patients. Long-term administration leads to drug resistance through acquired tyrosine kinase domain (TKD) mutations in FLT3-ITD, such as N676K, F691L, D835V, and Y842C. Here, our screen to detect inhibitors capable of overcoming resistance to FLT3 inhibitors identified heat shock protein (HSP) 90 inhibitors as potential candidates. Although Ba/F3 cells expressing FLT3-ITD with TKD mutations (Ba/F3-ITD+N676K, Ba/F3-ITD+F691L, Ba/F3-ITD+D835V, and Ba/F3-ITD+Y842C) showed various resistance patterns to FLT3 inhibitors compared with Ba/F3-ITD cells that express FLT3-ITD lacking TKD mutations, they were more sensitive to HSP90 inhibitors than Ba/F3 cells. Notably, the Ba/F3-ITD+D835V cells were the most sensitive to HSP90 inhibitors. Treatment with HSP90 inhibitors downregulated FLT3 and its downstream signaling and induced G1 arrest followed by apoptosis in Ba/F3-ITD+N676K, Ba/F3-ITD+F691L, Ba/F3-ITD+Y842C, and especially Ba/F3-ITD+D835V cells at lower concentrations compared with Ba/F3-ITD cells. The downregulation of FLT3-ITD+D835V was caused by rapid proteolysis in autophagy. Similar results were also observed in the quizartinib-resistant MV4-11 cells, QR1 and QR2, which were established by culturing cells in the presence of quizartinib and harbored FLT3-ITD+D835H and FLT3-ITD+D835V, respectively, in a single allele. Interestingly, the efficacies of HSP90 inhibitors in QR cells are reversely correlated with that of quizartib, but not to gilteritinib and midostaurin. Collectively, HSP90 inhibitors are good candidates to overcome drug resistance in AML with various FLT3-ITD TKD mutations.

We previously reported the upregulation of cellular Glu and glutathione levels in human ABCB5- and murine Abcb5-transfected cells. Here, we demonstrate the upregulation of STAT1 and glutaminase (GLS) in ABCB5/Abcb5-transfected cells. Among a total of four ABCB5/Abcb5 high-expressing clones with docetaxel resistance, three of the clones expressed STAT1 and GLS highly and showed resistance to docetaxel and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. Neither STAT1 nor GLS upregulation was observed in the remaining ABCB5 high-expressing clone, as well as in another two ABCB5 low-expressing clones; these three clones did not show BSO resistance. The ABCB5/STAT1 high-expressing clones showed higher cellular levels of Ala, Glu, and Asp and lower cellular levels of Phe, Trp, Leu, Ile, Gly, Met, Tyr, Val, and His compared to the ABCB5/STAT1 low-expressing clones. The former clones also showed a higher resistance to Glu. The STAT1-transfected clones expressed high levels of GLS and the corresponding mRNA, suggesting the transactivation of GLS by STAT1. These clones showed resistance to Glu and BSO, similar to the ABCB5/STAT1 high-expressing clones. The cellular glutathione levels of the STAT1-transfected clones were significantly higher than that of the control. The STAT1-transfected clones also showed greater resistance to the effect of BSO on the cellular glutathione depletion compared to the control. These results demonstrate that STAT1 upregulates GLS and modulates amino acids and glutathione metabolism. Although we were unable to directly prove STAT1 upregulation by ABCB5, our results suggest that ABCB5 expression, directly or indirectly, leads to the overexpression of STAT1.

After decades of research, multidrug resistance (MDR) remains a huge challenge in cancer treatment. In this study, the cytotoxic of 4-hydroxy-N-(naphthalen-1-yl)-2-oxo-2H-chromene-3-carboxamide (MCC1734) has been investigated towards multidrug-resistant cancer cell lines. MCC1734 exerted cytotoxicity on cell lines expressing different mechanisms of drug resistance (P-glycoprotein, BCRP, ABCB5, EGFR, p53 knockout) to a different extent. Interestingly, sensitive CCRF-CEM cells and multidrug-resistant P-gp-overexpressing CEM/ADR5000 cells represented similar sensitivity towards MCC1734, indicating MCC1734 can bypass P-gp-mediated resistance. Microarray-based mRNA expression revealed that MCC1734 affected cells by multiple pathways, including cell cycle regulation, mitochondrial dysfunction, apoptosis signaling, and EIF2 signaling. MCC1734 stimulated the generation of excessive reactive oxygen species and the collapse of mitochondria membrane potential in CCRF-CEM cells, companied by the arrest of the cell cycle in the G2M phase and apoptosis induction as determined by flow cytometry. In addition, our immunoblotting analysis highlighted that MCC1734 triggered endoplasmic reticulum (ER) stress, evidenced by the activation of p-PERK, p-eIF2α, ATF4 and CHOP. The anti-cancer effects of MCC1734 were further observed in vivo using human xenograft tumors transplanted to zebrafish, providing further support for MCC1734 as a promising new candidate for cancer drug development.

// Tetsuo Mashima 1 , Yoko Taneda 1, 2 , Myung-Kyu Jang 1, 2 , Anna Mizutani 1 , Yukiko Muramatsu 1 , Haruka Yoshida 1 , Ayana Sato 1, 2 , Noritaka Tanaka 1, 3 , Yoshikazu Sugimoto 3 and Hiroyuki Seimiya 1, 2 1 Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan 2 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan 3 Division of Chemotherapy, Faculty of Pharmacy, Keio University, Tokyo, Japan Correspondence to: Hiroyuki Seimiya, email: hseimiya@jfcr.or.jp Keywords: tankyrase, Wnt, colorectal cancer, resistance, mTOR Received: August 26, 2016 Accepted: May 03, 2017 Published: May 24, 2017 ABSTRACT Activation of Wnt/β-catenin signaling is essential for colorectal carcinogenesis. Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, is a positive regulator of the Wnt/β-catenin signaling. Accordingly, tankyrase inhibitors are under preclinical development for colorectal cancer (CRC) therapy. However, Wnt-driven colorectal cancer cells are not equally sensitive to tankyrase inhibitors, and cellular factors that affect tankyrase inhibitor sensitivity remain elusive. Here, we established a tankyrase inhibitor-resistant cell line, 320-IWR, from Wnt/β-catenin-dependent CRC COLO-320DM cells. 320-IWR cells exhibited resistance to tankyrase inhibitors, IWR-1 and G007-LK, but remained sensitive to a PARP-1/2 inhibitor, olaparib, and several anti-CRC agents. In 320-IWR cells, nuclear localization of active β-catenin was decreased and expression of β-catenin target genes was constitutively repressed, suggesting that these cells repressed the Wnt/β-catenin signaling and were dependent on alternative proliferation pathways. 320-IWR cells exhibited upregulated mTOR signaling and were more sensitive to mTOR inhibition than the parental cells. Importantly, mTOR inhibition reversed resistance to tankyrase inhibitors and potentiated their anti-proliferative effects in 320-IWR cells as well as in CRC cell lines in which the mTOR pathway was intrinsically activated. These results indicate that mTOR signaling confers resistance to tankyrase inhibitors in CRC cells and suggest that the combination of tankyrase and mTOR inhibitors would be a useful therapeutic approach for a subset of CRCs.

In most colorectal cancers, Wnt/β-catenin signaling is activated by loss-of-function mutations in the adenomatous polyposis coli (APC) gene and plays a critical role in tumorigenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize Axins, a negative regulator of β-catenin, and upregulate β-catenin signaling. Tankyrase inhibitors downregulate β-catenin and are expected to be promising therapeutics for colorectal cancer. However, colorectal cancer cells are not always sensitive to tankyrase inhibitors, and predictive biomarkers for the drug sensitivity remain elusive. Here we demonstrate that the short-form APC mutations predict the sensitivity of colorectal cancer cells to tankyrase inhibitors. By using well-established colorectal cancer cell lines, we found that tankyrase inhibitors downregulated β-catenin in the drug-sensitive, but not resistant, colorectal cancer cells. The drug-sensitive cells showed higher Tcf/LEF transcriptional activity than the resistant cells and possessed “short” truncated APCs lacking all seven β-catenin-binding 20-amino acid repeats (20-AARs). In contrast, the drug-resistant cells possessed “long” APC retaining two or more 20-AARs. Knockdown of the long APCs with two 20-AARs increased β-catenin, Tcf/LEF transcriptional activity and its target gene AXIN2 expression. Under these conditions, tankyrase inhibitors were able to downregulate β-catenin in the resistant cells. These results indicate that the long APCs are hypomorphic mutants, whereas they exert a dominant-negative effect on Axin-dependent β-catenin degradation caused by tankyrase inhibitors. Finally, we established 16 patient-derived colorectal cancer cells and confirmed that the tankyrase inhibitor–responsive cells harbor the short-form APC mutations. These observations exemplify the predictive importance of APC mutations, the most common genetic alteration in colorectal cancers, for molecular targeted therapeutics. Mol Cancer Ther; 16(4); 752–62. ©2017 AACR.