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Background: Cisplatin is an important anticancer agent in cancer chemotherapy, but when resistant cells appear, treatment becomes difficult, and the prognosis is poor. Objective: In this study, we investigated the gene expression profile in cisplatin sensitive and resistant cells, and identified the genes involved in cisplatin resistance. Methods: Comparison of gene expression profiles revealed that UBE2L6 mRNA is highly expressed in resistant cells. To elucidate whether UBE2L6 is involved in the acquisition of cisplatin resistance, UBE2L6- overexpressing cells established from cisplatin-sensitive cells and UBE2L6-silenced cells developed from cisplatin- resistant cells were generated, and the sensitivity of cisplatin was examined. Results: The sensitivity of the UBE2L6-overexpressing cells did not change compared with the control cells, but the UBE2L6-silenced cells were sensitized to cisplatin. To elucidate the mechanism of UBE2L6 in cisplatin resistance, we compared the gene expression profiles of UBE2L6-silenced cells and control cells and found that the level of ABCB6 mRNA involved in cisplatin resistance was decreased. Moreover, ABCB6 promoter activity was partially suppressed in UBE2L6-silenced cells. Conclusion: These results suggest that cisplatin-resistant cells have upregulated UBE2L6 expression and contribute to cisplatin resistance by regulating ABCB6 expression at the transcriptional level. UBE2L6 might be a molecular target that overcomes cisplatin resistance.

The development of potent and selective therapeutic approaches to glioblastoma (GBM), one of the most aggressive primary brain tumors, requires identification of molecular pathways that critically regulate the survival and proliferation of GBM. Previous studies have reported that deregulated expression of N-myc downstream regulated gene 1 (NDRG1) affects tumor growth and clinical outcomes of patients with various types of cancer including glioma. Here, we show that high level expression of NDRG1 in tumors significantly correlated with better prognosis of patients with GBM. Loss of NDRG1 in GBM cells upregulated GSK3β levels and promoted cell proliferation, which was reversed by selective inhibitors of GSK3β. In contrast, NDRG1 overexpression suppressed growth of GBM cells by decreasing GSK3β levels via proteasomal degradation and by suppressing AKT and S6 cell growth signaling, as well as cell-cycle signaling pathways. Conversely, GSK3β phosphorylated serine and threonine sites in the C-terminal domain of NDRG1 and limited the protein stability of NDRG1. Furthermore, treatment with differentiation inducing factor-1, a small molecule derived from Dictyostelium discoideum, enhanced NDRG1 expression, decreased GSK3β expression, and exerted marked NDRG1-dependent antitumor effects in vitro and in vivo. Taken together, this study revealed a novel molecular mechanism by which NDRG1 inhibits GBM proliferation and progression. Our study thus identifies the NDRG1/GSK3β signaling pathway as a key growth regulatory program in GBM, and suggests enhancing NDRG1 expression in GBM as a potent strategy toward the development of anti-GBM therapeutics. Significance: This study identifies NDRG1 as a potent and endogenous suppressor of glioblastoma cell growth, suggesting the clinical benefits of NDRG1-targeted therapeutics against glioblastoma.

Respirable organic chemicals were originally thought to cause allergic respiratory diseases, such as bronchial asthma and hypersensitivity pneumonitis, and believed not to cause lung disorders derived from inflammatory or fibrotic processes such as pulmonary fibrosis and interstitial pneumonitis. It has recently been reported, however, that exposure to organic chemicals can cause interstitial lung diseases. In this review, we discuss the clinical features of occupational asthma and hypersensitivity pneumonitis, as well as other lung disorders, including interstitial pneumonitis, caused by humidifier disinfectants in Korea and by a cross‐linked acrylic acid‐based polymer (CL‐PAA) in Japan.

Y-box binding protein 2 (YBX2) has been associated with the properties of both germ cells and cancer cells. We hypothesized that YBX2 might contribute to the characteristics of cancer stem cells (CSCs). In this study, we clarified the function of YBX2 in endometrial cancer stem cells. We established a human YBX2-expressing Ishikawa (IK) cell line (IK-YBX2 cells). We analyzed gene expression associated with stemness and isolated SP cells from IK-YBX2 cells. The SP population of IK-YBX2 cells, the expression of ALDH1 and serial sphere-forming capacity were associated with levels of YBX2 expression. IK-YBX2 cells were resistant to anti-cancer drugs. In gene expression analysis, a gene for cancer testis antigen, CT45, was generally overexpressed in IK-YBX2 cells. YBX2-mediated CT45 expression was associated with increased levels of self-renewal capacity and paclitaxel resistance. The level of CT45 expression was enhanced in high-grade and/or advanced stages of human endometrial cancer tissues. We conclude that expression of YBX2 is essential for the stem cell-like phenotype. CT45 contributes to stemness associated with YBX2 and might be related to the progression of endometrial cancer.

Antisense oligonucleotides (AS-ODNs) specifically hybridize with target mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. In our previous reports, we demonstrated that β-glucan schizophyllan (SPG) can form a complex with AS-ODNs attached with oligo deoxyadenosine dA40 (AS-ODN-dA40/SPG), and that this complex can be recognized by β-glucan receptor Dectin-1 on antigen presenting cells and lung cancer cells. In many types of cancer cell, activating K-ras mutations related to malignancy are frequently observed. In this study, we first designed 78 AS-ODNs for K-ras to optimize the sequence for highly efficient gene suppression. The selected AS-ODN (K-AS07) having dA40 made a complex with SPG. The resultant complex (K-AS07-dA40/SPG) showed an effect of silencing the ras gene in the cells (PC9: human adenocarcinoma differentiated from lung tissue) expressing Dectin-1, leading to the suppression of cell growth. Furthermore, the cytotoxic effect was enhanced when used in combination with the anticancer drug gemcitabine. Gemcitabine, a derivative of cytidine, was shown to interact with dA40 in a sequence-dependent manner. This interaction did not appear to be so strong, with the gemcitabine being released from the complex after internalization into the cells. SPG and the dA40 part of K-AS07-dA40 play roles in carriers for K-AS07 and gemcitabine, respectively, resulting in a strong cytotoxic effect. This combination effect is a novel feature of the AS-ODN-dA40/SPG complexes. These results could facilitate the clinical application of these complexes for cancer treatment.

Background Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is life-threatening. Several serum biomarkers, such as Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D), are clinically used for evaluating AE-IPF, but these biomarkers are not adequate for establishing an early and accurate diagnosis of AE-IPF. Recently, the protective roles of the members of the peroxiredoxin (PRDX) family have been reported in IPF; however, the role of PRDX4 in AE-IPF is unclear. Methods Serum levels of PRDX4 protein, KL-6, SP-D and lactate dehydrogenase (LDH) in 51 patients with stable IPF (S-IPF), 38 patients with AE-IPF and 15 healthy volunteers were retrospectively assessed using enzyme-linked immunosorbent assay. Moreover, as an animal model of pulmonary fibrosis, wild-type (WT) and PRDX4-transgenic (Tg) mice were intratracheally administered with bleomycin (BLM, 2 mg/kg), and fibrotic and inflammatory changes in lungs were evaluated 3 weeks after the intratracheal administration. Results Serum levels of PRDX4 protein, KL-6, SP-D and LDH in patients with S-IPF and AE-IPF were significantly higher than those in healthy volunteers, and those in AE-IPF patients were the highest among the three groups. Using receiver operating characteristic curves, area under the curve values of serum PRDX4 protein, KL-6, SP-D, and LDH for detecting AE-IPF were 0.873, 0.698, 0.675, and 0.906, respectively. BLM-treated Tg mice demonstrated aggravated histopathological findings and poor prognosis compared with BLM-treated WT mice. Moreover, PRDX4 expression was observed in alveolar macrophages and lung epithelial cells of BLM-treated Tg mice. Conclusions PRDX4 is associated with the aggravation of inflammatory changes and fibrosis in the pathogenesis of IPF, and serum PRDX4 may be useful in clinical practice of IPF patients.

Zinc‐finger protein 143 (ZNF143) is a transcription factor that is involved in anticancer drug resistance and cancer cell survival. In the present study, we identified a novel small molecule N‐(5‐bromo‐2‐methoxyphenyl)‐3‐(pyridine‐3‐yl) propiolamide (YPC‐21661) that inhibited ZNF143 promoter activity and down‐regulated the expression of the ZNF143‐regulated genes, RAD51, PLK1, and Survivin, by inhibiting the binding of ZNF143 to DNA. In addition, YPC‐21661 was cytotoxic and induced apoptosis in the human colon cancer cell line, HCT116 and human prostate cancer cell line, PC‐3. 2‐(pyridine‐3‐ylethynyl)‐5‐(2‐(trifluoromethoxy)phenyl)‐1,3,4‐oxadiazole (YPC‐22026), a metabolically stable derivative of YPC‐21661, induced tumor regression accompanied by the suppression of ZNF143‐regulated genes in a mouse xenograft model. The present study revealed that the inhibition of ZNF143 activity by small molecules induced tumor regression in vitro and in vivo; therefore, ZNF143 is a promising target of cancer therapeutics.

Endocrine therapies effectively improve the outcomes of patients with estrogen receptor (ER)-positive breast cancer. However, the emergence of drug-resistant tumors creates a core clinical challenge. In breast cancer cells rendered resistant to the antiestrogen fulvestrant, we defined causative mechanistic roles for the transcription factor YBX1 and the levels of ER and the ERBB2 receptor. Enforced expression of YBX1 in parental cells conferred resistance against tamoxifen and fulvestrant in vitro and in vivo. Furthermore, YBX1 overexpression was associated with decreased and increased levels of ER and ERBB2 expression, respectively. In antiestrogen-resistant cells, increased YBX1 phosphorylation was associated with a 4-fold higher degradation rate of ER. Notably, YBX1 bound the ER, leading to its accelerated proteasomal degradation, and induced the transcriptional activation of ERBB2. In parallel fashion, tamoxifen treatment also augmented YBX1 binding to the ERBB2 promoter to induce increased ERBB2 expression. Together, these findings define a mechanism of drug resistance through which YBX1 contributes to antiestrogen bypass in breast cancer cells. Cancer Res; 77(2); 545–56. ©2016 AACR.

Nuclear expression of Y-box–binding protein (YBX1) is closely correlated with clinical poor outcomes and drug resistance in breast cancer. Nuclear translocation of YBX1 is facilitated by YBX1 phosphorylation at serine 102 by AKT, p70S6K, and p90RSK, and the phosphorylated YBX1 (pYBX1) promotes expression of genes related to drug resistance and cell growth. A forthcoming problem to be addressed is whether targeting the phosphorylation of YBX1 overcomes antiestrogen resistance by progressive breast cancer. Here, we found that increased expression of pYBX1 was accompanied by acquired resistance to antiestrogens, fulvestrant and tamoxifen. Forced expression of YBX1/S102E, a constitutive phosphorylated form, resulted in acquired resistance to fulvestrant. Inversely, YBX1 silencing specifically overcame antiestrogen resistance. Furthermore, treatment with everolimus, an mTORC1 inhibitor, or TAS0612, a novel multikinase inhibitor of AKT, p70S6K, and p90RSK, suppressed YBX1 phosphorylation and overcame antiestrogen resistance in vitro and in vivo. IHC analysis revealed that expression of pYBX1 and YBX1 was augmented in patients who experienced recurrence during treatment with adjuvant endocrine therapies. Furthermore, pYBX1 was highly expressed in patients with triple-negative breast cancer compared with other subtypes. TAS0612 also demonstrated antitumor effect against triple-negative breast cancer in vivo. Taken together, our findings suggest that pYBX1 represents a potential therapeutic target for treatment of antiestrogen-resistant and progressive breast cancer.

We examine the role of the heterogenous ribonucleoprotein U (hnRNP U) as a G-quadruplex binding protein in human cell lines. Hypothesizing that hnRNP U is associated with telomeres, we investigate what other telomere-related functions it may have. Telomeric G-quadruplexes have been fully characterized in vitro, but until now no clear evidence of their function or in vivo interactions with proteins has been revealed in mammalian cells. Techniques used were immunoprecipitation, DNA pull-down, binding assay, and Western blots. We identified hnRNP U as a G-quadruplex binding protein. Immunoprecipitations disclosed that endogenous hnRNP U associates with telomeres, and DNA pull-downs showed that the hnRNP U C-terminus specifically binds telomeric G-quadruplexes. We have compared the effect of telomere repeat containing RNA (TERRA) on binding between hnRNP U and telomeric (Tel) or single- stranded Tel (ssTel) oligonucleotides and found that ssTel binds stronger to TERRA than to Tel. We also show that hnRNP U prevents replication protein A (RPA) accumulation at telomeres, and the recognition of telomeric ends by hnRNP suggests that a G-quadruplex promoting protein regulates its accessibility. Thus, hnRNP U-mediated formation has important functions for telomere biology.

The lectin Dectin-1 is a good target for β-glucan-mediated drug delivery. Although many murine studies of Dectin-1 have been performed, its human analog has not been studied well in terms of being a drug delivery target. We thus analyzed human Dectin-1 cDNA obtained from chronic myelogenous leukemia-derived cells, CML-1, and confirmed the findings of previous studies that there are many isoforms of human Dectin-1 due to exon skipping, although murine Dectin-1 has only two forms. When we transfected the Dectin-1 gene into a non-Dectin-1-expressing cell line and examined cellular uptake of the antisense DNA/β-glucan complex, we confirmed that expression of the target gene was effectively suppressed through β-glucan/Dectin-1-mediated uptake. The present results suggest that the β-glucan complex would be an effective tool to deliver antisense oligonucleotide (AS-ODN) to Dectin-1-expressing cells not only for mice but also for humans.

Silencing Y-box binding protein 1 (YB-1) can be an excellent target for cancer therapy and many lung cancer cells express the polysaccharide-recognition receptor Dectin-1. We designed a Dectin-1 targeting vehicle delivering YB-1-antisense DNA. First, we selected five optimal antisense DNA sequences to silence YB-1 from among 153 candidates. We chose the sequence closest to the start codon (AS014), and attached dA40 to the 3' end; dA40 promotes complex formation with a β-(1➝3)-d-glucan called schizophyllan (SPG). The resultant complexes were applied to 12 human-oriented lung cancer cell lines, and cell viability was examined. The cell lines exhibited decreased viability and showed strong affinity to bind SPG, suggesting the AS014/SPG complex entered the cells via the Dectin-1 mediated pathway.

Antisense oligonucleotides (AS-ODNs) hybridize with specific mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. We previously demonstrated that the β-glucan schizophyllan (SPG) can form a complex with AS-ODNs with attached dA40 (AS-ODNs/SPG), and this complex can be incorporated into cells, such as macrophages and dendritic cells, expressing the β-glucan receptor Dectin-1. We have achieved efficient gene silencing in animal models, but the uptake mechanism and intracellular distribution are unclear. In this study, we prepared the complex consisting of SPG and AS-ODNs (AS014) for Y-box binding protein-1 (YB-1). After treatment with endocytosis inhibitor Pitstop 2 and small interfering RNA targeting Dectin-1, we found that AS014/SPG complexes are incorporated into cells by Dectin-1-mediated endocytosis and inhibit cell growth in a Dectin-1 expression level-dependent manner. After treatment with AS014/SPG complexes, we separated the cell lysate into endosomal and cytoplasmic components by ultracentrifugation and directly determined the distribution of AS014 by reverse transcription PCR using AS014 ODNs as a template or a reverse transcription primer. In the cytoplasm, AS014 clearly hybridized with YB-1 mRNAs. This is the first demonstration of the distinct distribution of the complex in cells. These results could facilitate the clinical application of the complex.

Nitric oxide (NO), synthesized by NOSs (NO synthases), plays a role in the development of pulmonary hypertension (PH). However, the role of NO/NOSs in bone marrow (BM) cells in PH remains elusive.To determine the role of NOSs in BM cells in PH.Experiments were performed on 36 patients with idiopathic pulmonary fibrosis and on wild-type (WT), nNOS (neuronal NOS)In the patients, there was a significant correlation between higher pulmonary artery systolic pressure and lower nitrite plus nitrate levels in the BAL fluid. In the mice, hypoxia-induced PH deteriorated significantly in the n/i/eNOSsThese results demonstrate that myelocytic n/i/eNOSs play an important protective role in the pathogenesis of PH.

The polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts) family of enzymes regulates the critical initial steps of mucin-type O-glycosylation. Among GalNAc-Ts that may significantly influence cancer biology, thus affecting cell differentiation, adhesion, invasion, and/or metastasis, GalNAc-T3 exhibits a high expression in several human cancers, closely associated with tumor progression and a poor prognosis. However, the expression pattern of GalNAc-T3 in oral squamous cell carcinoma (OSCC) remains obscure. Since postoperative recurrence of even early stage OSCC (ESOSCC) occurs at an early phase, significantly affecting their clinical course and worse outcome, the identification of clinically significant accurate biomarkers is needed. Therefore, we investigated the correlation between the immunohistochemical GalNAc-T3 expression and various clinicopathological characteristics and recurrence using 110 paraffin-embedded tumor samples obtained from patients with surgically resected ESOSCC (T1–2N0). Recurrence was recognized in 37 of 110 (33.6 %) patients. The GalNAc-T3 expression was considered to be strongly positive when 20 % or more of the cancer cells showed positive cytoplasmic staining. Consequently, a strong expression of GalNAc-T3 was observed in 40 patients (36.4 %), showing a close relationship to poor differentiation, the presence of lymphatic and vascular invasion, and recurrence. Univariate and multivariate analyses further demonstrated that the patients with a strong GalNAc-T3+ status had markedly lower disease-free survival (DFS) rates, especially within the first 2 years postoperatively. Therefore, GalNAc-T3 might play a role in the pathogenesis of ESOSCC recurrence, and its immunohistochemical detection potentially predicts a shorter DFS and may be a useful parameter for providing clinical management against ESOSCC in the early postoperative phase. Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-3928-7) contains supplementary material, which is available to authorized users.

Mitochondria are important cellular organelles that function as control centers of the energy supply for highly proliferative cancer cells and regulate apoptosis after cancer chemotherapy. Cisplatin is one of the most important chemotherapeutic agents and a key drug in therapeutic regimens for a broad range of solid tumors. Cisplatin may directly interact with mitochondria, which can induce apoptosis. The direct interactions between cisplatin and mitochondria may account for our understanding of the clinical activity of cisplatin and development of resistance. However, the basis for the roles of mitochondria under treatment with chemotherapy is poorly understood. In this review, we present novel aspects regarding the unique characteristics of the mitochondrial genome in relation to the use of platinum-based chemotherapy and describe our recent work demonstrating the importance of the mitochondrial transcription factor A (mtTFA) expression in cancer cells.

The SMARCE1 (SWI / SNF-related, matrix-associated, and actin-dependent regulator of chromatin, subfamily e, member 1) encodes BAF57 protein. Previously, we reported that BAF57 is a predictive marker of endometrial carcinoma. In this study, we investigated BAF57 expression in ovarian cancer cell lines and their sensitivities to cisplatin, doxorubicin, paclitaxel, and 5-fluorouracil. BAF57 expression was strongly correlated with sensitivities to cisplatin, doxorubicin, and 5-fluorouracil in 10 ovarian cancer cell lines. Paclitaxel sensitivity was also correlated with BAF57 expression, but without significance. In A2780 ovarian cancer cells, knockdown of BAF57 using specific siRNA increased cell cycle arrest at G1 phase and the sensitivities to these anticancer agents. cDNA microarray analysis of A2780 cells transfected with BAF57 siRNA showed that 134 genes were positively regulated by BAF57, including ATP-binding cassette, sub-family G (WHITE), member 2 (ABCG2) encoding breast cancer resistance protein (BCRP). We confirmed that knockdown of BAF57 decreased BCRP expression in ovarian cancer cells by Western blot analysis, and that ABCG2 gene expression might be regulated transcriptionally. These results suggested that BAF57 is involved in ovarian cancer cell growth and sensitivity to anticancer agents, and that BAF57 may be a target for ovarian cancer therapy.

Background/aim Mitochondrial transcription factor A (mtTFA) is necessary for both the transcription and maintenance of mitochondrial DNA (mtDNA). The present study investigated the clinical significance of mtTFA in patients with right- and left-sided colorectal cancer (CRC). Patients and methods Surgical specimens from 237 CRC patients were immunohistochemically stained with polyclonal anti-mtTFA antibody. The relationships among the mtTFA expression, clinicopathological factors and prognosis were evaluated. Results Thirty-five (60.3%) of 58 right-sided CRC patients and 82 (45.8%) of 179 left-sided CRC patients showed high mtTFA expression. The mtTFA expression significantly correlated with lymph node metastasis, distant metastasis, the TNM stage and lymphatic invasion in left-sided CRC patients and did not correlate with any factors in right-sided CRC patients. Univariate and multivariate analyses revealed the mtTFA expression to be a significant prognostic factor in left-sided CRC patients but not in right-sided CRC patients. Conclusion These results suggest that a high mtTFA expression is a useful marker for tumor progression and a poor prognosis in left-sided CRC patients.

The aberrant glycosylation of mucin type O-glycans is thought to be associated with functional alteration of cancer cells, including adhesive properties, as well as their potential for invasion and metastasis. Positive expression of N-acetylgalactosaminyltransferase-6 (GalNAc-T6) may also be a marker for aberrant O-glycans in carcinogenesis. We previously reported that over-expression of GalNAc-T6 had a strong association with endometrial cell invasion ability in vitro.This study investigated the relationship between GalNAc-T6 expression and cell adhesion molecules in 218 endometrial carcinomas by immunohistochemistry.Expression of GalNAc-T6 was found to be significantly related to expression of E-cadherin. Positive expression of GalNAc-T6 was significantly associated with better histological grade and good clinical prognosis of patients, but positive E-cadherin and β-catenin expression were not significantly associated with improved overall survival.GalNAc-T6 might be related to cell-cell adhesion in the early phase of cancer invasion in endometrial carcinoma.

Background: The transcription factor, zinc finger protein 143 (ZNF143), positively regulates many cell-cycle-related genes. The ZNF143 would show high expression of multiple solid tumours related closely to cancer cell growth, similar to the widely accepted Ki67 (MIB-1) protein, but the underlying mechanisms for ZNF143 remain unclear. We investigated the association of ZNF143 expression with clinicopathological features and prognoses of patients with lung adenocarcinoma. Methods: Expressions of ZNF143 and MIB-1 were immunohistochemically analysed in 183 paraffin-embedded tumour samples of patients with lung adenocarcinoma. The ZNF143 expression was considered to be strong when >30% of the cancer cells demonstrated positive staining. Results: Strong ZNF143+ expression showed a significantly close relationship to pathologically moderate to poor differentiation and highly invasive characteristics. The ZNF143 positivity potentially induced cell growth of lung adenocarcinoma, correlated significantly with high MIB-1 labelling index (⩾10%). Univariate and multivariate analyses demonstrated that both strong ZNF143+ and the high MIB-1 index group have only and significantly worse survival rates. Conclusions: The combination of strong ZNF143 expression and high MIB-1 index potentially predicts high proliferating activity and poor prognosis in patients with lung adenocarcinoma, and may offer a therapeutic target against ZNF143.

The assembly of spliceosomal U snRNPs in metazoans requires nuclear export of U snRNA precursors. Four factors, nuclear cap-binding complex (CBC), phosphorylated adaptor for RNA export (PHAX), the export receptor CRM1 and RanGTP, gather at the m7G-cap-proximal region and form the U snRNA export complex. Here we show that the multifunctional RNA-binding proteins p54nrb/NonO and PSF are U snRNA export stimulatory factors. These proteins, likely as a heterodimer, accelerate the recruitment of PHAX, and subsequently CRM1 and Ran onto the RNA substrates in vitro, which mediates efficient U snRNA export in vivo. Our results reveal a new layer of regulation for U snRNA export and, hence, spliceosomal U snRNP biogenesis.

// Koichi Azuma 1 , Akihiko Kawahara 2 , Kahori Sonoda 3 , Kazutaka Nakashima 2 ,Kousuke Tashiro 4 , Kosuke Watari 3 , Hiroto Izumi 5 , Masayoshi Kage 2 , Michihiko Kuwano 6 , Mayumi Ono 3 and Tomoaki Hoshino 1 1 Division of Respirology, Neurology, and Rheumatology, Department of Internal Medicine, Kurume University School of Medicine, Kurume, Fukuoka, Japan 2 Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Fukuoka, Japan 3 Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 4 Department of Bioscience and Biotechnology, Kyushu University, Fukuoka, Japan 5 Department of Occupational Pneumology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan 6 Laboratory of Molecular Cancer Biology, Department of Clinical Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan Correspondence: Koichi Azuma, email: // Keywords : activating EGFR mutation, FGFR1, non-small cell lung cancer, afatinib Received : January 31, 2014 Accepted : March 24, 2014 Published : March 26, 2014 Abstract Most NSCLC patients with EGFR mutations benefit from treatment with EGFR-TKIs, but the clinical efficacy of EGFR-TKIs is limited by the appearance of drug resistance. Multiple kinase inhibitors of EGFR family proteins such as afatinib have been newly developed to overcome such drug resistance. We established afatinib-resistant cell lines after chronic exposure of activating EGFR mutation-positive PC9 cells to afatinib. Afatinib-resistant cells showed following specific characteristics as compared to PC9: [ 1 ] Expression of EGFR family proteins and their phosphorylated molecules was markedly downregulated by selection of afatinib resistance; [ 2 ] Expression of FGFR1 and its ligand FGF2 was alternatively upregulated; [ 3 ] Treatment with anti-FGF2 neutralizing antibody blocked enhanced phosphorylation of FGFR in resistant clone; [ 4 ] Both resistant clones showed collateral sensitivity to PD173074, a small-molecule FGFR-TKIs, and treatment with either PD173074 or FGFR siRNA exacerbated suppression of both afatinib-resistant Akt and Erk phosphorylation when combined with afatinib; [ 5 ] Expression of twist was markedly augmented in resistant sublines, and twist knockdown specifically suppressed FGFR expression and cell survival. Together, enhanced expression of FGFR1 and FGF2 thus plays as an escape mechanism for cell survival of afatinib-resistant cancer cells, that may compensate the loss of EGFR-driven signaling pathway.

The aurora kinases are serine/threonine kinases that are essential for mitosis and contribute to tumorigenesis. Therefore, aurora kinases hold promise for molecularly targeted therapy. In the present study, we demonstrated that aurora B kinase (AURKB) is overexpressed in both cisplatin- and oxaliplatin-resistant cells. Downregulation of AURKB sensitized cells to both cisplatin and oxaliplatin, but not to paclitaxel, 5-FU or hydrogen peroxide. Interestingly, we found that both cisplatin- and oxaliplatin-resistant cells were hypersensitive to the AURKB specific inhibitors, AZD1152 HQPA and ZM447439, suggesting that both cisplatin- and oxaliplatinresistant cells develop an addiction to AURKB. These data provide evidence that aurora kinase inhibitors can overcome both cisplatin and oxaliplatin resistance. Therefore, AURKB inhibitors could offer potential benefits if used after first-line platinum-based chemotherapy.

Background: The polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) family of enzymes regulates the initial steps of mucin-type O-glycosylation. N-acetylgalactosaminyltransferases might show novel patterns of GalNAc-T glycosylation on tumour-derived proteins, which could influence cancer biology, but its mechanisms are unclear. We investigated the association of GalNAc-T3 and -T6 expressions with clinicopathological features and prognoses of patients with renal cell carcinomas (RCCs). Methods: Expressions of GalNAc-T3/6 and cell-adhesion molecules were analysed immunohistochemically in 254 paraffin-embedded tumour samples of patients with RCC. Results: Of 138 GalNAc-T3+ cases, 46 revealed significant co-expression with GalNAc-T6. N-acetylgalactosaminyltransferases-3+ expression showed a close relationship to poor clinical performance and large tumour size, or pathologically high Fuhrman's grading, and presence of vascular invasion and necrosis. The GalNAc-T3-positivity potentially suppressed adhesive effects with a significantly low β-catenin expression. Univariate and multivariate analyses showed the GalNAc-T3+ group, but not the GalNAc-T6+ group, to have significantly worse survival rates. Conclusion: N-acetylgalactosaminyltransferases-3 expression independently predicts high-grade tumour and poor prognosis in patients with RCC, and may offer a therapeutic target against RCC.

This study aimed to elucidate the mechanisms underlying the resistance of breast cancer to eribulin. Seven eribulin-resistant breast cancer cell lines (MCF7/E, BT474/E, ZR75-1/E, SKBR3/E, MDA-MB-231/E, Hs578T/E, and MDA-MB-157/E) were established. mRNA and protein expression of ATP-binding cassette subfamily B member 1 (ABCB1) and subfamily C member 11 (ABCC11) increased in all eribulin-resistant cell lines compared to the parental cell lines. When ABCB1 or ABCC11 expression was inhibited by small interfering RNA in MCF7/E, BT474/E, and MDA-MB-231/E cells, eribulin sensitivity was partially restored. Moreover, eribulin resistance was attenuated additively by inhibiting ABCB1 and ABCC11 in MCF7/E cells. Additionally, overexpression of exogenous ABCB1 or ABCC11 in HEK293T cells conferred resistance to eribulin. MCF7/E and MDA-MB-231/E cells showed cross-resistance to paclitaxel, doxorubicin, and fluorouracil. Inhibition of ABCB1 partially restored paclitaxel and doxorubicin sensitivity. Partial restoration of fluorouracil sensitivity was induced by inhibiting ABCC11 in MCF7/E and MDA-MB-231/E cells. Both ABCB1 and ABCC11 are involved in the development of eribulin resistance in breast cancer cells in vitro regardless of the breast cancer subtype. Thus, ABCB1 and ABCC11 expression may be used as a biomarker for predicting the response to eribulin in patients with breast cancer. Concomitant inhibition of ABCB1 and ABCC11 might help enhance the antitumor effects of eribulin.

We investigated the effects of bicarbonate/lactate-buffered peritoneal dialysis fluid (B/L-PDF) and lactate-buffered PDF (L-PDF) on cell viability and apoptosis, focusing on monocarboxylate transporters (MCTs). MCT-1 transports lactate into cells. Cell viability and apoptosis of human peritoneal mesothelial cells (HPMCs) were examined by water-soluble tetrazolium salt-1 and TUNEL assays, respectively. The relative number of viable HPMCs was significantly decreased by L-PDF at 48 h (8.8 ± 0.4%) compared with cells cultured in M199, but not by B/L-PDF (66.7 ± 1.1%). Apoptosis was markedly induced by L-PDF at 48 h (69.3 ± 16.2%), but not by B/L-PDF (2.6 ± 0.3%). Knockdown of MCT-1 by small interfering RNA (siRNA) attenuated the L-PDF-induced reduction of viable cells and increased apoptosis compared with control siRNA, but MCT-4 knockdown had no effect. B/L-PDF had lesser effects on cell viability and apoptosis of HPMCs compared with L-PDF. These results suggest that B/L-PDF biocompatibility occurs by avoiding the induction of apoptosis in HPMCs.

Inhalation studies are the gold standard for the estimation of the harmful effects of respirable chemical substances, while there is limited evidence of the harmful effects of chemical substances by intratracheal instillation. We reviewed the effectiveness of intratracheal instillation studies for estimating the hazards of nanoparticles, mainly using papers in which both inhalation and intratracheal instillation studies were performed using the same nanoparticles. Compared to inhalation studies, there is a tendency in intratracheal instillation studies that pulmonary inflammation lasted longer in the lungs. A difference in pulmonary inflammation between high and low toxicity nanoparticles was observed in the intratracheal instillation studies, as in the inhalation studies. Among the endpoints of pulmonary toxicity, the kinetics of neutrophil counts, percentage of neutrophils, and chemokines for neutrophils and macrophages, heme oxygenase-1 (HO-1) in bronchoalveolar lavage fluid (BALF), reflected pulmonary inflammation, suggesting that these markers may be considered the predictive markers of pulmonary toxicity in both types of study. When comparing pulmonary inflammation between intratracheal instillation and inhalation studies under the same initial lung burden, there is a tendency that the inflammatory response following the intratracheal instillation of nanoparticles is greater than or equal to that following the inhalation of nanoparticles. If the difference in clearance in both studies is not large, the estimations of pulmonary toxicity are close. We suggest that intratracheal instillation studies can be useful for ranking the hazard of nanoparticles through pulmonary inflammation.

Objective: Expression of N-myc downstream-regulated gene 1 (NDRG1)/Cap43 is a prognostic indicator of human malignancies according to the tumor type in which it occurs. We investigated how NDRG1/Cap43 could affect tumor growth and angiogenesis in non–small-cell lung cancer (NSCLC) in vivo using an animal experimental model, and also how it could affect tumor angiogenesis and prognosis in NSCLC patients. Methods and Results: Knockdown of NDRG1/Cap43 in lung cancer cells using a specific small interfering RNA resulted in growth rates in culture that were similar to those of counterpart control cells, but decreased tumor growth rates in vivo markedly. Stable NDRG1/Cap43 knockdown did not induce consistent changes in the expression of Epidermal growth factor receptor (EGFR) family proteins and c-Met in two human lung cancer cell lines in vitro. However, cell lines with NDRG1/Cap43 knockdown showed markedly decreased production of the potent angiogenic factors vascular endothelial growth factor-A and interleukin-8. Cells with knockdown of NDRG1/Cap43 showed marked reduction of tumor-induced angiogenesis. Using immunohistochemistry, we examined 182 surgically resected specimens of NSCLC for expression of NDRG1/Cap43 and tumor angiogenesis. High microvessel density in the tumor was significantly associated with nuclear positivity for NDRG1/Cap43 in both adenocarcinoma ( p = 0.003) and squamous cell carcinoma ( p =0.041). For both adenocarcinoma ( p = 0.031) and squamous cell carcinoma ( p =0.034), the survival curve of patients negative for nuclear NDRG1/Cap43 expression differed significantly from that of patients who were positive. Conclusion: Therefore, the expression of NDRG1/Cap43 may be predictive of tumor angiogenesis and poor prognosis in NSCLC.

In the workplace, localized muscle fatigue can be a limiting factor for prolonged static work. It is important to study the etiology of fatigue in the muscle to prevent discomfort and potential muscle injury. The purpose of this study was to investigate the effect of sustained trunk flexion and loading on the erector spinae muscles (ESM) fatigue using electromyography (EMG) and muscle oxygenation (Mox) changes. Twelve healthy male volunteers, aged between 19 and 28 years (mean age 23 +/- 2.6), participated in this study. The subjects held a load (5, 10 and 15 kg) and no load in their hands whilst sustaining trunk flexion at 60 degrees and also in an upright standing posture for 30 seconds. Five minutes rest was given between the conditions, and each condition was randomly repeated three times. Surface EMG and near-infrared spectroscopy were recorded. Root mean square and median frequency (MF) of EMG, and Mox were calculated. The results showed a greater decrease in Mox with an increase in loads over time (P = 0.015). Using unloaded trunk flexion as a control to ascertain load effect resulted in a significant difference in Mox in the unloaded condition and in the 15 kg condition (P = 0.027). EMG results showed a significant decrease in the MF over time in trunk flexion while holding a load (P < 0.001). These results show objectively that trunk flexion while holding loads causes ESM fatigue.

Blockage of hepatic autophagic degradation system occurs in obesity and is associated with the development of nonalcoholic fatty liver disease. However, the mechanism of this blockage remains unclear. We found a high-fat diet induced accumulation of autophagosomes in the mice livers. However, autophagy substrates such as p62 and ubiquitinated proteins also accumulated in the livers in this model. These findings indicate the possibility that a high-fat diet impairs autophagic flux in the liver. Then, to assess the autophagic flux in more detail, we performed analyses of autophagic flux in cultured hepatocytes exposed to monounsaturated fatty acids (FAs) or saturated FAs (SFAs). SFAs but not monounsaturated FAs suppressed degradation of contents in the autophagosomes. We analyzed each stage of the autophagy pathway (ie, autophagosome formation, autophagosome-lysosome fusion, lysosomal degradation) in cultured hepatocytes treated with monounsaturated FAs or SFAs and found that SFAs impaired autophagosome-lysosome fusion. This impairment occurred in an endoplasmic reticulum stress-dependent manner. Moreover, ubiquitin and p62-positive inclusions observed in high-fat diet–fed mice livers and SFA-treated cells were sequestered within autophagosomes. We also found that SFA-induced accumulation of Ser351-phosphorylated p62, which is indispensable for selective autophagy, further increased on administration of a lysosomal proteinase inhibitor. Although lipid-induced endoplasmic reticulum stress interferes with the autophagosome-lysosome fusion, selective autophagic sequestration of aggregated proteins is not inhibited.

Background: The family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) is responsible for the altered glycosylation in cancer. The purpose of our study was to investigate the clinical significance of two isoforms, GalNAc-T6 and -T3, and their correlation with the prognosis of pancreatic cancer. Methods: Immunohistochemistry was used to analyse GalNAc-T6 and -T3 expressions in 70 clinicopathologically characterised pancreatic cancer cases. Results: Positive expressions of GalNAc-T6 and -T3 were immunohistochemically identified in 51% (36 of 70) and in 77% (54 of 70) of patients, respectively. A close relationship was noted between GalNAc-T6 positive expression and pathological well/moderate differentiated type (P=0.001), small tumour size (P=0.044), absence of vascular invasion (P=0.009), and low stage of the American Joint Committee on Cancer systems (P=0.043). The expression of GalNAc-T3 significantly correlated with good differentiation (P=0.001), but not with other clinicopathologic features. Furthermore, univariate and multivariate analyses revealed that GalNAc-T6 expression was an independent prognosis indicator for the disease, whereas GalNAc-T3 expression had no impact on clinical outcome, even though 33 of 36 GalNAc-T6-positive cases also had a positive expression of GalNAc-T3 (P=0.001, r=0.356). Conclusion: Both GalNAc-T6 and -T3 expressions correlated significantly with tumour differentiation, whereas only GalNAc-T6 expression predicted prognosis in pancreatic cancer.

Mitochondrial transcription factor A (mtTFA) is one of the high mobility group protein family and is required for both transcription from and maintenance of mitochondrial genomes. However, the roles of mtTFA have not been extensively studied in cancer cells. Here, we firstly reported the nuclear localization of mtTFA. The proportion of nuclear-localized mtTFA varied among different cancer cells. Some mtTFA binds tightly to the nuclear chromatin. DNA microarray and chromatin immunoprecipitation assays showed that mtTFA can regulate the expression of nuclear genes. Overexpression of mtTFA enhanced the growth of cancer cell lines, whereas downregulation of mtTFA inhibited their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). Knockdown of mtTFA expression induced p21-dependent G1 cell cycle arrest. These results imply that mtTFA functions in both nuclei and mitochondria to promote cell growth.

Cancer cells show constitutive upregulation of glycolysis, and the concentration of lactate thus produced correlates with prognosis. Here, we examined whether lactate concentration and lactate transporter expression are related to migration and invasion activity. We found that the expression of the monocarboxylate transporters MCT1 and MCT4, but not MCT5, in human lung cancer cell lines was significantly correlated with invasiveness. To clarify the effects of MCT1 and MCT4 expression on invasion, we performed migration and invasion assays after transfection with siRNA specific for MCT1 or MCT4. Knockdown of MCT1 or MCT4 did not influence cell migration but reduced invasion; this was also observed for knockdown of the lactate transporter-associated protein basigin. We also demonstrated that both expression and activity of MMP9 and MMP2 were not correlated with invasion activity and not regulated by MCT1, MCT4 and basigin. Furthermore, the addition of lactate did not increase migration and invasion activity, but low concentration of 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), a general anion channel blocker, as well as other MCT inhibitors quercetin and simvastatin, inhibited cell invasion without influencing migration activity and the cellular expression of MCT1 and MCT4. This is the first report suggesting that lactate transporters are involved in human cancer cell invasiveness. As such, these proteins may be promising targets for the prevention of cancer invasion and metastasis.

We previously reported that mitochondrial transcription factor A (mtTFA) preferentially recognizes cisplatin-damaged DNA via physical interaction with p53 and is upregulated by treatment with cisplatin and fluorouracil (5-FU). The aim of the present study was to evaluate whether expression of mtTFA predicts the clinical outcome in patients with metastatic colorectal cancer treated with modified 5-fluorouracil, leucovorin and oxaliplatin 6 (mFOLFOX6). Fifty-nine patients with metastatic lesions from colorectal cancer treated with mFOLFOX6 were included in this study. The subjects consisted of 25 women and 34 men with a median age of 62 years. The patients were treated with oxaliplatin (85 mg/m(2) ) plus leucovorin (200 mg/m(2) ) as a 2-h infusion on day 1, followed by 5-FU (400 mg/m(2) ) bolus and 46-h continuous infusion of 2400 mg/m(2) . The expressions of mtTFA and p53 of resected primary tumors were examined by immunohistochemical analysis. Of the 59 patients, 33 (complete response 1, partial response 32) achieved a confirmed response to therapy. The positive cytoplasmic staining rate for mtTFA was 44.1% and that for p53 was 59.3%, respectively. Strong expression of mtTFA was detected in eight of 33 complete response/partial response (24.2%) and in 18 of 26 SD/PD (69.2%), indicating that mtTFA expression was significantly correlated with response to chemotherapy (P

Peroxiredoxin 4 (PRDX4) is one of a newly discovered family of antioxidative proteins. We generated human PRDX4 (hPRDX4) transgenic (Tg) mice, displaying a high level of hPRDX4 expression in the pancreatic islets, and then focused on the functions of PRDX4 in a type 1 diabetes mellitus (T1DM) model using a single high dose of streptozotocin (SHDS). After SHDS-injection, Tg mice showed significantly less hyperglycemia and hypoinsulinemia and a much faster response on glucose tolerance test than wild-type (WT) mice. Morphologic and immunohistochemical observation revealed that the pancreatic islet areas of Tg mice were larger along with less CD3-positive lymphocyte infiltration compared with WT mice. Upon comparison between these two mouse models, β-cell apoptosis was also repressed, and reversely, β-cell proliferation was enhanced in Tg mice. Real-time RT-PCR demonstrated that the expression of many inflammatory-related molecules and their receptors and transcription factors were significantly downregulated in Tg mice. These data indicate that PRDX4 can protect pancreatic islet β-cells against injury caused by SHDS-induced insulitis, which strongly suggests that oxidative stress plays an essential role in SHDS-induced diabetes. This study, for the first time, implicates that PRDX4 has a pivotal protective function against diabetes progression in this T1DM model.

Gefitinib (Iressa) and erlotinib (Tarceva), which target the epidermal growth factor receptor (EGFR), are approved for treatment of patients with advanced non–small cell lung cancer (NSCLC). Patients whose tumors harbor mutations in the EGFR gene, including delE746-A750 in exon 19 and L858R in exon 21, may benefit in particular from gefitinib treatment. However, acquired resistance to gefitinib has been a serious clinical problem, and further optimization is needed for application of EGFR-targeted drugs in lung cancer patients. In this study, we established gefitinib-resistant NSCLC cells from PC-9 cell line, which harbors the delE746-A750 mutation, by exposing the cell line to gefitinib for over 7 months. Gefitinib-resistant PC-9/GEFs cell lines showed a marked downregulation of PTEN expression and increased Akt phosphorylation. In revertant, gefitinib-sensitive clones (PC-9/Rev) derived from PC-9/GEF1-1 and PC-9/GEF2-1, PTEN expression, as well as sensitivity to gefitinib and erlotinib, was restored. Knockdown of PTEN expression using small interfering RNA specific for PTEN in PC-9 cells resulted in drug resistance to gefitinib and erlotinib. Nuclear translocation of the EGR1 transcription factor, which regulates PTEN expression, was shown to be suppressed in resistant clones and restored in their revertant clones. Reduced PTEN expression was also seen in tumor samples from a patient with gefitinib-refractory NSCLC. This study thus strongly suggests that loss of PTEN expression contributes to gefitinib and erlotinib resistance in NSCLC. Our findings reinforce the therapeutic importance of PTEN expression in the treatment of NSCLC with EGFR-targeted drugs. Cancer Res; 70(21); 8715–25. ©2010 AACR.

Mina53, a novel target gene product of c-Myc, is overexpressed in various malignancies. We previously demonstrated that Mina53 is overexpressed in lung cancer patients from the early clinical stages. In this paper, the association between disease prognosis and Mina53 expression in lung cancer patients is analyzed; we found that overexpression of Mina53 in lung cancer patients is associated with favorable prognosis. Statistical analysis using the Kaplan–Meier method showed that patients with negative staining for Mina53 had significantly shorter survival than patients with positive staining for Mina53, especially in stage I or with squamous cell carcinoma. Because the major cause of death in lung cancer patients after surgery is distant metastasis, the effect on cancer cell invasiveness was analyzed for the mechanisms involved in the association with favorable outcome. Overexpression of Mina53 in H226B, a lung squamous cell carcinoma cell line, inhibited cancer cell invasion. Transfection with mina53 shRNA increased the number of invading cells. These results suggest that Mina53 immunostaining is a useful prognostic marker – especially in the early stage of lung cancer – and that Mina53 negative patients should be managed particularly carefully after surgery.

Patients with non-small cell lung cancer harboring mutations in the epidermal growth factor receptor gene, including delE746-A750 and L858R, are highly sensitive to therapy with epidermal growth factor receptor-targeting drugs, such as gefitinib and erlotinib, in comparison with those harboring wild-type epidermal growth factor receptor. It remains unclear how such epidermal growth factor receptor mutations are induced. In this study, we examined whether 8-hydroxy-2'-deoxyguanosine, a representative oxygen nucleotide of DNA, could play a role in activating mutations of the epidermal growth factor receptor gene and also whether Y-box binding protein-1 and 8-oxoguanine DNA glycosylase that are involved in repair of oxidative stimuli-induced DNA damages could play any role in epidermal growth factor receptor activating mutations. Immunohistochemistry was used to evaluate the expression of 8-hydroxy-2'-deoxyguanosine, Y-box binding protein-1, and 8-oxoguanine DNA glycosylase in patients with non-small cell lung cancer (N = 170). We analyzed mutations of delE746-A750 and L858R in the epidermal growth factor receptor gene using peptide nucleic acid-locked nucleic acid polymerase chain reaction clamping. In non-small cell lung cancer patients, nuclear 8-hydroxy-2'-deoxyguanosine expression was strongly associated with these epidermal growth factor receptor mutations. Furthermore, nuclear expression of Y-box binding protein-1 was inversely associated with epidermal growth factor receptor mutations; but nuclear expression of 8-oxoguanine DNA glycosylase was not. Among 51 patients who were treated with gefitinib, progression-free survival was substantially better when 8-hydroxy-2'-deoxyguanosine expression was positive, when epidermal growth factor receptor mutations were present, and when nuclear Y-box binding protein-1 expression was negative. Thus, activating mutations of the epidermal growth factor receptor gene in non-small cell lung cancer were closely associated with a decrease in the damage repair process for 8-hydroxy-2'-deoxyguanosine in oxidized DNA.

Histone acetyltransferase (HAT) regulates transcription. We have previously shown that two HAT genes, Clock and Tip60, are overexpressed, and upregulate glutathione biosynthesis and the expression of DNA repair genes in cisplatin-resistant cells. To better understand the mechanism of HAT-related drug resistance, we investigated the role of another HAT gene, p300/CBP-associated factor (PCAF), and found that PCAF was also overexpressed in cisplatin-resistant cells and endowed an antiapoptotic phenotype through enhanced E2F1 expression. PCAF-overexpressing cells showed enhanced expression of E2F1 and conferred cell resistance to chemotherapeutic agents. Downregulation of PCAF decreased E2F1 expression and sensitized cells to chemotherapeutic agents. Moreover, knockdown of PCAF induced G1 arrest and apoptosis. These results suggest that PCAF is one of pleiotropic factors for drug resistance and seems to be critical for cancer cell growth. Mol Cancer Res; 8(6); 864–72. ©2010 AACR.

We have recently reported that N-myc downstream-regulated gene 1 (NDRG1)/Ca(2+)-associated protein with a molecular mass of 43kDa (Cap43) suppresses angiogenesis and tumor growth of pancreatic cancer through marked decreases in both the expression of CXC chemokines and phosphorylation of a NF-kappaB signaling molecule, inhibitor of kappaB kinase (IkappaBalpha). NDRG1/Cap43 is phosphorylated at serine/threonine sites in its C-terminal domain by serum- and glucocorticoid-regulated kinase 1 (SGK1). In this study, we attempted to clarify the domain or site of NDRG1/Cap43 responsible for its suppression of CXC chemokine expression in pancreatic cancer cells. Expression of the deletion constructs CapDelta2 [deletion of amino acids (AA) 130-142] and CapDelta4 [deletion of AA 180-294] as well as the wild-type full sequence of NDRG1/Cap43 (F-Cap), suppressed the production of CXC chemokines such as Groalpha/CXCL1 and ENA-78/CXCL5, whereas no or low suppression was observed in cell expressing the CapDelta5 mutant [deletion of AA 326-350] and CapDelta6 mutant [deletion of AA 326-394]. We further introduced mutations at the serine and threonine sites at 328 [T328A], 330 [S330A] and 346 [T346A], which are susceptible to phosphorylation by SGK1, and also constructed double mutants [T328A, S330A], [T328A, T346A] and [S330A, T346A]. Expression of all these mutants, with the exception of [S330A, T346A], suppressed the production of CXC chemokine to similar levels as their wild-type counterpart. IkappaBalpha was found to be specifically phosphorylated by this double mutant [S330A, T346A] and the CapDelta5 mutant at levels comparable to that induced in their wild-type counterpart. Phosphorylation of NDRG1/Cap43 at both serine330 and threonine346 is required for its suppressive action on the NF-kappaB signaling pathway and CXC chemokine expression in pancreatic cancer cells.

Y-box binding protein-1 (YB-1) plays pivotal roles in acquisition of global drug resistance and cell growth promotion through transcriptional activation of genes for both drug resistance and growth factor receptors. In this study, we investigated whether YB-1 is involved in regulation of the cell cycle and cell proliferation of human cancer cells. Treatment with YB-1 siRNA caused a marked suppression of cell proliferation and expression of a cell cycle related gene, CDC6 by cancer cells. Of cell cycle of cancer cells, S phase content was specifically reduced by knockdown of YB-1. The overexpression of CDC6 abrogated this inhibition of both cell proliferation and S phase entry. ChIP assay demonstrated that YB-1 binds to a Y-box located in the promoter region of the CDC6 gene. Expression of cyclin D1, CDK1 and CDK2 was also reduced with increased expression of p21(Cip1) and p16(INK4A) when treated with YB-1 siRNA. Furthermore, the nuclear YB-1 expression was significantly associated with the level of CDC6 nuclear expression in patients with breast cancer. In conclusion, YB-1 plays an important role in cell cycle progression at G1/S of human cancer cells. YB-1 thus could be a potent biomarker for tumour growth and cell cycle in its close association with CDC6.

Mitochondrial transcription factor A (mtTFA) is necessary for both transcription and maintenance of mitochondrial DNA. This study was conducted to elucidate the clinicopathologic and prognostic significance of mtTFA in patients with endometrial carcinoma. This study investigated the relationship between the immunohistochemical expression of mtTFA and various clinicopathological variables in 276 endometrial carcinomas, including 245 endometrioid adenocarcinomas and 31 nonendometrioid carcinomas (21 serous carcinomas and 10 clear cell adenocarcinomas). Both uni- and multivariate regression analyses were performed. The mtTFA labeling index of endometrioid adenocarcinomas ranged from 0% to 98%, with a median value of 32%, which was selected as the cut-off point for mtTFA expression. The mtTFA expression in endometrioid adenocarcinomas was significantly associated with the surgical stage, myometrial invasion, lymphovascular space invasion, cervical invasion, and lymph node metastasis. In contrast, no correlation between clinicopathologic variables and mtTFA expression was found in nonendometrioid carcinomas. Correlation analysis between mtTFA and p53 expression by using the Pearson test showed significant correlation in endometrioid adenocarcinomas (P = 0.007), but no significant correlation in nonendometrioid carcinomas (P = 0.947). A univariate survival analysis showed that the 10-year overall survival rate of the patients with mtTFA-positive endometrioid adenocarcinoma was significantly worse than that of patients with mtTFA-negative endometrioid adenocarcinoma (80.8% vs. 93.8%, P = 0.012). However, the multivariate analysis revealed that mtTFA expression in endometrioid adenocarcinomas was no independent prognostic factor. The positive mtTFA expression is a useful maker for progression of the tumors and the poor prognosis of the patients in endometrioid adenocarcinomas.

Mina53, whose expression is directly induced by c-Myc, is overexpressed in various cancers and plays an important role in cell growth. To clarify the involvement of Mina53 in lung cancers, we investigated its expression in human lung cancer tissues as well as in various lung cancer cell lines. Mina53 expression was determined by real-time RT-PCR, western blotting, and immunohistochemistry using lung cancer cell lines, normal human bronchial epithelial cells, and lung cancer tissues. Biological effects of Mina53 were evaluated by soft agar colony formation assay and tumorigenicity in nude mice using Mina53-transfected NIH/3T3 cells. cDNA microarray analysis was performed to determine the gene alteration by Mina53 and confirmation was made using real-time RT-PCR with mina53 expression plasmid or mina53 shRNA-transfected NIH/3T3 cells. We observed that 62% of patients evidenced overexpression of Mina53 from the early clinical stages of lung cancer. Differences according to gender, smoking status, or histologic type were not statistically significant. Forced expression of Mina53 in NIH/3T3 cells induced cell transformation, and mina53-transfected NIH/3T3 clones produced tumors in nude mice, demonstrating that Mina53 has oncogenic potential. cDNA microarray revealed that 254 genes had altered expression in a mina53-transfected NIH/3T3 clone. Mina53 regulates several genes related to cell adhesion and metabolism, which have also been reported to be regulated by c-Myc. Genes regulated by Mina53, but not by c-Myc included cytokine/growth factor related genes such as EGFR, IL-6, and HGF. Our results suggest that Mina53 plays an important role in carcinogenesis and may be a target for cancer prevention.

Histone modification is important for maintaining chromatin structure and function. Recently, histone acetylation has been shown to have a critical regulatory role in both transcription and DNA repair. We report here that expression of histone acetyltransferase (HAT) genes is associated with cisplatin resistance. We found that Tip60 is overexpressed in cisplatin-resistant cells. The expression of two other HAT genes, HAT1 and MYST1, did not differ between drug-sensitive and -resistant cells. Knockdown of Tip60 expression rendered cells sensitive to cisplatin but not to oxaliplatin, vincristine, and etoposide. Tip60 expression is significantly correlated with cisplatin sensitivity in human lung cancer cell lines. Interestingly, the promoter region of the Tip60 gene contains several E boxes, and its expression was regulated by the E-box binding circadian transcription factor Clock but not by other E-box binding transcription factors such as c-Myc, Twist, and USF1. Hyperacetylation of H3K14 and H4K16 was found in cisplatin-resistant cells. The microarray study reveals that several genes for DNA repair are down-regulated by the knockdown of Tip60 expression. Our data show that HAT gene expression is required for cisplatin resistance and suggest that Clock and Tip60 regulate not only transcription, but also DNA repair, through periodic histone acetylation.

YB-1 controls gene expression through both transcriptional and translational mechanisms and is involved in various biological activities such as brain development, chemoresistance, and tumor progression. We have previously shown that YB-1 is overexpressed in cisplatin-resistant cells and is involved in resistance against DNA-damaging agents. Structural analysis of the YB-1 promoter reveals that several E-boxes may participate in the regulation of YB-1 expression. Here, we show that the E-box–binding transcription factor Twist is overexpressed in cisplatin-resistant cells and that YB-1 is a target gene of Twist. Silencing of either Twist or YB-1 expression induces G1 phase cell cycle arrest of tumor cell growth. Significantly, reexpression of YB-1 led to increase colony formation when Twist expression was down-regulated by small interfering RNA. However, cotransfection of Twist expression plasmid could not increase colony formation when YB-1 expression was down-regulated. Collectively, these data suggest that YB-1 is a major downstream target of Twist. Both YB-1 and Twist expression could induce tumor progression, promoting cell growth and driving oncogenesis in various cancers. Thus, both YB-1 and Twist may represent promising molecular targets for cancer therapy. [Cancer Res 2008;68(1):98–105]

The focus of the present study was whether and how infiltrating macrophages play a role in angiogenesis and the growth of cancer cells in response to the inflammatory cytokine interleukin (IL)-1β. Lewis lung carcinoma cells overexpressing IL-1β grew faster and induced greater neovascularization than a low IL-1β-expressing counterpart in vivo. When macrophages were depleted using clodronate liposomes, both neovascularization and tumor growth were reduced in the IL-1β-expressing tumors. Co-cultivation of IL-1β-expressing cancer cells with macrophages synergistically augmented neovascularization and the migration of vascular endothelial cells. In these co-cultures, production of the angiogenic factors vascular endothelial growth factor-A and IL-8, monocyte chemoattractant protein-1, and matrix metalloproteinase-9 were increased markedly. The production of these factors, induced by IL-1β-stimulated lung cancer cells, was blocked by a nuclear factor (NF)-κB inhibitor, and also by the knockdown of p65 (NF-κB) and c-Jun using small interference RNA, suggesting involvement of the transcription factors NF-κB and AP-1. These results demonstrated that macrophages recruited into tumors by monocyte chemoattractant protein-1 and other chemokines could play a critical role in promoting tumor growth and angiogenesis, through interactions with cancer cells mediated by inflammatory stimuli. (Cancer Sci 2007; 98: 2009–2018)

Modification of transcription factors by anticancer agents plays an important role in both apoptotic and survival signaling. Here we report that both DNA topoisomerase I and II inhibitors such as SN-38 and etoposide, but not cisplatin, 5-fluorouracil or actinomycin D, can induce phosphorylation of the transcription factor Sp1. Furthermore, DNA topoisomerase inhibitors were shown to transactivate GC-box-dependent promoters such as the SV40 and vascular endothelial growth factor promoters. The phosphorylated form of Sp1 was detectable within 30 min of etoposide treatment and was greatly diminished by the presence of the PI3K inhibitor wortmannin and by DNA-dependent protein kinase (DNA-PK) knockdown. We also confirmed that the phosphorylated form of DNA-PK was increased by treatment with both etoposide and SN-38. Taken together, these findings demonstrate a novel genomic response to anticancer agents that induce Sp1 phosphorylation, and might contribute to tumor progression and drug resistance.

Zinc-finger protein 143 (ZNF143) is a human homolog of Xenopus transcriptional activator staf that is involved in selenocystyl tRNA transcription. We previously showed that ZNF143 expression is induced by treatment with DNA-damaging agents and that it preferentially binds to cisplatin-modified DNA. In this study, the potential function of ZNF143 was investigated. ZNF143 was overexpressed in cisplatin-resistant cells. ZNF143 knockdown in prostate cancer caused increased sensitivity for cisplatin, but not for oxaliplatin, etoposide and vincristine. We also showed that ZNF143 is associated with tumor suppressor gene product p73 but not with p53. p73 could stimulate the binding of ZNF143 to both ZNF143 binding site and cisplatin-modified DNA, and modulate the function of ZNF143. We provide a direct evidence that both Rad51 and flap endonuclease-1 are target genes of ZNF143 and overexpressed in cisplatin-resistant cells. Taken together, these experiments demonstrate that an interplay of ZNF143, p73 and ZNF143 target genes is involved in DNA repair gene expression and cisplatin resistance.

Extrahepatic bile duct carcinomas (EBDCs) still result in an unfavorable prognostic outcome, and little is known about their biological aggressiveness. Recently, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl transferase-3 (GalNAc-T3) was reported to be associated with differentiation and malignant potential of human carcinomas. Here, we investigated 61 EBDCs for their detailed clinicopathological features and GalNAc-T3 expression by immunohistochemistry, and then evaluated the relationships between the clinicopathological features and GalNAc-T3 expression patterns. Most of the EBDCs were massively invasive tumors with frequent vascular or perineural invasion and lymph node metastases. GalNAc-T3 expression was detected in all 61 EBDCs, and the expression patterns could be classified into granular and diffuse types. All four noninvasive or minimally invasive EBDCs were the granular type. Among the 58 minimally or massively invasive EBDCs, the GalNAc-T3 expression pattern at the luminal surface was the granular type in 38 cases (66%) and diffuse type in 20 cases (34%), while the expression pattern at the invasive front was the granular type in 26 cases (45%) and diffuse type in 32 cases (55%). Among the 38 cases with granular-type expression at the luminal surface, 26 cases (68%) remained the granular type and 12 cases (32%) became the diffuse type at the invasive front. All 20 cases with diffuse-type expression at the luminal surface remained the diffuse type at the invasive front. Diffuse-type GalNAc-T3 expression at the invasive front was significantly associated with lymph node metastasis (P

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.