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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.

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.

We analyzed the mRNA expression of chemokines in rat lungs following intratracheal instillation of nanomaterials in order to find useful predictive markers of the pulmonary toxicity of nanomaterials. Nickel oxide (NiO) and cerium dioxide (CeO2) as nanomaterials with high pulmonary toxicity, and titanium dioxide (TiO2) and zinc oxide (ZnO) as nanomaterials with low pulmonary toxicity, were administered into rat lungs (0.8 or 4 mg/kg BW). C-X-C motif chemokine 5 (CXCL5), C-C motif chemokine 2 (CCL2), C-C motif chemokine 7 (CCL7), C-X-C motif chemokine 10 (CXCL10), and C-X-C motif chemokine 11 (CXCL11) were selected using cDNA microarray analysis at one month after instillation of NiO in the high dose group. The mRNA expression of these five genes were evaluated while using real-time quantitative polymerase chain reaction (RT-qPCR) from three days to six months after intratracheal instillation. The receiver operating characteristic (ROC) results showed a considerable relationship between the pulmonary toxicity ranking of nanomaterials and the expression of CXCL5, CCL2, and CCL7 at one week and one month. The expression levels of these three genes also moderately or strongly correlated with inflammation in the lung tissues. Three chemokine genes can be useful as predictive biomarkers for the ranking of the pulmonary toxicity of nanomaterials.

It was reported that statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase that are used to prevent hypercholesterolemia, have antitumor activity in several cancers. In this study, we investigated the cell viability of statins in Cisplatin-resistant HCP4 and PCDP5 cells compared with their parent Hela and PC3 cells, respectively, and found that HCP4 and PCDP5 cells were 37-fold and 18-fold more resistant to Cisplatin but 13-fold and 7-fold more sensitive to Lovastatin by cell proliferation assay. Lovastatin induced the apoptosis of HCP4 cells more rapidly and to greater extent than in Hela cells as assessed by flow cytometry and western blotting analyses. The MVA pathway was not involved in this acquired Cisplatin resistance. To elucidate the mechanism underlying the reduced viability to Lovastatin, we performed cDNA microarray analysis and identified 65 and 54 genes that were induced more than 2-fold by Lovastatin in HCP4 and PCDP5 cells, respectively. Of these, only three genes, KLF2, KLF6, and RHOB, were commonly induced between HCP4 and PCDP5 cells. These mRNAs were strongly induced by Lovastatin with transcriptional regulation in HCP4 cells. Consistent with transcription, the protein expression of RHOB also was induced by Lovastatin. The induction of these genes was associated with cell cycle arrest and apoptosis. Combination treatment with Cisplatin and Lovastatin resulted in an agonistic effect in Hela and PC3 cells and an antagonistic effect in HCP4 and PCDP5 cells. These results suggest that statins might have the potential to overcome Cisplatin resistance as single-agent therapy.

// Asumi Iesato 1 , Teruo Nakamura 1 , Hiroto Izumi 2 , Takeshi Uehara 3 and Ken-Ichi Ito 1 1 Division of Breast, Endocrine and Respiratory Surgery, Department of Surgery (II), Shinshu University School of Medicine, Matsumoto, Japan 2 Department of Occupational Pneumology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan 3 Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan Correspondence to: Ken-Ichi Ito, email: kenito@shinshu-u.ac.jp Keywords: thyroid cancer, PATZ1, dedifferentiation, invasion, migration Received: February 25, 2017 Accepted: June 28, 2017 Published: August 02, 2017 ABSTRACT This study was designed to examine the involvement of PATZ1 in carcinogenesis and dedifferentiation of thyroid cancer. Immunohistochemistry on clinical specimens indicated nuclear PATZ1 expression in all normal thyroid glands and adenomatous goiter, while nuclear PATZ1 expression decreased along with the dedifferentiation of thyroid cancer. Knockdown of nuclear PATZ1 by siRNA in an immortalized normal follicular epithelial cell line (Nthy-ori 3-1) altered cellular morphology and significantly increased cell proliferation, migration, and invasion. In addition, the expression of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP) 2, MMP9, and MMP11 was increased by PATZ1 knockdown in Nthy-ori 3-1 cells. When PATZ1 was silenced in differentiated thyroid cancer (DTC) cell lines (TPC-1 and FTC-133), proliferation, cellular motility, and expression of uPA and MMPs were significantly increased. Forced expression of exogenous PATZ1 decreased proliferation, cellular motility, and the expression of uPA and MMPs in ATC cell lines (ACT-1 and FRO). In thyroid cancer cell lines, PATZ1 functioned as a tumor suppressor regardless of p53 status. Moreover, the ratio of nuclear PATZ1 positive tumors was significantly decreased in ATC irrespective of p53 status. Our study demonstrates that PATZ1 knockdown enhances malignant phenotype both in thyroid follicular epithelial cells and thyroid cancer cells, suggesting that PATZ1 functions as a tumor suppressor in thyroid follicular epithelial cells and is involved in the dedifferentiation of thyroid cancer.

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.

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.

Background In order to examine whether myeloperoxidase (MPO) can be a useful marker for evaluating the pulmonary toxicity of nanomaterials, we analyzed MPO protein in bronchoalveolar lavage fluid (BALF) samples obtained from previous examinations of a rat model. In those examinations we performed intratracheal instillation exposures (dose: 0.2–1.0 mg) and inhalation exposures (exposure concentration: 0.32–10.4 mg/m3) using 9 and 4 nanomaterials with different toxicities, respectively. Based on those previous studies, we set Nickel oxide nanoparticles (NiO), cerium dioxide nanoparticles (CeO2), multi wall carbon nanotubes with short or long length (MWCNT (S) and MWCNT (L)), and single wall carbon nanotube (SWCNT) as chemicals with high toxicity; and titanium dioxide nanoparticles (TiO2 (P90) and TiO2 (Rutile)), zinc oxide nanoparticles (ZnO), and toner with external additives including nanoparticles as chemicals with low toxicity. We measured the concentration of MPO in BALF samples from rats from 3 days to 6 months following a single intratracheal instillation, and from 3 days to 3 months after the end of inhalation exposure. Results Intratracheal instillation of high toxicity NiO, CeO2, MWCNT (S), MWCNT (L), and SWCNT persistently increased the concentration of MPO, and inhalation of NiO and CeO2 increased the MPO in BALF. By contrast, intratracheal instillation of low toxicity TiO2 (P90), TiO2 (Rutile), ZnO, and toner increased the concentration of MPO in BALF only transiently, and inhalation of TiO2 (Rutile) and ZnO induced almost no increase of the MPO. The concentration of MPO correlated with the number of total cells and neutrophils, the concentration of chemokines for neutrophils (cytokine-induced neutrophil chemoattractant (CINC)-1 and heme oxygenase (HO)-1), and the activity of released lactate dehydrogenase (LDH) in BALF. The results from the receiver operating characteristics (ROC) for the toxicity of chemicals by the concentration of MPO proteins in the intratracheal instillation and inhalation exposures showed that the largest areas under the curves (AUC) s in both examinations occurred at 1 month after exposure. Conclusion These data suggest that MPO can be a useful biomarker for the ranking of the pulmonary toxicity of nanomaterials, especially at 1 month after exposure, in both intratracheal instillation and inhalation exposure. Electronic supplementary material The online version of this article (10.1186/s12989-018-0277-x) contains supplementary material, which is available to authorized users.

Aurora kinase B (AURKB) inhibitors are regarded as potential molecular-targeting drugs for cancer therapy. The present study evaluated the cytotoxic effect of a combination of AZD1152-hQPA, an AURKB inhibitor, and various anticancer agents on the HeLa human cervical cancer cell line, as well as its cisplatin-resistant equivalent HCP4 cell line. It was demonstrated that AZD1152-hQPA had an antagonistic effect on the cytotoxicity of cisplatin, etoposide and doxorubicin, but had a synergistic effect on that of all-trans-retinoic acid (ATRA), Am80 and TAC-101, when tested on HeLa cells. Cisplatin, etoposide and doxorubicin were shown to increase the cellular expression of AURKB, while ATRA, Am80 and TAC-101 downregulated its expression. These results suggested that AURKB expression is regulated by these anticancer agents at the transcriptional level, and that the level of expression of AURKB may influence the cytotoxic effect of AZD1152-hQPA. Therefore, when using anticancer agents, decreasing the expression of AURKB using a molecular-targeting drug may be an optimal therapeutic strategy.

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.

In order to examine whether intratracheal instillation studies can be useful for determining the harmful effect of nanoparticles, we performed inhalation and intratracheal instillation studies using samples of the same nanoparticles. Nickel oxide nanoparticles (NiO) and titanium dioxide nanoparticles (TiO2) were used as chemicals with high and low toxicities, respectively. In the intratracheal instillation study, rats were exposed to 0.2 or 1 mg of NiO or TiO2. Cell analysis and chemokines in bronchoalveolar lavage fluid (BALF) were analyzed from 3 days to 6 months following the single intratracheal instillation. In the inhalation study, rats were exposed to inhaled NiO or TiO2 (1.65, 1.84 mg/m(3), respectively) for 4 weeks. The same endpoints were examined from 3 days to 3 months after the end of exposure. Inhalation of NiO induced an increase in the number of neutrophils in BALF and concentrations of cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2 and heme oxygenase (HO)-1. Intratracheal instillation of NiO induced persistent inflammation and upregulation of these cytokines was observed in the rats. However, inhalation of TiO2 did not induce pulmonary inflammation, and intratracheal instillation of TiO2 transiently induced an increase in the number of neutrophils in BALF and the concentrations of CINC-1, CINC-2 and HO-1. Taken together, a difference in pulmonary inflammation was observed between the high and low toxicity nanomaterials in the intratracheal instillation studies, as in the inhalation studies, suggesting that intratracheal instillation studies may be useful for ranking the harmful effects of nanoparticles.

Wnt10a is a member of the WNT family. Although deficiency of this gene causes symptoms related to teeth, hair, nails, and skin, we recently demonstrated a new phenotype of Wnt10a knockout (KO) mice involving bone and fat. The in vivo effect of the Wnt10a gene on bone and fat is unclear, and the relationship between bone/fat and muscle in Wnt10a signaling is also interesting. We aimed to evaluate the tissue changes in Wnt10a KO mice compared to wild-type mice and show the findings as a starting point to unravel the underlying mechanisms of in vivo interactions involving Wnt10a in bone, fat and muscle. Trabecular bone loss in the lower limbs of Wnt10a mice and decreased bone mineralization were observed. The adipose tissue in bone marrow was also decreased, and adipocyte differentiation was reduced. The body fat mass in Wnt10a KO mice was decreased, and white adipocytes in subcutaneous fat were converted to beige adipocytes. The muscle weight of the lower limbs was not decreased despite trabecular bone loss, but Gdf8/myostatin expression was reduced in the subcutaneous fat and gastrocnemius muscles of Wnt10a KO mice. Thus, in vivo deletion of Wnt10a inhibited osteogenic activity, promoted beige adipogenesis of white adipocytes and maintained muscle mass. These results suggest that regulation of Gdf8 by Wnt10a may help maintain the muscle mass of Wnt10a KO mice. This study was the first to histologically evaluate the bone, fat and muscle phenotypes of Wnt10a KO mice. The results of this study, which were obtained by investigating the three tissues together, could influence the understanding of in vivo interactions involving the Wnt10a gene.

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.

BACKGROUND 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. MATERIALS AND METHODS This study investigated the relationship between GalNAc-T6 expression and cell adhesion molecules in 218 endometrial carcinomas by immunohistochemistry. RESULTS 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. CONCLUSION 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.

Itai-itai disease is thought to be the result of chronic cadmium (Cd) intoxication. Renal proximal tubules are a major target of Cd toxicity. The whole mechanism of the adverse effects of Cd remains unresolved, especially how renal damage is related to the development of bone lesions. Fibroblast growth factor 23 (FGF23) is a bone-derived phosphaturic factor that regulates vitamin D and inorganic phosphate metabolism in the kidney. To clarify the role of FGF23 on Cd toxicity, we investigated the mechanisms of Cd-induced FGF23 production in the bone. Cd injection into mice significantly increased plasma FGF23 concentrations, but did not change FGF23 mRNA expression in bone. GalNAc-T3 is involved in secreting intact FGF23. To determine potential roles of GalNAc-T3 in Cd-induced FGF23 production, we examined the effect of Cd on GalNAc-T3 mRNA expression in vivo and in vitro. GalNAc-T3 gene expression was significantly increased in the bones of Cd-injected mice. Cd also enhanced the expression of GalNAc-T3 in cultured osteosarcoma UMR106 cells and primary osteocytes. Cd activated aryl hydrocarbon receptors (AhR) and AhR were required for GalNAc-T3 gene expression induced by Cd. In addition, Cd-dependent FGF23 production was completely inhibited by an AhR antagonist. AhR siRNA markedly suppressed the stimulation of transcriptional activity by Cd. Furthermore, Cd induced AhR activation via phosphorylation of Ser-68 by p38 kinase in the nuclear export signal of AhR. Thus, Cd stimulated GalNAc-T3 gene transcription via enhanced AhR binding to the GalNAc-T3 promoter. These findings suggest that the Cd-induced increase in GalNAc-T3 suppresses proteolytic processing of FGF23 and increases serum FGF23 concentrations.

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.

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.

NiO nanoparticles were administered to rat lungs via intratracheal instillation or inhalation. During pulmonary toxicity caused by NiO nanoparticles, the induction of oxidative stress is a major factor. Both intratracheal instillation and inhalation of NiO nanoparticles induced pulmonary oxidative stress. The oxidative stress response protein, heme oxygenase-1 (HO-1), was induced by the administration of NiO nanoparticles at both the protein and gene expression level. Additionally, certain oxidative-stress markers in the lung, such as 8-iso-prostaglandin F2α, thioredoxin, and inducible nitric oxide synthase were increased. Furthermore, the concentration of myeloperoxidase (MPO) in the lung was also increased by the administration of NiO nanoparticles. When the amount of NiO in the lung is similar, the responses against pulmonary oxidative stress of intratracheal instillation and inhalation are also similar. However, the state of pulmonary oxidative stress in the early phase was different between intratracheal instillation and inhalation, even if the amount of NiO in the lung was similar. Inhalation causes milder oxidative stress than that caused by intratracheal instillation. On evaluation of the nanoparticle-induced pulmonary oxidative stress in the early phase, we should understand the different states of oxidative stress induced by intratracheal instillation and inhalation.

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.

Summary TLX is known as the orphan nuclear receptor indispensable for maintaining neural stem cells in adult neurogenesis. We report here that neuroblastoma cell lines express high levels of TLX, which further increase in hypoxia to enhance the angiogenic capacity of these cells. The proangiogenetic activity of TLX appears to be induced by its direct binding to the von Hippel-Lindau protein (pVHL), which stabilizes TLX. In turn, TLX competes with hydroxylated hypoxia-inducible factor (HIF-α) for binding to pVHL, which contributes to the stabilization of HIF-2α in neuroblastoma during normoxia. Upon hypoxia, TLX increases in the nucleus where it binds in close proximity of the HIF-response element on the VEGF-promoter chromatin, and, together with HIF-2α, recruits RNA polymerase II to induce VEGF expression. Conversely, depletion of TLX by shRNA decreases the expression of HIF-2α and VEGF as well as the growth-promoting and colony-forming capacity of the neuroblastoma cell lines IMR-32 and SH-SY5Y. On the contrary, silencing HIF-2α will slightly increase TLX, suggesting that TLX acts to maintain a hypoxic environment when HIF-2α is decreasing. Our results demonstrate TLX to play a key role in controlling angiogenesis by regulating HIF-2α. TLX and pVHL might counterbalance each other in important fate decisions such as self-renewal and differentiation, as well as angiogenesis and anti-angiogenesis.

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.

We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143) regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1), aurora kinase B (AURKB) and some minichromosome maintenance complex components (MCM). However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells) and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

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.

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.

Passage of transcribed U snRNA precursors through Cajal bodies ensures that they are properly bound to the PHAX adaptor protein required for nuclear exit., Phosphorylated adaptor for RNA export (PHAX) is the key export mediator for spliceosomal U small nuclear RNA (snRNA) precursors in metazoa. PHAX is enriched in Cajal bodies (CBs), nuclear subdomains involved in the biogenesis of small ribonucleoproteins. However, CBs’ role in U snRNA export has not been demonstrated. In this study, we show that U snRNA precursors microinjected into Xenopus laevis oocyte nuclei temporarily concentrate in CBs but gradually decrease as RNA export proceeds. Inhibition of PHAX activity by the coinjection of a specific anti-PHAX antibody or a dominant-negative PHAX mutant inhibits U snRNA export and simultaneously enhances accumulation of U snRNA precursors in CBs, indicating that U snRNAs transit through CBs before export and that binding to PHAX is required for efficient exit of U snRNAs from CBs. Similar results were obtained with U snRNAs transcribed from microinjected genes. These results reveal a novel function for CBs, which ensure that U snRNA precursors are properly bound by PHAX.

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]

Ras is a key regulator of the MAP kinase-signaling cascade and may cause morphologic change of Ras-transformed cells. Signal transducer and activator of transcription 3 (Stat3) can be activated by cytokine stimulation. In this study, we unravel that Ha- ras V12 overexpression can downregulate the expression of Stat3 protein at a posttranslational level in NIH3T3 cells. Furthermore, we demonstrate that Stat3 expression downregulated by Ha- ras V12 overexpression is through proteosome degradation and not through a mTOR/p70S6K-related signaling pathway. The suppression of Stat3 accompanied by the morphologic change induced by Ha- ras V12 was through mitogen extracellular kinase (MEK)/extracellular-regulated kinase (ERK) signaling pathway. Microtubule disruption is involved in Ha- ras V12 -induced morphologic change, which could be reversed by overexpression of Stat3. Taken together, we are the first to demonstrate that Stat3 protein plays a critical role in Ha- ras V12 -induced morphologic change. Oncogenic Ras-triggered morphologic change is through the activation of MEK/ERK to posttranslationally downregulate Stat3 expression. Our finding may shed light on developing novel therapeutic strategies against Ras-related tumorigenesis.

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

In order to investigate the pulmonary toxicity of titanium dioxide (TiO2) nanoparticles, we performed an intratracheal instillation study with rats of well-dispersed TiO2 nanoparticles and examined the pulmonary inflammation and histopathological changes in the lung. Wistar Hannover rats were intratracheally administered 0.2 mg (0.66 mg/kg) and 1.0 mg (3.3 mg/kg) of well-dispersed TiO2 nanoparticles (P90; diameter of agglomerates: 25 nm), then the pulmonary inflammation responses were examined from 3 days to 6 months after the instillation, and the pathological features were examined up to 24 months. Transient inflammation and the upregulation of chemokines in the broncho-alveolar lavage fluid were observed for 1 month. No respiratory tumors or severe fibrosis were observed during the recovery time. These data suggest that transient inflammation induced by TiO2 may not lead to chronic, irreversible legions in the lung, and that TiO2 nanoparticles may not have a high potential for lung disorder.

The eukaryotic Y-box-binding protein-1 (YB-1) is involved in the transcriptional and translational control of many biological processes, including cell proliferation. In clinical studies, the cellular level of YB-1 closely correlates with tumor growth and prognosis. To understand the role of YB-1 in vivo, especially in the developmental process, we generated YB-1 knock-out mice, which are embryonic lethal and exhibit exencephaly associated with abnormal patterns of cell proliferation within the neuroepithelium. beta-Actin expression and F-actin formation were reduced in the YB-1 null embryo and YB-1(-/-) mouse embryonic fibroblasts, suggesting that the neural tube defect is caused by abnormal cell morphology and actin assembly within the neuroepithelium. Fibroblasts derived from YB-1(-/-) embryos demonstrated reduced growth and cell density. A colony formation assay showed that YB-1(-/-) mouse embryonic fibroblasts failed to undergo morphological transformation and remained contact-inhibited in culture. These results demonstrate that YB-1 is involved in early mouse development, including neural tube closure and cell proliferation.