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IC50 values of fulvestrant, everolimus, AZD8055 and U0126 in ER(+)/HER2(-) and ER(-)/HER2(+) human breast cancer cell lines.

Comparison of body weights of 4 experimental groups on day 20 after inoculation. Body weights graph corresponds to Fig. 5A-D.

Supplementary Materials and Methods

PDF File - 66K, Sensitivity to lapatinib, erlotinib, SU11274, cisplatin and CPT-11 in human gastric cancer cell lines.

Supplementary Figure S1. Effect of YBX1 on ESR protein expression and ubiquitination of ESR. Supplementary Figure S2. Effect of ESR knockdown on ERBB2 expression.

ST1:Clinical characteristics of 28 patients with GBM, from whom tumor samples are analyzed.

Supplementary materials and methods

SF1: NDRG1 silencing upregulates GSK3β expression and cell growth signaling pathways; SF2: NDRG1 overexpression suppresses cell growth, GSK3β expression and cell growth signaling pathways; SF3: Effect of WT and Î"6 deletion mutant of NDRG1 on cell growth and cell growth signaling pathway in U251 cells; SF4: DIF-1 suppresses GBM cell growth accompanied with enhanced expression of NDRG1 and decreased expression of GSK3β; SF5: DIF-1 suppressed GBM cell growth by enhancing NDRG1 expression and decreasing GSK3β expression; SF6: Measurement of the DIF-1 concentration in mouse plasma and brain; SF7: DIF-1 suppresses tumor growth by U87MG cells in orthotopic therapeutic models; SF8: NDRG1 mRNA expression level does not predict overall survival of patients with GBM.

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.

Supplementary Figure Legends and Methods

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.

PDF- 199K, A, Heterodimerization of integrinα2 or α5 with integrinβ1. B, Effects of integrinβ1, α2, or α5 siRNA, with or without erlotinib treatment, on downstream signaling molecules in PC9/ER2-2 cells.

PDF - 133K, A, PC9 and PC9/ER2-2 cells were exposed to 1 μM erlotinib with or without 0.1 μM dasatinib for 5 hr. B, The sensitivity to dasatinib after exposure for 3 days was determined by proliferation assay.

PDF - 82K, Supplementary materials and methods.

PDF - 163K, A, Establishment of stable integrinβ1-overexpressing cell lines (PC9/ITG2 and PC9/ITG11) or control cDNA transfected cell line (PC9/Mock) from PC9 cells by transfection with integrinβ1 cDNA. B, The effect of integrinβ1 overexpression on erlotinib sensitivity. Transfectants were treated with erlotinib for 3 days, and the IC50 values (μM) for erlotinib were 0.04 (PC9/Mock), 0.09 (PC9/ITG2), and 0.10 (PC9/ITG11). C, Western blots showing the expression and phosphorylation of EGFR, integrinβ1, Akt, and Erk1/2 following exposure to erlotinib for 5 hr.

PDF - 76K, Summary of expression states of integrins and resistance-related molecules in EGFR-TKI sensitive and refractory tumors.

PDF - 83K, Supplementary figure legends.

[Image: see text] Covalent modification of disease-associated proteins with small molecules is a powerful approach for achieving an increased and sustained pharmacological effect. To reduce the potential risk of nonselective covalent modification, molecular design of covalent inhibitors is critically important. We report herein the development of a targeted covalent inhibitor for mutated epidermal growth factor receptor (EGFR) (L858R/T790M) using α-chlorofluoroacetamide (CFA) as the reactive group. The chemically tuned weak reactivity of CFA was suitable for the design of third-generation EGFR inhibitors that possess the pyrimidine scaffold. The structure–activity relationship study revealed that CFA inhibitor 18 (NSP-037) possessed higher inhibition selectivity to the mutated EGFR over wild-type EGFR when compared to clinically approved osimertinib. Mass-based chemical proteomics analyses further revealed that 18 displayed high covalent modification selectivity for the mutated EGFR in living cells. These findings highlight the utility of CFA as a warhead of targeted covalent inhibitors and the potential application of the CFA-pyrimidines for treatment of non-small-cell lung cancer.

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-1 (YBX1), a multifunctional oncoprotein containing an evolutionarily conserved cold shock domain, dysregulates a wide range of genes involved in cell proliferation and survival, drug resistance, and chromatin destabilization by cancer. Expression of a multidrug resistance-associated ATP binding cassette transporter gene, ABCB1, as well as growth factor receptor genes, EGFR and HER2/ErbB2, was initially discovered to be transcriptionally activated by YBX1 in cancer cells. Expression of other drug resistance-related genes, MVP/LRP, TOP2A, CD44, CD49f, BCL2, MYC, and androgen receptor (AR), is also transcriptionally activated by YBX1, consistently indicating that YBX1 is involved in tumor drug resistance. Furthermore, there is strong evidence to support that nuclear localization and/or overexpression of YBX1 can predict poor outcomes in patients with more than 20 different tumor types. YBX1 is phosphorylated by kinases, including AKT, p70S6K, and p90RSK, and translocated into the nucleus to promote the transcription of resistance- and malignancy-related genes. Phosphorylated YBX1, therefore, plays a crucial role as a potent transcription factor in cancer. Herein, a novel anticancer therapeutic strategy is presented by targeting activated YBX1 to overcome drug resistance and malignant progression.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1–10 μM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton’s tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI. Discovery and exploitation of inherent reaction features of chlorofluoroacetamide (CFA) as a warhead such as low off-target activity and reversible reactivity with cysteine enable specific covalent inhibition of targeted kinases.

Mitochondrial DNA (mtDNA) escaping stressed mitochondria provokes inflammation via cGAS-STING pathway activation and, when oxidized (Ox-mtDNA), it binds cytosolic NLRP3, thereby triggering inflammasome activation. However, it is unknown how and in which form Ox-mtDNA exits stressed mitochondria in non-apoptotic macrophages. We found that diverse NLRP3 inflammasome activators rapidly stimulated uniporter-mediated calcium uptake to open mitochondrial permeability transition pores (mPTP) and trigger VDAC oligomerization. This occurred independently of mtDNA or reactive oxygen species, which induce Ox-mtDNA generation. Within mitochondria, Ox-mtDNA was either repaired by DNA glycosylase OGG1 or cleaved by the endonuclease FEN1 to 500-650 bp fragments that exited mitochondria via mPTP- and VDAC-dependent channels to initiate cytosolic NLRP3 inflammasome activation. Ox-mtDNA fragments also activated cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA. Understanding this process will advance the development of potential treatments for chronic inflammatory diseases, exemplified by FEN1 inhibitors that suppressed interleukin-1β (IL-1β) production and mtDNA release in mice.

Many diseases, including cancer, have been associated with impaired regulation of angiogenesis, of which vascular endothelial growth factor (VEGF)-A is a key regulator. Here, we test the contribution of N-myc downstream regulated gene 1 (NDRG1) to VEGF-A-induced angiogenesis in vascular endothelial cells (ECs). Ndrg1−/− mice exhibit impaired VEGF-A-induced angiogenesis in corneas. Tumor angiogenesis induced by cancer cells that express high levels of VEGF-A was also reduced in a mouse dorsal air sac assay. Furthermore, NDRG1 deficiency in ECs prevented angiogenic sprouting from the aorta and the activation of phospholipase Cγ1 (PLCγ1) and ERK1/2 by VEGF-A without affecting the expression and function of VEGFR2. Finally, we show that NDRG1 formed a complex with PLCγ1 through its phosphorylation sites, and the inhibition of PLCγ1 dramatically suppressed VEGF-A-induced angiogenesis in the mouse cornea, suggesting an essential role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ1 signaling., Kosuke Watari et al. show that N-myc downstream-regulated gene 1 (NDRG1) stimulates new blood vessel formation that is induced by VEGF-A, using Ndrg1 knockout mice. They find that PLCγ1/ERK signaling mediates this regulation, providing mechanistic insights into how vascular endothelial cells form new vessels.

The enhanced expression of the Y-box binding protein YBX1 is consistently correlated with poor outcomes or reduced survival of breast cancer patients. However, the mechanism underlying the association between increased YBX1 expression and poor outcomes has yet to be revealed. We searched a database for the top 500 genes that are positively or negatively correlated with YBX1 and with ESR1 in breast cancer patients. We further examined the association between YBX1-correlated genes and breast cancer outcomes in patients at Kyushu University Hospital. More than 60% of genes that are positively correlated with YBX1 are also negatively correlated with ESR1. The enhanced expression levels of the top 20 positively correlated genes mostly predict negative outcomes, while the enhanced expression levels of the top 20 negatively correlated genes mostly predict positive outcomes. Furthermore, in breast cancer patients at Kyushu University Hospital, the expression levels of YBX1 and YBX1-positively correlated genes were significantly higher and the expression levels of genes negatively correlated with YBX1 were significantly lower in patients who relapsed after their primary surgery than in those who did not relapse. The expression of YBX1 together with the expression of its positively or negatively correlated genes may help to predict outcomes as well as resistance to endocrine therapies in breast cancer patients. Determining the expression of YBX1 and its closely correlated genes will contribute to the development of precision therapeutics for breast cancer.

// Yuichi Murakami 1, 2, * , Kahori Sonoda 1, * , Hideyuki Abe 3 , Kosuke Watari 1 , Daiki Kusakabe 1, 4 , Koichi Azuma 5 , Akihiko Kawahara 3 , Jun Akiba 3 , Chitose Oneyama 6 , Jonathan A. Pachter 7 , Kazuko Sakai 8 , Kazuto Nishio 8 , Michihiko Kuwano 2 and Mayumi Ono 1 1 Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 2 Cancer Translational Research Center, St. Mary’s Institute of Health Sciences, Fukuoka, Japan 3 Department of Diagnostic Pathology, Kurume University Hospital, Fukuoka, Japan 4 Physical Chemistry for Life Science Laboratory, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 5 Division of Respirology, Neurology and Rheumatology, Department of Internal Medicine, Kurume University School of Medicine, Fukuoka, Japan 6 Division of Microbiology and Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan 7 Verastem Inc., Cambridge, MA, USA 8 Department of Genome Biology, Kinki University Faculty of Medicine, Osaka, Japan * These authors have contributed equally to this work Correspondence to: Mayumi Ono, email: mono@phar.kyushu-u.ac.jp Keywords: afatinib resistance, SRC family kinase, focal adhesion kinase, non-small cell lung cancer Received: February 28, 2017 Accepted: July 18, 2017 Published: August 07, 2017 ABSTRACT Second- and third-generation inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase activity (EGFR-TKIs) are improving the treatment of patients with non-small cell lung cancer. Here we established two sublines (BR1-8 and BR2-3) resistant to a second-generation inhibitor, afatinib, from the human lung cancer cell line HCC827 that harbors a mutation that activates the tyrosine kinase activity of EGFR. These afatinib-resistant sublines were resistant to first-generation EGFR-TKIs, gefitinib and erlotinib, and a third-generation EGFR-TKI, osimertinib. These resistant sublines showed markedly reduced levels of multiple EGFR family proteins, including the activated mutant EGFR, and complete loss of EGFR amplification as compared with their parental HCC827 cells harboring amplification of EGFR gene. Treatment with the multikinase inhibitor dasatinib or transfection with a SRC small interfering RNA inhibited cell survival and AKT phosphorylation in drug-resistant sublines to a greater extent compared with HCC827 cells. Further, the migration of drug-resistant cells was greater compared with that of HCC827 cells and was inhibited by dasatinib or an FAK inhibitor. These findings indicate that compensatory activation of SRC family kinases (SFKs) and FAK supports the survival and migration of afatinib-resistant cells when the expression of multiple EGFR family proteins was mostly abrogated. Combinations of potent drugs that target SFKs and FAK may overcome the resistance of lung cancer cells to second-generation TKIs.

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.

// Kosuke Watari 1, * , Ayumi Nishitani 1, * , Tomohiro Shibata 1 , Masaki Noda 1 , Akihiko Kawahara 2 , Jun Akiba 3 , Yuichi Murakami 1, 4 , Hirohisa Yano 3 , Michihiko Kuwano 4 , Mayumi Ono 1 1 Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 2 Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan 3 Department of Pathology, Kurume University School of Medicine, Kurume, Japan 4 Cancer Translational Research Center, St. Mary’s Institute of Health Sciences, Kurume, Japan * These authors contributed equally to this work Correspondence to: Mayumi Ono, email: mono@phar.kyushu-u.ac.jp Keywords: mTOR Ser2481, mTORC1, Raptor, rapalogs, hepatocellular carcinoma Received: February 15, 2016 Accepted: June 07, 2016 Published: June 18, 2016 ABSTRACT Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although recent studies facilitate the identification of crucial genes and relevant regulatory pathways, therapeutic approaches against advanced HCC are insufficiently effective. Therefore, we aimed here to develop potent therapeutics to provide a reliable biomarker for the therapeutic efficacy in patients with HCC. To this end, we first compared the cytotoxic effects of various anti-cancer drugs between well differentiated (HAK-1A) and poorly differentiated (HAK-1B) cell lines established from a single HCC tumor. Of various drug screened, HAK-1B cells were more sensitive by a factor of 2,000 to the mTORC1 inhibitors (rapalogs), rapamycin and everolimus, than HAK-1A cells. Although rapalogs inhibited phosphorylation of mTOR Ser2448 in HAK-1A and HAK-1B cells, phosphorylation of mTOR Ser2481 was specifically inhibited only in HAK-1B cells. Silencing of Raptor induced apoptosis and inhibited the growth of only HAK-1B cells. Further, three other cell lines established independently from the tumors of three patients with HCC were also approximately 2,000-fold times more sensitive to rapamycin, which correlated closely with the inhibition of mTOR Ser2481 phosphorylation by rapamycin. Treatment with everolimus markedly inhibited the growth of tumors induced by poorly differentiated HAK-1B and KYN-2 cells and phosphorylation of mTOR Ser2481 in vivo . To our knowledge, this is the first study showing that the phosphorylation of mTOR Ser2481 is selectively inhibited by rapalogs in mTORC1-addicted HCC cells and may be a potential reliable biomarker for the therapeutic efficacy of rapalogs for treating HCC patients.

There are various receptor tyrosine kinase (TK)-targeted drugs that are currently used in the treatment of patients with non-small cell lung cancer (NSCLC). Among them, the epidermal growth factor receptor (EGFR) TK inhibitors (TKIs) are the most extensively studied. Receptor TKIs including EGFR TKIs have shown dramatic therapeutic efficacies in malignant tumors, which harbor activating mutations in the EGFR gene. However, within 1 or 2years after treatment, patients harboring these mutations often develop resistance to TKI therapy. This review article is aimed at drawing attention to the fact that we must first understand how receptor TKI resistance is acquired to develop strategies for overcoming resistance to TKIs. Furthermore, an insight into the specific molecules or signaling pathways that mediate resistance is a key factor for understanding and overcoming acquired drug resistance. Finally, we present our views on the continuing battle against "drug resistance," and provide further guidelines and strategies on how to minimize the development of drug-resistant tumors.

5-Azacytidine (5AC), a hypomethylating agent, is clinically used for the treatment of patients with myelodysplastic syndromes (MDS). Cytidine deaminase (CDA) is a key enzyme in the detoxification of 5AC. We investigated whether the CDA expression could predict response to 5AC in MDS. Among leukemia-derived cell lines, MDS-L, an MDS-derived cell line with a relatively low CDA expression level, was found to be the most sensitive to 5AC. Combination with tetrahydrouridine, an inhibitor of CDA, synergistically potentiated the cytotoxic effect of 5AC. Treatment with 5AC markedly enhanced the expression level of CDA mRNA and showed demethylation at CpG sites in the 5'-flanking region of the CDA gene. We further compared the protein expression levels of CDA in matched clinical samples before and after treatment with 5AC in bone marrow cells from 8 MDS patients by an immunohistochemical analysis. The CDA expression level showed an approximately 2- to 3-fold increase after 5AC treatment in 3 of these cases, and these three patients with relatively higher CDA expression levels after 5AC treatment all showed better clinical responses to 5AC. In contrast, the 5 remaining patients, whose CDA expression showed no augmentation, observed no clinical benefit. Taken together, the optimized determination of the CDA expression levels before and after 5AC treatment, and the methylation status at CpG sites of 5'-flanking region of the CDA gene, may contribute to the development of precise 5AC therapy for MDS.

// 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.

[Background] Vascular endothelial growth factor (VEGF)-A is a key regulator of tumor angiogenesis that is essential for tumor growth and progression. The unraveling of the precise mechanisms behind VEGF-A-induced tumor angiogenic process will further contribute to development of novel and potent anti-cancer therapeutics. N-myc downstream regulated gene 1 (NDRG1) has been shown to play essential roles in multiple biological processes including embryogenesis, tissue development, cell growth, differentiation, and tumorigenesis. We previously reported that NDRG1 expression levels in cancer cells are closely correlated with tumor angiogenesis and growth (Hosoi et al., Cancer Res., 2009; Murakami et al., J Biol Chem., 2013), and also that NDRG1 promotes tumor angiogenesis through enhanced VEGF-A production by tumor-associated macrophages (Watari et al., Sci Rep., 2016). However, it remains unclear whether NDRG1 expression in vascular endothelial cells (ECs) plays any crucial role in VEGF-A-induced tumor angiogenesis. In our present study, we further ask whether and how NDRG1 in ECs could specifically regulate tumor angiogenesis. We also present our finding of intrinsic importance that NDRG1 functions as an essential factor for VEGF-A-induced angiogenesis. [Methods] NDRG1 deficient mice: The NDRG1 deficient mice on C57BL6 background were purchased from Laboratory Animal Resource Bank, National Institutes of Biomedical Innovation, Health and Nutrition (Osaka, Japan). Isolation of mouse endothelial cells: CD31+ endothelial cells were isolated from mouse lung by magnetic sorting using CD31 MicroBeads. Aortic ring assay: 1 mm mouse aortic rings were embedded in 3-dimensional growth factor reduced Matrigel, treated with or without FGF-2 (50 ng/mL) or VEGF (25 ng/ml), and incubated at 37°C. Vascular length and branching point were measured at day 7. [Results] [1] Analysis of TCGA datasets revealed that NDRG1 expression was positively correlated with VEGF-A expression in patients with various cancer types. [2] In breast cancer patients in Kurume University hospital, NDRG1 was expressed in both cancer cells and ECs. In NDRG1 deficient mice, we observed following experimental results. [3] Both tumor growth and angiogenesis were all suppressed in syngeneic tumors. [4] VEGF-A-induced angiogenesis was specifically impaired in corneal micropocket assay and aortic ring assay, whereas FGF-2 could induce angiogenesis in both assays. [5] NDRG1 formed a complex with PLC-γ, and this complex formation was requisite for the VEGF-A-induced PLCγ/ERK activation in ECs. [Conclusion] We first present an indispensable role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ/ERK activation in ECs. The NDRG1 could be a novel candidate target for development of therapeutics for VEGF-A-induced tumor angiogenesis and other vascular diseases. Citation Format: Kosuke Watari, Tomohiro Shibata, Ai Shinoda, Hideyuki Abe, Akihiko Kawahara, Yuichi Murakami, Eiji Oki, Jun Akiba, Yoshihiko Maehara, Michihiko Kuwano, Mayumi Ono. N-myc downstream regulated gene 1 (NDRG1) is indispensable for VEGF-A-induced tumor angiogenesis through PLCγ/ERK signaling activation in vascular endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 183.

BACKGROUND Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids. METHODS Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers. RESULTS PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased. CONCLUSIONS These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future. Cancer (Cancer Cytopathol) 2014;122:145–52. © 2013 American Cancer Society

Gene amplification of HER2/ErbB2 occurs in gastric cancer and the therapeutic efficacy of the HER2-targeted antibody, trastuzumab, has recently been improved against HER2-positive advanced stomach cancer. Here, we examined whether Y-box-binding protein-1 (YB-1) could selectively control HER2 gene expression and cellular sensitivity to EGF receptor (EGFR) family protein-targeted drugs in human gastric cancer cells. HER2 expression was specifically downregulated by YB-1 silencing using its cognate siRNA, whereas there was less change in the expression of EGFR and HER3. A chromatin immunoprecipitation assay revealed the specific binding of YB-1 to its consensus sequence on the 5′-regulatory region of HER2. YB-1 knockdown induced drug resistance to lapatinib, a dual EGFR and HER2 kinase inhibitor, and also to erlotinib, an EGFR kinase inhibitor. Moreover, phosphorylation of protein kinase B (Akt) was not markedly affected by lapatinib or erlotinib when YB-1 was silenced. Nuclear YB-1 expression was significantly (P = 0.026) associated with HER2 expression, but not with EGFR or HER3, in patients with gastric cancer (n = 111). The YB-1-HER2 axis may therefore be useful for the further development of personalized therapeutics against gastric cancer by HER2-targeted drugs. Mol Cancer Ther; 12(5); 737–46. ©2013 AACR.

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.

N-myc downstream regulated gene-1 (NDRG1)/Cap43 regulates tumor growth and metastasis in various carcinomas. In this study we examined whether and how NDRG1/Cap43 modulates tumor growth by human hepatocellular carcinoma (HCC) cells. NDRG1/Cap43 cDNA was used to transfect HCC cell lines (KIM-1), and stable transfectants overexpressing NDRG1/Cap43 (KIM-1/Cap43) were obtained. Cell cycle analysis showed that KIM-1/Cap43 cells were arrested in the G0/G1 phase. Western blot analysis demonstrated an increase in p21 in KIM-1/Cap43 cells in culture under full confluency as compared with KIM-1/Mock. When KIM-1 cells, which are very low in NDRG1/Cap43 expression, were treated with mimosine, a G0/G1 cell cycle blocker, expression of NDRG1/Cap43 was induced in a dose dependent manner, together with p21 induction and CDK4 reduction. In vivo, KIM-1/Cap43 cells showed markedly decreased tumor growth rates compared with those of KIM-1/Mock. Immunohistochemical staining demonstrated markedly higher p21 labeling index in the KIM-1/Cap43 tumor than KIM-1/Mock tumor, and lower CDK4 and Ki-67 labeling index in the KIM-1/Cap43 than KIM-1/Mock. In order to confirm suppressive effects of NDRG1/Cap43, we further established a stable transfectant expressing NDRG1/Cap43 (HAK-1B/Cap43) using another HCC cell line, HAK-1B. Western blot analysis demonstrated an increase in p21 and a decrease in CDK4 in HAK-1B/Cap43 cells in culture under full confluency as compared with HAK-1B/Mock. HAK-1B/Cap43 also showed decreased tumor growth rates as compared with its control counterpart in vivo. NDRG1/Cap43 overexpression thus induced cell cycle arrest at the G0/G1 phase accompanied by increased p21 and decreased CDK4 expression in HCC cells. NDRG1/Cap43 might play a key role in the cell cycle control of G0/G1 in HCC cells.

Expression of N-myc downstream regulated gene 1 (NDRG1)/Ca(2+)-associated protein 43 (Cap43) in cancer cells is a predictive marker of good or poor prognosis depending on tumor type. In this study, we examined whether NDRG1/Cap43 is a marker of good or poor prognosis in gastric cancer patients, and whether it is associated with tumor stromal responses, including angiogenesis and macrophage infiltration. The expression levels of NDRG1/Cap43, the number of CD68-positive macrophages and the CD34-positive microvessel density were analyzed by immunohistochemistry in 129 gastric cancer patients, including 65 with the intestinal type and 64 with the diffuse type. The expression of NDRG1/Cap43 in the nucleus and the membrane was evaluated. Nuclear NDRG1/Cap43 expression was found in 20/65 (30.8%) patients with the intestinal type and in 9/64 (14.1%) patients with the diffuse type of gastric cancer. Nuclear NDRG1/Cap43 expression was significantly associated with pathological stage in the intestinal type (P=0.002), but not in the diffuse type (P=0.039). Nuclear NDRG1/Cap43 expression was also closely associated with infiltrating macrophages (P=0.001) and tumor angiogenesis (P=0.001) in the intestinal type. Furthermore, nuclear NDRG1/Cap43 expression was associated with poor prognosis in both the intestinal (P=0.001) and the diffuse types of gastric cancer (P=0.047). By contrast, membranous NDRG1/Cap43 expression was not associated with the overall survival of gastric cancer patients with either the intestinal or diffuse type of gastric cancer. The expression of NDRG1/Cap43 in the nucleus may be a predictive biomarker for malignant progression in the intestinal type of gastric cancer, preferable to the expression of NDRG1/Cap43 in the membrane.

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.

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)

N-myc downstream regulated gene 1 (NDRG1) is a responsible gene for a hereditary motor and sensory neuropathy-Lom (Charcot–Marie–Tooth disease type 4D). This is the first study aiming to assess the contribution of NDRG1 to differentiation of macrophage lineage cells, which has important implications for bone remodeling and inflammatory angiogenesis. Ndrg1 knockout (KO) mice exhibited abnormal curvature of the spine, high trabecular bone mass and reduced number of osteoclasts. We observed that serum levels of macrophage colony-stimulating factor (M-CSF) and macrophage-related cytokines were markedly decreased in KO mice. Differentiation of bone marrow (BM) cells into osteoclasts, M1/M2-type macrophages and dendritic cells was all impaired. Furthermore, KO mice also showed reduced tumor growth and angiogenesis by cancer cells, accompanied by decreased infiltration of tumor-associated macrophages. The transfer of BM-derived macrophages from KO mice into BM-eradicated wild type (WT) mice induced much less tumor angiogenesis than observed in WT mice. Angiogenesis in corneas in response to inflammatory stimuli was also suppressed with decreased infiltration of macrophages. Taken together, these results indicate that NDRG1 deficiency attenuates the differentiation of macrophage lineage cells, suppressing bone remodeling and inflammatory angiogenesis. This study strongly suggests the crucial role of NDRG1 in differentiation process for macrophages.

Objectives We have previously reported that decreased expression of PTEN in lung cancer PC9 cells harboring an EGFR-activating mutation (del E746–A750) results in acquisition of resistance to EGFR-TKIs, gefitinib and erlotinib, accompanied by enhanced phosphorylation of Akt and decreased nuclear translocation of a transcription factor EGR-1 [8] . In the present study, PTEN promoter methylation accounted for the decreased expression of PTEN in our gefitinib-resistant mutant. Material and methods DNA methylation status of the PTEN promoter in PC9 and gefitinib-resistant cells were examined using methylation-specific PCR. The effect of DNA methylation on PTEN expression was evaluated by treatment of lung cancer cell lines with 5-aza-2′-deoxycytidine (5AZA-CdR). Results We observed the characteristics of two gefitinib-resistant sublines, GEF1-1 and GEF2-1, derived from PC9 as follows. (1) PTEN overexpression suppressed AKT phosphorylation and restored the sensitivity to gefitinib and erlotinib in GEF1-1 cells. (2) EGR-1 siRNA mediated knockdown suppressed the expression of cyclin D1 and ICAM-1 genes but not of PTEN gene in PC9 cells. (3) Transfection of EGR-1 cDNA into a drug-resistant subline induced the expression of cyclin D1 and ICAM-1 but not of PTEN. (4) Treatment with 5AZA-CdR induced the expression of PTEN in resistant sublines but not in the parental line PC9. (5) A CpG site near the translational start point of the 5′-regulatory region was methylated in GEF1-1 and GEF2-1 but not in PC9. Conclusion Our results strongly suggest that CpG hypermethylation of the PTEN gene contributes to the decreased expression of PTEN during acquired resistance to gefitinib or erlotinib.

EGF receptor (EGFR) kinase inhibitors, including gefitinib and erlotinib, exert potent therapeutic efficacy in non–small cell lung cancers harboring EGFR-activating mutations. However, most patients ultimately develop resistance to these drugs. Here, we report a novel mechanism of acquired resistance to EGFR tyrosine kinase inhibitors and the reversal of which could improve clinical outcomes. In erlotinib-resistant lung cancer cells harboring activating EGFR mutations that we established, there was increased expression of Src, integrin β1, α2, and α5 along with enhanced cell adhesion activity. Interestingly, RNAi-mediated silencing of integrin β1 restored erlotinib sensitivity and reduced activation of Src and Akt after erlotinib treatment. Furthermore, Src silencing inhibited Akt phosphorylation and cell growth, with this inhibitory effect further augmented by erlotinib treatment. Increased expression of integrin β1, α5, and/or α2 was also observed in refractory tumor samples from patients with lung cancer treated with erlotinib and/or gefitinib. Together, our findings identify the integrin β1/Src/Akt signaling pathway as a key mediator of acquired resistance to EGFR-targeted anticancer drugs. Cancer Res; 73(20); 6243–53. ©2013 AACR.

The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with tumor angiogenesis and malignant progression together with poor prognosis in gastric cancer. However, the underlying mechanism for the role of NDRG1 in the malignant progression of gastric cancer remains unknown. Here we examined whether and how NDRG1 could modulate tumor angiogenesis by human gastric cancer cells. We established NU/Cap12 and NU/Cap32 cells overexpressing NDRG1 in NUGC-3 cells, which show lower tumor angiogenesis in vivo. Compared with parental NU/Mock3, NU/Cap12, and NU/Cap32 cells: 1) induced higher tumor angiogenesis than NU/Mock3 cells accompanied by infiltration of tumor-associated macrophages in mouse dorsal air sac assay and Matrigel plug assay; 2) showed much higher expression of CXC chemokines, MMP-1, and the potent angiogenic factor VEGF-A; 3) increased the expression of the representative inflammatory cytokine, IL-1α; 4) augmented JNK phosphorylation and nuclear expression of activator protein 1 (AP-1). Further analysis demonstrated that knockdown of AP-1 (Jun and/or Fos) resulted in down-regulation of the expression of VEGF-A, CXC chemokines, and MMP-1, and also suppressed expression of IL-1α in NDRG1-overexpressing cell lines. Treatment with IL-1 receptor antagonist (IL-1ra) resulted in down-regulation of JNK and c-Jun phosphorylation, and the expression of VEGF-A, CXC chemokines, and MMP-1 in NU/Cap12 and NU/Cap32 cells. Finally, administration of IL-1ra suppressed both tumor angiogenesis and infiltration of macrophages by NU/Cap12 in vivo. Together, activation of JNK/AP-1 thus seems to promote tumor angiogenesis in relationship to NDRG1-induced inflammatory stimuli by gastric cancer cells.

Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids.Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers.PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased.These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future.