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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.

Osimertinib, a third-generation EGFR-TKI, has nowadays been applied to non-small cell lung cancer harboring activated EGFR mutation with or without T790M, but ultimately develop resistance to this drug. Here we report a novel mechanism of acquired resistance to osimertinib and the reversal of which could improve the clinical outcomes. In osimertinib-resistant lung cancer cell lines harboring T790M mutation that we established, expression of multiple EGFR family proteins and MET was markedly reduced, whereas expression of AXL, CDCP1 and SRC was augmented along with activation of AKT. Surprisingly, AXL or CDCP1 expression was induced by osimertinib in a time-dependent manner up to 3 months. Silencing of CDCP1 or AXL restored the sensitivity to osimertinib with reduced activation of SRC and AKT. Furthermore, silencing of both CDCP1 and AXL increased the sensitivity to osimertinib. Either silencing of SRC or dasatinib, a SRC family kinase (SFK) inhibitor, suppressed AKT phosphorylation and cell growth. Increased expression of AXL and CDCP1 was observed in refractory tumor samples from patients with lung cancer treated with osimertinib. Together, this study suggests that AXL/SFK/AKT and CDCP1/SFK/AKT signaling pathways play some roles in acquired osimertinib resistance of non-small cell lung cancer.

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.

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.

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.

// 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.

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.

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)

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.

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.

Development of a novel type of angiogenesis inhibitor will be essential for further improvement of therapeutics against cancer patients. We examined whether an octahydronaphthalene derivative, AMF-26, which was screened as an inhibitor of intercellular adhesion molecule-1 (ICAM-1) production stimulated by inflammatory stimuli in vascular endothelial cells, could block angiogenesis in response to vascular endothelial growth factor (VEGF) and/or inflammatory cytokines. Low dose AMF-26 effectively inhibited the tumor necrosis factor-α (TNF-α)- or the interleukin-1β (IL-1β)-induced production of ICAM-1 in human umbilical vascular endothelial cells (HUVECs). We found that the TNF-α-induced phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and nuclear translocation of p65 were impaired by AMF-26 in both endothelial cells and cancer cells. AMF-26 was found to inhibit the phosphorylation of VEGF receptor 1 (VEGFR1), VEGFR2 and the downstream signaling molecules Akt, extracellular signal-regulated kinase (ERK)1/2 stimulated by VEGF in HUVECs. Therefore, the VEGF-induced proliferation, migration and tube formation of vascular endothelial cells was highly susceptible to inhibition by AMF-26. Oral administration of AMF-26 significantly blocked VEGF- or IL-1β-induced angiogenesis in the mouse cornea, and also tumor angiogenesis and growth. Together, our results indicate that AMF-26 inhibits angiogenesis through suppression of both VEGFR1/2 and nuclear factor-κB (NF-κB) signaling pathways when stimulated by VEGF or inflammatory cytokines. AMF-26 could be a promising novel candidate drug for cancer treatments.

An anti-inflammatory enone fatty acid, (E)-9-oxooctadec-10-enoic acid (C10), was previously isolated from red alga (Gracilaria verrucosa). Of the many cellular signaling pathways activated in response to the inflammatory stimulus, lipopolysaccharide, the extracellular signal-regulated kinase 1/2, the stress-activated protein kinase/Jun N-terminal kinase and the nuclear factor-κB pathways were specifically blocked by C10 in the macrophage-like cell line, RAW264.7. In this study, we investigated the anti-angiogenic and anti-inflammatory activities of C10 in endothelial cells. C10 only partially inhibited the proliferation of human cancer cell lines at relatively high concentrations of over 20 μg/ml. However, C10 inhibited the proliferation of RAW264.7 cells and human umbilical vein endothelial cells (HUVECs) with half-maximal inhibitory concentration (IC50) values of 4-8 μg/ml. Both the proliferation and the migration of HUVECs induced by the vascular endothelial growth factor (VEGF) were markedly blocked by C10 with IC50 values of 2-3 μg/ml. The activation of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α, by tumor necrosis factor-α or VEGF in these cells was also blocked by C10. Furthermore, in an in vivo model of angiogenesis in the mouse cornea, the neovascularization induced by VEGF was markedly inhibited by C10. The processes involved in inflammatory signaling, angiogenesis, and the development of malignancy in cancer are closely related, suggesting that C10 could be a useful lead compound for the development of novel anti-angiogenic therapies for cancer.

Ewing sarcoma–primitive neuroectodermal tumor (EWS) is associated with the most unfavorable prognosis of all primary musculoskeletal tumors. The objective of the present study was to investigate whether tumor-associated macrophages (TAMs) affect the development of EWS. TAMs were isolated from mouse xenografts using CD11b magnetic beads and examined for their cytokine expression and osteoclastic differentiation. To evaluate the role of TAMs in xenograft formation, liposome-encapsulated clodronate was used to deplete TAMs in mice. Macrophage infiltration and tumor microvascular density were histologically evaluated in 41 patients with EWS, and association with prognosis was examined using Kaplan-Meier survival analysis. In mouse EWS xenografts, TAMs expressed higher concentrations of cytokines including interleukin-6, keratinocyte-derived chemokine, and monocyte chemotactic protein-1. TAMs were more capable than normal monocytes of differentiating into tartrate-resistant acid phosphatase–positive giant cells. Depleting macrophages using liposome-encapsulated clodronate significantly inhibited development of EWS xenografts. In human EWS samples, higher levels of CD68-positive macrophages were associated with poorer overall survival. In addition, enhanced vascularity, increase in the amount of C-reactive protein, and higher white blood cell counts were also associated with poor prognosis and macrophage infiltration. TAMs seem to enhance the progression of EWS by stimulating both angiogenesis and osteoclastogenesis. Further investigation of the behavior of TAMs may lead to development of biologically targeted therapies for EWS.

Thymidine phosphorylase (TP), an enzyme catalyzing the reversible phospholysis of thymidine, deoxyuridine and their analogs at their respective bases and 2-deoxyribose-1-phosphate, thus promoting angiogenesis, is often expressed in macrophages present in tumor stroma. In this study, we investigated whether infiltration of TP-positive macrophages as well as tumor-associated macrophages affected tumor angiogenesis. TP was expressed in human macrophage-like cells, but not in gastric cancer cells in culture. The expression level of TP, the number of infiltrating CD68 + and CD163 + macrophages, and microvessel density (MVD) in the tumor were further analyzed by immunohistochemistry in 111 patients with gastric cancer. Biostatistical analysis of digitized data obtained by image analysis showed that TP expression was significantly correlated with the number of infiltrating macrophages and MVD in intestinal type gastric cancer (p

The close association of inflammation, angiogenesis and cancer progression is now highlighted, and in this study we especially focused on a close association of inflammation and lymphangiogenesis. We found that proinflammatory cytokine, interleukin-1beta (IL-1beta), could induce lymphangiogenesis in mouse cornea through enhanced production of potent lymphangiogenic factors, VEGF-A, VEGF-C and VEGF-D. IL-1beta-induced lymphangiogenesis, but not angiogenesis, was inhibited by administration of a selective anti-VEGF receptor-3 (VEGFR-3) neutralizing antibody. And in mouse cornea we observed recruitment of monocyte/macrophages and neutrophils by IL-1beta implanted cornea. Depletion of macrophages by a bisphosphonate encapsulated in liposomes inhibited this IL-1beta-induced lymphangiogenesis and also up-regulation of VEGF-A, VEGF-C, and VEGF-D. Furthermore, IL-1beta-induced lymphangiogenesis and angiogenesis were suppressed by NF-kappaB inhibition with marked suppression of VEGF-A, VEGF-C, and VEGF-D expression.

Tumors formed by a highly metastatic human lung cancer cell line are characterized by activated signaling via vascular endothelial growth factor (VEGF)-C through its receptor (VEGFR-3) and aggressive lymph node metastasis. In this study, we examined how these highly metastatic cancers acquired aggressive lymph node metastasis. Compared with their lower metastatic counterparts, the highly metastatic tumors formed by this cell line expressed higher amounts of interleukin (IL)-1α, with similarly augmented expression of IL-1α and IL-1β by tumor stromal cells and of VEGF-A and VEGF-C by tumor-associated macrophages. These tumor-associated macrophages were mainly of the M2 type. Administration of a macrophage-targeting drug suppressed the production of these potent angiogenic and lymphangiogenic factors, resulting in decreased tumor growth, angiogenesis, lymphangiogenesis, and lymph node metastasis. In Matrigel plug assays, the highly metastatic cells formed tumors that were extensively infiltrated by M2-type macrophages and exhibited enhanced angiogenesis and lymphangiogenesis. All of these responses were suppressed by the IL-1 receptor (IL-1R) antagonist anakinra. Thus, the IL-1α-driven inflammatory activation of angiogenesis and lymphangiogenesis seems to provide a highly metastatic tumor microenvironment favorable for lymph node metastasis through cross-talk with macrophages. Accordingly, the IL-1R/M2-type macrophage axis may be a good therapeutic target for patients with this form of lung cancer.