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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.

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.

[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.

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.

Widespread metastases including lymph node have often led to the failure of cancer patient treatments. Lymph node metastasis is closely associated with lymphangiogenesis and poor prognosis in lung cancer. Identifying the molecules involved in these processes could help to advance therapeutic strategies for lung cancer patients. We have asked how cancer cells acquire malignant characteristics including lymph node metastasis and lymphangiogenesis, and also how such highly malignant tumor could be therapeutically overcome. Human malignancies are often initiated and promoted by inflammation, in close association with angiogenesis and lymphangiogenesis, while the recruitment of macrophages and neutrophils to the tumor microenvironment activates cells to support cancer progression. Interleukin (IL-1), a representative inflammatory cytokine, is often expressed in human malignancies including lung cancers with poor prognosis. In this study we examined how IL-1 promotes lymph node metastasis and lymphangiogenesis by lung cancer cell lines expressing higher IL-lα. In our present study, we observed following experimental results. [1] Tumor growth, lymph node metastasis, lymphangiogenesis and enhanced infiltration of neutrophils and macrophage were all markedly augmented by the highly metastatic cancer cells. [2] In the highly metastatic tumors, we observed higher expression of IL-1α, IL-1β and CXC chemokines by cancer cells, and also that of VEGF-A and VEGF-C by tumor-associated macrophages, respectively. These tumor-associated macrophages were found to be mainly of the M2 type. [3] Treatment with an IL-1R antagonist (IL-1Ra) significantly blocked expression of CXC chemokines by cancer cells, and also expression of VEGFs by macrophages when co-cultured with cancer cells in vitro. [4] Administration of IL-1Ra significantly suppressed tumor growth, lymph node metastasis and lymphangiogenesis, accompanying by significantly decreased infiltration of M2-type macrophage in the highly metastatic tumors. Together, targeting IL-1/IL-1R signaling pathway and/or M2-type macrophages could be expected to provide useful therapeutic strategy against lymph node metastasis by inflammatory malignant cancer cells. Citation Format: Kosuke Watari, Tomohiro Shibata, Akihiko Kawahara, Yuichi Murakami, Hiroshi Nabeshima, Ai Shinoda, Koichi Azuma, Hiroto Izumi, Masayoshi Kage, Michihiko Kuwano, Mayumi Ono. Tumor-derived interleukin-1 promotes lymphangiogenesis and lymph node metastasis through activation of M2-type macrophages by lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3662. doi:10.1158/1538-7445.AM2014-3662

[Purpose] YB-1 containing a cold-shock domain is a potent oncogene for breast cancer (Bergmann et al., Cancer Res, 2005). Our previous studies have demonstrated that YB-1 plays pivotal roles not only in acquisition of global drug resistance and cell proliferation (Kuwano et al., Mol. Cancer Ther., 2004), but also in expression of HER2/ErbB2 in breast cancer cells (Fujii et al., Cancer Res., 2008) and stomach cancer cells (Shibata et al., Mol. Cancer Ther., 2013) in culture and clinical samples. In our present study, we first asked whether ERα affect YB-1-induced HER2 expression in breast cancer cells, and then asked whether YB-1 activation could be biostatistically linked to HER2 and/or ER expression in breast cancer patients. [Results] Is this study, we first examined the role of YB-1 in expression of HER2 and ERα by using ERα-positive and -negative breast cancer cell lines in culture, and we next examined whether YB-1 expression in nucleus could be biostatistically associated with HER2 or ERα expression by using clinical specimens of breast cancer. We observed novel findings as follows. [1] Knockdown of YB-1 markedly reduced HER2/ErbB2 expression in ERα positive human breast cancer cells, but only slightly reduced HER2/ErbB2 expression in ERα-negative breast cancer cells. [2] Nuclear YB-1 was forced expression by the tetracycline-induced YB-1/Tet-On system. Nuclear YB-1 expression enhanced HER2 expression and decreased ERα expression in ERα-positive cancer cells. By contrast, in ERα-negative breast cancer cells, there was no apparent increase in HER2 expression by YB-1. [3] Exogenous introduction of ERα cDNA resulted in suppression of HER2 expression dependent on YB-1 in ERα negative cells. Furthermore, treatment with tamoxifen stimulated YB-1-dependent HER2 expression in ERα-positive cells. [4] We further examined expression of YB-1, ERα, PgR and HER2 by immunohistochemical (IHC) analysis in clinical samples of breast cancer patients who had received no treatments by hormonal and chemical therapeutics. Based on expression levels of various factors, nuclear YB-1 expression was significantly correlated with HER2 expression, but negatively correlated with ERα expression in tumors of postmenopausal patients (n=114). By contrast, such significant association of YB-1 expression with ERα or HER2 expression was not seen in tumors of premenopausal patients (n=57). [Conclusions] YB-1 positively regulated HER2 expression and negatively ERα expression in ERα positive breast cancer cells, and the presence of ERα seems to be required for YB-1-dependent HER2 expression. Furthermore, in breast tumors of post-menopausal patients, nuclear localization of YB-1 was positively correlated with HER2 expression and negatively correlated with ERα expression. This study may contribute to further development of optimized endocrine- and HER2- targeted therapeutics against breast cancer. Citation Format: Tomohiro Shibata, Hiroto Izumi, Akihiko Kawahara, Satoshi Hattori, Chihiro Fukumitsu, Ryuji Takahashi, Kosuke Watari, Yuichi Murakami, Kimitoshi Kohno, Ken-ichi Ito, Masayoshi Kage, Michihiko Kuwano, Mayumi Ono. Y-box binding protein-1 YB-1 negatively regulates ERα expression accompanying by enhanced HER2/ErbB2 expression in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-183. doi:10.1158/1538-7445.AM2014-LB-183

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib and erlotinib have improved therapeutic efficacies on non-small cell lung cancer (NSCLC) harboring activating EGFR mutations. However, appearance of drug resistant mutation limits patients' outcome. A secondary T790M mutation and MET amplification are two major mechanisms of acquired EGFR-TKI resistance. To develop novel therapeutics to overcome drug resistant tumors, we should further understand other mechanisms at molecular basis of intrinsic importance. We established erlotinib or gefitinib resistant cell lines from PC9 cells that harbor an activating EGFR deletion mutation by chronic exposure to these TKIs. We have previously identified that loss of PTEN or loss of activated EGFR gene allele is responsible for acquired resistance (Cancer Res., 2010; Nat Med., 2011; PLoS ONE, 2012; Cancer Sci., 2013). In our present study, one erlotinib-resistant subline from PC9 cells revealed following interesting characteristics; [1] The increased expression of integrins β1, α2, and α5 was observed, along with enhanced adhesion and migratory activity in the resistant sublines. [2] The increased expression and activation of Src was also observed. [3] Interestingly, integrinβ1 knockdown markedly restored sensitivity to erlotinib and reduced the activation of Src and Akt upon treatment with erlotinib. [4] Furthermore, Src knockdown suppressed Akt phosphorylation and cell growth, and this inhibitory effect was further augmented by erlotinib. [5] The expression of integrins β1 and α5 was also observed in a refractory tumor sample from a lung cancer patient treated with erlotinib. Together, we will present our novel concept that integrinβ1/Src/Akt pathway represents a novel a bypass effector pathway for the acquisition of resistance to EGFR-TKIs. Citation Format: Mayumi Ono, Rina Kanda, Kosuke Watari, Akihiko Kawahara, Masayoshi Kage, Hidetaka Uramoto, Michihiko Kuwano. The Integrinβ1/Src/Akt-driven bypass signaling pathway contributes to Erlotinib resistance in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-220. doi:10.1158/1538-7445.AM2013-LB-220