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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.

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

Eribulin (ERI), an inhibitor of microtubule dynamics, is a key drug for metastatic breast cancer. While HDAC inhibitors have been shown to presents antitumor effect for various malignancies. Among HDAC family, HDAC6 is known as a deacetylase of tubulin. To develop a novel therapeutic strategy for triple negative breast cancer (TNBC), we tested the combinational effect of ERI and HDAC inhibitor for TNBC cells in vitro. Two TNBC cell lines (MDA-MB-231, Hs578T) and their ERI-resistant cells (MDA-MB-231/E, Hs578T/E) established in our laboratory were used in the study. Vorinostat (VOR), a pan-HDAC inhibitor, and ricolinostat (RICO), a selective HDAC6 inhibitor were used. The sensitivity to VOR or RICO was not different between the parental and the ERI-resistant cells. In the protein expression analysis, HDAC6 expression was not altered between parental and ERI-resistant cells. Prior treatment with VOR or RICO increased the expression of acetylatedα-tubulin in both parental and ERI-resistant TNBC cell lines. When low concentration of VOR (0.5 μM) or RICO (0.5 μM) was added simultaneously with ERI, the sensitivity to ERI was enhanced significantly in both parental MDA-MB-231 and Hs578T cell lines. Furthermore, when both parental cell lines were treated with VOR (5 μM) or RICO (5 μM) for 48 hrs prior to addition of ERI, the sensitivity to ERI was significantly enhanced. The enhancement of sensitivity to ERI both by the simultaneous addition with low concentration of VOR or RICO and by the prior treatment with VOR or RICO was observed in the ERI-resistant cells as well. Moreover, inhibition of HDAC6 by small interfering RNA partially enhanced the sensitivity to ERI in the both parental cell lines. Our study demonstrated the possibility that the combination of ERI with HDAC inhibitor might enhance the antitumor effect of ERI and overcome ERI resistance through acetylation of α-tubulin by HDAC6 inhibition in TNBC cells. Citation Format: Takaaki Oba, Hiroto Izumi, Ken-ichi Ito. Preclinical analysis of synergistic antitumor effect of eribulin and HDAC inhibitor for triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2924.

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

Background: PATZ1, a transcription regulator, has been reported to function as oncogene or tumor suppressor in several human malignant neoplasms. The aim of the study was to examine involvement of PATZ1 in carcinogenesis and progression of thyroid cancer. Materials and methods: PATZ1 expression in 165 clinical thyroid specimens obtained from 87 patients were evaluated by IHC. We examined the function of PATZ1 using an immortalized normal thyroid follicular epithelial cell line (Nthy-ori 3-1), and four thyroid cancer cell lines; TPC-1 (papillary cancer), FTC-133 (follicular cancer), FRO and ACT-1 (anaplastic cancer). Knockdown of PATZ1 in Nthy-ori 3-1 and thyroid cancer cell lines was performed with siRNA-PATZ1, and overexpression of PATZ1 in thyroid cancer cell lines was performed with pcDNA3-FLAG-PATZ1. Results: In the IHC analysis of clinical specimens, nuclear PATZ1 expression in anaplastic carcinoma was significantly less frequently than that in normal thyroid tissue or well-differentiated carcinoma (Table 1). Knockdown of PATZ1 in Nthy-ori 3-1 and differentiated thyroid cancer cell lines (TPC-1, FTC-133) induced morphological change of the cells and significant increase of cell proliferation and migration as well as moderate increase of invasion. Furthermore, the expression of u-PA, MMP2 and MMP11 was increased by inhibition of PATZ1 in TPC-1 and FTC-133. On the other hand, overexpression of PATZ1 in anaplastic cancer cell lines (FRO and ACT-1) decreased proliferation and migration of the cells along with the decreased expression of u-PA, MMP2, MMP9 and MMP11. Conclusion: Our study suggests that PATZ1 might play an important role as a tumor suppressor in thyroid follicular epithelial cells and might be involved in progression of thyroid cancer. Expression of nuclear PATZ1 in thyroid tumortypeNormal thyroid/ goiter (N=68)Follicular adenoma (N=5)Papillary cancer (N=39)Follicular cancer (N=8 )Poorly differentiated cancer (N=12)Anaplastic cancer (N=28)Medullary cancer (N=5)positive (%)68(100%)4(80.0%)35(89.7%)5(62.5%)7(58.3%)3(10.7%)4(80.0%)negative (%)0(0%)1(20.0%)4(10.3%)3(37.5%)5(41.7%)25(89.3%)1(20.0%) Citation Format: Asumi Iesato, Teruo Nakamura, Tsuyoshi Uehara, Hiroto Izumi, Ken-ichi Ito. PATZ1 promotes migration and invasion of thyroid cancer cells through upregulation of the activity of plasminogen activator and matrix metalloproteinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 906. doi:10.1158/1538-7445.AM2017-906

Programmed cell death protein 4 (PDCD4) has recently been shown to be involved in both transcription and translation, and to regulate cell growth. However, the mechanisms underlying PDCD4 function are not well understood. In this study, we show that PDCD4 interacts directly with the transcription factor Twist1 and leads to reduced cell growth through the down-regulation of the Twist1 target gene Y-box binding protein-1 (YB-1). PDCD4 interacts with the DNA binding domain of Twist1, inhibiting its DNA binding ability and YB-1 expression. Immunohistochemical analysis showed that an inverse correlation between nuclear PDCD4 and YB-1 expression levels was observed in 37 clinical prostate cancer specimens. Growth suppression by PDCD4 expression was completely recovered by either Twist1 or YB-1 expression. Moreover, PDCD4-overexpressing cells are sensitive to cisplatin and paclitaxel but not to etoposide or 5-fluorouracil. In summary, PDCD4 negatively regulates YB-1 expression via its interaction with Twist1 and is involved in cancer cell growth and chemoresistance. [Cancer Res 2009;69(7):3148–56]

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

Resistance to cisplatin is a major problem in the treatment of solid tumors. To investigate the determinants of cisplatin resistance, we have identified cisplatin-inducible genes by differential display of mRNA. One of the cisplatin-inducible genes was identified as activating transcription factor 4 (ATF4). Northern blot analysis demonstrated that expression of ATF4 is inducible at the transcriptional level. Its expression is also up-regulated in two cisplatin-resistant cell lines. We tested whether cellular levels of ATF4 are responsible for cisplatin sensitivity by examining 11 human lung cancer cell lines. Expression of ATF4 was found to correlate with cisplatin sensitivity (P = 0.01). We also evaluated the cisplatin sensitivity of two stable transfectants overexpressing ATF4. Both were less sensitive to cisplatin than the parental cells but equally sensitive to vincristine. Our findings suggest that levels of ATF4 expression could help to predict cisplatin sensitivity.

Mitochondrial transcription factor A (mtTFA; also designated Tfam) is necessary for both transcription and maintenance of mitochondrial DNA. mtTFA preferentially recognizes cisplatin-damaged DNA, as well as oxidized DNA. Increased apoptosis has been observed in mtTFA knockout animals, suggesting that mtTFA is involved in apoptosis. A fraction of p53 protein localizes to mitochondria at the onset of p53-dependent apoptosis, but not during p53-independent apoptosis. Using immunochemical coprecipitation, we observed binding of mtTFA and p53. Interaction between mtTFA and p53 required the high mobility group-box1 or high mobility group-box2 of mtTFA and amino acids 363-376 of p53. Binding of mtTFA to cisplatin-modified DNA was significantly enhanced by p53, whereas binding to oxidized DNA was inhibited. Our findings suggest that the interaction of p53 with mtTFA may play an important role in apoptosis.

Y-box binding protein-1 (YB-1) plays an important role in tumor progression and drug resistance. This study examined whether YB-1 is involved in the alteration of response to endocrine therapy in ER-positive breast cancer cells. MCF7 cells that stably expressed YB-1 (MCF7-YB-1) and vector control cells (MCF7-vector) were established. These cells were used to analyze the expression of the factors related to the ER and growth factor receptor signaling pathways, response to the antiestrogens (tamoxifen and fulvestrant), and estrogen responsive element (ERE) activity. The effect of knocking down endogenous YB-1 expression was tested in wild-type MCF7 cells. In addition, the expression of the YB-1 and the factors related to the ER and growth factor receptor signaling pathways were evaluated in the clinical breast cancers treated with preoperative chemotherapy. The expression of HER2, AIB1, p-Erk and c-Myc were increased in the MCF7-YB-1 cells. In contrast, knocking down of YB-1 decreased the expression of these factors, but increased the expression of ERα in the wild-type MCF7 cells. Furthermore, sensitivity to antiestrogens was decreased in the MCF7-YB-1 in comparison to those in the MCF7-vector cells. In the MCF7-YB-1 cells, the expression levels of p-Erk and c-Myc were continuously upregulated when the cells were treated either with tamoxifen or fulvestrant. The ERE activity was decreased in the MCF7-YB-1 cells in comparison to the MCF7-vector cells, and the ERE activity of the MCF7-YB-1 cells was inhibited by fulvestrant at a lower concentration than that which inhibited the ERE activity of the MCF7-vector cells. In the ER-positive clinical breast cancers treated with preoperative chemotherapy, significantly more of the specimens that showed increased or positive nuclear YB-1 expression after the chemotherapy were positive for HER2 expression. These data suggest that alteration of YB-1 may modify the crosstalk between the ER pathway and HER2 pathway in ER-positive breast cancer cells, and consequently may alter the response to endocrine therapy in these cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-15.

Purpose: The Y-box binding protein 1 (YB-1) regulates expression of P-glycoprotein (P-gp) encoded by the MDR1 gene. There have been no previous studies regarding the involvement of YB-1 in the development of resistance to paclitaxel. The present study was performed to examine how paclitaxel affects the localization and expression of YB-1 in breast cancer.Experimental Design: We evaluated the expression and localization of YB-1 and P-gp in breast cancer tissues obtained from 27 patients before and after treatment with paclitaxel. The effect of paclitaxel on localization of cellular YB-1 was examined by using GFP-YB-1. Interaction of YB-1 with the Y-box motif of the MDR1 promoters was studied by EMSA. The effects of paclitaxel on MDR1 promoter activity were examined by luciferase assay.Results: Of 27 breast cancer tissues treated with paclitaxel, nine (33%) showed translocation of YB-1 from the cytoplasm to the nucleus together with increased expression of P-gp during the course of treatment. Twelve breast cancer tissues (44%) showed neither translocation of YB-1 nor increased expression of P-gp. Nuclear translocation of YB-1 was correlated significantly with increased expression of P-gp (P=0.0037). Confocal analysis indicated that paclitaxel induced nuclear translocation of green fluorescent fused YB-1 in MCF7 cells. Furthermore, binding of YB-1 to the Y-box of MDR1 promoter was increased in response to treatment with paclitaxel. In addition, MDR1 promoter activity was significantly up-regulated by paclitaxel in MCF7 cells (p< 0.001).Conclusions: The results of the present study suggested that YB-1 may be involved in the development of resistance to paclitaxel in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1141.