Kei Daizumoto

Early growth response 4 is involved in cell proliferation of small cell lung cancer through transcriptional activation of its downstream genes.

Small cell lung cancer (SCLC) is aggressive, with rapid growth and frequent bone metastasis; however, its detailed molecular mechanism remains poorly understood. Here, we report the critical role of early growth factor 4 (EGR4), a DNA-binding, zinc-finger transcription factor, in cell proliferation of SCLC. EGR4 overexpression in HEK293T cells conferred significant upregulation of specific splice variants of the parathyroid hormone-related protein (PTHrP) gene, resulting in enhancement of the secretion of PTHrP protein, a known mediator of osteolytic bone metastasis. More importantly, depletion of EGR4 expression by siRNA significantly suppressed growth of the SCLC cell lines, SBC-5, SBC-3 and NCI-H1048. On the other hand, introduction of EGR4 into NIH3T3 cells significantly enhanced cell growth. We identified four EGR4 target genes, SAMD5, RAB15, SYNPO and DLX5, which were the most significantly downregulated genes upon depletion of EGR4 expression in all of the SCLC cells examined, and demonstrated the direct recruitment of EGR4 to their promoters by ChIP and luciferase reporter analysis. Notably, knockdown of the expression of these genes by siRNA remarkably suppressed the growth of all the SCLC cells. Taken together, our findings suggest that EGR4 likely regulates the bone metastasis and proliferation of SCLC cells via transcriptional regulation of several target genes, and may therefore be a promising target for the development of anticancer drugs for SCLC patients.

The Involvement of Hepatocyte Growth Factor-MET-Matrix Metalloproteinase 1 Signaling in Bladder Cancer Invasiveness and Proliferation. Effect of the MET Inhibitor, Cabozantinib (XL184), on Bladder Cancer Cells

OBJECTIVE To clarify the invasive mechanisms of muscle-invasive bladder cancer (BCa) would be useful for the determination of appropriate treatment strategies. We previously showed that hepatocyte growth factor (HGF)-MET signaling is correlated with invasiveness of BCa cells. Here, we investigated the effects of the MET inhibitor, cabozantinib (XL184), on BCa cells. METHODS We first conducted Western blot analysis to investigate MET expression in BCa cell lines. Next, we examined the effect of cabozantinib on their proliferation and invasive abilities using MTT and Matrigel invasion assays, respectively. Invasion assays were performed using the xCELLigence system. Additionally, to investigate the biological function of HGF-MET signaling, we analyzed gene expression profiles and performed real-time polymerase chain reaction analyses of 5637 cells that were cultivated with or without HGF stimulation, with or without cabozantinib. RESULTS MET was highly expressed in 4 of 5 BCa cell lines, and 5637 and T24 cells showed especially high protein expression of MET. Cabozantinib suppressed cell proliferation and invasion (cell index; mock, 1.49 vs HGF, 2.26 vs HGF + XL184, 1.47, P < .05). Gene expression profile analysis indicated that matrix metalloproteinase 1 (MMP1) was significantly elevated at the mRNA level with addition of HGF. Moreover, cabozantinib suppressed HGF-induced MMP1 expression in 5637 T24 cells. CONCLUSIONS These data indicate that cabozantinib suppressed MMP1 expression by blocking HGF-MET signaling and that HGF-MET-MMP1 signaling is involved in the invasiveness and proliferation of BCa cells. These results suggest that cabozantinib might prove useful for future treatment of muscle-invasive BCa.

Complications of Flexible Ureteroscopic Treatment for Renal and Ureteral Calculi during the Learning Curve

Background: The flexible ureterorenoscope (URS) and associated devices have developed rapidly. However, despite its therapeutic benefits, URS may be associated with some complications. To the best of our knowledge, there are no studies discussing the complications of flexURS during the learning curve. Methods: A retrospective review of the records of patients who underwent flexURS from January 2005 to June 2013 was performed. To compare the complications after the introduction of flexURS, patients were divided into four groups based on the surgeons training experience, that is, based on the number of cases performed by the surgeon. A total of 219 cases underwent flexURS. Groups 1, 2, 3, and 4 included 35, 50, 50, and 84 cases, respectively. The complications were classified using the Clavien system (I-IV). Results: The mean operation time and stone-free rate were significantly different (p < 0.001, p = 0.013, respectively). The total complication rates were 13.6, 10, 8.3, and 3.2%, respectively (p = 0.068). The more the surgeons experience, the less was the complication rate. Despite our best efforts, the incidence of urosepsis was not reduced (p = 0.902). Conclusions: To reduce severe complications, it is necessary to have performed about 100 cases. Increased surgeon experience tended to decrease the risk of severe complications, but the incidence of urosepsis was not reduced.

A DDX31/mutant-p53/EGFR axis promotes multistep progression of muscle invasive bladder cancer

The p53 and EGFR pathways are frequently altered in bladder cancer, yet their contributions to its progression remain elusive. Here we report that DEAD box polypeptide 31 (DDX31) plays a critical role in the multistep progression of muscle-invasive bladder cancer (MIBC) through its sequential interactions with mutant p53 (mutp53) and EGFR. In early MIBC cells, nuclear DDX31-bound mutp53/SP1 enhanced mutp53 transcriptional activation, leading to migration and invasion of MIBC. Cytoplasmic DDX31 also bound EGFR and phospho-nucleolin in advanced MIBC, leading to EGFR-Akt signaling activation. High expression of both cytoplasmic DDX31 and p53 proteins correlated with poor prognosis in patients with MIBC, and blocking the DDX31/NCL interaction resulted in downregulation of EGFR/Akt signaling, eliciting an in vivo antitumor effect against bladder cancer. These findings reveal that DDX31 cooperates with mutp53 and EGFR to promote progression of MIBC, and inhibition of DDX31/NCL formation may lead to potential treatment strategies for advanced MIBC.Significance: DDX31 cooperates with mutp53 and EGFR to promote progression of muscle invasive bladder cancer. Cancer Res; 78(9); 2233-47. ©2018 AACR.

MP72-06 CORRELATION OF THE INSULIN RECEPTOR EXPRESSION AND THE RESISTANCE TO VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR TYROSINE KINASE INHIBITORS IN ADVANCED CLEAR CELL RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) demonstrate the significant efficacy for advanced clear cell renal cell carcinoma (ccRCC), however, it eventually becomes resistant to VEGFR-TKIs during the treatment. So far, the mechanisms for the resistance to VEGFR-TKIs remain to be fully elucidated. Previously we have identified the gene set which could predict poor prognosis of ccRCC patients (Takahashi M et.al., Proc Natl Acad Sci U S A., 98: 9754, 2001). We examined whether the insulin receptor (INSR) expression in the gene set may correlate with the resistance to VEGFR-TKIs. METHODS: The INSR expression was examined immunohistochemically in the nephrectomy specimens of the RCC patients (n1⁄433) who then received axitinib as the VEGFR-TKI and correlated with their survival outcome. We compared the INSR expression of the nephrectomy or metastasectomy specimens before and after the administration of VEGFR-TKIs in 5 cases. The INSR expression of the human renal glomerular endothelial cells (HGEC) was compared before and after the administration of axitinib by Western blotting. In addition, we established patient derived Xenograft model (PDX) of ccRCC. Tumors of PDX were resected when it regrew and showed the resistance for axitinib and the INSR expression was compared before and after the treatment by Western blotting. RESULTS: The INSR was expressed at the vessels surrounding tumor cells. Progression-free survival (PFS) was significantly shorter in the INSR-negative group than in the INSR-positive group. Multivariate analysis revealed that the INSR expression was the significantly independent predictor of PFS. In the specimens resected after VEGFR-TKI, the INSR expression was more frequently decreased. As the concentration of axitinib increased, the INSR expression in the HGEC was decreased. The tumors of PDX that were resected after demonstrating the resistance to axitinib had the decreased INSR expression. CONCLUSIONS: The decreased INSR expression could be correlated with the resistance to VEGFR-TKI and its expression may be useful in selecting appropriate drugs for advanced ccRCC patients. Source of Funding: None

MP58-17 THE CONTRIBUTION OF HGF-MET-MMP1 SIGNALING IN BLADDER CANCER INVASION, AND MET INHIBITOR AS A POTENTIAL THERAPEUTIC OPTION FOR INVASIVE BLADDER CANCER

INTRODUCTION AND OBJECTIVES: The current standard for muscle-invasive bladder cancer (BC) patients is cisplatin-based neoadjuvant chemotherapy. However, the prognosis of the cancer patients has not been substantially improved and several different mechanisms could be involved. Previously, we showed that BC cells promote autophagy as a resistant mechanism upon cisplatin treatment. Cancer immunoresistance and immune escape are major obstacle in chemotherapy. However, the effects of cisplatin on the immune responses of BC cells are note clear. In the present studies, we investigate the expression of immunoresistance molecular PD-L1 on cisplatin-treated 5637 and T24 cells. METHODS: Human immortalized urothelial (SV-Huc-1) and bladder cancer (5637 and T24) cell lines were used in this study. The expression of PD-L1 mRNA and protein in control or cisplatin-treated cells were detected by quantitative RT-PCR and Western blot. PD-L1 knockdown in cisplatin treated cells was conducted using lenti-viral based shRNA. Cell viability in cisplatin-treated cells with or without PDL1 knockdown was monitored by tetrazolium salt WST-1. Cell signaling pathways, including AKT, ERK, MAPK and JNK, were detected by Western blot. PD98059 and Baf A1 were used as inhibitors for MAPK and autophagy, respectively. RESULTS: Up-regulation of PD-L1 expression was detected in 5637 and T24 cells treated with various concentrations of cisplatin. Increased levels of ERK, AKT, and JNK phosphorylation were observed in cisplatin-treated human BC cells, but only pretreatment of PD98059 in cisplatin-treated cells attenuated the expression of PD-L1, suggesting that cisplatin-induced PD-L1 expression in BC cells is dependent of ERK pathway. In addition, inhibition of autophagy using Baf A1 did not suppressed cisplatin-induced PD-L1, indicating cisplatin not only induces the intrinsic protective autophagy but up-regulates immune checkpoint, PD-L1, as an immune escape pro-survival mechanism in human bladder cancer cells. CONCLUSIONS: Cisplatin induces PD-L1 in human BC cells, suggesting an immune escape mechanism other than intrinsic protective autophagy. Targeting ERK pathway could be considered as therapeutic options in cisplatin resistant.

MP45-17 GALECTIN-3 PLAYS CRITICAL ROLES FOR THE GROWTH OF BENIGN PROSTATIC HYPERPLASIA

INTRODUCTION AND OBJECTIVES: Galectin-3, a multifunctional oncogenic protein, has been reported to play important roles of progression in a variety of cancer including prostate cancer through the regulation of cancer cell proliferation, apoptosis, invasion and metastasis. However, we also identified the frequent up-regulation of Galectin-3 in benign prostatic hyperplasia (BPH), yet its pathophysiological roles in BPH. Here, we report the involvement of Galectin-3 in the growth of BPH. METHODS: To investigate the association of Galectin-3 expression and prostate volume, we examined serum Galectin-3 (pg/ ml) with ELISA method in non-cancer cohort and analyzed the correlation between Galectin-3 expression and prostate volume with Speamans correlation coefficient. Next, to analyzed proliferation abilities and the effect of Galectin-3 on smooth muscle, we examined knockdown of Galectin-3 expression by siRNA in BPH1 cells (benign prostatic hyperplasia cell line) and a co-culture experiment of BPH1 and PrSMC (Normal Human Prostate Smooth Muscle. Cells). Moreover, to investigate biological function of Galectin-3, we examined the gene expression profiles in Galectin-3-depleted BPH1 cells with microarray and bioinformatics analyses. RESULTS: Correlation analysis revealed that serum Galectin-3 (ng/ml) were correlated with prostate volume (R1⁄40.643 p1⁄40.023) (Figure 1). Next, depletion of Galectin-3 suppressed cell proliferation in BPH1 cells. Moreover, depletion of Galectin-3 in BPH1 cells suppressed cell proliferation of PrSMC cells in a co-culture method, suggesting Galectin-3 enhanced proliferation of PrSMC cells. Bioinformatics analysis with GSEA revealed that depletion of Galectin-3 was involved in interferon a response and interferon a response and interferon ? response (FDR q value < 0.001) (Figure 2), suggesting Galectin-3 regulates the proliferation of PrSMC through interferon response. CONCLUSIONS: Our findings suggest that Galectin-3 is significantly involved in the growth of BPH and a promising therapeutic target for patients with BPH. Source of Funding: GSK Japan Research Grant 2016

Abstract 94: DDX31 cooperates with mutant p53 and EGFR to promote the multistep progression of invasive bladder cancer

Bladder cancer is the most common malignancy of the urinary tract worldwide. Approximately 70% of cases are diagnosed as non-muscle-invasive bladder cancer (NMIBC), while the remaining 30% of cases are classified as muscle-invasive bladder cancer (MIBC). Evidence based on molecular biology has highlighted that the aggressiveness of MIBC advances through a multistep mechanism due to many genomic alterations. Notably, alterations of TP53 and EGFR pathways frequently occur in bladder cancers and are associated with poor prognosis, respectively. However, the connection between the overexpression of EGFR and the p53 mutation in multistep carcinogenesis and the progression of MIBC remains unknown. Here we report the distinct critical roles of DEAD box polypeptide 31 (DDX31) in the multistep progression of muscle invasive bladder cancer (MIBC) through its sequential interaction with mutant p53 (mutp53) and EGFR. In early MIBC cells, nuclear DDX31 acts as a transcriptional co-activator that binds to mutp53/SP1 and enhances mutp53 transcriptional activation, thereby upregulating the EPB41L4B gene, which plays a critical role in metastatic behavior and promotes the invasion and migration of MIBC. Notably, in advanced MIBC, cytoplasmic DDX31, which is transported from the nucleus, functions as an adaptor scaffold protein that forms a complex with EGFR via its interaction with phosho-nucleolin (NCL), leading to constitutive activation of EGFR-Akt signaling. Significantly, high expression of both cytoplasmic DDX31 and p53 proteins is correlated with a poor prognosis in MIBC patients. More importantly, blocking the DDX31-NCL interaction via a dominant-negative peptide led to downregulation of EGFR-Akt signaling, resulting in a significant anti-tumoral effect of bladder cancer in vivo. Our findings reveal that DDX31 cooperates with mutp53 and EGFR to promote the multistep progression of MIBC and that inhibition of DDX31-NCL formation may lead to potential treatment strategies for advanced MIBC. Citation Format: Toyomasa Katagiri, Kei Daizumoto, Tetsuro Yoshimaru, Yosuke Matsushita, Tomoya Fukawa, Masaya Ono, Hiro-omi Kanayama. DDX31 cooperates with mutant p53 and EGFR to promote the multistep progression of invasive bladder cancer [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 94.

MP98-20 THE ROLE OF TOLL-LIKE RECEPTOR 4 IN BLADDER CANCER PROGRESSION.

INTRODUCTION AND OBJECTIVES: The molecular mechanisms of muscle invasion and distant metastasis in bladder cancer (BC) are not well understood. MicroRNA (miRNA) expression signature of various human cancers have reported that miR-26a-5p/-26b-5p were frequently downregulated in cancer tissues, suggesting these miRNAs act as tumor-suppressors. However, their functions in BC remains unknown. The aim of this study was to investigate the functional role of /-26b-5p and to identify molecular targets that contribute to metastasis in BC. METHODS: Expression of miR-26a-5p/-26b-5p and their target genes were evaluated in 69 BC clinical BC specimens and 23 normal bladder epithelia (NBE) by real-time PCR. We performed gain-of-function studies (cell proliferation, migration and invasion) by usingmiR-26a/b transfectants in BC cell lines (T24 and BOY). Putative target genes were listed by in-silico study using the gene expression omnibus (GEO) and TargetScan. We performed loss-of-function studies by using si-RNA transfectants to evaluate the functional role of the target gene. Luciferase reporter analyses were employed to validate direct binding between the target gene and miR-26a/b in BC cells. Overall survival (OS) of the BC patients was evaluated by the Kaplan-Meier analysis. RESULTS: The expression levels of miR-26a-5p/-26b-5p in clinical BCs were significantly downregulated compared to that in NBE (p < 0.0001 and p 1⁄4 0.0006, respectively). miR-26a-5p/-26b-5p transfectants significantly suppressed cell migration and invasion, suggesting these miRNAs act as tumor-suppressors. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was identified as a direct regulation of miR-26a-5p/-26b-5p by luciferase reporter assay. KaplanMeier analysis revealed that the patients with higher PLOD2 expression showed lower overall survival probabilities than those with lower expression (p 1⁄4 0.0153). Loss-of-function study showed that cell migration and invasion were significantly inhibited in si-PLOD2 transfectans. CONCLUSIONS: PLOD2 was directly regulated by tumor-suppressive miR-26a-5p/-26b-5p, and might be good prognostic markers for survival of BC patients. Recent studies showed that PLOD2 function as a collagen cross-linking enzyme associate with extracellular matrix (ECM) stiffness. Aberrant expression of PLOD2 by regulation of these miRNAs might cause extracellular matrix (ECM) disruption and promoting metastasis.