Seiji Naganuma

A NOTCH3-Mediated Squamous Cell Differentiation Program Limits Expansion of EMT-Competent Cells That Express the ZEB Transcription Factors

Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in TGF-β-mediated senescence, epithelial-to-mesenchymal transition (EMT), and cancer stem cell functions. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here, we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like 1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1-enriched EMT-competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage-independent growth and tumor formation in nude mice. RNA interference experiments suggested the involvement of ZEBs in anchorage-independent colony formation, invasion, and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation were recapitulated upon Notch inhibition by DNMAML1 in organotypic three-dimensional culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of esophageal squamous cancers.

Podocalyxin‐like protein, linked to poor prognosis of pancreatic cancers, promotes cell invasion by binding to gelsolin

The cell‐adhesion glycoprotein PODXL is associated with an aggressive tumor phenotype in several forms of cancer. Here, we report that high PODXL expression was an independent predictor of worse overall survival of pancreatic cancer patients, and that PODXL promoted pancreatic cancer cell motility and invasion by physically binding to the cytoskeletal protein gelsolin. Suppression of PODXL or gelsolin decreased membrane protrusions with abundant peripheral actin structures, and in turn inhibited cell motility and invasion. Transfection of a PODXL‐rescue construct renewed the expression of gelsolin bound to peripheral actin structures in cell protrusions, and abrogated the decreased cell protrusions caused by the knockdown of PODXL. Furthermore, transfection of a PODXL‐rescue construct into pancreatic cancer cells in which both PODXL and gelsolin were suppressed failed to increase the formation of the protrusions. Thus, PODXL enhances motility and invasiveness through an increase in gelsolin–actin interactions in cell protrusions.

Vav3 is linked to poor prognosis of pancreatic cancers and promotes the motility and invasiveness of pancreatic cancer cells

BACKGROUND/OBJECTIVESnThe aim of this study was to investigate the role of the guanine nucleotide exchange factor Vav3 in the motility and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells.nnnMETHODSnImmunohistochemistry was used to determine whether high Vav3 expression in human PDAC tissues is correlated with poor prognosis. Immunocytochemistry was used to determine the association and intracellular distribution of Vav3, Rac1 and Akt in PDAC cells. Phosphoprotein array analysis was performed to determine the Vav3-associated intracellular signaling pathways. Immunocytochemistry and Matrigel invasion assays were used to examine the effects of Vav3 on the formation of cell protrusions and PDAC cell invasion.nnnRESULTSnExpression of Vav3 in PDAC tissue was significantly correlated with overall survival. Vav3 was localized in cell protrusions of migrating PDAC cells. Knockdown of Vav3 inhibited the motility and invasiveness of PDAC cells through a decrease in cell protrusions. The levels of active Rac1 or active Akt were not associated with the concentration of Vav3 in cell protrusions. The Vav3-dependent promotion of motility and invasiveness was not modulated by Rac1 or Akt. Additionally, knockdown of Vav3 increased phosphorylated WNK1 in PDAC cells, and knockdown of WNK1 inhibited the motility and invasiveness. This study suggests that Vav3 can be a useful marker for predicting the outcome of patients with PDAC and that Vav3 can promote PDAC cell motility and invasion through association with dephosphorylation of WNK1.nnnCONCLUSIONSnVav3 was accumulated in cell protrusions, contributed to the formation of membrane protrusions, and thereby increased the motility and invasiveness of PDAC cells.

High load of Merkel cell polyomavirus DNA detected in the normal skin of Japanese patients with Merkel cell carcinoma

BACKGROUNDnAlthough Merkel cell polyomavirus (MCPyV) has the potential to cause Merkel cell carcinoma (MCC), it is also found in the normal skin of healthy individuals. However, the mechanism for transformation of MCPyV to an oncogenic form is unknown.nnnOBJECTIVESnTo investigate the levels of MCPyV infection in the normal skin patients with MCC compared with those in a control cohort.nnnSTUDY DESIGNnWe studied a total of six Japanese patients with cutaneous MCC. Sun-exposed and sun-unexposed skin swabs were obtained and analyzed for MCPyV loads using quantitative real-time polymerase chain reaction.nnnRESULTSnAt first, we found a patient with MCC carrying an extremely high load of MCPyV DNA in normal skin. This unique case prompted us to further explore the levels of MCPyV as skin microbiota in patients with MCC. We showed that MCPyV DNA levels were significantly higher in swabs obtained from normal skin samples of six patients with MCC compared with those from 30 age-matched healthy individuals and 19 patients with other cutaneous cancers. Whereas MCPyV strains obtained from the normal skin of patients with MCC had gene sequences without structural alterations, sequences of the tumor-derived strains showed truncating mutations or deletions.nnnCONCLUSIONSnAlthough the number of patients with MCC studied was small, our findings suggest that MCC may occur with a background of high MCPyV load in the skin, and are expected to stimulate further studies on whether such skin virome levels could be one of predictive markers for the development of MCC.

CCDC88A, a prognostic factor for human pancreatic cancers, promotes the motility and invasiveness of pancreatic cancer cells.

BackgroundCoiled-Coil Domain Containing 88A (CCDC88A) was identified as a substrate of the serine/threonine kinase Akt that is capable of binding to the actin cytoskeleton. The aim of this study was to investigate the potential role of CCDC88A in the migration and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells.MethodsImmunohistochemistry was performed to determine whether high CCDC88A expression in human PDAC tissues is correlated with poor prognosis. Immunoprecipitation, immunoblotting and immunocytochemistry were performed to determine the intracellular distribution of CCDC88A, and its association with the serine/threonine kinase Akt and actin-filaments in PDAC cells. Phosphoprotein array analysis was performed to determine CCDC88A-associated intracellular signaling pathways. Finally, immunofluorescence analyses and Matrigel invasion assays were performed to examine the effects of CCDC88A on the formation of cell protrusions and PDAC cell invasion.ResultsExpression of CCDC88A in PDAC tissue was significantly correlated with overall survival. CCDC88A was co-localized with peripheral actin structures in cell protrusions of migrating PDAC cells. Knockdown of CCDC88A inhibited the migration and invasiveness of PDAC cells through a decrease in cell protrusions. Although CCDC88A has been previously reported to be a binding partner and substrate of Akt, the level of active Akt was not associated with the translocation of CCDC88A towards cell protrusions. CCDC88A-dependent promotion of cell migration and invasiveness was not modulated by Akt signaling. Knockdown of CCDC88A decreased phosphorylated Src and ERK1/2 and increased phosphorylated AMPK1 in PDAC cells. Knockdown of AMPK1 inhibited the migration and invasiveness of PDAC cells. The combined data suggest that CCDC88A may be a useful marker for predicting the outcome of patients with PDAC and that CCDC88A can promote PDAC cell migration and invasion through a signaling pathway that involves phosphorylation of Src and ERK1/2 and/or dephosphorylation of AMPK1.ConclusionsCCDC88A was accumulated in cell protrusions, contributed to the formation of membrane protrusions, and increased the migration and invasiveness of PDAC cells.

Regulation of microRNA Expression by Growth Factors in Tumor Development and Progression

MicroRNAs (miRNAs) are a class of noncoding small RNAs (22–25 nucleotides) that regulate cell proliferation and various cellular functions by interfering with the translation of target messenger RNAs (mRNAs). Altered expression of miRNAs is found in various human malignancies, and indeed, we previously reported that the expression of miR-205 and miR-21 was altered in human head and neck squamous cell carcinoma (HNSCC), by miRNA microarray analysis. We also confirmed that the expression of miR-200c and miR-27b was directly regulated by hepatocyte growth factor (HGF) in HNSCC cell line, HSC3. These results suggest the significance of miRNAs as a key regulatory molecule for achieving various functions of growth factors. Altered miRNA expression might contribute enhanced progressive and invasive characteristics, such as epithelial-mesenchymal transition (EMT), of malignant tumors by regulating the translation of growth factor-induced functional molecules. There are a growing number of reports that describe the translational regulation of growth factors, their receptors, and intracellular signaling molecules by miRNAs in various tumors. However, less of the reports describe the regulation of miRNA expression by a growth factor itself. In this article, we review the relation of tumor development and progression by growth factors with miRNA expression, especially the regulation of miRNA expression by growth factors, and focus on the cooperative interactions of miRNAs, their mRNA targets, and growth factor signaling, in the context of tumor progression.

ARHGEF4 predicts poor prognosis and promotes cell invasion by influencing ERK1/2 and GSK-3α/β signaling in pancreatic cancer

Rho guanine nucleotide exchange factorxa04 (ARHGEF4) is a guanine nucleotide exchange factor that is specific for Rac1 and Cdc42. The aim of the present study was to investigate the role of ARHGEF4 in the motility and invasiveness of pancreatic cancer cells. Evaluation of an immunohistochemical staining of 102 resected pancreatic cancer samples demonstrated that high ARHGEF4 expression was correlated with an independent predictor of worse overall survival in univariate and multivariate analyses. Immunofluorescence analyses and Matrigel invasion assays demonstrated that suppression of ARHGEF4 inhibited the formation of membrane protrusions, and in turn inhibited cell motility and invasion. A phosphoprotein array analysis demonstrated that knockdown of ARHGEF4 decreased phosphorylated extracellular signal-regulated kinasexa0(ERK)1/2 and glycogen synthase kinase-3xa0(GSK-3)α/β in pancreatic cancer cells, and ERK1/2 and GSK-3α/β were associated with ARHGEF4-related motility and invasiveness through an increase in cell protrusions. These results suggested that ARHGEF4 stimulates ERK1/2 and GSK-3α/β, and provided evidence that ARHGEF4 promotes cell motility and invasiveness. Inhibition of ARHGEF4 may be a novel approach to a targeted molecular therapy, as any such therapy would limit the motility and invasiveness of pancreatic cancer cells.

Murine remote preconditioning increases glucose uptake and suppresses gluconeogenesis in hepatocytes via a brain-liver neurocircuit, leading to counteracting glucose intolerance

AIMSnOur previous study revealed that cyclic hindlimb ischaemia-reperfusion (IR) activates cardiac acetylcholine (ACh) synthesis through the cholinergic nervous system and cell-derived ACh accelerates glucose uptake. However, the mechanisms regulating glucose metabolism in vivo remain unknown. We investigated the effects and mechanisms of IR in mice under pathophysiological conditions.nnnMETHODSnUsing IR-subjected male C57BL/6J mice, the effects of IR on blood sugar (BS), glucose uptake, central parasympathetic nervous system (PNS) activity, hepatic gluconeogenic enzyme expression and those of ACh on hepatocellular glucose uptake were assessed.nnnRESULTSnIR decreased BS levels by 20% and increased c-fos immunoreactivity in the center of the PNS (the solitary tract and the dorsal motor vagal nucleus). IR specifically downregulated hepatic gluconeogenic enzyme expression and activities (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase) and accelerated hepatic glucose uptake. Transection of a hepatic vagus nerve branch decreased this uptake and reversed BS decrease. Suppressed gluconeogenic enzyme expression was reversed by intra-cerebroventricular administration of a choline acetyltransferase inhibitor. Moreover, IR significantly attenuated hyperglycaemia in murine model of type I and II diabetes mellitus.nnnCONCLUSIONSnIR provides another insight into a therapeutic modality for diabetes mellitus due to regulating gluconeogenesis and glucose-uptake and advocates an adjunctive mode rectifying disturbed glucose metabolism.

WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4

WAVE2 is a member of the WASP/WAVE family of actin cytoskeletal regulatory proteins; unfortunately, little is known about its function in pancreatic cancers. In this study, we report the role of WAVE2 in the motility and invasiveness of pancreatic cancer cells. High WAVE2 expression in human pancreatic cancer tissues was correlated with overall survival. WAVE2 accumulated in the cell protrusions of pancreatic cancer cell lines. Downregulation of WAVE2 by small interfering RNA decreased the cell protrusions and inhibited the motility and invasiveness of pancreatic cancer cells. WAVE2 promoted pancreatic cancer cell motility and invasion by forming a complex with the actin cytoskeletal protein alpha‐actinin 4 (ACTN4). Downregulation of ACTN4 by small interfering RNA also inhibited the motility and invasiveness of the cells through a decrease in cell protrusions. Further investigation showed that WAVE2/ACTN4 signaling selectively stimulated p27 phosphorylation and thereby increased the motility and invasiveness of the cells. These results suggest that WAVE2 and ACTN4 stimulate p27 phosphorylation and provide evidence that WAVE2 promotes the motility and invasiveness of pancreatic cancer cells.

Abstract 2678: Notch1 regulates epithelial-mesenchymal transition and tumor-initiating capability in esophageal squamous-cell carcinoma

Introduction: Notch signaling may act as a tumor suppressor during the development of squamous cell carcinomas (SCCs); yet, Notch activation promotes tumor growth in a subset of SCC cells. The roles of Notch in the pathogenesis of esophageal squamous cell carcinoma (ESCC) remain elusive. Methods: Notch1 activation and epithelial-mesenchymal transition (EMT) were determined in an esophageal epithelium-targeted cell-lineage traceable (K5CreERT2-Rosa26tdTomatolsl) mouse model of ESCC induced by 4-nitroquinoline 1-oxide (4NQO), which was coupled with flow cytometry and single cell-derived ESCC organoid formation assays. Tumor-initiating capability was assessed in xenograft transplantation experiments with TE11 human ESCC cells carrying either Crispr-Cas9-deleted Notch1 loci or tetracycline-inducible expression of the activated form of Notch1 (ICN1). Surgically resected primary tumors and adjacent normal mucosa from ESCC patients (n = 152) were analyzed by immunohistochemistry for Notch1 activation and the EMT marker ZEB1. Results: 4NQO-treated mice developed tdTomato-positive primary and metastatic ESCC tumors with EpCAM-negative ESCC cells displaying traits compatible with EMT. Notch1 activation and ZEB1 expression were co-localized in ESCC cells at the stromal interface, a finding that was further recapitulated in ESCC tumor organoids. Interestingly, Cre-mediated ex vivo Notch1 deletion in a single cell suspension from Notch1loxP/loxP mouse-derived ESCC tumors decreased organoid formation rate. TE11 xenograft tumors appeared to contain a unique ESCC cell fraction containing EpCAM-negative cells, where ICN1 conferred tumorigenicity upon serial transplantation. This population displayed upregulation of Notch1 target genes and the ESCC-lineage survival factor/oncogene SOX2. Moreover, Notch1 deletion in TE11 not only limited tumor formation, but also decreased EMT in culture. A subset of ESCC patients (49/140, 33%) showed ICN1-positive ESCC cells with concurrent ZEB1 expression at the tumor invasive front. The presence of such ESCC cells was associated with poor 5-year survival (P = 0.001), tumor depth (P = 0.01), lymphatic and venous invasion (P = 0.003) and distant metastasis (P = 0.002). Moreover, such ICN1-expressing cells were increased in ESCC patients (7/12, 58%) who received pre-surgical neoadjuvant therapy. Conclusions: Cell-lineage tracing experiments validate for the first time Notch1 activation and EMT in the 4NQO-induced mouse model of ESCC. Analyses of single cell-derived ESCC tumor organoids, xenograft and primary ESCC tumors reveal that Notch1 activation may be associated with tumor initiating capability, EMT and chemotherapy resistance, implicating Notch1 activation in the pathogenesis of ESCC and potentially other SCCs. Citation Format: Koji Tanaka, Kelly A. Whelan, Naryan L. Rustgi, Prasanna M. Chandramouleeswaran, Seiji Naganuma, Shingo Kagawa, Mitsuteru Natsuizaka, Yoshiaki Kita, Shoji Natsugoe, Que Jianwen, Devraj Basu, Andres J. Klein-Szanto, Adam Bass, J. Alan Diehl, Hiroshi Nakagawa. Notch1 regulates epithelial-mesenchymal transition and tumor-initiating capability in esophageal squamous-cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2678.