Control siRNA-transfected Cells Research Articles

Effects of transmembrane serine protease 4 on the survival in patients with pancreatic ductal adenocarcinoma undergoing surgery followed by adjuvant chemotherapy.

The transmembrane serine protease 4 (TMPRSS4) gene is upregulated in various human cancers. However, its biological functions in pancreatic ductal adenocarcinoma remain unclear. We examined the expression of TMPRSS4 in pancreatic ductal adenocarcinoma tissues and its correlation with clinicopathological parameters in patients with pancreatic ductal adenocarcinoma who underwent surgery. The TMPRSS4 expression was immunohistochemically examined in 81 PDAC patients with pancreatic ductal adenocarcinoma. We analyzed the association between the TMPRSS4 expression and clinicopathological factors, the recurrence-free survival (RFS), and the overall survival (OS) and examined the effect of TMPRSS4 expression on cell migration and sensitivity to 5-fluorouracil. The expression rate of TMPRSS4 in the samples was 62.9% (51/81). The TMPRSS4 expression was not correlated with any clinicopathological feature. The five-year overall and recurrence-free survival rates were significantly lower in the TMPRSS4-positive group than in the TMPRSS4-negative group. On a multivariate analysis, TMPRSS4 positivity, poorly differentiated histology, and non-adjuvant chemotherapy predicted a poor OS, while TMPRSS4 positivity and poorly differentiated histology predicted a poor RFS. TMPRSS4-silenced pancreatic ductal adenocarcinoma cells showed higher sensitivity to 5-fluorouracil than did the control siRNA-transfected cells. TMPRSS4 can be considered a prognostic factor and therapeutic target for pancreatic ductal adenocarcinoma.

Kinesin Family Member B18 Is Related to Gastric Mucin Phenotype and Contributes to Gastric Cancer Progression by Regulating Epithelial-Mesenchymal Transition

Introduction: Gastric cancer (GC) remains a common health concern worldwide and is the third leading cause of death in Japan. It can be broadly classified into gastric and intestinal mucin phenotypes using immunohistochemistry. We previously reported numerous associations of kinesin family member (KIF) genes and mucin phenotypes with GC. However, no previous studies have reported on the importance of KIF18B in GC using immunostaining. Thus, in this study, we investigated the expression and functions of KIF18B, which is highly expressed in gastric mucin phenotype GC. Methods: We performed RNA-seq of gastric and intestinal mucin type GCs, and clinicopathological studies of the KIF18B we found were performed using 96 GC cases. We also performed functional analysis using GC-derived cell lines. Result: RNA-seq showed the upregulation of matrisome-associated genes in gastric mucin phenotype GC and a high expression of KIF18B. KIF18B was detected in 52 of the 96 GC cases (54%) through immunohistochemistry. Low KIF18B expression was significantly associated with poor overall survival (p < 0.01). Other molecules that were significantly associated with KIF18B were MUC5AC and claudin 18; these were also significantly associated with the gastric mucin phenotype. KIF18B small interfering RNA (siRNA)-transfected GC cells showed greater growth and spheroid colony formation than the negative control siRNA-transfected cells. Furthermore, expression of snail family transcriptional repressor 1 and cadherin 2 was significantly increased and that of cadherin 1 was significantly decreased in KIF18B siRNA-transfected GC cells. Conclusion: These findings not only suggest that KIF18B may be a useful prognostic marker, but also provide insight into the pathogenesis of the GC phenotype.

Expression of kinesin family member C1 in pancreatic ductal adenocarcinoma affects tumor progression and stemness.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer and the third leading cause of cancer-related deaths. Therefore, there is an urgent need for a novel molecular target for the treatment of PDAC. Kinesin family member C1 (KIFC1) belongs to the kinesin superfamily proteins and has been reported to be involved in the pathogenesis of a wide variety of carcinomas. However, the role of KIFC1 in PDAC remains unknown. This study aimed to analyze the expression and biological function of KIFC1 in PDAC. Immunohistochemically, KIFC1 was found in 37 of 81 PDAC cases (46%). A high expression of KIFC1 was significantly related to tumor size (p=0.023) and poor overall survival (p=0.011). Univariate and multivariate analysis indicated that KIFC1 expression was a prognostic factor in PDAC cases. As for cancer stem cell markers, KIFC1 expression tended to co-express significantly with CD44 (p<0.01). The growth and spheroid colony formation of KIFC1 small interfering RNA (siRNA)-transfected PDAC cells were significantly lower than those of negative control siRNA-transfected cells. Therefore, our findings suggest that KIFC1 is an independent prognostic factor in PDAC and may represent a new promising therapeutic target in PDAC.

Streptococcus pneumoniae exerts oxidative stress, subverts antioxidant signaling and autophagy in human corneal epithelial cells that is alleviated by tert-Butylhydroquinone.

Streptococcus pneumoniae is one of the leading causes of bacterial keratitis in the developing world and globally. In the current study, we have determined oxidative stress as pathogenesis of S. pneumoniae infection in corneal tissues and human corneal epithelial cells (HCEC) and explored host immune response of HCEC towards S. pneumoniae. We also determined whether treatment with tert-Butylhydroquinone (tBHQ), a Nrf2 inducer, could alleviate oxidative stress and reduce bacterial cytotoxicity in these cells. Oxidative stress was determined in corneal tissues of patients and HCEC by immunohistochemistry and immunofluorescence analysis, respectively. The expression of antioxidant genes, cytokines and antimicrobial peptides was determined by quantitative PCR. Infection of HCEC by S. pneumoniae was determined by colony-forming units. The autophagy and cell death were determined by fluorescence microscopy. The phosphorylation of signaling proteins was evaluated by immunoblot analysis. S. pneumoniae induced oxidative stress during corneal infections and inhibited antioxidant signaling pathways and immune responses like autophagy. tBHQ aided in restoring Nrf2 activation, reduced reactive oxygen species generation and prevented cytotoxicity and cell death in S. pneumoniae-infected HCEC. tBHQ also induced autophagy in a Nrf2-dependent manner and reduced bacterial survival in HCEC. Increased expression of antimicrobial peptides by tBHQ might have contributed to a reduction of bacterial load and cytotoxicity, as exemplified in LL-37 depleted corneal epithelial cells exposed to S. pneumoniae compared to control siRNA-transfected cells. tBHQ mediates alleviation of oxidative stress induced by S. pneumoniae by activating Nrf2-mediated antioxidant signaling in corneal epithelial cells. tBHQ also enhances expression of antimicrobial peptides in corneal cells and aids in inhibition of bacterial survival and cytotoxicity of HCEC.

NLRP12 attenuates tumor necrosis factor-α production in Burkholderia pseudomallei-infected RAW264.7 macrophages.

NLRP12 has been shown to play an essential role as a negative regulator in several bacterial infection. The purpose of this study is to elucidate the role of NLRP12 in B. pseudomallei-infected RAW264.7 macrophages. The protein expression and the level of TNF-α production were determined by immunoblotting and ELISA assay, respectively. The results demonstrated that unlike the LPS-mutant strain which lacks O antigenic polysaccharide, the wild-type B. pseudomallei was able to upregulate NLRP12 protein expression in RAW264.7 macrophages. NLRP12 expression also correlated with the suppression of TNF-α production as demonstrated in wild-type B. pseudomallei-infected Nlrp12-depleted macrophages when compared to that of the control siRNA-transfected cells. The expression of NLRP12 was also inhibited in cytochalasin D treated cells. Our findings showed that wild-type B. pseudomallei can activate NLRP12 expression leading to the suppression of TNF-α production. It is possible that the regulation of NLRP12 may contribute to the pathogenesis of B. pseudomallei infection in melioidosis patients.

Utility of TMPRSS4 as a Prognostic Biomarker and Potential Therapeutic Target in Patients with Gastric Cancer

BackgroundTransmembrane serine protease 4 (TMPRSS4) belongs to the family of type II transmembrane serine proteases that are known to be upregulated in many malignant tumors. However, there is a paucity of studies documenting the clinical impact and biological effects of TMPRSS4 on gastric cancer (GC) patients who underwent surgery. MethodsTissues samples were obtained from 105 patients with GC who underwent gastrectomy followed by adjuvant chemotherapy, excluding those at stage I. The expression of TMPRSS4 was examined through immunohistochemical analysis. The association between TMPRSS4 expression and clinico-pathological features as well as prognosis was assessed. Moreover, the effects of TMPRSS4 expression on cell migration and sensitivity to 5-FU were investigated. ResultsThe expression rate of TMPRSS4 was 56.3% (59/105) in GC cases. The expression of TMPRSS4 was positively correlated with the depth of tumor (T) and venous (V) invasion. The 5-year overall survival (OS) and recurrence-free survival (RFS) rates of the TMPRSS4-positive group was significantly lower than that of the TMPRSS4-negative group (p=0.0001 and p=0.005, respectively). Especially, there was significant differences in OS and RFS of patients with stage III cancer between the two groups (p=0.0064 and 0.012, respectively). Multivariate analysis demonstrated that TMPRSS4 expression and the stage of cancer were crucial prognostic factors for RFS. TMPRSS4-silenced GC cells exhibited increased sensitivity to 5-FU when compared with the non-specific control siRNA-transfected cells. ConclusionTMPRSS4 can be considered as a potential prognostic biomarker, especially for stage III, and a promising therapeutic target for GC.

FoxM1: A New Therapeutic Target for Mantle Cell Lymphoma

IntroductionMantle cell lymphoma (MCL) is an aggressive B-cell lymphoma subtype with elevated B-cell receptor activity. Ibrutinib (IBN), the Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have an overall response rate of 68% in relapsed or refractory MCL patients. However, with the emergence of IBN resistance, novel therapies to thwart resistance are urgently needed. FOXM1 (Forkhead box M1) is a proliferation-associated transcription factor that stimulates cell proliferation and exhibits a proliferation-specific expression pattern. FOXM1 has recently been classified as a human proto-oncogene and we previously found this gene to be associated with IBN resistance in our gene expression analysis; therefore, the prognostic significance of FOXM1 and its potential as a new MCL therapeutic target was investigated.MethodsBone marrow aspirates and peripheral blood clinical specimens were obtained from MCL patients. FOXM1 gene expression was assessed in 50 primary MCL patient samples using Quantitative real-time PCR (Q-PCR). To conduct dose-dependent cell viability assays, MCL cell lines (ATCC) were cultured in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (Millipore). The drug screening was performed in a 96-well format in which MCL cells were seeded at 10,000 cells per well and were treated with the FOXM1 inhibitor thiostrepton or ibrutinib at the following concentrations: 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 uM. Cell viability was tested using the CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation period. To determine the effects of inhibiting FOXM1 on apoptosis, MCL cells (200,000 cells) were incubated with IBN or thiostrepton at varying doses (1, 3, 5 uM) for 24 hours, and apoptosis was detected using the Annexin V-binding assay. Furthermore, caspase-3/7 activity was assessed in MCL cells with the Caspase-Glo-3/7 Assay (Promega) after 24 hours of treatment with thiostrepton or IBN.ResultsTo evaluate the possibility that FOXM1 is important for MCL, we conducted an analysis of FOXM1 expression in MCL patient samples at the time of diagnosis using Q-PCR. FOXM1 expression was detected in 50 MCL patient samples, and FOXM1 expression levels were significantly higher (82.3%, fold change=2) than the levels observed in peripheral blood mononuclear cells (PBMCs). The association between FOXM1 expression in MCL and clinical pathology was also analyzed by examining the relationship between FOXM1 and Ki-67 expression (a cell-proliferation marker) in 40 diagnosed MCL patient samples, and a significant correlation was noted between the two markers (p<0.05).The effects of inhibiting FOXM1 on MCL cell lines (Jeko-1, Jeko-1-BTK-KO-#1, Jeko-1-BTK-KO-#2, Mino, Maver-1, Z-138, JVM-2, and JVM-13) in vitro were examined by transfection of siRNA targeting FOXM1 or treatment with the FOXM1 inhibitor thiostrepton. Thiostrepton reduced the cell viability of the MCL cell lines in a dose-dependent manner compared with the DMSO control. Likewise, siFOXM1-transfected MCL cell lines showed reduced cell viability compared with the control siRNA-transfected cells (P < 0.01). Annexin V/PI dual staining showed that thiostrepton induced apoptosis in the MCL cell lines in a dose-dependent manner. Furthermore, activated-caspase3-7 expression was increased in a dose-dependent manner in MCL cell lines after treatment with thiostrepton.ConclusionWe have shown that FOXM1 inhibition may be a potential candidate treatment for MCL based on the results of our clinicopathological assessment and in vitro studies. Clinical studies are warranted to further validate FOXM1 as a potential therapeutic target in high-risk MCL. DisclosuresWang:Celgene: Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Oncternal: Research Funding; BeiGene: Research Funding; Juno Therapeutic: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Consultancy, Honoraria, Research Funding; Oncoceutics: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Asana: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Karus: Research Funding.

Defective Rab1b Related Megakaryocytic ER-to-Golgi Transport in RUNX1 Haplodeficiency

RUNX1, a major hematopoietic transcription factor, plays a critical role in megakaryopoiesis, megakaryocytic (MK) maturation and platelet production. Patients with RUNX1 haplodeficiency have thrombocytopenia, abnormal platelet function and impaired granule secretion. Defective dense and/or -alpha granules are a major finding in these patients. We have reported a patient with inherited thrombocytopenia, decreased granules, and abnormal platelet function (Gabbeta et al, Blood 87: 1368-76, 1996) with a heterozygous RUNX1 mutation (c.508+1G>1) (Sun et al, Blood 103: 948-54, 2004). Platelet expression profiling revealed down-regulation of several genes including ALOX12, MYL9, PRKCQ, PF4, and PLDN (Sun et al J Thromb Haemost 5: 146-154, 2007) and we have shown these to be direct RUNX1 transcriptional targets. In addition, a small G protein, RAB1B, was significantly decreased on expression profiling of the patient platelets relative to control subjects (fold change: 0.32, p < 0.025; confirmed by qPCR). With real-time PCR platelet RAB1B mRNA was decreased by 50% compared to healthy 5 subjects. Immunofluorescence studies showed that Rab1b protein was decreased in patient platelets compared to that of normal subjects.RAB GTPases are key players in vesicle trafficking, granule targeting of proteins, and secretion. Rab1b has been implicated in ER-to-Golgi transport in COS cells, but cell biological roles of RAB1B in MK cells are unexplored. We addressed the hypothesis that RAB1B is a transcriptional target of RUNX1 and regulates ER-to-Golgi transport in MK cells. In silico analysis of the RAB1B promoter region (-1/-785 bp from the ATG) revealed 4 RUNX1 consensus sites: site-1 (-440/-435), site-2 (-622/-617), site-3 (-660/-655) and site-4 (-770/-765). Following phorbol 12-myristate 13-acetate treatment of human erythroleukemia (HEL) cells to induce MK transformation, chromatin immunoprecipitation (ChIP) revealed RUNX1 binding to RAB1B sites 1 and 4 but not to other sites. Electrophoretic mobility shift assays using HEL cell proteins showed RUNX1 binding to these sites. In luciferase reporter assays, mutation individually of sites 1 and 4 reduced the promoter activity by 90%, compared to that of full-length promoter. RUNX1 over-expression enhanced RAB1B promoter activity by 2-fold and RAB1B protein by ~30%. Enhancement of promoter activity was abrogated by mutations of sites 1 and 4, indicating that these sites were functional. RUNX1 siRNA reduced RAB1B promoter activity and RAB1B protein.We studied vesicle trafficking from ER-to-Golgi in HEL cells using vesicular stomatitis virus G envelope glycoprotein (VSVG) tagged with enhanced green fluorescence protein (EGFP) and GalT on siRNA downregulation of RUNX1 or RAB1B . (Presley JF et al, Nature 389: 81-85, 1997). VSVG-GFP colocalized with GalT in the Golgi in control siRNA-transfected cells, whereas in RUNX1 knockdown cells, VSVG was not colocalized with GalT; moreover, Golgi structures were disrupted. Similar results were obtained in RAB1B downregulation by siRNA. These effects of RUNX1 knockdown were reversed by RAB1B overexpression. Together these results suggest that RAB1B downregulation may be the primary cause of the impaired ER-to-Golgi transport by RUNX1 downregulation.Conclusions: These studies show that RAB1B is a key regulator of vesicle trafficking in the secretory and endocytic pathway in megakaryocytes and platelets. This is the first evidence that RAB1B is a direct transcriptional target of RUNX1 and provides a mechanism for RAB1B down regulation in our RUNX1 haplodeficient patient. One of the hallmarks of RUNX1 haplodeficiency is defective granule formation and secretion. We propose RAB1B reduction of ER-to-Golgi transport contributes to defects in granule formation and secretion in patients with RUNX1 deficiency. DisclosuresNo relevant conflicts of interest to declare.

The tumor necrosis factor family molecules LIGHT and lymphotoxins in sinus mucosa of patients with chronic rhinosinusitis with or without nasal polyps.

Little is known about the role of lymphotoxins (LTs) family in the sinonasal mucosa of patients with chronic rhinosinusitis (CRS). This study aims at investigating the expression of LIGHT, LTα, LTβ, and their receptors, LTβR and HVEM in normal and inflammatory sinus mucosa, and the effect of LIGHT and LTalpha1beta2 on chemokine secretion in epithelial cells, epithelial permeability, and leukocyte migration. The expression of LTs family in sinonasal mucosa was evaluated with real-time PCR, immunohistochemistry, and western blot. In LTβR, HVEM siRNA, or control siRNA-transfected epithelial cells treated with LIGHT or LTalpha1beta2, the expression of chemokines, the epithelial permeability, and the expression of junctional complex proteins were evaluated using real-time PCR, ELISA, western blot, confocal microscopy, and FITC-dextran. In cultured endothelial cells treated with LIGHT or LTalpha1beta2, the expression of ICAM-1 and VCAM-1, and leukocyte migration were elucidated. LTs family was expressed in normal mucosa and their levels were increased in inflammatory mucosa of CRS patients. Recombinant LIGHT and LTalpha1beta2 induced chemokine secretion, increased epithelial permeability, and promoted leukocyte migration. However, the activity of LIGHT and LTalpha1beta2 was attenuated in cells transfected with LTβR and HVEM siRNA. LIGHT and LTs may participate in the ongoing process of chronic inflammation, inducing chemokine secretion, leukocyte migration, and dysregulated epithelial barrier through LTβR and HVEM in sinonasal mucosa.

Porphyromonas gingivalis Mfa1 Induces Chemokine and Cell Adhesion Molecules in Mouse Gingival Fibroblasts via Toll-Like Receptors.

Porphyromonas gingivalis Mfa1 fimbriae are thought to act as adhesion factors and to direct periodontal tissue destruction but their immunomodulatory actions are poorly understood. Here, we investigated the effect of Mfa1 stimulation on the immune and metabolic mechanisms of gingival fibroblasts from periodontal connective tissue. We also determined the role of Toll-like receptor (TLR) 2 and TLR4 in Mfa1 recognition. Mfa1 increased the expression of genes encoding chemokine (C-X-C motif) ligand (CXCL) 1, CXCL3, intercellular adhesion molecule (ICAM) 1 and Selectin endothelium (E) in gingival fibroblasts, but did not have a significant effect on genes that regulate metabolism. Mfa1-stimulated up-regulation of genes was significantly suppressed in Tlr4 siRNA-transfected cells compared with that in control siRNA-transfected cells, which indicates that recognition by TLR4 is essential for immunomodulation by Mfa1. Additionally, suppression of Tlr2 expression partially attenuated the stimulatory effect of Mfa1. Overall, these results help explain the involvement of P. gingivalis Mfa1 fimbriae in the progression of periodontal disease.

Abstract 5916: The role of YAP signaling in KRAS driven non-small cell lung cancer

Abstract Background Oncogenic KRAS mutant non-small cell lung cancer (NSCLC) remains treatment refractory. Even with the novel KRAS-G12C inhibitors in clinical development, caution should be taken because tumors will likely develop resistance. Subgroups of KRAS mutant NSCLC, defined by co-occurring genetic alterations in STK11/LKB1 (KL) and TP53 (KP) tumor suppressor genes, have distinct biology and therapeutic vulnerabilities. The Hippo pathway effector YAP has been shown to substitute oncogenic KRAS in promoting the growth of lung adenocarcinomas and confer resistance to MAPK pathway inhibitors and immunotherapy. Here we have examined the role of YAP in KL, KP and K-only (intact STK11/LKB1 and TP53) NSCLC cells. Methods All the cell lines were obtained from ATCC. Cell viability was assessed by CellTiter-Glo Luminescent Assay (Promega) and protein levels by western blot. YAP silencing was achieved by siRNA transfection (20 nM) using Lipofectamine RNAiMAX. Results YAP is phosphorylated by the Hippo/LATS kinase cascade at serine 127 (S127), resulting in cytoplasmic sequestration. YAP phosphorylation at tyrosine 357 (Y357) results in its nuclear localization and is Hippo/LATS independent. We first assessed the status of YAP phosphorylation in our KRAS mutant NSCLC cell lines. Among 5 KL (H460, H2030, A549, H23 and H2122) cells, 3 (H460, H2030 and A549) had higher YAP(Y357) and lower YAP(S127) expression compared to KP (H1792, SK-LU1, H2009 and Calu-6) and K-only (H358 and Calu-1) cells, normal fibroblasts (IMR-90) and the Hippo/LATS inactive breast cancer cell line, MDA-MB-231. These findings suggest the nuclear retention of YAP in KL cells. In all 3 KL cells we detected the expression of CTGF and/or Cyr61, the bona fide YAP target genes. H460 (KL) cells had the highest PD-L1 expression, although high PD-L1 expression was also seen in KP (H2009) and K-only (Calu-1) cells. Then we sought to explore whether YAP serves a functional role in KL cells compared to KP or K-only cells. H460 (KL), H1792 (KP), and H358 (K-only) cells were transfected with YAP siRNA or control siRNA. YAP protein expression was reduced in all cells by 70-90% compared to control siRNA-transfected cells. Transfection with YAP siRNA reduced the viability of H460 (KL) cells more profoundly compared to H1792 (KP), and H358 (K-only) cells. The effect of YAP silencing on downstream signaling and immune cell composition and the co-targeting of YAP and MEK in vitro and in vivo are ongoing. Conclusions STK11/LKB1 alterations have been identified as a major driver of primary resistance to PD-1 or PD-L1 blockade in KRAS mutant NSCLC. YAP acts as a transcriptional driver of cytokines that orchestrate the immunosuppressive microenvironment within KRAS mutant tumors. Our results corroborate the role of YAP in KRAS mutant NSCLC and allow considering the synthetic lethality of YAP suppression with MEK inhibitors or immunotherapy as a promising strategy to enhance treatment response and patient survival. Citation Format: Marina Keil, Richard Schneider, Elisabeth Trivier, Christian Dillon, Dirk Wienke, Anita Seshire, Linda Pudelko, Andree Blaukat, Niki Karachaliou. The role of YAP signaling in KRAS driven non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5916.

Abstract 5306: Clinicopathological significance of KIFC1 in human colorectal cancer

Abstract Colorectal cancer (CRC) ranks third in terms of incidence and second in terms of mortality, more than 1.8 million new colorectal cancer cases and 881,000 deaths are reported in Global Cancer Statistics 2018. Therefore, there is an urgent need for a new molecular target in considering treatment strategy. In recent years, cancer has been recognized as a stem cell disease. Spheroid colony formation has been demonstrated as an effective method to characterize cancer stem cells (CSCs). Previously, we analyzed the gene expression profile of spheroid colonies from the gastric cancer cell lines by microarray analysis and found that expression of Kinesin Family Member C1 (KIFC1) was upregulated in gastric spheroid colonies. The kinesin superfamily is a group of proteins that share a motor domain and participate in several biological functions. KIFC1 (or HSET) has been reported to play a vital role in centrosome clustering in cancer cells and is related to pathogenesis in several kinds of cancers. However, the expression of KIFC1 in CRC has not been investigated. The aim of this study is to investigate the expression and the biological function of KIFC1 in CRC. We analyzed the expression and distribution of KIFC1 in 129 CRC cases by immunohistochemistry. In contrast to weak or no staining of KIFC1 in non-neoplastic mucosa, CRC tissue showed stronger, more extensive KIFC1 staining. In total, 67 (52%) of 129 CRC cases were positive for KIFC1. KIFC1-positive CRC cases were significantly associated with CD44-positive and aldehyde dehydrogenase 1 (ALDH1)-positive CRC cases. In the CRC cell proliferation analysis, KIFC1 siRNA-transfected cells showed significantly reduced cell growth ability relative to negative control siRNA-transfected cells. The expression level of phosphorylated Akt and Erk were lower in KIFC1 siRNA-transfected cells compared to negative control siRNA-transfected cells. In the sphere formation analysis, the number and the size of spheres from CRC cells were significantly reduced in KIFC1 siRNA transfected cells than in negative control siRNA-transfected cells. These results indicate that KIFC1 plays an important role in CRC pathogenesis and might be involved in cancer stemness. Although further in-depth studies to clarify the biological significance of KIFC1 in colorectal CSCs are needed, KIFC1 may be a promising therapeutic target for the treatment of CRC. Citation Format: Shintaro Akabane, Naohide Oue, Yuto Fujiki, Naohiro Uraoka, Naoya Sakamoto, Kazuhiro Sentani, Hiroyuki Egi, Hideki Ohdan, Wataru Yasui. Clinicopathological significance of KIFC1 in human colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5306.

Effects of Egr1 on pancreatic acinar intracellular trypsinogen activation and the associated ceRNA network.

Acute pancreatitis (AP) is a common digestive disorder with high morbidity and mortality. The present study aimed to investigate the expression of early growth response protein 1 (Egr1), and the effect of competing endogenous (ce)RNA network on trypsinogen activation. Pancreatic acinar intracellular trypsinogen activation (PAITA) is an important event in the early stage of AP; however, the underlying mechanisms remain unclear. The present study used taurolithocholic acid 3-sulfate (TLC-S)-treated AR42J cells (pancreatic cell line) to establish a PAITA model. A gene microarray and bioinformatics analysis was performed to identify the potential key targets in PAITA. The results demonstrated that Egr1, an important transcription factor, was significantly overexpressed in PAITA. In Egr1 small interfering (si)RNA-transfected cells, Egr1 expression was decreased and trypsinogen activation was significantly decreased compared with negative control siRNA-transfected cells, indicating that in TLC-S-induced PAITA, overexpression of Egr1 enhanced trypsinogen activation. A ceRNA network [mRNA-microRNA (miRNA/miR)-long non-coding (lnc)RNA] generated using the PAITA model revealed that the effects of Egr1 on PAITA may be regulated by multiple ceRNA pairs, and the lncRNAs (including NONRATT022624 and NONRATT031002) and miRNAs [including Rattus norvegicus (rno)-miR-214-3p and rno-miR-764-5p] included in the ceRNA pairs may serve roles in PAITA by regulating the expression of Egr1. The results of the present study may provide novel targets for researching the underlying mechanisms of, and developing treatments for AP.

2020-P: ß3-Adrenergic Receptors Regulate Lipolysis and Thermogenesis in Human Brown/Beige Adipocytes

Activation of rodent brown adipose tissue (BAT) via pharmacological stimulation of β3-adrenergic receptors (β3-AR) leads to metabolic benefit, however in humans, the physiological relevance of BAT and β3-AR remain controversial. To investigate the role of the β3-AR in human brown adipocytes, first, we established a human primary supraclavicular adipocyte in vitro model that upon differentiation preserved the physiological properties brown adipose tissue with increased β-ARs, adipogenic, thermogenic and brown/beige markers. Next, we selective silenced β3-AR expression in differentiated primary brown/beige adipocytes using a lipofectamine-based non-viral transfection. 48h of transfection of siRNA targeting ADRB3 (siRNA-ADRB3) resulted in a 90% reduction in ADRB3 mRNA levels, relative to control siRNA-transfected cells (siRNA-Ctrl). siRNA-ADRB3 also reduced genes of fatty acid metabolism, thermogenesis (UCP1, 90% decrease), and mitochondrial mass. Functionally, lower cAMP levels, lipolysis, oxygen consumption rates and higher glycolysis were caused by silencing β3-AR receptor. Immortalized brown adipocyte (hBA’s), from a separate subject, displayed a similar decrease in lipolysis and cellular respiration. Mirabegron, the selective human β3-AR agonist, stimulated BAT lipolysis and thermogenesis, that were lost after knockdown ADRB3. Similarly, selective subtype human β-AR agonists dobutamine (β1-AR agonist) and terbutaline (β2-AR agonist) treatment also increased lipolysis and thermogenesis, that were also attenuated when β3-AR was silenced. Thus, the β3-AR maintains multiple components of the human brown/beige adipocyte lipolytic and thermogenic cellular machinery. These findings indicate that ADRB3 and β3-AR agonists could be used to achieve metabolic benefit in humans. Disclosure C. Cero: None. H.J. Lea: None. K.Y. Zhu: None. A.M. Cypess: None.

Calponin 3 promotes invasion and drug resistance of colon cancer cells.

BACKGROUNDCalponin 3 (CNN3) is an actin-binding protein expressed in smooth muscle and non-smooth muscle cells. It is required for cytoskeletal rearrangement and wound healing.AIMTo dissect the role of CNN3 in carcinogenesis with a focus on colon cancer.METHODSA total of 20 cancer cell lines (8 breast, 11 colon, and HeLa cervical cancer cell as a positive control for mesenchymal phenotype) and 57 formalin-fixed, paraffin-embedded sections from archived sporadic colorectal carcinomas were included in this study. CNN3 expression analysis by western blot or immunohistochemistry was followed by functional analyses. The CNN3 gene was silenced by specific small interfering RNA (commonly known as siRNA), followed by confirmation of the silencing efficiency by western blotting. Then, the silenced cells and control siRNA-transfected cells were analyzed for changes in epithelial and mesenchymal markers, invasion, and response to 5-fluoruracil treatment. We also performed proteomics analysis using a phospho-kinase array-based panel of 45 proteins.RESULTSCNN3 showed positive expression in 6/8 breast and 9/11 colon cancer lines and in HeLa cells. Interestingly, the colorectal adenocarcinoma line SW480 was negative, while the cell line developed from its matching lymph node metastasis (SW620) was positive for CNN3. CNN3 expression was fairly consistent with the metastatic phenotype in colon cancer because it was absent in one other colon cell line from a primary site and expressed in all others. We selected SW620 for subsequent functional analyses. CNN3-silenced SW620 cells showed a reduction in collagen invasion and loss of mesenchymal markers. CNN3 silencing caused an increase in the SW620 colon cancer cell sensitivity to 5-fluorouracil. Phospho-kinase array-based proteomics analysis showed that CNN3 silencing in SW620 reduced extracellular signal-regulated kinase, β-Catenin, mutant p53, c-Jun, and heat shock protein 60 activities but increased that of checkpoint kinase 2. CNN3 was expressed in 20/57 (35%) colon cancer cases as shown by immunohistochemistry. CNN3 was associated with a decrease in overall survival in colon cancer in silico.CONCLUSIONThese results show the involvement of CNN3 in lymph node metastasis and resistance to chemotherapy in colon cancer and suggest that significant oncogenic pathways are involved in these CNN3-related actions.

Dysfunction of Poly (ADP-Ribose) Glycohydrolase Induces a Synthetic Lethal Effect in Dual Specificity Phosphatase 22-Deficient Lung Cancer Cells.

Poly (ADP-ribose) glycohydrolase (PARG) is the main enzyme responsible for catabolism of poly (ADP-ribose) (PAR), synthesized by PARP. PARG dysfunction sensitizes certain cancer cells to alkylating agents and cisplatin by perturbing the DNA damage response. The gene mutations that sensitize cancer cells to PARG dysfunction-induced death remain to be identified. Here, we performed a comprehensive analysis of synthetic lethal genes using inducible PARG knockdown cells and identified dual specificity phosphatase 22 (DUSP22) as a novel synthetic lethal gene related to PARG dysfunction. DUSP22 is considered a tumor suppressor and its mutation has been frequently reported in lung, colon, and other tumors. In the absence of DNA damage, dual depletion of PARG and DUSP22 in HeLa and lung cancer A549 cells reduced survival compared with single-knockdown counterparts. Dual depletion of PARG and DUSP22 increased the apoptotic sub-G1 fraction and upregulated PUMA in lung cancer A549, PC14, and SBC5 cells, and inhibited the PI3K/AKT/mTOR pathway in A549 cells, suggesting that dual depletion of PARG and DUSP22 induced apoptosis by upregulating PUMA and suppressing the PI3K/AKT/mTOR pathway. Consistently, the growth of tumors derived from double knockdown A549 cells was slower compared with those derived from control siRNA-transfected cells. Taken together, these results indicate that DUSP22 deficiency exerts a synthetic lethal effect when combined with PARG dysfunction, suggesting that DUSP22 dysfunction could be a useful biomarker for cancer therapy using PARG inhibitors. SIGNIFICANCE: This study identified DUSP22 as a novel synthetic lethal gene under the condition of PARG dysfunction and elucidated the mechanism of synthetic lethality in lung cancer cells.

Combination simvastatin and metformin synergistically inhibits endometrial cancer cell growth

ObjectiveRecent data show that simvastatin (SIM) and metformin (MET) have anti-proliferative effects in endometrial cancer cells. The combination (MET+SIM) inhibits tumor growth and metastasis in prostate cancer cells which possess similar molecular alterations to many early endometrial cancers. We tested the hypothesis that the anti-proliferative effects of MET+SIM in endometrial cancer cells would be greater than the effects of each agent alone. MethodsRL95-2, HEC1B, and Ishikawa endometrial cancer cell lines were treated with MET and/or SIM. Growth inhibition was measured by MTS cell proliferation assays. Apoptosis was evaluated by caspase-3, Annexin V, and TUNEL assays and by apoptosis markers (BAX, Bcl-2, Bim) using western blot. Bim was silenced using Bim siRNA to confirm this apoptotic pathway. Treatment effects on the mTOR pathway were investigated by western blot using antibodies to phosphorylated (phospho)-AMPK and phospho-S6. ResultsMET+SIM synergistically inhibited growth in all three cell lines. The combination induced apoptosis as measured by TUNEL, Annexin V, and caspase-3 assays. Bim siRNA transfection abrogated this effect—silencing Bim in MET+SIM-treated RL95-2 cells rescued cell viability in MTS assays and reduced caspase-3 activity compared with control siRNA-transfected cells. Combination treatment upregulated phosphorylated AMPK and downregulated downstream phosphorylated S6, suggesting mTOR inhibition as a mechanism for these anti-proliferative effects. ConclusionsMET+SIM treatment synergistically inhibits endometrial cancer cell viability. This may be mediated by apoptosis and mTOR pathway inhibition. Our results provide preclinical evidence that the combination of these well-tolerated drugs may warrant further clinical investigation for endometrial cancer treatment.

The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity.

How intracellular organelles acquire their characteristic sizes is a fundamental question in cell biology. Given stereotypical changes in nuclear size in cancer, it is important to understand the mechanisms that control nuclear size in human cells. Using a high-throughput imaging RNAi screen, we identify and mechanistically characterize ELYS, a nucleoporin required for post-mitotic nuclear pore complex (NPC) assembly, as a determinant of nuclear size in mammalian cells. ELYS knockdown results in small nuclei, reduced nuclear lamin B2 localization, lower NPC density, and decreased nuclear import. Increasing nuclear import by importin α overexpression rescues nuclear size and lamin B2 import, while inhibiting importin α/β-mediated nuclear import decreases nuclear size. Conversely, ELYS overexpression increases nuclear size, enriches nuclear lamin B2 at the nuclear periphery, and elevates NPC density and nuclear import. Consistent with these observations, knockdown or inhibition of exportin 1 increases nuclear size. Thus, we identify ELYS as a novel positive effector of mammalian nuclear size and propose that nuclear size is sensitive to NPC density and nuclear import capacity.

Protocadherin B9 promotes resistance to bicalutamide and is associated with the survival of prostate cancer patients.

Background Prostate cancer (PCa) is a common malignancy worldwide and is the second leading cause of cancer death in men. The standard therapy for advanced PCa is androgen deprivation therapy (ADT). Although ADT, including bicalutamide treatment, is initially effective, resistance to bicalutamide frequently occurs and leads to the development of castration-resistant PCa. Thus, clarifying the mechanisms of bicalutamide resistance is urgently needed. We designed this study to assess the expression and function of PCDHB9, which encodes the protocadherin B9 protein. Methods The expression of PCDHB9 was determined using immunohistochemistry and a qRT-PCR. The effects of the overexpression or knockdown of PCDHB9 on cell growth, migration, adhesion were evaluated. To evaluate the PCDHB9-mediated effects in PCa, we performed a gene expression analysis using DU145 transfected with PCDHB9. We examined the effects of PCDHB9 inhibition on bicalutamide resistance. Results The qRT-PCR revealed that the expression of PCDHB9 was much higher in PCa than that in non-neoplastic prostate tissues. In 152 clinically localized PCa cases immunohistochemistry showed that 59% of PCa cases were positive for protocadherin B9. A Kaplan-Meier analysis showed that the high expression of protocadherin B9 was associated with PSA recurrence after radical prostatectomy. A functional analysis showed that PCDHB9 modulated cell migration and adhesion. We also found that PCDHB9 induced the expression of ITGB6 based on a gene expression analysis. The effect of PCDHB9 inhibition on bicalutamide sensitivity was examined using MTT assays. The IC50 value of PCDHB9 siRNA-transfected PCa cells was significantly lower than that of negative control siRNA-transfected cells. Furthermore, immunohistochemical staining of protocadherin B9 in 74 PCa patients who were treated with androgen depletion therapy, including bicalutamide treatment, demonstrated that the high expression of protocadherin B9 was significantly associated with poor overall survival. Conclusions PCDHB9 plays an important role in the progression of PCa and bicalutamide resistance. Collectively, our results suggest that PCDHB9 targeted therapy may be more effective than bicalutamide alone.

A High-Fat/High-Sucrose Diet Induces WNT4 Expression in Mouse Pancreatic β-cells.

Mice were fed either standard mouse chow or a HF/HS diet from 8 weeks of age. At 20 weeks of age, intraperitoneal glucose tolerance tests were performed in both groups of mice, followed by euthanasia and isolation of pancreatic islets. WNT-related gene expression in islets and MIN6 cells was measured by quantitative real-time RT-PCR. To explore the direct effects of WNT signals on pancreatic β-cells, MIN6 cells were exposed to recombinant mouse WNT4 protein (rmWNT4) for 48 h, and glucose-induced insulin secretion was measured. Furthermore, Wnt4 siRNAs were transfected into MIN6 cells, and cell viability and insulin secretion were measured in control and Wnt4 siRNA-transfected MIN6 cells. Mice fed the HF/HS diet were heavier and their plasma glucose and insulin levels were higher compared with mice fed standard chow. Wnt4, Wnt5b, Ror1, and Ror2 expression was upregulated, while Fzd4, Fzd5, Fzd6, Lrp5, and Lrp6 expression was downregulated in the islets of mice fed the HF/HS diet. Wnt4 was the most abundantly expressed WNT ligand in β-cells, and its expression was increased by the HF/HS diet. Although exposure to recombinant mouse WNT4 protein for 48 h did not alter glucose-induced insulin secretion, it was significantly reduced by knockdown of Wnt4 in MIN6 cells. We demonstrated that the HF/HS diet-induced increase of WNT4 signaling in β-cells is involved in augmentation of glucose-induced insulin secretion and impaired β-cell proliferation. These results strongly indicate an essential role of WNT4 in the regulation of β-cell functions in mouse pancreatic islets.