Establishment and characterization of NCC-ASPS2-C1: a novel patient-derived cell line of alveolar soft part sarcoma.

Alveolar soft part sarcoma (ASPS) is a rare mesenchymal tumor characterized by rearrangement of the ASPSCR1 and TFE3 genes and a histologically distinctive pseudoalveolar pattern. ASPS progresses slowly, but is prone to late metastasis. As ASPS is refractory to conventional chemotherapy, the only curative treatment is complete surgical resection. The prognosis of advanced and metastatic cases is poor, highlighting the need for preclinical research to develop appropriate treatment options. However, ASPS is extremely rare, accounting for < 1% of all soft tissue sarcomas, and only one patient-derived ASPS cell line is available from public cell banks worldwide for research. This study reports the establishment of a novel ASPS cell line derived from the primary tumor tissue of an ASPS patient, named NCC-ASPS2-C1. This cell line retains the ASPSCR1-TFE3 fusion gene, which is characteristic of ASPS. The characterization of this cell line revealed stable growth, spheroid formation, and invasive properties. By screening a drug library using NCC-ASPS2-C1, we identified several drugs that inhibited the proliferation of ASPS cells. In conclusion, the establishment of NCC-ASPS2-C1 provides a valuable resource for advancing ASPS research and developing novel treatments for this challenging disease.

Exosomes secreted by ST3GAL5high cancer cells promote peritoneal dissemination by establishing a premetastatic microenvironment.

Peritoneal dissemination of cancer affects patient survival. The behavior of peritoneal mesothelial cells (PMCs) and immune cells influences the establishment of a microenvironment that promotes cancer cell metastasis in the peritoneum. Here, we investigated the roles of lactosylceramide alpha-2,3-sialyltransferase (ST3G5; also known as ST3GAL5 and GM3 synthase) in the exosome-mediated premetastatic niche in peritoneal milky spots (MSs). Exosomes secreted from ST3G5high cancer cells (ST3G5high -cExos) were found to contain high levels of hypoxia-inducible factor 1-alpha (HIF1α) and accumulated in MSs via uptake in macrophages (MΦs) owing to increased expression of sialic acid-binding Ig-like lectin 1 (CD169; also known as SIGLEC1). ST3G5high -cExos induced pro-inflammatory cytokines and glucose metabolic changes in MΦs, and the interaction of these MΦs with PMCs promoted mesothelial-mesenchymal transition (MMT) in PMCs, thereby generating αSMA+ myofibroblasts. ST3G5high -cExos also increased the expression of immune checkpoint molecules and T-cell exhaustion in MSs, which accelerated metastasis to the omentum. These events were prevented following ST3G5 depletion in cancer cells. Mechanistically, ST3G5high -cExos upregulated chemokines, including CC-chemokine ligand 5 (CCL5), in recipient MΦs and dendritic cells (DCs), which induced MMT and immunosuppression via activation of signal transducer and activator of transcription 3 (STAT3). Maraviroc, a C-C chemokine receptor type 5 (CCR5) antagonist, prevented ST3G5high -cExo-mediated MMT, T-cell suppression, and metastasis in MSs. Our results suggest ST3G5 as a suitable therapeutic target for preventing cExo-mediated peritoneal dissemination.

CCDC85A is regulated by miR-224-3p and augments cancer cell resistance to endoplasmic reticulum stress.

MicroRNAs (miRNAs) play pivotal roles in the tumor microenvironment. Here, we analyzed miRNAs in tumor stromal fibroblasts. Expression of miR-224-3p in cancer-associated fibroblasts (CAF) from scirrhous gastric cancer patients was lower than in normal fibroblasts (NF). Introduction of a miR-224-3p mimic attenuated migration and invasion of CAF. Coiled-coil domain containing 85A (CCDC85A), whose function in tumors is not understood, was the target gene of miR-224-3p. Immunohistological analysis revealed that CCDC85A is expressed to varying degrees by cancer cells and CAFs in gastric and pancreatic carcinomas. Downregulation of CCDC85A in cancer cells revealed that these cells are vulnerable to endoplasmic reticulum (ER) stress induced by thapsigargin or tunicamycin, which were ameliorated after addback of CCDC85A. Injection of NF-derived exosomes containing miR-224-3p into the xenograft tumor increased tumor shrinkage by cisplatin treatment. Mechanistically, CCDC85A associated with the molecular chaperone GRP78 and GRP94, thereby inhibiting association of these negative regulators of the unfolded protein response (UPR), leading to sustained activation of PERK and downstream eIF2〈 and ATF4 upon ER stress. These data suggest a novel miR-224-3p-mediated function for CCDC85A: protection from ER stress and cisplatin resistance.

TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells.

Long noncoding RNAs (lncRNAs) play pivotal roles in tumor development. To identify dysregulated lncRNAs in gastric cancer (GC), we analyzed genome-wide trimethylation of histone H3 lysine 4 (H3K4me3) to screen for transcriptionally active lncRNA genes in the non-tumorous gastric mucosa of patients with GC and healthy individuals. We found that H3K4me3 at TM4SF1-AS1 was specifically upregulated in GC patients and that the expression of TM4SF1-AS1 was significantly elevated in primary and cultured GC cells. TM4SF1-AS1 contributes to GC cell growth in vitro and in vivo, and its oncogenic function is mediated, at least in part, through interactions with purine-rich element-binding protein α (Pur-α) and Y-box binding protein 1 (YB-1). TM4SF1-AS1 also activates interferon signaling in GC cells, which is dependent on Pur-α and RIG-I. Chromatin isolation by RNA purification (ChIRP)-mass spectrometry demonstrated that TM4SF1-AS1 was associated with several stress granule (SG)-related proteins, including G3BP2, RACK1, and DDX3. Notably, TM4SF1-AS1 promoted SG formation and inhibited apoptosis in GC cells by sequestering RACK1, an activator of the stress-responsive MAPK pathway, within SGs. TM4SF1-AS1-induced SG formation and apoptosis inhibition are dependent on Pur-α and YB-1. These findings suggested that TM4SF1-AS1 contributes to tumorigenesis by enhancing SG-mediated stress adaptation.

Involvement of clusterin expression in the refractory response of pancreatic cancer cells to a MEK inhibitor.

Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway is essential for tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). To date, however, almost all clinical trials of inhibitor targeting this pathway have failed to improve the outcome of patients with PDAC. We found that implanted MIA Paca2, a human PDAC cell line sensitive to a MAPK inhibitor, PD0325901, became refractory within a week after treatment. By comparing the expression profiles of MIA Paca2 before and after acquisition of the refractoriness to PD0325901, we identified clusterin (CLU) as a candidate gene involved. CLU was shown to be induced immediately after treatment with PD0325901 or expressed primarily in more than half of PDAC cell lines, enhancing cell viability by escaping from apoptosis. A combination of PD0325901 and CLU downregulation was found to synergistically or additively reduce the proliferation of PDAC cells. In surgically resected PDAC tissues, overexpression of CLU in cancer cells was observed immunohistochemically in approximately half of the cases studied. Collectively, our findings highlight the mechanisms responsible for the rapid refractory response to MEK inhibitor in PDAC cells, suggesting a novel therapeutic strategy that could be applicable to patients with PDAC using inhibitor targeting the MAPK signaling pathway and CLU.

Cancer-associated fibroblast-dependent and -independent invasion of gastric cancer cells.

PURPOSE: Cancer cells are known to exhibit a cancer-associated fibroblast (CAF)-dependent invasive mode in the presence of CAFs. The purpose of this study was to investigate whether intrinsic factors of gastric cancer cells influence the CAF-dependent invasive mode of cancer cells. METHODS: We observed dynamic movement of CAFs, and cancer cells, by time-lapse imaging of 2-D and 3-D collagen invasion models, and evaluated invasion modes of gastric cancer cell lines (MKN-7, MKN-45, and HSC44PE). We further examined whether modification of invasive capacity of CAFs can alter invasive mode of MKN-7, and HSC44PE cells. RESULTS: When MKN-7 and MKN-45 cells were co-cultured with CAFs, CAFs first invade collagen matrix followed by cancer cells (CAF-dependent invasion), whereas HSC44PE cells invaded collagen matrix independent of CAFs' invasion. Overexpression or suppression of podoplanin in CAFs, respectively, increased or decreased the invasive capacity of CAFs, and significantly increased or decreased the number of invading MKN-7 cells, respectively. CAFs overexpressing a podoplanin mutant, lacking the cytoplasmic domain, had significantly reduced invasive capacity, compared to CAFs overexpressing wild-type podoplanin, and it also reduced the number of invading MKN-7 cells significantly. When HSC44PE cells, and CAFs were co-cultured, changes in the podoplanin expression in CAFs similarly altered the invasive capacity of CAFs, but it did not affect the number of invading HSC44PE cells. CONCLUSIONS: These results indicate that in presence of CAFs, gastric cancer cells exhibit both CAF-dependent and -independent modes of invasion, the determinants of which may depend on the intrinsic properties of the gastric cancer cells.

Tissue factor-induced fibrinogenesis mediates cancer cell clustering and multiclonal peritoneal metastasis.

Peritoneal metastasis is one of the most frequent causes of death in several types of advanced cancers; however, the underlying molecular mechanisms remain largely unknown. In this study, we exploited multicolor fluorescent lineage tracking to investigate the clonality of peritoneal metastasis in mouse xenograft models. When peritoneal metastasis was induced by intraperitoneal or orthotopic injection of multicolored cancer cells, each peritoneally metastasized tumor displayed multicolor fluorescence regardless of metastasis sites, indicating that it consists of multiclonal cancer cell populations. Multicolored cancer cell clusters form within the peritoneal cavity and collectively attach to the peritoneum. In vitro, peritoneal lavage fluid or cleared ascitic fluid derived from cancer patients induces cancer cell clustering, which is inhibited by anticoagulants. Cancer cell clusters formed in vitro and in vivo are associated with fibrin formation. Furthermore, tissue factor knockout in cancer cells abrogates cell clustering, peritoneal attachment, and peritoneal metastasis. Thus, we propose that cancer cells activate the coagulation cascade via tissue factor to form fibrin-mediated cell clusters and promote peritoneal attachment; these factors lead to the development of multiclonal peritoneal metastasis and may be therapeutic targets.

Enhanced phosphorylation of c-Jun by cisplatin treatment as a potential predictive biomarker for cisplatin response in combination with patient-derived tumor organoids.

Despite recent advances in sequencing technology and large-scale drug screenings employing hundreds of cell lines, the predictive accuracy of mutation-based biomarkers is still insufficient as a guide for cancer therapy. Therefore, novel types of diagnostic methods using alternative biomarkers would be highly desirable. We have hypothesized that sensitivity-specific changes in the phosphorylation of signaling molecules could be useful in this respect. Here, with the aim of developing a method for predicting the response of cancers to cisplatin using a combination of specific biomarker(s) and patient-derived tumor organoids (PDOs), we found that cisplatin-sensitive cell lines or PDOs showed enhanced phosphorylation of c-Jun (p-c-Jun) within 24 h after cisplatin treatment. We also compared the responses of 6 PDOs to cisplatin with the therapeutic effect of neoadjuvant chemotherapy (docetaxel/cisplatin/5-fluorouracil) in 6 matched patients. Mechanistically, the c-Jun induction was partly related to TNF signaling induced by cisplatin. Our data suggest that enhanced phosphorylation of c-Jun in response to cisplatin treatment could be a predictive biomarker for the efficacy of cisplatin in selected cancer patients.

Elevated METTL9 is associated with peritoneal dissemination in human scirrhous gastric cancers.

Methylation, the most common chemical modification of cellular components such as DNA, RNA, and proteins, impacts biological processes including transcription, RNA processing, and protein dynamics. Although abnormal expression of methyltransferase can lead to various diseases including cancers, little is known about the relationship between methyltransferase and cancers. Here we aimed to understand the role of methyltransferase in cancer metastasis. We found that elevated methyltransferase-like 9 (METTL9) is closely associated with the acquisition of metastatic activity in human scirrhous gastric cancers. The stable knockdown of METTL9 via an shRNA vector technique in our original metastatic cells from scirrhous gastric cancer patients significantly inhibited migration and invasion. In metastatic cells, METTL9 protein is predominantly localized in mitochondria, and the METTL9 knockdown significantly reduced mitochondrial Complex I activity. METTL9 can be a candidate of molecular targets to inhibit peritoneal dissemination of scirrhous gastric cancers. This report is the first to describe the relationship between METTL9 and cancer metastasis.

A Comparative Study of Patient-Derived Tumor Models of Pancreatic Ductal Adenocarcinoma Involving Orthotopic Implantation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is associated with very poor prognoses. Therefore, new therapies and preclinical models are urgently needed. In the present study, we sought to develop more realistic experimental models for use in PDA research. METHODS: We developed patient-derived xenografts (PDXs), established PDX-derived cell lines (PDCLs), and generated cell line-derived xenografts (CDXs), which we integrated to create 13 matched "trios" - i.e., patient-derived tumor models of PDA. We then compared and contrasted histological and molecular alterations between these three model systems. RESULTS: Orthotopic implantation (OI) of the PDCLs resulted in tumorigenesis and metastases to the liver and peritoneum. Morphological comparisons of OI-CDXs and OI-PDXs with passaged tumors revealed that the histopathological features of the original tumor were maintained in both models. Molecular alterations in PDX tumors (including those to KRAS, TP53, SMAD4, and CDKN2A) were similar to those in the respective PDCLs and CDX tumors. When gene expression levels in the PDCLs, ectopic tumors, and OI tumors were compared, the distant metastasis-promoting gene CXCR4 was specifically upregulated in OI tumors, whose immunohistochemical profiles suggested epithelial-mesenchymal transition and adeno-squamous trans-differentiation. CONCLUSION: These patient-derived tumor models provide useful tools for monitoring responses to antineoplastic agents and for studying PDA biology.

Cancer-associated fibroblasts educate normal fibroblasts to facilitate cancer cell spreading and T-cell suppression.

In some tumors, a small number of cancer cells are scattered in a large fibrotic stroma. Here, we demonstrate a novel mechanism for expansion of pro-tumor fibroblasts via cancer-associated fibroblast (CAF)-mediated education of normal fibroblasts (NFs). When NFs were incubated with conditioned medium from CAFs, the resulting CAF-educated fibroblasts (CEFs) generated reactive oxygen species, which induced NF-κB-mediated expression of inflammatory cytokines and the extracellular matrix protein asporin (ASPN), while expression of a common CAF marker gene, α-SMA, was not increased. ASPN further increased CEF expression of downstream molecules, including indoleamine 2,3-dioxygenase 1 (IDO-1), kynureninase (KYNU), and pregnancy-associated plasma protein-A (PAPP-A). These CEFs induce cytocidal effects against CD8+ T cells and IGF-I activation in cancer cells. CEFs were generated without cancer cells by the direct mixture of NFs and CAFs in mouse xenografts, and once CEFs were generated, they sequentially educated NFs, leading to continuous generation of CEFs. In diffuse-type gastric cancers, ASPNhigh /IDO-1high /KYNUhigh /α-SMA- CEFs were located at the distal invading front. These CEFs expanded in the fibrotic stroma and caused dissemination of cancer cells. ASPN may therefore be a key molecule in facilitating tumor spreading and T-cell suppression.

Integrin α5 mediates cancer cell-fibroblast adhesion and peritoneal dissemination of diffuse-type gastric carcinoma.

Diffuse-type gastric carcinoma (DGC) has a poor prognosis due to its rapid diffusive infiltration and frequent peritoneal dissemination. DGC is associated with massive fibrosis caused by aberrant proliferation of cancer-associated fibroblasts (CAFs). Previously, we reported that direct heterocellular interaction between cancer cells and CAFs is important for the peritoneal dissemination of DGC. In this study, we aimed to identify and target the molecules that mediate such heterocellular interactions. Monoclonal antibodies (mAbs) against intact DGC cells were generated and subjected to high-throughput screening to obtain several mAbs that inhibit the adhesion of DGC cells to CAFs. Immunoprecipitation and mass spectrometry revealed that all mAbs recognized integrin α5 complexed with integrin ß1. Blocking integrin α5 in DGC cells or fibronectin, a ligand of integrin α5ß1, deposited on CAFs abrogated the heterocellular interaction. Administration of mAbs or knockout of integrin α5 in DGC cells suppressed their invasion led by CAFs in vitro and peritoneal dissemination in a mouse xenograft model. Altogether, these findings demonstrate that integrin α5 mediates the heterotypic cancer cell-fibroblast interaction during peritoneal dissemination of DGC and may thus be a therapeutic target.

SHP2 as a Potential Therapeutic Target in Diffuse-Type Gastric Carcinoma Addicted to Receptor Tyrosine Kinase Signaling.

Diffuse-type gastric carcinoma (DGC) exhibits aggressive progression associated with rapid infiltrative growth, massive fibrosis, and peritoneal dissemination. Gene amplification of Met and fibroblast growth factor receptor 2 (FGFR2) receptor tyrosine kinases (RTKs) has been observed in DGC. However, the signaling pathways that promote DGC progression downstream of these RTKs remain to be fully elucidated. We previously identified an oncogenic tyrosine phosphatase, SHP2, using phospho-proteomic analysis of DGC cells with Met gene amplification. In this study, we characterized SHP2 in the progression of DGC and assessed the therapeutic potential of targeting SHP2. Although SHP2 was expressed in all gastric carcinoma cell lines examined, its tyrosine phosphorylation preferentially occurred in several DGC cell lines with Met or FGFR2 gene amplification. Met or FGFR inhibitor treatment or knockdown markedly reduced SHP2 tyrosine phosphorylation. Knockdown or pharmacological inhibition of SHP2 selectively suppressed the growth of DGC cells addicted to Met or FGFR2, even when they acquired resistance to Met inhibitors. Moreover, SHP2 knockdown or pharmacological inhibition blocked the migration and invasion of Met-addicted DGC cells in vitro and their peritoneal dissemination in a mouse xenograft model. These results indicate that SHP2 is a critical regulator of the malignant progression of RTK-addicted DGC and may be a therapeutic target.

A newly established monoclonal antibody against ERCC1 detects major isoforms of ERCC1 in gastric cancer.

Identifying patients resistant to cisplatin treatment is expected to improve cisplatin-based chemotherapy for various types of cancers. Excision repair cross-complementing group 1 (ERCC1) is involved in several repair processes of cisplatin-induced DNA crosslinks. ERCC1 overexpression is reported as a candidate prognostic factor and considered to cause cisplatin resistance in major solid cancers. However, anti-ERCC1 antibodies capable of evaluating expression levels of ERCC1 in clinical specimens were not fully optimized. A mouse monoclonal antibody against human ERCC1 was generated in this study. The developed antibody 9D11 specifically detected isoforms of 201, 202, 203 but not 204, which lacks the exon 3 coding region. To evaluate the diagnostic usefulness of this antibody, we have focused on gastric cancer because it is one of the major cancers in Japan. When ERCC1 expression was analyzed in seventeen kinds of human gastric cancer cell lines, all the cell lines were found to express either 201, 202, and/or 203 as major isoforms of ERCC1, but not 204 by Western blotting analysis. Immunohistochemical staining showed that ERCC1 protein was exclusively detected in nuclei of the cells and a moderate level of constant positivity was observed in nuclei of vascular endothelial cells. It showed a clear staining pattern in clinical specimens of gastric cancers. Antibody 9D11 may thus be useful for estimating expression levels of ERCC1 in clinical specimens.

Drug-exposed cancer-associated fibroblasts facilitate gastric cancer cell progression following chemotherapy.

BACKGROUND: Cancer progression following chemotherapy is a significant barrier to effective cancer treatment. We aimed to evaluate the role of drug-exposed cancer-associated fibroblasts (CAFs) in the growth and progression of drug-exposed gastric cancer (GC) cells and to explore the underlying molecular mechanism. METHODS: The human GC cell line 44As3 and CAFs were treated with 5-fluorouracil and oxaliplatin (5FU + OX). 5FU + OX-pretreated 44As3 cells were then cultured in a conditioned medium (CM) from 5FU + OX-pretreated CAFs, and the growth and migration/invasion ability of the cells were evaluated. We also compared the clinicopathological characteristics of the GC patients treated with S1 + OX in accordance with the properties of their resected specimens, focusing on the number of CAFs. Changes in gene expression in CAFs and 44As3 cells were comprehensively analyzed using RNA-seq analysis. RESULTS: The CM from 5FU + OX-pretreated CAFs promoted the migration and invasion of 5FU + OX-pretreated 44As3 cells. Although the number of cases was relatively small (n = 21), the frequency of positive cases of lymphovascular invasion and the recurrence rate were significantly higher in those with more residual CAF. RNA-seq analysis revealed 5FU + OX-pretreated CAF-derived glycoprotein 130 (gp130) as a candidate factor contributing to the increased migration of 5FU + OX-pretreated 44As3 cells. Administration of the gp130 inhibitor SC144 prevented the increased migration ability of 5FU + OX-pretreated 44As3 cells owing to drug-treated CAFs. CONCLUSIONS: Our findings provide evidence regarding the interactions between GC cells and CAFs in the tumor microenvironment following chemotherapy, suggesting that ligands for gp130 may be novel therapeutic targets for suppressing or preventing metastasis in GC.

PLEKHA5 regulates the survival and peritoneal dissemination of diffuse-type gastric carcinoma cells with Met gene amplification.

Met gene amplification has been found in a subset of malignant carcinomas, including diffuse-type gastric carcinoma (DGC), which has a poor prognosis owing to rapid infiltrative invasion and frequent peritoneal dissemination. Met is considered a promising therapeutic target for DGC. However, DGC cells with Met gene amplification eventually acquire resistance to Met inhibitors. Therefore, identification of alternate targets that mediate Met signaling and confer malignant phenotypes is critical. In this study, we conducted a phosphoproteomic analysis of DGC cells possessing Met gene amplification and identified Pleckstrin Homology Domain Containing A5 (PLEKHA5) as a protein that is tyrosine-phosphorylated downstream of Met. Knockdown of PLEKHA5 selectively suppressed the growth of DGC cells with Met gene amplification by inducing apoptosis, even though they had acquired resistance to Met inhibitors. Moreover, PLEKHA5 silencing abrogated the malignant phenotypes of Met-addicted DGC cells, including peritoneal dissemination in vivo. Mechanistically, PLEKHA5 knockdown dysregulates glycolytic metabolism, leading to activation of the JNK pathway that promotes apoptosis. These results indicate that PLEKHA5 is a novel downstream effector of amplified Met and is required for the malignant progression of Met-addicted DGC.

Polyvinyl alcohol scaffolds and supplementation support 3D and sphere culturing of human cancer cell lines by reducing apoptosis and promoting cellular proliferation.

Three-dimensional (3D) culturing mimics the heterogeneous cellular conditions of the in vivo tumor microenvironment compared to 2D monolayer-cultured cells and 3D cultures of established cancer cell lines (sphere culture) or patient-derived cancer cells (organoid culture) are frequently used for cancer research or drug screening and evaluation. To establish more cost and time-efficient 3D culture methods for cancer cell lines, we supplemented sphere culture medium with polyvinyl alcohol (PVA) and found that 3D sphere cultures of breast and pancreatic cancer cell lines were significantly increased. Mechanistically, we found that PVA prevented cell death and promoted cellular proliferation while maintaining levels of stemness-related gene expression. Furthermore, we showed that polyvinyl formal resin (PVF) 3D scaffolds made by cross-linked PVA can function in serum-free, long-term 3D cultures to support maintenance of sphere- or tumor-like cell masses for diverse cancer cell types. Taken together, we demonstrate the effectiveness of PVA and PVF in human cancer cell line culture protocols.

Expression of asporin reprograms cancer cells to acquire resistance to oxidative stress.

Asporin (ASPN), a small leucine-rich proteoglycan expressed predominantly by cancer associated fibroblasts (CAFs), plays a pivotal role in tumor progression. ASPN is also expressed by some cancer cells, but its biological significance is unclear. Here, we investigated the effects of ASPN expression in gastric cancer cells. Overexpression of ASPN in 2 gastric cancer cell lines, HSC-43 and 44As3, led to increased migration and invasion capacity, accompanied by induction of CD44 expression and activation of Rac1 and MMP9. ASPN expression increased resistance of HSC-43 cells to oxidative stress by reducing the amount of mitochondrial reactive oxygen species. ASPN induced expression of the transcription factor HIF1α and upregulated lactate dehydrogenase A (LDHA) and PDH-E1α, suggesting that ASPN reprograms HSC-43 cells to undergo anaerobic glycolysis and suppresses ROS generation in mitochondria, which has been observed in another cell line HSC-44PE. By contrast, 44As3 cells expressed high levels of HIF1α in response to oxidant stress and escaped apoptosis regardless of ASPN expression. Examination of xenografts in the gastric wall of ASPN-/- mice revealed that growth of HSC-43 tumors with increased micro blood vessel density was significantly accelerated by ASPN; however, ASPN increased the invasion depth of both HSC-43 and 44As3 tumors. These results suggest that ASPN has 2 distinct effects on cancer cells: HIF1α-mediated resistance to oxidative stress via reprogramming of glucose metabolism, and activation of CD44-Rac1 and MMP9 to promote cell migration and invasion. Therefore, ASPN may be a new therapeutic target in tumor fibroblasts and cancer cells in some gastric carcinomas.

Sixteen Different Types of Lipid-Conjugated siRNAs Containing Saturated and Unsaturated Fatty Acids and Exhibiting Enhanced RNAi Potency.

SiRNAs are strong gene-silencing agents that function in a target sequence-specific manner. Although siRNAs might one day be used in therapy for intractable diseases such as cancers, a number of problems with siRNAs must first be overcome. In this study, we developed 16 different types of lipid-conjugated siRNAs (lipid-siRNAs) that could effectively inhibit the expression of target genes. We determined the hybridization properties, cellular uptake efficacies, and RNAi potencies of the resulting lipid-siRNAs. The lipid-siRNAs exhibited a mild interaction with Lipofectamine RNAiMAX (LFRNAi) as a transfection reagent, and a high membrane permeability was observed in all lipid-siRNAs-LFRNAi complexes; the conjugate siRNAs composed of 16-18 carbon chains as fatty acids showed an especially good cellular uptake efficacy. The in vitro RNAi effect of lipid-siRNAs targeted to a ß-catenin gene exhibited a strong RNAi potency compared with those of unmodified siRNAs. In particular, the conjugate siRNAs composed of 16-18 carbon chains as fatty acids showed excellent RNAi potencies with prolonged effectivities. Interestingly, the RNAi potencies of conjugate siRNAs containing 18 carbon chains with a trans-form (elaidic acid and trans-vaccenic acid) were inferior to those of the carbon chains with a cis-form (oleic acid and cis-vaccenic acid). These lipid-siRNAs can solve the many problems hindering the clinical application of siRNAs.

Long Noncoding RNA HOTAIR Promotes Epithelial-Mesenchymal Transition and Is a Suitable Target to Inhibit Peritoneal Dissemination in Human Scirrhous Gastric Cancers.

OBJECTIVES: Scirrhous gastric cancer, which accounts for approximately 10% of all gastric cancers, often disseminates to the peritoneum, leading to intractable cases with poor prognosis. There is an urgent need for new treatment approaches for this difficult cancer. METHODS: We previously established an original cell line, HSC-60, from a scirrhous gastric cancer patient and isolated a peritoneal-metastatic cell line, 60As6, in nude mice following orthotopic inoculations. In the present study, we focused on the expression of long noncoding ribonucleic acid (RNA) (lncRNA) in the cell lines and investigated the mechanism on peritoneal dissemination. RESULTS: We demonstrated that an lncRNA, HOX transcript antisense RNA (HOTAIR), is expressed significantly more highly in 60As6 than HSC-60 cells. Then, using both HOTAIR knockdown and overexpression experiments, we showed that high-level expression of HOTAIR promotes epithelial-mesenchymal transition (EMT) in 60As6 cells. By luciferase assay, we found that HOTAIR directly targets and binds to miR-217, and that miR-217 directly binds to Zinc finger E-box-binding homeobox 1 (ZEB1). The knockdown of HOTAIR in 60As6 cells significantly reduced the invasion activity and peritoneal dissemination - and significantly prolonged the survival - in the orthotopic tumor mouse model. CONCLUSION: An EMT-associated pathway (the HOTAIR-miR-217-ZEB1 axis) appears to inhibit peritoneal dissemination and could lead to a novel therapeutic strategy against scirrhous gastric cancer in humans.