Tubulointerstitial nephritis antigen-like 1 from cancer-associated fibroblasts contribute to the progression of diffuse-type gastric cancers through the interaction with integrin ß1.

BACKGROUND: Tumor cells of diffuse-type gastric cancer (DGC) are discohesive and infiltrate into the stroma as single cells or small subgroups, so the stroma significantly impacts DGC progression. Cancer-associated fibroblasts (CAFs) are major components of the tumor stroma. Here, we identified CAF-specific secreted molecules and investigated the mechanism underlying CAF-induced DGC progression. METHODS: We conducted transcriptome analysis for paired normal fibroblast (NF)-CAF isolated from DGC patient tissues and proteomics for conditioned media (CM) of fibroblasts. The effects of fibroblasts on cancer cells were examined by transwell migration and soft agar assays, western blotting, and in vivo. We confirmed the effect of blocking tubulointerstitial nephritis antigen-like 1 (TINAGL1) in CAFs using siRNA or shRNA. We evaluated the expression of TINAGL1 protein in frozen tissues of DGC and paired normal stomach and mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue using RNA in-situ hybridization (RNA-ISH). RESULTS: CAFs more highly expressed TINAGL1 than NFs. The co-culture of CAFs increased migration and tumorigenesis of DGC. Moreover, CAFs enhanced the phosphorylation of focal adhesion kinase (FAK) and mesenchymal marker expression in DGC cells. In an animal study, DGC tumors co-injected with CAFs showed aggressive phenotypes, including lymph node metastasis. However, increased phosphorylation of FAK and migration were reduced by blocking TINAGL1 in CAFs. In the tissues of DGC patients, TINAGL1 was higher in cancer than paired normal tissues and detected with collagen type I alpha 1 chain (COL1A1) in the same spot. Furthermore, high TINAGL1 expression was significantly correlated with poor prognosis in several public databases and our patient cohort diagnosed with DGC. CONCLUSIONS: These results indicate that TINAGL1 secreted by CAFs induces phosphorylation of FAK in DGC cells and promotes tumor progression. Thus, targeting TINAGL1 in CAFs can be a novel therapeutic strategy for DGC.

Characterization of gastric cancer-stimulated signaling pathways and function of CTGF in cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME) that play an important role in cancer progression. Although the mechanism by which CAFs promote tumorigenesis has been well investigated, the underlying mechanism of CAFs activation by neighboring cancer cells remains elusive. In this study, we aim to investigate the signaling pathways involved in CAFs activation by gastric cancer cells (GC) and to provide insights into the therapeutic targeting of CAFs for overcoming GC. METHODS: Alteration of receptor tyrosine kinase (RTK) activity in CAFs was analyzed using phospho-RTK array. The expression of CAFs effector genes was determined by RT-qPCR or ELISA. The migration and invasion of GC cells co-cultured with CAFs were examined by transwell migration/invasion assay. RESULTS: We found that conditioned media (CM) from GC cells could activate multiple receptor tyrosine kinase signaling pathways, including ERK, AKT, and STAT3. Phospho-RTK array analysis showed that CM from GC cells activated PDGFR tyrosine phosphorylation, but only AKT activation was PDGFR-dependent. Furthermore, we found that connective tissue growth factor (CTGF), a member of the CCN family, was the most pronouncedly induced CAFs effector gene by GC cells. Knockdown of CTGF impaired the ability of CAFs to promote GC cell migration and invasion. Although the PDGFR-AKT pathway was pronouncedly activated in CAFs stimulated by GC cells, its pharmacological inhibition affected neither CTGF induction nor CAFs-induced GC cell migration. Unexpectedly, the knockdown of SRC and SRC-family kinase inhibitors, dasatinib and saracatinib, significantly impaired CTGF induction in activated CAFs and the migration of GC cells co-cultured with CAFs. SRC inhibitors restored the reduced expression of epithelial markers, E-cadherin and Zonula Occludens-1 (ZO-1), in GC cells co-cultured with CAFs, as well as CAFs-induced aggregate formation in a 3D tumor spheroid model. CONCLUSIONS: This study provides a characterization of the signaling pathways and effector genes involved in CAFs activation, and strategies that could effectively inhibit it in the context of GC. Video Abstract.

Targeting GAS6/AXL signaling improves the response to immunotherapy by restoring the anti-immunogenic tumor microenvironment in gastric cancer.

AIMS: Immunotherapy has shown remarkable effects on several malignancies; however, its impact on gastric cancers has been limited. Therefore, a novel strategy to overcome resistance to immunotherapy is required. In this study, we compared the gene expression profiles of two murine GC cell lines that exhibited different effects on tumor immunity. The functions of specific genes related to negative tumor immunity and the impact of a specific inhibitor were evaluated in syngeneic GC mouse models. MATERIALS AND METHODS: RT-PCR and Western blotting validated Gas6 and AXL expression in murine cell lines. RT-PCR compared YTN16 and YTN3 GC cell's impact on T cell activation. AXL, the receptor for GAS6 in YTN16, was validated by western blotting. Gas6 was inhibited in YTN16 cells using shRNA, and then the gene expression pattern, effects to T cell activation, and tumor growth were assessed. YTN16 cells were injected into mice and treated with CCB-3233, anti-PD-1 antibody, or both. Immunohistochemistry and flow cytometry evaluated tumor-infiltrating immune cells. KEY FINDINGS: YTN16 cells expressed more Gas6 and had reduced T cell activation compared to YTN3 cells. AXL activation was higher in YTN16. CCB-3233 reduced AXL phosphorylation. Knocking down Gas6 in YTN16 reduced immunosuppression-related genes and increased tumor-infiltrating T cells. Combined CCB-3233 and anti-PD-1 treatment reduced tumor growth and increased T-cell infiltration. Human GC data revealed a negative correlation between GAS6 and immune activation-related genes. SIGNIFICANCE: The GAS6/AXL pathway contributes to immunotherapy resistance in GC. Targeting this pathway may be a novel therapeutic strategy.

Molecular and Immune Profiling of Syngeneic Mouse Models Predict Response to Immune Checkpoint Inhibitors in Gastric Cancer.

PURPOSE: Appropriate preclinical mouse models are needed to evaluate the response to immunotherapeutic agents. Immunocompetent mouse models have rarely been reported for gastric cancer. Thus, we investigated immunophenotypes and responses to immune checkpoint inhibitor (ICI) in immunocompetent mouse models using various murine gastric cancer cell lines. MATERIALS AND METHODS: We constructed subcutaneous syngeneic tumors with murine gastric cancer cell lines, YTN3 and YTN16, in C57BL/6J mice. Mice were intraperitoneally treated with IgG isotype control or an anti-programmed death-ligand 1 (PD-L1) neutralizing antibody. We used immunohistochemistry to evaluate the tumor-infiltrating immune cells of formalin-fixed paraffin-embedded mouse tumor tissues. We compared the protein and RNA expression between YTN3 and YTN16 cell lines using a mouse cytokine array and RNA sequencing. RESULTS: The mouse tumors revealed distinct histological and molecular characteristics. YTN16 cells showed upregulation of genes and proteins related to immunosuppression, such as Ccl2 (CCL2) and Csf1 (M-CSF). Macrophages and exhausted T cells were more enriched in YTN16 tumors than in YTN3 tumors. Several YTN3 tumors were completely regressed by the PD-L1 inhibitor, whereas YTN16 tumors were unaffected. Although treatment with a PD-L1 inhibitor increased infiltration of T cells in both the tumors, the proportion of exhausted immune cells did not decrease in the non-responder group. CONCLUSION: We confirmed the histological and molecular features of cancer cells with various responses to ICI. Our models can be used in preclinical research on ICI resistance mechanisms to enhance clinical efficacy.

Secretome analysis reveals reduced expression of COL4A2 in hypoxic cancer-associated fibroblasts with a tumor-promoting function in gastric cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are major components of the tumor microenvironment (TME). Hypoxic TME is known to promote tumor progression. However, how a hypoxic condition regulates CAFs remains elusive. METHODS: To investigate the underlying mechanism involved in the regulation of gastric cancer (GC) progression by hypoxic CAFs, we performed secretome profiling. Normoxic or hypoxic CAFs conditioned media (CM) were filter-concentrated and in-gel trypsin digested. Resulting peptides were analyzed with LC-MS/MS. RESULTS: We observed that CM derived from hypoxic CAFs could promote migration of a panel of GC cell lines (AGS, SNU668, SNU638). Mass spectrometry analysis of hypoxic or normoxic CAFs CM identified 1595 proteins, of which 19 proteins (10 upregulated and 9 downregulated) were differentially expressed in the hypoxic secretome. We focused on COL4A2, whose expression was significantly decreased in hypoxic CAFs in HIF-1α-independent manner. Silencing of COL4A2 expression in normoxic CAFs phenocopied the effect of hypoxic CAFs in promoting GC cell migration. CONCLUSIONS: The reduced expression of COL4A2 in a hypoxic environment might be associated with the tumor-promoting role of hypoxic CAFs in GC.

Curcumin inhibits the cancer­associated fibroblast­derived chemoresistance of gastric cancer through the suppression of the JAK/STAT3 signaling pathway.

The present study aimed to investigate whether the Janus­activated kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is a critical mechanism underlying the cancer­associated fibroblast (CAF)­induced chemoresistance of gastric cancer (GC). In addition, the present study tried to suggest a natural product to compromise the effects of CAF on the chemoresistance of GC. The results of cell proliferation assay revealed that the conditioned medium (CM) collected from CAFs further increased resistance to 5­fluorouracil (5­FU) in GC cell lines. Secretome analysis revealed that the levels of several secreted proteins, including C­C motif chemokine ligand 2, C­X­C motif chemokine ligand 1, interleukin (IL)­6 and IL­8, were increased in the CM from CAFs co­cultured with cancer cells compared to CM from cancer cells. Western blot analysis revealed that CAFs activated the JAK/STAT3 signaling pathway in cancer cells. The experimental models revealed that curcumin abrogated the CAF­mediated activation of the JAK/STAT3 signaling pathway in GC cells. In vivo data revealed the synergistic effects of curcumin with 5­FU treatment in xenograft GC tumors. These data strongly suggest that the suppression of the JAK/STAT3 signaling pathway counteracts the CAF­induced chemoresistance of GC cells. It is suggested that curcumin may be a suitable natural product which may be used to overcome chemoresistance by inhibiting the CAF­induced activation of the JAK/STAT3 signaling pathway in GC.

Spatially Distinct Reprogramming of the Tumor Microenvironment Based On Tumor Invasion in Diffuse-Type Gastric Cancers.

PURPOSE: Histologic features of diffuse-type gastric cancer indicate that the tumor microenvironment (TME) may substantially impact tumor invasiveness. However, cellular components and molecular features associated with cancer invasiveness in the TME of diffuse-type gastric cancers are poorly understood. EXPERIMENTAL DESIGN: We performed single-cell RNA-sequencing (scRNA-seq) using tissue samples from superficial and deep invasive layers of cancerous and paired normal tissues freshly harvested from five patients with diffuse-type gastric cancer. The scRNA-seq results were validated by immunohistochemistry (IHC) and duplex in situ hybridization (ISH) in formalin-fixed paraffin-embedded tissues. RESULTS: Seven major cell types were identified. Fibroblasts, endothelial cells, and myeloid cells were categorized as being enriched in the deep layers. Cell type-specific clustering further revealed that the superficial-to-deep layer transition is associated with enrichment in inflammatory endothelial cells and fibroblasts with upregulated CCL2 transcripts. IHC and duplex ISH revealed the distribution of the major cell types and CCL2-expressing endothelial cells and fibroblasts, indicating tumor invasion. Elevation of CCL2 levels along the superficial-to-deep layer axis revealed the immunosuppressive immune cell subtypes that may contribute to tumor cell aggressiveness in the deep invasive layers of diffuse-type gastric cancer. The analyses of public datasets revealed the high-level coexpression of stromal cell-specific genes and that CCL2 correlated with poor survival outcomes in patients with gastric cancer. CONCLUSIONS: This study reveals the spatial reprogramming of the TME that may underlie invasive tumor potential in diffuse-type gastric cancer. This TME profiling across tumor layers suggests new targets, such as CCL2, that can modify the TME to inhibit tumor progression in diffuse-type gastric cancer.See related commentary by Huang and Brekken, p. 6284.

Cancer-Associated Fibroblast-Induced Resistance to Chemotherapy and Radiotherapy in Gastrointestinal Cancers.

In the past few decades, the role of cancer-associated fibroblasts (CAFs) in resistance to therapies for gastrointestinal (GI) cancers has emerged. Clinical studies focusing on GI cancers have revealed that the high expression of CAF-related molecules within tumors is significantly correlated with unfavorable therapeutic outcomes; however, the exact mechanisms whereby CAFs enhance resistance to chemotherapy and radiotherapy in GI cancers remain unclear. The cells of origin of CAFs in GI cancers include normal resident fibroblasts, mesenchymal stem cells, endothelial cells, pericytes, and even epithelial cells. CAFs accumulated within GI cancers produce cytokines, chemokines, and growth factors involved in resistance to therapies. CAF-derived exosomes can be engaged in stroma-related resistance to treatments, and several non-coding RNAs, such as miR-92a, miR-106b, CCAL, and H19, are present in CAF-derived exosomes and transferred to GI cancer cells. The CAF-induced desmoplastic reaction interferes with drug delivery to GI cancer cells, evoking resistance to chemotherapy. However, due to the heterogeneity of CAFs in GI cancers, identifying the exact mechanism underlying CAF-induced resistance may be difficult. Recent advancements in single-cell "omics" technologies could offer clues for revealing the specific subtypes and biomarkers related to resistance.

Inhibiting the GAS6/AXL axis suppresses tumor progression by blocking the interaction between cancer-associated fibroblasts and cancer cells in gastric carcinoma.

BACKGROUND: The effects of cancer-associated fibroblasts (CAF) on the progression of gastric carcinoma (GC) has recently been demonstrated. However, agents targeting the interaction between CAF and GC cells have not been applied in a clinical setting. Here, we examined if inhibition for Axl receptor tyrosine kinase (AXL) can suppress CAF-induced aggressive phenotype in GC. METHODS: We investigated the function of CAF-derived growth arrest-specific 6 (GAS6), a major ligand of AXL, on the migration and proliferation of GC cells. The effect of the AXL inhibitor, BGB324, on the CAF-induced aggressive phenotype of GC cells was also investigated. In addition, we performed immunohistochemistry to examine the expression of phosphorylated AXL protein in 175 GC tissues and evaluated its correlation with the prognosis. RESULTS: The qPCR and western blot analysis showed that GAS6 expression was higher in CAF relative to other cells. We found that co-culture with CAF increased the phosphorylation of AXL (P-AXL), differentiation into a mesenchymal-like phenotype, and cell survival in GC cell lines. When the expression of AXL was genetically inhibited in GC cells, the effect of CAF was reduced. BGB324, a small molecule inhibitor of AXL, suppressed the effects of CAF on GC cell lines. In GC tissues, high levels of P-AXL were significantly associated with poor overall survival (P = 0.022). CONCLUSIONS: We concluded that CAF are a major source of GAS6 and that GAS6 promotes an aggressiveness through AXL activation in GC. We suggested that an AXL inhibitor may be a novel agent for GC treatment.

Role of Cancer-Associated Fibroblast in Gastric Cancer Progression and Resistance to Treatments.

Although the survival of gastric cancer (GC) patients has gradually improved, the outcomes of advanced GC patients remain unsatisfactory despite standard treatment with conventional chemotherapy or targeted agents. Several studies have shown that cancer-associated fibroblasts (CAFs), a major component of tumor stroma in GC, may have significant roles in GC progression and resistance to treatments. CAFs are a major source of various secreted molecules in the tumor microenvironment, which stimulate cancer cells and other noncancerous components of GC. Surprisingly, these factors could be involved in gastric carcinogenesis. Cytokines, including interleukin-6 and interleukin-11, or growth factors, such as fibroblast growth factor produced from CAFs, can directly activate GC cells and consequently lead to the development of an aggressive phenotype. Galectin-1 or hepatocyte growth factor can be involved in CAF-derived neovascularization in GC. In addition, recent studies showed that CAFs can affect tumor immunity through M2 polarization of tumor-associated macrophages. Finally, the current study aimed to introduce several inhibitory agents and evaluate their suppressive effects on CAFs in patients with GC progression. However, further studies are required to evaluate their safety and select appropriate patients for application in clinical settings.

Targeting interleukin-6 as a strategy to overcome stroma-induced resistance to chemotherapy in gastric cancer.

BACKGROUND: Although the tumor stroma in solid tumors like gastric cancer (GC) plays a crucial role in chemo-resistance, specific targets to inhibit the interaction between the stromal and cancer cells have not yet been utilized in clinical practice. The present study aims to determine whether cancer-associated fibroblasts (CAFs), a major component of the tumor stroma, confer chemotherapeutic resistance to GC cells, and to discover potential targets to improve chemo-response in GC. METHODS: To identify CAF-specific proteins and signal transduction pathways affecting chemo-resistance in GC cells, secretome and transcriptome analyses were performed. We evaluated the inhibiting effect of CAF-specific protein in in vivo and in vitro models and investigated the expression of CAF-specific protein in human GC tissues. RESULTS: Secretome and transcriptome data revealed that interleukin-6 (IL-6) is a CAF-specific secretory protein that protects GC cells via paracrine signaling. Furthermore, CAF-induced activation of the Janus kinase 1-signal transducer and activator of transcription 3 signal transduction pathway confers chemo-resistance in GC cells. CAF-mediated inhibition of chemotherapy-induced apoptosis was abrogated by the anti-IL-6 receptor monoclonal antibody tocilizumab in various experimental models. Clinical data revealed that IL-6 was prominently expressed in the stromal portion of GC tissues, and IL-6 upregulation in GC tissues was correlated with poor responsiveness to chemotherapy. CONCLUSIONS: Our data provide plausible evidence for crosstalk between GC cells and CAFs, wherein IL-6 is a key contributor to chemoresistance. These findings suggest the potential therapeutic application of IL-6 inhibitors to enhance the responsiveness to chemotherapy in GC.

Scaffold-Assisted Ectopic Transplantation of Internal Organs and Patient-Derived Tumors.

Xenotransplantation of human tissues into immunodeficient mice has emerged as an invaluable preclinical model to study human biology and disease progression and predict clinical response. The most common anatomical site for tissue transplantation is the subcutaneous pocket due to simple surgical procedures and accessibility for gross monitoring and advanced imaging modalities. However, subcutaneously implanted tissues initially experience a sharp change in oxygen and nutrient supply and increased mechanical deformation. During this acute phase of tissue integration to the host vasculature, substantial cell death and tissue fibrosis occur limiting engraftment efficiency. Previously, we demonstrated that the implantation of inverted colloidal crystal hydrogel scaffolds triggers proangiogenic and immunomodulatory functions without characteristic foreign body encapsulation. In this study, we examine the use of this unique host response to improve the ectopic transplantation of tissues to the subcutaneous site. Scaffold-assisted tissues preserved morphological features and blood vessel density compared to native tissues, whereas scaffold-free tissues collapsed and were less vascularized. Notably, the supporting biomaterial scaffold modulated the foreign body response to reduce the localization of Ly6G+ cells within the transplanted tissues. Cotransplantation of patient-derived gastric cancer with a scaffold resulted in a comparable level of engraftment to conventional methods; however, detailed immunohistological characterization revealed significantly better retention of proliferative cells (Ki67+) and human immune cells (CD45+) by the end of the study. We envision that leveraging the immunomodulatory properties of biomaterial interfaces can be an attractive strategy to improve the functional engraftment of xenotransplants and accelerate individualized diagnostics and the development of novel therapeutic strategies.

Inhibition of Discoidin Domain Receptor 1 Prevents Stroma-Induced Peritoneal Metastasis in Gastric Carcinoma.

Discoidin domain receptor 1 (DDR1) is activated by fibrillar (triple-helical) collagens and collagen IV, which are major components of tumor stroma; thus, DDR1 might be a critical mediator of communication between cancer cells and stroma. The aim of this study was to investigate the effect of DDR1 inhibition on stroma-induced peritoneal metastasis in gastric carcinoma. We analyzed by immunohistochemistry the correlation between DDR1 expression and the pattern of recurrence in gastric carcinoma tissues from a previously characterized and established gastric carcinoma patient cohort. We also cocultured human gastric carcinoma cell lines with gastric cancer-associated fibroblasts (CAF) and investigated DDR1 expression and activation. We evaluated CAF-induced tumorigenic properties of gastric carcinoma cell lines and the effect of a DDR1-specific inhibitor in organotypic cultures and in a peritoneal seeding xenograft animal model. The expression of DDR1 in gastric cancer tissues was positively associated with early recurrence (P = 0.043) and a high incidence of peritoneal recurrence (P = 0.036). We confirmed that coculturing with CAFs elevated DDR1 protein expression in gastric carcinoma cell lines and enhanced gastric carcinoma cell line spheroid formation in organotypic cultures in a tumor cell DDR1-dependent manner. Coimplantation of CAFs with gastric carcinoma cells enhanced peritoneal tumor formation in vivo, an effect that was sensitive to pharmacologic inhibition of DDR1.Implications: This study highlights that CAF-induced elevation of DDR1 expression in gastric carcinoma cells enhances peritoneal tumorigenesis, and that inhibition of DDR1 is an attractive strategy for the treatment of gastric carcinoma peritoneal metastasis. Mol Cancer Res; 16(10); 1590-600. ©2018 AACR.

Gastrokine 1 protein is a potential theragnostic target for gastric cancer.

BACKGROUND: Gastrokine 1 (GKN1) plays important roles in maintaining mucosal homeostasis, and in regulating cell proliferation and differentiation. Here, we determined whether GKN1 is a potential theragnostic marker for gastric cancer. METHODS: We identified GKN1 binding proteins using the protein microarray assay and investigated whether GKN1 is one of the exosomal cargo proteins by western blot, immunoprecipitation, and immunofluorescent assays. Cell proliferation and apoptosis were analyzed by MTT, BrdU incorporation, flow cytometry, and western blot assays. We further validated the functional relevance of exosomal GKN1 in MKN1-injected xenograft mice. The possibility of serum GKN1 as a diagnostic marker for gastric cancer was determined by ELISA assay. RESULTS: In protein microarray assay, GKN1 binding to 27 exosomal proteins was clearly observed. GKN1 was expressed in exosomes derived from HFE-145 gastric epithelial cells by western blot and immunofluorescent assays, but not in exosomes from AGS and MKN1 gastric cancer cells. Exosomes carrying GKN1 inhibited cell proliferation and induced apoptosis in both AGS and MKN1 cells, and exosomes carrying GKN1-treated nude mice-bearing MKN1 xenograft tumors exhibited significantly reduced tumor volume and tumor weight. Silencing of clathrin markedly down-regulated the internalization of exosomal GKN1. Interestingly, serum GKN1 concentrations in patients with gastric cancer were significantly lower than those in healthy individuals and patients with colorectal and hepatocellular carcinomas. CONCLUSIONS: The GKN1 is secreted and internalized in the gastric epithelium by exosome-driven transfer, which inhibits gastric tumorigenesis and supports the clinical application of GKN1 protein in gastric cancer diagnosis and treatment.

AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells.

BACKGROUND: The At-rich interactive domain 1A (ARID1A) is frequently mutated in gastric cancers (GCs) with a poor prognosis. Growing evidence indicates that loss of ARID1A expression leads to activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by AKT phosphorylation. We aim to investigate the different sensitivity for the AKT inhibitor in ARID1A-deficient GC cells. METHODS: After transfection using siRNA or shRNA, the effect of ARID1A knockdown on the PI3K/AKT signaling pathway was evaluated by Western blot analysis. ARID1A-knockdown cells were treated with AKT inhibitor (GSK690693), 5-fluorouracil, or cisplatin, alone or in combination. Viability and apoptosis were analyzed using EZ-CYTOX cell viability assay and flow cytometry, respectively. RESULTS: ARID1A depletion accelerated the phosphorylation of AKT and S6 in a dose-dependent manner and led to an increased proliferation of MKN-1, MKN-28, and KATO-III GC cells (P<0.001). ARID1A-deficient cells were more vulnerable to GSK690693 in comparison to the controls (P<0.001), even at very low doses. Flow cytometry confirmed the increased apoptosis in ARID1A-deficient cells treated with GSK690693 (0.01 µmol/L; P<0.001). In contrast to our expectations, ARID1A depletion did not cause resistance to 5-fluorouracil or cisplatin. Addition of GSK690693 to the conventional chemotherapy induced more decreased cell viability in ARID1A-knockdown cells (P<0.01). CONCLUSION: Loss of ARID1A expression is a surrogate marker for the activation of the AKT signaling pathway and is also a reliable biomarker to predict the response for the AKT inhibitor. We anticipate that appropriate patient selection based on ARID1A expression in the tumor tissue will increase the drug sensitivity for the AKT inhibition and improve the clinical outcome.

HVC1 ameliorates hyperlipidemia and inflammation in LDLR-/- mice.

BACKGROUND: HVC1 consists of Coptidis Rhizoma (dried rhizome of Coptischinensis), Scutellariae Radix (root of Scutellariabaicalensis), Rhei Rhizoma (rhizome of Rheum officinale), and Pruni Cortex (cortex of Prunusyedoensis Matsum). Although the components are known to be effective in various conditions such as inflammation, hypertension, and hypercholesterolemia, there are no reports of the molecular mechanism of its hypolipidemic effects. METHODS: We investigated the hypolipidemic effect of HVC1 in low-density lipoprotein receptor-deficient (LDLR-/-) mice fed a high-cholesterol diet for 13 weeks. Mice were randomized in to 6 groups: ND (normal diet) group, HCD (high-cholesterol diet) group, and treatment groups fed HCD and treated with simvastatin (10 mg/kg, p.o.) or HVC1 (10, 50, or 250 mg/kg, p.o.). RESULTS: HVC1 regulated the levels of total cholesterol, triglyceride (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol in mouse serum. In addition, it regulated the transcription level of the peroxisome proliferator-activated receptors (PPARs), sterol regulatory element-binding proteins (SREBP)-2, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, lipoprotein lipase (LPL), apolipoprotein B (apo B), liver X receptor (LXR), and inflammatory cytokines (IL-1ß, IL-6, and TNF-α). Furthermore, HVC1 activated 5' adenosine monophosphate-activated protein kinase (AMPK). CONCLUSION: Our results suggest that HVC1 might be effective in preventing high-cholesterol diet-induced hyperlipidemia by regulating the genes involved in cholesterol and lipid metabolism, and inflammatory responses.

Discoidin domain receptor 1 activity drives an aggressive phenotype in gastric carcinoma.

BACKGROUND: Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that utilizes collagen as a ligand, is a key molecule in the progression of solid tumors as it regulates the interaction of cancer cells with the tumor stroma. However, the clinical relevance of DDR1 expression in gastric carcinoma is yet to be investigated. Here, we assessed the role of DDR1 in mediating the aggressive phenotype of gastric carcinoma and its potential as a therapeutic target. METHODS: We conducted DDR1 immunohistochemistry using a tissue microarray of 202 gastric carcinoma specimens. We examined the effect of collagen-induced activation of DDR1 on cell signaling, tumorigenesis, and cell migration in gastric cancer cell lines, and tumor growth in a xenograft animal model of gastric cancer. RESULTS: Our results showed that 50.5% of gastric cancer tissues are positive for DDR1 expression, and positive DDR1 expression was significantly correlated with a poor prognosis (P = 0.015). In a subgroup analysis, DDR1 expression was prognostically meaningful only in patients receiving adjuvant treatment (P = 0.013). We also demonstrated that collagen was able to activate DDR1 and increase the clonogenicity and migration of gastric cancer cells. We observed that a DDR1 inhibitor, 7rh benzamide, suppressed tumor growth in gastric cancer xenografts. CONCLUSIONS: Our findings suggest a key role for DDR1 signaling in mediating the aggressive phenotype of gastric carcinoma. Importantly, inhibition of DDR1 is an attractive strategy for gastric carcinoma therapy.

Clinicopathological Implication of Insulin-like Growth Factor-II mRNA-Binding Protein 3 (IMP3) Expression in Gastric Cancer.

BACKGROUND: The clinicopathological significance of oncofetal mRNA-binding protein, human insulin-like growth factor II mRNA-binding protein 3 (IMP3), in gastric carcinoma (GC) is not fully understood. MATERIALS AND METHODS: Tissue microarray blocks with specimens from 346 patients with GC were constructed to evaluate the clinicopathological role of IMP3 expression in GC. These results were validated with an online dataset of 876 patients from the Kaplan-Meier Plotter. Sera from 15 controls and 57 patients with GC were collected in order to compare the levels of serum IMP3 between groups. RESULTS: High expression of IMP3 was significantly associated with poor prognosis. Survival curves from the Kaplan-Meier Plotter showed that high IMP3 expression was significantly related to worse disease-free survival and overall survival. CONCLUSION: Tissue overexpression of IMP3 might be used as a predictor of advanced disease or lymph node metastasis, and is associated with poorer prognosis in GCs.

Intratumor stromal proportion predicts aggressive phenotype of gastric signet ring cell carcinomas.

OBJECTIVE: The aim of this study was to evaluate the prognostic significance of the intratumor stromal proportion in gastric signet ring cell (SRC) carcinomas. BACKGROUND: Cancer stroma, as exemplified by cancer-associated fibroblasts (CAFs), plays critical roles in cancer proliferation, invasion, and metastasis. METHODS: One hundred seventy-five SRC carcinoma cases were classified according to the intratumor desmoplastic stromal proportion to then analyze the clinicopathologic characteristics of stroma-rich cases. We also investigated the impact of CAFs on the migration as well as on the phenotypic changes of gastric SRC carcinomas in vitro. Furthermore, we performed RNA sequencing of a pair of CAFs and normal-tissue-associated fibroblasts. RESULTS: Stroma-rich SRC carcinomas (64 of 175 cases, 36.5%) were associated with female patients (P = 0.045), large tumor size (P = 0.007), higher T category (P < 0.001), and the presence of perineural invasion (P = 0.018). Patients with stroma-rich SRC carcinomas had a significantly shorter disease-free survival (P < 0.001) and overall survival (P < 0.001). However, in a subgroup analysis, the prognostic significance of the stromal proportion correlated only with patients with T3/4 disease. From multivariate analysis, the high stromal proportion is an independent prognostic factor to predict worse disease-free survival (hazard ratio 2.288; P = 0.001) and overall survival (hazard ratio 2.503; P = 0.001). We found that CAFs enhanced the migratory abilities of cancer cells through the epithelial-mesenchymal transition, and RNA sequencing results confirmed these findings. CONCLUSIONS: The intratumor stromal proportion could be a useful prognostic biomarker and a potential therapeutic target in gastric SRC carcinomas.

Preoperative serum levels of insulin-like growth factor-binding protein 2 predict prognosis of gastric cancer patients.

It has been reported that serum insulin-like growth factor-binding protein 2 (IGFBP2) levels are elevated in various types of cancers. However, the clinicopathologic and prognostic implications of circulating IGFBP2 have never been investigated in gastric cancer. We tested IGFBP2 levels in the sera of 118 gastric cancer patients and 34 healthy controls using enzyme-linked immunosorbent assay (ELISA). The mean serum IGFBP2 level was significantly elevated in the gastric cancer patients compared to controls (805.23 ± 590.56 ng/ml vs. 459.61 ± 277.01 ng/ml; P < 0.001). Serum IGFBP2 levels were significantly higher in larger (> 6 cm) tumors (956.8 ± 734.0 ng/ml vs. 548.6 ± 364.0 ng/ml; P = 0.007) and in higher (T3/4) T stages (854.8 ± 621.4 ng/ml vs. 546.5 ± 315.1 ng/ml; P = 0.037). Multivariate Cox analysis showed that higher serum IGFBP2 level (> 400.01 ng/ml) was an independent prognostic factor predicting worse overall survival in patients with gastric cancer (hazard ratio (HR): 3.749, P = 0.034). When we divided patients into four groups based on blood IGFBP2 levels, survival was stratified. The HRs for death in the 3rd and 4th quartiles of serum IGFBP2 levels in comparison to that in the 1st quartile were 2.527 (P = 0.043) and 3.092 (P = 0.012). In conclusion, circulating IGFBP2 has potential as a biomarker predicting prognosis for gastric cancer patients.