The chemokine CCL1 triggers an AMFR-SPRY1 pathway that promotes differentiation of lung fibroblasts into myofibroblasts and drives pulmonary fibrosis.

Recruitment of immune cells to the site of inflammation by the chemokine CCL1 is important in the pathology of inflammatory diseases. Here, we examined the role of CCL1 in pulmonary fibrosis (PF). Bronchoalveolar lavage fluid from PF mouse models contained high amounts of CCL1, as did lung biopsies from PF patients. Immunofluorescence analyses revealed that alveolar macrophages and CD4+ T cells were major producers of CCL1 and targeted deletion of Ccl1 in these cells blunted pathology. Deletion of the CCL1 receptor Ccr8 in fibroblasts limited migration, but not activation, in response to CCL1. Mass spectrometry analyses of CCL1 complexes identified AMFR as a CCL1 receptor, and deletion of Amfr impaired fibroblast activation. Mechanistically, CCL1 binding triggered ubiquitination of the ERK inhibitor Spry1 by AMFR, thus activating Ras-mediated profibrotic protein synthesis. Antibody blockade of CCL1 ameliorated PF pathology, supporting the therapeutic potential of targeting this pathway for treating fibroproliferative lung diseases.

Targeting Degradation of the Transcription Factor C/EBPß Reduces Lung Fibrosis by Restoring Activity of the Ubiquitin-Editing Enzyme A20 in Macrophages.

Although recent progress provides mechanistic insights into the pathogenesis of pulmonary fibrosis (PF), rare anti-PF therapeutics show definitive promise for treating this disease. Repeated lung epithelial injury results in injury-repairing response and inflammation, which drive the development of PF. Here, we report that chronic lung injury inactivated the ubiquitin-editing enzyme A20, causing progressive accumulation of the transcription factor C/EBPß in alveolar macrophages (AMs) from PF patients and mice, which upregulated a number of immunosuppressive and profibrotic factors promoting PF development. In response to chronic lung injury, elevated glycogen synthase kinase-3ß (GSK-3ß) interacted with and phosphorylated A20 to suppress C/EBPß degradation. Ectopic expression of A20 or pharmacological restoration of A20 activity by disturbing the A20-GSK-3ß interaction accelerated C/EBPß degradation and showed potent therapeutic efficacy against experimental PF. Our study indicates that a regulatory mechanism of the GSK-3ß-A20-C/EBPß axis in AMs may be a potential target for treating PF and fibroproliferative lung diseases.

Tumor-repopulating cells evade ferroptosis via PCK2-dependent phospholipid remodeling.

Whether stem-cell-like cancer cells avert ferroptosis to mediate therapy resistance remains unclear. In this study, using a soft fibrin gel culture system, we found that tumor-repopulating cells (TRCs) with stem-cell-like cancer cell characteristics resist chemotherapy and radiotherapy by decreasing ferroptosis sensitivity. Mechanistically, through quantitative mass spectrometry and lipidomic analysis, we determined that mitochondria metabolic kinase PCK2 phosphorylates and activates ACSL4 to drive ferroptosis-associated phospholipid remodeling. TRCs downregulate the PCK2 expression to confer themselves on a structural ferroptosis-resistant state. Notably, in addition to confirming the role of PCK2-pACSL4(T679) in multiple preclinical models, we discovered that higher PCK2 and pACSL4(T679) levels are correlated with better response to chemotherapy and radiotherapy as well as lower distant metastasis in nasopharyngeal carcinoma cohorts.

The phase separation of extracellular matrix protein matrilin-3 from cancer-associated fibroblasts contributes to gastric cancer invasion.

Cancer-associated fibroblast (CAF) has emerged as a key contributor to the remodeling of tumor microenvironment through the expression and secretion of extracellular matrix (ECM) proteins, thereby promoting carcinogenesis. However, the precise contribution of ECM proteins from CAFs to gastric carcinogenesis remains poorly understood. In this study, we find that matrilin-3 (MATN3), an upregulated ECM protein associated with poorer prognosis in gastric cancer patients, originates from CAFs in gastric cancer tissues. Ectopic expression of MATN3 in CAFs significantly promotes the invasion of gastric cancer cells, which can be attenuated by neutralizing MATN3 with its antibody. Notably, a portion of MATN3 protein is found to form puncta in gastric cancer tissues ECM. MATN3 undergoes phase separation, which is mediated by its low complexity (LC) and coiled-coil (CC) domains. Moreover, overexpression of MATN3 deleted with either LC or CC in CAFs is unable to promote the invasion of gastric cancer cells, suggesting that LC or CC domain is required for the effect of CAF-secreted MATN3 in gastric cancer cell invasion. Additionally, orthotopic co-injection of gastric cancer cells and CAFs expressing MATN3, but not its ΔLC and ΔCC mutants, leads to enhanced gastric cancer cell invasion in mouse models. Collectively, our works suggest that MATN3 is secreted by CAFs and undergoes phase separation, which promotes gastric cancer invasion.

Construction of Ultrafine PtIr Clusters Supported on Co3O4 Nanoflowers for Enhanced Overall Water Splitting.

Green hydrogen production through electrochemical overall water splitting has suffered from sluggish oxygen evolution reaction (OER) kinetics, inferior conversion efficiency, and high cost. Herein, ultrafine PtIr clusters are synthesized via an electrodeposition method and decorated on the Co3O4 nanoflowers assembled by nanowires (PtIr-Co3O4). The encouraging performances in electrochemical OER and hydrogen evolution reaction (HER) are achieved over the PtIr-Co3O4 catalyst, with the overpotentials as low as 410 and 237 mV at 100 mA cm-2, respectively, outperforming the commercial IrO2 and Pt/C catalysts. Due to the ultralow loading of PtIr clusters, the PtIr-Co3O4 catalyst exhibits 1270 A gIr -1 for OER at the overpotential of 400 mV. Our detailed analyses also show that the strong interactions between the ultrafine PtIr clusters and the Co3O4 nanoflowers enable the PtIr-Co3O4 catalyst to afford 10 mA cm-2 for the overall water splitting at the potential of 1.57 V, accompanied by high durability for 100 h.

Access to Chiral O,O-Acetals Enabled by Palladium-Catalyzed Asymmetric Addition of Oximes to Alkoxyallenes.

Enantiomerically pure acyclic O,O-acetal compounds (up to 97 % ee) have been accessed through chemo-, regio- and enantioselective palladium-catalyzed addition of oximes to alkoxyallenes. DFT calculations support that a protonative hydropalladation pathway is favourable, in which the hydrogen bonding interaction between the amide group of the diphosphine ligand and the alkoxyallene is critical for the highly stereoselective formation of the dioxygenated stereogenic center.

Regular gastroscopy and colonoscopy during the evaluation of urachal cancer: do we really need them?

PURPOSE: Urachal cancer is similar to gastrointestinal adenocarcinoma in histology, and gastroscopy/colonoscopy is often administered during perioperative evaluation. However, gastroscopy and colonoscopy have corresponding disadvantages. This study discusses whether gastroscopy/colonoscopy is truly necessary for patients with urachal cancer. PATIENTS AND METHODS: A total of 166 bladder adenocarcinoma cases diagnosed at Sun Yat-sen University Cancer Center were retrospectively reviewed and divided into two groups (urachal cancer and nonurachal cancer), and perioperative evaluations were retrieved. RESULTS: There were 78 patients with urachal cancer, the median age was 48 years, and 59 were male. Perioperative gastroscopy/colonoscopy revealed 5 intestinal polyps and 1 adenoma during these evaluations, and no primary gastrointestinal cancer was found. Meanwhile, preoperative imaging evaluation did not detect significant gastrointestinal lesions. For 88 patients with nonurachal cancer, including primary bladder adenocarcinoma and metastatic tumors from gastrointestinal cancer, the median age was 56 years, and 64 were male. Preoperative imaging evaluation demonstrated 36 cases of gastrointestinal lesions, and 32 were confirmed by gastroscopy/colonoscopy; the other 4 were negative. Another 4 cases of colon cancer were detected by regular colonoscopy for suspected primary bladder adenocarcinoma. In all, 35 cases of colon cancer and 1 case of gastric cancer were identified by endoscopic examination. The diagnostic consistency of imaging and gastrointestinal endoscopy was favorable (P < 0.001), and the negative predictive value and diagnostic efficiency of imaging were 96.9% and 94.6%, respectively. CONCLUSIONS: The vast majority of gastrointestinal cancer cases can be identified by assessment of the patient's clinical symptoms, meticulous physical examination, and imaging evaluation. We recommend that gastroscopy/colonoscopy only be applied to patients with urachal cancer when the above examinations are positive.

Immune-based combination therapy to convert immunologically cold tumors into hot tumors: an update and new insights.

As a breakthrough strategy for cancer treatment, immunotherapy mainly consists of immune checkpoint inhibitors (ICIs) and other immunomodulatory drugs that provide a durable protective antitumor response by stimulating the immune system to fight cancer. However, due to the low response rate and unique toxicity profiles of immunotherapy, the strategies of combining immunotherapy with other therapies have attracted enormous attention. These combinations are designed to exert potent antitumor effects by regulating different processes in the cancer-immunity cycle. To date, immune-based combination therapy has achieved encouraging results in numerous clinical trials and has received Food and Drug Administration (FDA) approval for certain cancers with more studies underway. This review summarizes the emerging strategies of immune-based combination therapy, including combinations with another immunotherapeutic strategy, radiotherapy, chemotherapy, anti-angiogenic therapy, targeted therapy, bacterial therapy, and stroma-targeted therapy. Here, we highlight the rationale of immune-based combination therapy, the biomarkers and the clinical progress for these immune-based combination therapies.

Roles of exosome-derived non-coding RNA in tumor micro-environment and its clinical application. / å¤–æ³Œä½“æ¥æºéžç¼–ç RNA在肿瘤微环境中的作用及临床应用价值.

Tumor-derived exosomes play an important role in the tumor micro-environment. The exosome-derived non-coding RNAs are transmitted in the tumor microenvironment in three ways, communication between tumor cells, normal cells affecting tumor cells, and tumor cells affecting normal cells. Through these three ways, exosomal non-coding RNAs are involved in the regulation of tumor progression, affecting tumor angiogenesis, tumor invasiveness, drug resistance, stemness, tumor metabolic repro-gramming and immune escape, resulting in dual roles in promoting or inhibiting tumor development. Exosomes have a membranous structure and their contents are resistant to degradation by extracellular proteases and remain highly stable in body fluids, thus exosome-derived non-coding RNAs are expected to serve as diagnostic and prognostic indicators for a variety of cancers. In addition, exosomes can be used to deliver non-coding RNAs for targeted therapy, or to knock down or modify tumor-promoting non-coding RNAs for tumor therapy. This article reviews the function and communication mechanism of exosomal non-coding RNAs in the tumor microenvironment, including their pathways of action, effects, potential values for tumor biomarkers and treatment targets. This article also points out the issues that need to be further studied in order to promote the progress of extracellular non-coding RNAs in cancer research and their application in tumor diagnosis and treatment.

Combining gene expression signature with clinical features for survival stratification of gastric cancer.

The AJCC staging system is considered as the golden standard in clinical practice. However, it remains some pitfalls in assessing the prognosis of gastric cancer (GC) patients with similar clinicopathological characteristics. We aim to develop a new clinic and genetic risk score (CGRS) to improve the prognosis prediction of GC patients. We established genetic risk score (GRS) based on nine-gene signature including APOD, CCDC92, CYS1, GSDME, ST8SIA5, STARD3NL, TIMEM245, TSPYL5, and VAT1 based on the gene expression profiles of the training set from the Asian Cancer Research Group (ACRG) cohort by LASSO-Cox regression algorithms. CGRS was established by integrating GRS with clinical risk score (CRS) derived from Surveillance, Epidemiology, and End Results (SEER) database. GRS and CGRS dichotomized GC patients into high and low risk groups with significantly different prognosis in four independent cohorts with different data types, such as microarray, RNA sequencing and qRT-PCR (all HR > 1, all P < 0.001). Both GRS and CGRS were prognostic signatures independent of the AJCC staging system. Receiver operating characteristic (ROC) analysis showed that area under ROC curve of CGRS was larger than that of the AJCC staging system in most cohorts we studied. Nomogram and web tool (http://39.100.117.92/CGRS/) based on CGRS were developed for clinicians to conveniently assess GC prognosis in clinical practice. CGRS integrating genetic signature with clinical features shows strong robustness in predicting GC prognosis, and can be easily applied in clinical practice through the web application.

Highly Modulated In-Fiber Mach-Zehnder Interferometer Based on an Ultracompact Leaky-Guided Liquid Core.

We proposed a novel sensor based on an ultracompact leaky-guided liquid core fiber Mach-Zehnder interferometer (LLCFMZI) for high modulation of an interference spectrum. The sensor structure is based on a micro-sized hollow-core fiber (HCF) splicing a tilt end face single-mode fiber (SMF) to create a miniature oblique gap for the effective access of different liquids. The liquid core with a relatively lower refractive index (RI) than the cladding can achieve a leaky-mode optical waveguide (LMOW) mechanism, and its volume is only approximately 7.85 pL. In addition, the utilized micro-length HCF can reduce the energy loss of core in the LMOW to obtain an acceptable extinction ratio (>30 dB) with high temperature (T) sensitivity in the interference spectra. Experimental results show that the interference spectra can be highly modulated within the wide measurement range of 1250-1650 nm with a steadily linear response for thermal effect. The measured temperature sensitivities (T-sensitivities) of various liquids of DI water, ethanol, and Cargille-liquid (nD = 1.305) are 0.8869, 4.4754, and 4.8229 nm/°C, and the corresponding measured thermal optics coefficient (TOC) are -4.16 × 10-5, -2.11 × 10-4, and -3.6 × 10-4 °C-1, respectively. Measurement results demonstrate that the used liquids with a higher TOC can obtain better T-sensitivity modulation. The highest experimental sensitivity of the liquid-core filled with Cargille-liquid (nD = 1.40) is up to +13.87 nm/°C with a corresponding TOC of -4.07 × 10-4 °C-1. Furthermore, the experimental and theoretical values are in good agreement according to FSR the measuring scheme that investigates the effectiveness of the proposed LLCFMZI.

MiR-129-5p induces cell cycle arrest through modulating HOXC10/Cyclin D1 to inhibit gastric cancer progression.

MicroRNAs (miRNAs) play important roles in posttranscriptional regulation and may serve as targets for the diagnosis and treatment of cancers. Nevertheless, a comprehensive understanding of miRNAs profiles in gastric cancer progression is still lacking. Here, we report that miR-129-5p is downregulated in gastric cancer by analyzing TCGA database (n = 41) and clinical tumor samples (n = 60). MiR-129-5p transfection suppressed gastric cancer cell proliferation through inducing G1 phase arrest in vitro and inhibit xenograft tumor growth in vivo. MiR-129-5p directly targeted the 3' untranslated regions (3' UTR) of HOXC10 mRNA and downregulated its expression. Importantly, miR-129-5p could reverse the oncogenic effect induced by HOXC10. We systemically screened the downstream target of HOXC10 by ChIP sequencing, and found that HOXC10 could transcriptionally regulate the expression of Cyclin D1 and facilitate G1/S cell cycle transition. Notably, high levels of HOXC10 and Cyclin D1 were related with poor prognosis of gastric cancer patients (n = 90). These findings reveal a novel role of miR-129-5p/HOXC10/Cyclin D1 axis in modulating cell cycle and gastric tumorigenesis, which might provide potential prognostic biomarkers and therapeutic targets for gastric cancer patients.

Radiotherapy plus chemotherapy versus chemotherapy alone in penile cancer patients with extracapsular nodal extension after inguinal lymph node surgery: a multi-institutional study.

PURPOSE: Because there is a lack of evidence, it is not generally recommended to use adjuvant radiotherapy plus chemotherapy to treat lymph node disease in penile cancer. The aim of this study was to determine the benefit of using adjuvant radiotherapy after inguinal surgery for penile cancer. METHODS: Multi-institutional data were obtained from a total of nine centers from April 2003 to April 2015 and retrospectively analyzed. pN3 patients with an extracapsular nodal extension who received adjuvant therapy after inguinal surgery were included. Cancer-specific survival (CSS) was estimated using the Kaplan-Meier method. The multivariate analysis was performed using a Cox proportional hazards model. RESULTS: A total of 93 pN3 patients met the inclusion criteria. During the study period, 32 (34.4%) and 61 (65.6%) of these patients received adjuvant radiotherapy plus chemotherapy (AR + AC) or adjuvant chemotherapy alone (AC). The median CSS in all patients was 12.0 months (interquartile range [IQR] 7.5-16.5). The Kaplan-Meier estimated 3-year CSS rate was significantly longer in the AR + AC group (28.5%) than the AC group (16.2%) (p = 0.036). AC + AR was associated with an improvement in CSS by 7.7 months (17.7 [IQR 3.8-31.6] vs. 10.0 [IQR 6.6-13.4] months). In the Cox regression analysis, AR + AC was an independent predictor of CSS [model a: HR 0.486 (95% CI 0.258-0.916), model b: HR 0.527 (95% CI 0.286-0.972)]. CONCLUSION: In conclusions, AR + AC was associated with improved CCS in patients with penile cancer who displayed an extracapsular nodal extension after inguinal surgery. This hypothesis requires further confirmation.

Paeoniflorin elicits the anti-proliferative effects on glioma cell via targeting translocator protein 18 KDa.

As a natural compound isolated from Paeoniae radix, Paeoniflorin (PF) has been shown the antitumor effects in various types of human cancers including glioma, which is one of the serious tumors in central nervous system. Translocator protein 18 KDa (TSPO) has been shown to be relevant to the glioma aetiology. However, the regulation of PF in TSPO and neurosteriods biosynthesis on glioma is still unclear. In the present study, the glioma cell (U87 and U251) were cultured and used to quantify the bindings of PF on TSPO. Results indicated that there was not significant different between IC50 of PF and TSPO ligand PK11195. Moreover, PF exerted the anti-proliferative effects in glioma cell with a dose dependent inhibition from 12.5 to 100 µM in vitro. Consistent with the effects of PK11195, lowered levels on progesterone, allopregnanolone, as well as TSPO mRNA were induced by PF (25 and 50 µM). Furthermore, a xenograft mouse model with U87 cell-derived was significant inhibited by PF treatment, as well as the PK11195 administration. These results demonstrate that PF exerts its antitumor effects associated with the TSPO and neurosteroids biosynthesis in glioma cells could be a promising therapeutic agent for glioma therapy.

Increased nuclear translation of YAP might act as a potential therapeutic target for NF1-related plexiform neurofibroma.

Plexiform neurofibroma (pNF) in the head and neck is a characteristic feature in patients with neurofibromatosis type 1 (NF1) and is associated with significant disfigurement and psychological distress. Yes-associated protein (YAP), the key molecule involved in the Hippo pathway, is a vital transductor that regulates the proliferation and remyelinating of Schwann cells. The functional status of YAP and its feasibility as a potential target are still unknown in pNF. A total of 17 pNF tumor tissue specimens from the head and neck were collected at the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. Histologically, diagnosis of the Schwann cell region in pNF was achieved with hematoxylin-eosin staining, positive reactions for S100, SOX10, ERK and p-ERK, and low identification of Ki67 and SMA. Compared with normal nerve tissue, obviously increased nuclear YAP was detected in the Schwann cell region of pNF, with a mean nuclear staining rate of 67.11%. Based on the shNF1 Schwann cell model (the RSC96 cell line), with upregulated expression of RAS, ERK and p-ERK, p-YAP (Ser127) and p-YAP (Ser397) were significantly decreased and total YAP and nuclear YAP were increased. According to a confocal assay, the interference of shNF1 substantially promoted YAP nuclear translocation. Compared with control Schwann cells, the YAP inhibitor CA3 might have a more sensitive effect (IC50: NC=0.96±0.04, shNF1=0.71±0.02, P<0.05) on the shNF1 Schwann cell model than the classic MEK1/2 inhibitor selumetinib (IC50: NC=14.36±0.95, shNF1=24.83±0.98, P>0.05). For in vivo inhibition, the CA3 group and the selumetinib group displayed a similar inhibition effect with no significant difference. Increased nuclear translation and the functional state of YAP implies that the YAP-Hippo pathway might play an important role in the formation and remyelination of pNF. Compared with selumetinib, the YAP inhibitor can exhibit a similar but more sensitive effect on NF1-/- Schwann cells. These observations imply that YAP as a novel or adjuvant therapy target in the treatment of pNF.

Autophagic Flux Detection: Significance and Methods Involved.

Macroautophagy is an important biological process in eukaryotic cells by which longevity proteins, misfolded proteins, and damaged organelles are degraded. The autophagy process consists of three key steps: (1) the formation of autophagosomes; (2) the fusion of the autophagosomes with lysosomes; and (3) the degradation of the contents of autolysosomes. If any of the three steps is impaired, autophagy will not be able to complete its biological function. Dysfunctional or blocked autophagy is closely involved in the pathogenesis of a variety of diseases. The accurate determination of the autophagy activity in vivo and in vitro has become a challenge in the field of autophagy research. At present, the most widely used detection method to determine autophagy activity in mammalian cells is to quantify LC3B in the cells by Western blot, or to observe the formation and changes of autophagosomes and autolysosomes by immunofluorescence and electron microscopy. However, ignoring the dynamic characteristics of autophagy and only evaluating the number of autophagosomes or the presence of LC3B cannot completely reflect the activation or a blockage of the autophagy system, and objectively analyze its real role in the occurrence and development of a disease. For example, the accumulation of autophagosomes and autolysosomes can occur through an increase in substrate to be degraded after the activation of autophagy, or it may be caused by the partial obstruction or blockage of autophagy. In this chapter, new and familiar ways to detect the autophagic flux are methodically summarized to provide researchers with a multi-angled viewpoint.

Comprehensive elaboration of the cGAS-STING signaling axis in cancer development and immunotherapy.

Cellular recognition of microbial DNA is an evolutionarily conserved mechanism by which the innate immune system detects pathogens. Cyclic GMP-AMP synthase (cGAS) and its downstream effector, stimulator of interferon genes (STING), are involved in mediating fundamental innate antimicrobial immunity by promoting the release of type I interferons (IFNs) and other inflammatory cytokines. Accumulating evidence suggests that the activation of the cGAS-STING axis is critical for antitumor immunity. The downstream cytokines regulated by cGAS-STING, especially type I IFNs, serve as bridges connecting innate immunity with adaptive immunity. Accordingly, a growing number of studies have focused on the synthesis and screening of STING pathway agonists. However, chronic STING activation may lead to a protumor phenotype in certain malignancies. Hence, the cGAS-STING signaling pathway must be orchestrated properly when STING agonists are used alone or in combination. In this review, we discuss the dichotomous roles of the cGAS-STING pathway in tumor development and the latest advances in the use of STING agonists.

Long Noncoding RNA GMAN, Up-regulated in Gastric Cancer Tissues, Is Associated With Metastasis in Patients and Promotes Translation of Ephrin A1 by Competitively Binding GMAN-AS.

BACKGROUND & AIMS: We aimed to identify long noncoding RNAs (lncRNAs) that are up-regulated in gastric cancer tissues from patients and study their function in gastric tumor metastasis. METHODS: We collected gastric tumor and nontumor tissues from patients in China and analyzed levels of lncRNAs by microarray analysis, proteins by immunohistochemistry, and RNAs by quantitative reverse-transcription polymerase chain reaction; we compared these with survival times of patients and tumor progression. RNA levels were knocked down or knocked out in BGC-823, SGC-7901, and MKN45 cell lines using small interfering or short hairpin RNAs or clustered regularly interspaced short palindromic repeats (ie, CRISPR)/CRISPR associated protein 9 (ie, Cas9) vectors. Genes were overexpressed from transfected plasmids in HGC-27 cells. Cells were analyzed by Northern blot and immunoblot, polysome profiling assay, and cell invasion assay. Cells were injected into the tail veins or spleens of nude mice or SCID mice; lung and liver tissues were collected, and metastases were counted. lncRNAs were cloned by using rapid amplification of complementary DNA ends. Their interactions with other genes were determined by RNA pulldown and mapping assays. RESULTS: In microarray analyses, we identified 151 lncRNAs expressed at significantly higher levels in gastric tumor vs nontumor tissues. Levels of an lncRNA that we called gastric cancer metastasis associated long noncoding RNA (GMAN) were increased in gastric tumor tissues, compared with nontumor tissues; its up-regulation was associated with tumor metastasis and shorter survival times of patients. The GMAN gene overlaps with the ephrin A1 gene (EFNA1) and was highly expressed in BGC-823 and MKN45 cells. Knockdown of GMAN in these cells did not affect proliferation, colony formation, or adhesion but did reduce their invasive activity in Transwell assays. Ectopic expression of GMAN increased the invasive activity of HGC-27 cells. BGC-823 and MKN45 cells with knockdown of GMAN formed fewer metastases after injection into tail veins of nude mice. Knockdown or knockout of GMAN also reduced levels of ephrin A1 protein in cells. We found that GMAN promoted translation of ephrin A1 messenger RNA into protein by binding to the antisense GMAN RNA (GMAN-AS)-this antisense sequence is also complementary to that of ephrin A1 mRNA. Levels of ephrin A1 protein were also increased in gastric tumors from patients with metastases than in those without metastases. Knockout of ephrin A1 in BGC-823 cells reduced their invasive activity in Transwell assays and ability to form metastases after injection into SCID mice. Ectopic expression of ephrin A1 in BGC-823 cells with knockdown or knockout of GMAN restored their invasive activities and ability form metastases in nude or SCID mice. A CRISPR/Cas9-based strategy to disrupt the GMAN gene significantly reduced the numbers of metastases formed from SGC-7901 cells in mice. CONCLUSIONS: We identified an lncRNA, which we call GMAN, that is increased in gastric tumors from patients and associated with survival and formation of metastases. It regulates translation of ephrin A1 mRNA by binding competitively to GMAN-AS. Knockdown or knockout of GMAN or ephrin A1 in gastric cancer cell lines reduces their invasive activity and ability to form metastases after injection into mice. These genes might be targeted to prevent or reduce gastric cancer metastasis.

TRIB3 Interacts With ß-Catenin and TCF4 to Increase Stem Cell Features of Colorectal Cancer Stem Cells and Tumorigenesis.

BACKGROUND & AIMS: Activation of Wnt signaling to ß-catenin contributes to the development of colorectal cancer (CRC). Expression of tribbles pseudo-kinase 3 (TRIB3) is increased in some colorectal tumors and associated with poor outcome. We investigated whether increased TRIB3 expression promotes stem cell features of CRC cells and tumor progression by interacting with the Wnt signaling pathway. METHODS: We performed studies with C57BL/6J-ApcMin/J mice injected with an adeno-associated virus vector that expresses a small hairpin RNA against Trib3 mRNA (ApcMin/J-Trib3KD) or a control vector (ApcMin/J-Ctrl). We created BALB/c mice that overexpress TRIB3 from an adeno-associated virus vector and mice with small hairpin RNA-mediated knockdown of ß-catenin. The mice were given azoxymethane followed by dextran sodium sulfate to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by histology, gene expression profiling, immunohistochemistry, and immunofluorescence. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-positive (LGR5Pos) and LGR5-negative (LGR5Neg) HCT-8 CRC cells, with or without knockdown or transgenic expression of TRIB3, were sorted and analyzed in sphere-formation assays. We derived organoids from human and mouse colorectal tumors to analyze the function of TRIB3 and test the effect of a peptide inhibitor. Wnt signaling to ß-catenin was analyzed in dual luciferase reporter, chromatin precipitation, immunofluorescence, and immunoblot assays. Proteins that interact with TRIB3 were identified by immunoprecipitation. CRC cell lines were grown in nude mice as xenograft tumors. RESULTS: At 10 weeks of age, more than half the ApcMin/J-Ctrl mice developed intestinal high-grade epithelial neoplasia, whereas ApcMin/J-Trib3KD mice had no intestinal polyps and normal histology. Colon tissues from ApcMin/J-Trib3KD mice expressed lower levels of genes regulated by ß-catenin and genes associated with cancer stem cells. Mice with overexpression of Trib3 developed more tumors after administration of azoxymethane and dextran sodium sulfate than BALB/c mice. Mice with knockdown of ß-catenin had a lower tumor burden after administration of azoxymethane and dextran sodium sulfate, regardless of Trib3 overexpression. Intestinal tissues from mice with overexpression of Trib3 and knockdown of ß-catenin did not have activation of Wnt signaling or expression of genes regulated by ß-catenin. LGR5Pos cells sorted from HCT-8 cells expressed higher levels of TRIB3 than LGR5Neg cells. CRC cells that overexpressed TRIB3 had higher levels of transcription by ß-catenin and formed larger spheroids than control CRC cells; knockdown of ß-catenin prevented the larger organoid size caused by TRIB3 overexpression. TRIB3 interacted physically with ß-catenin and transcription factor 4 (TCF4). TRIB3 overexpression increased, and TRIB3 knockdown decreased, recruitment of TCF4 and ß-catenin to the promoter region of genes regulated by Wnt. Activated ß-catenin increased expression of TRIB3, indicating a positive-feedback loop. A peptide (P2-T3A6) that bound ß-catenin disrupted its interaction with TRIB3 and TCF4. In primary CRC cells and HCT-8 cells, P2-T3A6 decreased expression of genes regulated by ß-catenin and genes associated with cancer stem cells and decreased cell viability and migration. Injection of C57BL/6J-ApcMin/J mice with P2-T3A6 decreased the number and size of tumor nodules and colon expression of genes regulated by ß-catenin. P2-T3A6 increased 5-fluorouracil-induced death of CRC cells and survival times of mice with xenograft tumors. CONCLUSION: TRIB3 interacts with ß-catenin and TCF4 in intestine cells to increase expression of genes associated with cancer stem cells. Knockdown of TRIB3 decreases colon neoplasia in mice, migration of CRC cells, and their growth as xenograft tumors in mice. Strategies to block TRIB3 activity might be developed for treatment of CRC.