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Tumor metastasis is a crucial impediment to the treatment of gastric cancer (GC), and the epithelial-to-mesenchymal transition (EMT) program plays a critical role for the initiation of GC metastasis. Thus, the aim of this study is to investigate the regulation of lnc-CTSLP4 in the EMT process during GC progression. We found that lnc-CTSLP4 was significantly downregulated in GC tumor tissues compared with adjacent non-tumor tissues, and its levels in GC tumor tissues were closely correlated with tumor local invasion, TNM stage, lymph node metastasis, and prognosis of GC patients. Loss- and gain-of-function assays indicated that lnc-CTSLP4 inhibited GC cell migration, invasion, and EMT in vitro, as well as peritoneal dissemination in vivo. Mechanistic analysis demonstrated that lnc-CTSLP4 could bind with Hsp90α/heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) complex and recruit E3-ubiquitin ligase ZFP91 to induce the degradation of HNRNPAB, thus suppressing the transcriptional activation of Snail and ultimately reversing EMT of GC cells. Taken together, our results suggest that lnc-CTSLP4 is significantly downregulated in GC tumor tissues and inhibits metastatic potential of GC cells by attenuating HNRNPAB-dependent Snail transcription via interacting with Hsp90α and recruiting E3 ubiquitin ligase ZFP91, which shows that lnc-CTSLP4 could serve as a prognostic biomarker and therapeutic target for metastatic GC., Graphical Abstract, lnc-CTSLP4 is significantly downregulated in GC tumor tissues and inhibits metastatic potential of GC cells by attenuating HNRNPAB-dependent Snail transcription via interacting with Hsp90α and recruiting E3-ubiquitin ligase ZFP91, which shows that lnc-CTSLP4 could serve as a prognostic biomarker and therapeutic target for metastatic GC.

Gastric cancer (GC) is characterized by extensive local invasion, distant metastasis and poor prognosis. In most cases, GC progression is associated with aberrant expression of cytokines or activation of signaling cascades mediated by tumor–stroma interactions. However, the mechanisms by which these interactions contribute to GC progression are poorly understood. In this study, we find that IL-33 and its receptor ST2L are upregulated in the human GC and served as prognostic markers for poor survival of GC patients. In a co-culture model with GC cells and cancer-associated fibroblasts (CAFs), we further demonstrate that CAFs-derived IL-33 enhances the migration and invasion of GC cells by inducing the epithelial–mesenchymal transition (EMT) through activation of the ERK1/2-SP1-ZEB2 pathway in a ST2L-dependent manner. Furthermore, the secretion of IL-33 by CAFs can be induced by the proinflammatory cytokines TNF-α that is released by GC cells via TNFR2-NF-κB-IRF-1 pathway. Additionally, silencing of IL-33 expression in CAFs or ST2L expression in GC cells inhibits the peritoneal dissemination and metastatic potential of GC cells in nude mice. Taken together, these results characterize a critical role of the interaction between epithelial-stroma mediated by the TNF-α/IL-33/ST2L signaling in GC progression, and provide a rationale for targeting this pathway to treat GC metastasis.

Oncostatin M receptor (OSMR) is a member of the interleukin 6 (IL-6) receptor family that transduces signaling events of Oncostatin M (OSM). OSM–OSMR signaling plays a key role in inflammation and cancer progression. However, the role of OSM–OSMR in gastric cancer (GC) is still unknown. OSMR expression in GC was determined by real-time PCR (RT-PCR), immunohistochemistry (IHC) and Western blot. The effects of OSM–OSMR on GC cell proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT) in vitro and metastasis in vivo were examined. The pathways underlying OSM–OSMR signaling were explored by Western blot. Regulatory mechanism between SP1 and OSMR was explored in vitro. OSMR was highly expressed in GC tissues and its expression level was closely associated with age, T stage, Lauren classification, lymph node metastasis, TNM stage and worse prognosis of patients with GC. Knockdown of OSMR expression in GC cells significantly inhibited cell proliferation, migration, invasion, and EMT in vitro, as well as tumorigenesis and peritoneal metastasis in vivo induced by OSM. These effects mediated by OSM–OSMR were dependent on the activation of STAT3/FAK/Src signaling. SP1 could bind to the promoter region of human OSMR gene from − 255 to − 246 bp, and transcriptionally regulated OSMR overexpression in GC cells. OSM–OSMR contributes to GC progression through activating STAT3/FAK/Src signaling, and OSMR is transcriptionally activated by SP1.

Adenosine triphosphate (ATP) in the tumor microenvironment serves a vital role during tumor progression. ATP synthase F1 β subunit (ATP5B) is one of the most important subunits of ATP synthase and increases cellular ATP levels. ATP5B reportedly participates in carcinogenesis in several tumors. However, the regulatory mechanisms of ATP5B remain poorly understood in gastric cancer (GC). Here, we determined that high ATP5B expression in tumor tissues of GC is positively correlated with age, the tumor size, the TNM stage, lymph node metastasis, and patients' poor prognosis. The overexpression of ATP5B in GC cells elevated the cellular ATP content and promoted migration, invasion and proliferation. The levels of MMP2 expression, phosphorylated FAK, and phosphorylated AKT were increased after ATP5B overexpression in GC cells. Additionally, ATP5B overexpression increased the extracellular ATP level through the secretion of intracellular ATP and activated the FAK/AKT/MMP2 signaling pathway. ATP5B-induced downstream pathway activation was induced through the plasma membrane P2X7 receptor. Inhibitors of P2X7, FAK, AKT, and MMP2 suppressed the proliferative, migratory, and invasive capabilities of GC cells. In conclusion, our experiments indicate that ATP5B contributes to tumor progression of GC via FAK/AKT/MMP2 pathway. ATP5B, therefore, may be a biomarker of poor prognosis and a potential therapeutic target for GC.

Acquired resistance to tyrosine kinase inhibitors (TKIs) is the major obstacle to improve clinical efficacy in cancer patients. The epithelial-stromal interaction in tumor microenvironment influences cancer drug response to TKIs. Anlotinib is a novel oral multi-targeted TKI, and has recently been proven to be effective and safe for several tumors. However, if and how the epithelial-stromal interaction in tumor microenvironment affects anlotinib response in gastric cancer (GC) is not known. In this study, we found that anlotinib inhibited GC cells growth by inducing GC cells apoptosis and G2/M phase arrest in a dose- and time-dependent manner. Reactive oxygen species (ROS) mediated anlotinib-induced apoptosis in GC cells, while cancer-associated fibroblasts (CAFs) significantly suppressed anlotinib-induced apoptosis and ROS in GC cells. Increased BDNF that was derived from CAFs activated TrkB-Nrf2 signaling in GC cells, and reduced GC cells response to anlotinib. We identified secreted lactate from GC cells as the key molecule instructing CAFs to produce BDNF in a NF-κB-dependent manner. Additionally, functional targeting BDNF-TrkB pathway with neutralizing antibodies against BDNF and TrkB increased the sensitivity of GC cells towards anlotinib in human patient-derived organoid (PDO) model. Taken together, these results characterize a critical role of the epithelial-stroma interaction mediated by the lactate/BDNF/TrkB signaling in GC anlotinib resistance, and provide a novel option to overcome drug resistance., Graphical abstract Image 1, Highlights • Anlotinib induced apoptosis through ROS accumulation. • CAFs drive acquired resistance of GC to anlotinib. • TrkB activation by CAFs-derived BDNF confer resistance to anlotinib-induced ROS. • GC cells-secreted lactate induced BDNF expression of CAFs through activating NF-κB. • Blocking BDNF and TrkB reversed CAFs-induced acquired resistance to anlotinib.

Gastric cancer (GC) is one of the most frequent malignant tumors worldwide and is associated with high invasiveness, high metastasis and poor prognosis. Cancer-associated fibroblasts (CAFs), residing around tumor cells in tumor stroma, are important modifiers of tumor initiation and progression. However, the molecular mechanisms by which CAF's modulate tumor development have yet not to be characterized in GC. Here we performed tissue assay analyses identifying that Lumican, an extracellular matrix protein, is highly expressed in human gastric CAFs and its expression is positively associated with depth of invasion, lymph node metastasis, TNM stage and poor survival rate of GC. Functional studies revealed that integrin β1-FAK signaling pathways mediate the promoting effect of Lumican on GC cell proliferation, migration and invasion in vitro. In accordance with these observations, in GC cells co-cultured with CAFs in which Lumican had been knocked down, decreased gastric tumor growth and metastasis in vivo was apparent. In summary, CAF-derived Lumican contributes to tumorigenesis and metastasis of GC by activating the integrin β1-FAK signaling pathway.

// Xiongyan Wu 1, * , Pan Tao 1, * , Quan Zhou 1, * , Jie Li 1 , Zhenjia Yu 1 , Xiaofeng Wang 1 , Jiaanfang Li 1 , Chen Li 1 , Min Yan 1 , Zhenggang Zhu 1 , Bingya Liu 1 , Liping Su 1 1 Department of Surgery, Shanghai Key Laboratory of Gastric Neoplasms, Shanghai Institute of Digestive Surgery, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, People’s Republic of China * These authors contributed equally to this work Correspondence to: Liping Su, email: suliping@shsmu.edu.cn Keywords: cancer-associated fibroblasts, interleukin-6, JAK/STAT3, gastric cancer Received: July 22, 2016 Accepted: January 23, 2017 Published: February 06, 2017 ABSTRACT Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. However, the molecular mechanisms underlying the tumor-promoting properties of CAFs in gastric cancer remain unclear. Here, we show that CAFs isolated from gastric cancer produce significant amounts of interleukin-6 (IL-6). CAFs enhances the migration and EMT of gastric cancer cells through the secretion of IL-6 that activates Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT3) pathway in gastric cancer cells, while deprivation of IL-6 using a neutralizing antibody or inhibition of JAK/STAT3 pathway with specific inhibitor AG490 markedly attenuates these phenotypes in gastric cancer cells induced by CAFs. Moreover, silencing IL-6 expression in CAFs or inhibiting JAK2/STAT3 pathway in gastric cancer cells impairs tumor peritoneal metastasis induced by CAFs in vivo . Taken together, these results suggest that CAFs in the tumor microenvironment promote the progression of gastric cancer through IL-6/JAK2/STAT3 signaling, and IL-6 targeted therapy could be a complementary approach against gastric cancer by exerting their action on stromal fibroblasts.

// Chenchen Wang 1, * , Quan Zhou 1, * , Xiaofeng Wang 1 , Xiongyan Wu 1 , Xuehua Chen 1 , Jianfang Li 1 , Zhenggang Zhu 1 , Bingya Liu 1 , Liping Su 1 1 Shanghai Key Laboratory of Gastric Neoplasms, Shanghai Institute of Digestive Surgery, Department of Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, People’s Republic of China * These authors have contributed equally to this work Correspondence to: Liping Su, e-mail: franesulp@hotmail.com Bingya Liu, e-mail: byliu@sjtu.edu.cn Keywords: TLR7, Treg cells, Immune suppression, Anti-tumor effect Received: October 08, 2014 Accepted: November 16, 2014 Published: December 15, 2014 ABSTRACT Treg-induced immunosuppression is now recognized as a key element in enabling tumors to escape immune-mediated destruction. Although topical TLR7 therapies such as imiquimod have been proved successful in the treatment of dermatological malignancy and a number of conditions beyond the FDA-approved indications, the mechanism behind the effect of TLR7 on effector T cell and Treg cell function in cancer immunosurveillance is still not well understood. Here, we found that Loxoribin, one of the TLR7 ligands, could inhibit tumor growth in xenograft models of colon cancer and lung cancer, and these anti-tumor effects of Loxoribin were mediated by promoting CD4 + T cell proliferation and reversing Treg-mediated suppression via dendritic cells (DCs). However, deprivation of IL-6 using a neutralizing antibody abrogated the ability of Loxoribin-treated DCs, which reversed the Treg cell-mediated suppression. Furthermore, adoptive transfer of Loxoribin-treated DCs inhibited the tumor growth in vivo . Thus, this study links TLR7 signaling to the functional control of effector T cells and Treg cells and identifies Loxoribin as a new therapeutic strategy in cancer treatment, which may offer new opportunities to improve the outcome of cancer immunotherapy.

Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. However, the mechanisms underlying stromal fibroblast activation and their promotion of tumor growth remain largely unknown in gastric cancer. Here, we show that normal fibroblasts (NFs) from non-cancerous regions of gastric cancer exhibit the traits of CAFs when grown together with gastric cancer cells in vivo. Activation of NFs can be induced by co-culture with gastric cancer cells, while deprivation of hepatocyte growth factor (HGF) using a neutralizing antibody inhibits the activation of NFs. Moreover, we identify HGF as an important factor from CAFs that acts in a paracrine manner to promote tumorigenesis in vitro and in vivo. Taken together, these results suggest that HGF may play a pivotal role in the regulatory circuit between gastric cancer cells and stromal fibroblasts, and neutralization of HGF inhibits both activation and tumor-promoting properties of CAFs.

The ALEX is a novel member of the armadillo family and ALEX1 was reported to be reduced or even lost in multiple solid tumors. However, its expression profile and oncogenic role in gastric cancer (GC) remains largely unknown. ALEX1 expression was detected in 161 GC samples by immunohistochemistry staining. NCI-N87 cells transfected by ALEX1 lentivirus vectors and MKN28 cells transfected by ALEX1 shRNA were used for biological function investigation. Western blot was applied to explore the molecular mechanism and pull-down assays were applied to measure the activity of Rho GTPases. In vivo tumorigenicity, peritoneal and lung metastasis experiments were performed by tumor cell engraftment into nude mice. Bisulfite genomic sequencing and methylation-specific PCR were applied to check the methylation status of the ALEX1 gene. The expression rate of ALEX1 was significantly reduced in gastric tumor samples compared to non-tumor samples (43.5 vs. 90.2%), and its expression was closely related to the tumor differentiation, TNM staging, and lymph nodes metastasis. ALEX1 overexpression in NCI-N87 cells significantly inhibited cell proliferation, migration, and invasion in vitro, and disrupted the structure of the cytoskeleton. ALEX1 overexpression attenuated xenografts growth, peritoneal, and lung metastasis in nude mice. Mechanistically, the overexpression of ALEX1 inhibits thrombin-induced metastasis and Rho GTPases activation. Bisulfite genomic sequencing and methylation-specific PCR revealed that the promoter of ALEX1 is highly methylated in GC cells and tissues. ALEX1 expression is reduced in GC and is involved in diverse cellular functions. ALEX1 inhibits metastasis through the PAR-1/Rho GTPase signaling pathway.

// Yao-fei Pei 1, * , Ran Tao 2, * , Jian-fang Li 1 , Li-ping Su 1 , Bei-qin Yu 1 , Xiong-yan Wu 1 , Min Yan 1 , Qin-long Gu 1 , Zheng-gang Zhu 1 , Bing-ya Liu 1 1 Shanghai Key Laboratory of Gastric Neoplasms, Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China 2 Department of Hepatobiliary-Pancreatic Surgery, Zhejiang Province People’s Hospital, Hangzhou 310014, PR China * These authors have contributed equally to this work Correspondence to: Bing-ya Liu, email: liubingya@sjtu.edu.cn Keywords: TET1, 5-methylcytosine, 5-hydroxymethylcytosine, gastric cancer, PTEN Received: February 17, 2016 Accepted: March 31, 2016 Published: April 21, 2016 ABSTRACT Ten-Eleven Translocation 1 (TET1) is a member of ten eleven translocation enzymes, which convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). TET1 can promote CpG islands demethylation in specific genes and often absent in various cancers. Herein, we found that TET1 expression and 5-hmC content were low in gastric tumors compared to its adjacent non-tumor tissues. Cell proliferation, migration and invasion were enhanced upon TET1 knockdown in gastric cancer cells in vitro . This phenomenon was confirmed by an animal xeongraft model. We also found that TET1 directly binds to the promoter region of PTEN and activates its transcription through demethylation of CpG islands. TET1 knockdown activated AKT and FAK pathways, which were suppressed by PTEN. The activation of AKT and FAK facilitated tumor migration, invasion and accelerated cell growth. In conclusion, we found a novel mechanism that TET1 suppresses tumor cell growth, migration and invasion through demethylation of CpG island in PTEN promoter by increasing 5-hmC content. The re-expressed PTEN subsequently down regulates AKT and FAK activity.

Gastric cancer (GC) is one of the most common types of cancer worldwide, and it involves extensive local tumour invasion, metastasis and poor prognosis. Understanding the mechanisms regulating the progression of GC is necessary for the development of effective therapeutic strategies. Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) is an important regulator controlling gene activation and repression, which has been thought to be involved in tumorigenesis. However, the role of TBL1XR1 in human GC remains largely unknown. Here, we find that TBL1XR1 is aberrantly expressed in human GC tissues, and TBL1XR1 levels are highly correlated with local tumour invasion, late tumor, lymph node, metastasis (TNM) stage and poor prognosis. Knockdown of TBL1XR1 by shRNA inhibits GC cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) in vitro, as well as tumorigenesis and peritoneal metastasis in vivo, whereas overexpression of TBL1XR1 produces the opposite effects. These effects are mediated by activation of the ERK1/2 signalling pathway, and inhibition of this pathway with a specific ERK1/2 inhibitor (U0126) significantly impairs the tumour-promoting effects induced by TBL1XR1. Moreover, TBL1XR1 mediated ERK1/2 activation is dependent on the β-catenin/MMP7/EGFR signalling pathway. In conclusion, TBL1XR1 contributes to GC tumorigenesis and progression through the activation of the β-catenin/MMP7/EGFR/ERK signalling pathway and may act as a new therapeutic target for GC.

// Xiaofeng Wang 1,* , Zhenjia Yu 1,* , Quan Zhou 1 , Xiongyan Wu 1 , Xuehua Chen 1 , Jianfang Li 1 , Zhenggang Zhu 1 , Bingya Liu 1 and Liping Su 1 1 Shanghai Key Laboratory of Gastric Neoplasms, Shanghai Institute of Digestive Surgery, Department of Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China * These authors have contributed equally to this work Correspondence to: Liping Su, email: // Keywords : TG2, gastric cancer, ERK1/2, tumor progression Received : August 26, 2015 Accepted : January 04, 2016 Published : January 11, 2016 Abstract Gastric cancer (GC) is one of the most common tumors worldwide and involves extensive local tumor invasion, metastasis, and poor prognosis. Understanding mechanisms regulating progression of GC is necessary for developing effective therapeutic strategies. Tissue transglutaminase-2 (TG2), a multifunctional member of the transglutaminase family, has been shown to be critical for tumor initiation and progression. However, how TG2 promotes the progression of GC is unknown. We report that TG2 was highly expressed in GC tissues and positively associated with depth of tumor invasion and late TNM stage. With gain- and loss-of-function approaches, we observed that TG2 promoted GC cell proliferation, migration, invasion, as well as tumorigenesis and peritoneal metastasis in vivo . These events were associated with the ERK1/2 pathway activation and an ERK1/2 inhibitor (U0126) inhibited cell proliferation, migration, and invasion induced by overexpression of TG2. In summary, TG2 contributes to tumorigenesis and progression of GC by activating the ERK1/2 signaling pathway and is a potential therapeutic target of metastatic gastric cancer.

Paired box gene 4 (PAX4) is a transcriptional modulator located on chromosome 7q32, and its expression is dysregulated in a variety of human cancers, suggesting that PAX4 may be important in multiple tumors as a driver gene. Here, we show that PAX4 promoted migration and invasion in human epithelial cancers by decreasing miR-144 and miR-451 (miR-144/451) expression levels. Accordingly, miR-144/451 suppressed the migratory and invasive phenotypes, even in PAX4-expressing cells. Mechanistically, miR-144/451 inhibits cancer metastasis by targeting the A disintegrin and metalloproteinase (ADAM) protein family members ADAMTS5 and ADAM10. Their dysregulation is associated with increased tumor invasiveness and metastasis, then reduced patient prognosis in certain epithelial cancers. This discovery suggests that a PAX4-miR-144/451-ADAMs axis regulates human epithelial cancer metastasis, thus opening up therapeutic possibilities and predicting prognosis for those cancer types.

Background Toll-like receptor 4 (TLR4) is a receptor of lipopolysaccharide in the signaling transduction of gastric epithelial cell. It plays a pivotal role in activation of innate immunity and pathogen recognition and thus acts as a modulator in the development and progression of gastric cancer. Growing studies explored the association of polymorphisms in TLR4 with susceptibility to gastric cancer, but the results have remained controversial and conflicting. To investigate the effect of two selected TLR4 (+896A/G and +1196C/T) polymorphisms on gastric cancer, we performed a meta-analysis. Methods A comprehensive search was conducted to identify all eligible case-control publications investigating the association between TLR4 polymorphisms and gastric cancer risk. Odds ratios (OR) and corresponding 95% confidence intervals (CI) were used to assess such association. Results Up to March 26 2014, 10 published case-control studies from PubMed and EMBase were available, involving a total of 1888 gastric cancer patients and 3433 control subjects. In the overall meta-analyses, a significantly increased gastric cancer risk was detected in TLR4 +896A/G polymorphism (heterozygous model, AG vs. AA: OR = 1.67, 95% CI, 1.39-2.01; additive model, G vs. A: OR = 1.64, 95% CI, 1.37-1.95) and TLR4 +1196C/T polymorphism (heterozygous model, CT vs. CC: OR = 1.42, 95% CI, 1.11-1.81; additive model, T vs. C: OR = 1.36, 95% CI, 1.08-1.72), similar results were obtained in the subgroup analyses of Caucasian, whereas no associations were detected in any genetic models of non-Caucasian. Conclusions The overall results suggest that TLR4 polymorphisms (+896A/G and +1196C/T) may be associated with a significantly increased gastric cancer risk in Caucasian.

Gastric cancer is one of the most common tumors worldwide with extensive local tumor invasion, metastasis and poor prognosis. Thus, understanding the mechanisms regulating progression of gastric cancer is the key for developing effective therapeutic strategies. Tissue tranglutaminase-2 (TG2), a multifunctional member of the transglutaminase (TGase) family, has already been proved to play an important role in tumor initiation and progression. However, the specific role and underlying molecular mechanisms of TG2 in the progression of gastric cancer (GC) remain unknown. Here we showed that TG2 was highly expressed in GC tissues and positively associated with depth of tumor invasion and late TNM stage. With gain and lose-of function approaches, we found that TG2 promotes GC cell proliferation, migration and invasion, as well as tumorigenesis and peritoneal metastasis in vivo. These events were associated with activating of ERK1/2 pathway, and ERK1/2 inhibitor U0126 inhibited cell proliferation, migration and invasion induced by overexpression of TG2. Taken together, these findings suggest that TG2 contributes to tumorigenesis and progression of GC by activating the ERK1/2 signaling pathway and is a potential therapeutic target of metastatic gastric cancer. Citation Format: Liping Su, Xiaofeng Wang, Quan Zhou, Xiongyan Wu, Bingya Liu. Tissue tranglutaminase-2 promotes gastric cancer progression via ERK1/2 pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 945.

Background: Cancer-associated fibroblasts (CAFs) are important modifiers of tumor progression, which can actively communicate with cancer cells through soluble factors. Interleukin-33 (IL-33) is a regulator of immune and inflammatory responses and has also been associated with certain epithelial tumors. Although the importance of CAFs in gastric cancer has been documented in experimental studies, the mechanisms by which CAFs and IL-33 promote gastric cancer remain unclear. Objectives: The aim of this study was to determine the role of IL-33 in the epithelial-stromal interaction in gastric cancer and if CAFs can promote the invasion of gastric cancer cells via ERK pathway through IL-33. Methods: CAFs and normal fibroblasts (NFs) were isolated from gastric cancer tissues and adjacent non-cancerous tissues respectively. The identification of CAFs was detected by immunofluorescence (IF) and qRT-PCR. IL-33 expression in CAFs and gastric cancer cells was detected by qRT-PCR. CAFs was co-cultured with gastric cancer cells in the presence of exogenous TNF-α, IL-1β and/or their corresponding neutralizing antibodies, the expression of IL-33 in CAFs was detected using qRT-PCR. EMT changes and activation of ERK pathway in gastric cancer cells after co-cultured with CAF-conditioned media were detected by qRT-PCR and western blot, and cell invasion assays were performed by using 8 μm transwell chambers. Results: Gastric CAFs expressed more α-SMA, a marker of myofibroblasts, and more fibroblast activation protein (FAP) when compared with patient-matched NFs. In comparison to gastric cancer cells and NFs, IL-33 was highly expressed in CAFs. Upregulation of IL-33 in CAFs can be induced by co-culture with gastric cancer cells or in the presence of TNF-α and IL-1β, while deprivation of TNF-α and IL-1β using their neutralizing antibodies inhibited the upregulation of IL-33. CAFs induced EMT changes of gastric cancer cells, which was demonstrated by decreased expression of E-cadherin and increased expression of Twist1, MMP2, ZEB1 and ZEB2. However, deprivation of IL-33 using a neutralizing antibody impaired the EMT changes of gastric cancer cells. Gastric cancer cells co-cultured with CAFs or CAFs conditioned media showed enhanced ability of invasion than gastric cancer cells alone. However, adding neutralizing IL-33 antibody or IL-33-siRNA-CAFs conditioned media or U0126 into the culture system led to significantly decreased invasion (P < 0.05) of gastric cancer cells. Moreover, IL-33 secreted from CAFs activated ERK pathway in gastric cancer cells, however, the invasion of gastric cancer cells was attenuated when IL-33-ERK pathway was blocked. Conclusions: TNF-α and IL-1β derived from gastric cancer cells can significantly enhance the expression of IL-33 in CAFs, and IL-33 secreted by CAFs promotes gastric cancer invasion via the ERK signaling pathway. Note: This abstract was not presented at the meeting. Citation Format: Liping Su, Quan Zhou, Chenchen Wang, Xiongyan Wu, Xiaofeng Wang, Jianfang Li, Zhenggang Zhu, Bingya Liu. IL-33 secreted by cancer-associated fibroblasts mediates epithelial-stromal interactions and promotes gastric cancer invasion via ERK signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5069. doi:10.1158/1538-7445.AM2015-5069

Objectives: The molecular mechanisms involved in the regulation of gastric cancer progression by CAFs are still unclear. The aim of the present study was to determine if CAFs can enhance the tumor epithelial-to-mesenchymal transition (EMT) and invasion of gastric cancer cells, and link CAFs with activation of the IL-6/JAK/ STAT3 signaling pathway in the progression of gastric cancer. Methods: Fibroblasts were isolated from gastric cancer. The identification of CAFs was defined by immunofluorescence staining and qRT-PCR. IL-6 expression in CAFs and gastric cancer cells was detected by qRT-PCR and ELISA. EMT and activation of Stat3 pathway in gastric cancer cells were detected by realtime PCR and western blot. Cell invasion assays were performed by using 8 μm transwell chambers. Results: IL-6 expression in gastric cancer tissue was significantly higher than that in adjacent non-tumor tissue (3.450ng/g vs 2.665ng/g,P Conclusions: CAFs induced EMT and invasion of gastric cancer cells through activation of the IL-6/JAK/STAT3 pathway. Citation Format: Liping Su, Xiongyan Wu, Quan Zhou, Chenchen Wang, Bingya Liu. Cancer-associated fibroblast promotes gastric cancer invasion and epithelial-mesenchymal transition via the IL-6/JAK/STAT3 signaling pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4803. doi:10.1158/1538-7445.AM2014-4803

Background Gastric cancer (GC) is one of the most malignant tumors and the second leading cause of cancer-related deaths in the world. Luteolin, a flavonoid present in many fruits and green plants, suppresses cancer progression. The effects of luteolin on GC cells and their underlying mechanisms remain unclear. Methods Effects of luteolin on cell proliferation, migration, invasion, and apoptosis were examined in vitro and in vivo by cell counting kit-8 (CCK-8), transwell assays, and flow cytometry, respectively. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blots were performed to evaluate Notch1 signaling and activation of epithelial-mesenchymal transition (EMT) in GC cells treated with or without luteolin. Immunohistochemistry was performed to examine proliferation and Notch1 expression in xenograft tumors. Results Luteolin significantly inhibited cell proliferation, invasion, and migration in a dose-dependent and time-dependent manner and promoted cell apoptosis. Luteolin reversed EMT by shrinking the cytoskeleton and by inducing the expression of epithelial biomarker E-cadherin and downregulating the mesenchymal biomarkers N-cadherin, vimentin and Snail. Furthermore, Notch1 signaling was inhibited by luteolin, and downregulation of Notch1 had similar effects as luteolin treatment on cell proliferation, migration, and apoptosis. In addition, luteolin suppressed tumor growth in vivo. A higher expression of Notch1 correlated with a poor overall survival and a poor time to first progression. Furthermore, co-immunoprecipitation analysis revealed that activated Notch1 and β-catenin formed a complex and regulated cell proliferation, migration, and invasion. Conclusions In this study, GC progression was inhibited by luteolin through suppressing Notch1 signaling and reversing EMT, suggesting that luteolin may serve as an effective anti-tumor drug in GC treatment.