Gastric Tumor Cells Research Articles

Immunohistochemical study of MUC5AC, MUC1, MUC2, Cdx-2 in hyperplastic polyps and intestinal-type gastric adenocarcinoma

Aim – to study the features of MUC1, MUC2, MUC5AC, Cdx-2 immunohistochemical expression in hyperplastic polyps and intestinal-type gastric adenocarcinoma, and their relationships with the proliferation and apoptosis markers expression levels, EGFR and HER-2/neu growth receptors. Materials and м ethods. Pathomorphological and immunohistochemical studies of hyperplastic polyps from 30 patients (the age ranged from 52 to 73 years) and intestinal-type gastric adenocarcinoma from 30 patients (the age ranged from 49 to 86 years) were carried out. Results. It has been established that the gastric hyperplastic polyps are characterized by the high MUC5AC expression level, the moderate MUC1expression level, as well as the low MUC2 and Cdx-2 expression levels. The tumor cells of the intestinal-type gastric adenocarcinoma express gastric mucins less often, and are characterized by the moderate expression levels of the examined markers. In doing so, the carcinoma is characterized by the inverse correlations between gastric and intestinal mucins expression levels, and also by the correlations between the mucins markers expression levels and the histological grade of the tumor. It has been found, that the decrease in the MUC1 expression level by the tumor cells is associated with the increase in their proliferative level, while the decrease in the MUC1 and MUC5AC expression levels – with the decrease in their apoptosis level and also with the increase in the ErbB family growth receptors expression levels by the tumor cells. In addition, the increase in MUC2 expression level is associated with the decrease in apoptosis level, while the increase in the MUC2 and Cdx-2 expression levels – with the increase in EGFR expression level. Conclusions. There are close relationships between MUC1, MUC2, MUC5AC, Cdx-2 immunohistochemical expression levels and the levels of proliferative and apoptotic activity of intestinal-type gastric adenocarcinoma tumor cells, and also their expression of ErbB growth receptors.

GRP78 positively regulates estrogen‑stimulated cell growth mediated by ER‑α36 in gastric cancer cells.

Estrogen receptor (ER)‑α36, a novel isoform of ER, primarily mediates non‑classical estrogen signaling. It has been reported that ER‑α36‑mediated growth stimulating signals are involved in the malignancy of gastric tumor cells. However, the mechanism underlying the regulation of ER‑α36 function in development of gastric cancer remains to be elucidated. The present study investigated the role of 78 kDa glucose‑regulated protein (GRP78) in the regulation of ER‑α36 expression and signaling during the growth of gastric tumor cells. It was demonstrated that GRP78 expression was detectable in gastric cancer tumor tissues, and was positively‑correlated with tumor stage, lymphatic metastasis and ER‑α36 expression (P<0.05). An increased growth rate, and increased expression of ER‑α36 and the cell cycle regulator cyclin D1 was detected in cells with GRP78 overexpression (SGC‑High78 cells). SGC‑High78 cells are more sensitive to estrogen compared with SGC‑Control cells. Therefore, the results of the present study demonstrated that GRP78 positively regulated ER‑α36 expression and signaling with cell growth in gastric cancer, which is involved in gastric carcinogenesis.

684P - Targeting c-met and DNA double-strand break (DSB) repair pathways for BRCA-mutated gastric carcinomas

Background: In the present study, we underline the importance of combining c-Met Inhibitor (Inh.) and DSB repair Inhibitor (PARP Inh.) with the goal of accomplishing a synergistic therapeutic strategy in BRCA-mutated gastric carcinomas. Methods: Firstly, to verify whether c-Met affects tumor response to PARP Inh., AGS (low c-Met expression) and Hs746T (high c-Met expression) cell lines, were subjected to knockdown c-Met expression, in the presence of PARP Inh. (NU1025). Subsequently, we examined the correlation between BRCA1/2 and c-Met. We knocked down BRCA1/2 expression in AGS and Hs746T cells and treated them with PARP Inh. Next, we examined the effects of combining c-Met Inh. (SU11274) and PARP Inh. (NU1025), measured by MTT, clonogenic cell survival and Annexin, assays. We also evaluated the effect of combining PARP Inh. and c-Met Inh. in AGS/Hs746T xenograft tumor models. AGS/Hs746T -pcDNA3 and AGS/Hs746T -siBRCA cells were injected into SCID mice. Tumor sizes were measured every 3 days. DNA damage was assessed by γ-H2AX staining. Results: Silence of Met expression promoted Hs746T cells more sensitive to PARP Inh. to similar levels as the AGS cells, as indicated by decreased cell viability. Silence of BRCA1/2 expression sensitized only AGS cells, signifying that increased expression of c-Met renders cells resistant to PARP Inh. in the context of inactivating BRCA1/2. Combining c-Met Inh. and PARP Inh. suppressed cell growth and clonogenicity and enhanced apoptosis in both AGS and Hs746T cells. The dual inhibition was demonstrated to be even more successful when cells were knockdown for BRCA1/2. Also, co-inhibition treatment substantially reduced tumor growth to AGS/Hs746T -pcDNA3 and more even effectively to AGS/Hs746T –siBRCA1/2 xenograft models, compared to either Inh. alone. DNA damage was higher in AGS/Hs746T –siBRCA1/2 compare to AGS/Hs746T -pcDNA3 xenograft models. Conclusions: BRCA deficiency renders gastric tumor cells sensitive to PARP inhibition. In addition, treatment with c-Met Inh. enhanced Hs746T sensitivity to the PARP Inh. Our data demonstrate that dual Met/PARP inhibition is synergistic providing an effective therapeutic strategy in BRCA-mutated gastric carcinomas. Legal entity responsible for the study: M.V. Karamouzis Funding: None Disclosure: All authors have declared no conflicts of interest.

The long noncoding RNA XIAP-AS1 promotes XIAP transcription by XIAP-AS1 interacting with Sp1 in gastric cancer cells.

Long noncoding RNAs (lncRNAs) play roles in the tumorigenesis, proliferation and metastasis of tumor cells. Previous studies indicate that the transcription factor Sp1 is responsible for transcription of the XIAP gene, but it is unknown whether lncRNAs are involved in XIAP transcription. Herein, we identified a novel lncRNA, denoted as XIAP-AS1, transcribed from the first intron of the complementary strand of the XIAP gene. Using RNA FISH, cell fractionation and qRT-PCR, XIAP-AS1 was determined to be located primarily in the nucleus. After various XIAP-AS1 deletion mutants were expressed, RIP assays showed that only the full-length XIAP-AS1 RNA interacted with Sp1 and thereby participated in XIAP transcription. ChIP assays showed that XIAP-AS1 knockdown decreased the binding of Sp1 to the promoter region of XIAP. XIAP-AS1 knockdown promoted tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in gastric tumor cells, as cleaved caspase-3 and caspase-9 was detected. Moreover, in an in vivo mouse xenograft model, tumor cell proliferation was inhibited by XIAP-AS1 knockdown in response to TRAIL administration. In conclusion, our results indicate that XIAP-AS1 is involved in XIAP transcription by interacting with Sp1. Additionally, XIAP-AS1 is a potential target for TRAIL-induced apoptosis in gastric cancer cells.

Design of theranostic nanomedicine (II): synthesis and physicochemical properties of a biocompatible polyphosphazene&amp;ndash;docetaxel conjugate

To prepare an efficient theranostic polyphosphazene–docetaxel (DTX) conjugate, a new drug delivery system was designed by grafting a multifunctional lysine ethylester (LysOEt) as a spacer group along with methoxy poly(ethylene glycol) (MPEG) to the polyphosphazene backbone ([NP]n), and then DTX was conjugated to the carrier polymer using acid-cleavable cis-aconitic acid (AA) as a linker. The resultant polyphosphazene–DTX conjugate, formulated as [NP(MPEG550)3(Lys-OEt)(AA)(DTX)]n and named “Polytaxel”, exhibited high water solubility and stability by forming stable polymeric micelles as shown in its transmission electron microscopy image and dynamic light scattering measurements. Another important aspect of Polytaxel is that it can easily be labeled with various imaging agents using the lysine amino group, enabling studies on various aspects, such as its organ distribution, tumor-targeting properties, pharmacokinetics, toxicity, and excretion. The pharmacokinetics of Polytaxel was remarkably improved, with prolonged elimination half-life and enhanced area under the curve. Ex vivo imaging study of cyanine dye-labeled Polytaxel showed that intravenously injected Polytaxel is long circulating in the blood stream and selectively accumulates in tumor tissues. Polytaxel distributed in other organs was cleared from all major organs at ~6 weeks after injection. The in vitro study of DTX release from the carrier polymer showed that >95% of conjugated DTX was released at pH 5.4 over a period of 7 days. Xenograft trials of Polytaxel using nude mice against the human gastric tumor cell line MKN-28 showed complete tumor regression, with low systemic toxicity. Polytaxel is currently in preclinical study.

Abstract LB-216: Stroma-induced up-regulation of discoidin domain receptor 1 enhances peritoneal metastasis of gastric carcinomas

Abstract Introduction: Discoidin domain receptor-1 (DDR1), a receptor tyrosine kinase, is expressed by about 50% of gastric tumor cells in previous our study and is activated by fibrillar collagens, which are major components of tumor stroma. Further, it has been proposed that peritoneal metastasis is associated with high stroma content in gastric carcinomas (GCs). In this study we investigated the contribution of DDR1 activity to peritoneal metastasis of GCs. Methods: We performed the immunohistochemistry for DDR1 and Masson’s trichrome staining for accumulation of collagen bundles in 202 human GC tissues. The human GC cell lines (MKN-28 and KATO-III) were co-cultured with GCs-associated fibroblasts (CAFs), and the expression of DDR1 and signal transduction pathways were investigated by Western blot. We evaluated CAF-induced tumorigenesis of GCs cell lines and the effects of DDR1 specific inhibitor in 3D co-culture system and peritoneal seeding in xenograft animal models. Results: High stromal collagen correlated with peritoneal recurrence (P=0.004) and was positively associated with expression of DDR1 in GC tissues (P&amp;lt;0.001). We confirmed that co-culture with CAFs elevated the expression of DDR1 in GC cell lines. CAFs also enhanced GC cell line spheroid formation in vitro in a tumor cell DDR1-dependent manner. Co-implantation of CAFs with GC cells enhanced peritoneal tumor formation in vivo, an effect that was sensitive to pharmacologic inhibition of DDR1. Conclusion: Our findings suggest that CAF-induced elevation of tumor cells DDR1 enhances peritoneal tumorigenesis of GCs and the inhibition of DDR1 signaling is an attractive strategy for the treatment of GC peritoneal metastasis. Citation Format: Hyejin Jin, In-Hye Ham, Hye Jung Oh, Dakeun Lee, Sang-Uk Han, Rolf A. Brekken, Hoon Hur. Stroma-induced up-regulation of discoidin domain receptor 1 enhances peritoneal metastasis of gastric carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-216. doi:10.1158/1538-7445.AM2017-LB-216

Abstract 3837: Primary tumor cell lines derived from gastric PDX tumors displays differences in chemosensitivity

Abstract Gastric cancer is a multifactorial and fatal disease with limited effective therapeutic options. Its overall mortality ranked third among cancer-related deaths worldwide. Patient-derived-xenograft (PDX) models have been widely used in the biomedical research. They recapitulate the genomic diversity/heterogeneity of patient tumors, maintain a lot of the characteristics of the original tumors and showed much better correlation with clinical outcome comparing to traditional cell line derived xenograft models. However, the relatively low throughput and time-consuming nature of PDX studies prevent them from being used in large scale screening or mechanistic studies. To facilitate drug development in gastric cancer, different cancer cell lines have been developed in our laboratory from gastric PDX tumors. We performed STR analyses to validate their identity and RNAseq data also showed high similarities to their parental PDX tumors. These primary gastric tumor cell lines displayed differences from each other in their chemo-sensitivities in vitro and in vivo. They also differ in their protein expression such as the HER-2 expression level. Pharmacology studies using different compounds showed similar drug responses of these cell lines in tissue culture, xenografts using these cell lines and the original PDX models. Our data demonstrated the high fidelity of these cell lines to their parental PDX tumors and serve as a bridge between the high-throughput drug screening and highly clinical relevant PDX models and will greatly facilitate the drug development in gastric cancer. Citation Format: Xiaomei Ge, Fulin Qiang, Yixin Zhang, Jibing Liu, Lei Yang, Yingying Qu, Fubo Xie, Xueting Li, Ying Gu, He Zhou. Primary tumor cell lines derived from gastric PDX tumors displays differences in chemosensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3837. doi:10.1158/1538-7445.AM2017-3837

PTTG3P promotes gastric tumour cell proliferation and invasion and is an indicator of poor prognosis.

Pseudogenes play a crucial role in cancer progression. However, the role of pituitary tumour‐transforming 3, pseudogene (PTTG3P) in gastric cancer (GC) remains unknown. Here, we showed that PTTG3P expression was abnormally up‐regulated in GC tissues compared with that in normal tissues both in our 198 cases of clinical samples and the cohort from The Cancer Genome Atlas (TCGA) database. High PTTG3P expression was correlated with increased tumour size and enhanced tumour invasiveness and served as an independent negative prognostic predictor. Moreover, up‐regulation of PTTG3P in GC cells stimulated cell proliferation, migration and invasion both in vitro in cell experiments and in vivo in nude mouse models, and the pseudogene functioned independently of its parent genes. Overall, these results reveal that PTTG3P is a novel prognostic biomarker with independent oncogenic functions in GC.

PPeOp from Omphalia lapidescens Schroet induces cell cycle arrest and inhibits the migration of MC-4 gastric tumor cells.

The aim of the present study was to investigate the effect of purified Omphalia lapidescens protein (pPeOp) extracted by polyvinylpyrrolidone from the fungus Omphalia lapidescens Schroet on the proliferation and cell cycle progression of MC-4 human gastric tumor cells. Using polyvinylpyrrolidone, pPeOp was extracted from O. lapidescens Schroet. MC-4 cells were cultured with 30, 60 or 90 µg/ml pPeOp, with 5-fluorouracil used as a positive control. Survival rates of treated cells were significantly decreased compared with those of the untreated control group in a dose-dependent manner. Using flow cytometric analysis, cells treated with pPeOp were demonstrated to arrest in S phase and exhibit abnormal G0/G1 and G2/M phase cell cycle distribution. In addition, a wound healing assay demonstrated that pPeOp significantly inhibited the migration of MC-4 cells. The mRNA and protein expression levels of cyclin D1/cyclin-dependent kinase (CDK) 4, cyclin B/CDK1, cyclin A/CDK2, matrix metalloproteinase (MMP)-2 and MMP-9 were determined using reverse transcription-quantitative polymerase chain reaction analysis and western blotting. The mRNA expression level of CDK4 and cyclin A was significantly increased compared with the untreated control; however, cyclin D1, CDK1, CDK2, cyclin B, MMP-2, and MMP-9 exhibited a significantly decreased mRNA expression level, indicating that there is a negative association between concentration and cyclin D1 expression levels. The expression of the cycle arrest-associated proteins and migration-associated proteins examined were similar to the observed mRNA expression levels. In conclusion, pPeOp was identified to inhibit migration of and cause S phase cell cycle arrest in MC-4 cells.

Characterization of Bitter Compounds via Modulation of Proton Secretion in Human Gastric Parietal Cells in Culture.

Humans perceive bitterness via around 25 different bitter receptors. Therefore, the identification of antagonists remains a complex challenge. We previously demonstrated several bitter-tasting compounds such as caffeine to induce acid secretion in the stomach and in a human gastric tumor cell line (HGT-1). Here, the results of a fluorescent-based in vitro assay using HGT-1 cells and a human sensory panel testing nine selected potential bitter modulators, with or without the bitter compounds caffeine or theobromine, were compared. Of the bitter-modulating compounds tested, eriodictyol, matairesinol, enterolacton, lariciresinol, and homoeriodictyol reduced the effect of caffeine on proton secretion by -163 ± 14.0, -152 ± 12.4, -74 ± 16.4, -58 ± 7.2, and -44.6 ± 16.5%, respectively, and reduced the bitter intensity of caffeine in the human sensory panel. In contrast, naringenin and 5,7-dihydroxy-4(4-hydroxyphenyl)chroman-2-one neither reduced the caffeine-induced proton secretion in HGT-1 cells nor showed an effect on bitter intensity perceived by the sensory panel. Results for theobromine were not as pronounced as those for caffeine, but followed a similar trend. The results demonstrate that the HGT-1 in vitro assay is a useful tool to identify potential bitter-masking compounds. Nevertheless, a sensory human panel is necessary to quantify the bitter-masking potency.

STRAIL-iRGD is a promising therapeutic agent for gastric cancer treatment

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells and augments chemotherapeutics in vivo. Here, we developed sTRAIL-iRGD, a recombinant protein consisting of sTRAIL fused to CRGDKGPDC, a C-terminal end binding peptide with an integrin-binding arginine-glycine-aspartic acid (iRGD) motif. CRGDKGPDC is a tumor-homing peptide with high penetration into tumor tissue and cells. We found that sTRAIL-iRGD internalized into cultured gastric cancer tumor cells and localized to both the tumor mass in vivo and three-dimensional multicellular spheroids in vitro. sTRAIL-iRGD had an antitumor effect in tumor cell lines, multicellular spheroids and nude mice with tumors. Repeated treatment with sTRAIL-iRGD reduced tumor growth and volume in vivo. Mice treated with sTRAIL-iRGD and paclitaxel (PTX) in combination showed no sign of sTRAIL-iRGD-related liver toxicity. Our data suggest that sTRAIL-iRGD is a promising anti-gastric cancer agent with high selectivity and limited systemic toxicity.

Laparoscopic and endoscopic cooperative surgery for gastrointestinal tumor.

With technological progress of endoscopic submucosal dissection (ESD) in the last decade, several laparoscopic and endoscopic cooperative surgeries (LECS) for gastrointestinal tumor have recently been developed. LECS is definitely favorable to the minimization of surgical margin, which leads to functional and anatomical preservation of gastrointestinal tract. LECS for gastrointestinal tumor is mainly sorted by two categories: exposure procedures and non-exposure procedures between endoluminal and extraluminal spaces. Exposure procedures have the potential risk of gastric contents or tumor cells spilling out over the abdominal cavity, because the stomach wall has to be perforated intentionally during the procedure. In order to avoid the potential these risks, non-exposure procedures have been developed. Currently, the LECS concept has rapidly permeated for treatment of gastrointestinal tumor due to its certainty and safety, although there is still room for improvement to lessen its technical difficulty. This review describes the current LECS for gastrointestinal tumor based on the several articles.

Curcumin suppresses gastric tumor cell growth via ROS-mediated DNA polymerase \u03b3 depletion disrupting cellular bioenergetics

BackgroundCurcumin, as a pro-apoptotic agent, is extensively studied to inhibit tumor cell growth of various tumor types. Previous work has demonstrated that curcumin inhibits cancer cell growth by targeting multiple signaling transduction and cellular processes. However, the role of curcumin in regulating cellular bioenergetic processes remains largely unknown.MethodsWestern blotting and qRT-PCR were performed to analyze the protein and mRNA level of indicated molecules, respectively. RTCA, CCK-8 assay, nude mice xenograft assay, and in vivo bioluminescence imaging were used to visualize the effects of curcmin on gastric cancer cell growth in vitro and in vivo. Seahorse bioenergetics analyzer was used to investigate the alteration of oxygen consumption and aerobic glycolysis rate.ResultsCurcumin significantly inhibited gastric tumor cell growth, proliferation and colony formation. We further investigated the role of curcumin in regulating cellular redox homeostasis and demonstrated that curcumin initiated severe cellular apoptosis via disrupting mitochondrial homeostasis, thereby enhancing cellular oxidative stress in gastric cancer cells. Furthermore, curcumin dramatically decreased mtDNA content and DNA polymerase γ (POLG) which contributed to reduced mitochondrial oxygen consumption and aerobic glycolysis. We found that curcumin induced POLG depletion via ROS generation, and POLG knockdown also reduced oxidative phosphorylation (OXPHOS) activity and cellular glycolytic rate which was partially rescued by ROS scavenger NAC, indiating POLG plays an important role in the treatment of gastric cancer. Data in the nude mice model verified that curcumin treatment significantly attenuated tumor growth in vivo. Finally, POLG was up-regulated in human gastric cancer tissues and primary gastric cancer cell growth was notably suppressed due to POLG deficiency.ConclusionsTogether, our data suggest a novel mechanism by which curcumin inhibited gastric tumor growth through excessive ROS generation, resulting in depletion of POLG and mtDNA, and the subsequent disruption of cellular bioenergetics.

Increased nuclear localization of substance P in human gastric tumor cells

Gastric cancer (GC) is an aggressive disease that remains the fourth most common type of cancer and is the second leading cause of cancer-related death worldwide. Treatment of advanced or metastatic GC has seen little progress and median overall survival in this group remains <1 year. It is urgent to investigate new mechanisms to understand GC progression. It is known that substance P (SP), after binding to the neurokinin-1 (NK-1) receptor, elicits GC proliferation; that GC cells and samples express NK-1 receptors; that NK-1 receptor antagonists, in a concentration dependent manner, inhibit the proliferation of GC cells and that these cells die by apoptosis. However, the presence of SP in GC and normal gastric cells is unknown. In order to know more on the involvement of the SP/NK-1 receptor system in GC, we studied in thirty human GC and normal gastric samples the immunolocalization of SP after using an immunohistochemical technique. SP was observed in the cytoplasm and in the nucleus of GC and normal gastric cells. The nuclear expression of SP was higher in GC cells than in normal cells. No significant difference was observed when the cytoplasmatic expression of SP in normal and GC cells was compared. The findings suggest that SP plays an important role in both nuclear function and GC.

Regulation of Gastric Carcinogenesis by InflammatoryCytokines.

Chronic inflammation caused by infection with Helicobacter pylori and autoimmune gastritis increases an individual’s risk of developing gastric cancer. More than 90% of gastric cancers are adenocarcinomas, which originate from epithelial cells in the chronically inflamed gastric mucosa. However, only a small subset of chronic gastritis patients develops gastric cancer, implying a role for genetic and environmental factors in cancer development. A number of DNA polymorphisms that increase gastric cancer risk have mapped to genes encoding cytokines. Many different cytokines secreted by immune cells and epithelial cells during chronic gastritis have been identified, but a better understanding of how cytokines regulate the severity of gastritis, epithelial cell changes, and neoplastic transformation is needed. This review summarizes studies in both human and mouse models, describing a number of different findings that implicate various cytokines in regulating the development of gastric cancer.

Grifolin exhibits anti-cancer activity by inhibiting the development and invasion of gastric tumor cells.

Grifolin, a natural product isolated from the mushroom Albatrellus confluens, has been reported to be a potent anti-cancer agent in nasopharyngeal carcinoma and osteosarcoma. The data obtained in this study revealed that grifolin is capable of inhibiting the growth and invasion of gastric cancer cells by inducing apoptosis and suppressing the ERK1/2 pathway. Our results support the potent utility of grifolin as an anti-tumor lead compound against gastric cancer cells.

Proteogenomic analysis of NCC-S1M, a gastric cancer stem cell-like cell line that responds to anti-PD-1

To elucidate signaling pathways that regulate gastric cancer stem cell (CSC) phenotypes and immune checkpoint, we performed a proteogenomic analysis of NCC-S1M, which is a gastric cancer cell line with CSC-like characteristics and is the only syngeneic gastric tumor cell line transplant model created in the scientific community. We found that the NCC-S1M allograft was responsive to anti-PD-1 treatment, and overexpressed Cd274 encoding PD-L1. PD-L1 was transcriptionally activated by loss of the TGF-β signaling. Il1rl1 protein was overexpressed in NCC-S1M cells compared with NCC-S1 cells that are less tumorigenic and less chemoresistant. Il1rl1 knockdown in NCC-S1M cells reduced tumorigenic potential and in vivo chemoresistance. Our proteogenomic analysis demonstrates a role of Smad4 loss in the PD-L1 immune evasion, as well as Il1rl1's role in CSC-like properties of NCC-S1M.

Blocking TGF-β1 by P17 peptides attenuates gastric cancer cell induced peritoneal fibrosis and prevents peritoneal dissemination in vitro and in vivo.

Our previous study demonstrated that the peritoneal stroma environment favors proliferation of tumor cells by serving as a rich source of growth factors and chemokines known to be involved in tumor metastasis. In this study, we investigated the interaction between gastric cancer cells and peritoneal mesothelial cells, and determined the effects of TGF-β1 in this processing. Human peritoneal tissues and peritoneal wash fluid were obtained, which examined by hematoxylin and eosin staining or ELISA for measurements of TGF-β1 levels. The peritoneal mesothelial cells were co-incubated with the supernatants of gastric cancer, the expression of TGF-β1, collagen and fibronectin was observed by ELISA and western blot. We then investigated the effects of serum-free conditioned media from HSC-39 gastric cancer cells on the peritoneum of nude mice, and the effects of peritoneal fibrosis on the development of peritoneal metastasis in vivo. The peritoneum from gastric patients were thickened and contained extensive fibrosis. After co-culture both gastric tumor cells and mesothelial cells, we found that TGF-β1 expression was greatly increased in the co-culture system compared to individual culture condition. Serum-free Conditioned Media from HSC-39 was able to induce extracellular matrix expression in vitro and in vivo, and tumorigenicity in mice with peritoneal fibrosis was greater than in mice with normal peritoneum, while blocking TGF-β1 by peptide P17 can partially inhibit these effects. In conclusion, these results indicated that the interaction of gastric cancer with peritoneal fibrosis and determined that TGF-β1 plays a key role in induction of peritoneal fibrosis, which in turn affected dissemination of gastric cancer.

5O - GBR1302-BEAT® bispecific antibody targeting CD3 and HER2 demonstrates a higher anti-tumor potential than current HER2-targeting therapies

Aim/Background: Current HER2-targeting therapies (such as Herceptin or TDM-1) have demonstrated efficacy only in cancers significantly overexpressing HER2 (cancers characterized as HER2 3+ according to the IHC HercepTest®) and are also limited by several possible resistance mechanisms. Bridging T lymphocytes to HER2 tumor cells has the potential to trigger a more efficient elimination of tumor cells. Using the Glenmark BEAT® platform, we have produced a bispecific antibody, GBR 1302, targeting human CD3ε and HER2 and designed to effectively recruit cytotoxic T lymphocytes against HER2 positive cancer cells. These studies aimed at comparing the potency of GBR 1302 and the current HER-2 targeting therapies. Methods: Short term (48 hours) in vitro killing assays, against different breast or gastric tumor cell lines characterized as HER2 3+ or 2+ by IHC HercepTest and using resting human PBMCs from healthy donors as effector cells. Metabolic or colony forming readouts were performed to quantify tumor cell death. In vivo tumor xenograft models, either subcutaneous or disseminated (intravenous injection of the cells). The tumor cells were grafted with human PBMCs to bring effector cells. The treatments were started the day of the tumor+PBMC graft. Results: Killing experiments comparing the potency of GBR 1302 to current HER2-targeting therapies such as trastuzumab or T-DM1 highlighted a more efficient, rapid and complete cytotoxic potential for GBR 1302 against a variety of tumor cells. In particular, killing assays on breast or gastric tumor cell lines characterized as HER2 3+ or 2+ by IHC HercepTest, followed by a clonogenic readout or an absolute count of viable target cells, revealed a significantly more complete killing by GBR 1302 as compared to T-DM1. Tumor xenograft models confirmed these data, by showing that T-DM1 could only slow subcutaneous tumor growth, or delay the time of sacrifice of mice in a metastatic model, while GBR 1302 was able to abrogate almost completely tumor development. Conclusions: These observations highlight that GBR 1302, consistent with its mechanism of action, is able to trigger a rapid and very potent mobilization of T lymphocyte cytotoxic functions against cancer cells. These features warrant for GBR 1302 a potential to elicit in patients a more complete response in a shorter period compared to current HER2 targeting treatments which are essentially cytostatic drugs, thereby limiting the probability of the emergence and Darwinian selection of resistant cells. Legal entity responsible for the study: Glenmark Pharmaceuticals S.A Funding: Glenmark Pharmaceuticals S.A Disclosure: J. Back: Employee of Glenmark Pharmaceuticals S.A

Depletion of FOXM1 via MET Targeting Underlies Establishment of a DNA Damage-Induced Senescence Program in Gastric Cancer.

Deregulated signaling via the MET receptor tyrosine kinase is abundant in gastric tumors, with up to 80% of cases displaying aberrant MET expression. A growing body of evidence suggests MET as a potential target for tumor radiosensitization. Cellular proliferation and DNA damage-induced senescence were studied in a panel of MET-overexpressing human gastric cancer cell lines as well as in xenograft models after MET inhibition and/or ionizing radiation. Pathways activation and protein expression were assessed by immunoblotting and immunohistochemistry. Tumor tissue microarrays (91 gastric cancer patients) were generated and copy number alteration (178 patients) and gene expression (373 patients) data available at The Cancer Genome Atlas were analyzed to assess the coalterations of MET and FOXM1. MET targeting administered before ionizing radiation instigates DNA damage-induced senescence (∼80%, P < 0.001) rather than cell death. MET inhibition-associated senescence is linked to the blockade of MAPK pathway, correlates with downregulation of FOXM1, and can be abrogated (11.8% vs. 95.3%, P < 0.001) by ectopic expression of FOXM1 in the corresponding gastric tumor cells. Cells with ectopic FOXM1 expression demonstrate considerable (∼20%, P < 0.001) growth advantage despite MET targeting, suggesting a novel clinically relevant resistance mechanism to MET inhibition as the copresence of both MET and FOXM1 protein (33%) and mRNA (30%) overexpression as well as gene amplification (24,7%) are common in patients with gastric cancer. FOXM1, a negative regulator of senescence, has been identified as a key downstream effector and potential clinical biomarker that mediates MET signaling following infliction of DNA damage in gastric tumors. Clin Cancer Res; 22(21); 5322-36. ©2016 AACR.