Positive Gastric Cancer Cells Research Articles

Fabrication of Metal Nuclear Acid Framework to Enable Carrier‐Free MNAzyme Self‐Delivery for Gastric Cancer Treatment

AbstractMulti‐component deoxyribozymes (MNAzymes) have shown extraordinary potential in precise gene therapy in vitro, however, the in vivo application is limited by complicated delivery systems. Herein, a novel DNA‐metal binding mechanism is discovered, and metal‐nucleic acid frameworks (MNFs) are built composed of MNAzymes and metal ions, which enable the carrier‐free self‐delivery of MNAzymes. Metal ions have a high affinity to DNA, however, the binding of metals with DNA at 20–30 base pair long (that normally a MNAzyme has) to form MNF structure is challenged by stringent high‐temperature synthesis conditions, poor stability of the products, and lack of targeting capabilities. While, it is discovered that through folding and entanglement of the MNAzyme with an aptamer tail, and prolonging the sequence to 71 base pair, the metal MNAzymes binding is significantly improved and stabilized to MNF structure even at room temperature. Moreover, the aptamer tail also endows MNFs with targeting capabilities. As proof of concept, a carrier‐free Ca/MNAzyme delivery system at room temperature, loaded with the model imaging protein BSA‐Cy5 is synthesized. This system can effectively target Her‐2 positive gastric cancer cells with the Her‐2 responsive aptamer tail and initiate dual gene regulation, thereby inducing energy depletion in cancer cells.

Development of Aptamer-DNAzyme based metal-nucleic acid frameworks for gastric cancer therapy.

The metal-nucleic acid nanocomposites, first termed metal-nucleic acid frameworks (MNFs) in this work, show extraordinary potential as functional nanomaterials. However, thus far, realized MNFs face limitations including harsh synthesis conditions, instability, and non-targeting. Herein, we discover that longer oligonucleotides can enhance the synthesis efficiency and stability of MNFs by increasing oligonucleotide folding and entanglement probabilities during the reaction. Besides, longer oligonucleotides provide upgraded metal ions binding conditions, facilitating MNFs to load macromolecular protein drugs at room temperature. Furthermore, longer oligonucleotides facilitate functional expansion of nucleotide sequences, enabling disease-targeted MNFs. As a proof-of-concept, we build an interferon regulatory factor-1(IRF-1) loaded Ca2+/(aptamer-deoxyribozyme) MNF to target regulate glucose transporter (GLUT-1) expression in human epidermal growth factor receptor-2 (HER-2) positive gastric cancer cells. This MNF nanodevice disrupts GSH/ROS homeostasis, suppresses DNA repair, and augments ROS-mediated DNA damage therapy, with tumor inhibition rate up to 90%. Our work signifies a significant advancement towards an era of universal MNF application.

Abstract 62: Intra-tumor persistence of activated anti-mesothelin hYP218 CAR T cells is associated with increased efficacy in gastric and colorectal cancers

Abstract Purpose: Patients with gastric and colorectal cancers that have progressed after standard therapies have poor prognosis. Given the high expression of mesothelin in these tumor types, we examined the potential utility of the anti-mesothelin hYP218 CAR T cells to treat these cancers. Experimental Design: Cell surface mesothelin expression in a panel of gastric and colorectal cancer cell-lines was analyzed using flow cytometry. In vitro efficacy of hYP218 CAR T cells against these cell lines was evaluated using cytotoxicity and cytokine release assays. In vivo efficacy of hYP218 CAR T cells was studied using gastric (HGC27) and colorectal (SW48) tumor xenografts in NSG mice. NSG mice were injected with 1 × 106 cancer cells and the tumors were allowed to grow until they reached a volume of 80-100mm³, after which the mice were treated with either 5 × 106 hYP218 CAR T cells, 5 × 106 untransduced T cells, or saline and monitored over time. Persistence of CAR T cells in the tumor was evaluated over time and expression of activation and exhaustion markers were studied. Results: hYP218 CAR T cells demonstrated strong cytotoxic activity against mesothelin positive gastric and colorectal cancer cell lines and exhibited increased pro-inflammatory cytokine production. In both the HGC27 and SW48 tumor models hYP218 CAR T cells demonstrated significant reduction in tumor volume and overall survival compared to mice treated with untransduced T cells or saline. CAR T cells isolated from tumors of both the xenograft models, at day 40 after treatment, displayed higher expression levels of activation markers CD39 and CD69 when compared to the infused product. Furthermore, a concomitant elevation in the levels of effector cytokines, TNF-α and IFN-γ, was detected in the blood of treated mice demonstrating the functional capacity of the CAR T cells to not only persist but also to maintain an activated state. Conclusions: hYP218 CAR T cells show anti-tumor efficacy against gastric and colorectal tumor xenografts due to increased accumulation of activated CAR T cells in the tumor and warrants further investigation for treatment of patients with these cancers. Citation Format: Sameer A. Mir, Abhilash Venugopalan, Jingli Zhang, Mana Kamana Khanal, Chaido Stathopoulou, Manjistha Sengupta, Qun Jiang, Raffit Hassan. Intra-tumor persistence of activated anti-mesothelin hYP218 CAR T cells is associated with increased efficacy in gastric and colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 62.

Molecular characterization and potential therapeutic roles of miR125a in HER-2 positive gastric cancer.

miR-125a-3p could have a role in gastric cancer by targeting HER2. This study aimed to investigate the expression pattern of miR-125a-3p, identify the expression level of its target gene in gastric carcinoma, and test its effect in HER-2 positive gastric carcinoma cells. The levels of miR-125a-3p in both cancer and noncancer tissues were measured by using Quantitative real-time polymerase chain in 70 gastric carcinomas. Immunohistochemical study was used to measure the expression of HER2 protein in these carcinomas. In addition, the level of expression of this miRNA is correlated to different pathological and clinical parameters. The effects of miR-125a-3p alone and in combination with 5-FU (fluorouracil) on the growth of HER2 positive (NUGC4) and HER2 negative (ECC10) gastric carcinoma cells were also analyzed by in vitro studies. Most gastric cancer tissues samples showed downregulation of miR-125a-3p (84%) when compared to their noncancer tissues. Significant correlations of downregulation of miR-125a-3p with cancer recurrence and pathological staging of gastric carcinoma (P = 0. 02 and 0.02, respectively) were noted. HER2 protein expression correlated significantly and inversely with miR-125a-3p expression (P < 0.05). A reduction in cell growth rate was noted significantly in miR-125a-3p transfected gastric carcinoma cells when 5-FU was added to them in comparison to other control cells (P < 0.01). When both gastric carcinoma cell lines were transfected with miR-125a-3p, a significantly higher growth inhibition percentage in HER2 positive (NUGC4) cell line was seen in comparison to the HER2 negative (ECC10) cells (P < 0.01). miR-125a-3p plays a significant role in the pathogenesis of gastric carcinoma. Therapeutic transfection of miR-125a-3p in HER2 positive gastric cancer cells resulted in reduced cell proliferation and potentiate the effect of 5-FU.

Mono-PEGylated thermostable Bacillus caldovelox arginase mutant (BCA-M-PEG20) induces apoptosis, autophagy, cell cycle arrest and growth inhibition in gastric cancer cells

Gastric cancer is one of the most common malignant solid tumors in the world, especially in Asia with high mortality due to a lack of effective treatment. The potential usage of the newly constructed arginine-depleting enzyme—mono-PEGylated Bacillus caldovelox arginase mutant (BCA-M-PEG20), an effective drug against multiple cancer cell lines such as cervical and lung cancers, for the treatment of gastric cancer was demonstrated. Our results indicated that BCA-M-PEG20 significantly inhibited argininosuccinate synthetase (ASS)-positive gastric cancer cells, MKN-45 and BGC-823, while another arginine-depleting enzyme, arginine deiminase (ADI, currently under Phase III clinical trial), failed to suppress the growth of gastric cancer cells. In vitro studies demonstrated that BCA-M-PEG20 inhibited MKN-45 cells by inducing autophagy and cell cycle arrest at the S phase under 0.58 U/mL (IC50 values). Significant caspase-dependent apoptosis was induced in MKN-45 after the treatment with 2.32 U/mL of BCA-M-PEG20. In vivo studies showed that administrations of BCA-M-PEG20 at 250 U/mouse twice per week significantly suppressed about 50% of tumor growth in the MKN-45 gastric cancer xenograft model. Taken together, BCA-M-PEG20 demonstrated a superior potential to be an anti-gastric cancer drug.

An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer

Gastric cancer is a worldwide health problem. Chemotherapy and radiotherapy are of great importance in the management of advanced gastric cancer. However, their therapeutic efficacy is limited by off-target side effects. Peptide-drug conjugates (PDCs) are a novel strategy for tumor-targeted drug delivery to overcome the existing drug resistance mechanisms and improve antitumor effects. Kita-Kyushu lung cancer antigen 1 (KK-LC-1) is exclusively expressed in several types of cancer including gastric cancer, representing a promising target for drug delivery. Here, we suggested KK-LC-1 as a potential target for PDC design for the first time and reported the first KK-LC-1-targeting PDC product 1131-MMAE, which is composed of a KK-LC-1-targeting peptide and an antimitotic drug conjugated by an enzymatically cleavable linker. We observed that 1131-MMAE could be efficiently endocytosed by KK-LC-1 positive gastric cancer cells for subsequent drug release and arrest the cell cycle at the most radiosensitive G2/M phase. We demonstrated that 1131-MMAE could significantly delay tumor growth with reduced toxicity than free drugs as a monotherapy. We further confirmed that 1131-MMAE was also a potent radiosensitizer. 1131-MMAE could selectively enhance the radiation response of KK-LC-1 positive tumor cells and achieve improved tumor control when combined with low-dose radiation. Overall, our study proposed an optimized therapeutic regimen for precision chemoradiotherapy, which has translational potential in multiple types of cancer.

Abstract 5487: An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer

Abstract Gastric cancer is a worldwide health problem. Concurrent chemoradiotherapy (CRT) represents an important strategy to reduce recurrence and prolong survival for gastric cancer. However, non-targeted cytotoxic drugs capable of enhancing radiosensitization remain the main choice to be combined with radiotherapy, which has limited the anti-tumor efficacy of CRT by off-target side effects. Monomethyl auristatin E (MMAE) is a highly potent anti-tubulin agent and has been reported to act as an effective radiosensitizer recently. Kita-Kyushu lung cancer antigen 1 (KK-LC-1) is a cancer/testis antigen highly and exclusively expressed in multiple types of cancers including gastric cancer, representing a promising target for drug delivery. Previously, we identified a KKLC-1-targeting cyclic nonapeptide (1131 peptide) by screening a phage display peptide library. Here, we described a novel peptide-drug conjugate (PDC) 1131-MMAE by connecting the 1131 peptide with MMAE via a protease-sensitive linker for tumor-selective radiosensitization. We demonstrated that 1131-MMAE could be efficiently internalized by gastric cancer cells through KKLC1-mediating endocytosis and exhibited a comparable cytotoxicity profile with free MMAE. 1131-MMAE could arrest the cell cycle at the most radiosensitive G2/M phase in a dose-dependent manner and selectively enhance the radiation response of KK-LC-1 positive gastric cancer cells by increasing DNA double-strand break as indicated by γH2Ax immunofluorescence staining. Furthermore,1131-MMAE was able to inhibit tumor growth as a monotherapy at 2mg/kg with a good safety profile in vivo. Increased tumor control was achieved when 1131-MMAE was combined with radiotherapy at a relatively low dose of 0.3mg/kg. Overall, we proposed an MMAE-based PDC targeting a novel tumor marker KK-LC-1 and demonstrated its tumor-selective radiosensitization in gastric cancer models for the first time. This anti-KKLC1 PDC may help to promote the clinical progress in precision chemoradiotherapy for multiple types of cancer. Citation Format: Xiaotong Chen, Fangcen Liu, Xiaoxiao Yu, Lin Li, Jiayao Yan, Baorui Liu. An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5487.

C-Met-Specific Chimeric Antigen Receptor T Cells Demonstrate Anti-Tumor Effect in c-Met Positive Gastric Cancer.

Simple Summaryc-Met is known to be overexpressed in gastric cancers. Here, we developed anti-c-Met CAR T cell and measured its anti-tumor efficacy in vitro and in vivo. Our anti c-Met CAR T cells have shown selective killing of c-Met overexpressed gastric cancer cells. Based on our results, we suggest that anti-c-Met CAR T cell therapy could be effective for gastric cancer patients.Chimeric antigen receptor (CAR) technology has been highlighted in recent years as a new therapeutic approach for cancer treatment. Although the impressive efficacy of CAR-based T cell adoptive immunotherapy has been observed in hematologic cancers, limited effect has been reported on solid tumors. Approximately 20% of gastric cancer (GC) patients exhibit a high expression of c-Met. We have generated an anti c-Met CAR construct that is composed of a single-chain variable fragment (scFv) of c-Met antibody and signaling domains consisting of CD28 and CD3ζ. To test the CAR construct, we used two cell lines: the Jurkat and KHYG-1 cell lines. These are convenient cell lines, compared to primary T cells, to culture and to test CAR constructs. We transduced CAR constructs into Jurkat cells by electroporation. c-Met CAR Jurkat cells secreted interleukin-2 (IL-2) only when incubated with c-Met positive GC cells. To confirm the lytic function of CAR, the CAR construct was transduced into KHYG-1, a NK/T cell line, using lentiviral particles. c-Met CAR KHYG-1 showed cytotoxic effect on c-Met positive GC cells, while c-Met negative GC cell lines were not eradicated by c-Met CAR KHYG-1. Based on these data, we created c-Met CAR T cells from primary T cells, which showed high IL-2 and IFN-γ secretion when incubated with the c-Met positive cancer cell line. In an in vivo xenograft assay with NSG bearing MKN-45, a c-Met positive GC cell line, c-Met CAR T cells effectively inhibited the tumor growth of MKN-45. Our results show that the c-Met CAR T cell therapy can be effective on GC.

Sijunzi Decoction Inhibits Stemness by Suppressing β-Catenin Transcriptional Activity in Gastric Cancer Cells.

To investigate a previously uncharacterized function of Sijunzi Decoction (SJZD) in inhibition of gastric cancer stem cells (GCSCs). MKN74 and MKN45, two CD44 positive gastric cancer cell lines with stem cell properties were used. The cells were divided into 2 groups. Treatment group was treated with SJZD (1-5 mg/mL) for indicated time (48 h-14 days). The control group was treated with equal volume of phosphate buffered saline. Cell Counting Assay Kit-8 were used to measure cell viability. Spheroid colony formation and GCSCs marker expression were performed to determine GCSCs stemness. Cell fractionation and chromatin immunoprecipitation assays were used to assess the distribution and DNA-binding activity of β-catenin after SJZD treatment, respectively. SJZD treatment repressed cell growth and induced apoptosis in MKN74 and MKN45 cell lines (P<0.05). Moreover, SJZD dramatically inhibited formation of spheroid colony and expression of GCSC markers in GC cells (P<0.05). Mechanistically, SJZD reduced nuclear accumulation and DNA binding activity of β-catenin (P<0.05), the key regulator for maintaining CSC stemness. SJZD inhibits GCSCs by attenuating the transcriptional activity of β-catenin.

Enhanced Antitumor Effect of Trastuzumab and Duligotuzumab or Ipatasertib Combination in HER-2 Positive Gastric Cancer Cells.

Simple SummaryThe ToGA trial has demonstrated, in HER2-expressing patients, that unresectable and advanced gastric cancer, chemotherapy and trastuzumab in combination increase overall survival, even if it is still unclear why after one year the same patients are non-responsive to trastuzumab treatment. Here, we have demonstrated that in HER2-positive gastric cancer cell lines, the addition of duligotuzumab, targeting HER3 receptor, or ipatasertib, targeting AKT protein, enhances the antitumor effect of trastuzumab in vitro through a full inhibition of the membrane signals, on HER2 and HER3, and of downstream signaling, including AKT, and MAPK pathways. Hence, this study suggests a novel and biomarker-driven therapeutic strategy supporting further evaluation of the anti-tumor efficacy of these combinations in HER2 human gastric cancer patients.The anti-HER2 monoclonal antibody trastuzumab is a key drug for the treatment of HER2-positive gastric cancer (GC); however, its activity is often limited by the onset of resistance and mechanisms of resistance are still poorly understood. Several targeted agents showed synergistic activity by concomitant use with trastuzumab in vitro and are under clinical investigation. The aim of this study was to assess the antitumor activity of duligotuzumab, an anti HER3/EGFR antibody or ipatasertib, an AKT inhibitor, combined with trastuzumab in a panel of HER2-positive human gastric cancer cells (GCC), and the efficacy of such combinations in HER2-resistant cells. We have assessed the efficacy of duligotuzumab or ipatasertib and trastuzumab in combination, analyzing proliferation, migration and apoptosis and downstream intracellular signaling in vitro on human HER2-positive GCC (NCI-N87, OE33, OE19) and in negative HER2 GCC (MKN28). We observed a reduction of proliferation, migration and apoptotic rate in HER2-positive OE33, OE19 and N87 cell lines with the combination of duligotuzumab or ipatasertib plus trastuzumab. In particular, in OE33 and OE19 cell lines, the same combined treatment inhibited the activation of proteins downstream of HER2, HER3, AKT and MAPK pathways. Targeting both HER2 and HER3, or HER2 and AKT, results in an improved antitumor effect on HER2-positive GCC.

Combined inhibition of PD-1/PD-L1, Lag-3, and Tim-3 axes augments antitumor immunity in gastric cancer\u2013T cell coculture models

BackgroundImmunotherapy targeting PD-1 provides a limited survival benefit in patients with unresectable advanced or recurrent gastric cancer (GC). Beside PD-L1, the expression of inhibitory ligands such as CEACAM-1 and LSECtin on GC cells account for this limitation. Here we assessed their expression and immune suppressive effect in GC patients.MethodsUsing multiplexed immunohistochemistry staining, we evaluated the distribution of different inhibitory ligands, including PD-L1, CEACAM-1, LSECtin, and MHC class II, in 365 GC patients. We analyzed their correlations and overall survival (OS) based on the expression of each inhibitory ligand and the independent prognostic factors that affect OS. Subsequently, we evaluated the additive effect of anti-PD-1 mAb or anti-PD-L1 mAb with/without anti-Lag-3 mAb with/without anti-Tim-3 mAb in cytotoxic assay using tumor-antigen specific CTL clones against GC cell lines.ResultsCo-expression of the inhibitory ligands for PD-1, Tim-3, and Lag-3 was observed in the largest proportion (34.7%). CEACAM-1, LSECtin, and MHC class II expression showed significant correlation with PD-L1 expression and OS. Multivariable analysis demonstrated that CEACAM-1 low is an independent prognostic factor. Furthermore, combining dual and triple ICIs yielded additive effect on cytotoxicity of CTL clones against each immune inhibitory ligand positive GC cell lines.ConclusionsOur findings suggested that the expression of inhibitory ligands for Tim-3 and Lag-3 on GC cells serve as potential biomarkers to predict the response to anti-PD-1 therapy and the combinatorial immunotherapy with ICIs targeting for PD-1, Tim-3, and Lag-3 has a therapeutic potential for GC patients.

Functional inhibition of fatty acid binding protein 4 ameliorates impaired ciliogenesis in GCs

Ciliogenesis is often impaired in some cancer cells, leading to acceleration of cancer phenotypes such as cell migration and proliferation. From the investigation of primary cilia of 16 gastric cancer cells (GCs), we found that GCs could be grouped into four primary cilia (PC)–positive GCs and 12 PC-negative GCs. The proliferation of the PC-positive GCs was lower than that of PC-negative GCs. To explore the role of fatty acid binding protein 4 (FABP4), which is a known oncogenic factor, in ciliogenesis, FABP4 expression and function were inhibited by transfection of cells with short interfering RNA targeting FABP4 (siFABP4) or FABP4 inhibitor treatment. Notably, the proliferation and migration of the cilia-forming GCs was effectively suppressed by inhibition of FABP4. In addition, the primary cilia in GCs were restored by a factor greater than two, suggesting a negative role of FABP4 in ciliogenesis in these GCs and FABP4 as a potential anticancer target.

Modulatory effect of Tagetes erecta flowers essential oils via Nrf2/HO-1/NF-κB/p65 axis mediated suppression of N-methyl-N'nitro-N-nitroguanidine (MNNG) induced gastric cancer in rats.

Protective effect of Tagetes erecta flowers essential oils was investigated on oxidative stress, immune response, inflammation, and apoptosis against N-methyl-N'nitro-N-nitroguanidine (MNNG) induced gastric cancer in rats. Essential oil were extracted from Tagetes erecta flowers and analyzed using gas chromatography-mass spectrometry (GC-MS). For observing a protective effect against MNNG induced gastric cancer, we divided rats into 4 groups (group A to D) having 10 rats in each group. Performed various experiments and measured a different parameters to investigate antioxidant activity, immune response, anti-inflammatory and anti-apoptotic activity. The levels of malondialdehyde were markedly increased in the presence of N-methyl-N'nitro-N-nitroguanidine, whereas, the antioxidant activities of superoxide dismutase, and catalase were lowered in the treated rats in contrast with the control. Intervention with TEEO to gastric cancer-induced rats upregulated the redox status and the activity of the immune system to decrease cancer risk. The proinflammatory cytokines (IL-6 and TNF-α) secretions that were induced by MNNG were markedly inhibited by TEEO. Administration of TEEO also significantly reduced terminal deoxynucleotidyl transferase dUTP nick end labeling positive gastric cancer cells, expression of mRNA of caspase-3, and Bax. Whereas, the expression of Bcl-2 was increased. Additionally, downregulation of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) and IκBα degradation and the nuclear factor-κB (NF-κB) p65 expression in tissues of the stomach of MNNG-induced-rats were markedly elevated due to TEEO. This suggested possession of TEEO with a protective shield against MNNG induced gastric cancer by the exertion of antioxidative stress, anti-apoptotic response, the anti-inflammatory response through Nrf2/HO-1, and NF-κB signaling pathways.

USP8 Inhibitor Suppresses HER-2 Positive Gastric Cancer Cell Proliferation and Metastasis via the PI3K/AKT Signaling Pathway.

PurposeReferring to global cancer statistics, the incidence of gastric cancer (GC) was ranked sixth; however, detailed mechanisms underlying its development were not thoroughly investigated. Previous studies have reported that inhibition of ubiquitin-specific peptidase 8 (USP8) induced degradation of several receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), embryonic stem cells (ESCs), etc. Nevertheless, the regulation of HER-2 by USP8 and the molecular mechanisms controlling their role in the pathogenesis of GC remain unknown.Patients and MethodsA total of 69 patients with histologically confirmed GC were recruited to satisfy the purpose of this study. Initially, tumor samples and GC cell lines were used to detect USP8 and HER-2 levels. Next, MTT and colony formation assays were applied to analyze cell proliferation capability. Cell migration and invasion ability were examined by transwell assays. To examine related mRNA and protein expressions, Western blot assays and quantitative real-time PCR (qRT-PCR) were performed. Immunofluorescence was used to detect the effect of USP8 inhibitor on GC cells. Finally, in vivo experiments were used to examine the effect of USP8 inhibitor.ResultsPatients with USP8 high-expression tumors have shown worse overall survival while opposite results found in patients with low USP8 expressions. Regarding disease prognosis, patients with low expression of USP8 and HER-2 were performed better prognosis, whereas those with overexpression of USP8 and HER-2 shown poor prognosis. USP8 inhibitor significantly inhibited HER-2 positive cell NCI-N87 proliferation and metastasis. In addition, USP8 stabilizes HER-2, preventing it from ubiquitin proteasome-mediated degradation. In vivo studies confirmed that the USP8 inhibitor inhibited HER-2 positive cell NCI-N87 tumor growth. However, it did not affect the HER2-negative cell MGC-803. Careful investigation unraveled that the USP8 inhibitor significantly inhibited NCI-N87 cell proliferation and metastasis via phosphatidylinositol-3-kinases/protein-serine-threonine kinase (PI3K/AKT) pathway.ConclusionThe USP8 inhibited HER-2 positive GC cell proliferation and migration in vivo and in vitro and probably served as a novel potential therapeutic biomarker for HER-2 positive GC.

Honokiol Suppression of Human Epidermal Growth Factor Receptor 2 (HER2)-Positive Gastric Cancer Cell Biological Activity and Its Mechanism.

BackgroundThe purpose of our study was to determine the effects and mechanisms of honokiol on human epidermal growth factor receptor 2 (HER2)-positive gastric cancer cells by in vitro study.Material/MethodsWe measured HER2 expression in different gastric cancer cell lines by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) assay. Cell proliferation, apoptosis, and cell cycle were evaluated by cell-counting kit 8 and flow cytometry assays. The invading cell numbers and wound-healing rates were measured by transwell and wound-healing assays. Phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), P21, and matrix metalloproteinase (MMP)-9 proteins and messenger ribonucleic acid (mRNA) expression were measured by WB and RT-qPCR assay. HER2 protein expression was evaluated by cellular immunofluorescence.ResultsHonokiol suppressed cell proliferation via increasing cell apoptosis, invasion, and migration with dose dependence. By WB and RT-qPCR assays, compared with the control group, PI3K, AKT, P21, and MMP-9 proteins and mRNA expression were significantly different (P<0.05). By cellular immunofluorescence, HER2 protein expression was significantly depressed in honokiol-treated groups compared with control groups (P<0.05).ConclusionsHonokiol has suppressive effects on HER2-positive gastric cancer cell biological activities via regulation of HER2/PI3K/AKT pathways in vitro.

Abstract 2218: High affinity and cleavage resistant CD16 enhances Trastuzumab mediated killing of gastric cancer targets by human placental CD34+-derived natural killer cells

Abstract Introduction: When targeting tumors with monoclonal antibodies, natural killer (NK) cells are key effectors of antibody dependent cellular cytotoxicity (ADCC) following recognition of antibody Fc by CD16. Celularity has developed a novel proprietary technology platform that enables production of an allogeneic, off-the-shelf, placental CD34+-derived NK cell therapy for the treatment of hematologic and solid tumor malignancies. Here we report the genetic modification of CD16 in NK cells for high binding affinity to IgG and resistance to protease cleavage, together with functional evaluation of anti-tumor ADCC activity against epidermal growth factor receptor 2 (HER2) positive gastric cancer cells in combination with Trastuzumab (anti-Her2). Methods: Human placental CD34+ cells were transduced with lentivirus expressing a high binding affinity (158Val) and protease resistant (197Pro) CD16 variant (CD16VP) and cultured for 35 days in the presence of cytokines, including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, to generate CD16VP cells. The tumorigenic activity of CD16VP cells against HER2-positive gastric cancer cell lines NCI-N87 and OE19 was assessed in combination with Trastuzumab. ADCC activity was measured by real-time xCELLigence, and cytokine secretion by Luminex. The kinase inhibitors PD98059 (MEK/ERK), SB203580 (p38 MAPK), SP600125 (JNK), and Wortmannin (PI3K) were used to study the molecular mechanisms underlying cytotoxicity of CD16VP cells against gastric cancer. Results and conclusion: High transduction efficiency was achieved in multiple placental CD34+ donors with an average of 64.6 ± 10.3% (n=8) CD16 expression on NK cells compared to 12.1 ± 3.3% (n=8) on non-transduced cells. These cells expanded [7095 ± 2998-fold (n=8)] and differentiated into NK cells with &amp;gt;90% purity (CD56+CD3-). While treatment of PMA/ionomycin for 4 hours resulted in &amp;gt;89% CD16 cleavage on non-transduced cells, &amp;lt;11% cleavage was observed on CD16VP cells, demonstrating shedding resistance. CD16VP cells in the presence of Trastuzumab compared to IgG control, showed enhanced lysis of NCI-N87 and OE19 targets (97 ± 7% and 95 ± 2% vs. 55 ± 19% and 42 ± 14% respectively, p&amp;lt;0.01), secreted higher levels of GM-CSF, IFN-γ, and TNF-α, and demonstrated increased degranulation against OE19. The ADCC mediated cytotoxicity and degranulation were PI3K and JNK pathway-dependent as Wortmannin decreased ADCC by 82.2 ± 17.7% and degranulation by 43.9 ± 1.9%, and SP600125 decreased degranulation by 72.3 ± 1.2%. Our results demonstrate that CD16VP cells have enhanced Trastuzumab-mediated ADCC activity against gastric cancer tumor cell lines with resistance to CD16 cleavage. Development of CD16VP cells provides a combination therapeutic option by harnessing the anti-tumor activities of both targeted biologics and innate cytotoxicity of NK cells. Citation Format: Irene Raitman, Salvatore Rotondo, Tanel Mahlakõiv, Xuan Guo, Andrea DiFiglia, Hemlata Rana, Qian Ye, Srinivas Somanchi, William van der Touw, Lin Kang, Robert Hariri, Xiaokui Zhang. High affinity and cleavage resistant CD16 enhances Trastuzumab mediated killing of gastric cancer targets by human placental CD34+-derived natural killer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2218.

Down-Regulation of USP8 Suppresses HER-3 Positive Gastric Cancer Cells Proliferation.

BackgroundUbiquitin specific peptidase 8 (USP8) has been reported to induce the degradation of several receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), among which human epidermal growth factor receptor-3 (HER-3) is one of them. However, the role and functional mechanisms of USP8 and HER-3 in gastric cancer (GC) remain unknown.ObjectiveTo explore the function and mechanism of USP8 and HER-3 in the progression of GC.Materials and MethodsEighty-eight patients with histologically confirmed GC were recruited for this study. Tumor samples and GC cell lines were used to detect USP8 and HER-3 levels. MGC803 (HER-3 negative GC cell) was selected as the control group and NCI-N87, MKN-45 and AGS (HER-3 positive GC cells) as the experimental group. USP8i and si-RNA were then used to down-regulate USP8 in each group. Apoptosis and cell-cycle experiments were performed to detect the effects of USP8 on GC cells. Cytotoxicity Assay Kit (MTT) and colony formation assays were used to analyze cell proliferation. Cell migration and invasion ability were examined by wound healing. The expression of related mRNA and protein was detected by Western blot and quantitative real-time PCR (qRT-PCR). In vivo experiments were used to examine the effect of USP8 and HER-3.ResultsPatients with high expression of USP8 or HER-3 tumors alone died earlier than those with low expression and the patients with both USP8 and HER-3 high expression had a shorter overall survival than those with the opposite pattern (both USP8 and HER-3 low expression). Down-regulation of USP8 inhibited cell proliferation and cell metastasis and also reduced the HER-3 expression. We also observed that down-regulation of USP8 inhibited the proliferation of GC cells which highly expressed HER-3. Moreover, down-regulation of USP8 could promote the apoptosis of HER3-positive GC cells and inhibit the proliferation of them by affecting the cell-cycle. In vivo studies demonstrated that down-regulation of USP8 inhibited HER-3 positive tumors growth.ConclusionDown-regulation of USP8 inhibits HER-3 positive GC cells proliferation in vivo and in vitro, which indicate that USP8 represents a feasible choice as a therapeutic target for HER-3 positive GC cells.

IntegrinB5 upregulated by HER2 in gastric cancer: a promising biomarker for liver metastasis.

BackgroundLiver is the most frequent metastatic site of gastric cancer (GC), especially in patients with HER2 positive GC. Exosomal integrin αvβ5 has been shown to promote liver metastasis (LM), and the cross talk between integrins and HER2 during breast cancer metastasis has been reported. However, whether there’s an association between HER2 and integrin αvβ5 (ITGAvB5), and whether their association has predictive value in GC liver metastasis (GCLM) remains unknown.MethodsThe association between ITGβ5 and HER2 were accessed by RT-PCR, western blot and ELISA. We tested the function of ITGβ5 on HER2 positive GC cells using Transwell assays and scratch assays. Besides, we detect ITGβ5 expression in tumor tissue of GC patients and exosomes derived from advanced GC to analyze the association between HER2 and LM.ResultsIn our study, we found that ITGβ5, rather than ITGAV, was highly upregulated by HER2 through PI3K-AKT pathways in HER2 positive GC. Overexpression of ITGβ5 promoted the migration and invasion of HER2 positive GC cells in vitro. ITGβ5 was found to be an independent prognostic factor for GC. Besides, ITGβ5 level was only associated with LM. Detection of exosomal ITGβ5 and HER2 in the serum of GC patients revealed that exosomal ITGβ5 and HER2 levels are in accordance with that in tissue, and exosomal ITGβ5 level was higher in GCLM than other metastasis.ConclusionsOur study demonstrated ITGβ5 is regulated and functions in accordance with HER2 in promoting GCLM. Exosomal ITGβ5 levels might be a potential liquid biopsy biomarker for GCLM.

CMIP promotes Herceptin resistance of HER2 positive gastric cancer cells

Gastric cancer remains one of the most malignant human cancers with poor prognosis. Herceptin is a well-received antibody drug for HER2 positive gastric cancer. Primary Herceptin resistance and acquired Herceptin resistance retarded the use of Herceptin for gastric cancer. We herein reported CMIP (C-Maf-inducing protein) was overexpressed in Herceptin-resistant gastric cancer cells MKN45-HR and NCI-N87-HR; CMIP promoted Herceptin resistance of HER2 positive gastric cancer cells. SOX2 was examined to be positively regulated by CMIP and also promoted Herceptin resistance of HER2 positive gastric cancer cells. SOX2 might mediate the Herceptin resistance promoting role of CMIP in gastric cancer cells. Elevated expression of CMIP was associated with poor clinicopathological features including tumor size, lymph node metastasis and clinical stage in HER2 positive gastric cancer patients. Inhibitors of CMIP could be used as potential adjuvant therapeutic drugs for HER2 positive gastric cancer.

Abstract 3558: Interrogation of onco-microRNA 125b reveals androgen receptor as a key upstream transcriptional factor that is targetable in female gastric cancer

Abstract Background &amp; Aims: Our prior study revealed a miRNA-regulatory network by an integrated analysis of microRNA and mRNA profiling of gastric cancer (GC). It defined a GC microRNA subtype that was associated with poor survival in GC cases. In this study, we further demonstrate that onco-microRNA miR-125b, a key node in this microRNA regulatory network, is upregulated in GC and associated with poor overall survival by interfering in the apoptosis pathway and this pathway can be regulated by androgen receptor (AR). Our study also clarifies the potential clinical utility of the AR antagonist Bicalutamide on GC treatment. Methods: To further investigate the role of AR-miR-125b axis in GC, we conducted both in vitro experiment and clinical study with a cohort of 373 GC samples. The expression of miR-125b and AR was analyzed and compared with patient survival, and its implications were evaluated between genders of GC cases. We explored the tumor suppressive effect of bicalutamide using in vitro assays and in vivo subcutaneous xenotransplanted tumor model of human GC cell lines in nude mice. Results: The markedly up regulated expression of AR-miR-125b axis was validated in 373 GC samples. Univariate and multivariate analyses find that the miR-125b and/or AR were associated with poor prognosis and this trend was more pronounced among female than male cases. Pathway analysis shows that the predicted targets of miR-125b are highly involved in apoptosis/program death pathway. The robust apoptosis genes, BIK and CASP6 are validated as the directed targets of miR-125b. miR-125b can suppress apoptosis and increase cell growth, migration and invasion in GC cell lines. Furthermore, low expression of BIK and CASP6 in GC were associated with poor disease-free survival. There was a notable positive correlation between miR-125b and AR level in GC samples. Chip, EMSA and luciferase-binding assay confirmed that AR can direct regulated miR-125b expression as a transcriptional factor. We examined AR antagonist bicalutamide to inhibit proliferation and increased apoptosis in AR positive gastric cancer cells. The effect of bicalutamide is more apparent in female than in male nude mice. Conclusions: Our findings suggest that miR-125b regulated apoptosis pathway is important to GC progression and that the AR/miR-125 may be an important clinical biomarker for GC survival and possible therapeutic target for GC treatment. Citation Format: Ben Liu, Meng Zhou, Da Yang, Qinghua Wang, Qiang Zhang, Xiangchun Li, Meng Yang, Qiong Wang, Lian Li, Luyang Liu, Hongji Dai, Fengju Song, Hong Zheng, Wei Zhang, Kexin Chen. Interrogation of onco-microRNA 125b reveals androgen receptor as a key upstream transcriptional factor that is targetable in female gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3558.