Exosomal transfer of macrophage-derived miR-223 confers oxaliplatin resistance in gastric cancer

Oxaliplatin is the first-line regime for advanced gastric cancer treatment, while its resistance is a major problem that leads to the failure of clinical treatments. Tumor cell heterogeneity has been considered as one of the main causes for drug resistance in cancer. In this study, the mechanism of oxaliplatin resistance was investigated through in vitro human gastric cancer organoids and gastric cancer oxaliplatin-resistant cell lines and in vivo subcutaneous tumorigenicity experiments. The in vitro and in vivo results indicated that CD133+ stem cell-like cells are the main subpopulation and PARP1 is the central gene mediating oxaliplatin resistance in gastric cancer. It was found that PARP1 can effectively repair DNA damage caused by oxaliplatin by means of mediating the opening of base excision repair pathway, leading to the occurrence of drug resistance. The CD133+ stem cells also exhibited upregulated expression of N6-methyladenosine (m6A) mRNA and its writer METTL3 as showed by immunoprecipitation followed by sequencing and transcriptome analysis. METTTL3 enhances the stability of PARP1 by recruiting YTHDF1 to target the 3'-untranslated Region (3'-UTR) of PARP1 mRNA. The CD133+ tumor stem cells can regulate the stability and expression of m6A to PARP1 through METTL3, and thus exerting the PARP1-mediated DNA damage repair ability. Therefore, our study demonstrated that m6A Methyltransferase METTL3 facilitates oxaliplatin resistance in CD133+ gastric cancer stem cells by Promoting PARP1 mRNA stability which increases base excision repair pathway activity.

YTHDF2-mediated m6A modification of ONECUT2 promotes stemness and oxaliplatin resistance in gastric cancer through transcriptionally activating TFPI.

Oxaliplatin-based chemotherapy is the main treatment regimen for gastric cancer (GC), but can fail because of drug resistance. We investigated the role of a recently identified drug-resistance gene, taxol-resistant gene 1 (Txr1), in oxaliplatin resistance. A retrospective study based on banked tissue was carried out. We collected clinical data from 95 patients with stage II-III GC who were treated with radical D2 surgery and standardized first-line chemotherapy with oxaliplatin; paraffin blocks of their tumor specimens were prepared for a tissue microarray in which Txr1 expression was analyzed immunohistochemically and compared with their clinical data and their 3-year disease-free survival (DFS) rate. The human GC cell line, SGC7901, was developed into the oxaliplatin-resistant cell line, SGC7901/L-OHP, using slowly increased oxaliplatin concentrations over 6months. The relationship between Txr1 expression and drug-resistance of oxaliplatin in GC was studied with drug intervention, gene silencing technology, real-time PCR and Western blot analysis. Of the 95 patients with GC, those with TXR1(-) GC had longer postoperative 3-year DFS (77.8%) than those with TXR1(+) GC (52.9%). In oxaliplatin-resistant SGC7901/L-OHP cells, the main expression location of Txr1 shifted from the nucleus to cytoplasm, and both the mRNA and protein expression of Txr1 were higher than that of the parental cells, whereas expression of thrombospondin-1 (TSP1) decreased. When the Txr1 gene was silenced, TSP1 expression increased and the oxaliplatin resistance was significantly reduced in SGC7901/L-OHP cells. Changed Txr1 expression in GC affects the efficacy of oxaliplatin-based chemotherapy. Increased Txr1 expression decreases TSP1 expression and inhibits apoptosis. Txr1 could be a target in reversing oxaliplatin resistance in GC.

The dysregulation of long non-coding RNAs (lncRNAs) and transcription factors (TFs) is closely related to the development and progression of drug resistance in cancer chemotherapy. However, their regulatory interactions in the multidrug resistance (MDR) of gastric cancer (GC) has largely remained unknown. In this study, we report a novel oncogenic role of lncRNA FENDRR in conferring MDR in GC by coordinated regulation of FOXC2 expression at the transcriptional and posttranscriptional levels. In vitro and in vivo experiments demonstrated that downregulation of FENDRR expression remarkably decreased drug resistant ability of GC MDR cells while upregulation of FENDRR expression produced the opposite effect. FENDRR overexpression was observed in MDR GC cell lines, patient-derived xenografts, and clinical samples. And the high levels of FENDRR expression were correlated with poor prognosis in GC patients. Regarding the mechanism, FENDRR was revealed to increase proto-oncogene FOXC2 transcription by performing an enhancer-like role in the nucleus and by sponging miR-4700-3p in the cytoplasm. Both FOXC2 and miR-4700-3p were shown to be functionally involved in the FENDRR-induced chemoresistance. In addition, there is a positive correlation between FENDRR and FOXC2 expression in clinic and the overexpressed FOXC2 indicated a poor prognosis in GC patients. Collectively, our findings provide a new perspective for the lncRNA-TF regulatory interaction involved in MDR, suggesting that targeting the FENDRR/FOXC2 axis may be an effective approach to circumvent GC chemoresistance.

BackgroundChemotherapy resistance is a major challenge in the treatment of intermediate and advanced gastric cancer (GC). This study aimed to recognize oxaliplatin resistance-related genes (OXARGs) in GC and to explore their role and mechanism in oxaliplatin resistance of GC.MethodsOXARGs with prognostic value in GC were analyzed using GC oxaliplatin resistance data from the GEO and TCGA databases. RT-qPCR and WB assay were applied to verify the expression of MT2A, NOTCH1 and SLC7A5 in oxaliplatin-resistant GC cells (HGC27R and MKN45R). The effect of SLC7A5 on the malignant phenotype of oxaliplatin-resistant GC cells was verified by CCK-8, EDU, TUNEL, colony formation, wound healing, transwell assay, tumor bearing experiments and WB assay.ResultsBioinformatics analysis and experimental validation indicate that SLC7A5 was a target for oxaliplatin-resistance in GC. Knockdown of SLC7A5 obviously decreased the viability, migration, and invasion of oxaliplatin-resistant GC cells in vitro and tumor growth in vivo. It also increased the apoptosis levels and BAX expression, and reduced the expression of BCL2, MMP 2 and MMP9. Additionally, the knockdown of SLC7A5 enhanced the sensitivity of oxaliplatin-resistant GC cells to oxaliplatin both in vitro and in vivo. Furthermore, knockdown of SLC7A5 downregulated the expression of HK2, LDHA, Glut1, and PDK1 both in vivo and in vitro, leading to increased extracellular glucose levels and decreased lactate levels. However, glutathione significantly attenuated the regulatory effect of SLC7A5 knockdown on the malignant phenotype of oxaliplatin-resistant GC cells.Trial registrationNot Applicable.ConclusionKnockdown of SLC7A5 inhibits malignant progression and attenuates oxaliplatin resistance in GC by suppressing glycolysis.

Activation of vitamin D receptor (VDR) in cancer-associated fibroblasts (CAFs) has been implicated in hesitating tumor progression and chemoresistance of several human malignancies. Yet, the role of VDR in CAF-induced chemotherapy resistance of gastric cancer (GC) cells remains elusive. In this study we first conducted immunohistochemistry analysis on tissue microarrays including 88 pairs of GC and normal mucosa samples, and provided clinical evidence that VDR was mainly expressed in gastric mucous cells but almost invisible in CAFs, and VDR expression was negatively correlated with malignant clinical phenotype and advanced stages, low VDR expression confers to poor overall survival rate of patients with GC. In a co-culture system of primary CAFs and cancer cells, we showed that treatment of HGC-27 and AGS GC cells with VDR ligand calcipotriol (Cal, 500 nM) significantly inhibited CAF-induced oxaliplatin resistance. By using RNA-sequencing and Human Cytokine Antibody Array, we demonstrated that IL-8 secretion from CAFs induced oxaliplatin resistance via activating the PI3K/AKT pathway in GC, whereas Cal treatment greatly attenuated the tumor-supportive effect of CAF-derived IL-8 on GC cells. Taken together, this study verifies the specific localization of VDR in GC tissues and demonstrates that activation of VDR abrogates CAF-derived IL-8-mediated oxaliplatin resistance in GC via blocking PI3K/Akt signaling, suggesting vitamin D supplementation as a potential strategy of enhancing the anti-tumor effect of chemotherapy in GC.

Currently, platinum-containing regimens are the most commonly used regimens for advanced gastric cancer patients, and chemotherapy resistance is one of the main reasons for treatment failure. Thus, it is important to reveal the mechanism of oxaliplatin resistance and to seek effective intervention strategies to improve chemotherapy sensitivity, thereby improving the survival and prognosis of gastric cancer patients. To understand the molecular mechanisms of oxaliplatin resistance, we generate an oxaliplatin-resistant gastric cancer cell line and conduct assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) for both parental and oxaliplatin-resistant AGS cells. A total of 3232 genomic regions are identified to have higher accessibility in oxaliplatin-resistant cells, and DNA-binding motif analysis identifies JUNB as the core transcription factor in the regulatory network. JUNB is overexpressed in oxaliplatin-resistant gastric cancer cells, and its upregulation is associated with poor prognosis in gastric cancer patients, which is validated by our tissue microarray data. Moreover, chromatin immunoprecipitation sequencing (ChIP-seq) analysis reveals that JUNB binds to the transcriptional start site of key genes involved in the MAPK signaling pathway. Knockdown of JUNB inhibits the MAPK signaling pathway and restores sensitivity to oxaliplatin. Combined treatment with the ERK inhibitor piperlongumine or MEK inhibitor trametinib effectively overcomes oxaliplatin resistance. This study provides evidence that JUNB mediates oxaliplatin resistance in gastric cancer by activating the MAPK pathway. The combination of MAPK inhibitors with oxaliplatin overcomes resistance to oxaliplatin, providing a promising treatment opportunity for oxaliplatin-resistant gastric cancer patients.

Targeting LINC00665/miR-199b-5p/SERPINE1 axis to inhibit trastuzumab resistance and tumorigenesis of gastric cancer via PI3K/AKt pathway

BackgroundAdvanced gastric cancers often metastasize to distant organs and the peritoneum, leading to a poor prognosis. Both invasiveness and resistance to anchorage-independent cell death (anoikis) are important factors in the process of metastasis. p600 (600-kDa protein), recently identified from a cervical cancer cell line, plays a role in both anoikis resistance and cell migration. In this study, we examined whether p600 is involved in the progression of gastric cancer.MethodsWe used both normal gastric mucosal cells and cancer cells laser-microdissected from 42 gastric cancers and their normal counterparts, and compared their p600 mRNA expression levels with quantitative reverse transcriptase–polymerase chain reaction. We inhibited p600 expression in two gastric cancer cell lines with siRNA and examined its effect on the invasiveness and anoikis resistance both in vitro and in vivo.ResultsExpression of p600 mRNA was significantly higher in gastric cancer cells than in normal mucosal cells (P = 0.027). The invasion assay revealed that invasiveness was significantly reduced by inhibition of p600 (P < 0.01). In vitro experiments revealed that cell viability and colony-formation capacity under anchorage-independent conditions were significantly reduced by inhibition of p600 (P < 0.05). In vivo experiments also showed that the establishment of intraperitoneal disseminated tumors was significantly suppressed by transient inhibition of p600 (P < 0.001).ConclusionsOur results strongly suggest that p600 is involved in gastric cancer progression, and has a potential to be a new molecular target for gastric cancer therapy.

Oxaliplatin is the main chemotherapy drug for gastric cancer (GC), but quite a few patients are resistant to oxaliplatin, which contributes to the poor prognosis of GC patients. There is therefore an urgent need to identify potential targets for reversing chemotherapy resistance in GC patients. In this study, we analyzed the tumor samples of GC patients who received neoadjuvant chemotherapy based on oxaliplatin through quantitative proteomics and identified the potential chemoresistance-related protein cellular retinoic acid binding protein 2 (CRABP2). CRABP2 was significantly upregulated in the tumor tissues of chemoresistant GC patients and was closely related to prognosis. The results of cell function experiments showed that CRABP2 can promote the oxaliplatin resistance of GC cells in vitro. Coimmunoprecipitation and GST pulldown assays showed that CRAPB2 expedited the binding of BAX and PARKIN in GC cells and facilitated the ubiquitination-mediated degradation of BAX. Furthermore, both the in vitro assay and cell-derived xenograft (CDX) in vivo model verified that CRABP2 promoted oxaliplatin resistance by inhibiting BAX-dependent cell apoptosis. Further experiments proved that the abnormally high expression of CRABP2 in oxaliplatin-resistant GC cells was affected by TET1-mediated DNA hydroxymethylation. The patient-derived xenograft (PDX) model suggested that interference with CRABP2 reversed oxaliplatin resistance in GC in vivo. In conclusion, the results of our study show that CRABP2 was a key molecule in oxaliplatin resistance regulation and could be a new target for reversing the chemoresistance of GC.

BACKGROUNDKeratin 80 (KRT80), a type I intermediate filament protein, is a member of the keratin family with specialized functions in epithelial tissues. While KRT80 has been implicated in both normal physiological processes and various diseases, its role in gastric cancer (GC), particularly its expression and prognostic significance, remains poorly understood. In this study, we investigated the role and underlying molecular mechanisms of KRT80 in oxaliplatin resistance in GC. Our analysis revealed that KRT80 is significantly upregulated in GC tissues and is associated with poor clinical prognosis. The role of KRT80 in GC cell proliferation was assessed through in vitro and in vivo assays.AIMTo explore the expression of KRT80 in GC and its impact on the prognosis of patients.METHODSKRT80 expression in GC tissues was analyzed using Western blotting, quantitative reverse transcription PCR, multiple immunofluorescence staining, and immunohistochemistry. Survival analysis was conducted using the Kaplan-Meier method with the log-rank test. The role of KRT80 in GC cell proliferation was assessed through in vitro and in vivo assays. Immunoprecipitation and mass spectrometry analyses identified elongation factor 1-alpha 1 (EEF1A1) as a binding protein of KRT80.RESULTSIntegrating our experimental findings with multiple published studies, we found that increased KRT80 expression is associated with poor prognosis in GC and promotes resistance to oxaliplatin. Moreover, we have preliminarily verified the interaction between KRT80 and EEF1A1. Therefore, this study provides a novel perspective on overcoming oxaliplatin resistance in GC.CONCLUSIONIncreased KRT80 expression predicts poor prognosis and promotes oxaliplatin resistance in GC, suggesting its potential as a novel prognostic biomarker.

643P - Cell Growth Inhibition of Her2 Positive Trastuzumab Resistant Gastric Cancer Cell Lines By Combined Inhibition of Pi3K/Akt/Mtor and Mapk

Decidual protein induced by progesterone (DEPP1) was identified to exert heterogeneous functions in several cancers, whereas its role in gastric cancer (GC) remains elusive. In the present study, differential expression analysis was conducted using three Gene Expression Omnibus datasets (GSE54129, GSE26942 and GSE3438). Validation of DEPP1 expression was performed using reverse transcription‑quantitative PCR, western blotting and immunofluorescence. Kaplan‑Meier survival and Cox regression analyses were employed to assess the association between DEPP1 expression and the prognosis of patients with GC. Immune infiltration analysis was conducted to explore the correlation between DEPP1 and the tumor microenvironment. The potential of DEPP1 to promote oxaliplatin resistance was assessed using flow cytometry, western blotting, and subcutaneous mouse models. DEPP1 was found to be significantly upregulated in the aforementioned cohorts, which was consistent with the clinical specimens of the present study, and it emerged as an independent risk factor for poor overall survival in patients with GC. A prognostic nomogram was developed to improve prognosis prediction. High DEPP1 expression correlated with increased infiltration of cancer‑associated fibroblasts, endothelial cells, and M2 macrophages, contributing to the development of a stroma‑rich and immunosuppressive microenvironment in GC. Furthermore, high DEPP1 expression was associated with reduced sensitivity to chemotherapy drugs in patients with GC. In vitro and in vivo experiments highlighted DEPP1's crucial role in promoting oxaliplatin resistance in GC. In conclusion, DEPP1 is identified as a promising prognostic biomarker linked to a stroma‑rich and immunosuppressive microenvironment, and it is critical in driving oxaliplatin resistance in GC. These findings may inform personalized therapeutic strategies for patients with GC.

IntroductionGastric cancer (GC) is one of the most malignancies leading to human mortality due to its development, progress, metastasis and poor prognosis, despite the development of remarkable chemotherapy and surgery. The 5-fluorouracil (5-Fu) is an effective anti-gastric cancer agent. However, a fraction of GC patients acquire 5-Fu chemoresistance.MethodsIn this study, the CagA protein was detected from CagA-positive gastric cancer patients by qRT-PCR and immunohistochemistry. The 5-Fu resistant gastric cancer cell line was generated from MKN45-CagA cells which was transfected with CagA overexpression vector. Cellular glucose metabolism was determined by measurements of glucose uptake, lactate product and glycolysis enzymes.ResultsWe report that the Helicobacter pylori (H. pylori)-secreted Cytotoxin-associated gene A (CagA) oncoprotein is positively correlated with 5-Fu resistance of gastric cancer. From totally 72 CagA-positive gastric cancer patients, CagA high-expressed patients showed more resistance to 5-Fu than CagA low-expressed patients. Moreover, statistical analysis revealed that CagA mRNA and protein expressions were significantly upregulated in 5-Fu resistant gastric cancer patients. We observed that CagA protein is upregulated in 5-Fu resistant gastric cancer cells compared with sensitive cells. Interestingly, cellular glucose metabolism was upregulated; the glucose uptake and lactate production were significantly higher in 5-Fu resistant gastric cancer cells. The Akt phosphorylation and expressions of glycolysis key enzymes, Hexokinase 2 and LDHA, were significantly upregulated in 5-Fu resistant gastric cancer cells. On the other way, inhibition of glycolysis or Akt pathway effectively overcame 5-Fu resistance from both in vitro and in vivo models. Finally, we report that the combination of Akt or glycolysis inhibitor with 5-Fu could synergistically enhance the cytotoxicity of 5-Fu to CagA-overexpressed gastric cancer cells.DiscussionIn summary, our study demonstrated a CagA-Akt-glycolysis-5-Fu resistance axis, contributing to the development of new therapeutic agents against chemoresistant human gastric cancer.

Nucleophosmin (NPM) and human epidermal growth factor receptor-2 (Her-2) are abnormally expressed in various types of human malignant tumors, including gastric cancer, and have been closely associated with cancer chemoresistance. However, their interaction and roles in oxaliplatin resistance are not fully understood. Therefore, the present study aimed to elucidate the relationship between NPM and Her-2 in gastric cancer cell lines and clinical samples, and further investigated their role in the resistance of gastric cancer to oxaliplatin. Western blotting and reverse transcription-quantitative polymerase chain reaction confirmed that NPM and Her-2 expression were significantly upregulated in gastric cancer cells and clinical samples, and that their expression levels were strongly correlated. However, Her-2 expression was not affected by upregulation or downregulation of NPM expression in gastric cancer cells. Cell counting kit-8 assays demonstrated that the cell sensitivity to oxaliplatin decreased simultaneously with an increase in NPM expression. Furthermore, inhibition of Her-2 expression using trastuzumab significantly increased the sensitivity of the cells to oxaliplatin, which occurred simultaneously with the downregulation of NPM. These results indicated that inhibition of NPM, as a Her-2 downstream signal, may be a novel strategy to overcome oxaliplatin-resistant gastric cancer, and that trastuzumab and oxaliplatin may exhibit a synergistic antitumor effect in Her-2-positive gastric cancer cells.