Targeting HER3 or MEK overcomes acquired Trastuzumab resistance in HER2-positive gastric cancer-derived xenograft.

Acquired Trastuzumab resistance is a complicated and disastrous event for HER2-positive gastric cancer (GC). In this study, we successfully established a GC PDX model with Trastuzumab sensitivity (176P) and induced a homologous model with acquired Trastuzumab resistance (176R), then comprehensively delineated the landscape of Trastuzumab resistance mechanisms using single-cell transcriptome sequencing, as well as protein profiling and genomic variation analysis. According to multi-omics study, different gene expression profiles, rather than genetic changes, contributed to acquired Trastuzumab resistance. The mechanisms underlying acquired Trastuzumab resistance present great complexity as multiple molecules and pathways were involved, including ERBB family, MAPK, PI3K/AKT, JAK/STAT, and cell cycle pathways. Through phenotypical and molecular validation, we found that Trastuzumab combined with HER3-targeted antibody or MEK inhibitor demonstrated excellent antitumor activity and good tolerance, which may serve as promising strategies for overcoming acquired Trastuzumab resistance.

Antimicrobial effects of thymol-loaded phytoglycogen/zein nanocomplexes against foodborne pathogens on fresh produce.

In this study, thymol-loaded hydrophobically modified phytoglycogen/zein nanocomplexes with a particle size around 100 nm were developed for improving microbial safety of fresh produce. The antimicrobial activities, including the determination of minimum inhibitory and bactericidal concentration, growth kinetic curves, and inhibition zone of the nanocomplexes against foodborne pathogens (Listeria monocytogenes, Salmonella enteritidis, and Escherichia coli) were evaluated. The results showed that the antimicrobial activities of the nanocomplexes were significantly stronger than that of free thymol control (without encapsulation), and the antimicrobial efficacy remained unchanged after storage at 4 °C for 60 days. The morphological results from atomic force microscope revealed that small micellar blebs were formed at the surface of bacteria after treatment with nanocomplexes and the gradual disappearance of the cell boundary indicated the occurrence of cytolysis. The potential applications of this nanocomplex as disinfectant agent in wash water were evaluated on different types of fresh produce (lettuce, cantaloupe, and strawberries). Notably, the nanocomplexes also demonstrated efficacy in biofilm removal. Findings from this study clearly demonstrated that the thymol-loaded nanocomplexes hold promising potential for the disinfection of fresh produce to improve their microbial safety and quality.

PPARγ induces PD-L1 expression in MSS+ colorectal cancer cells.

Only a small subset of colorectal cancer (CRC) patients benefits from immunotherapies, comprising blocking antibodies (Abs) against checkpoint receptor "programmed-cell-death-1" (PD1) and its ligand (PD-L1), because most cases lack the required mutational burden and neo-antigen load caused by microsatellite instability (MSI) and/or an inflamed, immune cell-infiltrated PD-L1+ tumor microenvironment. Peroxisome proliferator-activated-receptor-gamma (PPARγ), a metabolic transcription factor stimulated by anti-diabetic drugs, has been previously implicated in pre/clinical responses to immunotherapy. We therefore raised the hypothesis that PPARγ induces PD-L1 on microsatellite stable (MSS) tumor cells to enhance Ab-target engagement and responsiveness to PD-L1 blockage. We found that PPARγ-agonists upregulate PD-L1 mRNA/protein expression in human gastrointestinal cancer cell lines and MSS+ patient-derived tumor organoids (PDOs). Mechanistically, PPARγ bound to and activated DNA-motifs similar to cognate PPARγ-responsive-elements (PPREs) in the proximal -2 kb promoter of the human PD-L1 gene. PPARγ-agonist reduced proliferation and viability of tumor cells in co-cultures with PD-L1 blocking Ab and lymphokine-activated killer cells (LAK) derived from the peripheral blood of CRC patients or healthy donors. Thus, metabolic modifiers improved the antitumoral response of immune checkpoint Ab, proposing novel therapeutic strategies for CRC.

Docking protein-1 promotes inflammatory macrophage signaling in gastric cancer.

Docking protein-1 (DOK1) is a tumor suppressor frequently lost in malignant cells, however, it retains the ability to control activities of immune receptors in adjacent stroma cells of the tumor microenvironment. We therefore hypothesized that addressing DOK1 may be useful for cancer immunotherapy. DOK1 mRNA and DOK1 protein expression were downregulated in tumor cells of gastric cancer patients (n = 249). Conversely, its expression was up-regulated in cases positive for Epstein Barr Virus (EBV+) together with genes related to macrophage biology and targets of clinical immunotherapy such as programmed-cell-death-ligand-1 (PD-L1). Notably, high DOK1 positivity in stroma cells conferred poor prognosis in patients and correlated with high levels of inducible nitric oxide synthase in CD68+ tumor-associated macrophages. In macrophages derived from human monocytic leukemia cell lines, DOK1 (i) was inducible by agonists of the anti-diabetic transcription factor peroxisome proliferator-activated receptor-gamma (PPARγ), (ii) increased polarization towards an inflammatory phenotype, (iii) augmented nuclear factor-κB-dependent transcription of pro-inflammatory cytokines and (iv) reduced PD-L1 expression. These properties empowered DOK1+ macrophages to decrease the viability of human gastric cancer cells in contact-dependent co-cultures. DOK1 also reduced PD-L1 expression in human primary blood monocytes. Our data propose that the drugability of DOK1 may be exploited to reprogram myeloid cells and enforce the innate immune response against EBV+ human gastric cancer.

Chromosomal instability of circulating tumor DNA reflect therapeutic responses in advanced gastric cancer.

Gastric cancer is characterized by chromosomal instability. In this study, we investigated chromosomal instability quantified by copy number instability (CNI) score of circulating tumor DNA (ctDNA) during the drug treatment in advanced gastric cancer (AGC). A total of 55 pretherapeutic plasmas from 55 AGC patients and 75 plasmas during drug treatment of 26 AGC patients were collected. Plasma ctDNA was extracted and assessed by whole-genome sequencing (WGS) for somatic copy number alteration (SCNA), and according to which we calculated the CNI scores. We next assessed the correlations between chromosomal instability and therapeutic response. The cutoff value of chromosomal instability was defined as the mean + SD of the CNI scores (56.60) in cfDNA of plasmas from 100 healthy people. For 55 enrolled cases, chromosomal instability was observed in 27 (49%) prior to drug treatment, whose response rate (59%, 16/27) was higher than in 28 patients with stable chromosomes (32%, 9/28, P = 0.043). We also observed that CNI scores fluctuated during treatment in 26 patients. Specifically, the CNI scores in 93% (14/15) of patients sensitive to drug treatment reduced to the level of chromosomal stability and the CNI scores in 52% (13/25) of patients resistant to treatment elevated again. For ctDNA with developed resistance, the SCNA patterns were identical to those before treatment, whereas the CNI scores were lower than the pretherapeutic scores. We found that chromosomal instability based on ctDNA could predict and monitor therapeutic response in gastric cancer, although validation in a larger cohort will be necessary.

Dbx2 exhibits a tumor-promoting function in hepatocellular carcinoma cell lines via regulating Shh-Gli1 signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. HCC patients suffer from a high mortality-to-incidence ratio and low cure rate since we still have no specific and effective treatment. Although tremendous advances have been made in the investigation of HCC, the specific mechanisms of the progression of this disease are still only partially established. Hence, more research is needed to elucidate the underlying potential mechanisms to develop effective strategies for HCC. AIM: To determine the role of developing brain homeobox 2 (Dbx2) gene in promoting the development of HCC. METHODS: Dbx2 expression in clinical specimens and HCC cell lines was detected by Western blot (WB) and immunohistochemistry. Gain and loss of Dbx2 function assays were performed in vitro and in vivo. Cell viability assays were used to investigate cell growth, flow cytometry was employed to assess cell cycle and apoptosis, and trans-well assays were conducted to evaluate cell migration, invasion, and metastasis. The expression of key molecules in the sonic hedgehog (Shh) signaling was determined by WB. RESULTS: Compared to matched adjacent non-tumorous tissues, Dbx2 was overexpressed in 5 HCC cell lines and 76 surgically resected HCC tissues. Dbx2 overexpression was correlated with large tumor size. Both gain and loss of function assays indicated that Dbx2 promoted HCC cell proliferation by facilitating the transition from G1 to S phase, attenuating apoptosis and promoted HCC proliferation, migration, and invasion in vitro and in vivo. Mechanistically, Dbx2 modulated Shh signaling by enhancing FTCH1 and GLi1 expression in HCC cells that overexpressed Dbx2, which was reversed in HCC cells with Dbx2 knockdown. CONCLUSION: Our results indicate that Dbx2 is significantly upregulated in HCC tissues and plays significant roles in proliferation and metastasis of HCC cells by activating the Shh pathway.

Dual PI3K/mTOR inhibitor BEZ235 as a promising therapeutic strategy against paclitaxel-resistant gastric cancer via targeting PI3K/Akt/mTOR pathway.

Paclitaxel (PTX) is widely used in the front-line chemotherapy for gastric cancer (GC), but resistance limits its use. Due to the lack of proper models, mechanisms underlying PTX resistance in GC were not well studied. Using established PTX-resistant GC cell sublines HGC-27R, we for the first time integrated biological traits and molecular mechanisms of PTX resistance in GC. Data revealed that PTX-resistant GC cells were characterized by microtubular disorders, an EMT phenotype, reduced responses to antimitotic drugs, and resistance to apoptosis (marked by upregulated ß-tubulin III, vimentin, attenuated changes in G2/M molecules or pro-apoptotic factors in response to antimitotic drugs or apoptotic inducers, respectively). Activation of the phosphoinositide 3-kinase, the serine/threonine kinase Akt and mammalian target of rapamycin (PI3K/Akt/mTOR) and mitogen-activated protein kinase (MAPK) pathways were also observed, which might be the reason for above phenotypic alternations. In vitro data suggested that targeting these pathways were sufficient to elicit antitumor responses in PTX-resistant GC, in which the dual PI3K/mTOR inhibitor BEZ235 displayed higher therapeutic efficiency than the mTOR inhibitor everolimus or the MEK inhibitor AZD6244. Antitumor effects of BEZ235 were also confirmed in mice bearing HGC-27R tumors. Thus, these data suggest that PI3K/Akt/mTOR and MAPK pathway inhibition, especially PI3K/mTOR dual blockade, might be a promising therapeutic strategy against PTX-resistant GC.

HER2 copy number of circulating tumour DNA functions as a biomarker to predict and monitor trastuzumab efficacy in advanced gastric cancer.

BACKGROUND: HER2 status is significant to trastuzumab therapy; however, it is difficult to determine HER2 status accurately with few pieces of biopsies from advanced gastric cancer (AGC) due to highly heterogeneity and invasive behaviour, which will be investigated in this study. METHODS: Fifty-six patients with AGC were included in this study. Primary tumour tissues and matched plasmas before medication from 36 patients were retrospectively collected, and the other 20 patients with primary tumour tissues and paired plasmas were prospectively collected. HER2 expression and amplification in 56 tumour tissues were determined by immunohistochemistry (IHC) and dual in situ hybridisation (DISH), and HER2 copy number in 135 circulating tumour DNAs (ctDNAs) was judged by next-generation sequencing. RESULTS: For tumour tissues, HER2 amplification by DISH was most commonly found in patients with HER2 score 3+by IHC. For plasmas, HER2 amplification defined as HER2 copy number >2.22 was identified in 26 of 56 patients. There was a high concordance of HER2 amplification between ctDNA and tumour tissues, suggesting that ctDNA could function as an alternative to screen HER2-targeted population. Moreover, the changes of HER2 copy number in ctDNA could efficiently monitor trastuzumab efficacy, the power of which was superior to commonly used markers carcinoembryonic antigen (CEA) and CA199, suggesting its potential role in clinical practice. CONCLUSION: ctDNA for HER2 analysis was strongly recommended to serve as a surrogate to screen trastuzumab-suitable population and monitor trastuzumab efficacy.

Gimatecan exerts potent antitumor activity against gastric cancer in vitro and in vivo via AKT and MAPK signaling pathways.

BACKGROUND: We investigated antitumor activity and underlying mechanisms of DNA topoisomerase I (TopI) inhibitor gimatecan and irinotecan in gastric cancer (GC) in vitro cell lines and in vivo patient-derived xenograft (PDX) models. METHODS: GC cell lines SNU-1, HGC27, MGC803 and NCI-N87 were used to evaluate cell viability and apoptosis after gimatecan or irinotecan treatment, using a cell proliferation assay and flow cytometry, respectively. DNA TopI expression and critical molecules of PI3K/AKT, MAPK and apoptosis signaling pathways were analyzed with western blot. For in vivo studies, five PDXs models were treated with gimatecan or irinotecan to assess its antitumor activity. Immunohistochemistry staining of Ki-67 was performed after mice were sacrificed. RESULTS: Gimatecan inhibited the proliferation of GC cells in vitro in a dose- and time-dependent manner by inducing apoptosis, and gimatecan had greater inhibitory effects than irinotecan. In addition, both gimatecan and irinotecan demonstrated significant tumor growth inhibition in in vivo PDX models. Gimatecan treatment significantly inhibited the expression of DNA TopI, phosphorylated AKT (pAKT), phosphorylated MEK (pMEK) and phosphorylated ERK (pERK). Meanwhile, gimatecan could also activate the JNK2 and p38 MAPK pathway as indicated by upregulation of phosphorylated p38 MAPK (p-p38) and phosphorylated JNK2 (pJNK2). CONCLUSIONS: For the first time, we have shown that the antitumor activity of gimatecan in GC via suppressing AKT and ERK pathway and activating JNK2 and p38 MAPK pathway, which indicated that gimatecan might be an alternative to irinotecan in the treatment of GC.

Genomic alterations in advanced gastric cancer endoscopic biopsy samples using targeted next-generation sequencing.

Gastric cancer (GC) remains the second tumor caused death threat worldwide, and personalized medicine for GC is far from expectation. Finding novel, recurrently mutated genes through next-generation sequencing (NGS) is a powerful and productive approach. However, previous genomic data for GC are based on surgical resected samples while a large proportion of advanced gastric cancer (AGC) patients have already missed the chance for operation. The aim of this study is to assess frequent genomic alteration in AGC via biopsy samples. Here we performed targeted genomic sequencing of 78 AGC patients' tumor biopsies along with matched lymphocyte samples based on a 118 cancer related gene panel. In total, we observed 301 somatic nonsynonymous genomic alterations in 92 different genes, as well as 37 copy number gain events among 15 different genes (fold change 2-12), and validated the fold changes of ERBB2 copy number gains with IHC and FISH test showed an accuracy of 81.8%. Previously reported driver genes for gastric cancer (TP53, KMT2D, KMT2B, EGFR, PIK3CA, GNAQ, and ARID1A), and several unreported mutations (TGFBR2, RNF213, NF1, NSD1, and LRP2) showed high non-silent mutation prevalence (7.7%-34.6%). When comparing intestinal-type gastric cancer (IGC) with diffuse-type gastric cancer (DGC), TP53 and GNAQ appear to be more frequently mutated in IGC (P=0.028 and P=0.023, respectively), whereas LRP2, BRCA2 and FGFR3 mutations are not observed in IGC, but have 12.8%, 7.7% and 7.7% mutation rates, respectively, in DGC patients. Patients with one or more mutations in adherens junction pathway (CREBBP, EP300, CDH1, CTNNB1, EGFR, MET, TGFBR2 and ERBB2) or TGF-ß signaling pathway (CREBBP, EP300, MYST4, KRAS and TGFBR2) showed significantly better overall survival (P=0.007 and P=0.014, respectively), consistent with The Cancer Genome Atlas (TCGA) cohort data. Importantly, 57 (73.1%) patients harbored at least one genomic alteration with potential treatments, making NGS-based drug target screening a viable option for AGC patients. Our study established a comprehensive genomic portrait of AGC, and identified several mutation signatures highly associated with clinical features, survival outcomes, which may be used to design future personalized treatments.

Circulating tumor DNA functions as an alternative for tissue to overcome tumor heterogeneity in advanced gastric cancer.

Overcoming tumor heterogeneity is a major challenge for personalized treatment of gastric cancer, especially for human epidermal growth factor receptor-2 targeted therapy. Analysis of circulating tumor DNA allows a more comprehensive analysis of tumor heterogeneity than traditional biopsies in lung cancer and breast cancer, but little is known in gastric cancer. We assessed mutation profiles of ctDNA and primary tumors from 30 patients with advanced gastric cancer, then performed a comprehensive analysis of tumor mutations by multiple biopsies from five patients, and finally analyzed the concordance of HER2 amplification in ctDNA and paired tumor tissues in 70 patients. By comparing with a single tumor sample, ctDNA displayed a low concordance of mutation profile, only approximately 50% (138/275) somatic mutations were found in paired tissue samples, however, when compared with multiple biopsies, most DNA mutations in ctDNA were also shown in paired tumor tissues. ctDNA had a high concordance (91.4%, Kappa index = 0.784, P < 0.001) of HER2 amplification with tumor tissues, suggesting it might be an alternative for tissue. It implied that ctDNA-based assessment could partially overcome the tumor heterogeneity, and might serve as a potential surrogate for HER2 analysis in gastric cancer.