Comprehensive multi-omics analysis of resectable locally advanced gastric cancer: Assessing response to neoadjuvant camrelizumab and chemotherapy in a single-center, open-label, single-arm phase II trial.

BACKGROUND: The current standard of care for locally advanced gastric cancer (GC) involves neoadjuvant chemotherapy followed by radical surgery. Recently, neoadjuvant treatment for this condition has involved the exploration of immunotherapy plus chemotherapy as a potential approach. However, the efficacy remains uncertain. METHODS: A single-arm, phase 2 study was conducted to evaluate the efficacy and tolerability of neoadjuvant camrelizumab combined with mFOLFOX6 and identify potential biomarkers of response through multi-omics analysis in patients with resectable locally advanced GC. The primary endpoint was the pathological complete response (pCR) rate. Secondary endpoints included the R0 rate, near pCR rate, progression-free survival (PFS), disease-free survival (DFS), and overall survival (OS). Multi-omics analysis was assessed by whole-exome sequencing, transcriptome sequencing, and multiplex immunofluorescence (mIF) using biopsies pre- and post-neoadjuvant therapy. RESULTS: This study involved 60 patients, of which 55 underwent gastrectomy. Among these, five (9.1%) attained a pathological complete response (pCR), and 11 (20.0%) reached near pCR. No unexpected treatment-emergent adverse events or perioperative mortality were observed, and the regimen presented a manageable safety profile. Molecular changes identified through multi-omics analysis correlated with treatment response, highlighting associations between HER2-positive and CTNNB1 mutations with treatment sensitivity and a favourable prognosis. This finding was further supported by immune cell infiltration analysis and mIF. Expression data uncovered a risk model with four genes (RALYL, SCGN, CCKBR, NTS) linked to poor response. Additionally, post-treatment infiltration of CD8+ T lymphocytes positively correlates with pathological response. CONCLUSION: The findings suggest the combination of PD-1-inhibitor and mFOLFOX6 showed efficacy and acceptable toxicity for locally advanced GC. Extended follow-up is required to determine the duration of the response. This study lays essential groundwork for developing precise neoadjuvant regimens.

Exploring the Role of Active Functional Microbiota in Flavor Generation by Integrated Metatranscriptomics and Metabolomics during Niulanshan Baijiu Fermentation.

Active functional microbiota for producing volatile flavors is critical to Chinese baijiu fermentation. Microbial communities correlated with the volatile metabolites are generally explored using DNA-based sequencing and metabolic analysis. However, the active functional microbiota related to the volatile flavor compounds is poorly understood. In this study, an integrated metatranscriptomic and metabolomics analysis was employed to unravel the metabolite profiles comprehensively and the contributing active functional microbiota for flavor generation during Niulanshan baijiu fermentation. A total of 395, 83, and 181 compounds were annotated using untargeted metabolomics, including LC-MS, GC-MS, and HS-SPME-GC-MS, respectively. Significant variances were displayed in the composition of compounds among different time-point samples according to the heatmaps and orthogonal partial least-square discriminant analysis. The correlation between the active microbiota and the volatile flavors was analyzed based on the bidirectional orthogonal partial least squares discriminant analysis (O2PLS-DA) model. Six bacterial genera, including Streptococcus, Lactobacillus, Pediococcus, Campylobacter, Yersinia, and Weissella, and five fungal genera of Talaromyces, Aspergillus, Mixia, Rhizophagus, and Gloeophyllum were identified as the active functional microbiota for producing the volatile flavors. In summary, this study revealed the active functional microbial basis of unique flavor formation and provided novel insights into the optimization of Niulanshan baijiu fermentation.

Unveiling innovation in aroma attribute evaluation of Niulanshan Baijiu: An advanced exploration of two different processing methods via food sensory omics and penalty analysis.

Niulanshan Baijiu (NLS), a notable variety of Baijiu known for its light flavor and extensive historical legacy, was subjected to a comparative analysis using two different processes: Hunzheng Xucha (HX) and Qingzheng Qingcha (QQ). The study combined sensory-oriented flavor analysis and penalty analysis to assess the differences between the two processes. Aroma compounds in NLS were extracted using liquid-liquid extraction and headspace solid phase microextraction. Gas chromatography-olfactometry-mass spectrometry was employed to identify 46 aroma-active compounds, including the first-time discovery of ethyl isohexanoate and 2,4-nonadienal in NLS. Quantification of 35 compounds with odor activity value (OAV) ≥ 1 was achieved using internal standard curve methods. Sensory assessments by a cohort of 111 participants highlighted the preference for HX-NLS in terms of flavor, while QQ-NLS exhibited a sour-Chen aroma that required improvement. The study further revealed the significant impact of acetic acid, butyric acid, hexanoic acid, octanoic acid, and 3-methylbutanal on the sour-Chen aroma in liquor.

RNA-seq based elucidation of mechanism underlying the protective effect of Huangshui polysaccharide on intestinal barrier injury in Caco-2 cells.

Polysaccharides from Huangshui (HS) have the function of antioxidant and immunoregulation, but its intestinal barrier protection activity and the underlying mechanism remains unclear. The present work mainly studied the intestinal barrier protection function and its potential molecular mechanism of a heteropolysaccharide named NLS-2 with a molecular weight of 51.9 kDa. NLS-2 reduced intestinal permeability by decreasing the content of inflammatory cytokines and increasing the expression of tight junction (TJ) protein in LPS-damaged Caco-2 cells, thus protecting the intestinal barrier function. RNA-seq results showed that the differentially expressed genes (DEGs) were mainly enriched in the signaling pathways of MAPK, Toll-like receptor, and NF-κB. Subsequent western blot validation experiments proved that NLS-2 could indeed inhibit the two pathways of MAPK and NF-κB by reducing the expression of TRL4, thereby down-regulating the release of downstream pro-inflammatory cytokines and playing the role of intestinal barrier protection. Collectively, NLS-2 has beneficial effects on LPS-damaged intestinal barrier by inhibiting the TRL4/MyD88/NF-κB and MAPK signaling pathways.

An open, observational, three-arm clinical study of 2-3 cycles of treatment as neoadjuvant therapy in operable locally advanced non-small cell lung cancer: An interim analysis.

Background: An open, observational, three-arm clinical study aimed at investigating the efficacy of different neoadjuvant therapies (neoadjuvant immunotherapy with(out) chemotherapy, neoadjuvant chemotherapy, and neoadjuvant targeted therapy) in operable locally advanced non-small cell lung cancer (NSCLC) was conducted (NCT04197076). We report an interim analysis of 49 of 53 evaluable patients. Methods: This study was conducted at Shanghai Chest Hospital and included eligible NSCLC patients who were 18 years old and had clinical stage IIB-IIIB disease. All 49 patients had surgical resection within 4-6 weeks after 2-3 cycles of neoadjuvant treatment consisting of immunotherapy (24 patients), chemotherapy (16 patients), and a targeted therapy (9 patients) regimen starting on the first day of each 21-day cycle. Pathologic complete response (pCR) was evaluated as the primary endpoint. Major pathological response (MPR) and tumor regression rate (TRR) were also evaluated. Results: An improved pathologic complete response was achieved in the neoadjuvant immunotherapy arm compared with the neoadjuvant chemotherapy arm and neoadjuvant targeted therapy arm [20.8% (5/24) vs. 6.3% (1/16) vs. 0.0% (0/9); P = 0.089, 95% CI 0.138-0.151]. More importantly, we found that the curative effect of the neoadjuvant immunotherapy arm in pCR+MPR was better than that of the neoadjuvant chemotherapy arm and neoadjuvant targeted therapy arm [45.8% (11/24) vs. 18.8% (3/16) vs. 0.0% (0/9); P = 0.006, 95% confidence interval, 0.008-0.012]. Different neoadjuvant therapies had a statistically significant effect on postoperative pathological tumor downstaging (P = 0.017). Conclusions: Neoadjuvant immunotherapy was associated with a trend toward better pCR than the neoadjuvant chemotherapy arm and neoadjuvant targeted therapy. Curative effect (pCR + MPR) was significantly better with neoadjuvant immunotherapy (P = 0.006, 95% confidence interval, 0.008-0.012). Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT04197076?recrs=a&cond=NCT04197076&draw=2&rank=1.

Research on interaction regularities and mechanisms between lactic acid and aroma compounds of Baijiu.

This study investigated the interactions between lactic acid (LA) and odorants of Baijiu using headspace solid phase microextraction with gas chromatography-mass spectrometry (HS-SPME-GC-MS), ultraviolet absorption spectroscopy (UV) and nuclear magnetic resonance (NMR). The HS-SPME-GC-MS analysis results showed that LA promoted the volatilities of most of low boiling acids, esters, alcohols, aldehydes and ketones, especially short-chain branched esters were promoted by 41-49%. In contrast, LA suppressed the volatilities of most aromatic compounds. UV spectroscopy, thermodynamic analysis, and NMR combined with theoretical calculations further revealed that the interactions between LA and 4-ethyl-2-methoxyphenol (4-EP), 2-methoxy-4-methylphenol (2-MP) and 1-butanol were dominated by van der Waals forces and supplemented by electrostatic interactions, which included hydrogen bonds formed between the carboxyl group in LA and the hydroxyl and methoxy groups in 4-EP or 2-MP and π-hydrogen bonds between the hydrogen of the carboxyl group of LA and the benzene ring of 4-EP or 2-MP.

Metatranscriptomics Unravel Composition, Drivers, and Functions of the Active Microorganisms in Light-Flavor Liquor Fermentation.

The microbial community in the fermented pit determines the quantity and quality of light-flavor liquor. Genetic diversity and the potential functions of the microbial community are often analyzed by DNA-based omics sequencing. However, the features of the active microbial community have not been systematically studied. Here, metatranscriptomic analysis was performed to elucidate the active microbial composition, drivers, and their functions in light-flavor liquor fermentation. Bacterial genera, Lactobacillus, Streptococcus, Pediococcus, Thermotoga, and Faecalibacterium, and fungal genera, Saccharomyces, Talaromyces, Aspergillus, Clavispora, Rhizophagus, Cyberlindnera, and Wickerhamomyces, were the dominant active microorganisms during the fermentation process. Additionally, they dominated the three-stage fermentation successively. Redundancy analysis showed that pH, ethanol, moisture, and starch were the main driving forces of microbial succession. Among the genes for the respective carbohydrate-active enzyme families, those for the glycoside hydrolase family 23, the glycosyltransferase family 2, the carbohydrate-binding module family 50, the polysaccharide lyase family 4, the auxiliary activity family 1, and the carbohydrate esterase family 9 showed the highest expression level. Additionally, the highly expressed enzymes and their contributed microorganisms were found in the key KEGG pathways, including carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Based on these data, a functional model of carbohydrate hydrolysis, ethanol production, and flavor generation were proposed. Taken together, Saccharomyces, Lactobacillus, Wickerhamomyces, Pediococcus, Candida, and Faecalibacterium were suggested as the core active microorganisms. Overall, our findings provide new insights into the composition, drivers, and functions of the active microorganisms, which is crucial for improving the quality of light-flavor liquor. IMPORTANCE There is an urgent need for discovering the diversity and functions of the active microbial community in solid-state fermentation, especially in the pit of Chinese distilled liquor fermentation. Although the genetic composition of the microbial community has been clarified frequently by DNA-based sequencing, the composition and functions of the active microbial community have not been systematically revealed so far. Therefore, analysis of RNA-based data is crucial for discovering the functional microbial community. In this study, we employed metatranscriptomic analysis to elucidate the active microbial composition, successive drivers, and their functions in light-flavor liquor fermentation. The strategy can be broadly useful for discovering the active microbial community and exploring their functions in other types of flavor distilled liquor or other ecosystems. This study provides new insights into the understanding of the active microbial community composition and its functions.

Estimating tumor mutational burden across multiple cancer types using whole-exome sequencing.

BACKGROUND: Tumor mutational burden (TMB) is emerging as a promising biomarker in immune checkpoint inhibitor (ICI) therapy. Despite whole-exome sequencing (WES) being the gold standard for quantifying TMB, TMB is determined by selected targeted panels in most cases, and WES-derived TMB data are lacking due to the greater cost and complexity. Determining TMB thresholds is another issue that needs attention. METHODS: A total of 309 patients who had received ICI therapy, representing five cancers (listed in "Results"), were recruited. Among them, 269 patients were evaluable for survival analysis. Tumor and matched blood samples from the patients were analyzed using WES and somatic mutations were determined. TMB is defined as the total number of somatic nonsynonymous mutations in the tumor exome in our study. The patients were divided into different TMB subgroups according to a common fixed number (10 mutations/Mb) or the top tertile within each tumor type. RESULTS: The distribution of WES-derived median TMBs was highly variable across different tumor types, ranging from 2.71 (cholangiocarcinoma) to 2.97 (nervous system tumor), 3.69 (gastric cancer), 4.31 (hepatocellular carcinoma), and 4.64 [colorectal cancer (CRC)] mutations/Mb. In CRC, the survival benefit of TMB-high patients was significant using both the top tertile and the 10 mutations/Mb threshold. In hepatocellular carcinoma, the 10 mutations/Mb threshold showed an advantage over the top tertile threshold. Among patients with nervous system tumors, cholangiocarcinoma, and gastric cancer, no obvious survival differences were observed between the TMB-high and TMB-low groups with either TMB stratification approach. CONCLUSIONS: The TMB threshold criterion may vary for different cancers. Our data suggest that TMB is unable to predict ICI benefit across all cancer types in Chinese patients. However, it may be an effective biomarker for predicting the clinical benefit of ICI therapy for patients with CRC.

Characterization of the key odorants and their content variation in Niulanshan Baijiu with different storage years using flavor sensory omics analysis.

Niulanshan Baijiu (NLS) is a light-flavor Baijiu (LFB) with a long history. The aroma-active compounds in six NLSs with different storage years were analyzed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS), coupled with Osme and aroma extraction dilution analysis (AEDA). A total of 59 odorants were identified, 5 odorants of them were, for the first time, identified as aroma-active compounds of LFB. After quantifying and calculating their odorant activity values (OAVs), 34 compounds had OAVs > 1, and the recombination and omission experiments further confirmed that ethyl acetate, ethyl acrylate, ethyl 2-methylbutyrate, γ-nonalactone, ethyl isovalerate, ethyl butyrate, isoamyl acetate, ethyl caprylate, ethyl valerate, 3-methylbutanal, ß-damascenone, and geosmin have important contributions to the aroma of NLS. One-way ANOVA analysis further found that the contents of key odorants in NLS stored for four to five years were relatively stable. This study provides an important reference for product quality control in NLS.

Unique genomic features and prognostic value of COSMIC mutational signature 4 in lung adenocarcinoma and lung squamous cell carcinoma.

BACKGROUND: Analysis of mutational signatures is becoming routine in cancer genomics, with implications for pathogenesis, classification, and prognosis. Among the signatures cataloged at COSMIC, mutational signature 4 has been linked to smoking. However, the distribution of signature 4 in Chinese lung cancer patients has not been evaluated, and its clinical value has not been evaluated. Here we survey mutational signatures in Chinese lung cancer patients and explore the relationship between signature 4 and other genomic features in the patients. METHODS: We extracted mutational signatures from whole-exome sequencing data of Chinese non-small cell lung cancer patients. The data included 401 lung adenocarcinoma (LUAD) and 92 squamous cell carcinoma (LUSC). We then performed statistical analysis to search for genomic and clinical features that can be linked to mutation signatures. RESULTS: We found signature 4 is the most frequent mutational signature in LUSC and the second most frequent in LUAD. Fifty-six LUAD and thirty-five LUSC patients were named with high signature 4 similarities (cosine similarity >0.7). These patients have shorter survival and higher tumor mutational burden comparing to those with low signature 4 similarities. Dozens of genes with single nucleotide variation, index mutations, and copy number variations were differentially enriched in the patients with high signature 4 similarities. Among these genes, CSMD3, LRP1B, TP53, SYNE1, SLIT2, FGF4, and FGF19 are common in both LUADs and LUSCs with high signature 4 similarities, showing that these genes are tightly associated with signature 4. CONCLUSIONS: The present study is the first to report a comparison in Chinese NSCLC patients with or without COSMIC mutational signature 4. These results will help find the Signature 4 related mutational process in NSCLC.

Patient specific circulating tumor DNA fingerprints to monitor treatment response across multiple tumors.

BACKGROUND: Circulating tumor DNA (ctDNA) offers a convenient way to monitor tumor progression and treatment response. Because tumor mutational profiles are highly variable from person to person, a fixed content panel may be insufficient to track treatment response in all patients. METHODS: We design ctDNA fingerprint panels specific to individual patients which are based on whole exome sequencing and target to high frequency clonal population clusters in patients. We test the fingerprint panels in 313 patients who together have eight tumor types (colorectal, hepatocellular, gastric, breast, pancreatic, and esophageal carcinomas and lung cancer and cholangiocarcinoma) and exposed to multiple treatment methods (surgery, chemotherapy, radiotherapy, targeted-drug therapy, immunotherapy, and combinations of them). We also monitor drug-related mutations in the patients using a pre-designed panel with eight hotspot genes. RESULTS: 291 (93.0%) designed fingerprint panels harbor less than ten previously known tumor genes. We detected 7475 ctDNA mutations in 238 (76%) patients and 6196 (96.0%) of the mutations are detected in only one test. Both the level of ctDNA content fraction (CCF) and fold change of CCF (between the definitive and proceeding tests) are highly correlated with clinical outcomes (p-values 1.36e-6 for level and 5.64e-10 for fold change, Kruskal-Wallis test). The CCFs of PD patients are an order of magnitude higher than the CCFs of SD and OR patients (median/mean 2.22%/8.96% for SD, 0.18/0.21% for PD, and 0.31/0.54% for OR; pairwise p-values 7.8e-6 for SD ~ PD, 2.7e-4 for OR ~ PD, and 7.0e-3 for SD ~ OR, Wilcoxon rank sum test). The fold change of CCF distinguishes the patient groups even better, which increases for PD, remains stable for SD, and decreases for OR patients (p-values 0.002, ~ 1, and 0.0001 respectively, Wilcoxon signed-rank test). Eleven drug-related mutations are identified from nine out of the 313 patients. CONCLUSIONS: The ctDNA fingerprint method improves both specificity and sensitivity of monitoring treatment response across several tumor types. It can identify tumor relapse/recurrence potentially earlier than imaging-based diagnosis. When augmented with tumor hotspot genes, it can track acquired drug-related mutations in patients.

Stigmasterol alleviates cerebral ischemia/reperfusion injury by attenuating inflammation and improving antioxidant defenses in rats.

BACKGROUND/AIMS: The paper aimed to investigate the effects of Stigmasterol on inflammatory factors, antioxidant capacity, and apoptotic signaling pathways in brain tissue of rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: The neurological deficits of the rats were analyzed and HE staining was performed. The cerebral infarct volume was calculated by means of TTC staining, and neuronal apoptosis was detected by TUNEL staining. At the same time, the contents of glutathione peroxidase, glutathione, superoxide dismutase (SOD), nitric oxide, and malondialdehyde in brain tissue were measured. The expression of the relevant protein was detected by means of Western blotting. RESULTS: The results showed that the neurological deficit score and infarct area of the I/R rats in the soy sterol treatment group were significantly lower than those in the I/R group. Moreover, the levels of carbon monoxide and malondialdehyde in the soysterol group were significantly lower than those in the I/R group, and the expressions of cyclooxygenase-2 (Cox-2) and NF-κB (p65) in the soysterol group were also significantly lower than those in the I/R group. The expression of Nrf2 (nucleus) and heme oxygenase-1 (HO-1) increased significantly, and the activities of antioxidant enzymes and SOD were increased. In addition, the stigmasterol treatment can inhibit apoptosis, down-regulate Bax and cleaved caspase-3 expression, and up-regulate Bcl-Xl expression. CONCLUSION: Stigmasterol protects the brain from brain I/R damage by reducing oxidative stress and inflammation.

Comprehensive analysis of potential immunotherapy genomic biomarkers in 1000 Chinese patients with cancer.

BACKGROUND: Tumor mutation burden (TMB), DNA mismatch repair deficiency (dMMR), microsatellite instability (MSI), and PD-L1 amplification (PD-L1 AMP) may predict the efficacy of the PD-1/PD-L1 blockade. With the broadening landscape of immunotherapy use, it is important to identify patients who are likely to benefit from the therapy. This study aimed to characterize the distributions of these biomarkers and explore the relationships among these biomarkers for Chinese patients with cancer. METHODS: In this study, we examined the aforementioned biomarkers in more than 1000 Chinese patients with cancer. These biomarkers were determined based on whole-exome sequencing (WES) of tumor/blood samples. RESULTS: Of the 953 samples from Chinese cancer patients assessed in this study, 35% exhibited high TMB (TMB-H), 4% were positive for high MSI (MSI-H), dMMR occurred in 0.53%, and PD-L1 AMP was positive in 3.79%. We found higher rates of TMB-H among hepatocellular carcinoma, breast cancer, and esophageal cancer patients than was reported for The Cancer Genome Atlas (TCGA) data. Lung cancer patients with EGFR mutations had significantly lower TMB values than those with wild-type EGFR, and increased TMB was significantly associated with dMMR in colorectal cancer (CRC). The frequency of tumors with MSI-H was the highest in CRC and gastric cancer. PD-L1 AMP occurred most frequently in lung squamous cell carcinoma and HER2-positive breast cancer. While MSI and dMMR are associated with higher mutational loads, correlations between TMB-H and other biomarkers, between MSI-H and dMMR, and between PD-L1 AMP and other biomarkers were low, indicating different underlying causes of the four biomarkers. CONCLUSION: The results reveal the frequency of these biomarkers in different malignancies, with potential implications for PD-1/PD-L1 blockade use for Chinese patients with cancer.

Genome-Wide Association Study Identifies a Genetic Prediction Model for Postoperative Survival in Patients with Hepatocellular Carcinoma.

BACKGROUND As an important aspect of tumor heterogeneity, genetic variation may influence susceptibility and prognosis in different types of cancer. By exploring the prognostic value of genetic variation, this study aimed to establish a model for predicting postoperative survival and assessing the impact of variation on clinical outcomes in patients with hepatocellular carcinoma (HCC). MATERIAL AND METHODS A genome-wide association study of 367 patients with HCC was conducted to identify single nucleotide polymorphisms (SNPs) associated with prognosis. Identified predictors were further evaluated in 758 patients. Two prognostic models were established using Cox proportional hazards regression and Nomogram strategy, and validated in another 316 patients. The effect of the SNP rs2431 was analyzed in detail. RESULTS A prognostic model including 5 SNPs (rs10893585, rs2431, rs34675408, rs6078460, and rs6766361) was established and exhibited high predictive accuracy for HCC prognosis. The panel combined with tumor node metastasis (TNM) stage resulted in a significantly higher c-index (0.723) than the individual c-index values. Stratified by the Nomogram prediction model, the median overall survival for the low-risk and high-risk groups were 100.1 versus 30.8 months (P<0.001) in the training set and 82.2 versus 22.5 months (P<0.001) in the validation set. A closer examination of rs2431 revealed that it may regulate the expression of FNDC3B by disrupting a microRNA-binding site. CONCLUSIONS This study established prediction models based on genetic factors alone or in combination with TNM stage for postoperative survival in patients with HCC, and identified FNDC3B as a potential therapeutic target for combating HCC metastasis.

Using clinical genomic sequencing to guide personalized cancer therapy in China.

Aim: To evaluate whether clinical genomic sequencing may benefit Chinese patients with stage IV cancer. Patients & methods: Chinese patients with cancer and their oncologists were provided with genomic sequencing results and corresponding clinical treatment recommendations based on evidence-based medicine, defined as CWES (clinical whole-exome sequencing) analysis. Chinese patients with stage IV cancer who failed the previous treatment upon receiving the CWES reports were included for analyzing the impact of CWES on clinical outcomes in 1-year follow-ups. Results: A total of 88.6% of 953 Chinese patients with cancer had clinically actionable somatic genomic alterations. Eleven patients followed the CWES reports, and 11 patients did not follow the CWES suggestions. The median progression-free survival of two groups were 12 and 4 months, and 45 and 91% of patients failed this round of therapy, respectively. Conclusion: The current study suggested that CWES has the potential to increase clinical benefits for Chinese patients with stage IV cancer.

Mutated EPHA2 is a target for combating lymphatic metastasis in intrahepatic cholangiocarcinoma.

Exploring the genetic aberrations favoring metastasis is important for understanding and developing novel strategies to combat cancer metastasis. It remains lack of effective treatment for the dismal prognosis of intrahepatic cholangiocarcinoma (ICC). Here, we aimed to study genetic alternations during lymph node metastasis of ICC and investigate potential mechanisms and clinical strategy focused on mutations. We performed whole-exome sequencing and transcriptome sequencing on samples from 30 ICC patients, including lymph node metastases from five of the patients. We identified the alterations of genetic pattern related to lymph node metastases of ICC. EPHA2, a member of the tyrosine kinase family, was found to be frequently mutated in ICC. Correlation analysis indicated that EPHA2 mutations were closely associated with lymph node metastasis of ICC. In vitro and in vivo experiments revealed that EPHA2 mutations could lead to ligand independent phosphorylation of Ser897, and promote lymphatic metastasis of ICC, in which NOTCH1 signaling pathway played an important role. In both in vitro assays and patient-derived xenografts, an inhibitor of Ser897 phosphorylation effectively suppressed the metastasis of ICC with mutated EPHA2. Our findings demonstrated that EPHA2 mutants may be an attractive therapeutic target for lymphatic metastasis of ICC.

Osteopontin alters DNA methylation through up-regulating DNMT1 and sensitizes CD133+/CD44+ cancer stem cells to 5 azacytidine in hepatocellular carcinoma.

BACKGROUND: In hepatocellular carcinoma (HCC), CD133+/CD44+ cells are one subgroup with high stemness and responsible for metastatic relapse and resistance to treatment. Our previous studies have demonstrated that osteopontin (OPN) plays critical roles in HCC metastasis. We further investigated the molecular mechanism underlying the role of OPN in regulating the stemness of HCC epigenetically and explored possible targeting strategy. METHODS: CD133+/CD44+ subgroup sorting from HCC cell lines and HCC tissues was used to investigate the effects of OPN knockdown on stemness. iTRAQ and MedIP-sequencing were applied to detect the protein profile and epigenetic modification of CD133+/CD44+ subgroup with or without OPN knockdown. The antitumor effects of 5 Azacytidine were examined in cultured HCC cells and patient derived xenograft (PDX) models. RESULTS: OPN was accumulated in CD133+/CD44+ subgroup of HCC cells. Knocking down OPN significantly inhibited the sphere formation and stemness-related genes expression, and delayed tumor initiation of CD133+/CD44+ subgroup of HCC cells. Employing MedIP-sequencing, dot blot and iTRAQ analyses of CD133+/CD44+ SCR and CD133+/CD44+ shOPN cells, we found that OPN knockdown leaded to reduction in DNA methylation with particular enrichment in CGI. Meanwhile, DNA (cytosine-5)-methyltransferase 1 (DNMT1), the main methylation maintainer, was downregulated via proteomics analysis, which mediated OPN altering DNA methylation. Furthermore, DNMT1 upregulation could partially rescue the properties of CD133+/CD44+ shOPN cells. Both in vitro and in vivo assays showed that CD133+/CD44+ cells with high OPN levels were more sensitive to DNA methylation inhibitor, 5 Azacytidine (5 Aza). The above findings were validated in HCC primary cells, a more clinically relevant model. CONCLUSIONS: OPN induces methylome reprogramming to enhance the stemness of CD133+/CD44+ subgroup and provides the therapeutic benefits to DNMT1 targeting treatment in HCC.

MicroRNA-219-5p Promotes Tumor Growth and Metastasis of Hepatocellular Carcinoma by Regulating Cadherin 1.

MicroRNAs play significant roles in the development of cancer and may serve as promising therapeutic targets. In our previous work, miR-219-5p was identified as one of the important metastasis-related microRNAs in HCC. Here we demonstrated that miR-219-5p expression was elevated in HCC tissues and was associated with vascular invasion and dismal prognosis. In multivariate analysis, miR-219-5p was identified as an independent prognostic indicator for HCC patients. Functional mechanism analyses showed that miR-219-5p promoted HCC cell proliferation and invasion in in vitro, as well as in vivo, tumor growth and metastasis in nude mice models bearing human HCC tumors. In addition, cadherin 1 (CDH1) was revealed to be a downstream target of miR-219-5p in HCC cells. In conclusion, miR-219-5p promotes tumor growth and metastasis of HCC by regulating CDH1 and can serve as a prognostic marker for HCC patients.

Osteopontin promotes metastasis of intrahepatic cholangiocarcinoma through recruiting MAPK1 and mediating Ser675 phosphorylation of ß-Catenin.

The incidence and mortality of intrahepatic cholangiocarcinoma (ICC) are increasing worldwide in recent decades. Osteopontin (OPN) plays an important role in cancer metastasis, but its functional mechanism in ICC is not clear yet. In this study, we found that OPN level was elevated both in plasma and tumor tissues of ICC patients, which was closely related to a shorter overall survival (OS) and high probability of tumor relapse after curative resection. The gain- and loss-of-function studies determined that OPN could promote ICC growth and metastasis. OPN selectively interacted with ß-Catenin and knockdown of ß-Catenin abrogated the effects induced by OPN. OPN recruited MAPK1 and activated MEK-MAPK1 pathway to mediate the S675 phosphorylation of ß-Catenin and nucleus accumulation, which induced the activation of Wnt signaling. Moreover, a significant correlation between OPN and ß-Catenin was found in ICC tissues. OPN, ß-Catenin, and their combination were independent prognostic indicator for ICC patients. In conclusion, OPN promotes ICC progression through recruiting MAPK1 and activating the Wnt/ß-Catenin pathway and can serve as a novel prognostic marker and therapeutic target for ICC.