Dual role of cadmium in rat liver: Inducing liver injury and inhibiting the progression of early liver cancer.

The heavy metal cadmium (Cd) can induce damage in liver and liver cancer cells; however, the mechanism underlying its toxicity needs to be further verified in vivo. We daily administered CdCl2 to adult male rats at different dosages via gavage for 12 weeks and established rat liver injury model and liver cancer model to study the dual role of Cd in rat liver. Increased exposure to Cd resulted in abnormal liver function indicators, pathological degeneration, rat liver cell necrosis, and proliferation of collagen fibres. Using immunohistochemistry, we found that the area of GST-P-positive precancerous liver lesions decreased in a dose-dependent manner. Real-time quantitative polymerase chain reaction, western blot, immunohistochemistry, and transmission electron microscopy revealed that Cd induced mitophagy, as well as mitophagy blockade, as evidenced by the downregulation of TOMM20 and upregulation of LC3II and P62 with increasing Cd dose. Next, the expression of PINK1/Parkin, a classic signalling pathway protein that regulates mitophagy, was examined. Cd was found to promote PINK1/Parkin expression, which was proportional to the Cd dose. In conclusion, Cd activates PINK1/Parkin-mediated mitophagy in a dose-dependent manner. Mitophagy blockade likely aggravates Cd toxicity, leading to the dual role of inducing liver injury and inhibiting the progression of early liver cancer.

Epstein Barr virus-positive B-cell lymphoma is highly vulnerable to MDM2 inhibitors in vivo.

Epstein-Barr virus-positive (EBV-positive) B-cell lymphomas are common in immunocompromised patients and remain an unmet medical need. Here we report that MDM2 inhibitors (MDM2is) navtemadlin and idasanutlin have potent in vivo activity in EBV-positive B-cell lymphoma established in immunocompromised mice. Tumor regression was observed in all 5 EBV-positive xenograft-associated B-cell lymphomas treated with navtemadlin or idasanutlin. Molecular characterization showed that treatment with MDM2is resulted in activation of p53 pathways and downregulation of cell cycle effectors in human lymphoma cell lines that were either EBV-positive or had undetectable expression of BCL6, a transcriptional inhibitor of the TP53 gene. Moreover, treatment with navtemadlin resulted in tumor regression and prevented systemic dissemination of EBV-positive lymphoma derived from 2 juvenile patients with posttransplant lymphoproliferative diseases, including 1 whose tumor was resistant to virus-specific T-cell therapy. These results provide proof-of-concept for targeted therapy of EBV-positive lymphoma with MDM2is and the feasibility of using EBV infection or loss of BCL6 expression to identify responders to MDM2is.

Transcription profiling of cadmium-exposed livers reveals alteration of lipid metabolism and predisposition to hepatic steatosis.

1. Cadmium (Cd) is a ubiquitous environmental toxicant that can cause liver steatosis and nonalcoholic fatty liver disease (NAFLD) on long-term exposure.2. Sixteen Sprague Dawley rats were randomly divided into two groups, and were administered normal saline and 5 mg/(kg·d) cadmium chloride by gavage. In vitro, BRL3A cells, a rat normal liver cell line, were treated with different concentrations of Cd to verify the sequencing results.3. The RNA-seq revealed 146 upregulated genes and 127 downregulated genes in the Cd intervention group. The key genes of lipid metabolism were significantly overexpressed, such as Cyp1a1 and Pla2g2d. The GO enrichment analysis showed that the 'sterol biosynthetic process' was the most obvious difference. The KEGG analysis showed that six of the top 10 differential pathways were related to lipid metabolism. The expression of the essential genes in BRL3A was consistent with the sequencing results. The protein-protein interaction (PPI) yielded that Cyp1a1 is in the central region of the differentially expressed gene network.4. The chronic Cd exposure is still an important environmental health problem with a probable tendency to cause NAFLD. It may possibly act by affecting the lipid metabolism in the liver, especially the synthesis and decomposition of unsaturated fatty acids.

Structure-based classification predicts drug response in EGFR-mutant NSCLC.

Epidermal growth factor receptor (EGFR) mutations typically occur in exons 18-21 and are established driver mutations in non-small cell lung cancer (NSCLC)1-3. Targeted therapies are approved for patients with 'classical' mutations and a small number of other mutations4-6. However, effective therapies have not been identified for additional EGFR mutations. Furthermore, the frequency and effects of atypical EGFR mutations on drug sensitivity are unknown1,3,7-10. Here we characterize the mutational landscape in 16,715 patients with EGFR-mutant NSCLC, and establish the structure-function relationship of EGFR mutations on drug sensitivity. We found that EGFR mutations can be separated into four distinct subgroups on the basis of sensitivity and structural changes that retrospectively predict patient outcomes following treatment with EGFR inhibitors better than traditional exon-based groups. Together, these data delineate a structure-based approach for defining functional groups of EGFR mutations that can effectively guide treatment and clinical trial choices for patients with EGFR-mutant NSCLC and suggest that a structure-function-based approach may improve the prediction of drug sensitivity to targeted therapies in oncogenes with diverse mutations.

Cadmium causes hepatopathy by changing the status of DNA methylation in the metabolic pathway.

Toxicity caused by the heavy metal Cadmium leads to liver diseases; this finding has generated interest among researchers. We detected DNA methylation using Whole Genome Bisulfite Sequencing (WGBS) to study the relationship between Cadmium exposure and liver damage. Forty-eight Sprague-Dawley rats were randomly divided into six groups, and given normal saline or 2.5, 5, 10, 20, and 40 mg/kg body weight per day CdCl2 by gavage. Twelve weeks later, their liver tissues were collected for pathological examination and DNA extraction. Increased exposure to Cadmium led to a reduction in the amount of weight gain as well as pathological degeneration and necrosis of liver cells of the rats. Using WGBS, we found that DNA methylation changes in the high-dose exposure group were more remarkable, and most of the changes occurred in the gene promoter region. GO enrichment analysis showed that the genes were enriched in the biological process of "response to stimulus." KEGG analysis revealed that metabolic pathways, like MAPK, PI3K-Akt and cAMP, had the largest number of enriched genes. Using Integrative Genomics Viewer (IGV), the demethylation of F2rl3 after Cadmium poisoning was established. This finding may explain why there are changes in liver metabolism after Cadmium poisoning.

KRT-232 and navitoclax enhance trametinib's anti-Cancer activity in non-small cell lung cancer patient-derived xenografts with KRAS mutations.

Activating mutations of the KRAS gene are one of the major genomic alterations associated with tumorigenesis of non-small cell lung cancer (NSCLC). Thus far, treatment of KRAS-mutant NSCLC remains an unmet medical need. We determined the in vivo treatment responses of 13 KRAS mutant and 14 KRAS wild type NSCLC patient-derived xenografts (PDXs) to agents that target known NSCLC vulnerabilities: the MEK inhibitor trametinib, the MDM2 inhibitor KRT-232, and the BCL-XL/BCL-2 inhibitor navitoclax. The results showed that the tumor regression rate after single agent therapy with KRT-232, trametinib and navitoclax was 11%, 10% and 0%, respectively. Combination therapies of trametinib plus KRT-232 and trametinib plus navitoclax led to improved partial response rates over single-agent activity in a subset of PDX models. Tumor regression was observed in 23% and 50% of PDXs after treatment with trametinib plus KRT-232 and trametinib plus navitoclax, respectively. The disease control rates in KRAS-mutant PDXs tested were 90%-100% after treatment with trametinib plus KRT-232 or plus navitoclax. A correlation analysis of treatment responses and genomic and proteomic biomarkers revealed that sensitivity to KRT-232 was significantly associated with TP53 wild-type or STK11 mutant genotypes (P<0.05). The levels of several proteins, including GSK3b, Nrf2, LKB1/pS334, and SMYD3, were significantly associated with sensitivity to trametinib plus navitoclax. Thus, the combination of trametinib plus KRT-232 or navitoclax resulted in improved efficacy compared with the agents alone in a subgroup of NSCLC PDX model with KRAS mutations. Expanded clinical trials of these targeted drug combinations in NSCLC are warranted.

Tumor characteristics associated with engraftment of patient-derived non-small cell lung cancer xenografts in immunocompromised mice.

BACKGROUND: Patient-derived xenograft (PDX) models increasingly are used in translational research. However, the engraftment rates of patient tumor samples in immunodeficient mice to PDX models vary greatly. METHODS: Tumor tissue samples from 308 patients with non-small cell lung cancer were implanted in immunodeficient mice. The patients were followed for 1.5 to approximately 6 years. The authors performed histological analysis of PDXs and some residual tumor tissues in mice with failed PDX growth at 1 year after implantation. Quantitative polymerase chain reaction and enzyme-linked immunoadsorbent assay were performed to measure the levels of Epstein-Barr virus genes and human immunoglobulin G in PDX samples. Patient characteristics were compared for PDX growth and overall survival as outcomes using Cox regression analyses. Disease staging was based on the 7th TNM staging system. RESULTS: The overall engraftment rate for PDXs from patients with non-small cell lung cancer was 34%. Squamous cell carcinomas had a higher engraftment rate (53%) compared with adenocarcinomas. Tumor samples from patients with stage II and stage III disease and from larger tumors were found to have relatively high engraftment rates. Patients whose tumors successfully engrafted had worse overall survival, particularly those individuals with adenocarcinoma, stage III or stage IV disease, and moderately differentiated tumors. Lymphoma formation was one of the factors associated with engraftment failure. Human CD8-positive and CD20-positive cells were detected in residual samples of tumor tissue that failed to generate a PDX at 1 year after implantation. Human immunoglobulin G was detected in the plasma of mice that did not have PDX growth at 14 months after implantation. CONCLUSIONS: The results of the current study indicate that the characteristics of cancer cells and the tumor immune microenvironment in primary tumors both can affect engraftment of a primary tumor sample.

Glutathione Might Attenuate Cadmium-Induced Liver Oxidative Stress and Hepatic Stellate Cell Activation.

The liver is a major organ involved in cadmium (Cd)-induced oxidative damage. Following liver injury, hepatic stellate cells (HSCs) are activated to participate in the wound healing process, but also facilitate liver fibrosis. Previous studies have observed fibrogenic effects of Cd on liver. However, the oxidative stress mechanisms of Cd-induced HSC activation as well as whether administration of glutathione (GSH) alleviates this activation, remain unclear. In this study, 24 rats were divided randomly into four experimental groups: control, GSH-treated, Cd-treated, and Cd + GSH-treated. After 4 weeks, the liver injury index, HSC-specific activation markers, oxidative stress-related antioxidants, and enzyme activities and signals were measured. Cd uptake and the generation of reactive oxygen species (ROS) in hepatocytes were detected by mass cytometry and fluorescence microscopy, respectively. Levels of aspartate aminotransferase, xanthine oxidase, γ-glutamyl transpeptidase, and α-smooth muscle actin (αSMA) were significantly increased in Cd-treated rats. Activated HSCs positive for αSMA expression and excess collagen deposition were detected in the Cd-treated group. In contrast, activities of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase were reduced. Supplementation with GSH reversed some of the Cd-induced effects and increased the protein level of phosphorylated (p)-P65 while decreasing p-JNK. Pretreatment with GSH lowered Cd uptake and ROS generation in hepatocytes in vitro. These results indicate that administration of GSH was effective in attenuating Cd-induced oxidative stress via decreasing Cd uptake, restoring the activities of oxidative enzymes, activating NF-κB, inhibiting the JNK signaling pathway, and preventing excessive ROS generation and HSC activation.

Relationship between intestinal microbial dysbiosis and primary liver cancer.

BACKGROUND: Intestinal microbial dysbiosis is involved in liver disease pathogenesis. However, its role in primary liver cancer (PLC), particularly in hepatocarcinogenesis remains unclear. The present study aimed to study the changes in intestinal flora at various stages of PLC and clarify the relationship between intestinal microbes and PLC. METHODS: Twenty-four patients with PLC (PLC group), 24 patients with liver cirrhosis (LC group), and 23 healthy control individuals (HC group) were enrolled from October 2016 to October 2017. Stool specimens of the participants were collected and the genomic DNA of fecal bacteria was isolated. High-throughput pyrosequencing of 16S rDNA was used to identify differences in gut bacterial diversity among HC, LC, and PLC groups. We also analyzed the relationship between clinical factors and intestinal microorganisms in LC and PLC groups. RESULTS: Diversity of Firmicutes tended to decrease from the HC to LC and PLC groups at the phylum level. Among species, Enterobacter ludwigii displayed an increasing trend in the PLC group, wherein the relative abundance of Enterobacter ludwigii in the PLC group was 100 times greater than that in the HC and LC groups. The ratio of Firmicutes/Bacteroidetes was significantly decreased with the disease progression. In addition, the linear discriminant analysis effect size method indicated that Clostridia were predominant in the gut microbiota of the HC group, whereas Enterococcaceae, Lactobacillales, Bacilli and Gammaproteobacteria may be used as diagnostic markers of PLC. Redundancy analysis showed a correlation between intestinal microbial diversity and clinical factors AST, ALT, and AFP. Veillonella showed a significant positive correlation with AFP in the PLC group, whereas Subdoligranulum showed a negative correlation with AFP. CONCLUSIONS: This study indicates that dysbiosis of the gut microbiota might be involved in PLC development and progression.

Identification and characterization of RTVP1/GLIPR1-like genes, a novel p53 target gene cluster.

Our previous finding of RTVP1 (GLIPR1) as a p53 target gene with tumor suppressor functions prompted us to initiate a genome-wide sequence homology search for RTVP1/GLIPR1-like (GLIPR1L) genes. In this study we report the identification and characterization of a novel p53 target gene cluster that includes human RTVP1 (hRTVP-1) together with two GLIPR1L genes (GLIPR1L1 and GLIPR1L2) on human chromosome 12q21 and mouse Rtvp1 (mRTVP-1 or Glipr1) together with three Glipr1-like (Glipr1l) genes on mouse chromosome 10D1. GLIPR1L1 has two and GLIPR1L2 has five differentially spliced isoforms. Protein homology search revealed that hRTVP-1 gene cluster members share a high degree of identity and homology. GLIPR1L1 is testis-specific, whereas GLIPR1L2 is expressed in different types of tissues, including prostate and bladder. Like hRTVP-1, GLIPR1L1 and GLIPR1L2 are p53 target genes. The similarities of these novel p53 target gene cluster members in protein structure and their association with p53 suggest that these genes may have similar biological functions.

RTVP-1, a tumor suppressor inactivated by methylation in prostate cancer.

We previously identified and characterized a novel p53-regulated gene in mouse prostate cancer cells that was homologous to a human gene that had been identified in brain cancers and termed RTVP-1 or GLIPR. In this report, we document that the human RTVP-1 gene is also regulated by p53 and induces apoptosis in human prostate cancer cell lines. We show that the expression of the human RTVP-1 gene is down-regulated in human prostate cancer specimens compared with normal human prostate tissue at the mRNA and protein levels. We further document epigenetic changes consistent with RTVP-1 being a tumor suppressor in human prostate cancer.