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Abstract Chronic inflammation promotes development and progression of colorectal cancer (CRC). To comprehensively understand the molecular mechanisms underlying the development and progression of inflamed CRC, we perform in vivo screening and identify 142 genes that are frequently mutated in inflammation-associated colon tumors. These genes include senescence and TGFβ-activin signaling genes. We find that TNFα can induce stemness and activate senescence signaling by enhancing cell plasticity in colonic epithelial cells, which could act as a selective pressure to mutate senescence-related genes in inflammation-associated colonic tumors. Furthermore, we show the efficacy of the Cdk4/6 inhibitor in vivo for inflammation-associated colonic tumors. Finally, we functionally validate that Arhgap5 and Mecom are tumor suppressor genes, providing possible therapeutic targets for CRC. Thus, we demonstrate the importance of the inactivation of senescence pathways in CRC development and progression in an inflammatory microenvironment, which can help progress toward precision medicine.

Abstract Background Intra-tumor heterogeneity (ITH) encompasses cellular differences in tumors and is related to clinical outcomes such as drug resistance. However, little is known about the dynamics of ITH, owing to the lack of time-series analysis at the single-cell level. Mouse models that recapitulate cancer development are useful for controlled serial time sampling. Results We performed single-cell exome and transcriptome sequencing of 200 cells to investigate how ITH is generated in a mouse colorectal cancer model. In the model, a single normal intestinal cell is grown into organoids that mimic the intestinal crypt structure. Upon RNAi-mediated downregulation of a tumor suppressor gene APC, the transduced organoids were serially transplanted into mice to allow exposure to in vivo microenvironments, which play relevant roles in cancer development. The ITH of the transcriptome increased after the transplantation, while that of the exome decreased. Mutations generated during organoid culture did not greatly change at the bulk-cell level upon the transplantation. The RNA ITH increase was due to the emergence of new transcriptional subpopulations. In contrast to the initial cells expressing mesenchymal-marker genes, new subpopulations repressed these genes after the transplantation. Analyses of colorectal cancer data from The Cancer Genome Atlas revealed a high proportion of metastatic cases in human subjects with expression patterns similar to the new cell subpopulations in mouse. These results suggest that the birth of transcriptional subpopulations may be a key for adaptation to drastic micro-environmental changes when cancer cells have sufficient genetic alterations at later tumor stages. Conclusions This study revealed an evolutionary dynamics of single-cell RNA and DNA heterogeneity in tumor progression, giving insights into the mesenchymal-epithelial transformation of tumor cells at metastasis in colorectal cancer.

Abstract Extensive molecular analyses of ependymal tumors have revealed that supratentorial and posterior fossa ependymomas have distinct molecular profiles and are likely to be different diseases. The presence of C11orf95-RELA fusion genes in a subset of supratentorial ependymomas (ST-EPN) indicated the existence of molecular subgroups. However, the pathogenesis of RELA fusion-negative ependymomas remains elusive. To investigate the molecular pathogenesis of these tumors and validate the molecular classification of ependymal tumors, we conducted thorough molecular analyses of 113 locally diagnosed ependymal tumors from 107 patients in the Japan Pediatric Molecular Neuro-Oncology Group. All tumors were histopathologically reviewed and 12 tumors were re-classified as non-ependymomas. A combination of RT-PCR, FISH, and RNA sequencing identified RELA fusion in 19 of 29 histologically verified ST-EPN cases, whereas another case was diagnosed as ependymoma RELA fusion-positive via the methylation classifier (68.9%). Among the 9 RELA fusion-negative ST-EPN cases, either the YAP1 fusion, BCOR tandem duplication, EP300-BCORL1 fusion, or FOXO1-STK24 fusion was detected in single cases. Methylation classification did not identify a consistent molecular class within this group. Genome-wide methylation profiling successfully sub-classified posterior fossa ependymoma (PF-EPN) into PF-EPN-A (PFA) and PF-EPN-B (PFB). A multivariate analysis using Cox regression confirmed that PFA was the sole molecular marker which was independently associated with patient survival. A clinically applicable pyrosequencing assay was developed to determine the PFB subgroup with 100% specificity using the methylation status of 3 genes, CRIP1, DRD4 and LBX2. Our results emphasized the significance of molecular classification in the diagnosis of ependymomas. RELA fusion-negative ST-EPN appear to be a heterogeneous group of tumors that do not fall into any of the existing molecular subgroups and are unlikely to form a single category.

Single-cell sequencing is a promising technology that can address cancer cell evolution by identifying genetic alterations in individual cells. In a recent study, genome-wide DNA copy numbers of single cells were accurately quantified by single-cell sequencing in breast cancers. Phylogenetic-tree analysis revealed genetically distinct populations, each consisting of homogeneous cells. Bioinformatics methods based on population genetics should be further developed to quantitatively analyse the single-cell sequencing data. We developed a bioinformatics framework that was combined with molecular-evolution theories to analyse copy-number losses. This analysis revealed that most deletions in the breast cancers at the single-cell level were generated by simple stochastic processes. A non-standard type of coalescent theory, the multiple-merger coalescent model, aided by approximate Bayesian computation fit well with the data, allowing us to estimate the population-genetic parameters in addition to false-positive and false-negative rates. The estimated parameters suggest that the cancer cells underwent sweepstake evolution, where only one or very few parental cells produced a descendent cell population. We conclude that breast cancer cells successively substitute in a tumour mass, and the high reproduction of only a portion of cancer cells may confer high adaptability to this cancer.

Background: Spontaneous Reporting Systems (SRSs) are passive systems composed of reports of suspected Adverse Drug Events (ADEs), and are used for Pharmacovigilance (PhV), namely, drug safety surveillance. Exploration of analytical methodologies to enhance SRS-based discovery will contribute to more effective PhV. In this study, we proposed a statistical modeling approach for SRS data to address heterogeneity by a reporting time point. Furthermore, we applied this approach to analyze ADEs of incretin-based drugs such as DPP-4 inhibitors and GLP-1 receptor agonists, which are widely used to treat type 2 diabetes. Methods: SRS data were obtained from the Japanese Adverse Drug Event Report (JADER) database. Reported adverse events were classified according to the MedDRA High Level Terms (HLTs). A mixed effects logistic regression model was used to analyze the occurrence of each HLT. The model treated DPP-4 inhibitors, GLP-1 receptor agonists, hypoglycemic drugs, concomitant suspected drugs, age, and sex as fixed effects, while the quarterly period of reporting was treated as a random effect. Before application of the model, Fisher’s exact tests were performed for all drug-HLT combinations. Mixed effects logistic regressions were performed for the HLTs that were found to be associated with incretin-based drugs. Statistical significance was determined by a two-sided p-value