Genome-wide profiling in colorectal cancer identifies PHF19 and TBC1D16 as oncogenic super enhancers

Colorectal cancer is one of the most common cancers in the world. Although genomic mutations and single nucleotide polymorphisms have been extensively studied, the epigenomic status in colorectal cancer patient tissues remains elusive. Here, together with genomic and transcriptomic analysis, we use ChIP-Seq to profile active enhancers at the genome wide level in colorectal cancer paired patient tissues (tumor and adjacent tissues from the same patients). In total, we sequence 73 pairs of colorectal cancer tissues and generate 147 H3K27ac ChIP-Seq, 144 RNA-Seq, 147 whole genome sequencing and 86 H3K4me3 ChIP-Seq samples. Our analysis identifies 5590 gain and 1100 lost variant enhancer loci in colorectal cancer, and 334 gain and 121 lost variant super enhancer loci. Multiple key transcription factors in colorectal cancer are predicted with motif analysis and core regulatory circuitry analysis. Further experiments verify the function of the super enhancers governing PHF19 and TBC1D16 in regulating colorectal cancer tumorigenesis, and KLF3 is identified as an oncogenic transcription factor in colorectal cancer. Taken together, our work provides an important epigenomic resource and functional factors for epigenetic studies in colorectal cancer.

Capturing tumour heterogeneity in pre- and post-chemotherapy colorectal cancer ascites-derived cells using single-cell RNA-sequencing

Malignant ascites is an abnormal accumulation of fluid within the peritoneal cavity, caused by metastasis of several types of cancers, including colorectal cancer. Cancer cells in ascites reflect poor prognosis and serve as a good specimen to study tumour heterogeneity, as they represent a collection of multiple metastatic sites in the peritoneum. In this study, we have employed single-cell RNA-sequencing (scRNA-seq) to explore and characterise ascites-derived cells from a colorectal cancer patient. The samples were prepared using mechanical and enzymatic dissociations, and obtained before and after a chemotherapy treatment. Unbiased clustering of 19,653 cells from four samples reveals 14 sub-clusters with unique transcriptomic patterns in four major cell types: epithelial cells, myeloid cells, fibroblasts, and lymphocytes. Interestingly, the percentages of cells recovered from different cell types appeared to be influenced by the preparation protocols, with more than 90% reduction in the number of myeloid cells recovered by enzymatic preparation. Analysis of epithelial cell subpopulations unveiled only three out of eleven subpopulations with clear expansion or contraction after the treatment, suggesting that only parts of the heterogeneous ascites-derived cells were resistant to the treatment, potentially reflecting the poor treatment outcome observed in the patient. Overall, our study showcases highly heterogeneous cancer subpopulations at single-cell resolution, which respond differently to a particular chemotherapy treatment. All in all, this work highlights the potential benefit of single-cell analyses in planning appropriate treatments and real-time monitoring of therapeutic response in cancer patients through routinely discarded ascites samples.

Physical Activity after Colorectal Cancer Diagnosis and Mortality in a Nationwide Retrospective Cohort Study

Physical activity reduces the risk of colon cancer, but its prognostic impact after cancer diagnosis remains unclear. To evaluate the association between post-diagnosis activity and cause-specific mortality, we reconstructed a colorectal cancer patient cohort from the 2009–16 Korean National Health Insurance Service (NHIS) database. Subgroup analyses were performed by treatment group. In total, 27,143 colon cancer patients and 16,453 rectal cancer patients were included in the analysis (mean follow-up, 4.3 years; median 4.0 years). In the surgically treated group, a high level of activity (the weighted sum of the frequencies for walking, moderate, and vigorous activity greater than or equal to 3 times/week) was inversely associated with all-cause mortality (colon cancer: HR, 0.79; 95% CI, 0.72 to 0.88; rectal cancer: HR, 0.75; 95% CI, 0.66 to 0.86) and colorectal cancer-specific mortality (colon cancer: HR, 0.85; 95% CI, 0.76 to 0.97; rectal cancer: HR, 0.77; 95% CI, 0.66 to 0.90). No significant results were shown for cardiovascular disease-specific mortality. No association was shown in patients who received chemoradiotherapy without surgery. The present study may provide evidence for post-diagnosis physical activity as a prognostic factor in colorectal cancer, particularly in surgically treated early-stage patients.

Patients with mesenchymal tumours and high Fusobacteriales prevalence have worse prognosis in colorectal cancer (CRC)

ObjectivesTranscriptomic-based subtyping, consensus molecular subtyping (CMS) and colorectal cancer intrinsic subtyping (CRIS) identify a patient subpopulation with mesenchymal traits (CMS4/CRIS-B) and poorer outcome. Here, we investigated the relationship between prevalence of Fusobacterium nucleatum (Fn) and Fusobacteriales, CMS/CRIS subtyping, cell type composition, immune infiltrates and host contexture to refine patient stratification and to identify druggable context-specific vulnerabilities.DesignWe coupled cell culture experiments with characterisation of Fn/Fusobacteriales prevalence and host biology/microenviroment in tumours from two independent colorectal cancer patient cohorts (Taxonomy: n=140, colon and rectal cases of The Cancer Genome Atlas (TCGA-COAD-READ) cohort: n=605).ResultsIn vitro, Fn infection induced inflammation via nuclear factor kappa-light-chain-enhancer of activated B cells/tumour necrosis factor alpha in HCT116 and HT29 cancer cell lines. In patients, high Fn/Fusobacteriales were found in CMS1, microsatellite unstable () tumours, with infiltration of M1 macrophages, reduced M2 macrophages, and high interleukin (IL)-6/IL-8/IL-1β signalling. Analysis of the Taxonomy cohort suggested that Fn was prognostic for CMS4/CRIS-B patients, despite having lower Fn load than CMS1 patients. In the TCGA-COAD-READ cohort, we likewise identified a differential association between Fusobacteriales relative abundance and outcome when stratifying patients in mesenchymal (either CMS4 and/or CRIS-B) versus non-mesenchymal (neither CMS4 nor CRIS-B). Patients with mesenchymal tumours and high Fusobacteriales had approximately twofold higher risk of worse outcome. These associations were null in non-mesenchymal patients. Modelling the three-way association between Fusobacteriales prevalence, molecular subtyping and host contexture with logistic models with an interaction term disentangled the pathogen–host signalling relationship and identified aberrations (including NOTCH, CSF1-3 and IL-6/IL-8) as candidate targets.ConclusionThis study identifies CMS4/CRIS-B patients with high Fn/Fusobacteriales prevalence as a high-risk subpopulation that may benefit from therapeutics targeting mesenchymal biology.

Multi-Epitope-Based Vaccines for Colon Cancer Treatment and Prevention

BackgroundOverexpression of nonmutated proteins involved in oncogenesis is a mechanism by which such proteins become immunogenic. We questioned whether overexpressed colorectal cancer associated proteins found at higher incidence and associated with poor prognosis could be effective vaccine antigens. We explored whether vaccines targeting these proteins could inhibit the development of intestinal tumors in the azoxymethane (AOM)-induced colon model and APC Min mice.MethodsHumoral immunity was evaluated by ELISA. Web-based algorithms identified putative Class II binding epitopes of the antigens. Peptide and protein specific T-cells were identified from human peripheral blood mononuclear cells using IFN-gamma ELISPOT. Peptides highly homologous between mouse and man were formulated into vaccines and tested for immunogenicity in mice and in vivo tumor challenge. Mice treated with AOM and APC Min transgenic mice were vaccinated and monitored for tumors.ResultsSerum IgG for CDC25B, COX2, RCAS1, and FASCIN1 was significantly elevated in colorectal cancer patient sera compared to volunteers (CDC25B p=0.002, COX-2 p=0.001, FASCIN1 and RCAS1 p<0.0001). Epitopes predicted to bind to human class II MHC were identified for each protein and T-cells specific for both the peptides and corresponding recombinant protein were generated from human lymphocytes validating these proteins as human antigens. Some peptides were highly homologous between mouse and humans and after immunization, mice developed both peptide and protein specific IFN-γ-secreting cell responses to CDC25B, COX2 and RCAS1, but not FASCIN1. FVB/nJ mice immunized with CDC25B or COX2 peptides showed significant inhibition of growth of the syngeneic MC38 tumor compared to control (p<0.0001). RCAS1 peptide vaccination showed no anti-tumor effect. In the prophylactic setting, after immunization with CDC25B or COX2 peptides mice treated with AOM developed significantly fewer tumors as compared to controls (p<0.0002) with 50% of mice remaining tumor free in each antigen group. APC Min mice immunized with CDC25B or COX2 peptides developed fewer small bowel tumors as compared to controls (p=0.01 and p=0.02 respectively).ConclusionsImmunization with CDC25B and COX2 epitopes consistently suppressed tumor development in each model evaluated. These data lay the foundation for the development of multi-antigen vaccines for the treatment and prevention of colorectal cancer.

A qPCR-based method for rapid quantification of six intestinal homeostasis-relevant bacterial genera in feces

Aim: Developing efficient methods for monitoring the complex microbial community to rapidly assess the health status. Materials & methods: The qPCR-based method was developed, verified and in situ applied in fecal samples. Results: Six primer pairs with high specificity were designed to perform qPCR assays under a unified reaction condition within 2.5 h. The limits of detection, amplification efficiency and feasibility of the qPCR-based method established here were verified. In situ application of 18 fecal samples showed that the amounts of Bacteroides, Streptococcus and Bifidobacterium in colorectal cancer patient feces were obviously lower than those of healthy volunteers. Conclusion: This qPCR-based method was a reliable tool for rapid quantification of the six intestinal homeostasis relevant bacterial genera in feces.