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Prof Fuchou Tang’s and Prof Wei Fu’s groups uncovered pathogenesis of familial adenomatous polyposis (FAP)

时间:2019/11/21   放大字体 放小字体 打印

Colorectal cancer is the world's fourth most deadly cancer. Adenocarcinoma is the most common form of colorectal cancer (>95%) while rarer subtypes include carcinoid, sarcoma, and lymphoma. The progression of lung adenocarcinoma can be classified by histopathological grade and tumor stage. Familial adenomatous polyposis (FAP) is an inherited colorectal cancer syndrome and is caused by inherited mutations in adenomatous polyposis coli (APC). Patients with FAP develop hundreds to thousands of lesions, including pre-malignant adenomas and malignant carcinoma in the colon and rectum, which offers us a natural model to study the progression of colorectal cancer. Because we simultaneously collected adjacent normal tissue, adenomas at different stages and carcinomas from same FAP patient. In addition, we can obtain sufficient samples from limited patients. This will help us to avoid the interindividual variations, such as genetic background, eating habits and intestinal flora among lesions obtained from a large cohort.

On November 19, 2019, a research paper titled "Genomic and transcriptomic profiling of carcinogenesis in patients with familial adenomatous polyposis" was published in the internationally renowned academic journal GUT by Tang Fuchou's lab from Peking university and Fu Wei's lab from Third Hospital of Peking University. The study obtained 56 lesions from 6 typical FAP patients and 1 non-typical FAP patient (multiple colorectal adenoma associated with MUTYH mutation), including 6 peripheral blood samples, 12 adjacent normal tissues, 23 low-grade adenomas, 5 high-grade adenomas, and 10 carcinomas. For each lesion, three sets of data were simultaneously obtained, including WES, WGS and single-cell RNA- seq data. The genomic landscapes and clonal architecture of lesions at different evolutionary stages from the same patient with FAP were comprehensively investigated. In addition, single-cell RNA-seq data of 8,757 cells were used to investigate the transcriptomic heterogeneity among multi-regionally sampled lesions from the same patient with FAP and to explore the transcriptome dynamics in tumor cells during the initiation and carcinogenesis of colorectal adenomas (Figure 1).

Figure 1. Workflow. Adjacent normal tissue, adenomas and carcinomas from colon and rectum of FAPs were obtained for dissociation into single cells. Multiple-region sampling was used for adenocarcinomas larger than 10 mm. Parts of the dissociated cells were used for single-cell RNA-seq, and the remaining parts and matched peripheral blood were used for whole-exome sequencing and whole-genome sequencing.

The main findings of the paper are as follows:

1. Spatially separated tumors can originate from the same cancer-primed cell in patients with FAP, indicating that the pathogenic events may happen long before the appearance of clinically identifiable adenomas, even in a macroscopically normal epithelium.

By analyzing the genomic alterations of lesions from same patient, we found that adjacent lesions can share many somatic mutations and copy number variations, which indicates that these spatially separated lesions are monoclonal and originated from the same cell (Figure 2). This pattern was observed in two of the 7 patients. We propose that the process described in the previously reported field cancerization model also occurred in these two patients and contributed to physically separated lesions. In this model, a precancerous cell accumulates potential pathogenic mutations that endow it with a proliferative advantage, and its daughter cells then extend to adjacent areas by crypt fission. Next, with the accumulation of other pathogenic events, spatially separated lesions are formed. This phenomenon emphasizes that pathogenic events may occur long before the appearance of clinically identifiable adenomas, even in macroscopically normal epithelium.

Figure 2. (A) Phylogenetic tree of lesions at different evolutionary stages from FAP1 by using maximum parsimony algorithm. The colors of the lines in the phylogenetic tree correspond to the mutation types as mentioned previously. Potential driver mutations and cnLOH of APC genes are shown. (B) Schematic diagram indicating that all the lesions of FAP1 originated from the same cell. APC, adenomatous polyposis coli; Car, carcinoma; cnLOH, copy neutral loss of heterogeneity; FAP, familial adenomatous polyposis; HGIN, high- grade intra- epithelial neoplasia; Nor, adjacent normal tissue; R, region.

2. Enhanced metabolic processes and proliferative activity in the normal epithelium of patients with FAP.

Since patients with FAP are born with germline mutations in the APC gene, we speculated that the normal colon epithelium of patients with FAP may show transcriptomic signatures different from those of the normal colon epithelium of patients with sporadic CRCs. The transcriptomes of 707 single epithelial cells from adjacent normal tissues of six patients with FAP and 152 epithelial cells from adjacent normal tissues of two sporadic CRC specimens were profiled. We found that although these normal tissues did not share potential pathogenic mutations or CNAs with their corresponding adjacent lesions, they already tend to upregulated genes enriched in metabolic processes, including peptide biosynthetic, nucleotide metabolic, amino acid metabolic, lipid metabolic and carbohydrate metabolic processes (Figure 3). In addition, the genes involved in the cell cycle were also enriched, indicating that the proliferative potential of the cells from adjacent normal epithelium of patients with FAP has already been enhanced.

Figure 3. Transcriptome differences between the ‘normal’ epithelium of patients with FAP and the normal epithelium of sporadic patients with CRC. (A) Heatmap showing the top DEGs with the highest p- value between epithelial cells from adjacent normal tissue of patients with FAP (30 genes) and epithelial cells from adjacent normal tissue of patients with sporadic CRC (15 genes). (B) Gene ontology analysis of genes that show higher expression levels in FAP colon epithelium compared to CRC normal colon epithelium.

 

3. The metabolic signature of cancer has already been established in precancerous adenomas.

We constructed a pseudo-time map of carcinogenesis that could reflect the major transcriptomic changes during progression from normal epithelium to carcinoma. Cells from the adjacent normal epithelium and grade I adenomas were mainly distributed at the beginning of the pseudo-time trajectory, while cells from carcinomas were mainly distributed at the end of the pseudo-time trajectory. We found that the genes participated in the citric acid (tricarboxylic acid cycle (TCA)) cycle were downregulated in carcinoma compared to adjacent normal tissue. This result was consistent with previous findings that cancer cells mainly use glycolysis instead of the TCA pathway for energy production and generation of inter- mediate precursors for metabolite biosynthesis, a phenomenon called the Warburg effect. Interestingly, most of the genes involved in the TCA cycle were first downregulated from the adjacent normal epithelium to adenomas and were then slightly upregulated from adenomas to carcinomas, indicating that the TCA pathway is already strongly repressed in precancerous adenomas.

Figure 4. Transcriptome dynamics during carcinogenesis. (A) Heatmap showing scaled expression of dynamic genes along the pseudo-time. (B) Gene ontology analysis of genes from group 1 in (A).

In summary, we provided the first picture of the transcriptomic landscape of colorectal carcinogenesis in patients with FAP. The transcriptome dynamics during carcinogenesis were comprehensively investigated. In addition, we found that field cancerization occurs in patients with FAP. These findings will advance our understanding of the pathogenesis of CRC.

The co-first authors of the paper are Dr. Jingyun Li, Dr. Rui Wang Dr. Xin Zhou and Ph.D. student Wendong Wang. Professor Fuchou Tang from Beijing Advanced Innovation Center for Genomics and Professor Wei Fu from Peking University Third Hospital are the co-corresponding authors of the paper. This work was supported by grants from BIOPIC and Beijing Advanced Innovation Center for Genomics.