APC expression was attenuated in EC, which inhibited EC cell migration.
We analyzed the role of APC in endometrial cancer patients with the TCGA database using UALCAN (http://ualcan.path.uab.edu). The expression of APC decreased in EC, contrasted with normal tissue (Fig. 1A). In addition, the clinical EC tissue samples showed decreased APC expression levels in cancer tissue, compared with cancer adjacent tissue using Western blot (n = 4 per group) (Fig. 1B). Next, the expression levels of APC in EC cell lines were measured using western blot and RT-qPCR and KLE cell line showed a higher APC expression level among 7 EC cell lines (Fig. 1C and D). To research the function of the APC gene in EC, we constructed APC knockdown KLE cell line. Western blot and RT-qPCR confirmed that APC was downregulated in KLE (Fig. 1E and F). Next, we researched the effects of APC on cell proliferation and migration of KLE. Cell counting kit-8 assay showed that there was no difference in proliferation between NC and APC knockdown KLE cells (Fig. 1G). While knockdown of APC in EC cell line (KLE) presented increased migration ability compared to EC cells with empty vector (Fig. 1H).
The DEGs between the control cells and APC knockdown KLE cells.
RNA-seq was used to compare the transcriptomes of control cells and APC knockdown KLE cells. Compared with the control cells, the heatmap and volcano plot revealed that 3154 upregulated DEGs and 3653 downregulated DEGs were identified in APC knockdown KLE cells, using change fold FC > 1.2 or FC < 10/12 and P-value < 0.01 as criteria for the screening of upregulated or downregulated genes (Fig. 2A and B). The gene ontology (GO) analysis showed that DEGs were related to intracellular organelle and intracellular anatomical structure (Fig. 2C). Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis found TNF, mTOR and MAPK signaling pathways were enriched in APC knockdown KLE cells (Fig. 2D). Compared with the control cells, we revealed 377 increased DARs and 1579 decreased DARs in APC knockdown KLE cells. Besides, the DEGs in DARs were identified and presented in the volcano plot (Fig. 2E). The GO analysis revealed that DEGs were associated with protein serine/threonine kinase activity cell and cell communication (Fig. 2F). Additionally, the KEGG enrichment analysis found ECM receptor and p53 signaling pathways were enriched in APC knockdown KLE cells (Fig. 2G).
The chromosomal structure variations between the control cells and APC knockdown KLE cells.
To Compared with the control cells, 8 obvious structural variations regions (labeled with black boxes) of inter-chromosomal interactions were identified in APC knockdown KLE cells, indicating chromosomal translocation in endometrial cancer progression, such as chr3 and chr8 (Fig. 3A). For example, 3 obvious interaction regions R6 (between Chr3 R2 and Chr8 R5), R7 (between Chr3 R1 and Chr8 R4)and R8 (between Chr3 R1 and Chr8 R3) were observed in Hi-C maps. The interaction signal (B1) between position A1 (chr8:102270000) and A2 (chr8:12155000) suggested a large inversion region in Chr8 exactly close neighbor to the translocation region (Fig. 3B). In addition, another intra-chromosomal interaction signal (B2, CNV region) in chr8 (111550000-118550000bp, Fig. 3C and D) and an intra-chromosomal interaction signal in chr4 were also found (Fig. 3E). In the CNV region, the volcano plot revealed that 356 upregulated DEGs and 667 downregulated DEGs have been identified in APC knockdown KLE cells (Fig. 3F), including some tumorigenesis-associated genes, such as FGF12, VEGFA and MAPK11. The GO analysis revealed that genes in the CNV region were related to cell surface receptor signaling and cell migration (Fig. 3G). TNF, ECM-receptor interaction and VEGF signaling pathways were enriched in APC knockdown KLE cells by the KEGG analysis (Fig. 3H).
The variations of the compartment, TAD boundary, and loop between the control cells and APC knockdown KLE cells
During EC progression, dynamic switches occur in chromatin compartments. Compared to the control cells, switched compartments were found in every chromosome in APC knockdown KLE cells (Fig. 4A). We identified 56 DEGs with loci transformed from compartment A to B (22 downregulated and 34 upregulated) and 79 DEGs with loci switched from compartment B to A (54 downregulated and 25 upregulated; Fig. 4B). The GO and KEGG analysis revealed that they were associated with the cytoskeleton, phenylalanine, tyrosine and tryptophan biosynthesis, and nucleotide excision repair in cancer (Supplementary Fig. 1A and B). Compartments are composed of topologically associating domains (TADs), which are self-interacting regions and remain stable under different physiological conditions and cell types. The split or newly formed TAD boundaries might induce interactions between enhancers and promoters to alter gene expression. TAD boundaries presented higher GC content, and more chromosome-enriched open regions and genes than TADs interiors in KLE cells (Supplementary Fig. 1C and D). Compared to the control cells, we identified 790 TAD merges, 288 TAD splits, and 243 TAD rearrangements in APC knockdown KLE cells.
Next, the volcano plot showed 150 downregulated and 63 upregulated DEGs located in the APC knockdown KLE cell-specific boundaries, including FGF12, RAB3B and CA9 (Fig. 4E). The GO and KEGG analysis presented that these DEGs were associated with cell-cell adhesion plasma-membrane adhesion molecules, Hippo signaling, VEGF signaling and ECM receptor interaction (Supplementary Fig. 1E and F). Enhancers activate promoters via chromatin loops to regulate gene expression. We detected 2577 and 327 loops for the control cells and APC knockdown KLE cells. Loops related to enhancers upregulate gene expression. We identified 522 and 678 promoter-enhancer loops in the control cells and APC knockdown KLE cells, respectively (Fig. 4F). Besides, we revealed more promoter loops and enhancer loops in APC knockdown KLE cells than in the control cells (Fig. 4F). Moreover, we identified 446 control cell-specific loops and 602 APC knockdown KLE cells-specific loops, respectively (Fig. 4G). In control cells-specific loops, 50 APC knockdown KLE cells-upregulated genes and 74 APC knockdown KLE-downregulated genes were found, along with 49 APC knockdown KLE cells-upregulated genes and 34 APC knockdown KLE cells-downregulated genes in APC knockdown KLE cells-specific loops, such as FGF12, DIRC3, and ERBB4 (Fig. 4H). The GO and KEGG analysis indicated that these DEGs were associated with cell surface receptor, VEGF, ECM-receptor interaction and p53 signaling (Supplementary Fig. 1G and H).
APC knockdown in KLE cells upregulated FGF12 expression via rearranging the chromosomal structure, which predicts a worse outcome in EC.
To explore the potential downstream genes of APC, we identified the overlapped DEGs resided in the switched compartments, TADs, and loops in the control cells and APC knockdown KLE cells. 12 overlapped DEGs were identified, including FGF12, SLIT2, CDH6, SLIT3, SUGCT, CPED1, KCNU1, ZNF365, RAD52, GPC5, MX1 and SRPX (Fig. 4I). We found most significant changes in TADs and loops near FGF12 among 12 overlapped DEGs (Fig. 5A and Supplementary Fig. 2). In addition, FGF12 was highly expressed in APC knockdown KLE cells (Fig. 5B, C and D). P-AKT and P-ERK1/2 have been activated in APC knockdown KLE cells (Fig. 5C and D). Moreover, a high expression level of FGF12 predicts a worse prognosis in EC patients ((p = 0.0011, Fig. 5E).