Targeting KAT7 inhibits the progression of colorectal cancer

Abstract

Rationale: Colorectal cancer (CRC) is a leading cause of cancer-related mortality. Epigenetic modifications play a significant role in the progression of CRC. KAT7, a histone acetyltransferase, has an unclear role in CRC. Methods: In this research, we analyzed the expression of KAT7 in CRC patients and its correlation with prognosis using the GEO database, western blot, and immunohistochemistry. We assessed the impact of KAT7 on CRC cell functions through cell viability, colony formation, flow cytometry, scratch, and transwell assays. Mechanistic insights were obtained via RNA sequencing and ChIP-qPCR. Additionally, we evaluated the effects of KAT7 on CRC growth and metastasis in vivo using mouse subcutaneous tumor and lung metastasis models. Results: In this study, we discovered an upregulated KAT7 signaling pathway in CRC and its association with poor patient survival. Knockdown of KAT7 promotes apoptosis and inhibits proliferation, migration, and invasion of CRC cells. Conversely, KAT7 overexpression enhanced these cellular processes. In vivo assays confirmed that knockdown of KAT7 can inhibit CRC proliferation and lung metastasis. Mechanistically, KAT7 acetylated histone H3 at lysine 14 (H3K14) to enhance MRAS transcription, which activated the MAPK/ERK pathway and promoted tumorigenesis. The enzymatic function of KAT7 as an acetyltransferase is crucial for the advancement of colorectal cancer. In KAT7 knockdown CRC cells, re-expression of KAT7, but not an acetyltransferase-deficient mutant, rescued MRAS expression, ERK phosphorylation, and CRC tumorigenesis. Conclusion: We found that KAT7 is highly expressed in CRC patients, and those with high KAT7 expression have a worse prognosis. KAT7 enhances MRAS gene transcription by promoting H3K14 acetylation, thereby activating the MAPK/ERK pathway and promoting malignant phenotypes of CRC. In summary, KAT7 represents a promising target for CRC therapy.

Keywords: KAT7; MAPK; colorectal cancer; epigenetics; target therapy.

Figure 1

KAT7 expression is significantly up-regulated in CRC. (A-C) Expression levels of KAT7 mRNA in colorectal cancer (CRC) tissues and normal colorectal tissues were analyzed using data from the GSE9348 dataset (A), the GSE20916 dataset (B), and the GSE21510 dataset (C). (D-E) Prognostic analysis of CRC patients with high/low expression of KAT7 was conducted using the Kaplan-Meier plotter database, evaluating overall survival (OS) (D) and post-progression survival (PPS) (E). (F) The expression of the KAT7 protein was examined in normal colonic epithelial cells and various CRC cell lines. (G) KAT7 protein expression was determined in colorectal tumor tissues and adjacent normal tissues. (H) Immunohistochemical (IHC) analysis was performed to assess KAT7 expression in CRC tissues and adjacent normal tissues (Scale bar = 100 μm). (I) The TNM staging of CRC patients with high/low expression of KAT7 in our clinical samples. Error bars represent the mean ± standard deviation (SD). Statistical significance was determined using Two-tailed unpaired Student's t-tests (A-C) and Chi-square test (I). *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 2

KAT7 knockdown suppresses cell viability, proliferation, and promotes apoptosis. (A) Western blot analysis was performed to assess the knockdown of KAT7 expression using shRNA. (B) CCK-8 assay was conducted to evaluate the viability of CRC cells after KAT7 knockdown. (C) CRC cells were treated as described in (A), and cell proliferation was examined through cell counting. (D) The impact of KAT7 knockdown on the colony formation ability of cells was observed. (E) EdU assay was performed to investigate the effect of KAT7 knockdown on cell proliferation (left, Scale bar = 100 μm). The bar chart represents the percentage of EdU-positive cells (right). (F) Flow cytometry was employed to analyze apoptosis in CRC cells after KAT7 knockdown (left), and the percentage of apoptotic cells was quantified (right). (G) The expression of apoptosis-related proteins in CRC cells was treated as described in (A). Error bars represent the mean ± SD. Statistical significance was determined using one-way ANOVA (B-F). **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

KAT7 silencing impairs migration and invasion in CRC cells. (A-B) Wound-healing experiments were conducted to observe cell migration after shRNA-mediated knockdown of KAT7. (C-D) The impact of KAT7 knockdown on the migration (C) and invasion (D) abilities of CRC cells was assessed (Scale bar = 100 μm). (E) Statistical analysis of cell migration distance in (A) and (B). (F) Statistical analysis of cell migration quantity in (C). (G) Statistical analysis of cell invasion quantity in (D). (H) The effect of KAT7 knockdown on the expression of proteins associated with epithelial-mesenchymal transition (EMT). Error bars represent the mean ± SD. Statistical significance was assessed using one-way ANOVA (E-G). ***P < 0.001, ****P < 0.0001.

Figure 4

KAT7 overexpression drives oncogenic progression in CRC cells. (A) Overexpression of KAT7 was performed in SW620 and HT29 cells. (B) Impact of KAT7 overexpression on CRC cell viability. (C) Cell counting assessment of CRC cell proliferation after KAT7 overexpression. (D) Colony formation assay after KAT7 overexpression (left) and corresponding colony count (right). (E) EdU experiment evaluating the proliferation of CRC cells treated as in (A), fluorescence image (left, Scale bar = 100 μm), and a statistical bar graph (right). (F-H) Changes in migration ability of SW620 (F) and HT29 (G) cells after KAT7 overexpression, and migration distance statistics (H). (I) Transwell chamber experiment observing the effect of KAT7 overexpression on cell migration ability (Scale bar = 100 μm). (J) Enhanced invasion ability of CRC cells after KAT7 overexpression (Scale bar = 100 μm). (K) Expression changes of EMT-related proteins in CRC cells after being treated as in (A). Error bars represent the mean ± SD. Statistical significance was determined using Two-tailed unpaired Student's t-tests (B-E, H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

KAT7 knockdown attenuates MAPK/ERK signaling pathway activity. (A) Heatmap of differentially expressed genes in KAT7-knockdown and control HCT116 cells generated using R software. (B) Functional categorization based on gene ontology (GO) term enrichment. (C) Signaling pathway analysis based on KEGG enrichment of subcategories in signal transduction pathways. (D) Functionally grouped networks based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, showing genes in KAT7-knockdown and control cells. (E) Gene set enrichment analysis (GSEA) of the MAPK signaling pathway in HCT116 cells treated as described in (A). (F) Heatmap of differentially expressed genes related to the MAPK signaling pathway in HCT116 control and KAT7 knockdown cells, analyzed using R software. (G) Quantitative real-time PCR analysis of relative mRNA expression of indicated genes in control and KAT7-knockdown HCT116 cells. (H) Impact of KAT7 knockdown on the expression of proteins related to the MAPK pathway. (I) Detection of MAPK pathway-related protein expression levels after KAT7 overexpression. (J) Cell viability of CRC cells with or without KAT7 knockdown following 8 hours of treatment with 0.1% DMSO or the MAPK/ERK agonist C6 Ceramide (5 μM). (K) Cell proliferation measurement in cells treated as described in (J). (L-M) Flow cytometry scatter plot (L) and a bar graph (M) showing apoptosis analysis of CRC cells treated as described in (J). (N) Impact of treatment described in (J) on the expression of apoptosis-related proteins in CRC cells. Error bars represent the mean ± SD. Statistical significance was assessed using one-way ANOVA (G, J-K, M). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns indicates no significance.

Figure 6

KAT7 upregulates MRAS expression to activate the MAPK/ERK signaling pathway in CRC cells. (A) Pie chart displaying the distribution of KAT7 binding sites on genes in ChIP-seq, with data obtained from a public database. (B) Venn diagram showing the overlap between genes bound by KAT7 in ChIP-seq and genes downregulated in RNA-seq after KAT7 knockdown. (C) KEGG analysis reveals the genes overlapping between ChIP-seq and RNA-seq as shown in (B). (D) Peak plot illustrating the binding of KAT7 in the promoter region of the MRAS gene. (E) qRT-PCR measuring the impact of KAT7 knockdown on MRAS gene expression. (F) Expression profile of the MRAS gene in CRC tissues and normal individuals, with data obtained from a public database . (G) Expression of the MRAS protein in CRC tissues and adjacent normal tissues. (H) Correlation analysis between MRAS mRNA and KAT7 mRNA expression, with data obtained from a public database . (I-K) Kaplan-Meier plotter database analysis of the relationship between MRAS expression and prognosis in CRC patients, including OS (I), recurrence-free survival (RFS) (J), and PPS (K). (L) Expression profile of indicated proteins in KAT7-knockdown/untargeted HCT116 cells overexpressing/not overexpressing MRAS. (M-O) Effects on cell proliferation (M), colony formation (N), and apoptosis (O) in HCT116 cells treated as described in (L). Error bars represent the mean ± SD. Statistical significance was assessed using one-way ANOVA (E, M-O), Two-tailed unpaired Student's t-tests (F), and nonlinear regression analysis (H). *P < 0.05, **P < 0.01, ****P < 0.0001. ns indicates no significance. TSS: Transcription start site.

Figure 7

KAT7 acetyltransferase activity is required for activating MAPK/ERK signaling. (A) Impact of KAT7 knockdown on histone acetylation. (B) Expression changes of the MRAS gene in KAT7-knockdown CRC cells overexpressing wild-type KAT7 (KAT7-WT) or acetylation-deficient KAT7 mutant (KAT7-G485A). (C) Western blot analysis of protein expression in HCT116/COLO320-shKAT7 cells treated as described in (B). (D-I) Cell proliferation measurement (D-F), flow cytometry analysis of cell apoptosis (G-H), and expression changes of apoptosis-related proteins (I) in HCT116/COLO320-shKAT7 cells overexpressing KAT7-WT or KAT7-G485A. Scale bar = 100 μm (E). (J-K) ChIP-qPCR assays were conducted on CRC cells treated with either control shRNA or KAT7 shRNA. Chromatin DNA was immunoprecipitated using antibodies specific for KAT7 or acetylated H3K14. The results showed that KAT7 knockdown reduced the binding of KAT7 (K) and the acetylation of H3K14 (L) on the promoter region (-1243~-1066) of the MRAS gene (J). Error bars represent the mean ± SD. Statistical significance was evaluated using one-way ANOVA for experiments (B, D, F, H) and two-tailed unpaired Student's t-tests (K-L). *P < 0.05, **P < 0.01, ***P < 0.001, **P < 0.0001. ns indicates no significance.

Figure 8

Knockdown of KAT7 inhibits CRC growth and metastasis in vivo. (A) Observation of subcutaneous tumor growth rate in COLO320 cells after KAT7 knockdown (n = 7 per group). (B) Evaluation of average tumor growth rate 25 days after tumor cell inoculation. (C) Collection and photography of subcutaneous tumors 25 days after tumor cell inoculation. (D) Bar graph depicting the statistical analysis of subcutaneous tumor mass. (E) Immunohistochemical detection of Ki67, MRAS, and KAT7 expression in subcutaneous tumors after KAT7 knockdown (Scale bar = 100 μm). (F) Western blot analysis of protein expression in subcutaneous tumors after KAT7 knockdown. (G) Assessment of CRC lung metastasis in B-NDG mice through tail vein injection of COLO320 cells with or without KAT7 knockdown (n = 7 per group). (H) Statistical analysis of the number of lung metastatic tumors in (G). (I) Impact of KAT7 knockdown on the survival of CRC lung metastasis mice (n = 10 per group). Error bars represent the mean ± SD. Statistical significance was assessed using two-tailed unpaired Student's t-tests for experiments (A-B, D, H) and log-rank test for experiment (I). ****P < 0.0001.