G-CSF promotes H3K27ac-modified KLF5 to activate CXCR4 expression and drive colon cancer growth and metastasis

Abstract

Objective: This study investigated how granulocyte colony-stimulating factor (G-CSF) regulates colon cancer (CC) progression through epigenetic activation of the kruppel-like factor 5 (KLF5)/chemokine receptor type 4 (CXCR4) axis.

Methods: Human CC LoVo cells were exposed to G-CSF (20 ng/mL) alone in combination with si-KLF5, oe-CXCR4, or the CXCR4 antagonist AMD3100 for 24 h. Malignant behaviors were evaluated by CCK-8, colony formation, and Transwell assays. H3K27ac modification on the KLF5 promoter and KLF5 binding to the CXCR4 promoter were examined using immunofluorescence, dual-luciferase reporter, and ChIP assays. An orthotopic LoVo xenograft mouse model was used to assess tumor growth, metastasis, and epithelial-mesenchymal transition (EMT) marker expression. KLF5 and CXCR4 mRNA and protein levels were measured in CC cells and tissues via RT-qPCR and western blot.

Results: G-CSF enhanced LoVo cell proliferation, migration, and invasion in a dose-dependent manner, concomitant with increased H3 acetylation and histone H3 lysine 27 (H3K27ac) acetylation. Mechanistically, G-CSF upregulated KLF5 expression via H3K27ac modification, promoting CXCR4 transcriptional activation. Inhibition of KLF5 or CXCR4 partially reversed G-CSF-induced EMT and malignant phenotypes. In vivo, G-CSF accelerated tumor growth and metastasis through the KLF5/CXCR4 signaling pathway, confirming its pro-tumorigenic role.

Conclusions: G-CSF drives CC progression by enhancing H3K27ac-dependent upregulation of KLF5, which transactivates CXCR4 to promote EMT, proliferation and metastasis. Targeting the G-CSF/KLF5/CXCR4 axis may represent a potential therapeutic strategy for advanced CC.

Keywords: Chemokine receptor type 4; Epithelial-mesenchymal transition; Granulocyte colony; Histone 3 lysine 27 acetylation; Human colon cancer LoVo cells; Kruppel-like factor 5; Metastasis; Proliferation; Stimulating factor.

Fig. 1

G-CSF reinforced the malignant behaviors and EMT of human CC LoVo cells. A MTT assay to assess cell viability; B CCK-8 assay to evaluate cell viability; C Colony formation test to assess cell proliferation; D Transwell assay to assess cell migration and invasion; E RT-qPCR to measure the expression of epithelial and stromal marker proteins E-cadherin, Vimentin, Fibronectin, and ZEB1; F Immunofluorescence detection of the expression levels of epithelial and stromal marker proteins E-cadherin, Vimentin, Fibronectin and ZEB1. Cell experiments were independently repeated thrice. Data were presented as mean ± SD. Independent sample t test was used to compare the data between two groups. ns p > 0.05, ** p < 0.01, *** p < 0.001

Fig. 2

G-CSF induced histone H3K27ac modification to stimulate KLF5 expression. A RT-qPCR detection of KLF5 expression; B Western blot detection of the expression levels of H3K27ac and KLF5; C Western blot to assess CBP and p300 expression; D Histone H3 acetylation Kit was used to determine the level of H3 acetylation; E ChIP assay to detect H3K27ac binding at the KLF5 promoter. Cell tests were performed three times independently. Data were exhibited as mean ± SD. Independent sample t test was utilized to compare the data between two groups, * p < 0.05, ** p < 0.01

Fig. 3

Knockdown of KLF5 partly averted the promoting effects of G-CSF on EMT and malignant behaviors of LoVo cells. A Detection of KLF5 mRNA expression by RT-qPCR; B Assessment of KLF5 protein expression by western blot; C RT-qPCR detection of the expression of epithelial and stromal marker proteins E-cadherin, Vimentin, Fibronectin, and ZEB1; D Detection of E-cadherin, Vimentin, Fibronectin, and ZEB1 expression levels by immunofluorescence; E Assay of cell viability by CCK-8; F Assay of cell proliferation by colony formation test; G Examination of cell migration and invasion by Transwell assay. Cell tests were independently conducted thrice. Data were denoted as mean ± SD, and multi-group data comparisons were conducted using one-way ANOVA, followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01

Fig. 4

G-CSF facilitated the transcriptional expression of CXCR4 via regulation of KLF5 expression. A mRNA expression of CXCR4 assessed by RT-qPCR; B Protein expression of CXCR4 confirmed by western blot; C KLF5 binding sites in CXCR4 promoter region predicted by JASPAR database (https://jaspar.elixir.no/); D The dual-luciferase reporter system was used to detect the binding of CXCR4 and KLF5, and the activation fold was calculated based on pGL3-Basic (relative activity = 1.0); E Binding of KLF5 to CXCR4 promoter determined by ChIP assay. Independent repetition of cell experiments was carried out thrice. Data were expressed as mean ± SD. Comparisons between multiple sets of data were made using one-way ANOVA, with Tukey’s multiple comparison test performed afterward. ** p < 0.01, *** p < 0.001

Fig. 5

G-CSF facilitated EMT and malignant behaviors of human CC LoVo cells through the KLF5/CXCR4 axis. A RT-qPCR detection of CXCR4 mRNA expression; B Western blot to determine the protein expression of CXCR4; C CCK-8 detection of cell viability; D Colony formation assay to assess cell proliferation; E Transwell assay to evaluate cell migration and invasion; F RT-qPCR detection of the expression of epithelial and stromal marker proteins E-cadherin, Vimentin, Fibronectin, and ZEB1; G Immunofluorescence detection of the expression patterns of E-cadherin, Vimentin, Fibronectin and ZEB1. Cell experiment was independently replicated three times, and the data were presented as mean ± SD. One-way ANOVA was used to compare multiple-group data, and Tukey’s multiple comparison test was used for post hoc testing. * p < 0.05, ** p < 0.01

Fig. 5

G-CSF facilitated EMT and malignant behaviors of human CC LoVo cells through the KLF5/CXCR4 axis. A RT-qPCR detection of CXCR4 mRNA expression; B Western blot to determine the protein expression of CXCR4; C CCK-8 detection of cell viability; D Colony formation assay to assess cell proliferation; E Transwell assay to evaluate cell migration and invasion; F RT-qPCR detection of the expression of epithelial and stromal marker proteins E-cadherin, Vimentin, Fibronectin, and ZEB1; G Immunofluorescence detection of the expression patterns of E-cadherin, Vimentin, Fibronectin and ZEB1. Cell experiment was independently replicated three times, and the data were presented as mean ± SD. One-way ANOVA was used to compare multiple-group data, and Tukey’s multiple comparison test was used for post hoc testing. * p < 0.05, ** p < 0.01

Fig. 6

G-CSF accelerated the growth and metastasis of human CC LoVo cell orthotopic xenograft tumor model through the KLF5/CXCR4 pathway. A–B Volume size of orthotopic xenograft tumors; C The weight of xenograft tumors; D Measurement of KLF5 and CXCR4 protein levels in nude mouse tissue homogenate by western blot; E Numbers of Ki67, E-cadherin, Vimentin and Fibronectin positive cells ascertained by immunohistochemical staining; F Liver and lung morphology after 40 days of orthotopic xenograft tumor modeling; G HE staining of metastatic liver and lung tissues and quantification of metastatic nodules. n = 6. One-way ANOVA was used for comparing multiple sets of data, followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 6

G-CSF accelerated the growth and metastasis of human CC LoVo cell orthotopic xenograft tumor model through the KLF5/CXCR4 pathway. A–B Volume size of orthotopic xenograft tumors; C The weight of xenograft tumors; D Measurement of KLF5 and CXCR4 protein levels in nude mouse tissue homogenate by western blot; E Numbers of Ki67, E-cadherin, Vimentin and Fibronectin positive cells ascertained by immunohistochemical staining; F Liver and lung morphology after 40 days of orthotopic xenograft tumor modeling; G HE staining of metastatic liver and lung tissues and quantification of metastatic nodules. n = 6. One-way ANOVA was used for comparing multiple sets of data, followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001