EFHD1 Activates SIK3 to Limit Colorectal Cancer Initiation and Progression via the Hippo Pathway

Abstract

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, with high rates of metastasis and lethality. EF-hand domain-containing protein D1 (EFHD1) and salt-inducible kinase 3 (SIK3) have been studied in several cancer types. Aberrant expression of EFHD1 and SIK3 has been observed in CRC, but little research has addressed their regulatory abilities and signaling pathways. In this study, we aimed to explore the efficacy of EFHD1 in inhibiting CRC proliferation and metastasis and to elucidate the underlying mechanisms involved in the upregulation of SIK3 expression. Cell viability, colony formation, wound healing, Transwell assay, orthotopic xenograft, and pulmonary metastasis mouse models were used to detect the antiproliferative and anti-metastatic effects of EFHD1 against CRC in vitro and in vivo. The Gene Expression Profiling Interactive Analysis (GEPIA) database was used to determine EFHD1 and SIK3 expression in CRC. The regulatory roles of EFHD1 and SIK3 in mediating anti-metastatic effects in CRC were measured using western blotting, immunohistochemical, and immunofluorescence analyses. The results showed that EFHD1 expression was significantly repressed in the clinical CRC samples. EFHD1 markedly suppressed cell proliferation, migration, and invasion in vitro and inhibited tumor growth and metastasis in vivo. Analysis of the GEPIA database revealed that EFHD1 expression positively correlated with SIK3 expression. SIK3 overexpression inhibited the migration of CRC cells, and SIK3 knockdown partially eliminated the inhibitory effects of EFHD1 on CRC metastasis. EFHD1 exerted anti-metastatic effects against CRC via upregulating SIK3 and inhibiting epithelial-mesenchymal transition (EMT) processing through modulating the Hippo signaling pathway. Collectively, these findings identify EFHD1 as a potent SIK3 agonist and highlight the EFHD1-SIK3 axis as a key modulator of the Hippo signaling pathway in CRC. EFHD1 serves as a novel regulator and is worthy of further development as a novel therapeutic target in CRC.

Keywords: Colorectal cancer; EFHD1; EMT; Hippo pathway; SIK3.

Figure 1

(A) Scatter diagram derived from gene expression data in GEPIA comparing the expression of EFHD1 in the tumor tissue and the normal tissue in CRC. (B) EFHD1 protein expression in the indicated cancer tissues (T) and their corresponding adjacent nontumoral tissues (N) using western blot assay. (C) Representative IHC images of EFHD1 expression in CRC tissues and their corresponding normal tissues. (D) EFHD1 expression in HCT116, SW480, HT29, Caco2 and NCM460 cells were detected by western blot and q-PCR.

Figure 2

EFHD1 suppresses the proliferation of CRC cells in vitro. (A-B) The protein and mRNA levels of EFHD1 in HCT116 and SW480 cells stably overexpressing EFHD1 or silcencing EFHD1. (C) IC₅₀ of HCT116 and SW480 cells stably overexpressing EFHD1 or silcencing EFHD1 for 48h. (D) The colony-formation and morphological changes of HCT116 and SW480 were monitored in HCT116 and SW480 cells stably overexpressing EFHD1 or silcencing EFHD1. (E) The protein levels of MCM2 and PCNA in HCT116 and SW480 cells stably overexpressing EFHD1 or silcencing EFHD1. Data were represented as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to the control group.

Figure 3

EFHD1 inhibits the metastasis of CRC cells in vitro. (A) Wound healing assay. A scratch was made in monolayers of HCT116 and SW480 cells transduced with overexpressing EFHD1 or silcencing EFHD1 for 24 h and monitored with an inverted microscope. (B) Cell migration and invasion transwell assays. HCT116 and SW480 cell suspension was plated in the upper chamber of transwell insert, and migrated or invaded cells were fixed and stained with 0.1% crystal violet of cells. Representative photographs are presented (left) and the relative number of migratory and invasive cells (right) was counted. (C-D) Western blot analysis. Protein levels of EMT-related proteins in HCT116 and SW480 cells transduced with overexpressing EFHD1 or silcencing EFHD1. GAPDH was used as a loading control. Data were represented as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to the control group.

Figure 4

EFHD1 limits the tumor growth and tumor metastasis in CRC nude mouse models. (A) Bioluminescent imaging for HCT116-luc orthotopic xenograft colon tumors at different time points. (B) Bioluminescence signals recorded at the indicated time points were represented as total photon flux (left); Bioluminescence signals were analyzed on the 4^th week (right) in HCT116-luc orthotopic xenograft model. (C) The body weight was recorded every day of experimental animals throughout the study duration in HCT116-luc orthotopic xenograft model. (D) Analysis of tumor H & E staining in HCT116-luc orthotopic xenograft model. (E) Analysis of EFHD1 staining by IHC in HCT116-luc orthotopic xenograft model. (F) Bioluminescence imaging for HCT116-luc lung metastasis at different time points. (G) Bioluminescence signals recorded at the indicated time points were represented as total photon flux (left); Bioluminescence signals were analyzed on the 4^th week (right) in HCT116-luc lung metastasis model. (H) The body weight was recorded every day of experimental animals throughout the study duration in HCT116-luc lung metastasis model. (I) Analysis of lung H & E staining in HCT116-luc lung metastasis model. (J) Analysis of EFHD1 staining by IHC in HCT116-luc lung metastasis model. Data were represented as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to the control group.

Figure 5

Knockdown of SIK3 impairs the cell metastasis inhibitory effects of EFHD1 on CRC. (A) Spearman correlation analysis between EFHD1 and SIK3 of normal colon and CRC tissues in GAPIA. (B) Scatter diagram derived from gene expression data in GEPIA comparing the expression of SIK3 in the tumor tissue and the normal tissue in CRC. (C) Protein expression and statistical analysis of SIK3 in HCT116 and SW480 cells transduced with overexpressing EFHD1. (D) Protein expression and statistical analysis of EMT-related proteins in HCT116 and SW480 cells transduced with overexpressing SIK3. (E) Western blot analysis of the related markers of EMT processing in EFHD1-overexpressed HCT116 and SW480 cells treated with or without SIK3-knockdown plasmid for 24 h. GAPDH was used as a loading control. (F) Wound healing assay. A scratch was made in monolayers of HCT116 and SW480 cells transduced with overexpressing EFHD1 and treated with or without SIK3-knockdown plasmid (magnification, 50x) and monitored with an inverted microscope. Data were represented as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to the control group.

Figure 6

EFHD1 modulates Hippo pathway to upregulate SIK3 expression. (A) Western blot and statistical analysis of the specific protein expressions in EFHD1-overexpressed-transfected HCT116 and SW480 cells. GAPDH was used as a loading control. (B) Western blot and statistical analysis of the specific protein expressions in EFHD1-overexpressed-transfected HCT116 and SW480 cells in the presence or absence of SIK3-knockdown plasmid. GAPDH was used as a loading control. (C) Western blot analysis of nuclear-YAP1, nuclear-TAZ and nuclear-TEAD1 in EFHD1-overexpressed-transfected HCT116 cells in the presence or absence of SIK3-knockdown plasmid. H3 was used as a loading control. (D) Immunofluorescence staining of YAP1 (red) for EFHD1-overexpressed-transfected HCT116 cells in the presence or absence of SIK3-knockdown plasmid. Nuclei were stained with DAPI (blue). Representative images were shown. Data were represented as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to the control group.

Figure 7

Hypothetical model. EFHD1 possesses potent anti-proliferative and anti-metastatic effects on CRC, the underlying mechanisms involve the upregulation of SIK3 through modulating Hippo pathway.