Upregulation of ENAH by a PI3K/AKT/β-catenin cascade promotes oral cancer cell migration and growth via an ITGB5/Src axis

Abstract

Background: Oral cancer accounts for 2% of cancer-related deaths globally, with over 90% of cases being oral cavity squamous cell carcinomas (OSCCs). Approximately 50% of patients with OSCC succumb to the disease within 5 years, primarily due to the advanced stage at which it is typically diagnosed. This underscores an urgent need to identify proteins related to OSCC progression to develop effective diagnostic and therapeutic strategies.

Methods: To identify OSCC progression-related proteins, we conducted integrated proteome and transcriptome analyses on cancer tissues from patients and patient-derived xenograft (PDX) model mice. We investigated the role of protein-enabled homolog (ENAH), identified as an OSCC progression-associated protein, through proliferation, transwell migration, and invasion assays in OSCC cells. The mechanisms underlying ENAH-mediated functions were elucidated using gene knockdown and ectopic expression techniques in OSCC cells.

Results: ENAH was identified as a candidate associated with OSCC progression based on integrated analyses, which showed increased ENAH levels in primary OSCC tissues compared with adjacent noncancerous counterparts, and sustained overexpression in the cancer tissues of PDX models. We confirmed that level of ENAH is increased in OSCC tissues and that its elevated expression correlates with poorer survival rates in patients with OSCC. Furthermore, the upregulation of ENAH in OSCC cells results from the activation of the GSK3β/β-catenin axis by the EGFR/PI3K/AKT cascade. ENAH expression enhances cell proliferation and mobility by upregulating integrin β5 in oral cancer cells.

Conclusions: The upregulation of ENAH through a PI3K/AKT/β-catenin signaling cascade enhances oral cancer cell migration and growth via the ITGB5/Src axis. These findings offer a new interpretation of the ENAH function in the OSCC progression and provide crucial information for developing new OSCC treatment strategies.

Keywords: GSK3β/β-catenin signaling; Integrin β5 (ITGB5); Oral cavity squamous cell carcinoma (OSCC); Patient-derived xenograft (PDX); Protein-enabled homolog (ENAH); Proteomics.

Fig. 1

Proteome profiling of tumor tissues collected from patients with OSCC and PDX model mice. The PDX mouse models were established using tumor tissues from five patients with OSCC (#06, #29, #34, #44, and #48). The tissue proteomes of adjacent noncancerous epithelia (N), primary tumors (T), first (P1) and second (P2) generation PDX tumors were profiled using iTRAQ-based mass spectrometry. Protein ratios for T/N, P1/N, and P2/N were calculated. The means and standard deviations (SDs) for the ratios of all proteins in each comparison were determined. Proteins with ratios above the mean plus SD were considered overexpressed (A), while those below the mean minus SD were considered underexpressed (B). Venn diagrams illustrate the overlap between the differentially expressed proteins in the T, P1, and P2 groups

Fig. 2

Pathway analysis of proteins involved in OSCC progression. The OSCC progression-related proteins (18 upregulated and 45 downregulated proteins; see Supplementary Table S5) were functionally annotated with STRING v11.5 software. The biological process (A) and KEGG pathway (B) analyses were conducted and visualized with online SRplot software. C A protein–protein interaction network of the 18 upregulated proteins was constructed, revealing 21 interaction links between the 18 proteins/nodes (indicated by solid lines). Two modules identified within the network illustrate the interactions of proteins involved in actin cytoskeleton pathways (green nodes) and ECM organization regulation (red nodes)

Fig. 3

A higher level of tissue ENAH was associated with poor survival in patients with OSCC. The gene expression of ENAH in OSCC tissues was examined using RNA-seq data from Chen et al., Nat Commun. 2017, 8, 465 (A), and Yen et al., Front Oncol. 2022, 12, 792,297 (B). Differences between groups were determined using paired t-tests. (C) Representative images of ENAH IHC staining in paired adjacent noncancerous (upper panel) and OSCC (lower panel) tissues are shown. Scale bar: 100 μm. Original magnification: × 200. D The IHC scores of paired noncancerous and cancerous tissues are represented as box and whisker plots, where whiskers, boxes, and horizontal lines depict the distributions of the middle 90%, upper and lower quartiles, and medians of the IHC scores, respectively. Kaplan–Meier plots show the long-term overall (E), disease-specific (F), locoregional recurrence-free (G), and distant metastasis-free (H) survival of 304 patients with OSCC. The plots were stratified using the first tertile of the ENAH IHC scores, with p values obtained using log-rank tests

Fig. 4

ENAH enhances the proliferation, migration, and invasion of OSCC cells. A–C MTT cell proliferation (A), transwell migration (B), and Matrigel invasion (C) assays were conducted with control (siCtrl) and ENAH-knockdown (siENAH) SCC4 cells. D–F The growth (D), migration (E), and invasion (F) abilities of SCC4 cells transfected with either the control vector (vector) or the ENAH expression plasmid (pENAH) were evaluated. The representative images of transwell migration (lower panels of B, E) and matrigel invasion (lower panels of C, F) assays were shown (original magnification, × 200; scale bar: 200 μm). The efficacy of knockdown and overexpression was confirmed by ENAH immunoblotting. For MTT assays, a representative experiment with four replicates per group is shown, and the results of two independent replicate experiments must show similar trends. For transwell assays, comparisons were based on data from three independent experiments. The p values were determined using Student's t-test

Fig. 5

ENAH expression is regulated by the EGFR/PI3K/AKT signaling pathway in OSCC cells. After 16 h of serum starvation, SCC4 cells were treated with 40 ng/mL EGF (A, B) or 30 μg/mL cetuximab (C, D). Levels of gene expression were measured by qRT-PCR (A) and western blotting (B) after 12 h and 48 h of EGF treatment, respectively. After 24 h (C, D) and 48 h (D) of cetuximab treatment, ENAH expression was assessed by qRT-PCR (C) and western blot (D). E, F SCC4 cells were treated with PI3K inhibitors GDC0941 (9 μM) and GSK1059615 (3 μM) for 24 h for qRT-PCR (E) and 48–72 h for western blot (F) analysis. G, H SCC4 cells were transfected with the control vector (vector) or a β-catenin expression plasmid (pβ-catenin). After 48 h, qRT-PCR (G) and western blotting (H) were carried out. I SCC4 cells were transfected with pβ-catenin and then treated with 150 nM GDC0941. J The phosphorylation of GSK3β at Ser 9 (inhibition of GSK3β activity) was analyzed after treating cells with EGF (40 ng/mL) or GDC0941 (9 μM) for 2 h. Phosphorylation at Ser 9 (the 47-kDa protein band) was significantly reduced in the GDC0941-treated group and enhanced in the EGF-treated group. For qRT-PCR analysis, comparisons were based on data from three independent experiments. The p values were determined using Student’s t-test

Fig. 6

ITGB5 is a downstream target of ENAH in OSCC cells. A, B ENAH and ITGB5 expression levels were determined in SCC4 cells transfected with control siRNA (siCtrl), ENAH siRNA (siENAH), control vector (vector), or an ENAH-expression plasmid (pENAH) using qRT-PCR. C The levels of ITGB5 and p-Src (Tyr416) were analyzed in ENAH-knockdown and ENAH-overexpressing SCC4 cells. D ITGB5 protein expression in ENAH-knockdown SCC4 cells treated with or without EGF was assessed. E–G The growth (E), migration (F), and invasion (G) abilities of SCC4 cells transfected with the control vector (vector) or the ITGB5-expression plasmid (pITGB5) were evaluated. H The levels of p-Src were investigated in ITGB5-overexpressing SCC4 cells. For MTT assays, a representative experiment with four replicates per group is shown, and the results of two independent replicate experiments must show similar trends. For qRT-PCR and transwell assays, comparisons were based on data from three independent experiments. The p values were determined using Student’s t-test

Fig. 7

ITGB5 is a downstream effector of ENAH that promotes cell growth and migration. A–C MTT cell proliferation (A), transwell migration (B), and Matrigel invasion (C) assays were conducted using ENAH-overexpressing SCC4 cells with and without GDC0941 treatment (75 nM). D–F The growth (D), migration (E), and invasion (F) potentials were examined in ENAH-knockdown SCC4 cells with and without ITGB5 overexpression. For MTT assays, a representative experiment with four replicates per group is shown, and the results of two independent replicate experiments must show similar trends. For transwell assays, comparisons were based on data from three independent experiments. The p values were determined by one-way ANOVA

Fig. 8

Schematic representation of ENAH-mediated OSCC progression. The EGFR signaling pathway plays a pivotal role in OSCC progression, inducing the activation of PI3K/AKT. Activated PI3K/AKT leads to the inactivation of GSK3β by phosphorylating its Ser 9. Inactivation of GSK3β results in β-catenin accumulation, which subsequently upregulates ENAH expression. Functionally, the elevated expression of ENAH enhances ITGB5 upregulation, leading to cell proliferation and migration, probably by modulating Src phosphorylation