ABSTRACT
Either FOXO1 or HBP1 transcription factor is a downstream effector of the PI3K/Akt pathway and associated with tumorigenesis. However, the relationship between FOXO1 and HBP1 in oral cancer remains unclear. Analysis of 30 oral tumor specimens revealed that mean mRNA levels of both FOXO1 and HBP1 in non-invasive and invasive oral tumors were found to be significantly lower than that of the control tissues, and the status of low FOXO1 and HBP1 (< 0.3 fold of the control) was associated with invasiveness of oral tumors. To investigate if HBP1 is a direct transcription target of FOXO1, we searched potential FOXO1 binding sites in the HBP1 promoter using the MAPPER Search Engine, and two putative FOXO1 binding sites located in the HBP1 promoter -132 to -125 bp and -343 to -336 bp were predicted. These binding sites were then confirmed by both reporter gene assays and the in cellulo ChIP assay. In addition, Akt activity manipulated by PI3K inhibitor LY294002 or Akt mutants was shown to negatively affect FOXO1-mediated HBP1 promoter activation and gene expression. Last, the biological significance of the FOXO1-HBP1 axis in oral cancer malignancy was evaluated in cell growth, colony formation, and invasiveness. The results indicated that HBP1 knockdown potently promoted malignant phenotypes of oral cancer and the suppressive effect of FOXO1 on cell growth, colony formation, and invasion was alleviated upon HBP1 knockdown in invasive oral cancer cells. Taken together, our data provide evidence for HBP1 as a direct downstream target of FOXO1 in oral cancer malignancy.
Subject(s)
Carcinoma, Squamous Cell/secondary , Forkhead Box Protein O1/metabolism , Gene Expression Regulation, Neoplastic , High Mobility Group Proteins/genetics , Mouth Neoplasms/pathology , Repressor Proteins/genetics , Apoptosis , Biomarkers, Tumor/genetics , Blotting, Western , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Case-Control Studies , Cell Movement , Cell Proliferation , Cell Transformation, Neoplastic , Forkhead Box Protein O1/genetics , High Mobility Group Proteins/metabolism , Humans , Lymphatic Metastasis , Mouth/metabolism , Mouth Neoplasms/genetics , Mouth Neoplasms/metabolism , Neoplasm Invasiveness , Neoplasm Staging , Prognosis , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tumor Cells, CulturedABSTRACT
The squamous cell carcinomas of the head and neck (SCCHNs) with aberrant epidermal growth factor receptor (EGFR) signaling are often associated with poor prognosis and low survival. Therefore, efficient inhibition of the EGFR signaling could intervene with the development of malignancy. Quercetin appears to be antitumorigenesis, but the underlying mechanism remains unclear in oral cancer. Fork-head box O (FOXO) transcription factors, Akt downstream effectors, are important regulators of cell growth. Here, we hypothesized that FOXO1 might be crucial in quercetin-induced growth inhibition in EGFR-overexpressing oral cancer. Quercetin treatment suppressed cell growth by inducing G2 arrest and apoptosis in EGFR-overexpressing HSC-3 and TW206 oral cancer cells. Quercetin inhibited EGFR/Akt activation with a concomitant induction of FOXO1 activation. FOXO1 knockdown attenuated quercetin-induced p21 and FasL expression and subsequent G2 arrest and apoptosis, respectively. Likewise, quercetin suppressed tumor growth in HSC-3 xenograft mice. Taken together, our data indicate that quercetin is an effective anticancer agent and that FOXO1 is crucial in quercetin-induced growth suppression in EGFR-overexpressing oral cancer.
Subject(s)
Anticarcinogenic Agents/pharmacology , ErbB Receptors/genetics , Forkhead Transcription Factors/metabolism , Quercetin/pharmacology , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , ErbB Receptors/metabolism , Fas Ligand Protein/genetics , Fas Ligand Protein/metabolism , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , G2 Phase Cell Cycle Checkpoints/drug effects , Humans , Male , Mice , Mouth Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVES: Overexpression of the epidermal growth factor (EGF) receptor (EGFR) gene in the squamous cell carcinomas of the head and neck (SCCHN) is often associated with inauspicious prognosis and poor survival. N-acetylcysteine (NAC), a compound from some vegetables and allium species, appears anti-tumorigenesis, but the underlying mechanism is unclear. The objective of this study is to investigate the role of NAC in EGFR-overexpressing oral cancer. MATERIALS AND METHODS: Both HSC-3 and SCC-4 human tongue squamous carcinoma cell lines and an HSC-3 xenograft mouse model were used to test the anti-growth efficacy of NAC in vitro and in vivo, respectively. RESULTS: NAC treatment suppressed cell growth, with concomitantly increased expression of HMG box-containing protein 1 (HBP1), a transcription suppressor, and decreased EGFR/Akt activation, in EGFR-overexpressing HSC-3 oral cancer cells. HBP1 knockdown attenuated the growth arrest and apoptosis induced by NAC. Lastly, NAC and AG1478, an EGFR inhibitor, additively suppressed colony formation in HSC-3 cells. CONCLUSION: Taken together, our data indicate that NAC exerts its growth-inhibitory function through modulating EGFR/Akt signaling and HBP1 expression in EGFR-overexpressing oral cancer.
