Abstract
Homeobox B9 (HOXB9), a member of the homeobox gene family, is overexpressed in breast cancer and promotes tumor progression and metastasis by stimulating epithelial-to-mesenchymal transition and angiogenesis within the tumor microenvironment. HOXB9 activates the TGFβ-ATM axis, leading to checkpoint activation and DNA repair, which engenders radioresistance in breast cancer cells. Despite detailed reports of the role of HOXB9 in breast cancer, the factors that regulate HOXB9 transcription have not been extensively examined. Here we uncover an underlying mechanism that may suggest novel targeting strategies for breast cancer treatment. To identify a transcription factor binding site (TFBS) in the HOXB9 promoter region, a dual luciferase reporter assay was conducted. Protein candidates that may directly attach to a TFBS of HOXB9 were examined by Q-PCR, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and mutation analysis. A HOXB9 promoter region from −404 to −392 was identified as TFBS, and E2F1 was a potential binding candidate in this region. The induction of HOXB9 expression by E2F1 was observed by Q-PCR in several breast cancer cell lines overexpressing E2F1. The stimulatory effect of E2F1 on HOXB9 transcription and its ability to bind the TFBS were confirmed by luciferase, EMSA and ChIP assay. Immunohistochemical analysis of 139 breast cancer tissue samples revealed a significant correlation between E2F1 and HOXB9 expression (p<0.001). Furthermore, a CDK4/6 inhibitor suppressed E2F1 expression and also reduced expression of HOXB9 and its downstream target genes. Our in vitro analysis identified the TFBS of the HOXB9 promoter region and suggested that E2F1 is a direct regulator of HOXB9 expression; these data support the strong correlation we found between E2F1 and HOXB9 in clinical breast cancer samples. These results suggest that targeting the E2F1/HOXB9 axis may be a novel strategy for the control or prevention of cancer progression and metastasis.
Highlights
Diagnosis and treatment based on the molecular characteristics of breast tumors has significantly improved patients’ survival over the past few decades [1]
A non-specific probe not containing the transcription factor binding site (TFBS) was used as a negative control. (D) electrophoretic mobility shift assay (EMSA) was performed after a 40-min incubation with T47D nuclei extract in several concentrations of anti-E2F1 antibody (1:200, 1:100, and 1:33). (E) Mutation variants of the Homeobox B9 (HOXB9) TFBS, in which the GGC sequence was changed to ATT, cloned into pGL3 plasmid, and analyzed using a dual luciferase assay in MCF7 either with or without exogenous E2F1
Further experiments were performed to check for candidate transcription factors that could bind to this region of the promoter and could induce HOXB9 transcription
Summary
Diagnosis and treatment based on the molecular characteristics of breast tumors has significantly improved patients’ survival over the past few decades [1]. The identification of novel genes and genetic signatures has great potential in providing new approaches in clinical practice for controlling cancer progression. HOX genes play important role as developmental regulators and likely to be influenced by various factors [2]. Several studies have showed the influence of retinoic acid [3,4,5] and steroid hormones in regulation of HOX genes [6]. HOX genes are deregulated in solid tumors, including those of colorectal cancer [7], lung adenocarcinoma [8], small cell lung cancer [9] and ovarian cancer [10]. HOXB7 and HOXB13 are overexpressed [11], [12] and associated with increased cell proliferation. Differentiation and antagonism of these genes was recently shown to circumvent tamoxifen resistance [13]
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