Identification of transcription factor binding site (TFBS) of <i>HOXB9</i> promoter.

<p>(<b>A</b>) <i>HOXB9</i> promoter region and primer sets used for cloning. (<b>B</b>) Reporter plasmids with the <i>HOXB9</i> promoter region and deletion variants were constructed by inserting the PCR product of the putative HOXB9 promoter region. The numbers of the left side of graph indicate the full length of HOXB9 promoter and deletion variants, the right side shows transcriptional activity of each variant in MDA-MB231 cells. The difference in relative luciferase activity between pGL3-439 and pGL3-358 constructs was recorded. (*p<0.01) (<b>C</b>) Additional deletion plasmids between bps −439 and −358 of the promoter were constructed to identify the exact position of TFBS and gap of gene activity in that region was recorded each time when assay was repeated. The difference in relative luciferase activity between pGL3-404 and pGL3-392 constructs was significant (*p<0.01).</p

E2F1 as a possible candidate for regulating <i>HOXB9</i> expression.

<p>Bioinformatic analysis of transcription factor binding sites (TFBS) of the <i>HOXB9</i> region suggested several candidates as transcription factors: E2F1, PAX5, TP53, and SP1. (<b>A</b>) The mRNA expression of such candidates in MCF7 (HOXB9 expression low) and MDA-MB-231 (HOXB9 expression high) indicated the possible binding candidate by Q-PCR analysis. (<b>B</b>) The mRNA level in comparison with GAPDH of <i>HOXB9</i> and <i>E2F1</i> in MCF10A, MCF7, and MDA-MB-231 either with or without exogenous E2F1 displayed the positive correlation.</p

The correlation of E2F1 and HOXB9 in IHC staining.

<p>The correlation of E2F1 and HOXB9 in IHC staining.</p

Correlation between E2F1 and HOXB9 protein expression in breast cancer tissues.

<p>(<b>A</b>) A cohort of 139 breast cancer clinical samples stained with anti-E2F1 antibody was interrogated for HOXB9 expression. i- E2F1 positive, ii- HOXB9 positive, iii- E2F1 negative, iv- HOXB9 negative staining (original magnification, 100×). (<b>B</b>) The cross-link results of 139 breast cancer clinical samples to evaluate the correlation between E2F1 and HOXB9 staining. (<b>C,D</b>) The data was analyzed by hormone receptor status. (<b>E</b>) Kaplan–Meier plots of clinical outcomes of HR(+)/HER2(−) type breast cancer patients by E2F1 expression. (<b>F</b>) Kaplan–Meier plots of breast cancer patients' clinical outcomes by combination of HOXB9 and E2F1 expression. All p values were calculated using the log rank test.</p

An E2F1-HOXB9 Transcriptional Circuit Is Associated with Breast Cancer Progression

<div><p>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 <i>HOXB9</i> 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 <i>HOXB9</i> promoter region, a dual luciferase reporter assay was conducted. Protein candidates that may directly attach to a TFBS of <i>HOXB9</i> were examined by Q-PCR, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and mutation analysis. A <i>HOXB9</i> promoter region from −404 to −392 was identified as TFBS, and E2F1 was a potential binding candidate in this region. The induction of <i>HOXB9</i> expression by E2F1 was observed by Q-PCR in several breast cancer cell lines overexpressing E2F1. The stimulatory effect of E2F1 on <i>HOXB9</i> 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 <i>HOXB9</i> and its downstream target genes. Our in vitro analysis identified the TFBS of the <i>HOXB9</i> promoter region and suggested that E2F1 is a direct regulator of <i>HOXB9</i> 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.</p></div

HOXB9/E2F1 staining and breast cancer subtypes.

<p>HR: Hormone receptor.</p><p>HER2: Human Epidermal growth factor Receptor 2.</p><p>Triple neg: Triple negative.</p

<i>HOXB9</i> is a transcriptional target of E2F1.

<p>(<b>A</b>) A dual luciferase assay with plasmid including the <i>HOXB9</i> TFBS (pGL3-404) was analyzed in MCF7 wild type and MCF7 cells overexpressing E2F1 to indicate transcriptional activity. MCF7 cells without E2F1 overexpression and the pGL3 vector plasmid were used as negative control. (<b>B</b>) Genomic DNA extracted from MCF7 either with or without exogenous E2F1 overexpression was precipitated with anti-E2F1 antibody for ChIP analysis. The amplified DNA fragment level was confirmed by Q-PCR with a primer set containing TFBS of <i>HOXB9</i>. Another primer set that does not include <i>HOXB9</i> TFBS fragment was used as a negative control. (<b>C</b>) EMSA was performed with protein extract from T47D nuclei and a biotinylated probe coding the <i>HOXB9</i> promoter sequence from −404 to −392 bp (HOXB9-TFBS). A non-specific probe not containing the TFBS was used as a negative control. (<b>D</b>) 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). (<b>E</b>) Mutation variants of the <i>HOXB9</i> 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. The pGL3 vector plasmid was used as negative control. WT, wild type; MT, mutation variant.</p

CDK4/6 inhibitor decreases <i>HOXB9</i> expression by E2F1.

<p><i>HOXB9, E2F1</i> and <i>VEGF</i> expression in BT549 and MCF7 cell-lines was analyzed (<b>A</b>) by Q-PCR. (<b>B</b>) by Immunoblot analysis. GAPDH was used as a loading control. (<b>C</b>) MCF7 and (D) BT-549 cells were treated with PD-0332991 at a concentration of 5 µM for 48 h. Q-PCR analysis was used to determine the mRNA expression of <i>E2F1</i>, <i>HOXB9</i>, and its target genes (<i>VEGF</i>, <i>bFGF</i>, and <i>AREG</i>) in both cell-lines. (<b>E</b>) The expression of HOXB9 and VEGF was assessed in BT-549 cells in which E2F1 expression was knocked down with siE2F1. Non-targeted siRNA was used as negative control. Two different siRNA were used to avoid off-target effect.</p