The HOXB7 protein renders breast cancer cells resistant to tamoxifen through activation of the EGFR pathway

Breast Cancer Program, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21231-1000, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2011; 109(8):2736-41. DOI: 10.1073/pnas.1018859108
Source: PubMed


Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.

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Available from: Saraswati Sukumar, Sep 29, 2015
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    • "In breast cancer, HOXB7 and HOXB13 are overexpressed [11], [12] and associated with increased cell proliferation. Correspondingly, differentiation and antagonism of these genes was recently shown to circumvent tamoxifen resistance [13]. "
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    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.
    PLoS ONE 08/2014; 9(8):e105285. DOI:10.1371/journal.pone.0105285 · 3.23 Impact Factor
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    • "The indicated cell lines were grown in 6-well plates, treated as indicated, and lysed in lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate) containing protease and phosphatase inhibitors (Beyotime, Shanghai, China). Lysates were subjected to SDS-PAGE and western blotting with the indicated antibodies as previously described [62]. Briefly, cell lysates were resolved by SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). "
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    ABSTRACT: Selective estrogen receptor modulators, such as tamoxifen, play a pivotal role in the treatment of luminal-type breast cancer. However, in clinical applications, nearly half of breast cancer patients are insensitive to tamoxifen, a small number of whom have early recurrence or disease progression when receiving tamoxifen. The underlying mechanism of this resistance has not been determined. ZNF703 is a novel oncogene in the 15% of breast cancers that harbor 8p12 amplifications. Therefore, the goal of our study was to explore the role of ZNF703 in tamoxifen resistance. We used immunohistochemistry techniques to examine ZNF703 expression in stage I-III primary breast cancer specimens and found a positive expression rate of 91.3%. All patients were divided into either high or low ZNF703 expression groups. We found that high ZNF703 expression mainly occurred in ER+ and PR+ breast cancers. Furthermore, 4-hydroxytamoxifen had different modes of action in breast cancer cell lines with high or low ZNF703 expression. ZNF703 overexpression in MCF-7 breast cancer cells activated the Akt/mTOR signaling pathway, downregulated ERα, and reduced the antitumor effect of tamoxifen. Low-dose tamoxifen did not suppress, but rather, stimulated the growth of cells overexpressing ZNF703. ZNF703 knockdown in MDA-MB-134 and HCC1500 luminal B-type breast cancer cell lines by siRNA significantly decreased survival rates when cells were treated with tamoxifen. Furthermore, targeting ZNF703 with a mTOR inhibitor increased the inhibitory effects of tamoxifen in ZNF703-overexpressing cells. Our study suggests that ZNF703 expression levels may predict tamoxifen sensitivity. Tamoxifen should be administered with caution to those patients bearing tumors with ZNF703 overexpression. However, large clinical trials and prospective clinical studies are needed to verify these results.
    PLoS ONE 08/2013; 8(8):e72053. DOI:10.1371/journal.pone.0072053 · 3.23 Impact Factor
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    • "Intriguingly, a recent study implicated HOXB7, one of the genes up-regulated in luminal M1 MBC tumors, in rendering FBC cells resistant to tamoxifen. In addition, high expression of HOXB7 in tamoxifen treated FBC patients correlated with poor disease free survival [47]. "
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    ABSTRACT: Male breast cancer (MBC) is a rare and inadequately characterized disease. The aim of the present study was to characterize MBC tumors transcriptionally, to classify them into comprehensive subgroups, and to compare them with female breast cancer (FBC). A total of 66 clinicopathologically well-annotated fresh frozen MBC tumors were analyzed using Illumina Human HT-12 bead arrays, and a tissue microarray with 220 MBC tumors was constructed for validation using immunohistochemistry. Two external gene expression datasets were used for comparison purposes: 37 MBCs and 359 FBCs. Using an unsupervised approach, we classified the MBC tumors into two subgroups, luminal M1 and luminal M2, respectively, with differences in tumor biological features and outcome, and which differed from the intrinsic subgroups described in FBC. The two subgroups were recapitulated in the external MBC dataset. Luminal M2 tumors were characterized by high expression of immune response genes and genes associated with estrogen receptor (ER) signaling. Luminal M1 tumors, on the other hand, despite being ER positive by immunohistochemistry showed a lower correlation to genes associated with ER signaling and displayed a more aggressive phenotype and worse prognosis. Validation of two of the most differentially expressed genes, class 1 human leukocyte antigen (HLA) and the metabolizing gene N-acetyltransferase-1 (NAT1), respectively, revealed significantly better survival associated with high expression of both markers (HLA, hazard ratio (HR) 3.6, P = 0.002; NAT1, HR 2.5, P = 0.033). Importantly, NAT1 remained significant in a multivariate analysis (HR 2.8, P = 0.040) and may thus be a novel prognostic marker in MBC. We have detected two unique and stable subgroups of MBC with differences in tumor biological features and outcome. They differ from the widely acknowledged intrinsic subgroups of FBC. As such, they may constitute two novel subgroups of breast cancer, occurring exclusively in men, and which may consequently require novel treatment approaches. Finally, we identified NAT1 as a possible prognostic biomarker for MBC, as suggested by NAT1 positivity corresponding to better outcome.
    Breast cancer research: BCR 02/2012; 14(1):R31. DOI:10.1186/bcr3116 · 5.49 Impact Factor
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