HOXA1 binds RBCK1/HOIL-1 and TRAF2 and modulates the TNF/NF-κB pathway in a transcription-independent manner

HOX proteins define a family of key transcription factors regulating animal embryogenesis. HOX genes have also been linked to oncogenesis and HOXA1 has been described to be active in several cancers, including breast cancer. Through a proteome-wide interaction screening, we previously identified the TNFR-associated proteins RBCK1/HOIL-1 and TRAF2 as HOXA1 interactors suggesting that HOXA1 is functionally linked to the TNF/NF-κB signaling pathway. Here, we reveal a strong positive correlation between expression of HOXA1 and of members of the TNF/NF-κB pathway in breast tumor datasets. Functionally, we demonstrate that HOXA1 can activate NF-κB and operates upstream of the NF-κB inhibitor IκB. Consistently, we next demonstrate that the HOXA1-mediated activation of NF-κB is non-transcriptional and that RBCK1 and TRAF2 influences on NF-κB are epistatic to HOXA1. We also identify an 11 Histidine repeat and the homeodomain of HOXA1 to be required both for RBCK1 and TRAF2 interaction and NF-κB stimulation. Finally, we highlight that activation of NF-κB is crucial for HOXA1 oncogenic activity

HOXA1 functionally interacts with the NF-ĸB pathway in breast cancer

Over the last 30 years, HOX transcription factors were reported as dysregulated in a wide range of cancers. In that context, HOXA1 has been shown to act as an oncogene and its forced expression in normal mammary epithelial cells has been demonstrated to be sufficient to cause a switch to a breast cancer phenotype. However, the molecular interactions and the cellular pathways activated by HOXA1 to drive breast cancer remain scarcely understood. The goal of this thesis was therefore to dissect the molecular and functional interactions HOXA1 establishes with the TNF/NF-κB pathway. The transcription factor NF-κB is indeed known to be involved in mammary oncogenesis and HOXA1 has recently been shown to interact with RBCK1 and TRAF2, two TNF/NF-κB pathway modulators. We first highlighted that HOXA1 expression is strongly correlated to the NF-κB signaling pathway in public datasets of breast cancer transcriptomes. More particularly, exhaustive analysis of transcriptomic data revealed a tight connection between HOXA1 and the poor prognosis triple-negative mesenchymal subtype of breast cancer. At the functional level, we demonstrated in vitro that HOXA1 activates the NF-κB pathway in a transcription-independent fashion by interacting with RBCK1 and TRAF2. We showed that RBCK1 and HOXA1 display antagonistic effects whereas TRAF2 and HOXA1 both stimulate NF-κB. At the molecular level, we identified that an 11-histidine motif and the homeodomain of HOXA1 seem both required for the interaction with RBCK1 and TRAF2 and, in turn, for the HOXA1-mediated activation of NF-κB. Finally, cellular assays supported that the activation of the TNF/NF-κB pathway is crucial for the oncogenic activity of HOXA1. In addition to the HOXA1-to-NF-κB connection, we also revealed a possible mutual repression between HOXA1 and the estrogen receptor ERα in breast tumors. Taken together, this work revealed NF-κB as a new signaling pathway regulated by HOXA1 to mediate breast oncogenesis. Next, it will be worth investigating to what extent HOXA1 could be used as a prognostic marker and as a therapeutic target for triple negative mesenchymal breast tumors.(SC - Sciences) -- UCL, 201

HOXA1, a breast cancer oncogene

More than 25 years ago, the first literature records mentioned HOXA1 expression in human breast cancer. A few years later, HOXA1 was confirmed as a proper oncogene in mammary tissue. In the following two decades, molecular data about the mode of action of the HOXA1 protein, the factors contributing to activate and maintain HOXA1 gene expression and the identity of its target genes have accumulated and provide a wider view on the association of this transcription factor to breast oncogenesis. Large-scale transcriptomic data gathered from wide cohorts of patients further allowed refining the relationship between breast cancer type and HOXA1 expression. Several recent reports have reviewed the connection between cancer hallmarks and the biology of HOX genes in general. Here we take HOXA1 as a paradigm and propose an extensive overview of the molecular data centered on this oncoprotein, from what its expression modulators, to the interactors contributing to its oncogenic activities, and to the pathways and genes it controls. The data converge to an intricate picture that answers questions on the multi-modality of its oncogene activities, point towards better understanding of breast cancer aetiology and thereby provides an appraisal for treatment opportunities

Hoxa1^WT and Hoxa1^I-V relieve MCF7 cells from contact inhibition, while expressing Hoxa1^WM-AA does not.

<p>MCF7 cells were transiently transfected for Hoxa1^WT, Hoxa1^I-V and Hoxa1^WM-AA, together with Pbx1a and Prep1 cofactors, and grown for three weeks. Controls included cells transfected for the potent oncogene hRAS or cells transfected for Pbx1a and Prep1 only. Foci formation was observed for hRAS, Hoxa1^WT and Hoxa1^I-V transfected cells (arrowheads) while not for CTL and Hoxa1^WM-AA cells.</p

Activation of a Hoxa1 target reporter in mammary carcinoma cell clones.

<p>The Hoxa1 target reporter <i>EphA2-r42B-luc</i> is activated in cell clones for Hoxa1^WT and Hoxa1^I-V while not in Hoxa1^WM-AA clones. In each experiment, the pML-EphA2-r42B-luc reporter plasmid was transfected in combination with expression vectors for both Prep1 and Pbx1a. The constitutively active pCMV-LacZ reporter plasmid was added as a transfection control. Results were calculated by a luciferase/β-galactosidase ratio, pooled for each type of clones and represented as means ± S.D. of triplicates. *, p<0.05 and ***, p<0.001 (ANOVA 2).</p

Characterization of MCF7 clones for the constitutive expression of Hoxa1 variants.

<p>(A) Expression of Hoxa1, Neomycin resistance (Neo), Pbx1 and β-actin genes was detected by RT-PCR. While MCF7 cells do not express Hoxa1, clones obtained the stable transfection of Hoxa1^WT, Hoxa1^I-V and Hoxa1^WM-AA coding plasmids express the Hoxa1 variants at similar levels (β-actin used as reference). All cells express the endogenous Pbx1 gene. (B) The Hoxa1 and (C) PBX1B protein immunolocalisation reveals that both proteins localize into the cell nucleus.</p

The expression of Hoxa1^WM-AA in human mammary carcinoma cells does not result in increased anchorage independent cell growth.

<p>Cells were grown in soft agar and colonies were revealed by crystal violet staining (A) MCF7-Hoxa1^WT and MCF7-Hoxa1^I-V cells produced a lot of colonies in soft agar while CTL and MCF7-Hoxa1^WM-AA only provide a modest colony growth. (B) For each culture, colonies were counted in three random microscopic fields at 16X magnification. Results were pooled for each type of clones and represented as means ± S.D of triplicates. **, p<0.01; ***, p<0.001.</p

The expression of Hoxa1^WM-AA in human mammary carcinoma cells does not result in increased cell proliferation and growth.

<p>(A) WST-1 based proliferation assays were performed for MCF7-Hoxa1^WT, MCF7-Hoxa1^I-V, MCF7-Hoxa1^WM-AA and MCF7-CTL clones. The proliferation index was determined for each clone as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025247#s4" target="_blank">Materials and Methods</a>. Results were pooled for each type of clones and represented as means ± S.D. of triplicates. *, p<0.05 (ANOVA 2). (B) Cells for MCF7-Hoxa1^WT, MCF7-Hoxa1^I-V, MCF7-Hoxa1^WM-AA and MCF7-CTL clones were inoculated, kept in culture for 16 days and counted after day 4, 7, 9, 11, 14 and 16. Growth curves represent the mean of four independent experiments.</p

Transcriptional activity and relative expression of Hoxa1 variants.

<p>(A) The Hoxa1 target reporter <i>EphA2-r42B-luc</i> is activated in MCF7 cells in the presence of expression vectors for Hoxa1^WT, Hoxa1^I-V while not in the presence of Hoxa1^WM-AA or of an empty (CTL) plasmid. In each experiment, the pML-EphA2-r42B-luc reporter plasmid was transfected in combination with expression vectors for both Prep1 and Pbx1a. Results were calculated by a luciferase/β-galactosidase ratio and represented as means ± S.D. of triplicates. ***, p<0.001 (ANOVA2). (B) Detection of Hoxa1 variant proteins from whole cell lysates obtained from transiently transfected MCF7 cells reveal that Hoxa1^WT, Hoxa1^I-V and Hoxa1^WM-AA proteins are equally expressed and stable. No Hoxa1 protein could be detected from MCF7 cells or from cells transfected with an empty (CTL) expression vector. Detection of constitutively expressed β-actin protein was performed as control load.</p

HOXA1 Is an Antagonist of ERα in Breast Cancer

Breast cancer is a heterogeneous disease and the leading cause of female cancer mortality worldwide. About 70% of breast cancers express ERa. HOX proteins are master regulators of embryo development which have emerged as being important players in oncogenesis. HOXA1 is one of them. Here, we present bioinformatic analyses of genome-wide mRNA expression profiles available in large public datasets of human breast cancer samples. We reveal an extremely strong opposite correlation between HOXA1 versus ER expression and that of 2,486 genes, thereby supporting a functional antagonism between HOXA1 and ERa. We also demonstrate in vitro that HOXA1 can inhibit ERa activity. This inhibition is at least bimodal, requiring an intact HOXA1 DNA-binding homeodomain and involving the DNA-binding independent capacity of HOXA1 to activate NF-kB. We provide evidence that the HOXA1-PBX interaction known to be critical for the transcriptional activity of HOXA1 is not involved in the ERa inhibition. Finally, we reveal that HOXA1 and ERa can physically interact but that this interaction is not essential for the HOXA1-mediated inhibition of ERa. Like other HOX oncoproteins interacting with ERa, HOXA1 could be involved in endocrine therapy resistance