SUMMARY SIX1 interacts with EYA to form a bipartite transcription factor essential for development. Loss of function of this complex causes branchio-oto-renal syndrome (BOR), while re-expression of SIX1 or EYA promotes metastasis. Here we describe the 2.0 Å structure of SIX1 bound to EYA2, which suggests a novel DNA binding mechanism for SIX1 and provides a rationale for the effect of BOR syndrome mutations. The structure also reveals that SIX1 uses predominantly a single helix to interact with EYA. Substitution of a single amino acid in this helix is sufficient to disrupt the SIX1–EYA interaction, SIX1-mediated epithelial-mesenchymal transition and metastasis in mouse models. Given that SIX1 and EYA are co-overexpressed in many tumor types, our data indicate that targeting the SIX1–EYA complex may be a potent approach to inhibit tumor progression in multiple cancer types.
An association between lymph node metastasis and poor prognosis in breast cancer was observed decades ago. However, the mechanisms by which tumor cells infiltrate the lymphatic system are not completely understood. Recently, it has been proposed that the lymphatic system has an active role in metastatic dissemination and that tumor-secreted growth factors stimulate lymphangiogenesis. We therefore investigated whether SIX1, a homeodomain-containing transcription factor previously associated in breast cancer with lymph node positivity, was involved in lymphangiogenesis and lymphatic metastasis. In a model in which human breast cancer cells were injected into immune-compromised mice, we found that SIX1 expression promoted peritumoral and intratumoral lymphangiogenesis, lymphatic invasion, and distant metastasis of breast cancer cells. SIX1 induced transcription of the prolymphangiogenic factor VEGF-C, and this was required for lymphangiogenesis and lymphatic metastasis. Using a mouse mammary carcinoma model, we found that VEGF-C was not sufficient to mediate all the metastatic effects of SIX1, indicating that SIX1 acts through additional, VEGF-Cindependent pathways. Finally, we verified the clinical significance of this prometastatic SIX1/VEGF-C axis by demonstrating coexpression of SIX1 and VEGF-C in human breast cancer. These data define a critical role for SIX1 in lymphatic dissemination of breast cancer cells, providing a direct mechanistic explanation for how VEGF-C expression is upregulated in breast cancer, resulting in lymphangiogenesis and metastasis.
Introduction The SIX homeodomain proteins and the EYA family of co-activators form a bipartite transcription factor complex that promotes the proliferation and survival of progenitor cells during organogenesis and is down-regulated in most adult tissues. Abnormal over-expression of SIX1 and EYA in adult tissue is associated with the initiation and progression of diverse tumor types. Importantly, SIX1 and EYA are often co-overexpressed in tumors, and the SIX1-EYA2 interaction has been shown to be critical for metastasis in a breast cancer model. The EYA proteins also contain protein tyrosine phosphatase activity, which plays an important role in breast cancer growth and metastasis as well as directing cells to the repair pathway upon DNA damage. Areas covered This review provides a summary of the SIX1/EYA complex as it relates to development and disease and the current efforts to therapeutically target this complex. Expert opinion Recently, there have been an increasing number of studies suggesting that targeting the SIX1/EYA transcriptional complex will potently inhibit tumor progression. Although current attempts to develop inhibitors targeting this complex are still in the early stages, continued efforts towards developing better compounds may ultimately result in effective anti-cancer therapies.
Branchio-oto-renal syndrome (BOR) is an autosomal dominant developmental disorder characterized by hearing loss, branchial arch defects, and renal anomalies. Recently, eight mutations in the SIX1 homeobox gene were discovered in BOR patients. To characterize the effect of SIX1 BOR mutations on the EYA-SIX1-DNA complex, we expressed and purified six of the eight mutants in Escherichia coli. We demonstrate that only the most N-terminal mutation in SIX1 (V17E) completely abolishes SIX1-EYA complex formation, whereas all of the other mutants are able to form a stable complex with EYA. We further show that only the V17E mutant fails to localize EYA to the nucleus and cannot be stabilized by EYA in the cell. The remaining five SIX1 mutants are instead all deficient in DNA binding. In contrast, V17E alone has a DNA binding affinity similar to that of wild type SIX1 in complex with the EYA co-factor. Finally, we show that all SIX1 BOR mutants are defective in transcriptional activation using luciferase reporter assays. Taken together, our experiments demonstrate that the SIX1 BOR mutations contribute to the pathology of the disease through at least two different mechanisms that involve: 1) abolishing the formation of the SIX1-EYA complex or 2) diminishing the ability of SIX1 to bind DNA. Furthermore, our data demonstrate for the first time that EYA: 1) requires the N-terminal region of the SIX1 Six domain for its interaction, 2) increases the level of the SIX1 protein within the cell, and 3) increases the DNA binding affinity of SIX1. Branchio-oto-renal syndrome (BOR; Mendelian Inheritance in Man (MIM) 113650)5 is an autosomal dominant developmental disorder that is characterized by hearing loss, branchial fistulae, and renal anomalies. Although the penetrance of the syndrome is highly variable between and even within families (1), 70 -93% of BOR patients exhibit hearing loss (1). This hearing loss can be conductive, sensorineural, or mixed and ranges in severity. In total, BOR affects an estimated 1 in 40,000 children and accounts for 2% of profoundly deaf children (2).The most commonly mutated gene in BOR syndrome is EYA1 (3), with an estimated 40% of BOR patients exhibiting mutations in this gene (4). EYA1 belongs to the EYA gene family of transcriptional co-factors. There are four mammalian members (EYA1-4), each containing an N-terminal transactivation domain (5), and a highly conserved ϳ270-amino acid C-terminal Eya domain (ED), also referred to as the eya homologous region. The ED possesses phosphatase activity (6 -8) and is involved in protein-protein interactions with the SIX family of homeoproteins (9 -12). The SIX family of homeoproteins are characterized by a DNA-binding homeodomain (HD) and the protein-interaction Six domain (SD), which binds directly to the ED of EYA (9). As a complex, the SIX and EYA proteins are believed to form a bipartite transcription factor where SIX confers DNA binding and EYA confers transactivation activity.Recently, mutations in two SIX family members (SIX5 and SIX1) have also be...
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