Novel Small Molecule Compound disrupts the SIX1/EYA2 Complex and Inhibits Breast Cancer Metastasis

Abstract

The majority of targeted anti‐cancer drugs were developed to bind to active sites of enzymes or to bind protein receptors. These drugs generally inhibit upstream elements of signaling pathways, and thus resistance is easily developed. In contrast, targeting of transcription factors, which are the central nodes that respond to signaling pathways, has seldom been achieved but is appreciated to be a critical means by which tumors could be inhibited. In addition, few therapies are being developed that target metastasis specifically, which is the main cause of cancer‐related deaths. Thus, it is essential to investigate the more difficult, but perhaps more fruitful, task of targeting transcription factors that are involved in the metastatic process. The homeobox transcription factor, SIX1, partners with its transactivator, EYA, to regulate the development of numerous organs. Both SIX1 and EYA proteins are largely downregulated post‐embryogenesis, but are re‐expressed in a number of cancers where they are known to contribute to tumor growth and progression. Gain‐of‐function mutations in Six1 were recently described in Wilms tumors, with mutations increasing the ability of Six1 to transcriptionally activate glycolytic genes, thereby potentiating the Warburg effect. While gain‐of‐function Six1 mutations are not observed in breast cancer, it is amplified in a small percentage of breast tumors, and is overexpressed in up to 90% of all breast cancers regardless of subtype. In addition, we and others have shown that Six1 is a critical mediator of cancer onset and progression, and that it enhances metastasis via EMT through both cell‐autonomous and paracrine mechanisms. Thus, targeting Six1 may be a powerful means to inhibit tumor progression while conferring limited side effects. Here, via an alpha screen, we identified a novel small molecule compound, 8430, that disrupts the SIX1/EYA2 interaction and inhibits transactivating potential of SIX1/EYA2 complex. We show that 8430 partially reverses transcriptional and metabolic profiles influenced by SIX1 overexpression, indicating specificity of this compound. Additionally, SIX1‐mediated TGF‐β signaling and EMT, two major routes through which SIX1 contributes to metastasis, can be impeded by 8430 treatment. Importantly, we demonstrate that 8430 is well tolerated in mice, and that it suppresses breast cancer associated metastasis without significantly altering primary tumor growth. Taken together, we demonstrate for the first time that pharmacological inhibition of the SIX1/EYA2 complex is sufficient to suppress breast cancer metastasis.Support or Funding InformationNIH F99CA234940 (H. Zhou), NIH R01CA224867 (H. Ford) and Cancer League of Colorado Pilot Grant CTGG2 2017‐1386 (R. Zhao & H. Ford)To evaluate efficacy of 8430 in cells, MCF7 Ctrl/SIX1 overexpressing cells were treated by vehicle (DMSO) or 10μM 8430. Through Proximity Ligation Assay and Immunoprecipitation, we demonstrate that 8430 exhibits great potency in disrupting SIX1‐EYA2 interaction (a & b). Moreover, it is revealed that 8430 reverts SIX1‐induced epithelialmesenchymal transition transcriptional signature ( c). And this finding is validated by immunocytochemistry against E‐cadherin and Fibronectin (d).Figure 1To evaluate efficacy of 8430 in animal, MCF7 SIX1 overexpressing tumors (tagged with RFP) were treated by vehicle (NMP:PEG400:Solutol) or 8430 at 25mg/kg. Strikingly, 8430 significantly inhibits metastasis without affecting primary tumor, which is measured by fluorescence tracking (a). In addition, 8430 suppresses SIX1‐induced mesenchymal feature in tumors, shown by immunohistochemistry against E‐cadherin and Fibronectin (b).Figure 2