Background::Emerging evidence indicates that the sineoculis homeobox homolog 1-eyes absent homolog 1 (n SIX1-n EYA1) transcriptional complex significantly contributes to the pathogenesis of multiple cancers by mediating the expression of genes involved in different biological processes, such as cell-cycle progression and metastasis. However, the roles of the n SIX1-n EYA1 transcriptional complex and its targets in colorectal cancer (CRC) are still being investigated. This study aimed to investigate the roles of n SIX1-n EYA1 in the pathogenesis of CRC, to screen inhibitors disrupting the n SIX1-n EYA1 interaction and to evaluate the efficiency of small molecules in the inhibition of CRC cell growth.n Methods::Real-time quantitative polymerase chain reaction and western blotting were performed to examine gene and protein levels in CRC cells and clinical tissues (collected from CRC patients who underwent surgery in the Department of Integrated Traditional and Western Medicine, West China Hospital of Sichuan University, between 2016 and 2018, n n = 24). n In vivo immunoprecipitation and n in vitro pulldown assays were carried out to determine n SIX1-n EYA1 interaction. Cell proliferation, cell survival, and cell invasion were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, clonogenic assay, and Boyden chamber assay, respectively. The Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen) method was used to obtain small molecules that specifically disrupted n SIX1-n EYA1 interaction. CRC cells harboring different levels of n SIX1/n EYA1 were injected into nude mice to establish tumor xenografts, and small molecules were also injected into mice to evaluate their efficiency to inhibit tumor growth.n Results::Both n SIX1 and n EYA1 were overexpressed in CRC cancerous tissues (for n SIX1, 7.47 ± 3.54 n vs.1.88 ± 0.35, n t = 4.92, n P = 0.008; for n EYA1, 7.61 ± 2.03 n vs. 2.22 ± 0.45, n t = 6.73, n P = 0.005). The n SIX1/n EYA1 complex could mediate the expression of two important genes including cyclin A1 (n CCNA1) and transforming growth factor beta 1 (n TGFB1) by binding to the myocyte enhancer factor 3 consensus. Knockdown of both n SIX1 and n EYA1 could decrease cell proliferation, cell invasion, tumor growth, and n in vivo tumor growth (all n P < 0.01). Two small molecules, NSC0191 and NSC0933, were obtained using AlphaScreen and they could significantly inhibit the n SIX1-n EYA1 interaction with a half-maximal inhibitory concentration (ICn 50) of 12.60 ± 1.15 μmol/L and 83.43 ± 7.24 μmol/L, respectively. Administration of these two compounds could significantly repress the expression of n CCNA1 and n TGFB1 and inhibit the growth of CRC cells n in vitro and n in vivo.n Conclusions::Overexpression of the n SIX1/n EYA1 complex transactivated the expression of n CCNA1 and n TGFB1, causing the pathogenesis of CRC. Pharmacological inhibition of the n SIX1-n EYA1 interaction with NSC0191 and NSC0933 significantly inhibited CRC cell growth by affecting cell-cycle progression and metastasis.n “,”Background::Emerging evidence indicates that the sineoculis homeobox homolog 1-eyes absent homolog 1 (n SIX1-n EYA1) transcriptional complex significantly contributes to the pathogenesis of multiple cancers by mediating the expression of genes involved in different biological processes, such as cell-cycle progression and metastasis. However, the roles of the n SIX1-n EYA1 transcriptional complex and its targets in colorectal cancer (CRC) are still being investigated. This study aimed to investigate the roles of n SIX1-n EYA1 in the pathogenesis of CRC, to screen inhibitors disrupting the n SIX1-n EYA1 interaction and to evaluate the efficiency of small molecules in the inhibition of CRC cell growth.n Methods::Real-time quantitative polymerase chain reaction and western blotting were performed to examine gene and protein levels in CRC cells and clinical tissues (collected from CRC patients who underwent surgery in the Department of Integrated Traditional and Western Medicine, West China Hospital of Sichuan University, between 2016 and 2018, n n = 24). n In vivo immunoprecipitation and n in vitro pulldown assays were carried out to determine n SIX1-n EYA1 interaction. Cell proliferation, cell survival, and cell invasion were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, clonogenic assay, and Boyden chamber assay, respectively. The Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen) method was used to obtain small molecules that specifically disrupted n SIX1-n EYA1 interaction. CRC cells harboring different levels of n SIX1/n EYA1 were injected into nude mice to establish tumor xenografts, and small molecules were also injected into mice to evaluate their efficiency to inhibit tumor growth.n Results::Both n SIX1 and n EYA1 were overexpressed in CRC cancerous tissues (for n SIX1, 7.47 ± 3.54 n vs.1.88 ± 0.35, n t = 4.92, n P = 0.008; for n EYA1, 7.61 ± 2.03 n vs. 2.22 ± 0.45, n t = 6.73, n P = 0.005). The n SIX1/n EYA1 complex could mediate the expression of two important genes including cyclin A1 (n CCNA1) and transforming growth factor beta 1 (n TGFB1) by binding to the myocyte enhancer factor 3 consensus. Knockdown of both n SIX1 and n EYA1 could decrease cell proliferation, cell invasion, tumor growth, and n in vivo tumor growth (all n P < 0.01). Two small molecules, NSC0191 and NSC0933, were obtained using AlphaScreen and they could significantly inhibit the n SIX1-n EYA1 interaction with a half-maximal inhibitory concentration (ICn 50) of 12.60 ± 1.15 μmol/L and 83.43 ± 7.24 μmol/L, respectively. Administration of these two compounds could significantly repress the expression of n CCNA1 and n TGFB1 and inhibit the growth of CRC cells n in vitro and n in vivo.n Conclusions::Overexpression of the n SIX1/n EYA1 complex transactivated the expression of n CCNA1 and n TGFB1, causing the pathogenesis of CRC. Pharmacological inhibition of the n SIX1-n EYA1 interaction with NSC0191 and NSC0933 significantly inhibited CRC cell growth by affecting cell-cycle progression and metastasis.n