Two Naturally Derived Small Molecules Disrupt the SINEOCULIS Homeobox Homolog 1–Eyes Absent Homolog 1 (SIX1–EYA1) Interaction to Inhibit Colorectal Cancer Cell Growth

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Two Naturally Derived Small Molecules Disrupt the SINEOCULIS Homeobox Homolog 1–Eyes Absent Homolog 1 (SIX1–EYA1) Interaction to Inhibit Colorectal Cancer Cell Growth

Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality worldwide. Despite advancements in treatment modalities such as chemotherapy, radiotherapy, and targeted therapies, challenges like drug resistance and off-target effects persist. Recent studies have highlighted the oncogenic roles of transcriptional complexes in driving tumor progression and metastasis. Among these, the interaction between the sineoculis homeobox homolog 1 (SIX1) and eyes absent homolog 1 (EYA1) has emerged as a critical regulator of tumorigenesis. This study investigated the roles of the SIX1–EYA1 complex in CRC pathogenesis, identified small-molecule inhibitors disrupting this interaction, and evaluated their therapeutic potential.

Overexpression of SIX1 and EYA1 in Colorectal Cancer

The SIX1–EYA1 transcriptional complex was found to be significantly overexpressed in CRC tissues compared to adjacent non-cancerous tissues. In a cohort of 24 CRC patients, mRNA levels of SIX1 were 7.47 ± 3.54 in tumors vs. 1.88 ± 0.35 in controls (t = 4.92, P = 0.008), while EYA1 levels were 7.61 ± 2.03 vs. 2.22 ± 0.45 (t = 6.73, P = 0.005). Western blot analysis confirmed elevated protein levels of SIX1 (2.73 ± 0.24-fold), EYA1 (2.45 ± 0.17-fold), cyclin A1 (CCNA1; 2.68 ± 0.21-fold), and transforming growth factor-beta (TGF-β; 3.68 ± 0.44-fold) in CRC tissues. Similar overexpression patterns were observed in CRC cell lines, particularly HT29 and SW620. In HT29 cells, SIX1 and EYA1 mRNA levels were increased by 4.34 ± 0.35-fold and 4.13 ± 0.39-fold, respectively, compared to non-cancerous human colon epithelial cells (HCEC-1CT). Kaplan–Meier survival analysis of TCGA data revealed that high expression of SIX1 or EYA1 correlated with poorer overall survival in CRC patients.

The SIX1–EYA1 Complex Activates Oncogenic Targets CCNA1 and TGFB1

Co-immunoprecipitation (Co-IP) and immunofluorescence assays confirmed direct interaction and nuclear co-localization of SIX1 and EYA1 in CRC tissues and cells. The complex transcriptionally regulated two key oncogenes: CCNA1 (3.12-fold upregulated in tumors) and TGFB1 (6.04-fold upregulated). Chromatin immunoprecipitation (ChIP) assays demonstrated binding of the SIX1–EYA1 complex to the myocyte enhancer factor 3 (MEF3) consensus sites in the promoters of CCNA1 and TGFB1. Knockdown (KD) of SIX1 or EYA1 using lentiviral shRNAs reduced mRNA and protein levels of CCNA1 and TGF-β in CRC cells, while overexpression (OE) enhanced their expression. Luciferase reporter assays further validated that SIX1-dependent activation of these promoters required intact MEF3 binding sites.

Functional Consequences of SIX1/EYA1 Knockdown

Depleting SIX1 or EYA1 in HT29 cells significantly impaired oncogenic phenotypes. SIX1-KD cells exhibited a 70% reduction in proliferation (MTT assay), 80% fewer colonies (clonogenic assay), and 75% reduced invasion (Boyden chamber assay) compared to controls. Similarly, EYA1-KD cells showed 65%, 78%, and 70% reductions in these parameters. Overexpression of SIX1 or EYA1 modestly enhanced proliferation and invasion, underscoring their oncogenic roles. In vivo, xenograft tumors derived from SIX1-KD or EYA1-KD cells were 60–70% smaller in volume than controls. Rescue experiments overexpressing CCNA1 or TGFB1 in KD cells partially restored proliferative capacity, confirming their roles as downstream effectors.

Discovery of NSC0191 and NSC0933 as SIX1–EYA1 Interaction Inhibitors

A high-throughput AlphaScreen assay screened 2,000 compounds to identify inhibitors of the SIX1–EYA1 interaction. Recombinant GST-SIX1 and His-EYA1 proteins were used in optimized binding conditions (120 nM GST-SIX1, 100 nM His-EYA1). Two compounds, NSC0191 and NSC0933, demonstrated potent inhibition with half-maximal inhibitory concentrations (IC50) of 12.60 ± 1.15 µM and 83.43 ± 7.24 µM, respectively. In vitro pulldown assays confirmed dose-dependent disruption of SIX1–EYA1 binding by these molecules. At 20 µM, NSC0191 blocked 80.15% of interactions, while 160 µM NSC0933 inhibited 70.33%.

Anti-Tumor Effects of NSC0191 and NSC0933

In HT29 cells, NSC0191 (10–20 µM) and NSC0933 (80–160 µM) reduced CCNA1 and TGF-β protein levels by 50–80% without affecting SIX1 or EYA1 expression. Co-IP assays revealed that treatment with 20 µM NSC0191 decreased EYA1 binding to SIX1 by 75%, while 160 µM NSC0933 reduced it by 60%. Functionally, these inhibitors suppressed CRC cell proliferation (45–80% reduction), colony formation (60–85% fewer colonies), and invasion (50–75% fewer cells) in a dose-dependent manner.

In mouse xenograft models, weekly intraperitoneal injections of NSC0191 (10–20 µM) or NSC0933 (80–160 µM) reduced tumor volumes by 48–80% after 30 days. Immunohistochemistry of treated tumors showed diminished CCNA1 and TGF-β expression but unchanged SIX1/EYA1 levels, consistent with in vitro findings.

Mechanism and Therapeutic Implications

The SIX1–EYA1 complex promotes CRC progression by transactivating CCNA1 (a cell cycle regulator) and TGFB1 (a pro-metastatic cytokine). NSC0191 and NSC0933 disrupt this interaction, blocking transcriptional activation and downstream oncogenic signaling. These compounds exhibited minimal toxicity in non-cancerous cells, suggesting selective targeting of tumor-specific SIX1/EYA1 complexes.

Limitations include the unexplored roles of other EYA family members (EYA3/EYA4) in CRC and the need for structural optimization of NSC0191/NSC0933 to improve pharmacokinetics. Future studies will explore combinatorial therapies with existing chemotherapeutics and validate efficacy in patient-derived xenografts.

DOI: https://doi.org/10.1097/CM9.0000000000001736

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