RESUMEN
Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.
Asunto(s)
Movimiento Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Regulación Alostérica , Secuencia de Aminoácidos , Calorimetría , Línea Celular , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Magnesio/metabolismo , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Unión Proteica , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas/fisiología , Homología de Secuencia de Aminoácido , Espectrofotometría UltravioletaRESUMEN
The Eyes Absent (EYA) family of developmental proteins, often in partnership with the sine oculis (SIX) homeobox proteins, promote cancer metastasis and recurrence in numerous tumor types. In addition to being a transcriptional coactivator, EYA2 is a Tyr phosphatase that dephosphorylates H2AX which leads to repair instead of apoptosis upon DNA damage and ERß which inhibits the anti-tumor transcriptional activity of ERß. The SIX members of the EYA-SIX complex are difficult to target, therefore, we targeted the EYA2 to promote cell death and prevent cancer progression. We conducted structural optimization of a previously discovered allosteric inhibitor of EYA2, 9987, using the combination of inâ silico modeling, biochemical and cell-based assays. A new series of compounds was developed with significantly improved cellular activity and physiochemical properties desirable for brain targets. Specifically, compound 2 e showed >30-fold improvement in the medulloblastoma cell line D458, relative to 9987, while maintaining potent and selective inhibitory activity against EYA2 Tyr phosphatase activity and a good multiparameter optimization score for central nervous system drugs.
RESUMEN
Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFß signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFß signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis in vivo without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Homeodominio/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Estimación de Kaplan-Meier , Células MCF-7 , Ratones , Metástasis de la Neoplasia/prevención & control , Proteínas Nucleares/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Tirosina Fosfatasas/metabolismo , RNA-Seq , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: We previously reported preventive and therapeutic effects of Smad7, a multifunctional protein, on radiotherapy (RT)-induced mucositis in mice without promoting human oral cancer cell survival or migration in vitro. The current study aims to determine whether a Smad7-based biologic can treat existing oral mucositis during radiotherapy for oral cancer and whether this treatment compromises RT-induced cancer cell killing in neighboring oral cancer.Experimental Design: We transplanted human oral cancer cells into the tongues of mice and applied craniofacial irradiation to simultaneously kill tumor cells and induce oral mucositis, thus modeling RT and mucositis in oral cancer patients. We topically applied a recombinant human Smad7 protein fused with the cell-penetrating Tat tag (Tat-Smad7) to the oral mucosa of tumor-bearing mice post RT when oral mucositis began to develop. RESULTS: Topically applied Tat-Smad7 penetrated cells in both the oral mucosa and oral cancer, attenuating TGFß and NF-κB signaling as well as inflammation at both sites. Tat-Smad7 treatment alleviated oral mucositis with reductions in DNA damage and apoptosis in keratinocytes, but increased keratinocyte proliferation compared with vehicle-treated mucositis lesions. In contrast, adjacent oral cancer exposed to Tat-Smad7 did not show alterations in proliferation or direct DNA damage, but showed increased oxidative stress damage and apoptosis compared with tumors treated with vehicle. CONCLUSIONS: Our results suggest that short-course Tat-Smad7 application to oral mucositis promotes its healing but does not compromise the cytotoxic effect of RT on oral cancer and has context-specific effects on oral mucosa versus oral cancer.
Asunto(s)
Neoplasias de la Boca/complicaciones , Mucositis/etiología , Mucositis/patología , Proteína smad7/genética , Estomatitis/patología , Cicatrización de Heridas/genética , Animales , Biomarcadores , Línea Celular Tumoral , Daño del ADN , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Modelos Biológicos , Neoplasias de la Boca/radioterapia , Traumatismos Experimentales por Radiación , Transducción de Señal/efectos de la radiación , Proteína smad7/metabolismoRESUMEN
EYA proteins (EYA1-4) are critical developmental transcriptional cofactors that contain an EYA domain (ED) harboring Tyr phosphatase activity. EYA proteins are largely downregulated after embryogenesis but are reexpressed in cancers, and their Tyr phosphatase activity plays an important role in the DNA damage response and tumor progression. We previously identified a class of small-molecule allosteric inhibitors that specifically inhibit the Tyr phosphatase activity of EYA2. Herein, we determined the crystal structure of the EYA2 ED in complex with NCGC00249987 (a representative compound in this class), revealing that it binds to an induced pocket distant from the active site. NCGC00249987 binding leads to a conformational change of the active site that is unfavorable for Mg2+ binding, thereby inhibiting EYA2's Tyr phosphatase activity. We demonstrate, using genetic mutations, that migration, invadopodia formation, and invasion of lung adenocarcinoma cells are dependent on EYA2 Tyr phosphatase activity, whereas growth and survival are not. Further, we demonstrate that NCGC00249987 specifically targets migration, invadopodia formation, and invasion of lung cancer cells, but that it does not inhibit cell growth or survival. The compound has no effect on lung cancer cells carrying an EYA2 F290Y mutant that abolishes compound binding, indicating that NCGC00249987 is on target in lung cancer cells. These data suggest that the NCGC00249987 allosteric inhibitor can be used as a chemical probe to study the function of the EYA2 Tyr phosphatase activity in cells and may have the potential to be developed into an antimetastatic agent for cancers reliant on EYA2's Tyr phosphatase activity.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/farmacología , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/patología , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios Proteicos , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
The carboxyl-terminal binding proteins (CtBP) are transcriptional corepressors that regulate the expression of multiple epithelial-specific and pro-apoptotic genes. Overexpression of CtBP occurs in many human cancers where they promote the epithelial-to-mesenchymal transition, stem cell-like features, and cell survival, while knockdown of CtBP in tumor cells results in p53-independent apoptosis. CtBPs are recruited to their target genes by binding to a conserved PXDLS peptide motif present in multiple DNA-binding transcription factors. Disrupting the interaction between CtBP and its transcription factor partners may be a means of altering CtBP-mediated transcriptional repression and a potential approach for cancer therapies. However, small molecules targeting protein-protein interactions have traditionally been difficult to identify. In this study, we took advantage of the fact that CtBP binds to a conserved peptide motif to explore the feasibility of using peptides containing the PXDLS motif fused to cell-penetrating peptides (CPP) to inhibit CtBP function. We demonstrate that these peptides disrupt the ability of CtBP to interact with its protein partner, E1A, in an AlphaScreen assay. Moreover, these peptides can enter both lung carcinoma and melanoma cells, disrupt the interaction between CtBP and a transcription factor partner, and inhibit CtBP-mediated transcriptional repression. Finally, the constitutive expression of one such peptide, Pep1-E1A-WT, in a melanoma cell line reverses CtBP-mediated oncogenic phenotypes including proliferation, migration, and sphere formation and limits tumor growth in vivo. Together, our results suggest that CPP-fused PXDLS-containing peptides can potentially be developed into a research tool or therapeutic agent targeting CtBP-mediated transcriptional events in various biological pathways.
Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Péptidos de Penetración Celular/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Péptidos/farmacología , Activación Transcripcional/efectos de los fármacos , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Péptidos/química , Péptidos/genética , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Transcriptional corepressor proteins have emerged as an important facet of cancer etiology. These corepressor proteins are often altered by loss- or gain-of-function mutations, leading to transcriptional imbalance. Thus, research directed at expanding our current understanding of transcriptional corepressors could impact the future development of new cancer diagnostics, prognostics, and therapies. In this review, our current understanding of the CtBP corepressors, and their role in both development and disease, is discussed in detail. Importantly, the role of CtBP1 overexpression in adult tissues in promoting the progression of multiple cancer types through their ability to modulate the transcription of developmental genes ectopically is explored. CtBP1 overexpression is known to be protumorigenic and affects the regulation of gene networks associated with "cancer hallmarks" and malignant behavior, including increased cell survival, proliferation, migration, invasion, and the epithelial-mesenchymal transition. As a transcriptional regulator of broad developmental processes capable of promoting malignant growth in adult tissues, therapeutically targeting the CtBP1 corepressor has the potential to be an effective method for the treatment of diverse tumor types. Although efforts to develop CtBP1 inhibitors are still in the early stages, the current progress and the future perspectives of therapeutically targeting this transcriptional corepressor are also discussed. Mol Cancer Ther; 16(6); 981-90. ©2017 AACR.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/química , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Regulación de la Expresión Génica , Humanos , Terapia Molecular Dirigida , Complejos Multiproteicos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
INTRODUCTION: The SIX homeodomain proteins and the eyes absent (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 toward developing better compounds may ultimately result in effective anti-cancer therapies.
Asunto(s)
Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/genética , Proteínas Tirosina Fosfatasas/genética , Adulto , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Terapia Molecular Dirigida , Metástasis de la Neoplasia/genética , Neoplasias/genética , Neoplasias/patologíaRESUMEN
Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor that suppresses multiple proapoptotic and epithelial genes. CtBP is overexpressed in many human cancers, and its overexpression increases stem cell-like features, epithelial-mesenchymal transition, and cancer cell survival. Knockdown of CtBP also increases apoptosis independent of p53 in cell culture. Therefore, targeting CtBP with small molecules that disrupt its interaction with transcription factor partners may be an effective cancer therapy. To elicit its corepressing effect, CtBP binds to a conserved peptide motif in each transcription factor partner. We developed an AlphaScreen high-throughput screening assay to monitor the interaction between CtBP and E1A (which mimics the interaction between CtBP and its transcriptional partners). We screened the LOPAC library of 1280 bioactive compounds and identified NSC95397, which inhibits the CtBP-E1A interaction (IC50 = 2.9 µM). The inhibitory activity of NSC95397 was confirmed using two secondary assays and a counterscreen. NSC95397 also behaved as a weak substrate of CtBP dehydrogenase activity and did not inhibit another dehydrogenase, lactase dehydrogenase. Finally, NSC95397 was able to disrupt CtBP-mediated transcriptional repression of a target gene. These studies present a new possibility for the development of a therapeutic agent targeting tumors through disrupting the CtBP transcriptional complex.