RESUMEN

Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1's ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway.

Asunto(s)

Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Proteínas de Unión al ADN , Humanos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Fosforilación , Proteínas de Unión al ADN/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Proteínas Portadoras/metabolismo , Replicación del ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteína BRCA1/metabolismo , Transducción de Señal , Proteínas Nucleares/metabolismo , Fibroblastos/metabolismo , Puntos de Control del Ciclo Celular

RESUMEN

PURPOSE: This study evaluates the oncogenic role of PIBF1 in triple-negative breast cancer (TNBC). TNBC is considered to have a poorer prognosis than other types of breast cancer and is associated with high risk of recurrence and distant metastasis. Currently, there are no effective therapies for the TNBC patients with distant metastasis due to the lack of targeted therapeutic options. METHODS: The effects of PIBF1 knockdown on the cell viability and motility of TNBC cell lines were investigated. Effects of PIBF1 overexpression on tumorigenicity and cell motility were confirmed using Ba/F3 cell line and xenograft study on BALB/c nude mice. RESULTS: In TNBC cell lines that highly express PIBF1, knockdown of PIBF1 induces apoptosis and suppresses cell viability and motility with activation of the ATR/CHK1 signaling pathway. Moreover, the oncogenic function of PIBF1 was confirmed using the Ba/F3 cell line. CONCLUSION: For the first time, these findings clarify the role of PIBF1 in regulating ATR/CHK1 signaling pathway and inhibiting the proliferation and migration of TNBC cell lines. These results demonstrate the oncogenic roles of PIBF1 and provide new insights into the function and the molecular mechanism of PIBF1 in malignant TNBC.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Proteínas Gestacionales/metabolismo , Factores Supresores Inmunológicos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Apoptosis/fisiología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Gestacionales/biosíntesis , Proteínas Gestacionales/genética , Transducción de Señal , Factores Supresores Inmunológicos/biosíntesis , Factores Supresores Inmunológicos/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas

RESUMEN

Many cervical cancer (CC) patients suffer from cancer invasion and lymph node metastasis, resulting in poor therapeutic outcome. Evidence has indicated the involvement of misexpressed high-mobility group AT-hook 2 (HMGA2) in poor survival of cancer patients. This study hereby aims to investigate the role of HMGA2 in CC cell biological functions via the ATR/Chk1 signaling pathway. The cell line with the highest HMGA2 expression was selected to establish cell lines with wild-type and stable HMGA2 silencing. The underlying regulatory mechanisms of HMGA2 in CC cells were analyzed with the treatment of the ATR/Chk1 signaling pathway activator, inhibitor, shRNA against HMGA2 or pcDNA-HMGA2 plasmids, followed by quantification of expression levels of ATR, Chk1, Bcl-2, Bax, MMP-2, MMP-9, E-cadherin and N-cadherin. CC cell apoptosis, proliferation, migration, invasion and lymph node metastasis in nude mice were evaluated. The HeLa cell line with the highest HMGA2 expression was selected. HMGA2 inhibited the activation of the ATR/Chk1 signaling pathway. Notably, HMGA2 silencing or inhibition of the ATR/Chk1 signaling pathway inhibited epithelial mesenchymal transition (EMT), CC cell proliferation, invasion, migration, tumorigenicity and lymph node metastasis while promoting apoptosis, indicated by reduced expression of Bcl-2, MMP-2, MMP-9 and N-cadherin, with increased expression of E-cadherin and Bax. Collectively, our study provides evidence that HMGA2 gene silencing inhibits the activation of the ATR/Chk1 signaling pathway, whereby repressing EMT, proliferation, migration and invasion of CC cells and lymph node metastasis, and promoting CC cell apoptosis.