RESUMEN
Entry into mitosis has been classically attributed to the activation of a cyclin B/Cdk1 amplification loop via a partial pool of this kinase becoming active at the end of G2 phase. However, how this initial pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, depletion of the orthologue of FAM122A in C. elegans prevents entry into mitosis in germline stem cells. Moreover, data from Xenopus egg extracts strongly suggest that FAM122A-dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. Inhibition of this phosphatase allows subsequent phosphorylation of early mitotic substrates by cyclin A/Cdk, resulting in full cyclin B/Cdk1 and Greatwall (Gwl) kinase activation. Subsequent to Greatwall activation, Arpp19/ENSA become phosphorylated and now compete with FAM122A, promoting its dissociation from PP2A-B55 and taking over its phosphatase inhibition role until the end of mitosis.
Asunto(s)
Caenorhabditis elegans , Proteínas Serina-Treonina Quinasas , Animales , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Mitosis , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Ciclina B/metabolismoRESUMEN
While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Pirazinas/farmacología , Pirazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Nucleares/metabolismo , Fosfoproteínas/fisiología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Pirazinas/metabolismo , Pirazoles/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Family with sequence similarity 122a (FAM122A), identified as an endogenous inhibitor of protein phosphatase 2A (PP2A) previously, is involved in multiple important physiological processes, and essential for the growth of acute myeloid leukemia and hepatocellular carcinoma cells. However, the function of FAM122A in oral squamous cell carcinoma (OSCC) is undetermined. In this study, by analyzing TCGA and GEO databases, we found that the expression of FAM122A was significantly down-regulated in head and neck squamous cell carcinoma and OSCC patients, meanwhile this low expression was tightly associated with the poor prognosis and advanced clinical stage during OSCC development. The similar low expression pattern of FAM122A could also been seen in OSCC cell lines compared with normal human oral keratinocytes. Further, we demonstrated that FAM122A knockdown significantly promoted the growth, clonogenic potential as well as migration capabilities of OSCC cells, while these alterations could be rescued by the re-expression of FAM122A. Over-expression of FAM122A suppressed OSCC cell proliferation and migration. FAM122A also inhibited the epithelial-mesenchymal transition (EMT) in OSCC cells by the up-regulation of epithelial marker E-cadherin and down-regulation of mesenchymal markers Fibronectin and Vimentin, which is presumably mediated by transforming growth factor ß receptor 3 (TGFBR3), a novel tumor suppressor. In addition, FAM122A could induce T cell infiltration in OSCC, indicating that FAM122A might influence the immune cell activity of tumor environment and further interfere the tumor development. Collectively, our results suggest that FAM122A functions as a tumor suppressor in OSCC and possibly acts as a predictive biomarker for the diagnosis and/or treatment of OSCC.
Asunto(s)
Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Boca , Humanos , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Proliferación Celular/genética , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Línea Celular Tumoral , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Genes Supresores de Tumor , Pronóstico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , AnimalesRESUMEN
The serum- and glucocorticoid-induced kinase 1 (SGK1) promotes cell survival under stress conditions and facilitates the emergence of drug resistance in cancer. The underlying mechanisms of these observations are not fully understood. In this study, we found that SGK1 activity is suppressed by the action of the S/T phosphatases PP5 and PP2A, which constantly dephosphorylate SGK1. Using newly developed anti-phospho SGK1 antibodies and inhibitors of phosphatases, we determined that the high degree of dephosphorylation is caused by two factors: the tendency of SGK1 to unfold, which makes it dependent on Hsp90 chaperone complexes composed of four proteins, Hsp90/CDC37/PP5/SGK1, and where the phosphatase PP5 persistently dephosphorylates SGK1 within the complex. SGK1 binding to PP2A regulatory subunits B55γ and B55δ brings PP2A catalytic subunit close to exposed SGK1 phosphoresidues. A further association of phosphorylated pS37-FAM122A-an endogenous inhibitor of PP2A-to the holoenzyme diminishes dephosphorylation of SGK1 mediated by PP2A. Our study also reveals that genotoxic stress can reverse the dominant impact of phosphatases over kinases by activating the DNA-dependent protein kinase, which enhances mTORC2 activity directed to SGK1. Thus, our results provide insight into a molecular pathway that enables SGK1 to gain phosphorylation and catalytic activity and promote cell survival, potentially diminishing the efficacy of cancer treatments. As the DNA damage response operates in many cancer cells and is further induced by chemotherapies, the findings of this study could have significant implications for the development of novel cancer therapies targeting SGK1.
Asunto(s)
Daño del ADN , Fosfoproteínas Fosfatasas , Proteínas Serina-Treonina Quinasas , Daño del ADN/genética , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Línea Celular Tumoral , Proteínas Serina-Treonina Quinasas/metabolismo , Humanos , Activación Enzimática/fisiología , Supervivencia CelularRESUMEN
Mesendodermal specification and cardiac differentiation are key issues for developmental biology and heart regeneration medicine. Previously, we demonstrated that FAM122A, a highly conserved housekeeping gene, is an endogenous inhibitor of protein phosphatase 2A (PP2A) and participates in multifaceted physiological and pathological processes. However, the in vivo function of FAM122A is largely unknown. In this study, we observed that Fam122 deletion resulted in embryonic lethality with severe defects of cardiovascular developments and significantly attenuated cardiac functions in conditional cardiac-specific knockout mice. More importantly, Fam122a deficiency impaired mesendodermal specification and cardiac differentiation from mouse embryonic stem cells but showed no influence on pluripotent identity. Mechanical investigation revealed that the impaired differentiation potential was caused by the dysregulation of histone modification and Wnt and Hippo signaling pathways through modulation of PP2A activity. These findings suggest that FAM122A is a novel and critical regulator in mesendodermal specification and cardiac differentiation. This research not only significantly extends our understanding of the regulatory network of mesendodermal/cardiac differentiation but also proposes the potential significance of FAM122A in cardiac regeneration.
Asunto(s)
Células Madre Embrionarias , Procesamiento Proteico-Postraduccional , Animales , Ratones , Diferenciación Celular/fisiología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias de Ratones/metabolismoRESUMEN
FAM122A is a highly conserved protein in mammals, however its function is still largely unknown so far. In this study, we investigated the potential role of FAM122A in hepatocellular carcinoma (HCC). By analyzing HCC patient cohorts from RNA sequencing datasets, we found the expression level of FAM122A mRNA is significantly upregulated in HCC patients. Moreover, this abnormally higher expression pattern of FAM122A protein was also found in partial HCC tumor tissues, compared with the normal parts. Further, we demonstrated that CRISPR/Cas9-mediated FAM122A knockout significantly inhibits the growth, clonogenic potential and xenografts of HCC cells, induces cell cycle arrest and reduces the expression of proliferation-related genes. Interestingly, FAM122A deletion significantly enhances the cytotoxicity effect of Doxorubicin (Dox), a drug used in standard chemotherapy in HCC patients. In contrary, overexpression of FAM122A not only promotes HCC cell growth, but also inhibits Dox-induced DNA damage and cell death. Considering that FAM122A is previously identified as an endogenous inhibitor of PP2A, we asked whether FAM122A regulating HCC cell growth is associated with PP2A. The results showed FAM122A can also modulate PP2A activity in HCC cells although the modulated effect is relatively slight, however, treatment with a PP2A inhibitor okadaic acid did not rescue the inhibitory effects of cell growth and proliferation in FAM122A deletion cells, indicating that FAM122A may support HCC cell growth independent of its ability to modulate PP2A. Collectively, these results suggest that FAM122A is required for maintaining HCC cell growth, and its elimination combined with chemotherapy may represent a potential novel therapeutic strategy for HCC patients.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Proliferación Celular/genética , Doxorrubicina/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Fosfoproteínas/genética , Eliminación de Secuencia/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carcinoma Hepatocelular/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
FAM122A is a housekeeping gene and highly conserved in mammals. More recently, we have demonstrated that FAM122A is essential for maintaining the growth of hepatocellular carcinoma cells, in which we unexpectedly found that FAM122A deletion increases γH2AX protein level, suggesting that FAM122A may participate in the regulation of DNA homeostasis or stability. In this study, we continued to investigate the potential role of FAM122A in DNA damage and/or repair. We found that CRISPR/Cas9-mediated FAM122A deletion enhances endogenous DNA damages in cancer cells but not in normal cells, demonstrating a significant increase in γH2AX protein and foci formation of γH2AX and 53BP1, as well as DNA breaks by comet assay. Further, we found that FAM122A deletion greatly increases TOP2α protein level, and significantly and specifically enhances TOP2 poisons (etoposide and doxorubicin)-induced DNA damage effects in cancer cells. Moreover, FAM122A is found to be interacted with TOP2α, instead of TOP2ß. However, FAM122A knockout doesn't affect the intracellular ROS levels and the process of DNA repair after removal of etoposide with short-term stimulation, suggesting that FAM122A deletion-enhanced DNA damage does not result from endogenous overproduction of ROS and/or impairment of DNA repair ability. Collectively, our study provides the first demonstration that FAM122A is critical for maintaining DNA stability probably by modulating TOP2α protein, and FAM122A deletion combined with TOP2-targeted drugs may represent a potential novel chemotherapeutic strategy for cancer patients.
Asunto(s)
ADN-Topoisomerasas de Tipo II/genética , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Histonas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoproteínas/genética , Antineoplásicos/farmacología , Apoptosis/genética , Línea Celular Tumoral , Daño del ADN , Reparación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Neoplasias/metabolismo , Doxorrubicina/farmacología , Etopósido/farmacología , Fibroblastos , Eliminación de Gen , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Fosfoproteínas/deficiencia , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Inhibidores de Topoisomerasa II/farmacología , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
FAM122A is a highly conserved protein in mammals. Here, we identify that FAM122A can be sumoylated at lysine 89, which can be de-conjugated by SENP1. Furthermore, the sumoylation of FAM122A reduces the PP2A-Cα protein level together with the reduced phosphatase activity of PP2A, which suppresses cell proliferation. Collectively, our results suggest that the sumoylation of FAM122A may have a significant role in cellular function.
Asunto(s)
Fosfoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Fosfatasa 2/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis , Proteína SUMO-1/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Proliferación Celular , Células HEK293 , Humanos , Lisina/metabolismo , Fosfoproteínas/química , SumoilaciónRESUMEN
FAM122A is a highly conserved housekeeping gene, but its physiological and pathophysiological roles remain greatly elusive. Based on the fact that FAM122A is highly expressed in human CD71+ early erythroid cells, herein we report that FAM122A is downregulated during erythroid differentiation, while its overexpression significantly inhibits erythrocytic differentiation in primary human hematopoietic progenitor cells and erythroleukemia cells. Mechanistically, FAM122A directly interacts with the C-terminal zinc finger domain of GATA1, a critical transcriptional factor for erythropoiesis, and reduces GATA1 chromatin occupancy on the promoters of its target genes, thus resulting in the decrease of GATA1 transcriptional activity. The public datasets show that FAM122A is abnormally upregulated in patients with ß-thalassemia. Collectively, our results demonstrate that FAM122A plays an inhibitory role in the regulation of erythroid differentiation, and it would be a potentially therapeutic target for GATA1-related dyserythropoiesis or an important regulator for amplifying erythroid cells ex vivo.
Asunto(s)
Diferenciación Celular , Células Eritroides/citología , Células Eritroides/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Antígenos CD34/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , ADN/metabolismo , Regulación hacia Abajo/genética , Células Eritroides/efectos de los fármacos , Eritropoyetina/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Células K562 , Fosfoproteínas/química , Unión Proteica/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Dedos de ZincRESUMEN
The regulation of the ubiquitously expressed protein phosphatase 2A (PP2A) is essential for various cellular functions such as cell proliferation, transformation, and fate determination. In this study, we demonstrate that the highly conserved protein in mammals, designated FAM122A, directly interacts with PP2A-Aα and B55α rather than B56α subunits, and inhibits the phosphatase activity of PP2A-Aα/B55α/Cα complex. Further, FAM122A potentiates the degradation of catalytic subunit PP2A-Cα with the increased poly-ubiquitination. In agreement, FAM122A silencing inhibits while its overexpression enhances cell growth and colony-forming ability. Collectively, we identify FAM122A as a new endogenous PP2A inhibitor and its physiological and pathophysiological significances warrant to be further investigated.