RESUMEN
PTEN dysfunction plays a crucial role in the pathogenesis of hereditary and sporadic cancers. Here, we show that PTEN homodimerizes and, in this active conformation, exerts lipid phosphatase activity on PtdIns(3,4,5)P3. We demonstrate that catalytically inactive cancer-associated PTEN mutants heterodimerize with wild-type PTEN and constrain its phosphatase activity in a dominant-negative manner. To study the consequences of homo- and heterodimerization of wild-type and mutant PTEN in vivo, we generated Pten knockin mice harboring two cancer-associated PTEN mutations (PtenC124S and PtenG129E). Heterozygous Pten(C124S/+) and Pten(G129E/+) cells and tissues exhibit increased sensitivity to PI3-K/Akt activation compared to wild-type and Pten(+/-) counterparts, whereas this difference is no longer apparent between Pten(C124S/-) and Pten(-/-) cells. Notably, Pten KI mice are more tumor prone and display features reminiscent of complete Pten loss. Our findings reveal that PTEN loss and PTEN mutations are not synonymous and define a working model for the function and regulation of PTEN.
Asunto(s)
Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Transducción de Señal , Animales , Embrión de Mamíferos/citología , Femenino , Humanos , Pérdida de Heterocigocidad , Masculino , Ratones , Mutación , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Here, we present a unifying hypothesis about how messenger RNAs, transcribed pseudogenes, and long noncoding RNAs "talk" to each other using microRNA response elements (MREs) as letters of a new language. We propose that this "competing endogenous RNA" (ceRNA) activity forms a large-scale regulatory network across the transcriptome, greatly expanding the functional genetic information in the human genome and playing important roles in pathological conditions, such as cancer.
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Perfilación de la Expresión Génica , ARN/genética , ARN/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , MicroARNs/genética , Neoplasias/genética , Neoplasias/metabolismo , Seudogenes , ARN Mensajero/genética , ARN no Traducido/genéticaRESUMEN
PTEN is a frequently mutated tumor suppressor gene that opposes the PI3K/AKT pathway through dephosphorylation of phosphoinositide-3,4,5-triphosphate. Recently, nuclear compartmentalization of PTEN was found as a key component of its tumor-suppressive activity; however its nuclear function remains poorly defined. Here we show that nuclear PTEN interacts with APC/C, promotes APC/C association with CDH1, and thereby enhances the tumor-suppressive activity of the APC-CDH1 complex. We find that nuclear exclusion but not phosphatase inactivation of PTEN impairs APC-CDH1. This nuclear function of PTEN provides a straightforward mechanistic explanation for the fail-safe cellular senescence response elicited by acute PTEN loss and the tumor-suppressive activity of catalytically inactive PTEN. Importantly, we demonstrate that PTEN mutant and PTEN null states are not synonymous as they are differentially sensitive to pharmacological inhibition of APC-CDH1 targets such as PLK1 and Aurora kinases. This finding identifies a strategy for cancer patient stratification and, thus, optimization of targeted therapies. PAPERCLIP:
Asunto(s)
Cadherinas/metabolismo , Senescencia Celular , Fosfohidrolasa PTEN/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Animales , Antígenos CD , Aurora Quinasas , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Fosfohidrolasa PTEN/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Trasplante HeterólogoRESUMEN
Here, we demonstrate that protein-coding RNA transcripts can crosstalk by competing for common microRNAs, with microRNA response elements as the foundation of this interaction. We have termed such RNA transcripts as competing endogenous RNAs (ceRNAs). We tested this hypothesis in the context of PTEN, a key tumor suppressor whose abundance determines critical outcomes in tumorigenesis. By a combined computational and experimental approach, we identified and validated endogenous protein-coding transcripts that regulate PTEN, antagonize PI3K/AKT signaling, and possess growth- and tumor-suppressive properties. Notably, we also show that these genes display concordant expression patterns with PTEN and copy number loss in cancers. Our study presents a road map for the prediction and validation of ceRNA activity and networks and thus imparts a trans-regulatory function to protein-coding mRNAs.
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Regulación de la Expresión Génica , Fosfohidrolasa PTEN/genética , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Animales , Humanos , Ratones , MicroARNs/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Mensajero/genética , ARN no Traducido/genéticaRESUMEN
The importance of the physiological function of phosphatase and tensin homologue (PTEN) is illustrated by its frequent disruption in cancer. By suppressing the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway through its lipid phosphatase activity, PTEN governs a plethora of cellular processes including survival, proliferation, energy metabolism and cellular architecture. Consequently, mechanisms regulating PTEN expression and function, including transcriptional regulation, post-transcriptional regulation by non-coding RNAs, post-translational modifications and protein-protein interactions, are all altered in cancer. The repertoire of PTEN functions has recently been expanded to include phosphatase-independent activities and crucial functions within the nucleus. Our increasing knowledge of PTEN and pathologies in which its function is altered will undoubtedly inform the rational design of novel therapies.
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Regulación de la Expresión Génica , Neoplasias/enzimología , Fosfohidrolasa PTEN/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Núcleo Celular/enzimología , Humanos , Mutación , Neoplasias/patología , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/fisiología , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Conformación Proteica , Transducción de Señal , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome "fusion-fission" cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.
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Fibroblastos , Fosfatidilinositol 3-Quinasas , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Fibroblastos/metabolismo , Lisosomas/metabolismo , Ratones , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
Since its discovery as the elusive tumor suppressor gene at the frequently mutated 10q23 locus, PTEN has been identified as lost or mutated in several sporadic and heritable tumor types. A decade of work has established that PTEN is a nonredundant phosphatase that is essential for regulating the highly oncogenic prosurvival PI3K/AKT signaling pathway. This review discusses emerging modes of PTEN function and regulation, and speculates about how manipulation of PTEN function could be used for cancer therapy.
Asunto(s)
Fosfohidrolasa PTEN/metabolismo , Animales , Predisposición Genética a la Enfermedad , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fosfohidrolasa PTEN/genética , Transcripción GenéticaRESUMEN
PURPOSE: Mammographic density is a risk factor for breast cancer but the mechanism behind this association is unclear. The receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL) pathway has been implicated in the development of breast cancer. Given the role of RANK signaling in mammary epithelial cell proliferation, we hypothesized this pathway may also be associated with mammographic density. Osteoprotegerin (OPG), a decoy receptor for RANKL, is known to inhibit RANK signaling. Thus, it is of interest to evaluate whether OPG levels modify breast cancer risk through mammographic density. METHODS: We quantified serum OPG levels in 57 premenopausal and 43 postmenopausal women using an enzyme-linked immunosorbent assay (ELISA). Cumulus was used to measure percent density, dense area, and non-dense area for each mammographic image. Subjects were classified into high versus low OPG levels based on the median serum OPG level in the entire cohort (115.1 pg/mL). Multivariate models were used to assess the relationship between serum OPG levels and the measures of mammographic density. RESULTS: Serum OPG levels were not associated with mammographic density among premenopausal women (P ≥ 0.42). Among postmenopausal women, those with low serum OPG levels had higher mean percent mammographic density (20.9% vs. 13.7%; P = 0.04) and mean dense area (23.4 cm2 vs. 15.2 cm2; P = 0.02) compared to those with high serum OPG levels after covariate adjustment. CONCLUSIONS: These findings suggest that low OPG levels may be associated with high mammographic density, particularly in postmenopausal women. Targeting RANK signaling may represent a plausible, non-surgical prevention option for high-risk women with high mammographic density, especially those with low circulating OPG levels.
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Densidad de la Mama , Neoplasias de la Mama/patología , Osteoprotegerina/sangre , Adulto , Anciano , Estudios de Cohortes , Femenino , Humanos , Persona de Mediana Edad , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Factores de RiesgoRESUMEN
BACKGROUND: BRCA1 mutation carriers face a high lifetime risk of developing both breast and ovarian cancer. Haploinsufficiency is thought to predispose these women to cancer by reducing the pool of available BRCA1 transcript and protein, thereby compromising BRCA1 function. Whether or not cancer-free BRCA1 mutation carriers have lower messenger (m)RNA transcript levels in peripheral blood leukocytes has not been evaluated. The primary aim of this study was to characterize an association between BRCA1 mutation status and BRCA1 mRNA leukocyte expression levels among healthy women with a BRCA1 mutation. METHOD: RNA was extracted from freshly isolated peripheral blood leukocytes of 58 cancer-free, female participants (22 BRCA1 mutation carriers and 36 non-carriers). The expression levels of 236 cancer-associated genes, including BRCA1, were quantified using the Human Cancer Reference gene panel from the Nanostring Technologies nCounter Analysis System. RESULTS: Multivariate modeling demonstrated that carrying a BRCA1 mutation was the most significant predictor of BRCA1 mRNA levels. BRCA1 mRNA levels were significantly lower in BRCA1 mutation carriers compared to non-carriers (146.7 counts vs. 175.1 counts; P = 0.002). Samples with BRCA1 mutations within exon 11 had lower BRCA1 mRNA levels than samples with mutations within the 5' and 3' regions of the BRCA1 gene (122.1 counts vs. 138.9 and 168.6 counts, respectively; P = 0.003). Unsupervised hierarchical clustering of gene expression profiles from freshly isolated blood leukocytes revealed that BRCA1 mutation carriers cluster more closely with other BRCA1 mutation carriers than with BRCA1 wild-type samples. Moreover, a set of 17 genes (including BRCA1) previously shown to be involved in carcinogenesis, were differentially expressed between BRCA1 mutation carriers and non-carriers. CONCLUSION: Overall, these findings support the concept of BRCA1 haploinsufficiency wherein a specific mutation results in dosage-dependent alteration of BRCA1 at the transcriptional level. This study is the first to show a decrease in BRCA1 mRNA expression in freshly isolated blood leukocytes from healthy, unaffected BRCA1 mutation carriers.
Asunto(s)
Genes BRCA1 , Heterocigoto , Leucocitos/metabolismo , Mutación , Transcripción Genética , Adolescente , Adulto , Análisis por Conglomerados , Femenino , Dosificación de Gen , Perfilación de la Expresión Génica , Haploinsuficiencia , Voluntarios Sanos , Humanos , Persona de Mediana Edad , Degradación de ARNm Mediada por Codón sin Sentido , ARN Mensajero/genética , Transcriptoma , Adulto JovenRESUMEN
The canonical role of messenger RNA (mRNA) is to deliver protein-coding information to sites of protein synthesis. However, given that microRNAs bind to RNAs, we hypothesized that RNAs could possess a regulatory role that relies on their ability to compete for microRNA binding, independently of their protein-coding function. As a model for the protein-coding-independent role of RNAs, we describe the functional relationship between the mRNAs produced by the PTEN tumour suppressor gene and its pseudogene PTENP1 and the critical consequences of this interaction. We find that PTENP1 is biologically active as it can regulate cellular levels of PTEN and exert a growth-suppressive role. We also show that the PTENP1 locus is selectively lost in human cancer. We extended our analysis to other cancer-related genes that possess pseudogenes, such as oncogenic KRAS. We also demonstrate that the transcripts of protein-coding genes such as PTEN are biologically active. These findings attribute a novel biological role to expressed pseudogenes, as they can regulate coding gene expression, and reveal a non-coding function for mRNAs.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , MicroARNs/genética , Neoplasias/genética , Fosfohidrolasa PTEN/genética , Seudogenes/genética , ARN Mensajero/genética , Regiones no Traducidas 3'/genética , Unión Competitiva , Línea Celular , Genes Supresores de Tumor , Humanos , Modelos Genéticos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Proteínas ras/genéticaRESUMEN
Recent studies have shown that three major tumour-suppressor proteins undergo monoubiquitylation-mediated nuclear-cytoplasmic shuttling. Importantly, this mechanism has consequences for cancer and implies that proper localization is central to the function of tumour suppressors. This Progress article highlights recent efforts demonstrating that monoubiquitylation coupled to nuclear-cytoplasmic shuttling might be a novel regulatory mechanism that directly influences the function of tumour suppressors.
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Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Transporte Activo de Núcleo Celular , Citoplasma/metabolismo , Factores de Transcripción Forkhead/metabolismo , Humanos , Modelos Biológicos , Estrés Oxidativo , Fosfohidrolasa PTEN/metabolismoRESUMEN
Nuclear exclusion of the PTEN (phosphatase and tensin homologue deleted in chromosome 10) tumour suppressor has been associated with cancer progression. However, the mechanisms leading to this aberrant PTEN localization in human cancers are currently unknown. We have previously reported that ubiquitinylation of PTEN at specific lysine residues regulates its nuclear-cytoplasmic partitioning. Here we show that functional promyelocytic leukaemia protein (PML) nuclear bodies co-ordinate PTEN localization by opposing the action of a previously unknown PTEN-deubiquitinylating enzyme, herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), and that the integrity of this molecular framework is required for PTEN to be able to enter the nucleus. We find that PTEN is aberrantly localized in acute promyelocytic leukaemia, in which PML function is disrupted by the PML-RARalpha fusion oncoprotein. Remarkably, treatment with drugs that trigger PML-RARalpha degradation, such as all-trans retinoic acid or arsenic trioxide, restore nuclear PTEN. We demonstrate that PML opposes the activity of HAUSP towards PTEN through a mechanism involving the adaptor protein DAXX (death domain-associated protein). In support of this paradigm, we show that HAUSP is overexpressed in human prostate cancer and is associated with PTEN nuclear exclusion. Thus, our results delineate a previously unknown PML-DAXX-HAUSP molecular network controlling PTEN deubiquitinylation and trafficking, which is perturbed by oncogenic cues in human cancer, in turn defining a new deubiquitinylation-dependent model for PTEN subcellular compartmentalization.
Asunto(s)
Proteínas Nucleares/metabolismo , Fosfohidrolasa PTEN/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Ubiquitinas/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteínas Co-Represoras , Fibroblastos , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Masculino , Ratones , Chaperonas Moleculares , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Tretinoina/farmacología , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Peptidasa Específica de Ubiquitina 7RESUMEN
Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2(-/-) mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor.
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Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Células HEK293 , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos C57BL , Neoplasias/genética , Proteolisis , Proteínas Proto-Oncogénicas c-myc/genética , Tolerancia a Radiación , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genéticaRESUMEN
BACKGROUND: Lower levels of osteoprotegerin (OPG), the decoy receptor for receptor activator of NFκB (RANK)-ligand, have been reported among women with a BRCA1 mutation, suggesting OPG may be marker of cancer risk. Whether various reproductive, hormonal, or lifestyle factors impact OPG levels in these women is unknown. METHODS: BRCA1 mutation carriers enrolled in a longitudinal study, no history of cancer, and a serum sample for OPG quantification, were included. Exposure information was collected through self-reported questionnaire at study enrollment and every 2 years thereafter. Serum OPG levels (pg/mL) were measured using an ELISA, and generalized linear models were used to assess the associations between reproductive, hormonal, and lifestyle exposures at the time of blood collection with serum OPG. Adjusted means were estimated using the fully adjusted model. RESULTS: A total of 701 women with a median age at blood collection of 39.0 years (18.0-82.0) were included. Older age (Spearman r = 0.24; P < 0.001) and current versus never smoking (98.82 vs. 86.24 pg/mL; Pcat < 0.001) were associated with significantly higher OPG, whereas ever versus never coffee consumption was associated with significantly lower OPG (85.92 vs. 94.05 pg/mL; Pcat = 0.03). There were no other significant associations for other exposures (P ≥ 0.06). The evaluated factors accounted for 7.5% of the variability in OPG. CONCLUSIONS: OPG is minimally influenced by hormonal and lifestyle factors among BRCA1 mutation carriers. IMPACT: These findings suggest that circulating OPG levels are not impacted by non-genetic factors in high-risk women.
Asunto(s)
Genes BRCA1 , Osteoprotegerina , Adulto , Femenino , Humanos , Proteína BRCA1/genética , Estudios Longitudinales , Osteoprotegerina/genética , FumarRESUMEN
Aggressive solid malignancies, including pancreatic ductal adenocarcinoma (PDAC), can exploit lysosomal exocytosis to modify the tumor microenvironment, enhance motility, and promote invasiveness. However, the molecular pathways through which lysosomal functions are co-opted in malignant cells remain poorly understood. In this study, we demonstrate that inositol polyphosphate 4-phosphatase, Type II (INPP4B) overexpression in PDAC is associated with PDAC progression. We show that INPP4B overexpression promotes peripheral dispersion and exocytosis of lysosomes resulting in increased migratory and invasive potential of PDAC cells. Mechanistically, INPP4B overexpression drives the generation of PtdIns(3,5)P2 on lysosomes in a PIKfyve-dependent manner, which directs TRPML-1 to trigger the release of calcium ions (Ca2+). Our findings offer a molecular understanding of the prognostic significance of INPP4B overexpression in PDAC through the discovery of a novel oncogenic signaling axis that orchestrates migratory and invasive properties of PDAC via the regulation of lysosomal phosphoinositide homeostasis.
Asunto(s)
Carcinoma Ductal Pancreático , Movimiento Celular , Exocitosis , Lisosomas , Invasividad Neoplásica , Neoplasias Pancreáticas , Fosfatidilinositol 3-Quinasas , Monoéster Fosfórico Hidrolasas , Canales de Potencial de Receptor Transitorio , Animales , Humanos , Masculino , Ratones , Calcio/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Lisosomas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Canales de Potencial de Receptor Transitorio/metabolismo , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
PARP inhibitors (PARPi) are efficacious in BRCA1-null tumors; however, their utility is limited in tumors with functional BRCA1. We hypothesized that pharmacologically reducing BRCA1 protein levels could enhance PARPi effectiveness in BRCA1 wild-type tumors. To identify BRCA1 downregulating agents, we generated reporter cell lines using CRISPR-mediated editing to tag endogenous BRCA1 protein with HiBiT. These reporter lines enable the sensitive measurement of BRCA1 protein levels by luminescence. Validated reporter cells were used in a pilot screen of epigenetic-modifying probes and a larger screen of more than 6,000 compounds. We identified 7 compounds that could downregulate BRCA1-HiBiT expression and synergize with olaparib. Three compounds, N-acetyl-N-acetoxy chlorobenzenesulfonamide (NANAC), A-443654, and CHIR-124, were validated to reduce BRCA1 protein levels and sensitize breast cancer cells to the toxic effects of olaparib. These results suggest that BRCA1-HiBiT reporter cells hold promise in developing agents to improve the clinical utility of PARPi.
RESUMEN
Receptor tyrosine kinases such as EGF receptor (EGFR) stimulate phosphoinositide 3 kinases to convert phosphatidylinositol-4,5-bisphosophate [PtdIns(4,5)P2] into phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. PtdIns(3,4,5)P3 then remodels actin and gene expression, and boosts cell survival and proliferation. PtdIns(3,4,5)P3 partly achieves these functions by triggering activation of the kinase Akt, which phosphorylates targets like Tsc2 and GSK3ß. Consequently, unchecked upregulation of PtdIns(3,4,5)P3-Akt signaling promotes tumor progression. Interestingly, 50-70% of PtdIns and PtdInsPs have stearate and arachidonate at sn-1 and sn-2 positions of glycerol, respectively, forming a species known as 38:4-PtdIns/PtdInsPs. LCLAT1 and MBOAT7 acyltransferases partly enrich PtdIns in this acyl format. We previously showed that disruption of LCLAT1 lowered PtdIns(4,5)P2 levels and perturbed endocytosis and endocytic trafficking. However, the role of LCLAT1 in receptor tyrosine kinase and PtdIns(3,4,5)P3 signaling was not explored. Here, we show that LCLAT1 silencing in MDA-MB-231 and ARPE-19 cells abated the levels of PtdIns(3,4,5)P3 in response to EGF signaling. Importantly, LCLAT1-silenced cells were also impaired for EGF-driven and insulin-driven Akt activation and downstream signaling. Thus, our work provides first evidence that the LCLAT1 acyltransferase is required for receptor tyrosine kinase signaling.
Asunto(s)
Aciltransferasas , Factor de Crecimiento Epidérmico , Receptores ErbB , Fosfatos de Fosfatidilinositol , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Aciltransferasas/metabolismo , Receptores ErbB/metabolismo , Línea Celular Tumoral , Fosforilación , Proliferación CelularRESUMEN
BACKGROUND: Tumor progression is based on a close interaction between cancer cells and Tumor MicroEnvironment (TME). Here, we focus on the role that Cancer Associated Fibroblasts (CAFs), Mesenchymal Stem Cells (MSCs) and microRNAs (miRs) play in breast cancer and melanoma malignancy. METHODS: We used public databases to investigate miR-214 expression in the stroma compartment of primary human samples and evaluated tumor formation and dissemination following tumor cell injections in miR-214 overexpressing (miR-214over) and knock out (miR-214ko) mice. In addition, we dissected the impact of Conditioned Medium (CM) or Extracellular Vesicles (EVs) derived from miR-214-rich or depleted stroma cells on cell metastatic traits. RESULTS: We evidence that the expression of miR-214 in human cancer or metastasis samples mostly correlates with stroma components and, in particular, with CAFs and MSCs. We present data revealing that the injection of tumor cells in miR-214over mice leads to increased extravasation and metastasis formation. In line, treatment of cancer cells with CM or EVs derived from miR-214-enriched stroma cells potentiate cancer cell migration/invasion in vitro. Conversely, dissemination from tumors grown in miR-214ko mice is impaired and metastatic traits significantly decreased when CM or EVs from miR-214-depleted stroma cells are used to treat cells in culture. Instead, extravasation and metastasis formation are fully re-established when miR-214ko mice are pretreated with miR-214-rich EVs of stroma origin. Mechanistically, we also show that tumor cells are able to induce miR-214 production in stroma cells, following the activation of IL-6/STAT3 signaling, which is then released via EVs subsequently up-taken by cancer cells. Here, a miR-214-dependent pro-metastatic program becomes activated. CONCLUSIONS: Our findings highlight the relevance of stroma-derived miR-214 and its release in EVs for tumor dissemination, which paves the way for miR-214-based therapeutic interventions targeting not only tumor cells but also the TME.