RESUMO
Wnt signaling in mouse mammary development and tumorigenesis has been heavily studied and characterized, but its role in human breast cancer remains elusive. Although Wnt inhibitors are in early clinical development, it is unclear whether they will be of therapeutic benefit to breast cancer patients, and subsequently, to which ones. To address this, we generated a panel of Wnt reporting human breast cancer cell lines and identified a previously unrecognized enrichment for the ability to respond to Wnt in the basal B or claudin-low subtype, which has a poor prognosis and no available targeted therapies. By co-injecting Wnt3A expressing human mammary fibroblasts with human breast cancer cell lines into mouse mammary fat pads, we showed that elevated paracrine Wnt signaling was correlated with accelerated tumor growth. Using this heterotypic system and a dual lentiviral reporter system that enables simultaneous real-time measurement of both Wnt-responsive cells and bulk tumor cells, we analyzed the outcome of elevated Wnt signaling in patient-derived xenograft (PDX) models. Interestingly, the PDX models exhibited responses not observed in the cell lines analyzed. Exogenous WNT3A promoted tumor growth in one human epidermal growth factor receptor 2-overexpressing PDX line but inhibited growth in a second PDX line obtained from a patient with triple-negative breast cancer. Tumor suppression was associated with squamous differentiation in the latter. Thus, our work suggests that paracrine Wnt signaling can either fuel or repress the growth of human breast cancers depending on yet to be determined aspects of the molecular pathways they express.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/fisiopatologia , Comunicação Parácrina/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Fibroblastos/metabolismo , Fibroblastos/transplante , Humanos , Imuno-Histoquímica , Luciferases , Proteínas Luminescentes , Camundongos , Camundongos SCID , Receptor ErbB-2/metabolismo , Fatores de Tempo , Transplante Heterólogo , Proteína Wnt3A/metabolismo , Proteína Vermelha FluorescenteRESUMO
The mechanisms by which Mdm2 and Mdm4 (MdmX) regulate p53 remain controversial. We generated a mouse encoding p53 lacking the proline-rich domain (p53DeltaP). p53DeltaP exhibited increased sensitivity to Mdm2-dependent degradation and decreased transactivation capacity, correlating with deficient cell cycle arrest and reduced apoptotic responses. p53DeltaP induced lethality in Mdm2-/- embryos, but not in Mdm4-/- embryos. Mdm4 loss did not alter Mdm2 stability but significantly increased p53DeltaP transactivation to partially restore cycle control. In contrast, decreasing Mdm2 levels increased p53DeltaP levels without altering p53DeltaP transactivation. Thus, Mdm4 regulates p53 activity, while Mdm2 mainly controls p53 stability. Furthermore, Mdm4 loss dramatically improved p53DeltaP-mediated suppression of oncogene-induced tumors, emphasizing the importance of targeting Mdm4 in chemotherapies designed to activate p53.
Assuntos
Mutação/genética , Prolina/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose , Células Cultivadas , DNA/genética , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Prolina/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/deficiência , Proteínas Proto-Oncogênicas c-mdm2/genética , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
The stability and activity of tumor suppressor p53 are tightly regulated and partially depend on the p53 proline-rich domain (PRD). We recently analyzed mice expressing p53 with a deletion of the PRD (p53(DeltaP)). p53(DeltaP), a weak transactivator hypersensitive to Mdm2-mediated degradation, is unable to suppress oncogene-induced tumors. This phenotype could result from the loss of two motifs: Pin1 sites proposed to influence p53 stabilization and PXXP motifs proposed to mediate protein interactions. We investigated the importance of these motifs by generating mice encoding point mutations in the PRD. p53(TTAA) contains mutations suppressing all putative Pin1 sites in the PRD, while p53(AXXA) lacks PXXP motifs but retains one intact Pin1 site. Both mutant proteins accumulated in response to DNA damage, although the accumulation of p53(TTAA) was partially impaired. Importantly, p53(TTAA) and p53(AXXA) are efficient transactivators and potent suppressors of oncogene-induced tumors. Thus, Pin1 sites in the PRD may modulate p53 stability but do not significantly affect function. In addition, PXXP motifs are not essential, but structure dictated by the presence of prolines, PXXXXP motifs that may mediate protein interactions, and/or the length of this region appears to be functionally significant. These results may explain why the sequence of the p53 PRD is so variable in evolution.
Assuntos
Neoplasias/patologia , Prolina/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Sítios de Ligação , Ciclo Celular , Proliferação de Células , Transformação Celular Neoplásica , Sequência Conservada , Dano ao DNA , Fibroblastos/citologia , Marcação de Genes , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação Puntual/genética , Ligação Proteica , Estrutura Terciária de Proteína , Recombinação Genética/genética , Relação Estrutura-Atividade , Ativação Transcricional/genéticaRESUMO
The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Animais , Diferenciação Celular/fisiologia , Feminino , Humanos , Glândulas Mamárias Animais/citologia , Camundongos , Células-Tronco/metabolismo , TranscriptomaRESUMO
The replication initiation pattern of the murine beta-globin locus was analyzed in totipotent embryonic stem cells and in differentiated cell lines. Initiation events in the murine beta-globin locus were detected in a region extending from the embryonic Ey gene to the adult betaminor gene, unlike the restricted initiation observed in the human locus. Totipotent and differentiated cells exhibited similar initiation patterns. Deletion of the region between the adult globin genes did not prevent initiation in the remainder of the locus, suggesting that the potential to initiate DNA replication was not contained exclusively within the primary sequence of the deleted region. In addition, a deletion encompassing the six identified 5' hypersensitive sites in the mouse locus control region had no effect on initiation from within the locus. As this deletion also did not affect the chromatin structure of the locus, we propose that the sequences determining both chromatin structure and replication initiation lie outside the hypersensitive sites removed by the deletion.
Assuntos
Replicação do DNA , Globinas/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Diferenciação Celular , Linhagem Celular , DNA/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Fase S , Deleção de SequênciaRESUMO
Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL's ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL's ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Genes Reporter , Humanos , Luciferases de Vaga-Lume/biossíntese , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Recombinases/fisiologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The Hdmx protein restricts p53 activity in vivo and is overexpressed in a significant fraction of human tumors that retain the wild type p53 allele. An understanding of how Hdmx limits p53 activation and blocks apoptosis could therefore lead to development of novel therapeutic agents. We previously showed that Hdmx modulates tumor cell sensitivity to Nutlin-3a, a potent antagonist of the p53/Hdm2 interaction. In this report, we demonstrate that this also applies to MI-219, another Hdm2 antagonist. Thus, the inability to disrupt Hdmx/p53 complexes is a potential barrier to the efficacy of these compounds as single agents. We show that sensitivity to apoptosis in cells with high Hdmx levels is restored by combined treatment with Nutlin and a Bcl-2 family member antagonist to activate Bax. The data are consistent with a model in which Hdmx attenuates p53-dependent activation of the intrinsic apoptotic pathway, and that this occurs upstream of Bax activation. Thus, selectively inhibiting Hdm2 and activating Bax is one effective strategy to induce apoptosis in tumors with high Hdmx levels. Our findings also indicate that preferential induction of apoptosis in tumor versus normal cells occurs using appropriate drug doses.
Assuntos
Apoptose , Compostos de Bifenilo/farmacologia , Imidazóis/metabolismo , Nitrofenóis/farmacologia , Proteínas Nucleares/metabolismo , Piperazinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sulfonamidas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
P53 regulates numerous downstream targets to induce cell cycle arrest, senescence, apoptosis, and DNA repair in response to diverse stresses. Hdm2 and Hdmx are critical negative regulators of P53 because Hdm2 regulates P53 abundance, and both can antagonize P53 transactivation. Modest changes in Hdm2 or Hdmx abundance affect P53 regulation, yet quantitative information regarding their endogenous intracellular concentrations and subcellular distributions during a stress response are lacking. We analyzed these parameters in normal and cancer cells after DNA damage. Our data show that the nuclear abundance of Hdm2 and Hdmx relative to P53 limits P53 activity in cells growing in culture. Upon DNA damage, P53 nuclear abundance increases, whereas Hdm2 and Hdmx stability decreases, which greatly limits their ability to antagonize P53, regardless of their levels. These data indicate that the damage-activated switch in Hdm2 ubiquitin ligase preference from P53 to itself and Hdmx is central to P53 activation.