RESUMO
BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA deficiencies, the emergence of resistance mechanisms underscores the importance of seeking novel Synthetic Lethal (SL) targets for future drug development efforts. In this work, we performed a BRCA2-centric SL screen with a collection of plant-derived compounds from South America. We identified the steroidal alkaloid Solanocapsine as a selective SL inducer, and we were able to substantially increase its potency by deriving multiple analogs. The use of two complementary chemoproteomic approaches led to the identification of the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) as Solanocapsine's target responsible for its BRCA2-linked SL induction. Additional confirmatory evidence was obtained by using the highly specific dCK inhibitor (DI-87), which induces SL in multiple BRCA2-deficient and KO contexts. Interestingly, dCK-induced SL is mechanistically different from the one induced by PARP inhibitors. dCK inhibition generates substantially lower levels of DNA damage, and cytotoxic phenotypes are associated exclusively with mitosis, thus suggesting that the fine-tuning of nucleotide supply in mitosis is critical for the survival of BRCA2-deficient cells. Moreover, by using a xenograft model of contralateral tumors, we show that dCK impairment suffices to trigger SL in-vivo. Taken together, our findings unveil dCK as a promising new target for BRCA2-deficient cancers, thus setting the ground for future therapeutic alternatives to PARP inhibitors.
Assuntos
Antineoplásicos , Desoxicitidina Quinase , Humanos , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Nucleotídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteína BRCA2/genéticaRESUMO
Recent studies have described a DNA damage tolerance pathway choice that involves a competition between PrimPol-mediated repriming and fork reversal. Screening different translesion DNA synthesis (TLS) polymerases by the use of tools for their depletion, we identified a unique role of Pol ι in regulating such a pathway choice. Pol ι deficiency unleashes PrimPol-dependent repriming, which accelerates DNA replication in a pathway that is epistatic with ZRANB3 knockdown. In Pol ι-depleted cells, the excess participation of PrimPol in nascent DNA elongation reduces replication stress signals, but thereby also checkpoint activation in S phase, triggering chromosome instability in M phase. This TLS-independent function of Pol ι requires its PCNA-interacting but not its polymerase domain. Our findings unravel an unanticipated role of Pol ι in protecting the genome stability of cells from detrimental changes in DNA replication dynamics caused by PrimPol.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Dano ao DNA , Instabilidade Cromossômica , DNA Primase/genética , DNA Primase/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismoRESUMO
The trapping of Poly-ADP-ribose polymerase (PARP) on DNA caused by PARP inhibitors (PARPi) triggers acute DNA replication stress and synthetic lethality (SL) in BRCA2-deficient cells. Hence, DNA damage is accepted as a prerequisite for SL in BRCA2-deficient cells. In contrast, here we show that inhibiting ROCK in BRCA2-deficient cells triggers SL independently from acute replication stress. Such SL is preceded by polyploidy and binucleation resulting from cytokinesis failure. Such initial mitosis abnormalities are followed by other M phase defects, including anaphase bridges and abnormal mitotic figures associated with multipolar spindles, supernumerary centrosomes and multinucleation. SL was also triggered by inhibiting Citron Rho-interacting kinase, another enzyme that, similarly to ROCK, regulates cytokinesis. Together, these observations demonstrate that cytokinesis failure triggers mitotic abnormalities and SL in BRCA2-deficient cells. Furthermore, the prevention of mitotic entry by depletion of Early mitotic inhibitor 1 (EMI1) augmented the survival of BRCA2-deficient cells treated with ROCK inhibitors, thus reinforcing the association between M phase and cell death in BRCA2-deficient cells. This novel SL differs from the one triggered by PARPi and uncovers mitosis as an Achilles heel of BRCA2-deficient cells.
Assuntos
Dano ao DNA , Mutações Sintéticas Letais , Anáfase , Mitose , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Proteína BRCA2/genética , HumanosRESUMO
Chromosomal instability (CIN) refers to an increased rate of acquisition of numerical and structural changes in chromosomes and is considered an enabling characteristic of tumors. Given its role as a facilitator of genomic changes, CIN is increasingly being considered as a possible therapeutic target, raising the question of which variables may convert CIN into an ally instead of an enemy during cancer treatment. This review discusses the origins of structural chromosome abnormalities and the cellular mechanisms that prevent and resolve them, as well as how different CIN phenotypes relate to each other. We discuss the possible fates of cells containing structural CIN, focusing on how a few cell duplication cycles suffice to induce profound CIN-mediated genome alterations. Because such alterations can promote tumor adaptation to treatment, we discuss currently proposed strategies to either avoid CIN or enhance CIN to a level that is no longer compatible with cell survival.
RESUMO
p21Waf/CIP1 is a small unstructured protein that binds and inactivates cyclin-dependent kinases (CDKs). To this end, p21 levels increase following the activation of the p53 tumor suppressor. CDK inhibition by p21 triggers cell-cycle arrest in the G1 and G2 phases of the cell cycle. In the absence of exogenous insults causing replication stress, only residual p21 levels are prevalent that are insufficient to inhibit CDKs. However, research from different laboratories has demonstrated that these residual p21 levels in the S phase control DNA replication speed and origin firing to preserve genomic stability. Such an S-phase function of p21 depends fully on its ability to displace partners from chromatin-bound proliferating cell nuclear antigen (PCNA). Vice versa, PCNA also regulates p21 by preventing its upregulation in the S phase, even in the context of robust p21 induction by irradiation. Such a tight regulation of p21 in the S phase unveils the potential that CDK-independent functions of p21 may have for the improvement of cancer treatments.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Replicação do DNA/genética , Antígeno Nuclear de Célula em Proliferação/genética , Quinases Ciclina-Dependentes/genética , Humanos , Inibidores de Proteínas Quinases/metabolismo , Fase S/genéticaRESUMO
Emerging evidence shows that Rab11 recycling endosomes (REs Rab11) are essential for several neuronal processes, including the proper functioning of growth cones, synapse architecture regulation and neuronal migration. However, several aspects of REs Rab11 remain unclear, such as its sub-cellular distribution across neuronal development, contribution to dendritic tree organization and its consequences in memory formation. In this work we show a spatio-temporal correlation between the endogenous localization of REs Rab11 and developmental stage of neurons. Furthermore, Rab11-suppressed neurons showed an increase on dendritic branching (without altering total dendritic length) and misdistribution of dendritic proteins in cultured neurons. In addition, suppression of Rab11 in adult rat brains in vivo (by expressing shRab11 through lentiviral infection), showed a decrease on both the sensitivity to induce long-term potentiation and hippocampal-dependent memory acquisition. Taken together, our results suggest that REs Rab11 expression is required for a proper dendritic architecture and branching, controlling key aspects of synaptic plasticity and spatial memory formation.
Assuntos
Dendritos/metabolismo , Plasticidade Neuronal , Neurônios/fisiologia , Memória Espacial , Proteínas rab de Ligação ao GTP/genética , Animais , Giro Denteado/fisiologia , Fenômenos Eletrofisiológicos , Feminino , Hipocampo/fisiologia , Potenciação de Longa Duração , Masculino , Gravidez , Ratos , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The elimination of DNA polymerase eta (pol η) causes discontinuous DNA elongation and fork stalling in UV-irradiated cells. Such alterations in DNA replication are followed by S-phase arrest, DNA double-strand break (DSB) accumulation, and cell death. However, their molecular triggers and the relative timing of these events have not been fully elucidated. Here, we report that DSBs accumulate relatively early after UV irradiation in pol η-depleted cells. Despite the availability of repair pathways, DSBs persist and chromosome instability (CIN) is not detectable. Later on cells with pan-nuclear γH2AX and massive exposure of template single-stranded DNA (ssDNA), which indicate severe replication stress, accumulate and such events are followed by cell death. Reinforcing the causal link between the accumulation of pan-nuclear ssDNA/γH2AX signals and cell death, downregulation of RPA increased both replication stress and the cell death of pol η-deficient cells. Remarkably, DSBs, pan-nuclear ssDNA/γH2AX, S-phase arrest, and cell death are all attenuated by MRE11 nuclease knockdown. Such results suggest that unscheduled MRE11-dependent activities at replicating DNA selectively trigger cell death, but not CIN. Together these results show that pol η-depletion promotes a type of cell death that may be attractive as a therapeutic tool because of the lack of CIN.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA Polimerase Dirigida por DNA/genética , Histonas/genética , Proteína Homóloga a MRE11/genética , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Morte Celular/genética , Instabilidade Cromossômica/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , DNA de Cadeia Simples/efeitos da radiação , Humanos , Fase S/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
The development and maintenance of multicellular organisms require specialized coordination between external cellular signals and the proteins receiving stimuli and regulating responses. A critical role in the proper functioning of these processes is played by endosomal trafficking, which enables the transport of proteins to targeted sites as well as their return to the plasma membrane through its essential components, the endosomes. During this trafficking, signaling pathways controlling functions related to the endosomal system are activated both directly and indirectly. Although there are a considerable number of molecules participating in these processes, some are more known than others for their specific functions. Toward the end of the 1990s, Smad anchor for receptor activation (SARA) protein was described to be controlling and to facilitate the localization of Smads to transforming growth factor ß (TGF-ß) receptors during TGF-ß signaling activation, and, strikingly, SARA was also identified to be one of the proteins that bind to early endosomes (EEs) participating in membrane trafficking in several cell models. The purpose of this review is to analyze the state of the art of the contribution of SARA in different cell types and cellular contexts, focusing on the biological role of SARA in two main processes, trafficking and cellular signaling, both of which are necessary for intercellular coordination, communication, and development.
Assuntos
Endossomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transporte Proteico/fisiologia , Animais , Humanos , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
SARA (Smad Anchor for Receptor Activation) plays a crucial role in Rab5-mediated endocytosis in cell lines localizing to early endosomes where it regulates morphology and function. Here, we analyzed the role of SARA during neuronal development and tested whether it functions as a regulator of endocytic trafficking of selected axonal and membrane proteins. Suppression of SARA perturbs the appearance of juxtanuclear endocytic recycling compartments and the neurons show long axons with large growth cones. Furthermore, surface distribution of the cell adhesion molecule L1 in axons and the fusion of vesicles containing transferring receptor (TfR) in dendrites were increased in neurons where SARA was silenced. Conversely, SARA overexpression generated large early endosomes and reduced neurite outgrowth. Taken together, our findings suggest a significant contribution of SARA to key aspects of neuronal development, including neurite formation.