RESUMO
Despite the widespread deregulation of CDK4/6 activity in non-small cell lung cancer (NSCLC), the clinical trials with CDK4/6 inhibitors (CDK4/6is) as a monotherapy have shown poor antitumor activity. However, our preclinical studies have revealed a significant potential for CDK4/6is to collaborate by influencing DNA damage repair pathways during radiotherapy. Given the considerable upregulation of PARP1 expression in NSCLC, we analyzed the efficacy of combined PARP and CDK4/6 inhibition in NSCLC models. Our findings demonstrate that CDK4/6is synergize with PARP inhibitors (PARPis) to inhibit the clonogenic growth of RB-proficient NSCLC models. This synergy is associated with increased accumulation of DNA damage, interrupted cell-cycle checkpoints, and enhanced apoptotic cell death. We showed that CDK4/6is mechanically promote PARP1 protein degradation, leading to decreased availability of DNA repair factors involved in homologous recombination and suppression of DNA repair competency. Furthermore, we showed that PARP trapping is required for this synergy. We then confirmed that combining PARPi and CDK4/6i blocked the growth of NSCLC xenografts in vivo and patient-derived explant models ex vivo. These findings reveal a previously uncharacterized impact of CDK4/6i on PARP1 levels in RB-proficient NSCLC models and the requirement of PARP trapping to render synergy between CDK4/6i and PARPi. Our research suggests that combining CDK4/6i with PARPi could be a promising therapeutic strategy for patients with RB-proficient NSCLC, potentially opening up new and more effective avenues for treatment.
RESUMO
Triple-negative breast cancer (TNBC) has a poor clinical outcome, due to a lack of actionable therapeutic targets. Herein we define lysosomal acid lipase A (LIPA) as a viable molecular target in TNBC and identify a stereospecific small molecule (ERX-41) that binds LIPA. ERX-41 induces endoplasmic reticulum (ER) stress resulting in cell death, and this effect is on target as evidenced by specific LIPA mutations providing resistance. Importantly, we demonstrate that ERX-41 activity is independent of LIPA lipase function but dependent on its ER localization. Mechanistically, ERX-41 binding of LIPA decreases expression of multiple ER-resident proteins involved in protein folding. This targeted vulnerability has a large therapeutic window, with no adverse effects either on normal mammary epithelial cells or in mice. Our study implicates a targeted strategy for solid tumors, including breast, brain, pancreatic and ovarian, whereby small, orally bioavailable molecules targeting LIPA block protein folding, induce ER stress and result in tumor cell death.
Assuntos
Estresse do Retículo Endoplasmático , Neoplasias de Mama Triplo Negativas , Animais , Humanos , Lipase/química , Camundongos , Dobramento de Proteína , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails extensive epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer, although they can lead to muscular atrophy. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC and muscular atrophy.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Peptídeos/metabolismo , Receptores Androgênicos/genética , Androgênios , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Expressão Gênica/genética , Glutamina/metabolismo , Humanos , Masculino , Peptídeos/genética , Neoplasias da Próstata/genética , Neoplasias de Próstata Resistentes à Castração/genética , Domínios Proteicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/genética , Ativação Transcricional/genéticaRESUMO
Expression of the androgen receptor splice variant 7 (AR-V7) is frequently detected in castrate resistant prostate cancer and associated with resistance to AR-targeted therapies. While we have previously noted that homodimerization is required for the transcriptional activity of AR-V7 and that AR-V7 can also form heterodimers with the full-length AR (AR-FL), there are still many gaps of knowledge in AR-V7 stepwise activation. In the present study, we show that neither AR-V7 homodimerization nor AR-V7/AR-FL heterodimerization requires cofactors or DNA binding. AR-V7 can enter the nucleus as a monomer and drive a transcriptional program and DNA-damage repair as a homodimer. While forming a heterodimer with AR-FL to induce nuclear localization of unliganded AR-FL, AR-V7 does not need to interact with AR-FL to drive gene transcription or DNA-damage repair in prostate cancer cells that co-express AR-V7 and AR-FL. These data indicate that AR-V7 can function independently of its interaction with AR-FL in the true castrate state or "absence of ligand", providing support for the utility of targeting AR-V7 in improving outcomes of patients with castrate resistant prostate cancer.
Assuntos
Processamento Alternativo/genética , Neoplasias da Próstata/genética , Isoformas de Proteínas/genética , Receptores Androgênicos/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Próstata/patologia , Neoplasias da Próstata/patologiaRESUMO
The study of DNA damage repair response (DDR) in prostate cancer is restricted by the limited number of prostate cancer cell lines and lack of surrogates for heterogeneity in clinical samples. Here, we sought to leverage our experience with patient derived explants (PDEs) cultured ex vivo to study dynamics of DDR in primary tumors following application of clinically relevant doses of ionizing radiation (IR) to tumor cells in their native 3-dimensional microenvironment. We compared DDR dynamics between prostate cancer cell lines, PDEs and xenograft derived explants (XDEs) following treatment with IR (2Gy) either alone or in combination with pharmacological modulators of DDR. We have shown that following treatment with 2Gy, DDR can be consistently detected in PDEs from multiple solid tumors, including prostate, kidney, testes, lung and breast, as evidenced by γ-H2AX, 53BP1, phospho-ATM and phospho-DNA-PKcs foci. By examining kinetics of resolution of IR-induced foci, we have shown that DDR in prostate PDEs (complete resolution in 8â¯h) is much faster than in prostate cancer cell lines (<50% resolution in 8â¯h). The transcriptional profile of DDR genes following 2Gy IR appears to be distinct between PDEs and cell lines. Pre-treatment with drugs targeting DDR pathways differentially alter the kinetics of DDR in the PDEs and cell lines, as evidenced by altered kinetics of foci resolution. This study highlights the utility of PDEs as a robust model system for short-term evaluation of DDR in primary solid tumors in clinically relevant microenvironment.
RESUMO
Alternative splicing is emerging as an oncogenic mechanism. In prostate cancer, generation of constitutively active forms of androgen receptor (AR) variants including AR-V7 plays an important role in progression of castration-resistant prostate cancer (CRPC). AR-V7 is generated by alternative splicing that results in inclusion of cryptic exon CE3 and translation of truncated AR protein that lacks the ligand binding domain. Whether AR-V7 can be a driver for CRPC remains controversial as the oncogenic mechanism of AR-V7 activation remains elusive. Here, we found that KDM4B promotes AR-V7 and identified a novel regulatory mechanism. KDM4B is phosphorylated by protein kinase A under conditions that promote castration-resistance, eliciting its binding to the splicing factor SF3B3. KDM4B binds RNA specifically near the 5'-CE3, upregulates the chromatin accessibility, and couples the spliceosome to the chromatin. Our data suggest that KDM4B can function as a signal responsive trans-acting splicing factor and scaffold that recruits and stabilizes the spliceosome near the alternative exon, thus promoting its inclusion. Genome-wide profiling of KDM4B-regulated genes also identified additional alternative splicing events implicated in tumorigenesis. Our study defines KDM4B-regulated alternative splicing as a pivotal mechanism for generating AR-V7 and a contributing factor for CRPC, providing insight for mechanistic targeting of CRPC.
Assuntos
Processamento Alternativo/genética , Regulação Neoplásica da Expressão Gênica/genética , Histona Desmetilases com o Domínio Jumonji/genética , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Células HEK293 , Humanos , Masculino , Isoformas de Proteínas/genética , Receptores Androgênicos/metabolismo , Spliceossomos/genéticaRESUMO
Rapid and efficient synaptic vesicle fusion requires a pool of primed vesicles, the nearby tethering of Ca2+ channels, and the presence of the phospholipid PIP2 in the target membrane. Although the presynaptic active zone mediates the first two requirements, it is unclear how fusion is targeted to membranes with high PIP2 content. Here we find that the C2B domain of the active zone scaffold RIM is critical for action potential-triggered fusion. Remarkably, the known RIM functions in vesicle priming and Ca2+ influx do not require RIM C2B domains. Instead, biophysical experiments reveal that RIM C2 domains, which lack Ca2+ binding, specifically bind to PIP2. Mutational analyses establish that PIP2 binding to RIM C2B and its tethering to the other RIM domains are crucial for efficient exocytosis. We propose that RIM C2B domains are constitutive PIP2-binding modules that couple mechanisms for vesicle priming and Ca2+ channel tethering to PIP2-containing target membranes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Fosfatidilinositol 4,5-Difosfato/genética , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologiaRESUMO
Exocytosis of mammalian sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizing the exocytotic response of streptolysin O-permeabilized human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranylgeranylated, and active Rab3A elicited human sperm exocytosis per se. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino-terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.
Assuntos
Membrana Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Exocitose/fisiologia , Guanosina Trifosfato/metabolismo , Espermatozoides/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismo , Acrossomo/metabolismo , Adulto , Sequência de Aminoácidos , Cálcio/metabolismo , Imunofluorescência , Humanos , Masculino , Fusão de Membrana/fisiologia , Dados de Sequência Molecular , Receptores Androgênicos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Adulto Jovem , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. In general, exocytosis is initiated by a cytosolic calcium increase. In this report we show that calcium affects several factors during human sperm acrosomal exocytosis. By using an antibody that specifically recognizes synaptotagmin VI phosphorylated at the polybasic region of the C2B domain, we showed that a calcium-dependent dephosphorylation of this protein occurred at early stages of the acrosomal exocytosis in streptolysin O-permeabilized sperm. We identified the phosphatase as calcineurin and showed that the activity of this enzyme is absolutely required during the early steps of the secretory process. When added to sperm, an inhibitor-insensitive, catalytically active domain of calcineurin was able to rescue the effect of the specific calcineurin inhibitor cyclosporin A. This same domain dephosphorylated recombinant synaptotagmin VI C2B domain, validating this protein as a new substrate for calcineurin. When sperm were treated with catalytically active calcineurin before stimulation, exocytosis was inhibited, an effect that was rescued by the phosphomimetic synaptotagmin VI C2B-T418E,T419E mutant domain. These observations indicate that synaptotagmin must be dephosphorylated at a specific window of time and suggest that phosphorylated synaptotagmin has an active role at early stages of the acrosomal exocytosis.
Assuntos
Acrossomo/fisiologia , Calcineurina/metabolismo , Exocitose , Sinaptotagminas/metabolismo , Humanos , Masculino , Mutação , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Espermatozoides/metabolismo , Sinaptotagminas/genética , Fatores de TempoRESUMO
The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.
Assuntos
Exocitose/fisiologia , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas SNARE/metabolismo , Espermatozoides/fisiologia , Reação Acrossômica/fisiologia , Animais , Cálcio/metabolismo , Humanos , Masculino , Proteínas Sensíveis a N-Etilmaleimida/genética , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas SNARE/genética , Espermatozoides/citologia , Tirosina/metabolismo , Proteína rab3A de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/metabolismoRESUMO
Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. Regulated secretion requires SNARE proteins and, in neurons, also synaptotagmin I and complexin. Recent reports suggest that complexin imposes a fusion block that is released by Ca(2+) and synaptotagmin I. However, no direct evidence for this model in secreting cells has been provided and whether this complexin/synaptotagmin interplay functions in other types of secretion is unknown. In this report, we show that the C2B domain of synaptotagmin VI and an anti-complexin antibody blocked the formation of trans SNARE complexes in permeabilized human sperm, and that this effect was reversed by adding complexin. In contrast, an excess of complexin stopped exocytosis at a later step, when SNAREs were assembled in loose trans complexes. Interestingly, this blockage was released by the addition of the synaptotagmin VI C2B domain in the presence of Ca(2+). We have previously demonstrated that the activity of this domain is regulated by protein kinase C-mediated phosphorylation. Here, we show that a phosphomimetic mutation in the polybasic region of the C2B domain strongly affects its Ca(2+) and phospholipids binding properties. Importantly, this mutation completely abrogates its ability to rescue the complexin block. Our results show that the functional interplay between complexin and synaptotagmin has a central role in a physiological secretion event, and that this interplay can be modulated by phosphorylation of the C2B domain.
Assuntos
Acrossomo/metabolismo , Exocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Sinaptotagmina I/metabolismo , Sinaptotagminas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Fertilização/fisiologia , Humanos , Masculino , Proteínas do Tecido Nervoso/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sinaptotagmina I/farmacologia , Sinaptotagminas/farmacologiaRESUMO
The dynamics of SNARE assembly and disassembly during membrane recognition and fusion is a central issue in intracellular trafficking and regulated secretion. Exocytosis of sperm's single vesicle--the acrosome--is a synchronized, all-or-nothing process that happens only once in the life of the cell and depends on activation of both the GTP-binding protein Rab3 and of neurotoxin-sensitive SNAREs. These characteristics make acrosomal exocytosis a unique mammalian model for the study of the different phases of the membrane fusion cascade. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca2+ chelator we show that, in unactivated sperm, SNAREs are locked in heterotrimeric cis complexes. Upon Ca2+ entry into the cytoplasm, Rab3 is activated and triggers NSF/alpha-SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose trans complexes that are resistant to NSF/alpha-SNAP and differentially sensitive to cleavage by two vesicle-associated membrane protein (VAMP)-specific neurotoxins. Ca2+ must be released from inside the acrosome to trigger the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis.
Assuntos
Acrossomo/fisiologia , Cálcio/farmacologia , Exocitose/fisiologia , Proteínas SNARE/metabolismo , Espermatozoides/metabolismo , Proteína rab3A de Ligação ao GTP/fisiologia , Reação Acrossômica/fisiologia , Toxinas Botulínicas/farmacologia , Toxinas Botulínicas Tipo A , Calcimicina/farmacologia , Cálcio/metabolismo , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Etilenodiaminas/farmacologia , Imunofluorescência , Humanos , Masculino , Permeabilidade/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Espermatozoides/efeitos dos fármacos , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagminas/fisiologia , Toxina Tetânica/farmacologia , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
We have previously reported that synaptotagmin VI is present in human sperm cells and that a recombinant protein containing the C2A and C2B domains abrogates acrosomal exocytosis in permeabilized spermatozoa, an effect that was regulated by phosphorylation. In this report, we show that each individual C2 domain blocks acrosomal exocytosis. The inhibitory effect was completely abrogated by phosphorylation of the domains with purified PKCbetaII. We found by site-directed mutagenesis that Thr418 and/or Thr419 in the polybasic region (KKKTTIK) of the C2B domain--a key region for the function of synaptotagmins--are the PKC target that regulates its inhibitory effect on acrosomal exocytosis. Similarly, we showed that Thr284 in the polybasic region of C2A (KCKLQTR) is the target for PKC-mediated phosphorylation in this domain. An antibody that specifically binds to the phosphorylated polybasic region of the C2B domain recognized endogenous phosphorylated synaptotagmin in the sperm acrosomal region. The antibody was inhibitory only at early stages of exocytosis in sperm acrosome reaction assays, and the immunolabeling decreased upon sperm stimulation, indicating that the protein is dephosphorylated during acrosomal exocytosis. Our results indicate that acrosomal exocytosis is regulated through the PKC-mediated phosphorylation of conserved threonines in the polybasic regions of synaptotagmin VI.