RESUMO
The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Proteínas Serina-Treonina Quinases , Camundongos , Humanos , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citocinas , Inflamação/tratamento farmacológico , Isoformas de Proteínas , Anti-Inflamatórios/farmacologia , Imunidade Inata , Fatores de TranscriçãoRESUMO
Although recent efforts have led to the development of highly effective androgen receptor (AR)-directed therapies for the treatment of advanced prostate cancer, a significant subset of patients will progress with resistant disease including AR-negative tumors that display neuroendocrine features [neuroendocrine prostate cancer (NEPC)]. On the basis of RNA sequencing (RNA-seq) data from a clinical cohort of tissue from benign prostate, locally advanced prostate cancer, metastatic castration-resistant prostate cancer and NEPC, we developed a multi-step bioinformatics pipeline to identify NEPC-specific, overexpressed gene transcripts that encode cell surface proteins. This included the identification of known NEPC surface protein CEACAM5 as well as other potentially targetable proteins (e.g., HMMR and CESLR3). We further showed that cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) knockdown results in reduced NEPC tumor cell proliferation and migration in vitro. We provide in vivo data including laser capture microdissection followed by RNA-seq data supporting a causal role of CELSR3 in the development and/or maintenance of the phenotype associated with NEPC. Finally, we provide initial data that suggests CELSR3 is a target for T-cell redirection therapeutics. Further work is now needed to fully evaluate the utility of targeting CELSR3 with T-cell redirection or other similar therapeutics as a potential new strategy for patients with NEPC. Significance: The development of effective treatment for patients with NEPC remains an unmet clinical need. We have identified specific surface proteins, including CELSR3, that may serve as novel biomarkers or therapeutic targets for NEPC.
Assuntos
Tumores Neuroendócrinos , Neoplasias da Próstata , Humanos , Masculino , Neoplasias da Próstata/genética , Tumores Neuroendócrinos/genética , Próstata/metabolismo , Membrana Celular/metabolismo , Caderinas/genéticaRESUMO
New A-ring modified betulin and dihydrobetulin derivatives possessing the 2-cyano-1-en-3-one moiety were prepared and tested for cytotoxicity in seven cancer cell lines. The most active agent 9a synthesized in this account was further demonstrated to induce apoptosis and to activate caspases in malignant melanoma cells.
Assuntos
Inibidores do Crescimento/síntese química , Inibidores do Crescimento/toxicidade , Triterpenos/síntese química , Triterpenos/toxicidade , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Triterpenos Pentacíclicos , Ácido BetulínicoRESUMO
The serine/threonine kinase Akt inhibits mitochondrial cytochrome c release and apoptosis induced by a variety of proapoptotic stimuli. The antiapoptotic activity of Akt is coupled, at least in part, to its effects on cellular metabolism. Here, we provide genetic evidence that Akt is required to maintain hexokinase association with mitochondria. Targeted disruption of this association impairs the ability of growth factors and Akt to inhibit cytochrome c release and apoptosis. Targeted disruption of mitochondria-hexokinase (HK) interaction or exposure to proapoptotic stimuli that promote rapid dissociation of hexokinase from mitochondria potently induce cytochrome c release and apoptosis, even in the absence of Bax and Bak. These effects are inhibited by activated Akt, but not by Bcl-2, implying that changes in outer mitochondrial membrane (OMM) permeability leading to apoptosis can occur in the absence of Bax and Bak and that Akt inhibits these changes through maintenance of hexokinase association with mitochondria.
Assuntos
Apoptose , Hexoquinase/química , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Ligação Competitiva , Linhagem Celular , Proliferação de Células , Células Cultivadas , Clotrimazol/farmacologia , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Técnicas de Transferência de Genes , Inibidores do Crescimento/farmacologia , Substâncias de Crescimento/metabolismo , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Membranas Intracelulares/metabolismo , Potenciais da Membrana , Camundongos , Microscopia de Fluorescência , Fosfocreatina/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Tapsigargina/farmacologia , Fatores de Tempo , Raios UltravioletaRESUMO
The serine/threonine kinase Akt/protein kinase B inhibits apoptosis induced by a variety of stimuli, including overexpression or activation of proapoptotic Bcl-2 family members. The precise mechanisms by which Akt prevents apoptosis are not completely understood, but Akt may function to maintain mitochondrial integrity, thereby preventing cytochrome c release following an apoptotic insult. This effect may be mediated, in part, via promotion of physical and functional interactions between mitochondria and hexokinases. Here we show that growth factor deprivation induced proteolytic cleavage of the proapoptotic Bcl-2 family member BID to yield its active truncated form, tBID. Activated Akt inhibited mitochondrial cytochrome c release and apoptosis following BID cleavage. Akt also antagonized tBID-mediated BAX activation and mitochondrial BAK oligomerization, two downstream events thought to be critical for tBID-induced apoptosis. Glucose deprivation, which impaired the ability of Akt to maintain mitochondrion-hexokinase association, prevented Akt from inhibiting BID-mediated apoptosis. Interestingly, tBID independently elicited dissociation of hexokinases from mitochondria, an effect that was antagonized by activated Akt. Ectopic expression of the amino-terminal half of hexokinase II, which is catalytically active and contains the mitochondrion-binding domain, consistently antagonized tBID-induced apoptosis. These results suggest that Akt inhibits BID-mediated apoptosis downstream of BID cleavage via promotion of mitochondrial hexokinase association and antagonism of tBID-mediated BAX and BAK activation at the mitochondria.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/genética , Linhagem Celular , Meios de Cultura , Ativação Enzimática , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2RESUMO
In B lymphocytes, induction of apoptosis or programmed cell death (PCD) by Fas (CD95/APO-1) is suppressed by the triggering of CD40. This suppression controls various aspects of the humoral immune response, including antibody affinity maturation. The opposing effects of these receptors are also crucial to B-cell homeostasis, autoimmune disease, and cancer. Cytoprotection by CD40 involves activation of protective genes mediated by NF-kappa B transcription factors; however, its basis remains poorly understood. Here, we report that, in B cells, Gadd45 beta is induced by CD40 through a mechanism that requires NF-kappa B and that this induction suppresses Fas-mediated killing. Importantly, up-regulation of Gadd45 beta by CD40 precedes Fas-induced caspase activation, as well as up-regulation of other NF-kappa B-controlled inhibitors of apoptosis such as Bcl-xL and c-FLIPL. In the presence of Gadd45 beta, the Fas-induced apoptotic cascade is halted at mitochondria. However, in contrast to Bcl-xL, Gadd45 beta is unable to hamper the "intrinsic" pathway for apoptosis and in fact appears to block Fas cytotoxicity herein by suppressing a mitochondria-targeting mechanism activated by this receptor. These findings identify Gadd45 beta as a critical mediator of the prosurvival response to CD40 stimulation and provide important new insights into the apoptotic mechanism that is triggered by Fas in B cells.
Assuntos
Antígenos de Diferenciação/fisiologia , Apoptose , Antígenos CD40/fisiologia , Receptor fas/fisiologia , Antígenos de Diferenciação/biossíntese , Linfócitos B , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , NF-kappa B/fisiologia , Transdução de SinaisRESUMO
Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G(2)/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G(2)/M to the G(1) phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G(2) arrest that is alleviated by activated Akt. Furthermore, the transition from the G(2)/M to the G(1) phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G(2)/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G(2)/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.