RESUMO
Malignant transformation involves an orchestrated rearrangement of cell cycle regulation mechanisms that must balance autonomic mitogenic impulses and deleterious oncogenic stress. Human papillomavirus (HPV) infection is highly prevalent in populations around the globe, whereas the incidence of cervical cancer is 0.15%. Since HPV infection primes cervical keratinocytes to undergo malignant transformation, we can assume that the balance between transforming mitogenic signals and oncogenic stress is rarely attained. We showed that highly transforming mitogenic signals triggered by HRasG12V activity in E6E7-HPV-keratinocytes generate strong replication and oxidative stresses. These stresses are counteracted by autophagy induction that buffers the rapid increase of ROS that is the main cause of genotoxic stress promoted by the oncoprotein. As a result, autophagy creates a narrow window of opportunity for malignant keratinocytes to emerge. This work shows that autophagy is crucial to allow the transition of E6E7 keratinocytes from an immortalized to a malignant state caused by HRasG12V.
Assuntos
Alphapapillomavirus/patogenicidade , Autofagia , Transformação Celular Viral , Dano ao DNA , Queratinócitos/virologia , Infecções por Papillomavirus/virologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias do Colo do Útero/virologia , Alphapapillomavirus/genética , Alphapapillomavirus/metabolismo , Linhagem Celular , Proliferação de Células , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular , Interações Hospedeiro-Patógeno , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Mitose , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Estresse Oxidativo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Malignant transformation involves an orchestrated rearrangement of cell cycle regulation mechanisms that must balance autonomic mitogenic impulses and deleterious oncogenic stress. Human papillomavirus (HPV) infection is highly prevalent in populations around the globe, whereas the incidence of cervical cancer is 0.15%. Since HPV infection primes cervical keratinocytes to undergo malignant transformation, we can assume that the balance between transforming mitogenic signals and oncogenic stress is rarely attained. We showed that highly transforming mitogenic signals triggered by HRasG12V activity in E6E7–HPV–keratinocytes generate strong replication and oxidative stresses. These stresses are counteracted by autophagy induction that buffers the rapid increase of ROS that is the main cause of genotoxic stress promoted by the oncoprotein. As a result, autophagy creates a narrow window of opportunity for malignant keratinocytes to emerge. This work shows that autophagy is crucial to allow the transition of E6E7 keratinocytes from an immortalized to a malignant state caused by HRasG12V.
RESUMO
In malignant transformation, cellular stress-response pathways are dynamically mobilized to counterbalance oncogenic activity, keeping cancer cells viable. Therapeutic disruption of this vulnerable homeostasis might change the outcome of many human cancers, particularly those for which no effective therapy is available. Here, we report the use of fibroblast growth factor 2 (FGF2) to demonstrate that further mitogenic activation disrupts cellular homeostasis and strongly sensitizes cancer cells to stress-targeted therapeutic inhibitors. We show that FGF2 enhanced replication and proteotoxic stresses in a K-Ras-driven murine cancer cell model, and combinations of FGF2 and proteasome or DNA damage response-checkpoint inhibitors triggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressed the malignant phenotype and prevented these synergic toxicities in these murine cells. Moreover, in a panel of human Ewing's sarcoma family tumor cells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE-821 (ATR inhibitor) induced cell death when combined with FGF2. Sustained MAPK-ERK1/2 overactivation induced by FGF2 appears to underlie these synthetic lethalities, as late pharmacological inhibition of this pathway restored cell homeostasis and prevented these described synergies. Our results highlight how mitotic signaling pathways which are frequently overridden in malignant transformation might be exploited to disrupt the robustness of cancer cells, ultimately sensitizing them to stress-targeted therapies. This approach provides a new therapeutic rationale for human cancers, with important implications for tumors still lacking effective treatment, and for those that frequently relapse after treatment with available therapies.
Assuntos
Antineoplásicos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Estresse Fisiológico , Animais , Bortezomib/farmacologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Inibidores de Proteassoma/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Protein disulfide isomerases (PDIs) support endoplasmic reticulum redox protein folding and cell-surface thiol-redox control of thrombosis and vascular remodeling. The family prototype PDIA1 regulates NADPH oxidase signaling and cytoskeleton organization, however the related underlying mechanisms are unclear. Here we show that genes encoding human PDIA1 and its two paralogs PDIA8 and PDIA2 are each flanked by genes encoding Rho guanine-dissociation inhibitors (GDI), known regulators of RhoGTPases/cytoskeleton. Evolutionary histories of these three microsyntenic regions reveal their emergence by two successive duplication events of a primordial gene pair in the last common vertebrate ancestor. The arrangement, however, is substantially older, detectable in echinoderms, nematodes, and cnidarians. Thus, PDI/RhoGDI pairing in the same transcription orientation emerged early in animal evolution and has been largely maintained. PDI/RhoGDI pairs are embedded into conserved genomic regions displaying common cis-regulatory elements. Analysis of gene expression datasets supports evidence for PDI/RhoGDI coexpression in developmental/inflammatory contexts. PDIA1/RhoGDIα were co-induced in endothelial cells upon CRISP-R-promoted transcription activation of each pair component, and also in mouse arterial intima during flow-induced remodeling. We provide evidence for physical interaction between both proteins. These data support strong functional links between PDI and RhoGDI families, which likely maintained PDI/RhoGDI microsynteny along > 800-million years of evolution.
Assuntos
Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Sintenia , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/genética , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Animais , Sequência de Bases , Sequência Conservada , Citoesqueleto/metabolismo , Evolução Molecular , Genômica , Humanos , Filogenia , Regiões Promotoras Genéticas/genética , Ligação ProteicaRESUMO
Phospholipases A2 (PLA2s) overexpression is closely associated with the malignant potential of breast cancers. Here, we showed for the first the antitumoral effects of γCdcPLI, a PLA2 inhibitor from Crotalus durissus collilineatus via PI3K/Akt pathway on MDA-MB-231 cell. Firstly, γCdcPLI was more cytotoxic to MDA-MB-231 breast cancer cells than other cell lines (MCF-7, HeLa, PC3 and A549) and did not affect the viability of non-tumorigenic breast cell (MCF 10A). In addition, γCdcPLI induced modulation of important mediators of apoptosis pathways such as p53, MAPK-ERK, BIRC5 and MDM2. γCdcPLI decreased MDA-MB-231 adhesion, migration and invasion. Interestingly, the γCdcPLI also inhibited the adhesion and migration of endothelial cells and blocked angiogenesis by inhibiting tube formation by HUVECs in vitro and sprouting elongation on aortic ring assay ex vivo. Furthermore, γCdcPLI reduced the production of vascular endothelial growth factor (VEGF). γCdcPLI was also able to decrease PGE2 levels in MDA-MB-231 and inhibited gene and protein expression of the PI3K/Akt pathway. In conclusion, γCdcPLI showed in vitro antitumoral, antimestatatic and anti-angiogenic potential effects and could be an attractive approach for futures studies in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama , Lipoproteínas/farmacologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfolipase A2/farmacologia , Antineoplásicos/isolamento & purificação , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Venenos de Crotalídeos/química , Células Endoteliais/efeitos dos fármacos , Humanos , Lipoproteínas/isolamento & purificação , Modelos Biológicos , Neovascularização Patológica , Inibidores de Fosfolipase A2/isolamento & purificaçãoRESUMO
We present in this article a methodology for designing kinetic models of molecular signaling networks, which was exemplarily applied for modeling one of the Ras/MAPK signaling pathways in the mouse Y1 adrenocortical cell line. The methodology is interdisciplinary, that is, it was developed in a way that both dry and wet lab teams worked together along the whole modeling process.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Transdução de Sinais , Proteínas ras/metabolismo , Algoritmos , Animais , Biomarcadores , Linhagem Celular , Biologia Computacional/métodos , Ensaio de Imunoadsorção Enzimática , Cinética , Camundongos , Fosforilação , Reprodutibilidade dos TestesRESUMO
Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization. This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions.
Assuntos
Células Endoteliais da Veia Umbilical Humana/enzimologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Fenômenos Biomecânicos , Células Cultivadas , Complexo de Golgi/enzimologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina alfa5/metabolismo , Mecanotransdução Celular , Oxirredução , Transporte ProteicoRESUMO
Proteomics has emerged as an invaluable tool in the quest to unravel the biochemical changes that give rise to the hallmarks of cancer. In this review, we present the advances and challenges facing proteomics technology as applied to cancer research, and address how the information gathered so far has helped to enhance understanding of the mechanisms underlying the disease and contributed to the discovery of biomarkers and new drug targets. We conclude by presenting a perspective on how proteomics could be applied in the future to determine prognostic biomarkers and direct strategies for effective cancer treatment.
Assuntos
Descoberta de Drogas , Neoplasias/metabolismo , Proteômica , Animais , Biomarcadores Tumorais/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Mapeamento de Interação de ProteínasRESUMO
We recently reported that paracrine Fibroblast Growth Factor 2 (FGF2) triggers senescence in Ras-driven Y1 and 3T3(Ras) mouse malignant cell lines. Here, we show that although FGF2 activates mitogenic pathways in these Ras-dependent malignant cells, it can block cell proliferation and cause a G2/M arrest. These cytostatic effects of FGF2 are inhibited by PD173074, an FGF receptor (FGFR) inhibitor. To determine which downstream pathways are induced by FGF2, we tested specific inhibitors targeting mitogen-activated protein kinase (MEK), phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC). We show that these classical mitogenic pathways do not mediate the cytostatic activity of FGF2. On the other hand, the inhibition of Src family kinases rescued Ras-dependent malignant cells from the G2/M irreversible arrest induced by FGF2. Taken together, these data indicate a growth factor-sensitive point in G2/M that likely involves FGFR/Ras/Src pathway activation in a MEK, PI3K and PKC independent manner.