RESUMEN
CAR-T cell therapy offers a promising way for prolonged cancer remission, specifically in the case of blood cancers. However, its application in the treatment of solid tumors still faces many limitations. This review paper provides a comprehensive overview of the challenges and strategies associated with CAR-T cell therapy for solid tumors, with a focus on gynecological cancer. This study discusses the limitations of CAR-T therapy for solid tumor treatment, such as T cell exhaustion, stromal barrier, and antigen shedding. Additionally, it addresses possible approaches to increase CAR-T efficacy in solid tumors, including combination therapies with checkpoint inhibitors and chemotherapy, as well as the novel approach of combining CAR-T with oncolytic virotherapy. Given the lack of comprehensive research on CAR-T combination therapies for treating gynecological cancers, this review aims to provide insights into the current landscape of combination therapies for solid tumors and highlight the potential of such an approach in gynecology.
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Neoplasias de los Genitales Femeninos , Inmunoterapia Adoptiva , Viroterapia Oncolítica , Humanos , Femenino , Neoplasias de los Genitales Femeninos/terapia , Inmunoterapia Adoptiva/métodos , Terapia Combinada/métodos , Viroterapia Oncolítica/métodos , Receptores Quiméricos de Antígenos/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfocitos T/inmunologíaRESUMEN
Extensive application of technologies like phage display in screening peptide and protein combinatorial libraries has not only facilitated creation of new recombinant antibodies but has also significantly enriched repertoire of the protein binders that have polypeptide scaffolds without homology to immunoglobulins. These innovative synthetic binding protein (SBP) platforms have grown in number and now encompass monobodies/adnectins, DARPins, lipocalins/anticalins, and a variety of miniproteins such as affibodies and knottins, among others. They serve as versatile modules for developing complex affinity tools that hold promise in both diagnostic and therapeutic settings. An optimal scaffold typically has low molecular weight, minimal immunogenicity, and demonstrates resistance against various challenging conditions, including proteolysis - making it potentially suitable for peroral administration. Retaining functionality under reducing intracellular milieu is also advantageous. However, paramount to its functionality is the scaffold's ability to tolerate mutations across numerous positions, allowing for the formation of a sufficiently large target binding region. This is achieved through the library construction, screening, and subsequent expression in an appropriate system. Scaffolds that exhibit high thermodynamic stability are especially coveted by the developers of new SBPs. These are steadily making their way into clinical settings, notably as antagonists of oncoproteins in signaling pathways. This review surveys the diverse landscape of SBPs, placing particular emphasis on the inhibitors targeting the oncoprotein KRAS, and highlights groundbreaking opportunities for SBPs in oncology.
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Lipocalinas , Péptidos , Péptidos/química , Proteínas Recombinantes/química , Lipocalinas/química , Lipocalinas/uso terapéutico , Clonación Molecular , Biblioteca de Péptidos , Unión ProteicaRESUMEN
Autophagy is a highly conserved process of cellular self-digestion that involves the formation of autophagosomes for the delivery of intracellular components and dysfunctional organelles to lysosomes. This process is induced by different signals including starvation, mitochondrial dysfunction, and DNA damage. The molecular link between autophagy and DNA damage is not well understood yet. Importantly, tumor cells utilize the mechanism of autophagy to cope with genotoxic anti-cancer drug therapy. Another mechanism of drug resistance is provided to cancer cells via the execution of the EMT program. One of the critical transcription factors of EMT is Zeb1. Here we demonstrate that Zeb1 is involved in the regulation of autophagy in several breast cancer cell models. On the molecular level, Zeb1 likely facilitates autophagy through the regulation of autophagic genes, resulting in increased LC3-II levels, augmented staining with Lysotracker, and increased resistance to several genotoxic drugs. The attenuation of Zeb1 expression in TNBC cells led to the opposite effect. Consequently, we propose that Zeb1 augments the resistance of breast cancer cells to genotoxic drugs, at least partially, via autophagy. Collectively, we have uncovered a novel function of Zeb1 in the regulation of autophagy in breast cancer cells.
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Autofagia , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos , Mutágenos/toxicidad , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Autofagia/efectos de los fármacos , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Daño del ADN , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
Autophagy is a special catabolic cellular program that is induced in response to deprivation of nutrients and energy starvation. During the execution of this program, cellular components, including aggregates, as well as damaged organelles and some proteins are encapsulated in special vesicles known as autophagosomes and subsequently are degraded after fusion of autophagosomes with lysosomes. Importantly, at late stages of tumorigenesis cancer cells employ autophagy to sustain proliferation in unfavorable conditions, including anti-cancer drug therapy. E3 ubiquitin ligases play an important role in controlling autophagy. Here we demonstrate that the E3 ligase, a p53-induced RING-H2 protein (Pirh2), is involved in the regulation of autophagy in non-small cell lung cancer cells. Knockdown of Pirh2 decreased the expression of genes involved in all steps of autophagy. Concomitantly, Pirh2 knockdown cell lines exhibited much less of the processed form of LC3 compared to the respective cell lines with normal levels of Pirh2. These results were confirmed by the immune fluorescence microscopy using LC3 antibody and the LysoTracker dye. In agreement with the protective role of autophagy, cells with attenuated expression of Pirh2 were more sensitive to the treatment with doxorubicin. Collectively, we have uncovered a novel function of Pirh2 in the regulation of autophagy in lung cancer cells.
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Autofagia , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Resistencia a Antineoplásicos , Humanos , Neoplasias Pulmonares/patología , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Heat-shock factor 1 (HSF1) is the master transcription factor that regulates the response to proteotoxic stress by controlling the transcription of many stress-responsive genes including the heat-shock proteins. Here, we show a novel molecular mechanism controlling the activation of HSF1. We demonstrate that transglutaminase type 2 (TG2), dependent on its protein disulphide isomerase activity, triggers the trimerization and activation of HSF1 regulating adaptation to stress and proteostasis impairment. In particular, we find that TG2 loss of function correlates with a defect in the nuclear translocation of HSF1 and in its DNA-binding ability to the HSP70 promoter. We show that the inhibition of TG2 restores the unbalance in HSF1-HSP70 pathway in cystic fibrosis (CF), a human disorder characterized by deregulation of proteostasis. The absence of TG2 leads to an increase of about 40% in CFTR function in a new experimental CF mouse model lacking TG2. Altogether, these results indicate that TG2 plays a key role in the regulation of cellular proteostasis under stressful cellular conditions through the modulation of the heat-shock response.
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Fibrosis Quística/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al GTP/genética , Factores de Transcripción del Choque Térmico/genética , Transglutaminasas/genética , Animales , Fibrosis Quística/patología , Regulación de la Expresión Génica , Respuesta al Choque Térmico/genética , Humanos , Ratones , Regiones Promotoras Genéticas , Unión Proteica , Proteína Disulfuro Isomerasas/genética , Proteína Glutamina Gamma Glutamiltransferasa 2 , Procesamiento Proteico-Postraduccional/genética , Proteostasis/genética , Transducción de SeñalRESUMEN
Proteasome-mediated proteolysis is critical for regulation of vast majority of cellular processes. In addition to their well-documented functions in the nucleus and cytoplasm proteasomes have also been found in extracellular space. The origin and functions of these proteasomes, dubbed as circulating/plasmatic or extracellular proteasomes, are unclear. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ-based quantitative proteomics (iTRAQ LC/MS-MS). Our analysis of purified from K562 cells or conditioned medium intact proteasome complexes led to an identification and quantification of 114 proteins, out of which 19 were 26S proteasome proteins (all subunits of the 20S proteasome and a small number of the 19S regulatory particle proteins), and 3 belonged to the ubiquitin system. Sixty-two of proteasome interacting proteins (PIPs) were differentially represented in CP versus EP, with folds difference ranging from 1.5 to 4.8. The bioinformatics analysis revealed that functionally most of EP-PIPs were associated with protein biosynthesis and, unlike CP-PIPs, were under represented by chaperon/ATP-binding proteins. Identities of some of the proteasome proteins and PIPs were verified by Western blotting. Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes. Specifically, the EP prep contained only three 19S subunits versus at least 18 in the CP one, suggesting that the extracellular proteasomes are deficient in 19S complexes, which may imply that they have special biological functions. J. Cell. Physiol. 232: 842-851, 2017. © 2016 Wiley Periodicals, Inc.
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Espacio Extracelular/metabolismo , Marcaje Isotópico/métodos , Subunidades de Proteína/metabolismo , Proteómica/métodos , Medios de Cultivo Condicionados/farmacología , Humanos , Células K562 , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Espectrometría de Masas en TándemRESUMEN
p53 is arguably one of the most important tumor suppressor genes in humans. Due to the paramount importance of p53 in the onset of cell cycle arrest and apoptosis, the p53 gene is found either silenced or mutated in the vast majority of cancers. Furthermore, activated wild-type p53 exhibits a strong bystander effect, thereby activating apoptosis in surrounding cells without being physically present there. For these reasons, p53-targeted therapy that is designed to restore the function of wild-type p53 in cancer cells seems to be a very appealing therapeutic approach. Systemic delivery of p53-coding DNA or RNA using nanoparticles proved to be feasible both in vitro and in vivo. In fact, one p53-based therapeutic (gendicine) is currently approved for commercial use in China. However, the broad use of p53-based therapy in p53-inactivated cancers is severely restricted by its inadequate efficacy. This review highlights the current state-of-the-art in this area of biomedical research and also discusses novel approaches that may help overcome the shortcomings of p53-targeting nanomedicine.
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Antineoplásicos , Nanopartículas , Neoplasias , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Terapia Genética , Neoplasias/patología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
BACKGROUND: ACTN4 is an actin-binding protein involved in many cellular processes, including cancer development. High ACTN4 expression is often associated with a poor prognosis. However, it has been identified as a positive marker for platinum-based adjuvant chemotherapy for non-small cell lung cancer (NSCLC). The goal of our study was to investigate the involvement of ACTN4 in the NSCLC cells' response to the genotoxic drugs. RESULTS: We generated H1299 cells with the ACTN4 gene knock-out (ACTN4 KO), using the CRISPR/Cas9 system. The resistance of the cells to the cisplatin and etoposide was analyzed with the MTT assay. We were also able to estimate the efficiency of DNA repair through the DNA comet assay and gamma-H2AX staining. Possible ACTN4 effects on the non-homologous end joining (NHEJ) and homologous recombination (HR) were investigated using pathway-specific reporter plasmids and through the immunostaining of the key proteins. We found that the H1299 cells with the ACTN4 gene knock-out did not show cisplatin-resistance, but did display a higher resistance to the topoisomerase II inhibitors etoposide and doxorubicin, suggesting that ACTN4 might be somehow involved in the repair of DNA strand breaks. Indeed, the H1299 ACTN4 KO cells repaired etoposide- and doxorubicin-induced DNA breaks more effectively than the control cells. Moreover, the ACTN4 gene knock-out enhanced NHEJ and suppressed HR efficiency. Supporting the data, the depletion of ACTN4 resulted in the faster assembly of the 53BP1 foci with a lower number of the phospho-BRCA1 foci after the etoposide treatment. CONCLUSIONS: Thus, we are the first to demonstrate that ACTN4 may influence the resistance of cancer cells to the topoisomerase II inhibitors, and affect the efficiency of the DNA double strand breaks repair. We hypothesize that ACTN4 interferes with the assembly of the NHEJ and HR complexes, and hence regulates balance between these DNA repair pathways.
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Carcinoma de Pulmón de Células no Pequeñas , Carcinoma , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Inhibidores de Topoisomerasa II , Doxorrubicina , Pulmón , ActininaRESUMEN
Originally discovered by Nielsen in 1991, peptide nucleic acids and other artificial genetic polymers have gained a lot of interest from the scientific community. Due to their unique biophysical features these artificial hybrid polymers are now being employed in various areas of theranostics (therapy and diagnostics). The current review provides an overview of their structure, principles of rational design, and biophysical features as well as highlights the areas of their successful implementation in biology and biomedicine. Finally, the review discusses the areas of improvement that would allow their use as a new class of therapeutics in the future.
RESUMEN
Understanding the mechanisms that regulate cancer progression is pivotal for the development of new therapies. Although p53 is mutated in half of human cancers, its family member p73 is not. At the same time, isoforms of p73 are often overexpressed in cancers and p73 can overtake many p53 functions to kill abnormal cells. According to the latest studies, while p73 represses epithelial-mesenchymal transition and metastasis, it can also promote tumour growth by modulating crosstalk between cancer and immune cells in the tumor microenvironment, M2 macrophage polarisation, Th2 T-cell differentiation, and angiogenesis. Thus, p73 likely plays a dual role as a tumor suppressor by regulating apoptosis in response to genotoxic stress or as an oncoprotein by promoting the immunosuppressive environment and immune cell differentiation.
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Carcinogénesis/genética , Neoplasias/genética , Proteína Tumoral p73/genética , Proteína p53 Supresora de Tumor/genética , Apoptosis/genética , Diferenciación Celular/genética , Transición Epitelial-Mesenquimal/genética , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Metástasis de la Neoplasia , Neoplasias/terapia , Células Th2/metabolismo , Microambiente Tumoral/genéticaRESUMEN
During oncogenesis, cells become unrestrictedly proliferative thereby altering the tissue homeostasis and resulting in subsequent hyperplasia. This process is paralleled by resumption of cell cycle, aberrant DNA repair and blunting the apoptotic program in response to DNA damage. In most human cancers these processes are associated with malfunctioning of tumor suppressor p53. Intriguingly, in some cases two other members of the p53 family of proteins, transcription factors p63 and p73, can compensate for loss of p53. Although both p63 and p73 can bind the same DNA sequences as p53 and their transcriptionally active isoforms are able to regulate the expression of p53-dependent genes, the strongest overlap with p53 functions was detected for p73. Surprisingly, unlike p53, the p73 is rarely lost or mutated in cancers. On the contrary, its inactive isoforms are often overexpressed in cancer. In this review, we discuss several lines of evidence that cancer cells develop various mechanisms to repress p73-mediated cell death. Moreover, p73 isoforms may promote cancer growth by enhancing an anti-oxidative response, the Warburg effect and by repressing senescence. Thus, we speculate that the role of p73 in tumorigenesis can be ambivalent and hence, requires new therapeutic strategies that would specifically repress the oncogenic functions of p73, while keeping its tumor suppressive properties intact.
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Proteínas de Unión al ADN , Proteínas de Unión al ADN/genética , Genes Supresores de Tumor , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Proteína Tumoral p73/genética , Proteína p53 Supresora de Tumor , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Mycoplasmas are the smallest free-living organisms. Reduced sizes of their genomes put constraints on the ability of these bacteria to live autonomously and make them highly dependent on the nutrients produced by host cells. Importantly, at the organism level, mycoplasmal infections may cause pathological changes to the host, including cancer and severe immunological reactions. At the molecular level, mycoplasmas often activate the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) inflammatory response and concomitantly inhibit the p53-mediated response, which normally triggers the cell cycle and apoptosis. Thus, mycoplasmal infections may be considered as cancer-associated factors. At the same time, mycoplasmas through their membrane lipoproteins (LAMPs) along with lipoprotein derivatives (lipopeptide MALP-2, macrophage-activating lipopeptide-2) are able to modulate anti-inflammatory responses via nuclear translocation and activation of the Nrf2 (nuclear factor-E2-related anti-inflammatory transcription factor 2). Thus, interactions between mycoplasmas and host cells are multifaceted and depend on the cellular context. In this review, we summarize the current information on the role of mycoplasmas in affecting the host's intracellular signaling mediated by the interactions between transcriptional factors p53, Nrf2, and NF-κB. A better understanding of the mechanisms underlying pathologic processes associated with reprogramming eukaryotic cells that arise during the mycoplasma-host cell interaction should facilitate the development of new therapeutic approaches to treat oncogenic and inflammatory processes.
RESUMEN
The actin-binding protein ACTN4 belongs to a family of actin-binding proteins and is a non-muscle alpha-actinin that has long been associated with cancer development. Numerous clinical studies showed that changes in ACTN4 gene expression are correlated with aggressiveness, invasion, and metastasis in certain tumors. Amplification of the 19q chromosomal region where the gene is located has also been reported. Experimental manipulations with ACTN4 expression further confirmed its involvement in cell proliferation, motility, and epithelial-mesenchymal transition (EMT). However, both clinical and experimental data suggest that the effects of ACTN4 up- or down-regulation may vary a lot between different types of tumors. Functional studies demonstrated its engagement in a number of cytoplasmic and nuclear processes, ranging from cytoskeleton reorganization to regulation of different signaling pathways. Such a variety of functions may be the reason behind cell type and cell line specific responses. Herein, we will review research progress and controversies regarding the prognostic and functional significance of ACTN4 for tumorigenesis.
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Actinina/genética , Transformación Celular Neoplásica/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias/etiología , Neoplasias/patología , Actinina/metabolismo , Biomarcadores de Tumor , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/metabolismo , Proteoma , Transducción de SeñalRESUMEN
Major tumor suppressor and transcription factor p53 coordinates expression of many genes hence affecting critical cellular functions including cell cycle, senescence, and apoptosis. The NR4A family of orphan receptors (NR4A1-3) belongs to the superfamily of nuclear receptors. They regulate genes involved in proliferation, cell migration, and apoptosis. In this study, we report an identification of NR4A3 as a direct transcriptional target of p53. Using various techniques, we showed that p53 directly bound the promoter of NR4A3 gene and induced its transcription. Functionally, over-expression of NR4A3 attenuated proliferation of cancer cells and promoted apoptosis by augmenting the expression of pro-apoptotic genes, PUMA and Bax. Knockdown of NR4A3 reversed these phenotypes. Importantly, NR4A3 exhibited tumor suppressive functions both in p53-dependent and independent manner. In addition, NR4A3 physically interacted with an anti-apoptotic Bcl-2 protein hence sequestering it from blunting apoptosis. These observations were corroborated by the bioinformatics analysis, which demonstrated a correlation between high levels of NR4A3 expression and better survival of breast and lung cancer patients. Collectively, our studies revealed a novel transcriptional target of p53, NR4A3, which triggers apoptosis and thus likely has a tumor suppressive role in breast and lung cancers.
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Apoptosis , Proteínas de Unión al ADN/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias de la Mama/genética , Proliferación Celular , Transformación Celular Neoplásica , Citocromos c/metabolismo , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Células MCF-7 , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Análisis de Supervivencia , Transcripción GenéticaRESUMEN
Under physiological conditions, a finely tuned system of cellular adaptation allows the intestinal mucosa to maintain the gut barrier function while avoiding excessive immune responses to non-self-antigens from dietary origin or from commensal microbes. This homeostatic function is compromised in cystic fibrosis (CF) due to loss-of-function mutations in the CF transmembrane conductance regulator (CFTR). Recently, we reported that mice bearing defective CFTR are abnormally susceptible to a celiac disease-like enteropathy, in thus far that oral challenge with the gluten derivative gliadin elicits an inflammatory response. However, the mechanisms through which CFTR malfunction drives such an exaggerated response to dietary protein remains elusive. Here we demonstrate that the proteostasis regulator/transglutaminase 2 (TGM2) inhibitor cysteamine restores reduced Beclin 1 (BECN1) protein levels in mice bearing cysteamine-rescuable F508del-CFTR mutant, either in homozygosis or in compound heterozygosis with a null allele, but not in knock-out CFTR mice. When cysteamine restored BECN1 expression, autophagy was increased and gliadin-induced inflammation was reduced. The beneficial effects of cysteamine on F508del-CFTR mice were lost when these mice were backcrossed into a Becn1 haploinsufficient/autophagy-deficient background. Conversely, the transfection-enforced expression of BECN1 in human intestinal epithelial Caco-2 cells mitigated the pro-inflammatory cellular stress response elicited by the gliadin-derived P31-43 peptide. In conclusion, our data provide the proof-of-concept that autophagy stimulation may mitigate the intestinal malfunction of CF patients.
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Autofagia/efectos de los fármacos , Cisteamina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Gliadina/inmunología , Fragmentos de Péptidos/inmunología , Animales , Autofagia/genética , Beclina-1/genética , Beclina-1/metabolismo , Células CACO-2 , Cisteamina/uso terapéutico , Fibrosis Quística/inmunología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Citocinas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/metabolismo , Gliadina/toxicidad , Heterocigoto , Homocigoto , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/toxicidad , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/antagonistas & inhibidores , Transglutaminasas/metabolismoRESUMEN
Alpha-actinin 4 (ACTN4) is an actin-binding protein of the spectrin superfamily. ACTN4 is found both in the cytoplasm and nucleus of eukaryotic cells. The main function of cytoplasmic ACTN4 is stabilization of actin filaments and their binding to focal contacts. Nuclear ACTN4 takes part in the regulation of gene expression following by activation of certain transcription factors, but the mechanisms of regulation are not completely understood. Our previous studies have demonstrated the interaction of ACTN4 with the RelA/p65 subunit of NF-kappaB factor and the effect on its transcriptional activity in A431 and HEK293T cells. In the present work, we investigated changes in the composition of nuclear ACTN4-interacting proteins in non-small cell lung cancer cells H1299 upon stable RELA overexpression. We showed that ACTN4 was present in the nuclei of H1299 cells, regardless of the RELA expression level. The presence of ectopic RelA/p65 in H1299 cells increased the number of proteins interacting with nuclear ACTN4. Stable expression of RELA in these cells suppressed cell proliferation, which was further affected by simultaneous ACTN4 overexpression. We detected no significant effect on cell cycle but the apoptosis rate was increased in cells with a double RELA/ACTN4 overexpression. Interestingly, when expressed individually ACTN4 promoted proliferation of lung cancer cells. Furthermore, the bioinformatics analysis of gene expression in lung cancer patients suggested that overexpression of ACTN4 correlated with poor survival prognosis. We hypothesize that the effect of RELA on proliferation and apoptosis of H1299 cells can be mediated via affecting the interactome of ACTN4.
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Actinina/metabolismo , Apoptosis , Factor de Transcripción ReIA/metabolismo , Actinina/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Factor de Transcripción ReIA/genéticaRESUMEN
The product of RCHY1 human gene, Pirh2, is a RING-finger containing E3 ligase that modifies p53 with ubiquitin residues resulting in its subsequent degradation in proteasomes. Transcription of RCHY1 is regulated by p53 itself thus forming a negative regulatory feedback loop. Functionally, by eliminating p53, Pirh2 facilitates tumorigenesis. However, the role of Pirh2 in cancer cells lacking p53 is yet not well understood. Therefore, we decided to elucidate the role of Pirh2 in p53-negative human non-small cell lung carcinoma cells, H1299. We found that ectopic expression of Pirh2 enhanced cell proliferation, resistance to doxorubicin, and increased migration potential. Ablation of Pirh2 by specific shRNA reversed these phenotypes. Mechanistically, Pirh2 increased mRNA and protein levels of the c-Myc oncogene. The bioinformatics data indicate that co-expression of both c-Myc and Pirh2 strongly correlated with poor survival of lung cancer patients. Collectively, our results suggest that Pirh2 can be considered as a potential pharmacological target for developing anticancer therapies to treat p53-negative cancers.
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TAp73 is a tumor suppressor transcriptional factor, belonging to p53 family. Alteration of TAp73 in tumors might lead to reduced DNA damage response, cell cycle arrest and apoptosis. Carcinogen-induced TAp73(-/-) tumors display also increased angiogenesis, associated to hyperactivition of hypoxia inducible factor signaling. Here, we show that TAp73 suppresses BNIP3 expression, directly binding its gene promoter. BNIP3 is a hypoxia responsive protein, involved in a variety of cellular processes, such as autophagy, mitophagy, apoptosis and necrotic-like cell death. Therefore, through different cellular process altered expression of BNIP3 may differently contribute to cancer development and progression. We found a significant upregulation of BNIP3 in human lung cancer datasets, and we identified a direct association between BNIP3 expression and survival rate of lung cancer patients. Our data therefore provide a novel transcriptional target of TAp73, associated to its antagonistic role on HIF signaling in cancer, which might play a role in tumor suppression.
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Proteínas de Unión al ADN/genética , Proteínas de la Membrana/biosíntesis , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/biosíntesis , Transcripción Genética/genética , Proteínas Supresoras de Tumor/genética , Apoptosis/genética , Sitios de Unión/genética , Línea Celular , Genes Supresores de Tumor , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Neovascularización Patológica/genética , Proteína Tumoral p73RESUMEN
The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation, playing important functions in various biological processes. The C-terminus of the ß7 (PSMB4) proteasome subunit was tagged with EGFP and with a composite element for affinity purification and TEV cleavage elution (HTBH). When the construct was retrovirally delivered into HeLa cells, virtually all of the ß7-EGFP-HTBH fusion protein was found to be incorporated into fully functional proteasomes. This ensured that subcellular localization of the EGFP signal in living HeLa cells could be attributed to ß7-EGFP-HTBH within the proteasome complex rather than to free protein. The ß7-EGFP-HTBH fusion can, therefore, serve as a valuable tool for in vivo imaging of proteasomes as well as for high-affinity purification of these complexes and associated molecules for subsequent analyses.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas Fluorescentes Verdes/metabolismo , Imagen Molecular , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal/metabolismo , Supervivencia Celular , Células HeLa , Humanos , Subunidades de Proteína/metabolismo , Proteolisis , Proteínas Recombinantes de FusiónRESUMEN
Genotoxic stress inflicted by anti-cancer drugs causes DNA breaks and genome instability. DNA double strand breaks induced by irradiation or pharmacological inhibition of Topoisomerase II activate ATM (ataxia-telangiectasia-mutated) kinase signalling pathway that in turn triggers cell cycle arrest and DNA repair. ATM-dependent gamma-phosphorylation of histone H2Ax and other histone modifications, including ubiquitnylation, promote exchange of histones and recruitment of DNA damage response (DDR) and repair proteins. Signal transduction pathways, besides DDR itself, also control expression of genes whose products cause cell cycle arrest and/or apoptosis thus ultimately affecting the sensitivity of cells to genotoxic stress. In this study, using a number of experimental approaches we provide evidence that lysine-specific methyltransferase (KMT) Set7/9 affects DDR and DNA repair, at least in part, by regulating the expression of an E3 ubiquitin ligase, Mdm2. Furthermore, we show that Set7/9 physically interacts with Mdm2. Several cancer cell lines with inverse expression of Set7/9 and Mdm2 displayed diminished survival in response to genotoxic stress. These findings are signified by our bioinformatics studies suggesting that the unleashed expression of Mdm2 in cancer patients with diminished expression of Set7/9 is associated with poor survival outcome.