RESUMEN
Colorectal cancer (CRC) is a leading cause of cancer-related death. There is an urgent need for new methods of early CRC detection and monitoring to improve patient outcomes. Extracellular vesicles (EVs) are secreted, lipid-bilayer bound, nanoparticles that carry biological cargo throughout the body and in turn exhibit cancer-related biomarker potential. RNA binding proteins (RBPs) are posttranscriptional regulators of gene expression that may provide a link between host cell gene expression and EV phenotypes. Insulin-like growth factor 2 RNA binding protein 1 (IGF2BP1/IMP1) is an RBP that is highly expressed in CRC with higher levels of expression correlating with poor prognosis. IMP1 binds and potently regulates tumor-associated transcripts that may impact CRC EV phenotypes. Our objective was to test whether IMP1 expression levels impact EV secretion and/or cargo. We used RNA sequencing, in vitro CRC cell lines, ex vivo colonoid models, and xenograft mice to test the hypothesis that IMP1 influences EV secretion and/or cargo in human CRC. Our data demonstrate that IMP1 modulates the RNA expression of transcripts associated with extracellular vesicle pathway regulation, but it has no effect on EV secretion levels in vitro or in vivo. Rather, IMP1 appears to affect EV regulation by directly entering EVs in a transformation-dependent manner. These findings suggest that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.NEW & NOTEWORTHY This work demonstrates that the RNA binding protein IGF2BP1/IMP1 alters the transcript profile of colorectal cancer cell (CRC) mRNAs from extracellular vesicle (EV) pathways. IMP1 does not alter EV production or secretion in vitro or in vivo, but rather enters CRC cells where it may further impact EV cargo. Our work shows that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.
Asunto(s)
Neoplasias Colorrectales , Vesículas Extracelulares , Humanos , Ratones , Animales , Vesículas Extracelulares/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Mensajero/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/patologíaRESUMEN
The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.
Asunto(s)
Proteína 1 Similar a ELAV/química , Fenantrenos/química , Fenantrenos/farmacología , Regiones no Traducidas 3' , Elementos Ricos en Adenilato y Uridilato , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Proteína 1 Similar a ELAV/antagonistas & inhibidores , Proteína 1 Similar a ELAV/genética , Proteína 1 Similar a ELAV/metabolismo , Furanos , Humanos , Espectroscopía de Resonancia Magnética , Ratones Mutantes Neurológicos , Simulación de Dinámica Molecular , Fenantrenos/metabolismo , Mutación Puntual , Conformación Proteica , Dominios Proteicos , Quinonas , ARN Mensajero/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cigarette smoking is a crucial factor in the development and progression of multiple cancers including breast. Here, we report that repeated exposure to a fixed, low dose of cigarette smoke condensate (CSC) prepared from Indian cigarettes is capable of transforming normal breast epithelial cells, MCF-10A, and delineate the biochemical basis for cellular transformation. CSC transformed cells (MCF-10A-Tr) were capable of anchorage-independent growth, and their anchorage dependent growth and colony forming ability were higher compared to the non-transformed MCF-10A cells. Increased expression of biomarkers representative of oncogenic transformation (NRP-1, Nectin-4), and anti-apoptotic markers (PI3K, AKT, NFκB) were also noted in the MCF-10A-Tr cells. Short tandem repeat (STR) profiling of MCF-10A and MCF-10A-Tr cells revealed that transformed cells acquired allelic variation during transformation, and had become genetically distinct. MCF-10A-Tr cells formed solid tumors when implanted into the mammary fat pads of Balb/c mice. Data revealed that CSC contained approximately 1.011µg Cd per cigarette equivalent, and Cd (0.0003µg Cd/1×10(7) cells) was also detected in the lysates from MCF-10A cells treated with 25µg/mL CSC. In similar manner to CSC, CdCl2 treatment in MCF-10A cells caused anchorage independent colony growth, higher expression of oncogenic proteins and increased PI3K-AKT-NFκB protein expression. An increase in the expression of PI3K-AKT-NFκB was also noted in the mice xenografts. Interestingly, it was noted that CSC and CdCl2 treatment in MCF-10A cells increased ROS. Collectively, results suggest that heavy metals present in cigarettes of Indian origin may substantially contribute to tumorigenesis by inducing intercellular ROS accumulation and increased expression of PI3K, AKT and NFκB proteins.
Asunto(s)
Transformación Celular Neoplásica/inducido químicamente , Células Epiteliales/efectos de los fármacos , Metales Pesados/toxicidad , FN-kappa B/biosíntesis , Fosfatidilinositol 3-Quinasa/biosíntesis , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Humo/efectos adversos , Animales , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Transformada , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Fumar/efectos adversos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
We previously reported that quinacrine (QC) has anticancer activity against breast cancer cells. Here, we examine the mechanism of action of QC and its ability to inhibit Wnt-TCF signaling in two independent breast cancer cell lines. QC altered Wnt-TCF signaling components by increasing the levels of adenomatous polyposis coli (APC), DAB2, GSK-3ß and axin and decreasing the levels of ß-catenin, p-GSK3ß (ser 9) and CK1. QC also reduced the activity of the Wnt transcription factor TCF/LEF and its downstream targets cyclin D1 and c-MYC. Using a luciferase-based Wnt-TCF transcription factor assay, it was shown that APC levels were inversely associated with TCF/LEF activity. Induction of apoptosis and DNA damage was observed after treatment with QC, which was associated with increased expression of APC. The effects induced by QC depend on APC because the inhibition of Wnt-TCF signaling by QC is lost in APC-knockdown cells, and consequently, the extent of apoptosis and DNA damage caused by QC is reduced compared with parental cells. Because we previously showed that QC inhibits topoisomerase, we examined the effect of another topoisomerase inhibitor, etoposide, on Wnt signaling. Interestingly, etoposide treatment also reduced TCF/LEF activity, ß-catenin and cyclin D1 levels commensurate with induction of DNA damage and apoptosis. Lycopene, a plant-derived antioxidant, synergistically increased QC activity and inhibited Wnt-TCF signaling in cancer cells without affecting the MCF-10A normal breast cell line. Collectively, the data suggest that QC-mediated Wnt-TCF signal inhibition depends on APC and that the addition of lycopene synergistically increases QC anticancer activity.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Carotenoides/farmacología , Quinacrina/farmacología , Transducción de Señal/efectos de los fármacos , Factor 1 de Transcripción de Linfocitos T/antagonistas & inhibidores , Proteínas Wnt/antagonistas & inhibidores , Proteína de la Poliposis Adenomatosa del Colon/antagonistas & inhibidores , Proteína de la Poliposis Adenomatosa del Colon/genética , Anticarcinógenos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular , Proliferación Celular , Ensayo Cometa , Ciclina D1/metabolismo , Sinergismo Farmacológico , Etopósido/farmacología , Femenino , Citometría de Flujo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Licopeno , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Factor 1 de Transcripción de Linfocitos T/metabolismo , Factores de Transcripción TCF , Transactivadores/metabolismo , Transcripción Genética , Células Tumorales Cultivadas , Ensayo de Tumor de Célula Madre , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Based on known heterocyclic topoisomerase II inhibitors and anticancer agents, various indenoindolone derivatives were predicted as potential topoisomerase II-inhibiting anticancer agents. They are hydrazones, (thio)semicarbazones, and oximes of indenoindolones, and indenoindolols. These derivatives with suitable substitutions exhibited potent specific inhibition of human DNA TopoIIα while not showing inhibition of topoisomerase I and DNA intercalation, despite the fact that parent indenoindolones are known poor/moderate inhibitors of topoisomerase II. The potent topoisomerase II inhibitor indenoindolone derivatives exhibited good anticancer activities compared to etoposide and 5-fluorouracil, and relatively low toxicity to normal cells. These derivatizations of indenoindolones were found to result in enhancement of anticancer activities.
Asunto(s)
Antineoplásicos/farmacología , Indoles/química , Indoles/farmacología , Neoplasias/tratamiento farmacológico , Inhibidores de Topoisomerasa II/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/metabolismo , Humanos , Indoles/uso terapéutico , Modelos Moleculares , Neoplasias/enzimología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/uso terapéuticoRESUMEN
The small molecule Quinacrine (QC, a derivative of 9-aminoacridine), an anti-malaria drug, displays activity against cancer cell lines and can simultaneously suppress nuclear factor-κB (NF-κB) and activate p53 signaling. In this study, we investigated the anticancer mechanism underlying these drug activities in breast cancer cell lines. QC caused a dose-dependent decrease of both anchorage dependent and independent growth of breast cancer cells (MCF-7 and MDA-MB-231) without affecting normal breast epithelial cells (MCF-10A), as evident from clonogenic cell survival, [3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide] viability, wound healing and soft agar growth. QC activated the proapoptotic marker Bax, PARP cleavage, p53 and its downstream target, p21 (Cip1/Waf1) and downregulated the antiapoptotic marker Bcl-xL and relative luciferase activity of NF-κB in MCF-7 cells. Results of DAPI nuclear staining and FACS analysis show that QC increased apoptosis in a dose-dependent manner. QC caused apoptosis by increasing the cell population in S-phase and simultaneously decreasing the G1 and G2/M populations. A dose-dependent increase of DNA damage as measured by the comet assay was seen in MCF-7 cells after exposure to QC. With regards to the mechanism of DNA damage, we found that QC inhibited topoisomerase activity in MCF-7 cells by increasing the unwinding of supercoiled DNA. Collectively, the results demonstrate that QC has efficient anticancer potential against breast cancer cells via not only an induction of p53 and p21 but also an induction of S phase arrest, DNA damage and inhibition of topoisomerase activity.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , ADN-Topoisomerasas/metabolismo , Quinacrina/farmacología , Inhibidores de Topoisomerasa/farmacología , Adulto , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN/efectos de los fármacos , ADN Superhelicoidal/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , FN-kappa B/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
We previously showed that quinacrine (QC), a small molecule antimalarial agent, also presented anticancer activity in breast cancer cells through activation of p53, p21, and inhibition of topoisomerase activity. Here we have systematically studied the detailed cell death mechanism of this drug using three colon cancer cell lines (HCT-116 parental, isogenic HCT-116 p53-/-, and HCT-116 p21-/- sublines). QC caused a dose-dependent reduction in cell viability in all three cell lines. However, the parental cells were more susceptible to QC-mediated cell death, suggesting that p53- and p21-dependent processes were involved. QC-mediated cell death was measured with the following endpoints: the Bax/Bcl-xL ratio, cleaved PARP, apoptotic nuclei visualized by DAPI staining, and COMET formation. In addition, markers of autophagy were measured. Acridine orange staining revealed increased accumulation of autophagic vacuoles (AVs) after QC treatment in a dose-dependent manner in parental cells, and decreased staining in isogenic HCT-116 p53-/- and HCT-116 p21-/- cells. Immunofluorescence of LC3B was significantly lowered in QC-treated cells lacking p53 or p21, compared to the parental cells. Interestingly, the expression of the autophagy marker LC3B-II after exposure to QC was decreased in either p53 or p21 null cells compared to parental cells. After deletion of p21 in HCT-116 p53-/- cells, no change in LC3B-II expression was noted following QC treatment. Collectively, the results suggest that QC-mediated autophagy and apoptosis dependent on p53 and p21.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinacrina/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Células Tumorales CultivadasRESUMEN
Scaffold hybridization of several natural and synthetic anticancer leads led to the consideration of indenoindolones as potential novel anticancer agents. A series of these compounds were prepared by a diversity-feasible synthetic method. They were found to possess anticancer activities with higher potency compared to etoposide and 5-fluorouracil in kidney cancer cells (HEK 293) and low toxicity to corresponding normal cells (Vero). They exerted apoptotic effect with blocking of cell cycle at G2/M phase.
Asunto(s)
Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Indenos/síntesis química , Indoles/síntesis química , Animales , Antineoplásicos/farmacología , Biomarcadores/metabolismo , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Etopósido/farmacología , Citometría de Flujo , Fluorouracilo/farmacología , Células HEK293 , Humanos , Indenos/farmacología , Indoles/farmacología , Concentración 50 Inhibidora , Células VeroRESUMEN
HuR overexpression is related to poor survival in patients with colon cancer. HuR overexpression leads to stabilization of tumor-promoting mRNAs by binding to 3'UTR-resident AREs. Exosomes, nanosized lipid bilayer vesicles, mediate many steps in cancer progression. The potential role of exosomal HuR in colon cancer lung metastasis is unclear. HuR expression was assessed immunohistochemically in tumor tissue samples from 20 patients with metastatic or nonmetastatic colon cancer and colon cancer lung metastasis and benign lung disease samples from ten patients. Exosomes were isolated from HCT116 WT and HuR KO colon cancer cells, and uptake of PKH67- and PKH26-labeled exosomes by BEAS-2B cells was evaluated using fluorescence and confocal microscopy. C-Myc and p21protein and mRNA levels were measured by western blotting and RT-qPCR, respectively. In clinical patients, HuR overexpression was significantly enhanced in colon tissues of patients with lung metastasis. HuR expression was higher in lung tissue with metastasis of colonic origin than with benign lung disease. The effect of HuR-containing CRC exosomes compared to HuR-deficient exosomes on wound closure was observed as enhanced proliferation. BEAS-2B cell migration and invasion were enhanced after HuR-containing exosomes treatment. BEAS-2B cells showed similar uptake of PKH67 (HCT116 WT)- and PKH26 (HCT116 HuR KO)-labeled exosomes. Exosomal HuR stabilized c-Myc mRNA and downregulated p21 expression, leading to G1/S transition, in human bronchial epithelial cells. HuR overexpression is associated with lung metastasis in colon cancer patients. Exosomal HuR derived from colon cancer cells alter the biological effect on normal lung epithelial cells.
Asunto(s)
Neoplasias del Colon , Proteína 1 Similar a ELAV , Exosomas , Neoplasias Pulmonares , MicroARNs , Proteínas Proto-Oncogénicas c-myc , ARN Mensajero , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Proteína 1 Similar a ELAV/genética , Exosomas/genética , Exosomas/metabolismo , Células HCT116 , Humanos , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Resveratrol (Res) can modulate multiple cellular pathways relevant for tumorigenesis but is less effective in colon cancer compared to breast cancer. To increase the chemopreventive potential of Res in combination with 5-fluorouracil (5-FU), a systematic study was carried out in colon cancer cells. HCT-116 cells were treated with Res and 5-FU and several cell-based assays, such as MTT, clonogenic, wound healing, DAPI, comet assay, and Western blot, were performed. A significant inhibition of cell proliferation, migration, and increased apoptosis were observed when moderate concentration of Res (15 microM) was associated with very low concentration of 5-FU (0.5 microM). This combination caused apoptosis by blocking the cells at S phase and enhanced the DNA damage. Expression levels of p-JNK and p-p38 were increased without affecting pERK. 5-FU could be used as a therapeutic modality to improve efficacy of Res-based chemotherapy against colon cancer.
Asunto(s)
Anticarcinógenos/farmacología , Antimetabolitos Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Daño del ADN , Fluorouracilo/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Estilbenos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células HCT116 , Humanos , ResveratrolRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a lethal aggressive cancer, in part due to elements of the microenvironment (hypoxia, hypoglycemia) that cause metabolic network alterations. The FDA-approved antihelminthic pyrvinium pamoate (PP) has previously been shown to cause PDAC cell death, although the mechanism has not been fully determined. We demonstrated that PP effectively inhibited PDAC cell viability with nanomolar IC50 values (9-93 nmol/L) against a panel of PDAC, patient-derived, and murine organoid cell lines. In vivo, we demonstrated that PP inhibited PDAC xenograft tumor growth with both intraperitoneal (IP; P < 0.0001) and oral administration (PO; P = 0.0023) of human-grade drug. Metabolomic and phosphoproteomic data identified that PP potently inhibited PDAC mitochondrial pathways including oxidative phosphorylation and fatty acid metabolism. As PP treatment reduced oxidative phosphorylation (P < 0.001), leading to an increase in glycolysis (P < 0.001), PP was 16.2-fold more effective in hypoglycemic conditions similar to those seen in PDAC tumors. RNA sequencing demonstrated that PP caused a decrease in mitochondrial RNA expression, an effect that was not observed with established mitochondrial inhibitors rotenone and oligomycin. Mechanistically, we determined that PP selectively bound mitochondrial G-quadruplexes and inhibited mitochondrial RNA transcription in a G-quadruplex-dependent manner. This subsequently led to a 90% reduction in mitochondrial encoded gene expression. We are preparing to evaluate the efficacy of PP in PDAC in an IRB-approved window-of-opportunity trial (IND:144822).
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antihelmínticos/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Metabolómica/métodos , Compuestos de Pirvinio/uso terapéutico , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Animales , Antihelmínticos/farmacología , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Humanos , Ratones , Compuestos de Pirvinio/farmacología , Análisis de Supervivencia , Estados Unidos , United States Food and Drug AdministrationRESUMEN
DNA replication is the fundamental process for accurate duplication and transfer of genetic information. Its fidelity is under constant stress from endogenous and exogenous factors which can cause perturbations that lead to DNA damage and defective replication. This can compromise genomic stability and integrity. Genomic instability is considered as one of the hallmarks of cancer. In normal cells, various checkpoints could either activate DNA repair or induce cell death/senescence. Cancer cells on the other hand potentiate DNA replicative stress, due to defective DNA damage repair mechanism and unchecked growth signaling. Though replicative stress can lead to mutagenesis and tumorigenesis, it can be harnessed paradoxically for cancer treatment. Herein, we review the mechanism and rationale to exploit replication stress for cancer therapy. We discuss both established and new approaches targeting DNA replication stress including chemotherapy, radiation, and small molecule inhibitors targeting pathways including ATR, Chk1, PARP, WEE1, MELK, NAE, TLK etc. Finally, we review combination treatments, biomarkers, and we suggest potential novel methods to target DNA replication stress to treat cancer.
Asunto(s)
Replicación del ADN/genética , Neoplasias/genética , Animales , Daño del ADN/genética , Reparación del ADN/genética , Inestabilidad Genómica/genética , Humanos , Transducción de Señal/genéticaRESUMEN
Altered gene expression is a characteristic feature of many disease states such as tumorigenesis, and in most cancers, it facilitates cancer cell survival and adaptation. Alterations in global gene expression are strongly impacted by post-transcriptional gene regulation. The RNA binding protein (RBP) HuR (ELAVL1) is an established regulator of post-transcriptional gene regulation and is overexpressed in most human cancers. In many cancerous settings, HuR is not only overexpressed, but it is "overactive" as denoted by increased subcellular localization within the cytoplasm. This dysregulation of HuR expression and cytoplasmic localization allows HuR to stabilize and increase the translation of various prosurvival messenger RNA (mRNAs) involved in the pathogenesis of numerous cancers and various diseases. Based on almost 20 years of work, HuR is now recognized as a therapeutic target. Herein, we will review the role HuR plays in the pathophysiology of different diseases and ongoing therapeutic strategies to target HuR. We will focus on three ongoing-targeted strategies: (1) inhibiting HuR's translocation from the nucleus to the cytoplasm; (2) inhibiting the ability of HuR to bind target RNA; and (3) silencing HuR expression levels. In an oncologic setting, HuR has been demonstrated to be critical for a cancer cell's ability to survive a variety of cancer relevant stressors (including drugs and elements of the tumor microenvironment) and targeting this protein has been shown to sensitize cancer cells further to insult. We strongly believe that targeting HuR could be a powerful therapeutic target to treat different diseases, particularly cancer, in the near future. This article is categorized under: RNA in Disease and Development > RNA in Disease NRA Turnover and Surveillance > Regulation of RNA Stability Translation > Translation Regulation.
Asunto(s)
Proteína 1 Similar a ELAV/genética , Neoplasias/genética , Citoplasma/genética , Citoplasma/metabolismo , Proteína 1 Similar a ELAV/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Death Receptor 5 (DR5) is known to be an important anti-cancer drug target. TRAIL is a natural ligand of DR5, but its drug action is limited because of several factors. A few agonistic ligands were identified as TRAIL-DR5 axis modulators, which enhance the cellular apoptosis. Literature suggest that quinacrine (QC) acts as a DR5 agonistic ligand. However, the detailed mechanism explaining how QC interacts with TRAIL-DR5 axis has not been established. Also focused in vitro and in vivo experimental analysis to validate the hypothesis is not yet performed. In this work, extensive studies have been carried out using in silico analysis (molecular dynamics), in vitro analysis (cell based assays) and in vivo analysis (based on mice xenograft model), to delineate the mechanism of QC action in modulating the TRAIL-DR5 signaling. The MD simulations helped in identifying the important residues contributing to the formation of a QC-TRAIL-DR5 complex, which provide extra stability to it, consequently leading to the enhanced cellular apoptosis. QC caused a dose dependent increase of DR5 expression in cancer cells but not in normal breast epithelial cells, MCF-10A. QC showed a synergistic effect with TRAIL in causing cancer cell apoptosis. In DR5-KD MCF-10A-Tr (DR5 knocked down) cells, TRAIL+ QC failed to significantly increase the apoptosis but over expression of full length DR5 in DR5-silence cells induced apoptosis, further supporting DR5 as a drug target for QC. An increase in the release of reactive species (ROS and RNS) and activation of enzymes (FADD, CASPASES 3, 8, 9 and cytochrome-C) indicated the involvement of mitochondrial intrinsic pathway in TRAIL+QC mediated apoptosis. In vivo study pointed out that TRAIL+QC co-administration increases the expression of DR5 and reduce the tumor size in xenograft mice. This combined in silico, in vitro and in vivo analysis revealed that QC enhances the cellular apoptosis via the modulation of TRAIL-DR5 complexation and the mitochondrial intrinsic pathway.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Mitocondrias/metabolismo , Quinacrina/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Mitocondrias/efectos de los fármacos , Simulación de Dinámica Molecular , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States, whereas colorectal cancer is the third most common cancer. The RNA-binding protein HuR (ELAVL1) supports a pro-oncogenic network in gastrointestinal (GI) cancer cells through enhanced HuR expression. Using a publically available database, HuR expression levels were determined to be increased in primary PDA and colorectal cancer tumor cohorts as compared with normal pancreas and colon tissues, respectively. CRISPR/Cas9 technology was successfully used to delete the HuR gene in both PDA (MIA PaCa-2 and Hs 766T) and colorectal cancer (HCT116) cell lines. HuR deficiency has a mild phenotype, in vitro, as HuR-deficient MIA PaCa-2 (MIA.HuR-KO(-/-)) cells had increased apoptosis when compared with isogenic wild-type (MIA.HuR-WT(+/+)) cells. Using this isogenic system, mRNAs were identified that specifically bound to HuR and were required for transforming a two-dimensional culture into three dimensional (i.e., organoids). Importantly, HuR-deficient MIA PaCa-2 and Hs 766T cells were unable to engraft tumors in vivo compared with control HuR-proficient cells, demonstrating a unique xenograft lethal phenotype. Although not as a dramatic phenotype, CRISPR knockout HuR HCT116 colon cancer cells (HCT.HuR-KO(-/-)) showed significantly reduced in vivo tumor growth compared with controls (HCT.HuR-WT(+/+)). Finally, HuR deletion affects KRAS activity and controls a subset of pro-oncogenic genes.Implications: The work reported here supports the notion that targeting HuR is a promising therapeutic strategy to treat GI malignancies. Mol Cancer Res; 15(6); 696-707. ©2017 AACR.
Asunto(s)
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Neoplasias del Colon/genética , Proteína 1 Similar a ELAV/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Neoplasias del Colon/patología , Proteína 1 Similar a ELAV/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Ratones Desnudos , Neoplasias Experimentales/genética , Neoplasias Pancreáticas/patología , Fenotipo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Quinacrine (QC) causes apoptosis in breast cancer cells by induction of DNA damage, arrest of cells in S-phase, and by topoisomerase inhibition. Here, we show that QC-mediated apoptosis is not only due to increased DNA damage but also by compromising cell cycle checkpoints and base excision repair (BER) capacity in breast cancer cells. QC decreased CHK1, CDKs (CDC2, MDM2, CDC6), cyclins (B1, E1) and CDC25-A in a dose dependent manner. The expression of basal ATR remains unaltered but pATR (Ser-428) increased after QC treatment. A CHK1 inhibitor, SB218078, was also tested alone and in combination with QC. Like QC, SB218078 caused apoptosis by DNA damage and S-phase arrest. The combination of QC and SB218078 increased apoptosis by blocking the cell cycle in G2/M, which caused a mitotic catastrophe, and induced DNA damage at a higher level in comparison to individual compound treatments. Both drugs individually or in combination decreased the levels of replication protein A (RPA). Measurement of the expression of BER (SP- and LP-BER) proteins and direct in vivo BER activity revealed that the QC/SB218078 combination caused apoptosis in cancer cells by disrupting the induction of BER, which represents a novel means of potentially treating breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/fisiología , Neoplasias de la Mama/enzimología , Reparación del ADN/fisiología , Proteínas Quinasas/metabolismo , Quinacrina/farmacología , Alcaloides/farmacología , Alcaloides/uso terapéutico , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Reparación del ADN/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Quinacrina/uso terapéuticoRESUMEN
Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality. Observed during CRC tumorigenesis is loss of post-transcriptional regulation of tumor-promoting genes such as COX-2, TNFα and VEGF. Overexpression of the RNA-binding protein HuR (ELAVL1) occurs during colon tumorigenesis and is abnormally present within the cytoplasm, where it post-transcriptionally regulates genes through its interaction with 3'UTR AU-rich elements (AREs). Here, we examine the therapeutic potential of targeting HuR using MS-444, a small molecule HuR inhibitor. Treatment of CRC cells with MS-444 resulted in growth inhibition and increased apoptotic gene expression, while similar treatment doses in non-transformed intestinal cells had no appreciable effects. Mechanistically, MS-444 disrupted HuR cytoplasmic trafficking and released ARE-mRNAs for localization to P-bodies, but did not affect total HuR expression levels. This resulted in MS-444-mediated inhibition of COX-2 and other ARE-mRNA expression levels. Importantly, MS-444 was well tolerated and inhibited xenograft CRC tumor growth through enhanced apoptosis and decreased angiogenesis upon intraperitoneal administration. In vivo treatment of MS-444 inhibited HuR cytoplasmic localization and decreased COX-2 expression in tumors. These findings provide evidence that therapeutic strategies to target HuR in CRC warrant further investigation in an effort to move this approach to the clinic.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Proteína 1 Similar a ELAV/antagonistas & inhibidores , Furanos/farmacología , Naftoles/farmacología , Animales , Carcinogénesis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Proteína 1 Similar a ELAV/metabolismo , Células HCT116 , Células HT29 , Humanos , Ratones , Ratones Desnudos , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLß and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Reparación del ADN/efectos de los fármacos , Fluorouracilo/farmacología , Proteína de la Poliposis Adenomatosa del Colon/química , Proteína de la Poliposis Adenomatosa del Colon/genética , Secuencia de Aminoácidos , Antimetabolitos Antineoplásicos/farmacología , Línea Celular Tumoral/efectos de los fármacos , Neoplasias del Colon/genética , Endonucleasas de ADN Solapado/metabolismo , Técnicas de Silenciamiento del Gen , Células HCT116/efectos de los fármacos , Humanos , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Terciaria de ProteínaRESUMEN
A new family of andrographolide analogues were synthesized and screened in vitro against kidney (HEK-293) and breast (MCF-7) cancer cells. The anti-cancer effects of the active analogues (2b, 2c and 4c) were determined by multiple cell based assays such as MTT, immunostaining, FACS, western blotting and transcriptional inhibition of NF-κB activity. Importantly, these compounds were found to possess higher anti-cancer potency than andrographolide and low toxicity to normal (VERO and MCF-10A) cells. Increased level of Bax/Bcl-xL ratio, caspase 3, and sub G1 population, higher expression level of tumor suppressor protein p53 and lower expression level of NF-κB suggested potent apoptotic property of the active analogues. Data revealed that the andrographolide derivative-mediated cell death in cancer cells was p53 dependent.