RESUMO
Colorectal cancer (CRC) is the third most prevalent cancer worldwide. Current studies have demonstrated that tumor-derived extracellular vesicles (EVs) from different cancer cell types modulate the fibroblast microenvironment to contribute to cancer development and progression. Here, we isolated and characterized circulating large EVs (LEVs), small EVs (SEVs) and non-EV entities released in the plasma from wild-type (WT) mice and the APCMin/+ CRC mice model. Our results showed that human colon fibroblasts exposed from APC-EVs, but not from WT-EVs, exhibited the phenotypes of cancer-associated fibroblasts (CAFs) through EV-mediated NF-κB pathway activation. Cytokine array analysis on secreted proteins revealed elevated levels of inflammatory cytokine implicated in cancer growth and metastasis. Finally, non-activated cells co-cultured with supernatant from fibroblasts treated with APC-EVs showed increased mRNA expressions of CAFs markers, the ECM, inflammatory cytokines, as well as the expression of genes controlled by NF-κB. Altogether, our work suggests that EVs and non-EV components from APCMin/+ mice are endowed with pro-tumorigenic activities and promoted inflammation and a CAF-like state by triggering NF-κB signaling in fibroblasts to support CRC growth and progression. These findings provide insight into the interaction between plasma-derived EVs and human cells and can be used to design new CRC diagnosis and prognosis tools.
Assuntos
Vesículas Extracelulares , Fibroblastos , NF-kappa B , Transdução de Sinais , Animais , Humanos , Camundongos , Proteína da Polipose Adenomatosa do Colo/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinogênese/patologia , Carcinogênese/genética , Carcinogênese/metabolismo , Colo/patologia , Colo/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Fibroblastos/metabolismo , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Microambiente TumoralRESUMO
The RNA-binding protein human antigen R (HuR) is a well-established regulator of gene expression at the posttranscriptional level. Its dysregulation has been implicated in various human diseases, particularly cancer. In cancer, HuR is considered "active" when it shows increased subcellular localization in the cytoplasm, in addition to its normal nuclear localization. Cytoplasmic HuR plays a crucial role in stabilizing and enhancing the translation of prosurvival mRNAs that are involved in stress responses relevant to cancer progression, such as hypoxia, radiotherapy, and chemotherapy. In general, due to HuR's abundance and function in cancer cells compared with normal cells, it is an appealing target for oncology research. Exploiting the principles underlying HuR's role in tumorigenesis and resistance to stressors, targeting HuR has the potential for synergy with existing and novel oncologic therapies. This review aims to explore HuR's role in homeostasis and cancer pathophysiology, as well as current targeting strategies, which include silencing HuR expression, preventing its translocation and dimerization from the nucleus to the cytoplasm, and inhibiting mRNA binding. Furthermore, this review will discuss recent studies investigating the potential synergy between HuR inhibition and traditional chemotherapeutics.
Assuntos
Proteína Semelhante a ELAV 1 , Neoplasias , Humanos , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas ELAV/genéticaRESUMO
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.
Assuntos
Neoplasias Colorretais , Vesículas Extracelulares , Humanos , Camundongos , Animais , Vesículas Extracelulares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Mensageiro/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologiaRESUMO
Yes-associated protein 1 (YAP1) is indispensable for the development of mutant KRAS-driven pancreatic ductal adenocarcinoma (PDAC). High YAP1 mRNA is a prognostic marker for worse overall survival in patient samples; however, the regulatory mechanisms that mediate its overexpression are not well understood. YAP1 genetic alterations are rare in PDAC, suggesting that its dysregulation is likely not due to genetic events. HuR is an RNA-binding protein whose inhibition impacts many cancer-associated pathways, including the "conserved YAP1 signature" as demonstrated by gene set enrichment analysis. Screening publicly available and internal ribonucleoprotein immunoprecipitation (RNP-IP) RNA sequencing (RNA-Seq) data sets, we discovered that YAP1 is a high-confidence target, which was validated in vitro with independent RNP-IPs and 3' untranslated region (UTR) binding assays. In accordance with our RNA sequencing analysis, transient inhibition (e.g., small interfering RNA [siRNA] and small-molecular inhibition) and CRISPR knockout of HuR significantly reduced expression of YAP1 and its transcriptional targets. We used these data to develop a HuR activity signature (HAS), in which high expression predicts significantly worse overall and disease-free survival in patient samples. Importantly, the signature strongly correlates with YAP1 mRNA expression. These findings highlight a novel mechanism of YAP1 regulation, which may explain how tumor cells maintain YAP1 mRNA expression at dynamic times during pancreatic tumorigenesis.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Regiões 3' não Traduzidas/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Sinalização YAP , Neoplasias PancreáticasRESUMO
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.
Assuntos
Neoplasias do Colo , Proteína Semelhante a ELAV 1 , Exossomos , Neoplasias Pulmonares , MicroRNAs , Proteínas Proto-Oncogênicas c-myc , RNA Mensageiro , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteína Semelhante a ELAV 1/genética , Exossomos/genética , Exossomos/metabolismo , Células HCT116 , Humanos , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
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).
Assuntos
Adenocarcinoma/tratamento farmacológico , Anti-Helmínticos/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Metabolômica/métodos , Compostos de Pirvínio/uso terapêutico , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Animais , Anti-Helmínticos/farmacologia , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Humanos , Camundongos , Compostos de Pirvínio/farmacologia , Análise de Sobrevida , Estados Unidos , United States Food and Drug AdministrationRESUMO
Stress granules (SGs) represent important non-membrane cytoplasmic compartments, involved in cellular adaptation to various stressful conditions (e.g., hypoxia, nutrient deprivation, oxidative stress). These granules contain several scaffold proteins and RNA-binding proteins, which bind to mRNAs and keep them translationally silent while protecting them from harmful conditions. Although the role of SGs in cancer development is still poorly known and vary between cancer types, increasing evidence indicate that the expression and/or the activity of several key SGs components are deregulated in colorectal tumors but also in pre-neoplastic conditions (e.g., inflammatory bowel disease), thus suggesting a potential role in the onset of colorectal cancer (CRC). It is therefore believed that SGs formation importantly contributes to various steps of colorectal tumorigenesis but also in chemoresistance. As CRC is the third most frequent cancer and one of the leading causes of cancer mortality worldwide, development of new therapeutic targets is needed to offset the development of chemoresistance and formation of metastasis. Abolishing SGs assembly may therefore represent an appealing therapeutic strategy to re-sensitize colon cancer cells to anti-cancer chemotherapies. In this review, we summarize the current knowledge on SGs in colorectal cancer and the potential therapeutic strategies that could be employed to target them.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Neoplasias Colorretais/tratamento farmacológico , Grânulos Citoplasmáticos , Humanos , RNA Mensageiro , Proteínas de Ligação a RNA , Estresse FisiológicoRESUMO
Pancreatic cancer has poor prognosis and treatment outcomes due to its highly metastatic nature and resistance to current treatments. The RNA-binding protein (RBP) Hu-antigen R (HuR) is a central player in posttranscriptional regulation of cancer-related gene expression, and contributes to tumorigenesis, tumor growth, metastasis, and drug resistance. HuR has been suggested to regulate pancreatic cancer epithelial-to-mesenchymal transition (EMT), but the mechanism was not well understood. Here, we further elucidated the role HuR plays in pancreatic cancer cell EMT, and developed a novel inhibitor specifically interrupting HuR-RNA binding. The data showed that HuR binds to the 3'-UTR of the mRNA of the transcription factor Snail, resulting in stabilization of Snail mRNA and enhanced Snail protein expression, thus promoted EMT, metastasis, and formation of stem-like cancer cells (CSC) in pancreatic cancer cells. siRNA silencing or CRISPR/Cas9 gene deletion of HuR inhibited pancreatic cancer cell EMT, migration, invasion, and inhibited CSCs. HuR knockout cells had dampened tumorigenicity in immunocompromised mice. A novel compound KH-3 interrupted HuR-RNA binding, and KH-3 inhibited pancreatic cancer cell viability, EMT, migration/invasion in vitro KH-3 showed HuR-dependent activity and inhibited HuR-positive tumor growth and metastasis in vivo.
Assuntos
Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Transição Epitelial-Mesenquimal/genética , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Genes Reporter , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Estabilidade de RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein Hu antigen R (HuR) is upregulated in breast cancer, and elevated cytoplasmic HuR correlates with high-grade tumors and poor clinical outcome of breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors, and cytokines that support major tumor hallmarks including invasion and metastasis. Here, we report a HuR inhibitor KH-3, which potently suppresses breast cancer cell growth and invasion. Furthermore, KH-3 inhibits breast cancer experimental lung metastasis, improves mouse survival, and reduces orthotopic tumor growth. Mechanistically, we identify FOXQ1 as a direct target of HuR. KH-3 disrupts HuR-FOXQ1 mRNA interaction, leading to inhibition of breast cancer invasion. Our study suggests that inhibiting HuR is a promising therapeutic strategy for lethal metastatic breast cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Fatores de Transcrição Forkhead/metabolismo , Neoplasias Pulmonares/prevenção & controle , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Proteína Semelhante a ELAV 1/genética , Neoplasias/genética , Citoplasma/genética , Citoplasma/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
PURPOSE: Circulating adipose stromal cells (CASC) are thought to be increased in obesity and facilitate angiogenesis, and tumor metastases. METHODS: CASC were identified from buffy coat peripheral blood mononuclear cells (PBMCs) by flow cytometry as CD34brightCD31- CD45- and CASC frequency was compared to adiposity measures in 33 women at increased risk for breast cancer. Feasibility of CASC as a response biomarker for a diet and exercise intervention in ten breast cancer survivors was then explored. RESULTS: For 33 high-risk women, median CASC frequency was 9.7 per million PBMCs and trended positively with body mass index, fat mass index (FMI), and percent android fat. Correlation was significant when BMI was dichotomized at > versus < 35 kg/m2 (p = 0.02). For ten breast cancer survivors with a median BMI of 37 kg/m2, median CASC frequency was 16.4 per million PBMCs. In univariate analyses, change in BMI, total fat and visceral fat were significantly correlated with change in CASC frequency. On multivariate analysis, change in visceral adipose had the strongest association with change in CASC frequency (p < 0.00078). CONCLUSIONS: The association between the reduction in visceral adipose tissue and the decrease in frequency of circulating adipose stromal cells suggests that the latter might be a useful biomarker in clinical trials of obese breast cancer survivors undergoing a weight loss intervention.
Assuntos
Tecido Adiposo/imunologia , Biomarcadores/sangue , Neoplasias da Mama/sangue , Obesidade/terapia , Tecido Adiposo/citologia , Idoso , Antígenos CD34/metabolismo , Neoplasias da Mama/imunologia , Sobreviventes de Câncer , Estudos Transversais , Dietoterapia , Terapia por Exercício , Feminino , Humanos , Antígenos Comuns de Leucócito/metabolismo , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Pós-Menopausa , Pré-Menopausa , Células Estromais/citologia , Células Estromais/imunologiaRESUMO
Elevated prostaglandin E2 (PGE2) levels are observed in colorectal cancer (CRC) patients, and this increase is associated with poor prognosis. Increased synthesis of PGE2 in CRC has been shown to occur through COX-2-dependent mechanisms; however, loss of the PGE2-catabolizing enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH, HPGD), in colonic tumors contributes to increased prostaglandin levels and poor patient survival. While loss of 15-PGDH can occur through transcriptional mechanisms, we demonstrate that 15-PGDH can be additionally regulated by a miRNA-mediated mechanism. We show that 15-PGDH and miR-21 are inversely correlated in CRC patients, with increased miR-21 levels associating with low 15-PGDH expression. 15-PGDH can be directly regulated by miR-21 through distinct sites in its 3' untranslated region (3'UTR), and miR-21 expression in CRC cells attenuates 15-PGDH and promotes increased PGE2 levels. Additionally, epithelial growth factor (EGF) signaling suppresses 15-PGDH expression while simultaneously enhancing miR-21 levels. miR-21 inhibition represses CRC cell proliferation, which is enhanced with EGF receptor (EGFR) inhibition. These findings present a novel regulatory mechanism of 15-PGDH by miR-21, and how dysregulated expression of miR-21 may contribute to loss of 15-PGDH expression and promote CRC progression via increased accumulation of PGE2.
Assuntos
Neoplasias do Colo/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hidroxiprostaglandina Desidrogenases/genética , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Sítios de Ligação/genética , Células CACO-2 , Proliferação de Células/genética , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Células HCT116 , Células HT29 , Células HeLa , Humanos , Hidroxiprostaglandina Desidrogenases/metabolismoRESUMO
We previously reported that ionizing radiation (IR) mediates cell death through the induction of CUGBP elav-like family member 2 (CELF2), a tumor suppressor. CELF2 is an RNA binding protein that modulates mRNA stability and translation. Since IR induces autophagy, we hypothesized that CELF2 regulates autophagy-mediated colorectal cancer (CRC) cell death. For clinical relevance, we determined CELF2 levels in The Cancer Genome Atlas (TCGA). Role of CELF2 in radiation response was carried out in CRC cell lines by immunoblotting, immunofluorescence, autophagic vacuole analyses, RNA stability assay, quantitative polymerase chain reaction and electron microscopy. In vivo studies were performed in a xenograft tumor model. TCGA analyses demonstrated that compared to normal tissue, CELF2 is expressed at significantly lower levels in CRC, and is associated with better overall 5-year survival in patients receiving radiation. Mechanistically, CELF2 increased levels of critical components of the autophagy cascade including Beclin-1, ATG5, and ATG12 by modulating mRNA stability. CELF2 also increased autophagic flux in CRC. IR significantly induced autophagy in CRC which correlates with increased levels of CELF2 and autophagy associated proteins. Silencing CELF2 with siRNA, mitigated IR induced autophagy. Moreover, knockdown of CELF2 in vivo conferred tumor resistance to IR. These studies elucidate an unrecognized role for CELF2 in inducing autophagy and potentiating the effects of radiotherapy in CRC.
Assuntos
Autofagia/fisiologia , Proteínas CELF/metabolismo , Sobrevivência Celular/efeitos da radiação , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Proteínas do Tecido Nervoso/metabolismo , Animais , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Proteínas CELF/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Células HCT116 , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Proteínas do Tecido Nervoso/genética , Prognóstico , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Radiação Ionizante , Transplante HeterólogoRESUMO
Trans-acting factors controlling mRNA fate are critical for the post-transcriptional regulation of inflammation-related genes, as well as for oncogene and tumor suppressor expression in human cancers. Among them, a group of RNA-binding proteins called "Adenylate-Uridylate-rich elements binding proteins" (AUBPs) control mRNA stability or translation through their binding to AU-rich elements enriched in the 3'UTRs of inflammation- and cancer-associated mRNA transcripts. AUBPs play a central role in the recruitment of target mRNAs into small cytoplasmic foci called Processing-bodies and stress granules (also known as P-body/SG). Alterations in the expression and activities of AUBPs and P-body/SG assembly have been observed to occur with colorectal cancer (CRC) progression, indicating the significant role AUBP-dependent post-transcriptional regulation plays in controlling gene expression during CRC tumorigenesis. Accordingly, these alterations contribute to the pathological expression of many early-response genes involved in prostaglandin biosynthesis and inflammation, along with key oncogenic pathways. In this review, we summarize the current role of these proteins in CRC development. CRC remains a major cause of cancer mortality worldwide and, therefore, targeting these AUBPs to restore efficient post-transcriptional regulation of gene expression may represent an appealing therapeutic strategy.
RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with dismal patient outcomes. The underlying core genetic drivers of disease have been identified in human tumor specimens and described in genetically engineered mouse models. These genetic drivers of PDAC include KRAS signaling, TP53 mutations, and genetic loss of the SMAD4 tumor suppressor protein. Beyond the known mutational landscape of PDAC genomes, alternative disrupted targets that extend beyond conventional genetic mutations have been elusive and understudied in the context of PDAC cell therapeutic resistance and survival. This last point is important because PDAC tumors have a unique and complex tumor microenvironment that includes hypoxic and nutrient-deprived niches that could select for cell populations that garner therapeutic resistance, explaining tumor heterogeneity in regards to response to different therapies. We and others have embarked in a line of investigation focused on the key molecular mechanism of posttranscriptional gene regulation that is altered in PDAC cells and supports this pro-survival phenotype intrinsic to PDAC cells. Specifically, the key regulator of this mechanism is a RNA-binding protein, HuR (ELAVL1), first described in cancer nearly two decades ago. Herein, we will provide a brief overview of the work demonstrating the importance of this RNA-binding protein in PDAC biology and then provide insight into ongoing work developing therapeutic strategies aimed at targeting this molecule in PDAC cells. This article is categorized under: RNA in Disease and Development > RNA in Disease.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Humanos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/tratamento farmacológicoRESUMO
Human antigen R (ELAVL1; HuR) is perhaps the best-characterized RNA-binding protein. Through its overexpression in various tumor types, HuR promotes posttranscriptional regulation of target genes in multiple core signaling pathways associated with tumor progression. The role of HuR overexpression in pancreatic tumorigenesis is unknown and led us to explore the consequences of HuR overexpression using a novel transgenic mouse model that has a >2-fold elevation of pancreatic HuR expression. Histologically, HuR-overexpressing pancreas displays a fibroinflammatory response and other pathological features characteristic of chronic pancreatitis. This pathology is reflected in changes in the pancreatic gene expression profile due, in part, to genes whose expression changes as a consequence of direct binding of their respective mRNAs to HuR. Older mice develop pancreatic steatosis and severe glucose intolerance. Elevated HuR cooperated with mutant K-rasG12D to result in a 3.4-fold increase in pancreatic ductal adenocarcinoma (PDAC) incidence compared to PDAC presence in K-rasG12D alone. These findings implicate HuR as a facilitator of pancreatic tumorigenesis, especially in the setting of inflammation, and a novel therapeutic target for pancreatitis treatment.
Assuntos
Proteína Semelhante a ELAV 1/genética , Neoplasias Pancreáticas/etiologia , Pancreatite/patologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Citoplasma/genética , Citoplasma/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Feminino , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pâncreas/patologia , Pâncreas/fisiologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Pancreatite/genética , Papiloma/etiologia , Papiloma/genética , Papiloma/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias PancreáticasRESUMO
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.
Assuntos
Proteína Semelhante a ELAV 1/química , Fenantrenos/química , Fenantrenos/farmacologia , Regiões 3' não Traduzidas , Elementos Ricos em Adenilato e Uridilato , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Furanos , Humanos , Espectroscopia de Ressonância Magnética , Camundongos Mutantes Neurológicos , Simulação de Dinâmica Molecular , Fenantrenos/metabolismo , Mutação Puntual , Conformação Proteica , Domínios Proteicos , Quinonas , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Neoplasias do Colo/genética , Proteína Semelhante a ELAV 1/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/patologia , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias do Colo/patologia , Proteína Semelhante a ELAV 1/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Camundongos Nus , Neoplasias Experimentais/genética , Neoplasias Pancreáticas/patologia , Fenótipo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.