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Endocrine therapy that blocks estrogen signaling is the most effective treatment for patients with estrogen receptor positive (ER+) breast cancer. However, the efficacy of agents such as tamoxifen (Tam) is often compromised by the development of resistance. Here we report that cytokines-activated nuclear IKKα confers Tam resistance to ER+ breast cancer by inducing the expression of FAT10, and that the expression of FAT10 and nuclear IKKα in primary ER+ human breast cancer was correlated with lymphotoxin ß (LTB) expression and significantly associated with relapse and metastasis in patients treated with adjuvant mono-Tam. IKKα activation or enforced FAT10 expression promotes Tam-resistance while loss of IKKα or FAT10 augments Tam sensitivity. The induction of FAT10 by IKKα is mediated by the transcription factor Pax5, and coordinated via an IKKα-p53-miR-23a circuit in which activation of IKKα attenuates p53-directed repression of FAT10. Thus, our findings establish IKKα-to-FAT10 pathway as a new therapeutic target for the treatment of Tam-resistant ER+ breast cancer.
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Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Quinase I-kappa B , Transdução de Sinais , Tamoxifeno , Animais , Feminino , Humanos , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Células MCF-7 , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
We have previously shown that the extracellular matrix and basement membrane protein Nidogen1 (NID1) is secreted by more malignant, mesenchymal-like CRC cells and induces the epithelial-mesenchymal transition (EMT) and promotes the migration and invasion of less malignant, epithelial-like CRC cells. Here, we performed a comprehensive bioinformatics analysis of multiple datasets derived from CRC patients and showed that elevated expression of NID1 and the genes ITGA3, ITGB1, and ITGAV, which encode NID1 receptors, is associated with poor prognosis and advanced tumor stage. Accordingly, the expression of NID1, ITGA3, ITGB1, and ITGAV was associated with an EMT signature, which included SNAIL/SNAI1, an EMT-inducing transcription factor. In CRC cells, ectopic SNAIL expression induced NID1 and SNAIL occupancy was detected at an E-box upstream of the NID1 transcription start site. Therefore, NID1 represents a direct target of SNAIL. Ectopic expression of NID1 or treatment with NID1-containing medium endowed non-metastatic CRC cells with the capacity to form lung metastases after xenotransplantation into mice. Suppression of the NID1 receptor ITGAV decreased cell viability, particularly in CMS/consensus molecular subtype 4 CRC cells. Taken together, our results show that NID1 is a direct target of EMT-TF SNAIL and is associated with and promotes CRC progression and metastasis. Furthermore, the NID1 receptor ITGAV represents a candidate therapeutic target in CMS4 colorectal tumors.
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Aspirin and its active metabolite salicylate have emerged as promising agents for the chemoprevention of colorectal cancer (CRC). Moreover, aspirin suppresses the progression of established CRCs. However, the underlying molecular mechanisms are not completely understood. Here we found that salicylate induces the expression of the miR-34a and miR-34b/c genes, which encode tumor suppressive microRNAs, in a p53-independent manner. Salicylate activated AMPK, thereby activating NRF2, which directly induced miR-34a/b/c expression via ARE motifs. In addition, salicylate suppressed c-MYC, a known repressor of NRF2-mediated transactivation, via activating AMPK. The suppression of c-MYC by salicylate was necessary for NRF2-mediated activation of miR-34a/b/c. Inactivation of miR-34a/b/c largely abrogated the inhibitory effects of salicylate on migration, invasion and metastasis formation by CRC cells. In the future, aspirin and its derivates may be used therapeutically to activate miR-34a and miR-34b/c in tumors that have lost p53.
Assuntos
Neoplasias Colorretais , MicroRNAs , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Salicilatos/farmacologia , Linhagem Celular Tumoral , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Aspirina/farmacologia , Regulação Neoplásica da Expressão GênicaRESUMO
Elevated expression of c-MYC and inactivation of p53 represent two of the most common alterations in colorectal cancer (CRC). However, c-MYC and defective p53 are difficult to target therapeutically. Therefore, effectors downstream of both c-MYC and p53 may represent attractive, alternative targets for cancer treatment. In a bioinformatics screen we identified Squalene epoxidase/SQLE as a candidate therapeutic target that appeared to be especially relevant for cell survival in CRCs, which display elevated c-MYC expression and loss of p53 function. SQLE is a rate-limiting enzyme in the cholesterol synthesis. Here, we show that p53 supresses SQLE expression, cholesterol levels, and cell viability via the induction of miR-205, which directly targets SQLE. Furthermore, c-MYC induced SQLE expression directly and via its target gene AP4. The transcription factor AP4/TFAP4 directly induced SQLE expression and cholesterol levels, whereas inactivation of AP4 resulted in decreased SQLE expression and caused resistance to Terbinafine, an inhibitor of SQLE. Inhibition of SQLE decreased viability of CRC cells. This effect was enhanced in CRCs cells with p53 inactivation and/or enhanced c-MYC/AP4 expression. Altogether, our results demonstrate that SQLE represents a vulnerability for CRCs with p53 inactivation and elevated c-MYC activity.
Assuntos
Neoplasias Colorretais , Esqualeno Mono-Oxigenase , Humanos , Esqualeno Mono-Oxigenase/genética , Proteína Supressora de Tumor p53/genética , Mutação , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , ColesterolRESUMO
Currently, there is no consensus about the use of adjuvant chemotherapy for patients with stage II colorectal cancer. Here, we aimed to identify and validate a prognostic mRNA expression signature for the stratification of patients with stage II colorectal cancer according to their risk for relapse. First, publicly available mRNA expression profiling datasets from 792 primary, stage II colorectal cancers from six different training cohorts were analyzed to identify genes that are consistently associated with patient relapse-free survival (RFS). Second, the identified gene expression signature was experimentally validated using NanoString technology and computationally refined on primary colorectal cancer samples from 205 patients with stage II colorectal cancer. Third, the refined signature was validated in two independent publicly available cohorts of 166 patients with stage II colorectal cancer. Bioinformatics analysis of training cohorts identified a 61-gene signature that was highly significantly associated with RFS (HR = 37.08, P = 2.68*10-106, sensitivity = 89.29%, specificity = 89.61%, and AUC = 0.937). The experimental validation and refinement revealed a 15-gene signature that robustly predicted relapse in three independent cohorts: an in-house cohort (HR = 20.4, P = 8.73*10-23, sensitivity = 90.32%, specificity = 80.99%, AUC = 0.812), GSE161158 (HR = 5.81, P = 3.57*10-4, sensitivity = 64.29%, specificity = 81.67%, AUC = 0.796), and GSE26906 (HR = 7.698, P = 7.26*10-8, sensitivity = 61.54%, specificity = 78.33%, AUC = 0.752). In the pooled training cohort, the 15-gene signature (HR = 4.72, P = 7.76*10-25, sensitivity = 75%, specificity = 67.44%, AUC = 0.784) was superior to the Oncotype DX colon 7-gene signature (HR = 2.698, P = 6.3*10-8, sensitivity = 62.16%, specificity = 55.5%, AUC = 0.633). We report the identification and validation of a novel mRNA expression signature for robust prognostication and stratification of patients with stage II colorectal cancer, with superior performance in the analyzed validation cohorts when compared with clinicopathologic biomarkers and signatures currently used for stage II colorectal cancer prognostication. Significance: We identified and validated a 15-gene expression signature for robust prognostication and stratification of patients with stage II colorectal cancer, with superior performance when compared with currently used biomarkers. Therefore, the 15-gene expression signature has the potential to improve the prognostication and treatment decisions for patients with stage II colorectal cancer.
Assuntos
Neoplasias Colorretais , Transcriptoma , Humanos , Prognóstico , Transcriptoma/genética , Neoplasias Colorretais/genética , RNA MensageiroRESUMO
Curcumin, a natural phytochemical isolated from tumeric roots, represents a candidate for prevention and therapy of colorectal cancer/CRC. However, the exact mechanism of action and the downstream mediators of curcumin's tumor suppressive effects have remained largely unknown. Here we used a genetic approach to determine the role of the p53/miR-34 pathway as mediator of the effects of curcumin. Three isogenic CRC cell lines rendered deficient for the p53, miR-34a and/or miR-34b/c genes were exposed to curcumin and subjected to cell biological analyses. siRNA-mediated inhibition and ectopic expression of NRF2, as well as Western blot, qPCR and qChIP analyses of its target genes were performed. CRC cells were i.v. injected into NOD/SCID mice and lung-metastases formation was determined by longitudinal, non-invasive imaging. In CRC cells curcumin induced apoptosis and senescence, and suppressed migration and invasion in a p53-independent manner. Curcumin activated the KEAP1/NRF2/ARE pathway by inducing ROS. Notably, curcumin induced miR-34a and miR-34b/c expression in a ROS/NRF2-dependent and p53-independent manner. NRF2 directly induced miR-34a and miR-34b/c via occupying multiple ARE motifs in their promoter regions. Curcumin reverted repression of miR-34a and miR-34b/c induced by IL6 and hypoxia. Deletion of miR-34a and miR-34b/c significantly reduced curcumin-induced apoptosis and senescence, and prevented the inhibition of migration and invasion by curcumin or ectopic NRF2. In CRC cells curcumin induced MET and prevented the formation of lung-metastases in mice in a miR-34a-dependent manner. In addition, we found that curcumin may enhance the therapeutic effects of 5-FU on CRC cells deficient for p53 and miR-34a/b/c. Activation of the KEAP1/NRF2/miR-34a/b/c axis mediates the tumor suppressive activity of curcumin and suggests a new approach for activating miR-34 genes in tumors for therapeutic purposes.
Assuntos
Neoplasias Colorretais , Curcumina , Neoplasias Pulmonares , MicroRNAs , Animais , Camundongos , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/terapia , Curcumina/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Camundongos Endogâmicos NOD , Camundongos SCID , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Tumor-associated hypoxia, i.e., decreased availability of oxygen, results in a poor clinical outcome since it promotes EMT, metastasis, and chemotherapy-resistance. We have previously identified p53 and its target miR-34a, as critical determinants of the effect of hypoxia on colorectal cancer (CRC). Here, we aimed to characterize mechanisms that contribute to the selective advantage of cells with loss of p53/miR-34a function in a hypoxic environment. Using in silico prediction, we identified XBP-1 and IRE1A as potential miR-34a targets. IRE1A and XBP-1 are central components of the unfolded protein response that is activated by ER stress, which is also induced in tumor cells as a response to harsh conditions surrounding tumors such as hypoxia and a limited supply of nutrients. Here we characterized the XBP-1(S) transcription factor and its regulator IRE1A as direct, conserved miR-34a targets in CRC cells. After hypoxia and DNA damage, IRE1A and XBP-1 were repressed by p53 in a miR-34a-dependent manner, whereas p53-deficient cells showed induction of IRE1A and XBP-1(S). Furthermore, miR-34a expression was directly suppressed by XBP-1(S). In p53-deficient CRC cells, hypoxia-induced EMT, migration, invasion, metastases formation, and resistance to 5-FU were dependent on IRE1A/XBP-1(S) activation. Hypoxia-induced autophagy was identified as an XBP-1(S)-dependent mediator of 5-FU resistance and was reversed by ectopic miR-34a expression. The HIF1A/IRE1A/XBP-1(S)/p53/miR-34a feedback loop described here represents a central regulator of the response to hypoxia and ER stress that maintains cellular homeostasis. In tumors, the inactivation of p53 and miR-34a may result in IRE1A/XPB-1(S)-mediated EMT and autophagy, which ultimately promotes metastasis and chemoresistance.
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Members of the microRNA-34/miR-34 family are induced by the p53 tumor suppressor and themselves possess tumor suppressive properties, as they inhibit the translation of mRNAs that encode proteins involved in processes, such as proliferation, migration, invasion, and metastasis. Here we performed a comprehensive integrative meta-analysis of multiple computational and experimental miR-34 related datasets and developed tools to identify and characterize novel miR-34 targets. A miR-34 target probability score was generated for every mRNA to estimate the likelihood of representing a miR-34 target. Experimentally validated miR-34 targets were strongly enriched among mRNAs with the highest scores providing a proof of principle for our analysis. We integrated the results from the meta-analysis in a user-friendly METAmiR34TARGET website (www.metamir34target.com/) that allows to graphically represent the meta-analysis results for every mRNA. Moreover, the website harbors a screen function, which allows to select multiple miR-34-related criteria/analyses and cut-off values to facilitate the stringent and comprehensive prediction of relevant miR-34 targets in expression data obtained from cell lines and tumors/tissues. Furthermore, information on more than 200 miR-34 target mRNAs, that have been experimentally validated so far, has been integrated in the web-tool. The website and datasets provided here should facilitate further investigation into the mechanisms of tumor suppression by the p53/miR-34 connection and identification of potential cancer drug targets.
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Colorectal cancer is one of the most common cancers and a major cause of mortality. Proinflammatory and antitumor immune responses play critical roles in colitis-associated colon cancer. CCL17, a chemokine of the C-C family and ligand for CCR4, is expressed by intestinal dendritic cells in the steady state and is upregulated during colitis in mouse models and inflammatory bowel disease patients. In this study, we investigated the expression pattern and functional relevance of CCL17 for colitis-associated colon tumor development using CCL17-enhanced GFP-knockin mice. CCL17 was highly expressed by dendritic cells but also upregulated in macrophages and intermediary monocytes in colon tumors induced by exposure to azoxymethane and dextran sodium sulfate. Despite a similar degree of inflammation in the colon, CCL17-deficient mice developed fewer tumors than did CCL17-competent mice. This protective effect was abrogated by cohousing, indicating a dependency on the microbiota. Changes in microbiota diversity and composition were detected in separately housed CCL17-deficient mice, and these mice were more susceptible to azoxymethane-induced early apoptosis in the colon affecting tumor initiation. Immune cell infiltration in colitis-induced colon tumors was not affected by the lack of CCL17. Taken together, our results indicate that CCL17 promotes colitis-associated tumorigenesis by influencing the composition of the intestinal microbiome and reducing apoptosis during tumor initiation.
Assuntos
Colite , Neoplasias do Colo , Microbioma Gastrointestinal , Camundongos , Animais , Carcinogênese , Transformação Celular Neoplásica , Azoximetano/toxicidade , Neoplasias do Colo/patologia , Quimiocina CCL17RESUMO
Growth and metastasis of colorectal cancer is closely connected to the biosynthetic capacity of tumor cells, and colorectal cancer stem cells that reside at the top of the intratumoral hierarchy are especially dependent on this feature. By performing disease modeling on patient-derived tumor organoids, we found that elevated expression of the ribosome biogenesis factor NLE1 occurs upon SMAD4 loss in TGFß1-exposed colorectal cancer organoids. TGFß signaling-mediated downregulation of NLE1 was prevented by ectopic expression of c-MYC, which occupied an E-box-containing region within the NLE1 promoter. Elevated levels of NLE1 were found in colorectal cancer cohorts compared with normal tissues and in colorectal cancer subtypes characterized by Wnt/MYC and intestinal stem cell gene expression. In colorectal cancer cells and organoids, NLE1 was limiting for de novo protein biosynthesis. Upon NLE1 ablation, colorectal cancer cell lines activated p38/MAPK signaling, accumulated p62- and LC3-positive structures indicative of impaired autophagy, and displayed more reactive oxygen species. Phenotypically, knockout of NLE1 inhibit.ed proliferation, migration and invasion, clonogenicity, and anchorage-independent growth. NLE1 loss also increased the fraction of apoptotic tumor cells, and deletion of TP53 further sensitized NLE1-deficient colorectal cancer cells to apoptosis. In an endoscopy-guided orthotopic mouse transplantation model, ablation of NLE1 impaired tumor growth in the colon and reduced primary tumor-derived liver metastasis. In patients with colorectal cancer, NLE1 mRNA levels predicted overall and relapse-free survival. Taken together, these data reveal a critical role of NLE1 in colorectal cancer growth and progression and suggest that NLE1 represents a potential therapeutic target in colorectal cancer patients. SIGNIFICANCE: NLE1 limits de novo protein biosynthesis and the tumorigenic potential of advanced colorectal cancer cells, suggesting NLE1 could be targeted to improve the treatment of metastatic colorectal cancer.
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Neoplasias Colorretais , Genes myc , Proteínas dos Microfilamentos , Proteína Smad4 , Animais , Camundongos , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Proteínas dos Microfilamentos/genética , Biossíntese de Proteínas , Proteína Smad4/genética , Regulação para Cima , HumanosRESUMO
BACKGROUND: AP4 (TFAP4) encodes a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor and is a direct target gene of the oncogenic transcription factor c-MYC. Here, we set out to determine the relevance of AP4 in human colorectal cancer (CRC) cells. METHODS: A CRISPR/Cas9 approach was employed to generate AP4-deficient CRC cell lines with inducible expression of c-MYC. Colony formation, ß-gal staining, immunofluorescence, comet and homologous recombination (HR) assays and RNA-Seq analysis were used to determine the effects of AP4 inactivation. qPCR and qChIP analyses was performed to validate differentially expressed AP4 targets. Expression data from CRC cohorts was subjected to bioinformatics analyses. Immunohistochemistry was used to evaluate AP4 targets in vivo. Ap4-deficient APCmin/+ mice were analyzed to determine conservation. Immunofluorescence, chromosome and micronuclei enumeration, MTT and colony formation assays were used to determine the effects of AP4 inactivation and target gene regulation on chromosomal instability (CIN) and drug sensitivity. RESULTS: Inactivation of AP4 in CRC cell lines resulted in increased spontaneous and c-MYC-induced DNA damage, chromosomal instability (CIN) and cellular senescence. AP4-deficient cells displayed increased expression of the long non-coding RNA MIR22HG, which encodes miR-22-3p and was directly repressed by AP4. Furthermore, Mediator of DNA damage Checkpoint 1 (MDC1), a central component of the DNA damage response and a known target of miR-22-3p, displayed decreased expression in AP4-deficient cells. Accordingly, MDC1 was directly induced by AP4 and indirectly by AP4-mediated repression of miR-22-3p. Adenomas and organoids from Ap4-deficient APCmin/+ mice displayed conservation of these regulations. Inhibition of miR-22-3p or ectopic MDC1 expression reversed the increased senescence, DNA damage, CIN and defective HR observed in AP4-deficient CRC cells. AP4-deficiency also sensitized CRC cells to 5-FU treatment, whereas ectopic AP4 conferred resistance to 5-FU in a miR-22-3p and MDC1-dependent manner. CONCLUSIONS: In summary, AP4, miR-22-3p and MDC1 form a conserved and coherent, regulatory feed-forward loop to promote DNA repair, which suppresses DNA damage, senescence and CIN, and contributes to 5-FU resistance. These findings explain how elevated AP4 expression contributes to development and chemo-resistance of colorectal cancer after c-MYC activation.
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Neoplasias Colorretais , MicroRNAs , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Instabilidade Cromossômica , Neoplasias Colorretais/genética , Dano ao DNA , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND & AIMS: Patient-derived tumor organoids recapitulate the characteristics of colorectal cancer (CRC) and provide an ideal platform for preclinical evaluation of personalized treatment options. We aimed to model the acquisition of chemotolerance during first-line combination chemotherapy in metastatic CRC organoids. METHODS: We performed next-generation sequencing to study the evolution of KRAS wild-type CRC organoids during adaptation to irinotecan-based chemotherapy combined with epidermal growth factor receptor (EGFR) inhibition. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 protein (Cas9)-editing showed the specific effect of KRASG12D acquisition in drug-tolerant organoids. Compound treatment strategies involving Aurora kinase A (AURKA) inhibition were assessed for their capability to induce apoptosis in a drug-persister background. Immunohistochemical detection of AURKA was performed on a patient-matched cohort of primary tumors and derived liver metastases. RESULTS: Adaptation to combination chemotherapy was accompanied by transcriptomic rather than gene mutational alterations in CRC organoids. Drug-tolerant cells evaded apoptosis and up-regulated MYC (c-myelocytomatosis oncogene product)/E2F1 (E2 family transcription factor 1) and/or interferon-α-related gene expression. Introduction of KRASG12D further increased the resilience of drug-persister CRC organoids against combination therapy. AURKA inhibition restored an apoptotic response in drug-tolerant KRAS-wild-type organoids. In dual epidermal growth factor receptor (EGFR)- pathway blockade-primed CRC organoids expressing KRASG12D, AURKA inhibition augmented apoptosis in cases that had acquired increased c-MYC protein levels during chemotolerance development. In patient-matched CRC cohorts, AURKA expression was increased in primary tumors and derived liver metastases. CONCLUSIONS: Our study emphasizes the potential of patient-derived CRC organoids in modeling chemotherapy tolerance ex vivo. The applied therapeutic strategy of dual EGFR pathway blockade in combination with AURKA inhibition may prove effective for second-line treatment of chemotolerant CRC liver metastases with acquired KRAS mutation and increased AURKA/c-MYC expression.
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Neoplasias Colorretais , Neoplasias Hepáticas , Aurora Quinase A/genética , Aurora Quinase A/farmacologia , Aurora Quinase A/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Organoides/metabolismoRESUMO
Although the 14-3-3σ gene was initially identified as a p53 target gene in colorectal cancer cells, its potential role in intestinal tumorigenesis has remained unknown. Here we determined that 14-3-3σ expression is significantly downregulated in primary human colorectal cancer when compared with adjacent normal colonic tissue in patient samples. Downregulation of 14-3-3σ in primary colorectal cancers was significantly associated with p53 mutation, increasing tumor stage, distant metastasis, and poor patient survival. Poor survival was more significantly associated with decreased 14-3-3σ expression in p53 wild-type than in p53-mutant colorectal cancers. 14-3-3σ expression was detected in enterocytes of the transit amplifying zone and gradually increased towards the apical villi in the small intestinal epithelium. In small and large intestinal epithelia and adenomas, 14-3-3σ expression was upregulated in differentiated areas. Deletion of 14-3-3σ in ApcMin mice increased the number and size of adenomas in the small intestine and colon, shortening the median survival by 64 days. 14-3-3σ-deficient adenomas displayed increased proliferation and decreased apoptosis, as well as increased dysplasia. In adenomas, loss of 14-3-3σ promoted acquisition of a mesenchymal-like gene expression signature, which was also found in colorectal cancers from patients with poor relapse-free survival. The transcriptional programs controlled by the 14-3-3σ-interacting factors SNAIL, c-JUN, YAP1, and FOXO1 were activated by deletion of 14-3-3σ, potentially contributing to the enhanced tumor formation and growth. Taken together, these results provide genetic evidence of a tumor-suppressor function of 14-3-3σ in the intestine. SIGNIFICANCE: Downregulation of 14-3-3σ in colorectal cancer is associated with metastasis and poor survival of patients, and its inactivation in a murine tumor model drives intestinal tumor formation and epithelial-mesenchymal transition.
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Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinogênese/patologia , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal , Exorribonucleases/metabolismo , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/patologia , Proteínas 14-3-3/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Exorribonucleases/genética , Feminino , Genes APC , Genes Supressores de Tumor , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prognóstico , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: The miR-34a gene is a direct target of p53 and is commonly silenced in colorectal cancer (CRC). Here we identified the receptor tyrosine kinase CSF1R as a direct miR-34a target and characterized CSF1R as an effector of p53/miR-34a-mediated CRC suppression. METHODS: Analyses of TCGA-COAD and three other CRC cohorts for association of mRNA expression and signatures with patient survival and molecular subtypes. Bioinformatics identification and experimental validation of miRNA and transcription factor targets. Functional analysis of factors/pathways in the regulation of epithelial-mesenchymal transition (EMT), invasion, migration, acquired chemo-resistance and metastasis. Analyses of protein expression and CpG methylation within primary human colon cancer samples. RESULTS: In primary CRCs increased CSF1R, CSF1 and IL34 expression was associated with poor patient survival and a mesenchymal-like subtype. CSF1R displayed an inverse correlation with miR-34a expression. This was explained by direct inhibition of CSF1R by miR-34a. Furthermore, p53 repressed CSF1R via inducing miR-34a, whereas SNAIL induced CSF1R both directly and indirectly via repressing miR-34a in a coherent feed-forward loop. Activation of CSF1R induced EMT, migration, invasion and metastasis of CRC cells via STAT3-mediated down-regulation of miR-34a. 5-FU resistance of CRC cells was mediated by CpG-methylation of miR-34a and the resulting elevated expression of CSF1R. In primary CRCs elevated expression of CSF1R was detected at the tumor invasion front and was associated with CpG methylation of the miR-34a promoter as well as distant metastasis. CONCLUSIONS: The reciprocal inhibition between miR-34a and CSF1R and its loss in tumor cells may be relevant for therapeutic and prognostic approaches towards CRC management.
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Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Linhagem Celular Tumoral , Colectomia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Ilhas de CpG/genética , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética , Transição Epitelial-Mesenquimal/genética , Retroalimentação Fisiológica , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Estimativa de Kaplan-Meier , Masculino , MicroRNAs/genética , Protectomia , Prognóstico , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Reto/patologia , Reto/cirurgia , Fator de Transcrição STAT3/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND & AIMS: Intratumor heterogeneity is a common feature of colorectal cancer (CRC). Here, we analyzed whether mesenchymal-like CRC cells promote the progression of epithelial-like CRC cells via paracrine mechanisms. METHODS: Six CRC cell lines that show an epithelial phenotype were treated with conditioned media (CM) from CRC cell lines that show a mesenchymal phenotype, and effects on epithelial-mesenchymal transition (EMT), migration, invasion, and chemoresistance were determined. Secreted factors potentially mediating these effects were identified by using cytokine arrays. Associations of these factors with tumor progression and patient survival were determined. RESULTS: CM obtained from mesenchymal-like CRC cells induced EMT associated with increased migration, invasion, and chemoresistance in epithelial-like CRC cell lines. Notably, activation of p53 in mesenchymal-like CRC cells prevented these effects of CM. Increased concentrations of several cytokines were identified in CM from mesenchymal-like CRC cell lines and a subset of these cytokines showed repression by p53. The down-regulation of nidogen-1 (NID1) was particularly significant and was owing to p53-mediated induction of microRNA-192 and microRNA-215, which directly target the NID1 messenger RNA. NID1 was found to be required and sufficient for inducing EMT, invasion, and migration in epithelial-like CRC cells. In primary CRCs, increased NID1 expression was associated with p53 mutation and microRNA-192/215 down-regulation. Importantly, increased NID1 expression in CRCs correlated with enhanced tumor progression and poor patient survival. CONCLUSIONS: Taken together, our results show that CRC cells promote tumor progression via secreting NID1, which induces EMT in neighboring tumor cells. Importantly, the interference of p53 with this paracrine signaling between tumor cells may critically contribute to tumor suppression.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal , Glicoproteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Comunicação Parácrina , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Meios de Cultivo Condicionados/farmacologia , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/genética , PrognósticoRESUMO
BACKGROUND & AIMS: Combined inactivation of the microRNA 34a gene (MIR34A, by methylation) and the TP53 gene (by mutation or deletion) is observed in 50% of colorectal tumors that progress to distant metastases. We studied mice with intestinal disruption of Mir34a and Tp53 to investigate mechanisms of colorectal carcinogenesis and identify strategies to block these processes. METHODS: Mice with disruption of Mir34a and/or Tp53 specifically in intestinal epithelial cells (IECs) (Mir34aΔIEC mice, Tp53ΔIEC mice, and Mir34aΔIEC/Tp53ΔIEC mice) and controls (Mir34aFl/Fl/Tp53Fl/Fl) were given azoxymethane to induce colorectal carcinogenesis. Some mice were given intraperitoneal injections of an antibody against mouse interleukin 6 receptor (IL6R), or received an inhibitor of PAI1 (tiplaxtinin) in their chow. Intestinal tissues were collected and analyzed by immunohistochemistry; gene expression profiles were analyzed by RNA sequencing. We determined the expression and localization of PAI1 in 61 human primary colon cancers and compared them to MIR34A methylation and inactivating mutations in TP53. Data on mRNA levels, methylation, and clinical features of 628 colon and rectal adenocarcinomas were obtained from The Cancer Genome Atlas portal. RESULTS: Mir34aΔIEC/Tp53ΔIEC mice developed larger and more colorectal tumors, with increased invasion of surrounding tissue and metastasis to lymph nodes, than control mice or mice with disruption of either gene alone. Cells in tumors from the Mir34aΔIEC/Tp53ΔIEC mice had decreased apoptosis and increased proliferation compared to tumor cells from control mice, and expressed higher levels of genes, that regulate inflammation (including Il6r and Stat3) and epithelial-mesenchymal transition. The gene expression pattern of the tumors from Mir34aΔIEC/Tp53ΔIEC mice was similar to that of human colorectal tumor consensus molecular subtype 4 (mesenchymal, invasive). We identified the Pai1 messenger RNA as a target of Mir34a; levels of PAI1 protein were increased in primary colon cancer samples, that displayed methylation of MIR34A and mutational inactivation of TP53. Administration of tiplaxtinin or anti-IL6R antibody to Mir34aΔIEC/Tp53ΔIEC mice decreased proliferation of cancer cells, and reduced colorectal tumor invasion and metastasis. CONCLUSIONS: In mice, we demonstrated that combined inactivation of Mir34a and Tp53 promotes azoxymethane-induced colorectal carcinogenesis and tumor progression and metastasis by increasing levels of IL6R and PAI1. Strategies to inhibit these processes might be developed to slow progression of colorectal cancer.
Assuntos
Neoplasias Colorretais/genética , Inativação Gênica , Genes p53 , MicroRNAs/genética , Receptores de Interleucina-6/metabolismo , Serpina E2/metabolismo , Animais , Azoximetano , Carcinógenos , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Progressão da Doença , Células Epiteliais/fisiologia , Mucosa Intestinal/citologia , CamundongosRESUMO
Epithelial-mesenchymal transition (EMT) plays an important role in tumor invasion and metastasis. A comprehensive, bioinformatics analysis of CCLE and TCGA datasets of seven tumor types allowed us to identify a novel pan-cancer EMT-associated gene expression signature consisting of 16 epithelial and 4 mesenchymal state-associated mRNAs. Among the identified epithelial cell state-associated factors, down-regulation of the RBM47 (RNA binding motif protein 47) mRNA displayed the most significant association with metastasis and poor survival in multiple cohorts of colorectal cancer (CRC) patients. Moreover, decreased RBM47 protein expression was associated with metastasis in a cohort of primary CRCs. RBM47 was directly suppressed during EMT induced by IL6-activated STAT3 or ectopic SNAIL and SLUG expression via conserved binding motifs of these factors within the RBM47 promoter. Moreover, RNAi-mediated down-regulation of RBM47 in CRC lines resulted in increased cell migration, invasion and metastases formation. As demonstrated by the example of RBM47, the EMT-associated signature characterized here allows to identify biomarkers for predicting clinical outcome of CRC and presumably other cancer entities. In addition, our functional analysis of RBM47 shows that the down-regulation of RBM47 during CRC progression may promote EMT and metastasis.
Assuntos
Neoplasias Colorretais/patologia , Regulação para Baixo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Bases de Dados Genéticas , Progressão da Doença , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Camundongos , Metástase Neoplásica , Estadiamento de Neoplasias , Transplante de Neoplasias , Proteínas de Ligação a RNA/química , Análise de Sequência de RNA , Análise de SobrevidaRESUMO
BACKGROUND & AIMS: In colorectal tumors, hypoxia causes resistance to therapy and promotes metastasis. Loss of the tumor suppressor p53 (encoded by TP53) provides cancer cells with a selective advantage under conditions of hypoxia, but little is known about the mediators of this effect. METHODS: Isogenic colorectal cancer (CRC) cell lines with different TP53 genotypes were placed under conditions of hypoxia. We examined the effects on levels and activity of microRNA-34a (MIR34A) in CRC cells. We determined the expression and localization of protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11, also called INH3, HCGV, IPP3, HCGV, TCTE5, TCTEX5, or CFAP255) in 82 human colon cancers. We analyzed data on human colorectal carcinomas from the Cancer Genome Atlas collection to determine whether expression of PPP1R11 was affected by altered level or activity of p53, markers of epithelial-to-mesenchymal transition (EMT), or MIR34A or was associated with metastasis. We determined the effects of disruption Mir34a, Mir34b, and Mir34c in ApcMin/+ mice. DLD-1 cells were transfected with small inhibitor RNAs against PPP1R1, injected into the tail veins of immune-compromised mice, and followed by noninvasive bioluminescence imaging. RESULTS: The hypoxia inducible factor 1 alpha subunit (HIF1A) directly repressed the MIR34A gene in p53-defective CRC cells, whereas expression of MIR34A was induced in p53-proficient CRC cells exposed to hypoxia. Down-regulation of MIR34A was required for hypoxia-induced EMT, invasion and migration, and activation of STAT3 in CRC cells. We identified PPP1R11, whose product inhibits PP1, as a target of MIR34A. PPP1R11 mediates phosphorylation (activation) of STAT3, so expression of MIR34A reduced activation of STAT3 in p53-deficient CRC cells. Ectopic expression of PPP1R11 in CRC cells induced EMT, invasion, and migration, which all required STAT3. Increased expression of PPP1R11 in p53-deficient CRC cells was required for hypoxia-induced EMT, invasion, migration, and resistance to 5-fluorouracil, as well as metastasis of xenograft tumors to lungs of mice. Adenomas and derived tumoroids of ApcMin/+ mice with disruption of Mir34a, Mir34b, and Mir34c had increased levels of PPP1R11. Colorectal tumors from patients had increased levels of PPP1R11 at areas of invasion, compared with other areas of the tumor; increased level PPP1R11 associated with TP53 mutations and metastasis to the liver. CONCLUSIONS: HIF1A represses, whereas p53 increases, expression of MIR34A in CRC cells. MIR34A reduces expression of PPP1R11 to prevent activation of STAT3 and inhibit the EMT and metastasis. Strategies to target this pathway might be developed to inhibit CRC metastasis and overcome resistance to therapy associated with hypoxia.
Assuntos
Neoplasias Colorretais/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Genes p53/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , MicroRNAs/metabolismo , Hipóxia Tumoral/fisiologia , Adenoma/genética , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Genótipo , Humanos , Hipóxia/genética , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , MicroRNAs/genética , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/fisiologia , Fator de Transcrição STAT3/genética , Hipóxia Tumoral/genética , Ubiquitina-Proteína LigasesRESUMO
Here, we developed and comprehensively characterized a cellular model of colon cancer progression consisting of four defined derivatives of a colon cancer cell line that resulted from consecutive epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT/MET) and phenotypically recapitulate the metastatic cascade. Initial EMT was induced by prolonged exposure to IL6, a cytokine also generated by the tumor-stroma. Genome-wide characterization of transcriptional (mRNA, miRNA, and lncRNA) and epigenetic (DNA methylation, H3K4me3, H3K79me3, and H3K27me3 histone modifications) profiles of the cell derivatives, combined with correlative analyses of expression, methylation, and clinical data from the TCGA-COAD database gave insights into the molecular basis of their phenotypic changes. The signatures characterizing invasive, mesenchymal-like cell states as well as the metastases-derived epithelial-like state showed significant association with metastasis, positive nodal status, and poor survival of colon cancer patients. Global hypomethylation of gene-regulatory regions was observed during tumor progression, with the lowest degree of methylation present in cells isolated from metastases. Upregulation of an axon-guidance-related gene signature was the most significant feature of metastatic tumor cells and was also found in primary tumors from colon cancer patients with distant metastases. Furthermore, the microRNAs miR-99a, miR-100, and miR-125b showed elevated expression in mesenchymal-like cells, associated with poor survival, and promoted migration and invasion. Finally, elevated expression of H19 lncRNA due to promoter demethylation was observed in cells isolated from metastases and was associated with poor survival of colon cancer patients. In the future, our results may be further exploited for the discovery and evaluation of novel metastasis-associated mechanisms and biomarkers. Cancer Res; 77(8); 1854-67. ©2017 AACR.