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1.
Nat Commun ; 11(1): 3214, 2020 06 25.
Article in English | MEDLINE | ID: mdl-32587247

ABSTRACT

Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMR-depleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.


Subject(s)
Gene Expression Regulation, Neoplastic , Nucleotides/metabolism , RNA, Long Noncoding/genetics , Ribonucleoside Diphosphate Reductase , Y-Box-Binding Protein 1 , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Gene Silencing , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Ribonucleoside Diphosphate Reductase/genetics , Ribonucleoside Diphosphate Reductase/metabolism , Y-Box-Binding Protein 1/genetics , Y-Box-Binding Protein 1/metabolism
2.
Int J Cancer ; 141(9): 1879-1890, 2017 11 01.
Article in English | MEDLINE | ID: mdl-28656629

ABSTRACT

The tumor suppressor P53 is a critical regulator of normal cellular homeostasis whose function is either distorted or lost in several cancer types including colorectal cancer (CRC). A small group of microRNAs have come to be recognized as essential mediators of P53 function. In a genome-wide systematic approach, we explored miRNAs that are substantially altered by P53 loss and found miR-30e to be the most significantly deregulated miRNA in P53-knockout human CRC cells. We identified miR-30e-5p to be a novel direct transcriptional target of P53 with gain and loss of function experiments revealing miR-30e-5p to be a significant regulator of tumor cell migration, invasion and in vivo metastasis mediated in part by integrins alpha-6 and beta-1 as novel targets. MiR-30e-5p also significantly reduced tumor cell proliferation by causing G1/S cell cycle arrest, which was achieved by inducing P21 and P27 expression. Finally, we found miR-30e-5p to be lost in resected CRC tumors as compared to normal colon tissues. Taken together, miR-30e-5p is a novel effector of P53-induced suppression of migration, invasion and metastasis.


Subject(s)
Colorectal Neoplasms/genetics , Integrin alpha6/genetics , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , MicroRNAs/genetics , Tumor Suppressor Protein p53/genetics , Adaptor Proteins, Signal Transducing , Animals , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Colorectal Neoplasms/pathology , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p27/genetics , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Mice , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasm Metastasis , Xenograft Model Antitumor Assays
3.
Int J Cancer ; 125(4): 894-901, 2009 Aug 15.
Article in English | MEDLINE | ID: mdl-19480010

ABSTRACT

The identification of high-risk patients with thyroid cancer and the preoperative differentiation between follicular adenoma and carcinoma remain clinically challenging. Our study was conducted to analyze whether the quantification of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator receptor (u-PAR) and transcription factor binding to the u-PAR promoter improve prognostic predictability and differential diagnosis of thyroid tumors. Tumor/normal tissue was collected from 69 prospectively followed patients with thyroid carcinomas (papillary, medullary, follicular and anaplastic, PTC, MTC, FTC and ATC) or follicular adenomas. U-PAR, MMP-1, MMP-7 and MMP-9 amounts were determined by ELISA, and transcription factor binding was determined by electrophoretic mobility shift assay. Binding of transcription factors to the u-PAR promoter was observed, but not associated with u-PAR expression. Carcinomas except MTC expressed significantly more u-PAR/MMPs than adenomas/normal tissues, this being associated with advanced pT- or M-stages. MMP-1 and MMP-9 were significantly higher in follicular carcinomas than in adenomas. In carcinomas, high u-PAR-gene expression correlated significantly with high MMP-9, the latter being associated with MMP-7 in normal tissues. Poor survival in differentiated tumors was associated in trend (p = 0.07); poor survival of all patients (p = 0.043) and especially of patients with carcinomas of follicular origin (including ATC), but not medullary carcinomas, were significantly associated with high u-PAR-protein (p = 0.015). Quantification of u-PAR is of prognostic relevance in thyroid carcinomas of non-c-cell origin, and u-PAR in part may be regulated nontranscriptionally in thyroid cancers. This is the first study to suggest MMP-1/-9 as significant differentiation markers between follicular adenoma and follicular carcinoma.


Subject(s)
Adenoma/metabolism , Biomarkers, Tumor/metabolism , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 9/metabolism , Receptors, Urokinase Plasminogen Activator/metabolism , Thyroid Neoplasms/metabolism , Adenocarcinoma, Follicular/metabolism , Adenocarcinoma, Follicular/pathology , Adenoma/pathology , Adult , Aged , Blotting, Western , Carcinoma, Papillary/metabolism , Carcinoma, Papillary/pathology , Electrophoretic Mobility Shift Assay , Female , Humans , Immunoenzyme Techniques , Luciferases , Male , Matrix Metalloproteinase 7/metabolism , Middle Aged , Prognosis , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Urokinase Plasminogen Activator/genetics , Reverse Transcriptase Polymerase Chain Reaction , Thyroid Neoplasms/pathology , Transfection
4.
Cancer ; 110(8): 1697-707, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-17849461

ABSTRACT

BACKGROUND: Programmed cell death 4 (Pdcd4) inhibits malignant transformation, and initial studies of Pdcd4 suggested the regulation of Pdcd4 localization by protein kinase B (Akt). However, supporting patient tissue data are missing, and the diagnostic/prognostic potential of Pdcd4 rarely has been studied. The objectives of the current were 1) to determine Pdcd4 as a diagnostic marker in the adenoma-carcinoma sequence, 2) to support phosphorylated Akt (pAkt)-mediated Pdcd4 regulation in vivo, and 3) to obtain the first prognostic evidence of Pdcd4 in colorectal cancer. METHODS: Tumor samples and normal tissues from 71 patients with colorectal cancer who were followed prospectively (median follow-up, 36 months) and 42 adenomas were analyzed for Pdcd4, Akt, and pAkt in immunohistochemical and Western blot analyses. RESULTS: A significant reduction in Pdcd4 was observed between normal mucosa and adenomas and between adenomas and tumor samples (P < .01 and P < .01, respectively). Normal mucosa demonstrated strong nuclear Pdcd4, which was reduced significantly in adenomas (P < .01) and almost was lost in tumors (P < .01). pAkt was correlated inversely with Pdcd4 and with the transition of Pdcd4 from nucleus to cytoplasm (P < .01). Kaplan-Meier analysis (using the Mantel-Cox log-rank test) indicated a significant correlation between the loss of total and nuclear Pdcd4 in tumors and overall survival (P < .05 and P < .02, respectively) and disease-specific survival (P < .01 and P < .01, respectively). In multivariate analysis, loss of total or nuclear Pdcd4 was an independent predictor of disease-specific or overall survival. CONCLUSIONS: To the authors' knowledge, this is the first study to demonstrate an independent prognostic impact of Pdcd4 and its expression pattern in colorectal cancer. Data from this study support the regulation of Pdcd4 localization by pAkt in vivo. Pdcd4 immunohistochemistry may be useful as a supportive diagnostic tool for the transition between normal, adenoma, and tumor tissues.


Subject(s)
Adenocarcinoma/metabolism , Adenoma/metabolism , Apoptosis Regulatory Proteins/metabolism , Biomarkers, Tumor/metabolism , Colorectal Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA-Binding Proteins/metabolism , Adenocarcinoma/pathology , Adenocarcinoma/surgery , Adenoma/pathology , Adenoma/surgery , Colon/metabolism , Colon/pathology , Colorectal Neoplasms/pathology , Colorectal Neoplasms/surgery , Humans , Neoplasm Recurrence, Local/metabolism , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Phosphorylation , Prognosis , Prospective Studies , Rectum/metabolism , Rectum/pathology , Survival Rate
5.
Clin Cancer Res ; 13(4): 1123-32, 2007 Feb 15.
Article in English | MEDLINE | ID: mdl-17317820

ABSTRACT

PURPOSE: Prognostic studies on transcription factors acting at specific promoter elements have never been done so far. However, in tumors with long necessary follow-up, such as colorectal cancer, early-risk predictors would be needed. The invasion-related gene u-PAR is regulated via an activator protein 2 (AP-2)/Sp1 (-152/-135) and an AP-1 binding promoter motif (-190/-171), mediating u-PAR induction by K-Ras and Src. The present study was done to give first evidence for early prognostic relevance of transcription factors differentially bound to the u-PAR promoter, and their molecular inducers, in colorectal cancer. EXPERIMENTAL DESIGN: Tumor/normal tissues of 92 prospectively followed (median = 26.3 months) patients were analyzed for Src activity/protein, K-ras mutations, and transcription factor binding to both u-PAR promoter motifs (in vivo gel shift, kinase assay, and PCR). RESULTS: Kaplan-Meier/Mantel-Cox analysis showed a significant correlation among elevated Sp1/Sp3 binding to region -152/-135 (P = 0.002 and P = 0.006), the combinations of Sp1/AP-2 and Sp1/AP-1 binding to both motifs (P = 0.010 and P = 0.005), and Sp1 binding/high Src protein in tumors (P < 0.001), with poor survival. Survival decreased with the number of bound transcription factors to both motifs, with binding of three factors defining a high-risk group (P = 0.021). In multivariate analysis, elevated Sp1 binding, combinations of Sp1/AP-2 binding and Sp1/AP-1 binding, or Sp1 binding/high Src were independent prognostic variables; u-PAR expression itself being not yet prognostic. A first molecular staging model (CART) was defined, providing novel early high-risk groups (mean survival time as low as for non-curatively resected patients) from these variables. CONCLUSIONS: This study defines transcription factors acting at specific promoter elements of an invasion-related gene, mediating specific signaling, as novel, independent, early predictors of prognosis in colorectal cancer.


Subject(s)
Colorectal Neoplasms/genetics , Transcription Factors/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/surgery , Female , Genes, ras , Genes, src , Humans , Male , Mutation , Prognosis , Promoter Regions, Genetic , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, Urokinase Plasminogen Activator , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Survival Rate , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Transcription Factor AP-2/genetics , Transcription Factor AP-2/metabolism , Transcription Factors/metabolism , Transcription, Genetic , src-Family Kinases/genetics , src-Family Kinases/metabolism
6.
Clin Cancer Res ; 11(24 Pt 1): 8538-48, 2005 Dec 15.
Article in English | MEDLINE | ID: mdl-16361535

ABSTRACT

PURPOSE: Studies on the transactivation of genes via promoter elements have mostly been done on cell lines rather than resected tissues. This, however, is essential to address an in vivo or clinical relevance. We have previously shown tumor-specific binding of Sp1 and an activator protein (AP)-2-related factor to promoter region -152/-135 of the metastasis-related u-PAR gene in 60% of in vivo-resected cancer tissues. Cell lines have implicated an additional role, and potential synergism, of an AP-1 region (-190/-171) in u-PAR regulation. This study was done to (a) analyze AP-1 binding to this region in resected tumor and normal tissues, and define subgroups in which it is tumor-specific, and (b) to analyze transcription factor-binding patterns to both promoter motifs in resected tissues, supporting synergism, and draw first prognostic conclusions. EXPERIMENTAL DESIGN: In 103 patients with colorectal cancer, electrophoretic mobility shift assay/supershift analysis for u-PAR promoter region -190/-171 was done in tumors and normal tissues. In 71 patients, region -152/-135 was also analyzed. U-PAR protein was measured by ELISA. RESULTS: Tumor-specific AP-1 binding to region -190/-171 of the u-PAR promoter was found in 40% of patients. Subgroup analysis showed tumor-specific binding for c-Fos in 58%, for c-Jun in 50%, for JunD in 39%, and for Fra-1 in 4% of cases. AP-1 binding correlated significantly with u-PAR protein amounts in both normal and tumor tissues (P<0.001), in contrast to a tumor-specific correlation with u-PAR of the AP-2/Sp1 region. In analyses for both promoter regions, 62% of cancers showed simultaneous binding for AP-1, AP-2, and Sp1, 11% for AP-1 and AP-2, 16% for AP-2 and Sp1, 4% for AP-2 only, 3% for AP-1 only, and 0% for Sp1 only. The binding of AP-1, AP-2, and Sp1 correlated significantly with each other (P<0.001), the combination of AP-1 and AP-2 showing the highest correlation with u-PAR (P=0.008). Preliminary survival analysis indicated a trend for poorer prognosis for binding of all three transcription factors. CONCLUSION: This is the first study differentiating transcription factor-binding to two important u-PAR promoter regions in a large series of resected tumors and normal tissues. The AP-1 site seems to be a less tumor-specific regulator than the Sp1/AP-2 motif. Nevertheless, data corroborate the hypothesis of synergism between both elements in resected tumors.


Subject(s)
Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/genetics , Receptors, Cell Surface/genetics , Transcription Factors/metabolism , Adult , Aged , Aged, 80 and over , Colorectal Neoplasms/surgery , Electrophoretic Mobility Shift Assay , Female , Humans , Male , Middle Aged , Prognosis , Promoter Regions, Genetic/genetics , Receptors, Cell Surface/analysis , Receptors, Urokinase Plasminogen Activator , Sp1 Transcription Factor/analysis , Sp1 Transcription Factor/metabolism , Transcription Factor AP-1/analysis , Transcription Factor AP-1/metabolism , Transcription Factor AP-2/analysis , Transcription Factor AP-2/metabolism
7.
Clin Cancer Res ; 9(6): 2267-76, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12796395

ABSTRACT

PURPOSE: Evidence for transactivation of genes via specific promoter elements has been derived from studies on tumor cell lines but rarely on resected tumors. However, the proof of an in vivo relevance and the identification of patients with a potential tumor-specific gene expression is essential to transfer molecular-targeting strategies into clinical applications. This study gives the first clinical evidence that urokinase-type plasminogen activator receptor (u-PAR) gene expression is tumor-specifically regulated via an activator protein (AP)-2/Sp1 promoter element in a large patient subpopulation. EXPERIMENTAL DESIGN: In 145 gastrointestinal cancer patients, electrophoretic mobility shift analysis and supershift assays for u-PAR-promoter region -152/-135 were performed in tumors and corresponding normal tissues. u-PAR protein levels were measured by ELISA. RESULTS: Binding of Sp1 to region -152/-135 in tumors in contrast to corresponding normal mucosae was observed in 55% of colorectal and in 52% of gastric cancer patients. Tumor-specific binding of an AP-2-related factor was seen in 59% of colorectal and in 63% of gastric cancer patients. A significant correlation between AP-2 (P < 0.0001) and Sp1 (P = 0.0003) binding with a high u-PAR expression was observed in tumors but not in normal mucosae. Tissues of five nontumor patients did not show transcription factor binding to this region. CONCLUSIONS: This is the first study to show the tumor-specific binding of trans-activators to the u-PAR promoter region (-152/-135) biochemically in a large series of resected tumors. For the subpopulation of approximately 60% of patients with tumor-restricted u-PAR-transactivation, a molecular targeting of this region or its activating pathways should be pursued as a new antimetastasis therapy approach.


Subject(s)
DNA-Binding Proteins/metabolism , Gastrointestinal Neoplasms/metabolism , Promoter Regions, Genetic , Receptors, Cell Surface/genetics , Sp1 Transcription Factor/metabolism , Transcription Factors/metabolism , Adult , Aged , Aged, 80 and over , Electrophoretic Mobility Shift Assay , Female , Gastrointestinal Neoplasms/genetics , Humans , Male , Middle Aged , Receptors, Urokinase Plasminogen Activator , Transcription Factor AP-2
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