| 1. |
- Elander, Nils, et al.
(författare)
-
Association Between Adenomatosis Polyposis Coli Functional Status and Microsomal Prostaglandin E Synthase-1 Expression in Colorectal Cancer
- 2009
-
Ingår i: Molecular Carcinogenesis. - : Wiley. - 0899-1987 .- 1098-2744. ; 48:5, s. 401-407
-
Tidskriftsartikel (refereegranskat)abstract
- Bioactive metabolites downstream of cyclooxygenase-2 (COX-2) generated prostaglandin H-2 (PGH(2)), in particular prostaglandin E-2 (PGE(2)), are thought to play critical roles during the development of colorectal tumors. Previous reports reveal that defects of the tumor suppressor adenomatosis polyposis coli (APC) contribute to COX-2 upregulation in colon tumor cells. We investigated whether a similar relation was present between APC functional status and the expression of microsomal prostaglandin E synthase-1 (mPGES-1), which acts downstream of COX-2 and catalyses the terminal conversion of PGH(2) into PGE(2). Surprisingly, mPGES-1 mRNA and protein levels were upregulated upon induction of a wild type-APC carrying vector in HT29 colon cancer cells, and downregulated following siRNA silencing of APC in HCT-116 cells. mPGES-1 was overall enhanced in human colorectal tumor specimens versus corresponding non-tumor mucosa and, in accordance with data on HT29 and HCT116 cells, higher levels of mPGES-1 were observed among tumors carrying wild type versus mutant APC. RNAi silencing of beta-catenin and luciferase assays regarding the mPGES-1 promoter region did not reveal a role for APC or beta-catenin/Tcf in controlling mPGES-1 gene transcription. However, RNA degradation assays in HT29 cells revealed a suppressed degradation of mPGES-1 in the presence of wild type APC, implying that mPGES-1 mRNA is stabilized in the APC wild type state. Collectively, we demonstrate a novel association between APC functional status and mPGFS-1 expression in colorectal tumor cells, being most likely related to reduced mPGES-1 mRNA degradation rate in the APC wild type state.
|
|
| 2. |
- Elander, Nils, 1980-, et al.
(författare)
-
Genetic deletion of mPGES-1 accelerates intestinal tumorigenesis in APCMin/+ mice
- 2008
-
Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 372:1, s. 249-253
-
Tidskriftsartikel (refereegranskat)abstract
- The induced synthesis of bioactive prostanoids downstream of cyclooxygenase-2 (COX-2) and prostaglandin H2 (PGH2) exerts a critical event in colorectal carcinogenesis. Here we demonstrate that APCMin/+ mice with genetic deletion of microsomal prostaglandin E synthase-1 (mPGES-1), which catalyses the terminal conversion of PGH2 into PGE2, surprisingly develop more and generally larger intestinal tumors than do mPGES-1 wild type littermates (mean number of tumors/intestine 80 vs. 38, p < 0.0005, mean tumor diameter 1.64 vs. 1.12 mm, p < 0.0005). No deviation regarding the expression of other PGE2 related enzymes (COX-1, COX-2, mPGES-2, cPGES, and 15-PGDH) or receptors (EP1-4) was obvious among the mPGES-1 deficient mice. PGE2 levels were suppressed in tumors of mPGES-1 deficient animals, but the concentrations of other PGH2 derived prostanoids were generally enhanced, being most prominent for TxA2 and PGD2. Thus, we hypothesise that a redirected synthesis towards other lipid mediators might (over)compensate for loss of mPGES-1/PGE2 during intestinal tumorigenesis. Nevertheless, our results question the suitability for mPGES-1 targeting therapy in the treatment or prevention of colorectal cancer. © 2008 Elsevier Inc. All rights reserved.
|
|
| 3. |
- Karlsson, Anneli, et al.
(författare)
-
Notch1 is a frequent mutational target in chemically induced lymphoma in mouse
- 2008
-
Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 123:11, s. 2720-2724
-
Tidskriftsartikel (refereegranskat)abstract
- Activating Notch1 mutations have been reported in human T-lineage acute lymphoblastic leukemia (T-ALL) and lymphomas from genetically modified mice. We report that Notch1 is a prevalent and major mutational target in chemically induced mouse lymphoma. The regions of the gene that are frequently mutated are the hterodimerization domain and the N-terminal ligand-binding region, important for protein stability, and (he polypeptide rich in proline, glutamate, serine and threonine WEST) domains, which is critical for protein degradation. Another gene, CDC4, is also involved in Notch1 degradation and shows frequent mutations. Mutations in the heterodimerization and the ligand-binding regions may cause ligand-independent signaling. whereas mutations preventing protein degradation result in accumulation of intracellular Notch1. We analyzed 103 chemical-induced mouse lymphomas for mutations in the Notch1 gene using single strand conformation analysis (SSCA) and DNA sequencing. Genetic alterations resulting in premature truncation of Notch I were identified in 28 tumors, whereas 8 revealed alterations in the heterodimerization and 16 harbored deletions in the ligand-binding region. Dideoxycytidine-induced lymphomas displayed the highest frequency of Notch1 mutations (49%). whereas in butadiene- and phenolphthalein-indced tumors showed lower frequencies (26 10%, respectively). In total, 26 novel and 3 previously reported mutations were detected. This report shows that Notch1 is a prevalent and major mtational target for 2,3-dideoxycytidine and butadiene-induced lymphoma..
|
|
| 4. |
- Ungerbäck, Jonas, et al.
(författare)
-
Analysis of VEGF polymorphisms, tumor expression of VEGF mRNA and colorectal cancer susceptibility in a Swedish population
- 2009
-
Ingår i: MOLECULAR MEDICINE REPORTS. - : Spandidos Publications. - 1791-2997. ; 2:3, s. 435-439
-
Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial growth factor (VEGF) plays a significant role in tumor angiogenesis and is found to be overexpressed and involved in the development and progression of colorectal cancer (CRC). The VEGF gene contains several polymorphic sites known to influence VEGF expression. We examined the possible association between five polymorphisms, located in the promoter/5-untranslated region [-2578 (C/A), -2549 (del/ins 18 bp) -1154 (G/A), -634 (G/C)] or 3-untranslated region [+936 (C/T)] of the VEGF gene, and CRC Susceptibility and clinicopathological characteristics in 302 Swedish CRC patients and 336 healthy randomly selected controls. Both genotypes and combined haplotypes were analyzed. No significant differences were observed when VEGF genotype/haplotype frequencies in the CRC cases and controls were compared, nor were any associations found between the genotypes/haplotypes and clinicopathological characteristics. However, when the -2578 C and +936 T alleles were combined, a small but significant association with CRC susceptibility was detected (OR=1.6, 95% CI 1.3-1.9, p=0.01). In addition, VEGF mRNA expression was determined in a Subset of patients, revealing a 2-fold VEGF upregulation in CRC tissue compared to normal colonic mucosa, but no association between the genotypes or haplotypes and VEGF mRNA levels. Linkage analysis was performed, revealing that the polymorphisms in the promoter and 5-untranslated region were in tight linkage disequilibrium (LD) (vertical bar Dvertical bar=0.91-1.00), while the +936 C/T polymorphism was only weakly associated with the others (vertical bar Dvertical bar=0.05-0.19). In conclusion, VEGF is generally upregulated in colorectal tumors. However, the single nucleotide polymorphisms examined do not appear to influence the mRNA expression of VEGF in colorectal tumors, and most likely play a limited role in CRC development and progression.
|
|
| 5. |
- Ungerbäck, Jonas, et al.
(författare)
-
Genetic variation and alterations of genes involved in NFκB/TNFAIP3- and NLRP3-inflammasome signaling affect susceptibility and outcome of colorectal cancer
- 2012
-
Ingår i: Carcinogenesis. - Oxford, United kingdom : Oxford University Press. - 0143-3334 .- 1460-2180. ; 33:11, s. 2126-2134
-
Tidskriftsartikel (refereegranskat)abstract
- Colorectal tumors are continuously exposed to an inflammatory environment, which together with mitogenic signals sustain several cancer hallmarks. Nuclear factor-kappa B (NFκB) is a major regulator of inflammation and variation in NFκB-associated genes could potentially be used as biomarkers to identify patients with increased risk of colorectal cancer (CRC) development, and/or a rapidly progressing disease. In this study, 348 CRC cases and 806 randomly selected healthy individuals from southeastern Sweden were examined with regard to seven polymorphisms in NFκB pathway-associated genes. Log-rank-tests and Cox proportional hazard regression analysis examined the association between the polymorphisms and CRC-specific survival, whereas chi-square tests and logistic regression analysis were used to test for associations between the polymorphisms and CRC susceptibility. Gene expression and loss of heterozygosity analyses of TNFAIP3 were carried out in a subset of tumors to assess its role as a tumor suppressor in CRC. Heterozygous and polymorphic TNFAIP3 (rs6920220), heterozygous NLRP3 (Q705K) and polymorphic NFκB -94 ATTG ins/del genotypes were found to be associated with poorer survival in patients diagnosed with invasive CRC (aHR = 5.2, 95% CI: 2.5-10.9, P < 0.001). TNFAIP3 mRNA levels were significantly decreased in tumors compared with adjacent non-neoplastic mucosa (P < 0.0001) and loss of heterozygosity of 6q23.3 (TNFAIP3) was detected in 17% of cases, whereas only 2.5% of the investigated specimens displayed TNFAIP3 gene mutations. We propose that TNFAIP3 (rs6920220), NLRP3 (Q705K) and NFκB -94 ATTG ins/del polymorphisms are associated with poor survival in patients with advanced CRC and may be used as prognostic markers. Experimental results indicate that TNFAIP3 may act as a tumor suppressor in CRC.
|
|
| 6. |
- Ungerbäck, Jonas, et al.
(författare)
-
Genetic variation in NFκB signaling pathway genes in colorectal cancer susceptibility and survival
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- PURPOSE: Variations in genes orchestrating inflammatory responses, such as those being connected with NFκB and NLRP3 inflammasome signaling, are associated with chronic inflammatory bowel diseases, which are well-known risk factors for colorectal cancer (CRC). The purpose of this study was to investigate the association between genetic variation and alterations in genes involved in NFκB and NLRP3 inflammasome signaling and their possible influence on susceptibility and clinical outcome of colorectal cancer. EXPERIMENTAL DESIGN: 344 CRC cases and 793 randomly selected healthy individuals from southeastern Sweden were examined with regard to seven polymorphisms in NFκB, TNFAIP3, NLRP3, CARD8 and TLR4 genes. Chi-square tests and multiple logistic regression analysis were used to test for associations between the SNPs and CRC susceptibility, while log-rank tests and Cox proportional hazard regression analysis were used to examine the association between the SNPs and CRC-specific survival. Gene expression assay and loss of heterozygosity analyzes of TNFAIP3 were carried out in a subset of tumors to assess its role as a potential tumor suppressor in CRC. RESULTS: Adjusted for age, gender and polypoid/ulcerative CRC phenotype, a panel of heterozygous and mutant TNFAIP3 (rs6920220), mutant NFκB -94 ATTG ins/del and heterozygous NLRP3 (Q705K) genotypes were found to be associated with poorer survival in patients diagnosed with invasive CRC (aHR = 5.2 95% CI 2.5-10.9, P < 0.001). TNFAIP3 mRNA levels were significantly decreased in tumors compared to adjacent non-neoplastic mucosa (P < 0.0001) and LOH of 6q23.3, (TNFAIP3), was detected in 17% of cases, while only 2.5% of the investigated specimens displayed TNFAIP3 gene mutations. CONCLUSIONS: A panel of the TNFAIP3 (rs6920220), NFκB -94 ATTG ins/del and NLRP3 (Q705K) polymorphisms are associated with poor survival in patients with advanced CRC and may be used as a prognostic marker. Experimental results indicate that TNFAIP3 may act as a tumor suppressor in CRC.
|
|
| 7. |
- Ungerbäck, Jonas
(författare)
-
Inflammation and Intestinal Homeostasis-Associated Genes in Colorectal Cancer
- 2012
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Colorectal cancer (CRC) is a global ‘killer’ and every year more than 1.2 million new individuals are affected and approximately 600 000 succumb to the disorder. Several mechanisms such as inactivation of tumor suppressor genes, activation of oncogenes and dysregulation of cell fate determinating pathways e.g. Wnt and Notch can initiate a cancerous cell growth and promote colorectal tumorigenesis. In addition, most tumors are exposed to an inflammatory environment, which together with the presence of mitogenic and angiogenic signals may sustain several hallmarks of cancer. Genetic alterations in inflammatory genes are associated with chronic inflammatory bowel disease, which is a strong risk factor of developing CRC. Scientists have for a long time looked for ‘the Key’ that would unlock the ‘cancer door’ but more likely cancer should be considered as not one but many diseases where almost every single patient is genetically and clinically unique. Hence recent research has turned to identify such inter-individual discrepancies and to find disease markers and strategies for guiding clinicians when tailoring individual management and optimized therapy. A deeper understanding of the regulation and genetic variation of inflammation and intestinal-homeostasis associated genes is pivotal to find potential targets for future therapies.The present thesis focuses on genetic variation and alterations in inflammatory genes as well as genes specifically involved in maintaining intestinal homeostasis. The most common anti-inflammatory drugs, NSAIDs, inhibit the prostanoid-generating COX-enzymes and are associated with decreased CRC risk when administered for a long time. Unfortunately, continuous NSAID treatment may lead to severe side-effects such as gastrointestinal bleeding, possibly through the ablation of non-PGE2 prostanoids. Therefore, a more specific inhibition of PGE2 has been suggested to be superior to classical NSAIDs. In papers I and II, the terminal PGE2 generating enzyme mPGES1 was studied in the context of intestinal cancer. Unexpectedly, ApcMin/+ mice with a targeted deletion of the mPGES1 encoding gene displayed significantly more and larger intestinal adenomas as compared to their wilde-type (wt) littermates. Probably this was due to the redirected generation of PGE2 towards non-PGE2 prostanoids seen in the murine tumors, resulting in enhanced pro-tumorigenic activity of these transmitter substances. Next, with a battery of functional and descriptive assays we investigated whether the outcome of mPGES1 expression and activity could depend on the genetic profile of the tumor e.g. the Apc mutational status. Indeed, high expression of mPGES1 was associated with the presence of wt-Apc, both in vitro and in vivo, most likely depending on mPGES1 mRNA stabilization rather than upregulation through β–catenin/Lef/Tcf4 signaling.NFκB is a major regulator of inflammation e.g. through the production of inflammatory cytokines. Variations in genes controlling inflammation and angiogenesis could potentially be used as biomarkers to identify patients with increased risk of CRC development, and/or to identify those with high risk of a rapidly progressing disease. Further, such analyzes have been suggested to select patients, which may benefit from specific anti-inflammatory or anti-angiogenic therapies. In paper III, genetic alterations in NFκB associated genes were studied among CRC patients and healthy controls. The NFκB negative regulator TNFAIP3 was found to exert tumor suppressive functions in CRC and moreover, homozygous mutant TNFAIP3 (rs6920220), homozygous mutant NFκB -94 ATTG ins/del and heterozygous NLRP3 (Q705K) were identified as prognostic markers for identifying CRC patients with a high risk of rapid progression. Further studies, which focus on the potential to treat such patients with anti-inflammatory IL-1β targeting therapies, are warranted.In the intestinal epithelium, Notch and Wnt signaling function in synergy to maintain homeostasis and together these pathways promote stem cell renewal and drive proliferation. Thus, dysregulation and/or overactivation of one of the two pathways could potentially lead to simultaneous activation of the other. While the genetic mechanisms explaining aberrant Wnt signaling in CRC are well-known, the reasons for the Notch pathway activation are less so. Further, relatively little is known about the mechanisms linking the two pathways in CRC. In paper IV, we addressed this question with a set of experimental in vitro assays, hereby identifying Notch2 together with several additional genes classically belonging to the Notch pathway, as putative targets for canonical and non-canonical Wnt signaling. We therefore suggest that aberrant Notch signaling in colon cancer cells may be the result of dysregulated Wnt signaling.In summary, the results here presented add a couple of pieces to the immensely complex jigsaw puzzle connecting intestinal homeostasis, inflammation and CRC. These results may aid in identifying future biomarkers or potential drug targets that could take us to the next level in the war against cancer.
|
|
| 8. |
- Ungerbäck, Jonas, et al.
(författare)
-
Notch-2 and the Notch signaling pathway are regulated by Wnt signaling in colorectal cancer
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Both Notch and Wnt pathways are key regulators of intestinal homeostasis and alterations in these pathways can lead to development of colorectal cancer, where the Apc/β-catenin-genes in the Wnt signaling pathway are frequently mutated, and active Notch signaling contributes to tumorigenesis by keeping the epithelial cells in a proliferative state. These pathways are simultaneously active in proliferative adenoma cells and a crosstalk between them has been indicated. Using bioinformatics, we identified and screened proximal Notch pathway gene promoters for putative TCF/LEF1 sites, targets for β-catenin. Wild type (wt)-Apc negatively regulates β-catenin and by using semi-quantitative PCR, induction of wt-Apc or β-catenin silencing in HT29 cells, we observed that several genes in the Notch pathway, including Notch-2, were downregulated. Electrophoretic mobility-shift assay (EMSA) confirmed binding of Lef-1 to Notch-2 as well as other Notch pathway gene promoters and luciferase assays showed an increased activity for the LEF1/TCF-site at position -110 in the Notch-2 promoter upon cotransfection of HT29 or HCT116 cells with mutated β-catenin. Taken together, these results indicate that activation of the Wnt pathway with increased levels of β-catenin can function as a transcriptional regulator of the Notch pathway in colon cancer cell lines.
|
|
| 9. |
- Ungerbäck, Jonas, 1982-
(författare)
-
Notch signalling in carcinogenesis : With special emphasis on T-cell lymphoma and colorectal cancer
- 2009
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Notch signalling pathway is an evolutionary conserved pathway, named after the Notch receptors, Notch1-4 in mammals, which upon cell-cell contact and ligand binding releases the intracellular domain (NICD). NICD translocates into the nucleus where it binds the transcriptional repressor RBP-Jk, which together with co-activators belonging to the Mastermind-like family of proteins form a transcriptional activation complex. This complex activates genes controlling cell fate decision, embryonic development, proliferation, differentiation, adult homeostasis and stem cell maintenance. On the other hand, disrupted Notch signalling may result in pathological conditions like cancer, although the mechanisms behind the disruption are often complex and in many cases largely unknown.Notch1 drives the lymphocyte differentiation towards a T-cell fate and activating mutations in the gene have been suggested to be involved in T-cell lymphoma. In paper I, genetic alterations in Notch1 and the Notch1 regulating gene CDC4 were investigated in tumours from murine T-cell lymphoma induced with phenolphthalein, 1,3-butadiene or 2’,3’-dideoxycytidine. We identified activating Notch1 mutations in 39% of the lymphomas, suggesting that Notch1 is an important target gene for mutations in chemically induced lymphomas.While it is known that constitutively activated Notch signalling has a clear oncogenic function in several solid malignancies as well, the molecular mechanisms are less known in this context. Unpublished data of our lab, together with other recent studies, suggest that mutations of Notch and Notch-related genes per se are uncommon in solid malignancies including colorectal cancer, while a growing body of evidence indicates that aberrant Wnt/b-catenin signalling may result in pro-tumoural Notch activation in these contexts. In paper II, we therefore investigated potential transcriptional interactions between the Notch and Wnt signalling pathways in colorectal cancer cell lines. The proximal Notch and Wnt pathway gene promoters were bioinformatically identified and screened for putative TCF/LEF1 and RBP-Jk sites. In canonical Wnt signalling, Apc negatively regulates b-catenin leading to repression of TCF/LEF1 target genes. Upon repression of the Wnt pathway we observed that several genes in the Notch pathway, including Notch2, were transcriptionally downregulated. We also confirmed binding of Lef1 to Notch2 as well as other Notch pathway gene promoters and luciferase assays showed an increased activity for at least one LEF1/TCF-site in the Notch2 promoter upon co-transfection of HT29 or HCT116 cells with mutated b-catenin. HT29 cell lines were also treated with the g-secretase inhibitor DAPT, leading to inactivation of the Notch pathway by preventing release of NICD. However, results showed no effects on Apc, b-catenin or their target cyclin D1. Taken together, these results indicate that the Wnt pathway may function as a regulator of the Notch pathway through the TCF/LEF1 target gene program in colon cancer cell lines.In summary, Notch pathway deregulation is of importance in both murine T-cell lymphoma and human colorectal cancer, although the mechanisms differ. The current results give new insights in Notch pathway alterations as well as the signalling networks in which the Notch pathway interacts, and thus increase the understanding of Notch’s involvement in malignant diseases.
|
|
| 10. |
|
|
