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Molecular Signatures of Cancer
KTH, School of Biotechnology (BIO), Gene Technology.
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Cancer is an important public health concern in the western world, responsible for around 25% of all deaths. Although improvements have been made in the diagnosis of cancer, treatment of disseminated disease is inefficient, highlighting the need for new and improved methods of diagnosis and therapy. Tumours arise when the balance between proliferation and differentiation is perturbed and result from genetic and epigenetic alterations.

Due to the heterogeneity of cancer, analysis of the disease is difficult and a wide range of methods is required. In this thesis, a number of techniques are demonstrated for the analysis of genetic, epigenetic and transcriptional alterations involved in cancer, with the purpose of identifying a number of molecular signatures. Pyrosequencing proved to be a valuable tool for the analysis of both point mutations and CpG methylation. Using this method, we showed that oncogenes BRAF and NRAS, members of the Ras-Raf-MAPK pathway, were mutated in 82% of melanoma tumours and were mutually exclusive. Furthermore, tumours with BRAF mutations were more often associated with infiltrating lymphocytes, suggesting a possible target for immunotherapy. In addition, methylation of the promoter region of the DNA repair gene MGMT was studied to find a possible correlation to clinical response to chemotherapy. Results showed a higher frequency of promoter methylation in non-responders as compared to responders, providing a possible predictive role and a potential basis for individually tailored chemotherapy. Microarray technology was used for transcriptional analysis of epithelial cells, with the purpose of characterization of molecular pathways of anti-tumourigenic agents and to identify possible target genes. Normal keratinocytes and colon cancer cells were treated with the antioxidant N-acetyl L-cysteine (NAC) in a time series and gene expression profiling revealed that inhibition of proliferation and stimulation of differentiation was induced upon treatment. ID-1, a secreted protein, was proposed as a possible early mediator of NAC action. In a similar study, colon cancer cells were treated with the naturally occurring bile acid ursodeoxycholic acid (UDCA) in a time series and analysed by microarray and FACS analysis. Results suggest a chemopreventive role of UDCA by G1 arrest and inhibition of cell proliferation, possibly through the secreted protein GDF15.

These investigations give further evidence as to the diversity of cancer and its underlying mechanisms. Through the application of several molecular methods, we have found a number of potential targets for cancer therapy. Follow up studies are already in progress and may hopefully lead to novel methods of treatment.

Place, publisher, year, edition, pages
Stockholm: KTH , 2006. , 59 p.
Keyword [en]
: BRAF, NRAS, MGMT, methylation, pyrosequencing, microarray technology, gene expression analysis
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-3954ISBN: 91-7178-348-2 (print)OAI: oai:DiVA.org:kth-3954DiVA: diva2:10176
Public defence
2006-05-24, Sal FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20110121Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2011-01-21Bibliographically approved
List of papers
1. Gene expression analysis of human epidermal keratinocytes after N-acetyl L-cysteine treatment demonstrates cell cycle arrest and increased differentiation
Open this publication in new window or tab >>Gene expression analysis of human epidermal keratinocytes after N-acetyl L-cysteine treatment demonstrates cell cycle arrest and increased differentiation
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2005 (English)In: Pathobiology (Basel), ISSN 1015-2008, E-ISSN 1423-0291, Vol. 72, no 4, 203-212 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: Several cancer prevention programmes have previously been executed using treatment of antioxidant compounds. The antioxidant N-acetyl L-cysteine (NAC), a membrane-permeable aminothiol, is a sulfhydryl reductant reducing oxidised glutathione, as well as being a precursor of intracellular cysteine and glutathione. A previous report based on the cellular response to NAC treatment showed that NAC induced a 10-fold more rapid differentiation in normal primary keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation. In order to investigate molecular events underlying the changes in proliferation and differentiation induced by NAC treatment, we performed global gene expression analysis of normal human epidermal keratinocytes in a time series. Methods: Treated samples were compared to untreated samples through a reference design using a spotted cDNA array comprising approximately 30,000 features. B statistics was used to identify differentially expressed genes, and RT-PCR of a selected set of genes was performed to verify differential expression. Results: The number of differentially expressed genes increased over time, starting with 0 at 30 min, 73 at 3 h and increasing to 952 genes at 48 h. Results of the expression analysis showed arrest of the cell cycle and an upregulation of cytoskeletal reorganisation, implicating increased differentiation. A comparison to gene ontology groups indicated downregulation of a large number of genes involved in cell proliferation and regulation of the cell cycle. Conclusions: A significant fraction of the differentially expressed genes could be classified according to their role in the differentiation process, demonstrating that NAC regulates the conversion from proliferation to differentiation at a transcriptional level.

Keyword
Cell cycle, Gene expression, Keratinocytes, Microarray; N-acetyl L-cysteine, NHEK, Real-time, RT-PCR
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5716 (URN)10.1159/000086790 (DOI)000231700000005 ()2-s2.0-24044509171 (Scopus ID)
Note
QC 20100915Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2012-03-20Bibliographically approved
2. Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro
Open this publication in new window or tab >>Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro
2005 (English)In: BMC Cancer, ISSN 1471-2407, Vol. 5, 75- p.Article in journal (Refereed) Published
Abstract [en]

Background: Cancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation [1]. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process. Methods: In order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point. Results: Microarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action. The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform. Conclusion: NAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action.

Keyword
acetylcysteine, animal cell, article, cancer cell, cell differentiation, cell proliferation, cell strain CACO 2, cell type, computer program, controlled study, DNA microarray, down regulation, drug mechanism, epithelium, gene activation, gene control, gene expression, gene identification, gene repression, gene sequence, genetic transcription, growth inhibition, human, human cell, in vitro study, keratinocyte, nonhuman, nucleotide sequence, reverse transcription polymerase chain reaction, time series analysis, validation process
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5717 (URN)10.1186/1471-2407-5-75 (DOI)000230966200001 ()2-s2.0-26844509194 (Scopus ID)
Note
QC 20100916Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2012-03-20Bibliographically approved
3. Investigating the chemopreventive role of ursodeoxycholic acid in colorectal cancer cells
Open this publication in new window or tab >>Investigating the chemopreventive role of ursodeoxycholic acid in colorectal cancer cells
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(English)Manuscript (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5718 (URN)
Note
QC 20110121Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2011-01-21Bibliographically approved
4. NRAS and BRAF mutations in melanoma turnours in re ation to clinical characteristics: a study based on mutation screening by pyrosequencing
Open this publication in new window or tab >>NRAS and BRAF mutations in melanoma turnours in re ation to clinical characteristics: a study based on mutation screening by pyrosequencing
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2006 (English)In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 16, no 6, 471-478 p.Article in journal (Refereed) Published
Abstract [en]

We have previously demonstrated the use of pyrosequencing to investigate NRAS [neuroblastoma RAS viral (v-ras) oncogene homolog] mutations in melanoma biopsies. Here, we expanded the analysis to include BRAF (V-raf murine sarcoma viral oncogene homolog 1311), another member of the Ras-Raf-mitogen-activated protein kinase (MAPK) signalling pathway, and analysed a total of 294 melanoma tumours from 219 patients. Mutations in BRAF exons 11 and 15 were identified in 156 (53%) tumours and NRAS exon 2 mutations in 86 (29%) tumours. Overall, mutations in NRAS or BRAF were found in 242 of 294 tumours; (82%) and were found to be mutually exclusive in all but two cases (0.7%). Multiple metastases were analysed in 57 of the cases and mutations were identical in all except three, indicating that BRAF and NRAS mutations occur before metastasis. Association with preexisting nevi was significantly higher in BRAF mutated tumours (P=0.014). In addition, tumours with BRAF mutations showed a significantly more frequent moderate to pronounced infiltration of lymphocytes (P=0.013). NRAS mutations were associated with a significantly higher Clark level of invasion (P=0.022) than BRAF mutations. Age at diagnosis was significantly higher in tumours with NRAS mutations than in those with BRAF mutations (P=0.019). NRAS and BRAF mutations, however, did not influence the overall survival from time of diagnosis (P=0.7). In conclusion, the separate genotypes were associated with differences in several key clinical and pathological parameters, indicating differences in the biology of melanoma tumours with different proto-oncogene mutations.

Keyword
BRAF, melanoma, NRAS, pyrosequencing, n-ras mutations, malignant melanocytic lesions, human cutaneous melanoma, tumor progression, point mutations, cell-lines, cancer, skin, nevi, activation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-16231 (URN)10.1097/01.cmr.0000232300.22032.86 (DOI)000243159000001 ()2-s2.0-33845592531 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
5. Hypermethylation status of the MGMT promoter in melanoma tumours in relation to clinical response to chemotherapy
Open this publication in new window or tab >>Hypermethylation status of the MGMT promoter in melanoma tumours in relation to clinical response to chemotherapy
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(English)Manuscript (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5720 (URN)
Note
QC 20110121Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2011-01-21Bibliographically approved

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