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Molecular understanding of KRAS- and BRAF-mutated colorectal cancers
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy in both men and women, and one of the leading causes of cancer-related deaths worldwide. One frequently mutated pathway involved in oncogenesis in CRC is the RAS/RAF/MAP kinase pathway. Oncogenic activation of KRAS and BRAF occur in 30‒40% and 5‒15% of all CRCs, respectively, and the mutations are mutually exclusive. Even though KRAS and BRAF are known to act in the same pathway, KRAS- and BRAF-mutated CRCs have different clinical and histopathological features. For example, BRAF mutation in CRC is tightly linked to microsatellite instability (MSI) and a CpG island methylator phenotype (CIMP), which is not seen in KRAS-mutated tumours. BRAF-mutated CRCs are also more often found in right-sided tumours. However, the underlying molecular reasons for these differences have not yet been defined.

The overall aim of this thesis was to investigate molecular differences between KRAS- and BRAF-mutated CRCs to understand how KRAS and BRAF mutations differentially affect tumour progression. We used an in vitro cell culture system to explore molecular differences between KRAS- and BRAF-mutated CRCs and verified our findings using CRC tissue specimens from the Colorectal Cancer in Umeå Study (CRUMS).

We found that BRAF mutation, but not KRAS mutation, was associated with expression of the stem cell factor SOX2. Furthermore, SOX2 was found to be correlated to a poor patient prognosis, especially in BRAF-mutated cancers. We further investigated the role of BRAF in regulation of SOX2 expression and found that SOX2 is at least partly regulated by BRAF in vitro. We continued by investigating the functional role of SOX2 in CRC and found that SOX2-expressing cells shared several characteristics with cancer stem cells, and also had down-regulated expression of the intestinal epithelial marker CDX2. There was a strong correlation between loss of CDX2 expression and poor patient prognosis, and patients with SOX2 expression were found to have a particularly poor prognosis when CDX2 levels were down-regulated. In conclusion, in these studies we identified a subgroup of BRAF-mutated CRCs with a particularly poor prognosis, and having a cancer stem cell-like appearance with increased expression of SOX2 and decreased expression of CDX2.

Tumour progression is regulated by interactions with cells of the immune system. We found that BRAF-mutated CRCs were more highly infiltrated by Th1 lymphocytes than BRAF wild-type tumours, while the opposite was true for KRAS-mutated CRCs. Interestingly, we found that part of this difference is probably caused by differences in secreted chemokines and cytokines between KRAS- and BRAF-mutated CRCs, stimulating different arms of the immune response.

Altered levels of expression of miRNAs have been seen in several malignancies, including CRC. We found that BRAF- and KRAS-mutated CRCs showed miRNA signatures different from those of wild-type CRCs, but the expression of miRNAs did not distinguish KRAS-mutated tumours from BRAF-mutated tumours.

In summary, our findings have revealed possible molecular differences between KRAS- and BRAF-mutated CRCs that may explain some of the differences in their clinical and histopathological behaviour.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2017. , 44 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1885
Keyword [en]
Colorectal cancer, BRAF, KRAS, SOX2, CDX2, cancer stem cell, Th1 lymphocytes, miRNA, prognosis
National Category
Cancer and Oncology Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-133410ISBN: 978-91-7601-677-0 (print)OAI: oai:DiVA.org:umu-133410DiVA: diva2:1087745
Public defence
2017-05-05, Hörsal E04, byggnad 6E, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-04-12 Created: 2017-04-10 Last updated: 2017-04-12Bibliographically approved
List of papers
1. SOX2 expression is regulated by BRAF and contributes to poor patient prognosis in colorectal cancer
Open this publication in new window or tab >>SOX2 expression is regulated by BRAF and contributes to poor patient prognosis in colorectal cancer
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 7, e101957Article in journal (Refereed) Published
Abstract [en]

Sporadic colorectal cancer (CRC) is a common malignancy and also one of the main causes of cancer deaths worldwide. Aberrant expression of the transcription factor SOX2 has recently been observed in several cancer types, but its role in CRC has not been fully elucidated. Here we studied the expression of SOX2 in 441 CRC patients by immunohistochemistry and related the expression to clinicopathological and molecular variables and patient prognosis. SOX2 was expressed in 11% of the tumors and was significantly associated to BRAF(V600E) mutation, but not to KRAS mutations (codon 12 and 13). SOX2 positivity was correlated to poor patient survival, especially in BRAF(V600E) mutated cases. In vitro studies showed that cells expressing the constitutively active BRAF(V600E) had increased SOX2 expression, a finding not found in cells expressing KRAS(G12V). Furthermore, blocking downstream BRAF signalling using a MEK-inhibitor resulted in a decreased expression of SOX2. Since SOX2 overexpression has been correlated to increased migration and invasion, we investigated the SOX2 expression in human CRC liver metastasis and found that a SOX2 positive primary CRC also had SOX2 expression in corresponding liver metastases. Finally we found that cells overexpressing SOX2 in vitro showed enhanced expression of FGFR1, which has been reported to correlate with liver metastasis in CRC. Our novel findings suggest that SOX2 expression is partly regulated by BRAF signalling, and an increased SOX2 expression may promote CRC metastasis and mediate a poor patient prognosis.

Place, publisher, year, edition, pages
Public library of science, 2014
Keyword
island methylator phenotype, transcription factor sox2, dna copy number, gene-expression, microsatellite instability, v600e mutation, lung-cancer, stem-cells, carcinoma, growth
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-91747 (URN)10.1371/journal.pone.0101957 (DOI)000338763800054 ()
Available from: 2014-08-20 Created: 2014-08-15 Last updated: 2017-04-10Bibliographically approved
2. SOX2 expression is associated with a cancer stem cell state and down-regulation of CDX2 in colorectal cancer
Open this publication in new window or tab >>SOX2 expression is associated with a cancer stem cell state and down-regulation of CDX2 in colorectal cancer
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2016 (English)In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 16, 471Article in journal (Refereed) Published
Abstract [en]

Background: To improve current treatment strategies for patients with aggressive colorectal cancer (CRC), the molecular understanding of subgroups of CRC with poor prognosis is of vast importance. SOX2 positive tumors have been associated with a poor patient outcome, but the functional role of SOX2 in CRC patient prognosis is still unclear. Methods: An in vitro cell culture model expressing SOX2 was used to investigate the functional role of SOX2 in CRC. In vitro findings were verified using RNA from fresh frozen tumor tissue or immunohistochemistry on formalin fixed paraffin embedded (FFPE) tumor tissue from a cohort of 445 CRC patients. Results: Using our in vitro model, we found that SOX2 expressing cells displayed several characteristics of cancer stem cells; such as a decreased proliferative rate, a spheroid growth pattern, and increased expression of stem cell markers CD24 and CD44. Cells expressing SOX2 also showed down-regulated expression of the intestinal epithelial marker CDX2. We next evaluated CDX2 expression in our patient cohort. CDX2 down-regulation was more often found in right sided tumors of high grade and high stage. Furthermore, a decreased expression of CDX2 was closely linked to MSI, CIMP-high as well as BRAF mutated tumors. A decreased expression of CDX2 was also, in a stepwise manner, strongly correlated to a poor patient prognosis. When looking at SOX2 expression in relation to CDX2, we found that SOX2 expressing tumors more often displayed a down-regulated expression of CDX2. In addition, SOX2 expressing tumors with a down-regulated CDX2 expression had a worse patient prognosis compared to those with retained CDX2 expression. Conclusions: Our results indicate that SOX2 expression induces a cellular stem cell state in human CRC with a decreased expression of CDX2. Furthermore, a down-regulated expression of CDX2 results in a poor patient prognosis in CRC and at least part of the prognostic importance of SOX2 is mediated through CDX2 down-regulation.

Keyword
SOX2, CDX2, Colorectal cancer, Prognosis, Cancer stem cell
National Category
Cell and Molecular Biology Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-124507 (URN)10.1186/s12885-016-2509-5 (DOI)000380091600001 ()27411517 (PubMedID)
Available from: 2016-08-19 Created: 2016-08-15 Last updated: 2017-04-10Bibliographically approved
3. The infiltration, and prognostic importance, of Th1 lymphocytes vary in molecular subgroups of colorectal cancer
Open this publication in new window or tab >>The infiltration, and prognostic importance, of Th1 lymphocytes vary in molecular subgroups of colorectal cancer
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2016 (English)In: The Journal of Pathology: Clinical Research, ISSN 2056-4538, Vol. 2, no 1, 21-31 p.Article in journal (Refereed) Published
Abstract [en]

Giving strong prognostic information, T-cell infiltration is on the verge of becoming an additional component in the routine clinical setting for classification of colorectal cancer (CRC). With a view to further improving the tools for prognostic evaluation, we have studied how Th1 lymphocyte infiltration correlates with prognosis not only by quantity, but also by subsite, within CRCs with different molecular characteristics (microsatellite instability, CpG island methylator phenotype status, and BRAF and KRAS mutational status). We evaluated the Th1 marker T-bet by immunohistochemistry in 418 archival tumour tissue samples from patients who underwent surgical resection for CRC. We found that a high number of infiltrating Th1 lymphocytes is strongly associated with an improved prognosis in patients with CRC, irrespective of intratumoural subsite, and that both extent of infiltration and patient outcome differ according to molecular subgroup. In brief, microsatellite instability, CpG island methylator phenotype-high and BRAF mutated tumours showed increased infiltration of Th1 lymphocytes, and the most pronounced prognostic effect of Th1 infiltration was found in these tumours. Interestingly, BRAF mutated tumours were found to be more highly infiltrated by Th1 lymphocytes than BRAF wild-type tumours whereas the opposite was seen for KRAS mutated tumours. These differences could be explained at least partly by our finding that BRAF mutated, in contrast to KRAS mutated, CRC cell lines and tumour specimens expressed higher levels of the Th1-attracting chemokine CXCL10, and reduced levels of CCL22 and TGFB1, stimulating Th2/Treg recruitment and polarisation. In conclusion, the strong prognostic importance of Th1 lymphocyte infiltration in CRC was found at all subsites evaluated, and it remained significant in multivariable analyses, indicating that T-bet may be a valuable marker in the clinical setting. Our results also indicate that T-bet is of value when analysed in molecular subgroups of CRC, allowing identification of patients with especially poor prognosis who are in need of extended treatment.

Place, publisher, year, edition, pages
John Wiley & Sons, 2016
Keyword
colorectal cancer, Th1 lymphocytes, intratumoural subsites, molecular subgroups, BRAF, KRAS, prognosis
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-121095 (URN)10.1002/cjp2.31 (DOI)
Available from: 2016-05-26 Created: 2016-05-26 Last updated: 2017-04-10Bibliographically approved
4. MicroRNA expression in KRAS- and BRAF-mutated colorectal cancers
Open this publication in new window or tab >>MicroRNA expression in KRAS- and BRAF-mutated colorectal cancers
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(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-133409 (URN)
Available from: 2017-04-10 Created: 2017-04-10 Last updated: 2017-04-10

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