Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines: Role of cytokines
von Sajjad Khan
Datum der mündl. Prüfung:2013-05-14
Betreuer:Prof. Dr. Dr. h.c. Giuliano Ramadori
Gutachter:Prof. Dr. Ahmed Mansouri
Gutachter:Prof. Dr. Tomas Pieler
Gutachter:Prof. Dr. Ernst A. Wimmer
Gutachter:Prof. Dr. Heidi Hahn
Gutachter:Prof. Dr. Silvio Rizzoli
Name:Thesis 13-06-05 Ist Final.pdf
Format:PDFDescription:Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines: Role of cytokines
EnglischBackground: Worldwide, colorectal cancer is the third most common malignancy. The development of the colorectal cancer (CRC) is a multistep process that involves e.g. an accumulation of mutations in tumor-growth promoting genes. In CRC, the most frequently found mutations are in the KRAS (30-50%) and BRAF (~10%) genes. Recent studies have shown that KRAS and BRAF mutations represent an important step in the development of carcinoma from the adenoma stage in colon cancer by affecting multiple pathways linked to the MAPK1, JAK-STAT, and PI3K pathways. A role of inflammatory cells within the tumor microenvironment and tumorigenesis is well established. However, the mechanism of how inflammation promotes carcinogenesis remains unresolved. As the survival and proliferation of tumor cells is influenced by immune cells within the tumor environment, the aim of our study was to investigate whether pro-inflammatory cytokines (TNFα, IL-1β and IFNγ) can induce pro- (CXCL1 and CXCL8) and anti-angiogenic (CXCL10) chemokines and in mutated CRC cell lines compared to wild type. Furthermore, the behaviour of these chemokines in the presence/absence of the KRAS by siRNA silencing in KRAS-mutated (DLD1) and wild type (Caco2) was analysed. Methods: Six colonic cell lines were investigated: DLD1 (KRAS G13D), HT-29 and Colo205 (BRAF V600E) as well as the wild type (Wt) cell lines Caco-2, Colo-320 and CX-1. The presence of KRAS mutations was analysed in the cell lines by using specific mismatch primers to amplify genomic DNA fragments through the PCR-RFLP assay, containing the hot spots of codons G12D and G13D. The BRAF mutation for codon V600E was detected by real time PCR. DLD1, HT-29 and Caco-2 cell lines were treated with cytokines (TNFα 50ng, IL-1β 1ng and IFNγ 50ng) and harvested at different time points (1h-24h). KRAS inhibition was performed by the siRNA approach using specific nucleotide sequences in KRAS-mutant and wild type cell lines. Total RNA was isolated from cultured cells. Isolated RNA was converted into cDNA and further used for RT-PCR analysis. Similarly, protein was extracted from the cells to perform Western blotting. Results: RT-PCR analysis in non-stimulated cells showed a low basal expression of TNFα and IL-1ß in the KRAS mutated (DLD1) cell line, compared to wild type (Caco2). No detection was found for IL-6 and IFNγ in any of the studied cell lines. In contrast, pro-angiogenic chemokines (CXCL1, CXCL8) showed a high constitutive expression in mutated cell lines DLD1 (KRAS), HT-29 and Colo205 (BRAF), compared to wild type (Caco2). However, the anti-angiogenic chemokine (CXCL10) showed a high basal expression in wild type, compared to mutated cell lines. Treatment with pro-inflammatory cytokines showed an induction of CXCL1 gene expression in mutated, and to a lesser extent in wild type cell lines at mRNA and protein level. The most pronounced and quick induction of CXCL1 gene expression was detected after TNFα stimulation in DLD1 (KRAS; 310±2.18 fold) followed by HT-29 (BRAF; 36.15±3.28 fold) compared to wild type (Caco2; 29.45±0.82 fold). Similar results were found after treatment with IL-1ß which induced the maximum gene expression of CXCL1 in HT-29 (BRAF; 46.42±5.98 fold) followed by DLD1 (KRAS; 21.19±0.37 fold); a minor but significant increase was found in Caco2 (Wt; 2.6±1.6 fold). Likewise, CXCL8 mRNA and protein level was significantly induced by TNFα and IL-1ß in KRAS mutated cell line (DLD1) and wild type (Caco2). The maximum increase was observed in wild type (Caco2) cell line after IL-1ß treatment (806.41±19.76fold). In addition, administration of IFNγ significantly enhanced CXCL10 at mRNA and protein level in mutated cell lines HT-29 (BRAF; 15361.19±2974.33 fold) followed by DLD1 (KRAS; 597.71±64.62 fold) in comparison to wild type (Caco2; 45.75±1.44 fold). In order to determine the factors responsible for chemokine induction in the downstream-signalling pathway of pro-inflammatory cytokines, protein expression of transcription factors (NF-κB, MAPK1 and STAT3) involved in KRAS-mutant (DLD1) and wild type (Caco2) cell lines were studied. An increase in protein level of NF-κB and MAPK1 was found in both, mutated and wild type cell lines after cytokine stimulation. However, p-STAT-3 was only detected in the KRAS mutated cell line (DLD1) after IFNγ stimulation. The protein expression of p-STAT-3 showed a time-dependent increase up to 24 h. To understand the possible role of KRAS and the consequences of inhibiting its activity or expression in colorectal cancer cell lines, a KRAS knockdown experiment was performed in KRAS-mutant (DLD1) and wild type (Caco2) cell lines. KRAS was successfully knocked down by the siRNA technique. This down-regulation of KRAS showed a significant effect on chemokine gene expression: A decreased CXCL1 and CXCL10 gene expression was detected in the DLD1 (KRAS) cell line in comparison to wild type (Caco2) at 72h after KRAS silencing. In contrast, the specific KRAS inhibition resulted in an up-regulation of CXCL1 and CXCL10 and induction of the NF-κB pathway in wild type (Caco2) cell line. To summarize, basal chemokine gene expression for pro-angiogenic chemokines was high in mutated as compared to wild type cell lines. Furthermore, cytokine treatment induces the expression of pro-angiogenic (CXCL1, CXCL8) and anti-angiogenic (CXCL10) chemokines differentially in mutated cell lines compared to wild type. The inhibition of the KRAS resulted in induction of chemokines gene expression through the NF-κB pathway in wild-type cell line. Conclusion: This reflects the likely existence of a totally different microenvironment in tumors consistent of wild type or mutated cells. This may help to rationalize the choice of molecular targets for suitable therapeutic investigation in clinical studies. Key words: KRAS, BRAF, CXCL1 (GROα), CXCL10 (IP-10), CXCL8 (IL-8), TNFα, IL-1ß, IFNγ, siRNA.
Keywords: Key words: KRAS, BRAF, CXCL1 (GROα), CXCL10 (IP-10), CXCL8 (IL-8), TNFα, IL-1ß, IFNγ, siRNA.