Analyse der Statin-vermittelten Regulation der PIM1 Kinase in Krebszellen

The constitutively active serine/threonine kinase PIM1 is a prosurvival kinase, which contributes to the viability of cancer cells by regulating essential cellular processes such as cell cycle, cell growth, differentiation and apoptosis. This proto-oncogene is overexpressed in many types of cancer, including lymphoma, leukemia and solid tumors of the liver and colon. In addition, PIM1 can induce the formation of metastases by influencing the migration of cancer cells. Thus, high PIM1 levels are often associated with poor tumor prognosis. Since PIM1 acts as a master regulator of many signaling pathways and its knockout in mice only leads to a slightly altered phenotype, it is a promising target molecule for cancer therapy. Preliminary studies have shown that PIM1 can be downregulated by statins in cancer cells. Statins function as competitive inhibitors of HMG-CoA reductase, the key enzyme of the mevalonate pathway. The drug is available on the market for lowering cholesterol levels in the blood as it inhibits cholesterol synthesis. In addition, statins act by reducing the production of isoprenoids, which results in an attenuation of membrane anchoring of GTPases like Ras, Rac and Rho. This influences important signaling pathways and engenders in the promotion of apoptosis as well as the inhibition of proliferation, angiogenesis, tumor invasion and metastasis formation in various tumor types. Furthermore, clinical studies have shown that the risk of undesirable, harmful side effects from statins appears to be relatively low compared to the benefits that can be obtained from statin therapy. For this reason, statins may have also the potential to become promising drugs for the treatment of tumors. The molecular regulatory mechanisms induced by statin treatment in cancer cells are not well understood. Consequently,our oberservations that statins can mediate a PIM1 knockdown, provide the basis for the first project in this thesis, in which the regulatory mode of action of simvastatin on PIM1 was investigated. Therefore, different analyses were performed on transcriptional, post-transcriptional, translational and posttranslational levels in the cancer cell lines HepG2, LS174T and Skov3. Statin-induced PIM1 knockdown appears to be the result of altered regulatory mechanisms, as evidenced by the relatively late onset of action of simvastatin after 36 hours at the mRNA level and 48 hours at the protein level. The RT-qPCR experiments showed a similar decrease in Pim-1 mRNA levels in liver (HepG2) and colon (LS174T) carcinoma cell line, respectively. Whereas the Western blot results indicated that the protein levels of PIM1 in HepG2 cells are significantly lower than in LS174T cells after simvastatin treatment. They further revealed that simvastatin-mediated inhibition of PIM1 is not regulated by proteasomal or lysosomal protein degradation in both cell lines. Furthermore, statin-induced down-regulation of Pim-1 can also be excluded at the transcriptional level, as demonstrated by the dual luciferase assay performed after transfection of Pim-1 promoter constructs in HepG2 cells and the Western blot after ectopic expression of PIM1 in Skov3 cells. Instead, the RT-qPCR experiment on the inhibition of transcriptional initiation in the liver carcinoma cell line suggests regulation via the posttranscriptional level by Pim-1 mRNA turnover. In addition, in HepG2 and LS174T cells a statin-mediated knockdown of PIM1 seems to be regulated at the translational level, illustrated by the approaches on the inhibition of translation elongation. Further analyses indicated that the statin effect at this level is specific for PIM1 in HepG2 cells, whereas a non-specific mode of action of simvastatin can be suggested in the colon cancer cell line. In the second project, the role of PIM1 during differentiation of blood monocytes into macrophages was analyzed. Therefore, monocytes were isolated from donor blood and differentiated into M1 macrophages, which trigger an anti-tumor response of the immune system. To analyze the function of PIM1 as a putative regulator of differentiation, the Pim-1 mRNA and protein levels were investigated at the various developmental stages. The RT-qPCR experiments showed that the Pim-1 mRNA levels oscillate over the entire analyzed period of time of monocytes to the complete maturity of macrophages. An increase in mRNA levels could be observed at the beginning of differentiation process, which decreases towards the end of it. The investigations of the Western blots revealed that, along with the Pim-1 mRNA levels, the protein levels were also enhanced during the differentiation period of the macrophages. This confirms the assumption that PIM1 may play a role in regulating the differentiation of monocytes.