gms | German Medical Science

Objective: For glial tumor cells, the mechanisms used by integrins, especially α_vβ₃ integrin, to promote tumor cell proliferation are still a matter of discussion. Krüppel-like factor 8 (KLF-8) has been identified only recently as an integrin-dependent signaling molecule which stimulates cyclin D1 and subsequently G₁/S-progression in mouse fibroblast NIH3T3 tumor cells. In the present study we investigate the expression and the functional impact of suspected downstream signaling molecules of α_vβ₃ integrin with special interest on KLF-8.

Methods: Sections and single tumor cells from primary diagnosed glioblastomas (WHO°IV, n=5) and low grade gliomas (WHO°II, n=5) as well as glioma cell line U87 MG were subjected to immunohistochemical and/or immunocytochemical staining for integrin α_vβ₃ and downstream signaling molecules: the phosphorylated focal adhesion kinase (pFAK), KLF-8 and cyclin D1. To determine whether these molecules are expressed in a WHO grade dependent manner we also performed Western blot analysis in the same entities (n=5 each). Additionally, quantitative PCR was carried out for integrin subunits α_v and β₃, KLF-8 and cyclin D1. For analysis of the functional impact of KLF-8 on cell cycle progression in gliomas, U87 MG were transduced with sh-RNA for KLF-8 knockdown.

Results: Integrin α_vβ₃ and the signaling molecules pFAK and cyclin D1 as well as the transcription factor KLF-8 could be detected in low grade gliomas and glioblastomas but were much more prominent in high grade gliomas as confirmed by Western blot analysis and immunohistochemistry. In U87 MG cells α_vβ₃-integrin and pFAK were found to be co-localised on cell membranes, whereas cyclin D1 and KLF-8 were detected preferentially in the nucleus of isolated cells. Transduction of U87 MG cells with sh-RNA-expressing adenoviral vectors at a low multiplicity of infection led to a >80% knockdown of KLF-8 mRNA as quantified by qRT-PCR. Moreover, preliminary data showed a reduction of the proliferation rate of KLF-8 knockdown – U87 MG cells of approximately 25% compared to cells transduced with a scrambled sh-RNA.

Conclusions: KLF-8 is expressed grade-dependently in glial brain tumors. Inhibiting downstream integrin signaling by blocking KLF-8 may represent a novel antiproliferative treatment strategy for malignant brain tumors.