Background and relevance for cancer research:
Dissemination of lymphomas is regulated by signaling pathways that are poorly understood but appear to involve multiple proteins. The absence of any of these proteins may be the reason that a tumor does not disseminate, and these components are therefore potential prognostic markers, which are scarce for lymphomas. Our long-term aim is to elucidate these pathways, and thus to identify a comprehensive set of such potential markers.
We have analyzed the pathways in four different murine cell lines with defined dissemination behavior. The most detailed information was so far obtained for T-cell hybridomas, a model for T-lymphoma. Their invasion and dissemination requires cooperative signals from chemokine receptors and from the integrin LFA-1. The chemokine signal activates LFA-1, that can then bind its ligand, and this triggers the LFA-1 signal. This process can be mimicked in an assay of chemotaxis through filters coated with the LFA-1 ligand ICAM-1, using low levels of SDF-1 as chemoattractant. This is an appropriate model because all invasion inhibitors also block this chemotaxis. In contrast, several invasion inhibitors do not affect chemotaxis towards higher concentrations of SDF-1, which is LFA-1-independent and requires only the chemokine signal.
We observed that dominant-negative mutants of the tyrosine kinases ZAP-70 and Pyk2 blocked invasion and dissemination . LFA-1-dependent chemotaxis was also completely inhibited, but LFA-1-independent chemotaxis towards high SDF-1 levels was not affected at all, and thus the LFA-1 signal was blocked quite specifically. Both SH2 domains of ZAP-70 are required for inhibition. It is not yet clear whether the Pyk2 mutant interferes with Pyk2 itself or rather with its homologue FAK. BW5147 cells, from which the T-cell hybridomas were made, are not invasive, do not disseminate and do not migrate in the low-SDF-1/ICAM-1 chemotaxis assay. LFA-1 and the SDF-1 receptor are expressed in BW5147 cells, but ZAP-70 is not. This suggests that in these cells, ZAP-70 is one of the missing components responsible for the lack of dissemination.
For the other three cell lines: GROB large B-cell lymphoma, MDAY-D2 acute myeloid leukemia and ESb T-cell lymphoma, the mechanisms are in part similar and in part distinct. Similarly as in the T-cell hybridoma, introduction of the S1 subunit of pertussis toxin, which blocks Gi protein signals of chemokine receptors, blocks dissemination of GROB and MDAY-D2 cells, although for the latter only to the liver and spleen. To prevent dissemination of MDAY-D2 cells to the bone marrow, Gq proteins had to be inhibited as well. ESb cells, which use the integrin alpha4beta1 rather than LFA-1, do not require Gi proteins. The three cell lines express the ZAP-70 homologue Syk, but not ZAP-70. The role of tyrosine kinases in these cells remains to be established, but a role for Syk in ESb invasion is suggested by inhibitor effects. Comparison of all four cell lines should reveal common and divergent parts of pathways that are operative in different hematopoietic tumor types.
Purpose and possible results:
Our main aim is to elucidate the signal pathway that is blocked by the Pyk2 and ZAP-70 mutants. We will establish whether Pyk2 or FAK is involved, and identify the proteins to which the dominant-negative Pyk2 binds. The relevance of thus identified known proteins for invasion and dissemination will be tested using known or putative dominant-negative mutants. If none of these proteins is relevant, we will search for novel binding partners and substrates. Furthermore, we will study whether Pyk2 and/or FAK are involved in invasion and dissemination of GROB, MDAY-D2 and ESb cells.
The ZAP-70 tandem SH2 domains are so far only known to bind to ITAM sequences in the T-cell receptor/CD3 complex, which are phosphorylated by Src family kinases. However, the TCR/CD3 complex is not present in our T-cell hybridomas, and Src-like kinases are not involved in their invasion. The relevant ZAP-70 binding partner must therefore be novel and we aim to identify this protein. Finally, we will study the involvement of known effectors of ZAP-70 and Syk, such as Vav and PLC-p, and adaptors that link these molecules, in all four cell lines. For identified relevant proteins we will assess expression levels in a well-defined set of human non-Hodgkin lymphomas. This should indicate their potential usefulness as part of a set of prognostic markers. |
