RESUMO
Internal tandem duplications within the juxtamembrane region of the FMS-like tyrosine kinase receptor FLT3 (FLT3-ITD) represents one of the most common mutations in patients with acute myeloid leukemia (AML) which results in constitutive aberrant activation, increased proliferation of leukemic progenitors and is associated with an aggressive clinical phenotype. The expression of CD97, an EGF-TM7 receptor, has been linked to invasive behavior in thyroid and colorectal cancer. Here, we have investigated the association of CD97 with FLT3-ITD and its functional consequences in AML.Higher CD97 expression levels have been detected in 208 out of 385 primary AML samples. This was accompanied by a significantly increased bone marrow blast count as well as by mutations in the FLT3 gene. FLT3-ITD expressing cell lines as MV4-11 and MOLM-13 revealed significantly higher CD97 levels than FLT3 wildtype EOL-1, OCI-AML3 and HL-60 cells which were clearly decreased by the tyrosine kinase inhibitors PKC412 and SU5614. CD97 knock down by short hairpin RNA in MV4-11 cells resulted in inhibited trans-well migration towards fetal calf serum (FCS) and lysophosphatidic acid (LPA) being at least in part Rho-A dependent. Moreover, knock down of CD97 led to an altered mechanical phenotype, reduced adhesion to a stromal layer and lower wildtype FLT3 expression.Our results, thus, constitute the first evidence for the functional relevance of CD97 expression in FLT3-ITD AML cells rendering it a potential new theragnostic target.
Assuntos
Antígenos CD/biossíntese , Leucemia Mieloide Aguda/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Antígenos CD/genética , Linhagem Celular Tumoral , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HL-60 , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Inibidores de Proteínas Quinases/farmacologia , Receptores Acoplados a Proteínas G , Sequências de Repetição em Tandem , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Autotaxin (ATX) has been reported to act as a motility and growth factor in a variety of cancer cells. The ATX protein acts as a secreted lysophospholipase D by converting lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which signals via G-protein-coupled receptors and has important functions in cell migration and proliferation. This study demonstrates that ATX expression is specifically upregulated and functionally active in acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) mutation of the FLT3 receptor gene. Moreover, ATX expression was also found in normal human CD34+ progenitor cells and selected myeloid and lymphoid subpopulations. Enforced expression of mutant FLT3-ITD by retroviral vector transduction increased ATX mRNA in selected cell lines, whereas inhibition of FLT3-ITD signaling by sublethal doses of PKC412 or SU5614 led to a significant downregulation of ATX mRNA and protein levels. In the presence of LPC, ATX expression significantly increased proliferation. LPA induced proliferation, regardless of ATX expression, and induced chemotaxis in all tested human leukemic cell lines and human CD34(+) progenitors. LPC increased chemotaxis only in cells with high expression of endogenous ATX by at least 80%, demonstrating the autocrine action of ATX. Inhibition of ATX using a small molecule inhibitor selectively induced killing of ATX-expressing cell lines and reduced motility in these cells. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration during hematopoiesis and that deregulation of ATX contributes to the pathogenesis of AML.