We were the first to demonstrate that c Abl and Arg also are activated in solid tumors, downstream of constitutively activated receptor tyrosine kinases and Src kinases, VEGFR inhibition and promote invasion and proliferation. Arlinghaus and colleagues subsequently showed that c Abl and Arg also are activated in non small cell lung cancer cells, and Maina and colleagues demonstrated that c Abl is activated downstream of c Met in gastric carcinoma cells. Several lines of evidence suggest that c Abl and Arg may contribute to melanoma development/progression: 1) MDA MB 435s, originally thought to be of breast origin, was recently identified as melanoma M14, 2) imatinib inhibits proliferation of some melanoma cell lines.

However, the activities of c Abl and Arg were not examined, and the mechanism of STI571 mediated inhibition of proliferation was not determined, and 3) imatinib inhibits murine melanoma tumor growth in a model that lacks expression E7080 clinical trial of c Kit and PDGFR,B. These data prompted us to examine whether cAbl and Arg play a role in human melanoma progression. Here, we demonstrate that cAbl/Arg kinase activities are increased in primary melanomas and in some human melanoma cell lines, their activation is required for proliferation, survival, and invasion, cAbl and Arg promote melanoma invasion via distinct molecular pathways, and c Abl and Arg drive melanoma metastatic progression. Therefore, c Abl and Arg are important clinical targets in melanoma, and represent an unexplored avenue for targeted treatment. Expression of c Abl and Arg was dramatically elevated in all melanoma cell lines examined relative to primary melanocytes.

Inguinal canal To determine whether c Abl and Arg are activated in melanoma cell lines, their basal activities were directly assessed by in vitro kinase assay utilizing the known c Abl/Arg target, Crk, as substrate. Interestingly, several melanoma cell lines had high c Abl and/or Arg activity. With the exception of WM278, phosphorylation of Crk/CrkL, c Abl/Arg targets, paralleled c Abl/Arg activities. To test whether c Abl and Arg are activated in primary melanomas, we performed immunohistochemistry on melanoma tissue microarrays. Phospho specific antibodies to c Abl cross react with phospho PDGFR and phospho EGFR, and thus, cannot be used to assess activity by IHC, and phospho specific Arg antibodies are not available.

Therefore, we stained melanoma tissue microarrays with an antibody to the c Abl/ Arg phosphorylation order BI-1356 sites on c Abl/Arg substrates, Crk and CrkL. We and others previously showed that Crk/CrkL phosphorylation on Y221/Y207 correlates with c Abl/Arg activity in cancer cell lines. An advantage to this approach is that activation of c Abl and Arg can be assessed simultaneously. In normal skin, pCrk/CrkL staining was limited to the cytoplasm and nuclei of keratinocytes and nuclei of lymphocytes. Most benign nevi demonstrated weak nuclear pCrk/CrkL staining, although some exhibited moderate strong staining and P_proportion of positively staining tumor cells.