Purpose: Epidermal growth factor receptor (EGFR) is commonly overexpressed in lung cancer.Cetuximab is a chimeric mouse-human antibody targeted against EGFR. Compared with its inhibitory properties, its immunologic mechanisms have not been well studied. In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC) activity of cetuximab against lung cancer cell lines. Experimental Design: We studied the correlation between EGFR expression in lung cancer cell lines and the ADCC activity of cetuximab as well as the influence of interleukin-2 and chemotherapy on the ADCC activity. EGFR expression was measured by a quantitative flow cytometric analysis and immunohistochemistry. The ADCC activity was assessed by a 4-h 51 Cr release assay. Peripheral blood mononuclear cells, purified Tcells, natural killer (NK) cells, and monocytes from healthy donors or lung cancer patients were used as effector cells. Results: Fresh peripheral blood mononuclear cells exhibited cetuximab-mediated ADCC activity against lung cancer cell lines at a low concentration of cetuximab (0.25 Ag/mL). A logarithmic correlation was observed between the number of EGFRs and ADCC activity. Even low EGFR expression, which was weakly detectable by immunohistochemistry, was sufficient for maximum ADCC activity, and further increases in EGFR expression on the target cells had no further effect on the ADCC activity. In addition, ADCC activity was enhanced by interleukin-2 mainly through activation of NK cells and was less susceptible to immunosuppression by chemotherapy than NK activity in lung cancer patients. Conclusions: These observations suggest the importance of ADCC activity as an immunologic mechanism of cetuximab in biological therapy for lung cancer patients.

A putative guanine nucleotide exchange factor (GEF), termed leukemia-associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/ RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G proteincoupled receptors (GPCRs) and heterotrimeric G proteins of the GK K 12 family stimulate Rho-dependent signaling pathways. ß

A recently identified family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115RhoGEF exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G protein ␣ subunits of the G␣ 12 family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from G␣ 12 and G␣ 13 to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of G protein-coupled receptors (GPCRs) to stimulate Rho, although the underlying molecular mechanisms are still unknown. Here, we found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within 1 min (11-fold) and a more limited but sustained activation (4-fold) thereafter, which lasts even for several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. As thrombin receptors stimulate focal adhesion kinase (FAK) potently, we explored whether this non-receptor tyrosine kinase participates in the activation of Rho by GPCRs. We obtained evidence that FAK can be activated by thrombin, G␣ 12 , G␣ 13 , and G␣ q through both Rho-dependent and Rho-independent mechanisms and that PDZ-RhoGEF and LARG can in turn be tyrosine-phosphorylated through FAK in response to thrombin, thereby enhancing the activation of Rho in vivo. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through the tyrosine phosphorylation of RGL-containing RhoGEFs.