Enhanced EGFR inhibition and distinct epitope recognition by EGFR antagonistic mAbs C225 and 425.

Monoclonal antibodies (mAbs) that inhibit activation of the epidermal growth factor receptor (EGFR) have shown therapeutic potential in select malignancies including breast cancer. Here, we describe that combined use of two such mAbs, C225 (Cetuximab) and 425 (EMD55900), reduced growth and survival of EGFR overexpressing MDA-MB-468 breast cancer cells more effectively than either antibody alone. Similarly, the C225/425 antibody combination more effectively inhibited AKT and MAPK phosphorylation in MDA-MB-468 cells. Surface plasmon resonance, size exclusion chromatography and analytical ultracentrifugation demonstrated that mAbs C225 and 425 simultaneously bind to distinct antigenic epitopes on domain III of the soluble wild-type EGFR. Furthermore, neither mAb competed with the other for binding to cells expressing either wild-type EGFR or a mutant EGFR (EGFRvIII) associated with neoplasia. Mutagenesis experiments revealed that residues S460/G461 in EGFR domain III are essential components of the 425 epitope and clearly distinguish it from the EGF/ TGFalpha binding site and the C225 interaction interface. Collectively, these results support the conclusion that therapeutic EGFR blockade in cancer patients by combined use of mAbs C225 and 425 could provide advantages over the use of the two antibodies as single agents.

Malignant transformation of immortalized HaCaT keratinocytes through deregulated nuclear factor kappaB signaling.

Previous studies addressing functional aspects of nuclear factor kappaB (NF-kappaB) activation in normal and transformed keratinocytes revealed complex and seemingly contradictory roles of this transcription factor in this cell type. In normal skin, NF-kappaB signaling seems to inhibit squamous cell carcinoma development whereas, in squamous cell carcinoma themselves, deregulated NF-kappaB expression and/or signaling is frequently observed. To further investigate this paradox, we focused on NF-kappaB activation as it relates to the transformed phenotype of immortalized but nontumorigenic human keratinocytes (HaCaT cells). We observed that NF-kappaB activity contributed to survival and growth of cultured HaCaT keratinocytes as shown by use of pharmacologic NF-kappaB inhibitors, RNA interference, and inducible overexpression of a dominant interfering IkappaB construct. NF-kappaB activation was largely provided through interaction with extracellular matrix components because preventing cell attachment by forced suspension culture markedly reduced NFkappaB signaling associated with cell death (anoikis); conversely, anoikis was partially reversed by NF-kappaB activation induced either by tumor necrosis factor-alpha treatment or by overexpressing the NF-kappaB p65 subunit in HaCaT cells. Furthermore, overexpression of NF-kappaBp65 in HaCaT cells induced colony formation in soft agar and tumorigenicity in nude mice. In summary, as opposed to normal keratinocytes, immortalized HaCaT keratinocytes provide a cellular context in which deregulated NF-kappaB signaling supports multiple malignant traits in vitro and in vivo.

Complex regulation of signal transducers and activators of transcription 3 activation in normal and malignant keratinocytes.

Previous work implicated activation of the signal transducer and activator of transcription (STAT)3 downstream of the epidermal growth factor receptor (EGFR) in the malignant phenotype of squamous carcinoma cells (SCC). Here, we show that EGFR-dependent STAT3 activation is restricted to malignant keratinocytes. Specifically, constitutive and epidermal growth factor-induced phosphorylation of STAT3 on Y705 was observed only in SCC but not in either immortalized (HaCaT) or normal keratinocyte strains. Furthermore, STAT3 activation as determined by DNA binding assays was restricted to SCC and dependent on EGFR activation. Forced expression of EGFR in immortalized keratinocytes (HaCaT cells) was associated with enhanced EGFR activation but not STAT3-Y705 phosphorylation. EGFR-dependent activation of mitogen-activated protein kinase (MAPK) kinase 1 negatively regulated STAT3-Y705 phosphorylation in normal and malignant keratinocytes. Together, these results underscore that EGFR activation is required but not sufficient for STAT3 activation to occur in malignant keratinocytes. They also highlight complex regulation of STAT3 phosphorylation through EGFR activation including negative regulation via the MAPK kinase/MAPK signaling pathway.