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.

EGFR-dependent downregulation of Bim in epithelial cells requires MAPK and PKC-delta activities.

Activation of the epidermal growth factor receptor (EGFR) provides a measure of protection to immortalized epidermal keratinocytes (HaCaT cells) against apoptosis induced by diverse cellular stressors. This effect is due, in part, to sustained MAPK-dependent Bcl-xL expression. Here, we report a second EGFR/MAPK-dependent signaling event that protects HaCaT cells against apoptosis incurred during forced suspension culture (anoikis). This pathway targets Bim, a pro-apoptotic BH3-only Bcl-2 family member. Bim expression was functionally relevant to HaCaT cell survival as demonstrated by partial protection against anoikis provided by siRNA-induced Bim downregulation. Growth factor starvation of attached and suspended cells was associated with enhanced Bim expression whereas EGFR activation reduced Bim expression by inducing Bim phosphorylation and proteasomal degradation. EGFR-dependent Bim phosphorylation required MAPK activation. Furthermore, PKC-delta activity contributed to both MEK/MAPK phosphorylation and Bim phosphorylation as demonstrated using both pharmacological inhibitors of PKC-delta and siRNA-mediated PKC-delta knockdown. In addition to HaCaT cells, EGFR activation supported survival and induced Bim phosphorylation in several squamous carcinoma cell lines in a strictly MAPK-dependent fashion. These results establish that EGFR activation attenuates susceptibility of immortalized and malignant keratinocytes to apoptosis by post-translational control of Bim-EL expression through a pathway requiring PKC-delta and MEK/MAPK activation.

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.

Neuropathogenic murine leukemia virus TR1.3 induces selective syncytia formation of brain capillary endothelium.

Exposure of newborn BALB/c mice to murine leukemia virus (MLV) TR1.3 induces fusion of brain capillary endothelial cells (BCEC), loss of cerebral vessel integrity, hemorrhagic stroke, and death. Although TR1.3 infects endothelial cells in multiple organs, syncytia are only observed in BCEC. To determine if viral and cellular factors are responsible for selective syncytia formation, capillary endothelial cells (CEC) from multiple organs were assayed in vitro for MLV infection and cell fusion. Following incubation with virus, all CEC were infected to an equal extent as determined by expression of MLV envelope and infectious virus production; however, MLV-induced syncytia were only observed in TR1.3-infected BCEC cultures. These in vitro results mirror the in vivo pattern of TR1.3 MLV infection and neuropathology, and definitively show that selective fusion and pathology of BCEC by MLV is determined by properties unique to BCEC as contrasted to other endothelial cell types.

Inhibition of cell survival and invasive potential of colorectal carcinoma cells by the tyrosine kinase inhibitor STI571.

Inhibiting tyrosine kinases has recently emerged as a therapeutic modality in several forms of neoplasia. The tyrosine kinase inhibitor STI571 (IMATINIB MESYLATE; GLEEVEC; GLIVEC) is a case in point as it has shown promise in the treatment of malignancies expressing the BCR/ABL fusion protein. In addition to BCR/ABL, STI571 inhibits the tyrosine kinase moieties of several cell surface receptors including the platelet-derived growth factor (PDGF) receptors and c-Kit. Previous work demonstrated that c-Kit activation supports migration, invasion and, survival of certain colorectal carcinoma cells including DLD-1. Here we describe that blocking c-Kit with STI571 inhibits these malignant traits not only in DLD-1 cells but also in two early passage colorectal carcinoma cell strains. Specifically, STI571 inhibited anchorage-independent colony formation and cell scattering in semi-solid medium. Furthermore, it enhanced apoptosis susceptibility and abrogated invasion of DLD-1 cells through Matrigel. In addition, STI571 treatment affected the balance of the Bcl-2 family of apoptosis regulators on favor of a pro-apoptotic phenotype. Specifically, STI571 treatment of DLD-1 cells was associated with lower levels of Bcl-2 expression accompanied by de novo expression of Bcl-xS. Finally, STI571 acted as a chemosensitizing agent in DLD-1 cells when used in combination with 5-fluorouracil.

Targeting the epidermal growth factor receptor in cancer: apoptosis takes center stage.

Aberrant activation of the epidermal growth factor receptor (EGFR) is frequently observed in neoplasia,notably in tumors of epithelial origin. Attempts to treat such tumors with EGFR antagonists have met with remarkable initial successes, particularly when EGFR antagonists were used in combination with chemotherapy or ionizing radiation. Considering the almost ubiquitous expression of the EGFR in normal epithelial tissues, these clinical trials also revealed a surprisingly low rate of adverse side effects associated with EGFR blockade. This review highlights antiapoptotic effects of EGFR activation as they relate to therapeutic efficacy of EGFR blockade. We introduce the concept that control of cell survival through EGFR activation is conditional in the sense that it is rate limiting to tumor cell survival but not to survival of normal epithelial cells. Specifically, normal epithelial cells are provided with a full complement of physiological cell-cell contacts and cell-matrix interactions that lessen their dependence on survival signals provided by the EGFR. By contrast, malignant tumor cells faced with inadequate cell-matrix contacts critically depend on EGFR activation for survival, rendering them more susceptible to apoptosis induction by EGFR blockade. Redundant control of cell survival by the EGFR and extracellular matrix/cell adhesion receptors is enabled, in part, by shared signal transduction pathways that control expression and activation states of members of the Bcl-2 family of apoptosis regulators.