Matrix-independent Survival of Human Keratinocytes through an EGF Receptor/MAPK-Kinase-dependent Pathway

Abstract

Normal epithelial cells undergo apoptosis when they are denied contact with the extracellular matrix, in a process termed "anoikis." Conversely, malignant epithelial cells typically acquire anchorage independence, i.e., the capacity to survive and grow in the absence of matrix interaction. Here we asked the question whether anoikis is affected by signaling through the EGF receptor (EGFR). We focused on the EGFR because EGFR signaling is frequently deregulated in malignant epithelial cells. We demonstrate that EGFR activation markedly alleviated the requirement of matrix engagement for survival of primary and immortalized human keratinocytes in suspension culture. Protection of epithelial cells through EGFR activation against anoikis was associated with and required sustained MAPK phosphorylation during the early phase of suspension culture. Interestingly, high levels of MAPK phosphorylation were not only required for EGFR-mediated protection against anoikis but also occurred as a consequence of caspase activation at later stages of suspension culture. These results demonstrate that EGFR activation contributes to anchorage-independent epithelial cell survival and identify MAPK activation as an important mechanism in this process. INTRODUCTIONNormal epithelial cells require contact with extracellular matrix components to survive. In the absence of matrix attachment, these cells die exhibiting molecular characteristics of programmed cell death or apoptosis (Meredith et al., 1993;Frisch and Francis, 1994). This form of cell death has been termed anoikis (Frisch and Francis, 1994) and is likely to preclude dissemination of normal epithelial cells to inappropriate sites. Malignant transformation by oncogenic forms of Ras and Src confers resistance of cultured epithelial cells to anoikis (Frisch and Francis, 1994;Rosen et al., 2000). Activation of proto-oncogenic forms of Ras and Src in epithelial cells occurs not only during integrin engagement but also in response to soluble growth factors, including ligands for the EGF receptor (EGFR) (Luttrell et al., 1994;Walker et al., 1998). Our previous work has implicated the EGFR in protecting normal human keratinocytes against apoptosis of adherent cells in vitro (Rodeck et al., 1997a,b;Jost et al., 1999). Together, these findings led us to investigate whether signaling from the EGFR may also affect survival of human keratinocytes in the absence of matrix engagement. This is an important question because effective growth factor receptor signaling has been shown in fibroblasts to be contingent on adhesion receptor signaling (Lin et al., 1997;Renshaw et al., 1997;Bottazzi et al., 1999;Roovers et al., 1999). Thus, it may be argued that EGFR activation is not likely to affect survival of cells in forced suspension culture.By contrast, we demonstrate here that activation of the EGFR by endogenous and exogenous ligands substantially delayed death of normal human keratinocytes held in forced suspension. We describe that EGFR-dependent protection of keratinocytes against anoikis req...

Metastasis-associated protein (MTA)1 enhances migration, invasion, and anchorage-independent survival of immortalized human keratinocytes

“…We described previously that EGFR-dependent Bcl-x L expression is required for matrix-independent HaCaT cell survival (Jost et al, 2001b;Rodeck et al, 1997). Furthermore, Bcl-x L expression in these cells is controlled, in part, by MEK/ MAPK signaling (Jost et al, 2001a). Consistent with a role of MTA1 in EGFR-dependent Bcl-x L regulation we observed that inhibition of EGFR activation was associated with reduced MTA1 expression in HaCaT keratinocytes.…”

“…Importantly, survival of HaCaT-MTA1-S cells was similarly diminished by EGFR blockade suggesting that MTA1 expression alone is not su cient to relieve the requirement for EGFRderived signals for cell survival in the anchorageindependent state. Consistent with earlier results (Jost et al, 2001a) addition of EGF to the culture medium during suspension culture markedly improved survival of HaCaT-MTA-S cells (Figure 5b). However, overexpression of MTA1 antisense sequences obviated EGFdependent HaCaT cell survival in forced suspension culture indicating that MTA1 expression is essential to EGFR-dependent HaCaT cell survival in suspension culture.…”

“…However, overexpression of MTA1 antisense sequences obviated EGFdependent HaCaT cell survival in forced suspension culture indicating that MTA1 expression is essential to EGFR-dependent HaCaT cell survival in suspension culture. The concentrations of mAb 425 and EGF have been optimized for this assay as previously published (Jost et al, 2001a). The di erences in clonogenic growth after suspension culture were not due to di erences in cellular proliferation in the suspended state because forced suspension of HaCaT cells induced cell cycle arrest as assessed by BrdU incorporation (DeHoratius and Rodeck, unpublished results).…”

“…We observed marked downregulation of Bcl-x L protein levels in HaCaT-MTA1-AS cells as compared to HaCaT-MTA1-S cells ( Figure 7). As our previous work implicated MEK/MAPK signals in regulation of Bcl-x L expression levels in HaCaT cells (Jost et al, 2001a), we asked whether inhibition of MEK activity a ected MTA1 expression. Neither pharmacological inhibition of MEK activity by PD98059 nor over- (Jost et al, 2001a;Mansour et al, 1994) in HaCaT cells a ected MTA1 expression as determined by immunoblot analysis (not shown).…”

Expression and transcriptional regulation of caspase-14 in simple and complex epithelia

“…This was accomplished by maintaining keratinocytes for up to 72 h in suspension on top of a 0.9% agarose gel. We have shown previously that this treatment induces extensive apoptosis of primary human keratinocytes within 24 to 48 h. 25 This was confirmed by assessing PARP cleavage, which occurred within 48 h of forced suspension culture ( Figure 4B). However, no cleavage of procaspase-14 was observed throughout the observation period.…”