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In metastatic cancer, high expression levels of vitronectin (VN) receptors (integrins), FAK, and ERK5 are reported. We hypothesized that integrin-mediated ERK5 activation via FAK may play a pivotal role in cell adhesion, motility, and metastasis. ERK5 and FAK phosphorylation when metastatic MDA-MB-231 and PC-3 cells were plated on VN was enhanced. Further experiments showed co-immunoprecipitation of integrins beta1, alpha V beta 3, or alpha V beta 5 with ERK5 and FAK. To gain better insight into the mechanism of ERK5, FAK, and VN receptors in cell adhesion and motility, we performed loss-of-function experiments using integrin blocking antibodies, and specific mutants of FAK and ERK5. Ectopic expression of dominant negative ERK5/AEF decreased ERK5 and FAK (Y397) phosphorylation, cell adhesion, and haptotactic motility (micromotion) on VN. Additionally, DN FAK expression attenuated ERK5 phosphorylation, cell adhesion, and motility. This study documents the novel finding that in breast and prostate cancer cells, ERK5 is a critical target of FAK in cell adhesion signaling. Using different cancer cells, our experiments unveil a novel mechanism by which VN receptors and FAK could promote cancer metastasis via ERK5 activation.

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Despite constitutive expression of autocrine transforming growth factor-␣ (TGF-␣) in growth factor-independent colon carcinoma cells, the epidermal growth factor receptor (EGFr) is not saturated and can be further activated by exogenous EGFr ligand. Given that the activation of EGFr by exogenous growth factor has no further effect on DNA synthesis, the question arises as to what function this additional EGFr activation might have. We report that EGF induces integrin ␣ 2 expression, integrin-mediated adhesion, and micromotility of HCT116 cells. The stimulatory effect of ligand on these biological functions is abrogated by treatment with AG1478-and EGFr-blocking monoclonal antibody. This provides evidence that the biological responses are EGFr-mediated and EGFr is located upstream of integrin ␣ 2 expression. Therefore, although exogenous EGF has no effect on DNA synthesis beyond that induced by autocrine TGF-␣ (at subsaturating levels of EGFr occupation) exogenous growth factor does induce integrin ␣ 2 expression, cell adhesion, and micromotion. An important finding revealed by this study is the documentation of biological responses of EGFr-mediated functions, including DNA synthesis, cell adhesion, and micromotion, which differ in sensitivity with respect to different degrees of EGFr activation at the basal state and in response to exogenous ligand.The human colon carcinoma cell line HCT116 is aggressively tumorigenic, invasive, undifferentiated and growth factor-independent (1-5). The HCT116 cell line is representative of growth factor-independent carcinoma cell lines (2, 6). Constitutive expression of a full-length TGF-␣ 1 antisense cDNA has shown that the basis for the growth factor independence of these cells is the constitutive expression of TGF-␣ and, consequently, a low level constitutive activation of EGFr even when the cells are growth-arrested in G 0 (6, 7). We hypothesize that the relatively low level of EGFr activation resulting from autocrine TGF-␣ may be sufficient to fulfill one or more highly sensitive responses to EGFr signal transduction but would require augmentation by exogenously activated EGFr to optimally enable less sensitive functions. In HCT116 cells DNA synthesis from the G 0 state is fully activated by autocrine TGF-␣ such that exogenous EGF (or other growth factors) has no effect on this EGFr function despite the availability of unoccupied EGFr and signal transduction intermediates such as ERK (2, 7). Thus, the objective of this work was to identify whether there are any important functions mediated by the unoccupied EGFr in response to exogenous EGF and to determine whether this function is shared in part by autocrine TGF-␣ as well. We report that EGFr activation by exogenous ligand results in increased integrin expression, cell adhesion, and cell micromotion with less sensitivity than that exhibited by the optimal mitogenic function stimulated through relatively low level receptor occupation generated by autocrine TGF-␣ activity. In contrast, low level receptor occupation of EGFr by...

Recently, we showed that autocrine transforming growth factor ␣ (TGF␣) controls the epidermal growth factor receptor (EGFR)-mediated basal expression of integrin ␣ 2 , cell adhesion and motility in highly progressed HCT116 colon cancer cells. We also reported that the expression of basal integrin ␣ 2 and its biological effects are critically controlled by the constitutive activation of the ERK/MAPK pathway (Sawhney, R. S., Sharma, B., Humphrey, L. E., and Brattain, M. G. (2003)