The importance of normal cell dependence on the ECM in linking cyclin D1 expression to cell cycle progression and growth in vitro is well documented (Miranti and Brugge, 2002
; Walker and Assoian, 2005
). In the absence of attachment to ECM substrates, growth factor-stimulated cells are unable to induce cyclin D1. Our study with HNSCC cells, cultured as anchorage-independent MCA show a serum-dependent cyclin D1 expression and proliferation. Notably, analysis of EGFR signaling in serum-stimulated and -starved MCA revealed that depending on the mode of activation, EGFR differentially modulate the Gab1/PI3K/AKT signaling pathway and regulate cyclin D1 expression and proliferation.
Previous work reported that formation of MCAs by HNSCC cells induces adhesion-mediated ligand-independent EGFR/ERK activation (Shen and Kramer, 2004
). Indeed, in serum-deprived MCAs, EGFR/ERK was constitutively activated while AKT remained inactive. However, the presence of adhesion-mediated ligand-independent active EGFR and ERK1/2 was not sufficient to promote the cell proliferative response as suggested by the diminished cyclin D1 and increased p27 protein expressions. EGFR signaling through endocrine, autocrine and juxtacrine mechanism are known (Schneider and Wolf, 2009
; Singh and Harris, 2005
). For example, HB-EGF-mediated juxtacrine signaling is sufficient to trigger strong AKT activation (Singh et al., 2007
). Though such mechanisms are possible, the EGFR activation in serum-deprived MCA was unable to trigger active AKT or promote cyclin D1 expression or proliferation.
Blocking EGFR function inhibits the expression of cyclin D1 and proliferation (Lenferink et al., 2001
). It is also known that EGFR-mediated cell proliferation requires integrin-mediated cell adhesion to the ECM (Bill et al., 2004
; Kuwada and Li, 2000
). We found that in HNSCC cell aggregates, EGFR-mediated serum-dependent cyclin D1 expression. In this study, we focused on the HSC-3 cells since this HNSCC cell line, derived from a cervical lymph node metastasis of a tongue primary lesion, has been shown to retain both its in vivo tumorigenicity and metastatic potential in the nude mouse model, and has been extensively characterized for its in vitro and in vivo phenotype and responsiveness to EGFR and other growth factors (Bourguignon et al., 2006
; Kawano et al., 2001
; Nurmenniemi et al., 2009
; Onishi et al., 2008
). The results obtained in the current studies seem relevant to HNSCC since serum-stimulation enhanced EGFR-dependent cyclin D1 induction in a panel of four other well-characterized HNSCC cell lines. Other studies in anchorage-independent cultures, using MCF10A and Ewing tumor sarcoma cells have shown that AKT but not ERK signaling is essential in intercellular adhesion-mediated cyclin D1 expression and growth (Fournier et al., 2008
; Kang et al., 2007
; Lawlor et al., 2002
). Our study in HNSCC cell aggregates show that EGFR-dependent ERK and AKT activation are both important in that they act synergistically to regulate cyclin D1 expression.
What is the mechanism by which activated EGFR acts differentially under the two conditions (serum-stimulated versus serum-starved)? We identified EGFR-Y1086 as a unique residue inefficiently autophosphorylated in the serum-deprived cell aggregates. Cells that form cell aggregates do so by forming a complex 3-D network of intercellular adhesions. The role of E-cadherin in mediating these cell-cell interactions and transactivation of EGFR has been established (Pece and Gutkind, 2000
; Shen and Kramer, 2004
). Employing several approaches involving E-cadherin and EGFR-chimera constructs, we were able to confirm the unique pattern of ligand-independent EGFR autophosphorylation.
EGFR-Y1086 is one of the major tyrosine residues located at the cytoplasmic region that is readily phosphorylated during EGFR kinase activation. This, in turn, can mediate downstream signaling events by binding with molecules such as Grb2, Shc, Gab1, STAT3 and c-Abl (Jorissen et al., 2003
; Thelemann et al., 2005
). In line with our observation of persistence ERK activity, ligand-independent EGFR activation associated with the signaling adaptor proteins, Grb2 and Shc. EGFR is known to associate with the PI3K/p85 regulator subunit and induce PI3K-mediated AKT activation (Hu et al., 1992
). However, our findings clearly suggest that though competent in Grb2 and Shc binding and ERK activation, ligand-independent EGFR activation is unable to complex with the PI3K/AKT signaling module.
Specificity of signaling by receptor tyrosine kinases depends on interaction with specific substrates as well as with substrates shared by other signaling-competent receptors. For example, Gab1 is a docking protein involved in the downstream signaling of many types of tyrosine receptor kinases, including EGFR (Holgado-Madruga et al., 1996
; Weidner et al., 1996
). Upon EGFR activation, Gab1 undergo tyrosine phosphorylation and potentiate EGF-mediated activation of the PI3K/AKT pathway (LeVea et al., 2004
; Mattoon et al., 2004
). To our knowledge, we show here for the first time that ligand-independent EGFR activation is unable to phosphorylate Gab1. Since residue Y1068 and Y1086 of EGFR have been shown to be important for EGFR/Gab1 association (Rodrigues et al., 2000
), this finding highlights the significance of inefficient EGFR-Y1086 autophosphorylation. It warrants future work to specifically examine mutated EGFR-Y1086F and its various signaling responses. Nevertheless, it is conceivable that phosphorylation at both Y1068 and Y1086 residues is critical and essential for efficient EGFR/Gab1 associations and subsequent Gab1 phosphorylation. In serum-deprived MCAs, the lack of efficient Gab1 phosphorylation may uncouple the EGFR/PI3K/AKT activation, thereby fail to promote cyclin D1 expression and proliferation. This possibility is likely because the expression of constitutively active AKT was able to by-pass inefficient EGFR activation and permit cyclin D1 induction and proliferation.
EGFR mediates PI3K/AKT signaling and regulates VEGF expression in hypoxic conditions (Jiang et al., 2001
; Pore et al., 2006
). Similarly, loss of EGFR function has been suggested to contribute during stress signaling induction, which in turn might influence tumor dormancy (Aguirre-Ghiso, 2007
). Under serum-deprivation, tumor cell-derived factors like Clusterin can modulate the PI3K/AKT pathway to determine growth and dormancy (Jo et al., 2008
). Thus, under unfavorable growth conditions, the ability of EGFR to regulate PI3K/AKT activation may bear important physiological implications. We hypothesize that during limited availability of nutrients/serum (e.g., due to poor vascularization), tumor islands of compact cell aggregates utilize differential EGFR signaling to uncouple the PI3K/AKT pathway yet still maintaining the ERK survival pathway. This may in turn act as an alternate mechanism to prevent cells from entering active cell-cycle progression and promote growth arrest. The presence of dormant tumor cell clusters in non-permissive microenvironment may be resistant to chemotherapy or irradiation and present significant challenges to treatment of disseminated disease. Thus, understanding such microenvironmental-derived survival pathways may suggest novel therapeutic approaches that could target dormant tumor cell in micrometastases.