Abstract
AbstractTransforming Growth Factor-[beta] (TGF-[beta]) and Epidermal Growth Factor (EGF) signaling pathways are both independently implicated as key regulators in tumor formation and progression. Here, we demonstrate that activation of the tumor-associated and over-expressed EGFR desensitizes TGF-[beta] signaling and its cytostatic regulation through specific Stat3 activation and Smad7 induction. In normal and tumor human cell lines, reduction of TGF-[beta]-mediated Smad2 phosphorylation, nuclear translocation and Smad3 target gene activation were observed where EGFR is over-expressed, but not in cells which expressed EGFR at normal levels. The EGFR downstream signaling molecules phosphatidyinositol-3 Kinase (PI3K) or mitogen-activated protein kinase/ERK kinase (MEK) are not responsible for the down-regulation of TGF-[beta] signaling since blockade of them by specific pharmacological inhibitors LY294002 and U0126 had little effects on the sensitivity of TGF-[beta] signaling. We identified Stat3 as a signaling molecule activated specifically and persistently by over-expressed EGFR, but not by normal levels. Importantly, Stat3 is responsible for the reduced TGF-[beta] sensitivity, since its knockdown by siRNA restored TGF-[beta] signaling sensitivity. Furthermore, over-expressed EGFR, through Stat3 activates Smad7 promoter activity, increasing its protein levels, which is a negative regulator of TGF-[beta] signaling. Consequently, cells were re-sensitized to TGF-[beta] when Smad7 expression was reduced using siRNA. Therefore we establish a novel EGFR-Stat3-Smad7-TGF-[beta] signaling molecular axis where tumor-associated over-expression of EGFR in epithelial cells results in hyperactivation of Stat3, which activates Smad7 expression, compromising the TGF-[beta]'s cytostatic regulation of epithelium and consequent tumor formation.
Highlights
Growth factor and cytokine signaling networks control many aspects of cell behaviour such as proliferation, survival, migration, invasive capabilities, transformation and differentiation
Stimulation with Transforming Growth Factor-E (TGF-E) activated pCAGA12-luc activity in all 5 cell lines used (Fig. 1b). This increased pCAGA12luc activity was significantly reduced in EGFR over-expressing cells (A431, HN5 and 293TEGFR), but not in the low EGFR expressing cells lines (293T and A549) when cells were treated with Epidermal Growth Factor (EGF) (Fig. 1b), suggesting that EGF inhibited pCAGA12-luc activity in cells with high levels of EGFR expression
As we have previously shown that hyper-activation of Stat[3] induced expression of the negative regulator of TGF-ȕ signaling, Smad[737], we examined whether the EGFR-Stat[3] mediated desensitization of the TGF-ȕ signaling is dependant on Smad[7]
Summary
Growth factor and cytokine signaling networks control many aspects of cell behaviour such as proliferation, survival, migration, invasive capabilities, transformation and differentiation. In normal cells, these complex signaling pathways are tightly regulated. These complex signaling pathways are tightly regulated Alterations of these signals are often found to cause, directly or indirectly, tumor formation. That role of EGFR in carcinogenesis led to the development and evaluation of EGFR blocking agents for cancer treatment[1]. The successful development of EGFR-specific TKIs gave rise to high hope that EGFR-blocking reagents could be the generation of “magic bullets” in treating human cancers[14]
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