Functional interaction between mouse erbB3 and wild-type rat c-neu in transgenic mouse mammary tumor cells

INTRODUCTION: Co-expression of several receptor tyrosine kinases (RTKs), including erbB2 and erbB3, is frequently identified in breast cancers. A member of the RTK family, the kinase-deficient erbB3 can activate downstream signaling via heterodimer formation with erbB2. We studied the expression of RTK receptors in mammary tumors from the wild-type (wt) rat c-neu transgenic model. We hypothesized that physical and functional interactions between the wt rat neu/ErbB2 transgene and mouse ErbB3-encoded proteins could occur, activating downstream signaling and promoting mammary oncogenesis. METHODS: Immunohistochemical and Western blot analyses were performed to study the expression of rat c-neu/ErbB2 and mouse erbB3 in mammary tumors and tumor-derived cell lines from the wt rat c-neu transgenic mice. Co-immunoprecipitation methods were employed to quantitate heterodimerization between the transgene-encoded protein erbB2 and the endogenous mouse erbB3. Tumor cell growth in response to growth factors, such as Heregulin (HRG), epidermal growth factor (EGF), or insulin-like growth factor-1 (IGF-1), was also studied. Post-HRG stimulation, activation of the RTK downstream signaling was determined by Western blot analyses using antibodies against phosphorylated Akt and mitogen-activated protein kinase (MAPK), respectively. Specific inhibitors were then used with cell proliferation assays to study the phosphoinositide-3 kinase (PI-3K)/Akt and MAPK kinase (MEK)/MAPK pathways as possible mechanisms of HRG-induced tumor cell proliferation. RESULTS: Mammary tumors and tumor-derived cell lines frequently exhibited elevated co-expression of erbB2 and erbB3. The transgene-encoded protein erbB2 formed a stable heterodimer complex with endogenous mouse erbB3. HRG stimulation promoted physical and functional erbB2/erbB3 interactions and tumor cell growth, whereas no response to EGF or IGF-1 was observed. HRG treatment activated both the Akt and MAPK pathways in a dose- and time-dependent manner. Both the PI-3K inhibitor LY 294002 and MEK inhibitor PD 98059 significantly decreased the stimulatory effect of HRG on tumor cell proliferation. CONCLUSION: The co-expression of wt rat neu/ErbB2 transgene and mouse ErbB3, with physical and functional interactions between these two species of RTK receptors, was demonstrated. These data strongly suggest a role for erbB3 in c-neu (ErbB2)-associated mammary tumorigenesis, as has been reported in human breast cancers

Nicotine induces cell proliferation by β-arrestin–mediated activation of Src and Rb–Raf-1 pathways

Recent studies have shown that nicotine, a component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. While nicotine is not carcinogenic by itself, it has been shown to induce cell proliferation and angiogenesis. Here we find that mitogenic effects of nicotine in non–small cell lung cancers (NSCLCs) are analogous to those of growth factors and involve activation of Src, induction of Rb–Raf-1 interaction, and phosphorylation of Rb. Analysis of human NSCLC tumors show enhanced levels of Rb–Raf-1 complexes compared with adjacent normal tissue. The mitogenic effects of nicotine were mediated via the α(7)-nAChR subunit and resulted in enhanced recruitment of E2F1 and Raf-1 on proliferative promoters in NSCLC cell lines and human lung tumors. Nicotine stimulation of NSCLC cells caused dissociation of Rb from these promoters. Proliferative signaling via nicotinic acetylcholine receptors (nAChRs) required the scaffolding protein β-arrestin; ablation of β-arrestin or disruption of the Rb–Raf-1 interaction blocked nicotine-induced proliferation of NSCLCs. Additionally, suppression of β-arrestin also blocked activation of Src, suppressed levels of phosphorylated ERK, and abrogated Rb–Raf-1 binding in response to nicotine. It appears that nicotine induces cell proliferation by β-arrestin–mediated activation of the Src and Rb–Raf-1 pathways