The biology of Notch3 signalling pathway in human lung cancers

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Notch3 is a transmembrane receptor and a member of the Notch signaling pathway, essential for cellular differentiation in variety of developing tissues in both invertebrates and vertebrates. We previously have observed that the overexpression of Notch3 is associated with a balance t(15;19) in an aggressive lung tumors. Using IHC in microtissue arrays containing 222 lung tumors, we further demonstrated that approximately 20% of carcinomas of the lung showed increase Notch3 expression, suggesting that Notch3 plays an important role in lung cancer oncogenesis. To determine whether Notch3 contributes to the oncogenic phenotype, we stably transfected Notch3 overexpressing cell line HCC2429 and H1819 with a dominant-negative construct which retains the ligand-binding capability but lacks the signaling domain. Our preliminary data showed that the inhibition of the Notch3 pathway by the dominant-negative construct decrease the ability of Notch3 expressing cell lines to form colonies in soft agar colony formation assay and renders the cells more dependent on growth factors. Data from Drosophila literature suggest that there is crosstalk between Notch and mitogen-activated protein kinase (MAPK) pathway. Since extracellular-signal regulated kinase (ERK) plays an important role in cancer, we examined whether the inhibition of Notch3 alters ERK1 (p42) and ERK2 (p44) activation. Our data demonstrated that Notch3 inhibition renders the cells more dependent on growth factors. Furthermore, when exposed continuously with serum, p44/p42 phosphorylation was attenuated significantly compared to vector control, suggesting that the observed growth factor dependency from Notch3 inhibition is through the modulation of the MAPK pathway. To examine the regulation of p44/p42 phosphorylation, we investigated the expression of MKP1 by real-time PCR method. MKP1 expression in HCC2429 transfected with Notch3 dominant negative construct was increased compared to that with empty vector, suggesting that increased expression of MKP1 is one mechanism of down regulating ERK phosphorylation. Our data support a role of Notch3 in lung cancer, and the mechanism of Notch3-mediated oncogenesis is potentially through the activation of the MAPK pathway through MKP1 expression.