Abstract A084: TGFβ induces Chordin-like 1 (Chrdl1) expression in ErbB2 expressing breast cancer cells with attenuated ShcA signaling

Abstract

Abstract Background: In 25 to 30% of human primary breast cancer, the ErbB2 receptor is overexpressed and is associated with poor prognosis. Transgenic mouse models have demonstrated that ErbB2 co-operates with TGFβ signaling pathway to promote the invasive and metastatic behavior of breast cancer cells. By transient knockdown approaches, a role for ShcA in mediating cooperation between the ErbB2 and TGFβ pathways has been established. Furthermore, a stable knockdown of ShcA in NMuMG cells overexpressing oncogenic ErbB2 significantly reduced tumor growth, which was found to be the consequence of reduced proliferation and endothelial cell recruitment coupled with increased apoptosis. Many of the migratory and invasive effects induced by TGFβ are secondary to the induction of a transcriptional program and TGFβ is known to uniquely induce the expression of specific genes in cells overexpressing ErbB2. Given that ShcA is required for TGFβ induced migration and invasion of ErbB2 expressing cells, we hypothesize that the signaling molecules engaged downstream of ShcA will ultimately modulate gene expression induced by TGFβ stimulation. Experimental Approach: To characterize ShcA dependent gene expression changes induced by TGFβ in ErbB2-expressing cells, we generated NMuMG-ErbB2 cells harboring dox-inducible ShcA shRNAs. These cell populations were divided into two groups treated with (ShcAlow) or without (ShcAhigh) Doxycycline, and each group was subjected to three conditions: one was left untreated and the others stimulated for 3 and 24 hours with TGFβ. Using gene expression profiling, we have identified a list of differentially expressed genes that are regulated by TGFβ signaling in the context of acute ShcA loss. Subsequently, we identified genes that have functions associated with migratory and invasive phenotypes. Results: Among the candidates identified by these approaches, Chrdl1, which has been demonstrated to interact with and neutralize several different members of the TGFβ family including BMP4, BMP5, BMP6, TGFβ1 and TGFβ2, displayed elevated transcript levels in ShcAlow cells following TGFβ treatment. ELISA assays reveal that reduced ShcA signaling results in the up-regulation of secreted Chrdl1 protein in ErbB2-expressing cells following TGFβ stimulation. Similar results were also obtained using additional human and mouse breast cancer cell lines. We next determined whether Chrdl1 found in the conditioned media of our NMuMG-ErbB2 ShcAlow cells, is functional. We observed that conditioned media from ShcAlow cells, which was harvested from cells stimulated with TGFβ (high Chrdl1), was able to decrease the activation of Smad1/5/8 following BMP4 stimulation, as measured by immunblot analysis for phospho-Smad1/5/8. Furthermore, immunohistochemical analysis confirmed that Chrdl1 induction, in response to reduced ShcA expression, is also observed in mammary tumors in vivo. Moreover, high levels of Chrdl1 correlated with diminished pSmad2 and pSmad1/5/8 in these tumor lysates. Conclusion: We have identified Chrdl1, a BMP inhibitor, as an interesting candidate that may account for the reduced growth and metastasis that accompany ShcA loss in ErbB2-expressing breast cancer cells. Citation Format: Chanèle Cyr-Depauw, Jason Northey, Zhifeng Dong, Sean Cory, Michael Hallett, Peter M. Siegel. TGFβ induces Chordin-like 1 (Chrdl1) expression in ErbB2 expressing breast cancer cells with attenuated ShcA signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A084.