Abstract
83% of non-invasive breast cancers are diagnosed as ductal carcinoma in situ (DCIS). While some DCIS tumors remain confined in the mammary duct, others progress into invasive ductal carcinoma (IDC). The mechanisms underlying invasion are not well understood. Current diagnostic methods cannot accurately predict which DCIS cases will progress to IDC, and unnecessary treatment affects long-term health and quality of life, with radiation potentially promoting malignancy-inducing mutations. One possible regulator of invasion may be extracellular matrix (ECM) stiffness. Increased ECM stiffness has been correlated with invasion and 3D culture models of normal mammary epithelium show that enhanced stiffness induces an invasive phenotype. However, the mechanisms underlying stiffness-induced invasion remain unclear. Studies have converged upon the finding that YAP, a transcriptional regulator that is deregulated in diverse cancers, is the transducer of ECM stiffness. However, these studies were primarily performed in 2D culture and involved col-1, a ligand that activates distinct signaling pathways and is not normally found in the BM. Here, we examined the gene expression profiles of 3D cultured MCF10A cells during stiffness-induced invasion with and without col-1. We generated interpenetrating networks (IPNs) of reconstituted basement membrane (rBM) and alginate, which allow stiffness to be tuned in the absence of col-1 and independently of cell adhesion ligand concentration and matrix pore size. Traditionally used hydrogels composed of rBM and col-1, with stiffness tuned by increasing concentrations of col-1, were also produced for 3D culture. Our results show that enhanced 3D stiffness increases MCF10A cell invasion and proliferation in both the presence and absence of col-1. However, in contrast to results from 2D culture, invasive phenotypes in 3D cultured cells did not correlate with nuclear localization of YAP, indicating lack of YAP activity. The dispensible nature of YAP in 3D stiffness sensing was supported by RNA-seq analysis, which showed a lack of increased gene expression of YAP downstream targets in conditions of enhanced stiffness. RNA-seq identified 389 differentially expressed genes in response to enhanced 3D culture stiffness. By relating these genes to transcription factors using ChIP-Seq data provided by ENCODE, we identified p300, FOS, STAT3, NELFE, and TAF1 as potential mechanotransducers of stiffness-induced invasion. Furthermore, highly expressed stiffness-induced genes were validated by immunofluorescence to identify potential indicators for invasion. Our RNA-seq results may lead to the development of a PCR-based prognostic that allows accurate determination of cancer invasion risk in breast cancer patients, informing course of treatment.
Note: This abstract was not presented at the meeting.
Citation Format: Joanna Y. Lee, Jessica Chang, Sungmin Nam, Ovijit Chaudhuri. Cancer invasion of mammary epithelial cells in 3D culture shows YAP-independent mechanotransduction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2956A. doi:10.1158/1538-7445.AM2017-2956A
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