Abstract
Abstract Basal-like carcinoma is a subtype of breast cancer with poor prognosis that is characterized by pronounced cell-to-cell non-uniformities. Intratumor heterogeneity could involve sporadic single-cell activation of key pathways implicated in cancer progression, but it has not been possible to identify such pathways in an unbiased way. Our lab has developed a new approach for globally identifying coregulated, heterogeneously expressed genes among single cells (Nat Methods 7:311 [2010]). In a 3D in vitro culture model of a basal-like breast epithelial cell line (MCF10A), we stochastically profiled 4557 transcripts for heterogeneity and identified 547 that were predicted to be strongly dichotomous among extracellular matrix (ECM)-attached cells in the developing acinus. Hierarchical clustering of the heterogeneous transcripts revealed a group of ∼15 biosynthetic genes that contained the transcription factor, JUND, and the basal-cell cytokeratin, KRT5. In the same dataset, we identified a second group of genes-containing a transforming growth factor-beta (TGF-beta) family ligand (GDF11), a TGF-beta receptor (TGFBR3), and a TGF-beta marker protein (TGFBI)-with strong sampling fluctuations that were opposite to those of JUND cluster. Here, we show that perturbation of JUND or TGFBR3 expression profoundly disrupts 3D morphogenesis. Constitutive JUND expression causes stable cribiform-like bridges across the acinar lumen, whereas TGFBR3 knockdown induces a ductal-branching phenotype. These perturbations further revealed that JUND and TGFBR3 are reciprocally coupled by interlinked positive and negative feedback loops that could give rise to asynchronous oscillations during morphogenesis. Live-cell imaging with a fluorescently tagged Smad2 revealed sporadic pulses of TGF-betaR signaling in matrix-attached cells during morphogenesis. Last, in clinical specimens of breast cancer with dichotomous basal cytokeratin expression, we find that junD and Krt5 proteins are correlated in ECM-attached cells. Interestingly, junD and Krt5 are anticorrelated in cells not directly in contact with stromal ECM, and this regulation is mimicked in 3D culture of MCF10A cells. Together, our results raise the possibility that heterogeneous basal-like breast cancers switch between two states that depend critically on the ECM context. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4922. doi:10.1158/1538-7445.AM2011-4922
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