Breast cancer remains the second leading cause of cancer related death in women, in spite of advancements in detection, diagnosis and treatment. The heterogeneity of breast cancer hindered our understanding of the mechanisms of this disease. The tumor microenvironment, composed of stromal cells and non-cellular extracellular matrix, represents one of the heterogeneous features of breast cancer. One stromal cell type, fibroblasts, has been proposed to be important in the regulation of the development of normal mammary glands and tumors. However, the exact signaling pathways governing the crosstalk between the fibroblasts and tumor cells remain poorly understood.
In the present study, we examined functions of Pten and Ets2 in stromal fibroblasts using conditional knockout mouse models. Deletion of Pten from the stromal fibroblasts resulted in an acceleration of mammary tumorigenesis. Inactivation of Pten in the fibroblasts promoted collagen deposition, inflammation and angiogenesis in the mammary stroma, which in turn supported tumor growth. In addiction, the absence of Pten also resulted in the activation of Ets2, a transcription factor and proto-oncogene, in the stromal fibroblasts. Ets2 acted as a critical downstream effector molecule of Pten, as deletion of Ets2 in fibroblasts reduced tumor growth caused by Pten-absent fibroblasts. Furthermore, Ets2 ablation resulted in a reversal of inflammation and angiogenesis caused by Pten deletion in the fibroblasts.
Cre/loxP mediated conditional inactivation of Ets2 in the stromal fibroblasts and mammary epithelial cells revealed that Ets2 was necessary for tumor growth in the fibroblasts, but not the mammary epithelium, in the MMTV-PyMT mouse model. Gene expression profiling revealed that Ets2 controls a tumor fibroblast specific transcriptional program to mediate angiogenesis and ECM remodeling in the tumor microenvironment. As a transcription factor, Ets2 directly controlled the transcription of target genes, such as Mmp9, in the tumor associated fibroblasts. Ets2-null tumor associated fibroblasts had impaired ability to recruit endothelial cells and produce MMP9. Importantly, both the mouse Pten fibroblast gene signature and Ets2 tumor fibroblast signature were able to separate human breast cancer stroma from normal stroma, suggesting the PTEN-ETS2 axis represents a major pathway deregulated in breast cancer stroma.
Post-translational regulation of Ets2 by PTEN has been shown to be mediated by AKT, ERK and JNK pathways. However, the transcriptional regulation of Ets2 downstream of PTEN was unknown. With a panel of chemical inhibitors and ShRNA knockdowns, we have found that the activation of the JNK pathway was critical for the upregulation of Ets2 mRNA in Pten-deficient fibroblasts. Additional results suggest JNK is the main functional pathway controlling Ets2 expression in Pten-deficient stromal fibroblasts.
In summary, we have identified that PTEN and ETS2 as key regulators in the stromal fibroblasts to modulate mammary epithelial tumor growth. Understanding how the PTEN-ETS2 axis acts in the stroma provided novel mechanisms of cancer epithelium-stroma interactions.