Abstract 4560: Induced Sprouty4 expression in breast invasive ductal carcinoma cells downregulates ERK/MAPK activity and restores an epithelial-like phenotype
Authors: Brock Ethan J. , Jackson Ryan , Shah Seema , Li Quanwen , Sameni Mansoureh , Krawetz Stephen A. , Mao Shihong , Sloane Bonnie F. , Mattingly Raymond R. 
Published: July 2016

Breast cancer is one of the most common cancers in U.S. women, with approximately 25% of these cases being in situ disease. Sprouty proteins are currently recognized as important regulators of ERK/MAPK signaling, and have been studied in various cancer types. By employing RNA-seq, previous studies from our laboratories determined specific gene expression changes common to three models of ductal carcinoma in situ (DCIS)—MCF10.DCIS, SUM 102, and SUM 225—when compared to MCF10A cells (a model of non-transformed breast epithelium). All cell lines were grown in three-dimensional (3D) reconstituted basement membrane overlay culture because research has shown that the behavior of cancer cells in 3D matrices is more reflective of an in vivo response when exposed to drugs and radiotherapy than if they are cultured on plastic. Among the three models of DCIS, 63 genes were consistently upregulated. We identified 244 promoters associated with these 63 genes and performed bioinformatic data mining that revealed a high level of enrichment for a promoter framework shared by three of these genes: one of which encoded for the protein Sprouty4. We hypothesize that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling in DCIS, and its loss or reduced expression is a mechanism by which triple-negative lesions progress toward invasive ductal carcinoma (IDC). Using immunohistochemistry we found that Sprouty4 was highly expressed in certain human premalignant breast tissue samples, and that this expression was reduced in malignant triple-negative samples. These results correspond with immunoblot data from our 3D culture model of breast cancer progression in which Sprouty4 expression was higher during DCIS than in the IDC stage. Efficient over-expression of Sprouty4 reduced both ERK/MAPK activity as well as the aggressive phenotype of MCF10.CA1d IDC cells. Immunofluorescence experiments revealed data consistent with the relocation of E-cadherin back to the cell surface and the restoration of adherens. To determine if these effects were due to changes in ERK/MAPK signaling IDC cells were treated with MEK162, an allosteric MEK inhibitor. Nanomolar concentrations of drug produced a restoration of an epithelial-like phenotype similar to Sprouty4 over-expression. From these data we conclude that Sprouty4 may act to control ERK/MAPK signaling in a subset of DCIS, thus limiting the progression of these premalignant breast cancers.

Citation Format: Ethan J. Brock, Ryan Jackson, Seema Shah, Quanwen Li, Mansoureh Sameni, Stephen A. Krawetz, Shihong Mao, Bonnie F. Sloane, Raymond R. Mattingly. Induced Sprouty4 expression in breast invasive ductal carcinoma cells downregulates ERK/MAPK activity and restores an epithelial-like phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4560.