Abstract S6-9: Expression of Hedgehog Proteins in the Epithelial and Stromal Compartments of Primary Breast Tumors and Its Prognostic Significance
Authors: Ramaswamy B , Li X , Nuovo G , Lu Y , Shapiro CL , Majumder S. 
Published: April 2011

Abstract
Purpose: Aberrant hedgehog (Hh) signaling has been reported in various cancers including breast cancers and clinical trials targeting this pathway are currently ongoing. Preclinical evidence suggests that increased ligand-dependent Hh signaling in breast tumor epithelial (E) cells cross-talks and activates Hh pathway in the stroma (S) but the clinical implication of this E/S interaction is unknown. We sought to determine the expression of Hh proteins in the E and S compartments of primary breast tumor samples and correlate with clinical outcomes.
Methods: Using tissue microarray (TMA) of ∼500 primary breast cancer samples (OSU 2009C009 A) that includes both primary breast tumor and axillary metastasis, the expression pattern of the key Hh signaling proteins (Shh, Gli-1, SMO, Ptch1) and a target gene of the pathway (BMI1) was determined by immunohistochemistry (IHC) in both the epithelial and stromal tissues and compared to other known prognostic/predictive markers such as age, stage, estrogen/progesterone receptor (ER/PR)/HER2 status and to disease-free survival (DFS). IHC was performed using Benchmark LT automated system from Ventana Medical Systems using previously published methods (Nuovo GJ, Nature protocols, 2009). Scoring was done by combining the intensity and percentage of cells staining with respective antibodies by three independent measurements.
Results: Analysis of a total of 315 primary breast tumors revealed the following percentage of tumors with high expression of Hh proteins in the E/S compartments respectively: Shh (72/31%), Gli-1 (69/53%), SMO (40/64%), and Ptch-1 (93/74%). Analyzing the interaction between the epithelial and stromal expressions of these proteins, we found positive correlation between i) high Shh and SMO in the epithelial cells and Gli-1 in the stroma, and ii) high Gli-1 in the epithelial cells and target protein Bmi1 in the stroma. High intensity staining of Gli-1 in the tumor epithelial cells was significantly associated with higher stage, grade, ER negative status and worse DFS. Adjusting for stage and ER status, tumors with increased epithelial Gli-1 expression was still significantly associated with worse DFS (p=0.0131, survival analysis) when compared to tumors with low expression. In particular, among the ER negative and node positive patients higher Gli-1 expression in epithelial cells was very significantly associated with poor DFS (p= &lt;0.0001). Staining for other target proteins in these tumors is currently ongoing.
Conclusions: We report here for the first time a positive correlation between expression of Hh proteins in the stromal and epithelial compartment of primary breast tumors. Furthermore, we observed that higher Gli-1 expression in the tumor epithelial cells was associated with worse DFS, particularly in the ER negative, node-positive tumors suggesting that aberrant Hh signaling may contribute to drug-resistance and hence a rationale therapeutic target. Further correlative studies between clinical parameters and additional Hh target genes in these tumors are in progress.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S6-9.