Abstract P1-07-09: SMAD nuclear staining patterns in inflammatory breast cancer suggest non-canonical TGFβ-signalling establishing collective, sheet-like invasion SMAD nuclear staining patterns in inflammatory breast cancer suggest non-canonical TGFβ-signalling es
Authors: Marsan Melike , Van den Eynden Gert , Neven Patrick , Vergote Ignace , Vermeulen Peter , Dirix Luc , Van Laere Steven 
Published: May 2015

Abstract
Introduction: Inflammatory Breast Cancer (IBC) is an aggressive and highly metastatic form of breast cancer. Recent expression profiling studies revealed IBC-specific modulation of TGFβ-signalling. In the present study, we aim to validate these findings by evaluating nuclear SMAD staining patterns.
Materials and Methods: Immunohistochemistry (IHC) was performed for SMAD2, -3, -4 and -6 on tissue sections from patients with (N=79) and without (N=133) IBC. Protocols for IHC were established on cell blocks from breast cancer cell lines with and without SMAD staining identified by western blotting on nuclear protein extracts. A staining score was assigned by multiplying the percentage of stained cancer cell nuclei by the staining intensity evaluated on a three-scale basis (1=weak, 2=intermediate, 3=strong). Expression data were subjected to unsupervised hierarchical cluster analysis (UHCA) and statistical assessment of staining differences was done using uni- and multivariate models. Additionally, we looked at the correlation between nuclear SMAD expression and gene expression in 11 patients. For each gene, we generated a spearman correlation coefficient, and selected only those genes that were positively correlated with nuclear SMAD expression in 11 patients (p&lt;0.05).
Results: UHCA for SMAD2, -3 and -4 nuclear protein expression data identified two sample clusters downstream of the first bifurcation. IBC samples were significantly (OR=60.34, P&lt;0.001) enriched in the cluster characterized by increased nuclear SMAD2 protein expression and absence of both nuclear SMAD3 and -4 protein expression. Univariate analysis revealed that these staining patterns are significant (all Ps&lt;0.01). Multivariate analysis demonstrated that gain of SMAD2 nuclear expression and loss of SMAD3 nuclear expression in IBC are unrelated observations and independent of histological grade, hormone receptor expression, ErbB2 amplification and tumour stage. SMAD correlation analysis showed us that genes positively correlated with nuclear SMAD2 expression included CD44 and REL, both markers that represent crucial pathways in IBC. Interestingly, nuclear SMAD3 expression was positively correlated with ZEB1.
Discussion: In line with the expectations, our data show that TGFβ-signalling indeed differs between samples from patients with and without IBC. In addition, this study does offer novel insights on IBC biology. First, SMAD2 nuclear staining is gained in IBC in the absence of its canonical binding partners SMAD3 and -4, suggesting non-canonical TGFβ-signalling in IBC. Second, it has been shown that SMAD2 induces an invasive response program in cancer cells without affecting their epithelial morphology, while SMAD3 accounts for invasion by induction of Epithelial-to-Mesenchymal transition (EMT). Thus our results indicate that IBC cell invasion occurs in a collective, sheet-like fashion instead of EMT, which is probably responsible for the invasion of non-IBC cells.
Citation Format: Melike Marsan, Gert Van den Eynden, Patrick Neven, Ignace Vergote, Peter Vermeulen, Luc Dirix, Steven Van Laere. SMAD nuclear staining patterns in inflammatory breast cancer suggest non-canonical TGFβ-signalling establishing collective, sheet-like invasion SMAD nuclear staining patterns in inflammatory breast cancer suggest non-canonical TGFβ-signalling es [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-09.