Stromal Expression of β-Arrestin-1 Predicts Clinical Outcome and Tamoxifen Response in Breast Cancer  Katja Lundgren, Nicholas P. Tobin, Sophie Lehn, Olle Stål, Lisa Rydén, Karin Jirström, Göran Landberg  The Journal of Molecular Diagnostics  Volume 13, Issue 3, Pages 340-351 (May 2011) DOI: 10.1016/j.jmoldx.2011.01.009 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 β-Arrestin-1 antibody validation and migratory propensity of breast cancer cells (MDA-MB-468 and MDA-MB-231 cell lines) after β-arrestin-1 overexpression and silencing. A: β-Arrestin-1 overexpression was detected with anti-GFP antibody on Western blot, 48 hours after transfection. V, vector. B: Protein expression was significantly decreased 48 hours after transfection with siRNA against β-arrestin-1 in both cell lines, analyzed by immunocytochemistry. Magnification ×40. C: Overexpression of β-arrestin-1 reduced migration of both cell lines, compared with control, 48 hours after transfection. *P < 0.001; **P < 0.001. D: Knockdown of β-arrestin-1 resulted in an increased migratory propensity of both cell lines, compared with control, 48 hours after transfection. *P < 0.001; **P < 0.001. The Journal of Molecular Diagnostics 2011 13, 340-351DOI: (10.1016/j.jmoldx.2011.01.009) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Immunohistochemical staining of β-arrestin-1 in primary breast tumors. Cytoplasmic tumor cell β-arrestin-1 expression was evaluated as negative (A), low (B), moderate (C), or high (D). Stromal expression (Neg-High) was observed to be high (B) in some tumors in which tumor cell expression was quite low, and the opposite was also observed: low (D), when tumor cell expression was high. Scale bar applies to all images. The Journal of Molecular Diagnostics 2011 13, 340-351DOI: (10.1016/j.jmoldx.2011.01.009) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Kaplan-Meier curves showing the effect of β-arrestin-1 on RFS. A: In cohort I, only stromal expression (right panel) had an effect on overall survival. B: In the subgroup of untreated control patients of cohort II, negative or high expression of stromal β-arrestin-1 was associated with a shorter RFS, compared with low or moderate expression (right panel). There was a trend indicating that also high tumor cell expression was linked to a reduced RFS in cohort II (left panel). C: ERα-positive patients with tumors of low or moderate stromal β-arrestin-1 expression (right panel) did not benefit from tamoxifen treatment, in contrast to patients with tumors exhibiting negative or high expression (left panel). The Journal of Molecular Diagnostics 2011 13, 340-351DOI: (10.1016/j.jmoldx.2011.01.009) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions