Abstract 4968: Msi2 directly linked to Hedgehog and Wnt signaling in non-triple negative breast cancer
Authors: Abdalla Moustafa , Kanwar Nisha , Zhu Yanglong , Nair Ranju , Youngson Bruce J. , Miller Naomi A. , Liu Tianyu , Done Susan J. 
Published: May 2015

Introduction: Previously, we have shown that a gain on 17q22.24.2 was a predictor of invasiveness when found in duct carcinoma in situ (DCIS) and of nodal metastasis when found in invasive duct carcinoma (IDC). Within this amplicon, we have identified a gene that is a likely driver of breast tumorigenesis and progression: Musashi homolog 2 (Msi2). Msi2 has a functional role in neural stem cell maintenance and regulation of hematopoietic stem cells. Previously, Msi2 has been implicated in Notch and Hedgehog signaling in leukemia, and here we link Msi2 to an increase in Wnt activation.

Methodology: To understand the expression of Msi2 in patient tissue, tissue microarrays (TMAs) containing 232 breast cancers were analyzed by immunohistochemistry. MDA-MB-231 and MCF7 cells expressing Msi2-GFP and GFP clones were plated on transwells to examine cell migration and invasiveness. Proliferation was analyzed by the MTS assay. TOP-FOP and Gli1 Luciferase assays were used to confirm activation of Hedgehog and Wnt signaling. Western blots were used to assess total beta-catenin levels in the cell lines. Analysis of publicly available expression microarray databases and RT-PCR of the over-expression cell lines validated the proposed mechanism of action.

Results: Tissue microarrays confirmed that Allred scores for Msi2 significantly correlated with grade, size stage, and non-triple negative receptor status. Over-expression of Msi2, in MDA-MB-231 (triple negative) and MCF7 (luminal cell type with epithelial morphology) cell lines, caused an increase in proliferation, migration and invasion. The knockdowns demonstrated the reverse effect. As determined by the TOP-FOP luciferase assay and RT-PCR, Msi2 leads to increased signaling of Hedgehog and Wnt. Furthermore, Msi2-mediated activation of Wnt occurs independently of total beta-catenin levels. Microarray analysis of breast cancer cell lines and patients' tumors supports this mechanism of action for Msi2 in non-triple negative cell lines (as assessed by Pearson's correlation coefficient of pathway interactions).

Conclusion: When overexpressed in a cancerous cell system in vitro, Msi2 levels correlate with increased migratory and invasive capabilities. Furthermore, its implication in Notch, Hedgehog, and Wnt Signaling, make Msi2 a likely driver of the 17q22-24.2 amplicon in breast cancer. This study furthers our understanding of Msi2, and hints at the possibility that Msi2 plays a more significant role in cellular events and tumorigenesis than previously thought.

Citation Format: Moustafa Abdalla, Nisha Kanwar, Yanglong Zhu, Ranju Nair, Bruce J. Youngson, Naomi A. Miller, Tianyu Liu, Susan J. Done. Msi2 directly linked to Hedgehog and Wnt signaling in non-triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4968. doi:10.1158/1538-7445.AM2014-4968