Abstract 3051: The miR-200 family as a potential therapeutic target in inflammatory breast cancer.
Authors: Debeb Bisrat G. , Lacerda Lara , Larson Richard , Li Li , Xu Wei , Huo Lei , Wei Caimiao , Krishnamurthy Savitri , Reuben James , Ueno Naoto , Buchholz Thomas , Woodward Wendy A. ,   Morgan Welch Inflammatory Breast Cancer Research Program and Clinic.
Published: August 2014

Purpose: Inflammatory breast cancer (IBC) is the most lethal, aggressive variant of breast cancer characterized by rapid progression, local and distant metastases, younger age of onset, and poor overall survival. Despite advances in multimodal breast cancer care, no IBC specific therapy is available clinically, demonstrating a critical need to identify novel therapeutics that target the distinct biology of IBC. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and have been implicated as potential therapeutic targets. Although the oncogenic or tumor-suppressor functions of a large number of miRNAs have been described in various solid tumors including breast cancer, the role of miRNAs in the biology of IBC is poorly understood. Herein, we investigated miRNAs differentially expressed in IBC, particularly miR-200 family miRNAs as potential therapeutic targets in IBC.

Methods: MicroRNA microarray profiling was conducted in IBC and non-IBC cell lines, and microarray results were validated using qRT-PCR. MirZip lentiviral based mirRNA inhibition was used to stably knockdown miRNAs and the effects of specific miRNA knockdown on proliferation, EMT, colony and mammosphere formation was evaluated. For tumor progression and metastasis in vivo experiments, we injected 500,000 SUM149 miR-200a, miR-141 knockdown and control (N=15 for each group) cells into the cleared mammary fatpad of SCID/Beige mice. We also validated the expression of miR-200 miRNAs in IBC patient samples.

Results: Differential expression of mirRNAs was observed in IBC vs. non-IBC cell lines with a significantly higher expression of all miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-479) in IBC cell lines. These results were validated using qRT-PCR, with the IBC cell lines showing a &gt;1,000-fold higher expression of miR-200s vs non-IBC aggressive cell lines (P&lt;0.0001). Moreover, two known targets of the miR-200 family, Zeb1 and Zeb2, were significantly downregulated while E-cadherin was upregulated in the IBC samples. Importantly, these observations were validated in IBC patient samples which showed a significantly higher expression of all miRNA 200s compared to the normal breast tissues (P&lt;0.001). In vitro, miR-200a and miR-141 knockdown significantly reduced both colony and mammosphere formation in the triple negative SUM149 cells (P&lt;0.001) but did not have a significant effect on the HER2+ IBC cell lines SUM190 and MDA-IBC-3 (P&gt;0.05) while it upregulated the EMT markers N-cadherin, Fibronectin, TGFβ, and Smad4 and reduced E-cadherin expression compared to transduced controls. In vivo, miR-200 knockdown resulted in an increase in tumor progression vs. controls (P&lt;0.05, Day 63) while metastasis studies are undergoing.

Conclusions: These data suggest that miR-200 family microRNAs play an important role in IBC tumor progression and altering miR-200 levels could be an efficacious approach to target IBC.

Citation Format: Bisrat G. Debeb, Lara Lacerda, Richard Larson, Li Li, Wei Xu, Lei Huo, Caimiao Wei, Savitri Krishnamurthy, James Reuben, Naoto Ueno, Thomas Buchholz, Wendy A. Woodward, Morgan Welch Inflammatory Breast Cancer Research Program and Clinic. The miR-200 family as a potential therapeutic target in inflammatory breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3051. doi:10.1158/1538-7445.AM2013-3051