Open Access Highly Accessed Research article

Identification of PADI2 as a potential breast cancer biomarker and therapeutic target

John L McElwee1, Sunish Mohanan1, Obi L Griffith2, Heike C Breuer1, Lynne J Anguish1, Brian D Cherrington3, Ashley M Palmer1, Louise R Howe4, Venkataraman Subramanian5, Corey P Causey6, Paul R Thompson5, Joe W Gray7 and Scott A Coonrod1*

Author Affiliations

1 Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, 122 Hungerford Hill Road, Ithaca, NY, 14853, USA

2 Lawrence Berkeley National Laboratory, Life Sciences Division, Cancer and DNA Damage Responses, One Cyclotron Road, Berkeley, CA, 94720, USA

3 Department of Zoology and Physiology, University of Wyoming, 1000 E. University Ave, Laramie, WY, 82071, USA

4 Department of Cell and Developmental Biology, Weill Cornell Medical College, Box 60, 1300 York Avenue, New York, NY, 10065, USA

5 Department of Chemistry, The Scripps Research Institute, Scripps Florida, 120 Scripps Way, Jupiter, FL, 33458, USA

6 Department of Chemistry & Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, SC, 29208, USA

7 Department of Biomedical Engineering, Oregon Health and Science University, 3303 SW Bond Ave, Portland, OR 97239, USA

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BMC Cancer 2012, 12:500  doi:10.1186/1471-2407-12-500

Published: 30 October 2012

Abstract

Background

We have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects.

Methods

RNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes.

Results

We show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 × 106) in luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45α, and Ki67.

Conclusion

Together, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy.

Keywords:
Peptidylarginine deiminase; PAD2/PADI2; HER2/ERBB2; Breast cancer; Luminal; Cl-amidine; Citrullination