title: Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, 122 Hungerford Hill Road, Ithaca, NY 14853, USA.
authors.forename: John L
authors.initials: JL
authors.lastname: McElwee
authors.forename: Sunish
authors.initials: S
authors.lastname: Mohanan
authors.forename: Obi L
authors.initials: OL
authors.lastname: Griffith
authors.forename: Heike C
authors.initials: HC
authors.lastname: Breuer
authors.forename: Lynne J
authors.initials: LJ
authors.lastname: Anguish
authors.forename: Brian D
authors.initials: BD
authors.lastname: Cherrington
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Palmer
authors.forename: Louise R
authors.initials: LR
authors.lastname: Howe
authors.forename: Venkataraman
authors.initials: V
authors.lastname: Subramanian
authors.forename: Corey P
authors.initials: CP
authors.lastname: Causey
authors.forename: Paul R
authors.initials: PR
authors.lastname: Thompson
authors.forename: Joe W
authors.initials: JW
authors.lastname: Gray
authors.forename: Scott A
authors.initials: SA
authors.lastname: Coonrod
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000037:antagonists & inhibitors / Q000502:physiology*; D001943:Breast Neoplasms / Q000201:enzymology*; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D018450:Disease Progression; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000502:physiology*; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D006867:Hydrolases; C000620352:PADI2 protein, human; C000620353:Padi2 protein, mouse; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases