Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration

BACKGROUND: Penetration of the mammary gland basement membrane by cancer cells is a crucial first step in tumor invasion. Using a mouse model of ductal carcinoma in situ, we previously found that inhibition of peptidylarginine deiminase 2 (PAD2, aka PADI2) activity appears to maintain basement membrane integrity in xenograft tumors. The goal of this investigation was to gain insight into the mechanisms by which PAD2 mediates this process. METHODS: For our study, we modulated PAD2 activity in mammary ductal carcinoma cells by lentiviral shRNA-mediated depletion, lentiviral-mediated PAD2 overexpression, or PAD inhibition and explored the effects of these treatments on changes in cell migration and cell morphology. We also used these PAD2-modulated cells to test whether PAD2 may be required for EGF-induced cell migration. To determine how PAD2 might promote tumor cell migration in vivo, we tested the effects of PAD2 inhibition on the expression of several cell migration mediators in MCF10DCIS.com xenograft tumors. In addition, we tested the effect of PAD2 inhibition on EGF-induced ductal invasion and elongation in primary mouse mammary organoids. Lastly, using a transgenic mouse model, we investigated the effects of PAD2 overexpression on mammary gland development. RESULTS: Our results indicate that PAD2 depletion or inhibition suppresses cell migration and alters the morphology of MCF10DCIS.com cells. In addition, we found that PAD2 depletion suppresses the expression of the cytoskeletal regulatory proteins RhoA, Rac1, and Cdc42 and also promotes a mesenchymal to epithelial-like transition in tumor cells with an associated increase in the cell adhesion marker, E-cadherin. Our mammary gland organoid study found that inhibition of PAD2 activity suppresses EGF-induced ductal invasion. In vivo, we found that PAD2 overexpression causes hyperbranching in the developing mammary gland. CONCLUSION: Together, these results suggest that PAD2 plays a critical role in breast cancer cell migration. Our findings that EGF treatment increases protein citrullination and that PAD2 inhibition blocks EGF-induced cell migration suggest that PAD2 likely functions within the EGF signaling pathway to mediate cell migration

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

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 x 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, GADD45alpha, 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

Design, synthesis, and biological evaluation of tetrazole analogs of Cl-amidine as protein arginine deiminase inhibitors.

Protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of arginine residues to form citrulline. This once obscure modification is now known to play a key role in the etiology of multiple autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, lupus, and ulcerative colitis) and in some forms of cancer. Among the five human PADs (PAD1, -2, -3, -4, and -6), it is unclear which isozyme contributes to disease pathogenesis. Toward the identification of potent, selective, and bioavailable PAD inhibitors that can be used to elucidate the specific roles of each isozyme, we describe tetrazole analogs as suitable backbone amide bond bioisosteres for the parent pan PAD inhibitor Cl-amidine. These tetrazole based analogs are highly potent and show selectivity toward particular isozymes. Importantly, one of the compounds, biphenyl tetrazole tert-butyl Cl-amidine (compound 13), exhibits enhanced cell killing in a PAD4 expressing osteosarcoma bone marrow (U2OS) cell line and can also block the formation of neutrophil extracellular traps. These bioisosteres represent an important step in our efforts to develop stable, bioavailable, and selective inhibitors for the PADs

Potential Role for PAD2 in Gene Regulation in Breast Cancer Cells

<div><p>The peptidylarginine deiminase (PAD) family of enzymes post-translationally convert positively charged arginine residues in substrate proteins to the neutral, non-standard residue citrulline. PAD family members 1, 2, 3, and 6 have previously been localized to the cell cytoplasm and, thus, their potential to regulate gene activity has not been described. We recently demonstrated that PAD2 is expressed in the canine mammary gland epithelium and that levels of histone citrullination in this tissue correlate with PAD2 expression. Given these observations, we decided to test whether PAD2 might localize to the nuclear compartment of the human mammary epithelium and regulate gene activity in these cells. Here we show, for the first time, that PAD2 is specifically expressed in human mammary gland epithelial cells and that a portion of PAD2 associates with chromatin in MCF-7 breast cancer cells. We investigated a potential nuclear function for PAD2 by microarray, qPCR, and chromatin immunoprecipitation analysis. Results show that the expression of a unique subset of genes is disregulated following depletion of PAD2 from MCF-7 cells. Further, ChIP analysis of two of the most highly up- and down-regulated genes (PTN and MAGEA12, respectively) found that PAD2 binds directly to these gene promoters and that the likely mechanism by which PAD2 regulates expression of these genes is via citrullination of arginine residues 2–8–17 on histone H3 tails. Thus, our findings define a novel role for PAD2 in gene expression in human mammary epithelial cells.</p> </div

Reovirus Infection or Ectopic Expression of Outer Capsid Protein μ1 Induces Apoptosis Independently of the Cellular Proapoptotic Proteins Bax and Bak ▿

Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein μ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed μ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of μ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for μ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of μ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing μ1, indicating that caspases were not required. Additionally, μ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax−/−Bak−/− mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax−/−Bak−/− MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak

Knockdown of endogenous PAD2 in MCF-7 cells alters cellular proliferation rates versus control cells.

<p>Equal numbers of shRNA control and PAD2 knockdown MCF-7 cells were plated in 96-well plates and allowed to grow for 24, 48, or 72 hours. At time of harvest, dye solution from MTT assay kit was allowed to incubate with cells for one hour at which point solubilization reagent was added and resulting formazan product measured at 570 nm. Data represent the means ± SEM of three independent experiments performed in triplicate. Values represent the means ± SEM.</p