Brian D. Cherrington

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

BackgroundWe 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.MethodsRNA-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.ResultsWe 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.ConclusionTogether, 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.

Potential Role for PAD2 in Gene Regulation in Breast Cancer Cells

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.

Dysregulation of PAD4-mediated citrullination of nuclear GSK3β activates TGF-β signaling and induces epithelial-to-mesenchymal transition in breast cancer cells

Peptidylarginine deiminase 4 (PAD4) is a Ca2+-dependent enzyme that converts arginine and methylarginine residues to citrulline, with histone proteins being among its best-described substrates to date. However, the biological function of this posttranslational modification, either in histones or in nonhistone proteins, is poorly understood. Here, we show that PAD4 recognizes, binds, and citrullinates glycogen synthase kinase-3β (GSK3β), both in vitro and in vivo. Among other functions, GSK3β is a key regulator of transcription factors involved in tumor progression, and its dysregulation has been associated with progression of human cancers. We demonstrate that silencing of PAD4 in breast cancer cells leads to a striking reduction of nuclear GSK3β protein levels, increased TGF-β signaling, induction of epithelial-to-mesenchymal transition, and production of more invasive tumors in xenograft assays. Moreover, in breast cancer patients, reduction of PAD4 and nuclear GSK3β is associated with increased tumor invasiveness. We propose that PAD4-mediated citrullination of GSK3β is a unique posttranslational modification that regulates its nuclear localization and thereby plays a critical role in maintaining an epithelial phenotype. We demonstrate a dynamic and previously unappreciated interplay between histone-modifying enzymes, citrullination of nonhistone proteins, and epithelial-to-mesenchymal transition.

Anti–Citrullinated Protein Antibodies Are Associated With Neutrophil Extracellular Traps in the Sputum in Relatives of Rheumatoid Arthritis Patients

Studies suggest that rheumatoid arthritis (RA)–related autoimmunity is initiated at a mucosal site. However, the factors associated with the mucosal generation of this autoimmunity are unknown, especially in individuals who are at risk of future RA. Therefore, we tested anti–cyclic citrullinated peptide (anti‐CCP) antibodies in the sputum of RA‐free first‐degree relatives (FDRs) of RA patients and patients with classifiable RA.

Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK

We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3-1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3-1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca(2+) influx via the L-type Ca(2+) channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca(2+) influx through L-type Ca(2+) channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca(2+) influx via L-type channels to facilitate ERK phosphorylation.

Comparative analysis of peptidylarginine deiminase-2 expression in canine, feline and human mammary tumours.

The peptidylarginine deiminase (PAD) enzyme family converts arginine residues in proteins to citrulline. In the canine mammary gland, PAD2 expression is first detected in epithelial cells in oestrus and becomes more widely expressed during dioestrus. PAD2 appears to modify nuclear histones, suggesting a role for the enzyme in chromatin remodelling and gene regulation. Recent evidence suggests that PAD2 plays a role in gene regulation in primary human breast epithelial cells. PAD2 may therefore be involved in gene regulation as it relates to mammary development, the oestrus cycle and potentially to neoplasia. The aim of the present study was to determine whether PAD2 expression was increased or decreased in mammary carcinoma compared with normal mammary tissue. A human mammary tissue microarray and archival surgical biopsy tissues from canine and feline mammary tumours were used to demonstrate differential expression of PAD2 in mammary carcinoma that appeared to be consistent across species. Normal human and canine mammary epithelium showed strong cytoplasmic and nuclear expression of PAD2, but there was reduced PAD2 expression in mammary carcinomas from both species. Feline mammary carcinomas had complete loss of nuclear PAD2 expression. Loss of nuclear PAD2 expression may therefore represent a marker of progression towards more aggressive neoplasia.

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 μg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.

Warmed Winter Water Temperatures Alter Reproduction in Two Fish Species

We examined the spawning success of Fathead Minnows (Pimephales promelas) and Johnny Darters (Etheostoma nigrum) exposed to elevated winter water temperatures typical of streams characterized by anthropogenic thermal inputs. When Fathead Minnows were exposed to temperature treatments of 12, 16, or 20 °C during the winter, spawning occurred at 16 and 20 °C but not 12 °C. Eggs were deposited over 9 weeks before winter spawning ceased. Fathead Minnows from the three winter temperature treatments were then exposed to a simulated spring transition. Spawning occurred at all three temperature treatments during the spring, but fish from the 16° and 20 °C treatment had delayed egg production indicating a latent effect of warm winter temperatures on spring spawning. mRNA analysis of the egg yolk protein vitellogenin showed elevated expression in female Fathead Minnows at 16 and 20 °C during winter spawning that decreased after winter spawning ceased, whereas Fathead Minnows at 12 °C maintained comparatively low expression during winter. Johnny Darters were exposed to 4 °C to represent winter temperatures in the absence of thermal inputs, and 12, 16, and 20 °C to represent varying degrees of winter thermal pollution. Johnny Darters spawned during winter at 12, 16, and 20 °C but not at 4 °C. Johnny Darters at 4 °C subsequently spawned following a simulated spring period while those at 12, 16, and 20 °C did not. Our results indicate elevated winter water temperatures common in effluent-dominated streams can promote out-of-season spawning and that vitellogenin expression is a useful indicator of spawning readiness for fish exposed to elevated winter temperatures.

PAD Enzymes in Female Reproductive Tissues and Cancer Pathogenesis

Increasing clinical evidence now suggests that peptidyl-arginine deiminase (PAD) enzymes, especially PAD4 and possibly PAD2, have important roles in tumor progression. Further linking PADs and cancer are recent findings showing that treatment of cancer cell lines and mouse models of disease with PAD inhibitors significantly suppresses tumor growth and, interestingly, also inflammation. Current functional target proteins for PAD-catalyzed citrullination in cancer include transcription factors, co-regulators, and histones, the latter of which alter gene expression patterns in multiple cancerous cell lines. As the novel relationship between PADs, inflammation, and cancer unfolds, next-generation isozyme-specific PAD inhibitors may have therapeutic potential to regulate both the inflammatory tumor microenvironment and tumor cell growth. In this chapter we first discuss expression patterns of PADs in reproductive tissues, focusing on their endocrine regulation, as this appears to have major implication for expression, catalytic activity, and tumorigenesis. Next, the chapter details our current understanding of the molecular pathophysiological roles of PADs in cancer. Finally, we discuss the evolving role of PADs in inflammation, in the context of tumorigenesis, with discussion of the potential of new isozyme-specific PAD inhibitors to serve as adjuvant therapy for malignancies.