Anjana Saxena

Placental overgrowth in mice lacking the imprinted gene Ipl

The Ipl (Tssc3) gene lies in an extended imprinted region of distal mouse chromosome 7, which also contains the Igf2 gene. Expression of Ipl is highest in placenta and yolk sac, where its mRNA is derived almost entirely from the maternal allele. Ipl encodes a small cytoplasmic protein with a pleckstrin-homology (PH) domain. We constructed two lines of mice with germ-line deletions of this gene (Iplneo and IplloxP) and another line deleted for the similar but nonimprinted gene Tih1. All three lines were viable. There was consistent overgrowth of the Ipl-null placentas, with expansion of the spongiotrophoblast. These larger placentas did not confer a fetal growth advantage; fetal size was normal in Ipl nulls with the Iplneo allele and was decreased slightly in nulls with the IplloxP allele. When bred into an Igf2 mutant background, the Ipl deletion partially rescued the placental but not fetal growth deficiency. Neither fetal nor placental growth was affected by deletion of Tih1. These results show a nonredundant function for Ipl in restraining placental growth. The data further indicate that Ipl can act, at least in part, independently of insulin-like growth factor-2 signaling. Thus, genomic imprinting regulates multiple pathways to control placental size.

Placental growth retardation due to loss of imprinting of Phlda2.

The maternally expressed/paternally silenced genes Phlda2 (a.k.a. Ipl/Tssc3), Slc22a1l, Cdkn1c, Kcnq1, and Ascl2 are clustered in an imprinted domain on mouse chromosome 7. Paternal deletion of a cis-acting differentially methylated DNA element, Kvdmr1, causes coordinate loss of imprinting and over-expression of all of these genes and the resulting conceptuses show intrauterine growth restriction (IUGR). To test the specific contribution of Phlda2 to IUGR in the Kvdmr1-knockout, we crossed Kvdmr1(+/-) males with Phlda2(+/-) females. Conceptuses with the (Phlda2(+/+); Kvdmr1(+/-)) genotype showed fetal and placental growth retardation. Restoration of Phlda2 dosage to normal, as occurred in the conceptuses with the (Phlda2(-/+); Kvdmr1(+/-)) genotype, had a marginally positive effect on fetal weights and no effect on post-natal weights, but significantly rescued the placental weights. As we previously reported, loss of Phlda2 expression in the wild-type background (Phlda2(-/+); Kvdmr1(+/+) genotype) caused placentomegaly. Thus Phlda2 acts as a true rheostat for placental growth, with overgrowth after gene deletion and growth retardation after loss of imprinting. Consistent with this conclusion, we observed significant placental stunting in BAC-transgenic mice that over-expressed Phlda2 and one flanking gene, Slc22a1l, but did not over-express Cdkn1c.

Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein a complex formation.

ABSTRACT Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.

Nucleolin inhibits Hdm2 by multiple pathways leading to p53 stabilization

Nucleolin is a c-Myc-induced gene product with defined roles in ribosomal RNA processing and the inhibition of chromosomal DNA replication following stress. Here we find that changes in nucleolin protein levels in unstressed cells cause parallel changes in the amount of p53 protein. Alterations in p53 levels arise from nucleolin binding to the p53 antagonist Hdm2, resulting in the inhibition of both p53 ubiquitination and Hdm2 auto-ubiquitination. Nucleolin does not alter p53 ubiquitination by human papillomavirus E6, indicating that the effect is specific for Hdm2. Although the inhibition of ligase activity would be expected to stabilize Hdm2, we instead find that nucleolin also reduces Hdm2 protein levels, demonstrating that nucleolin inhibits Hdm2 using multiple mechanisms. Increases in nucleolin levels in unstressed cells led to higher expression of p21cip1/waf1, a reduced rate of cellular proliferation, and an increase in apoptosis. Thus, nucleolin has a number of properties in common with the tumor suppressor ARF (alternate reading frame). We propose that nucleolin, like ARF, responds to hyperproliferative signals by upregulation of p53 through Hdm2 inhibition.

Phosphoinositide Binding by the Pleckstrin Homology Domains of Ipl and Tih1

The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C δ1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the β1-loop1-β2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 arebona fide PH domain proteins, with broad specificity and moderate affinity for PIPs.

Specific domains of nucleolin interact with Hdm2 and antagonize Hdm2-mediated p53 ubiquitination.

Nucleolin is an abundant multifunctional nucleolar protein with defined roles in ribosomal RNA processing, RNA polymerase I catalyzed transcription and the regulation of apoptosis. Earlier we reported that human nucleolin binds to the p53 antagonist human double minute 2 (Hdm2) as determined by reciprocal co‐immunoprecipitation assays using cell lysates. We also demonstrated that nucleolin antagonizes Hdm2‐mediated degradation of p53. Here, we identify specific domains of nucleolin and Hdm2 proteins that support mutual interaction and investigate the implications of complex formation on p53 ubiquitination and protein levels. Our data indicate that the nucleolin N‐terminus as well as the central RNA‐binding domain (RBD) are predominantly involved in binding to Hdm2. The nucleolin RBD robustly bound to the NLS/NES (nuclear localization and export signals) domain of Hdm2 in vitro, while the N‐terminus of nucleolin preferentially associated with the Hdm2 RING (really interesting new gene) domain expressed in cells. We further demonstrate that the C‐terminal glycine‐arginine rich domain of nucleolin serves as the predominant binding domain for direct interaction with p53. While overexpression of nucleolin or its various domains had no significant effect on Hdm2 auto‐ubiquitination, the nucleolin RBD antagonized the Hdm2 E3 ligase activity against p53, leading to p53 stabilization. Conversely, the adjacent glycine‐arginine rich domain of nucleolin interacted with p53 causing a modest stimulatory effect on p53 ubiquitination. These data suggest that changes in nucleolin conformation can alter the availabilities of such domains in vivo to modulate the overall impact of nucleolin on Hdm2 activity and hence on p53 stability.

The Pancreatic Cancer Microbiome Promotes Oncogenesis by Induction of Innate and Adaptive Immune Suppression

We found that the cancerous pancreas harbors a markedly more abundant microbiome compared with normal pancreas in both mice and humans, and select bacteria are differentially increased in the tumorous pancreas compared with gut. Ablation of the microbiome protects against preinvasive and invasive pancreatic ductal adenocarcinoma (PDA), whereas transfer of bacteria from PDA-bearing hosts, but not controls, reverses tumor protection. Bacterial ablation was associated with immunogenic reprogramming of the PDA tumor microenvironment, including a reduction in myeloid-derived suppressor cells and an increase in M1 macrophage differentiation, promoting TH1 differentiation of CD4+ T cells and CD8+ T-cell activation. Bacterial ablation also enabled efficacy for checkpoint-targeted immunotherapy by upregulating PD-1 expression. Mechanistically, the PDA microbiome generated a tolerogenic immune program by differentially activating select Toll-like receptors in monocytic cells. These data suggest that endogenous microbiota promote the crippling immune-suppression characteristic of PDA and that the microbiome has potential as a therapeutic target in the modulation of disease progression.Significance: We found that a distinct and abundant microbiome drives suppressive monocytic cellular differentiation in pancreatic cancer via selective Toll-like receptor ligation leading to T-cell anergy. Targeting the microbiome protects against oncogenesis, reverses intratumoral immune tolerance, and enables efficacy for checkpoint-based immunotherapy. These data have implications for understanding immune suppression in pancreatic cancer and its reversal in the clinic. Cancer Discov; 8(4); 403-16. ©2018 AACR.See related commentary by Riquelme et al., p. 386This article is highlighted in the In This Issue feature, p. 371.

Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

Nucleolin (NCL) is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase 1 (CDK1). Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT) or a phosphorylation-deficient NCL-mutant, 6/S*A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2) homology 3 (BH3)-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation.

Amiloride-sensitive epithelial sodium channel subunits are expressed in human and mussel immunocytes.

In this study, we examined the expression of epithelial Na(+) channel (ENaC) subunits in human peripheral blood lymphocytes, human lymph nodes and molluscan immunocytes using non-radioactive in situ hybridization. The results showed that T lymphocytes express the ENaC gamma subunit mRNA, and B lymphocytes the ENaC beta subunit mRNA. Yet, the alpha subunit mRNA was not detected in either cell type. In molluscan immunocytes, all three homologous ENaC subunit mRNAs are present, and these data were also confirmed by RT-PCR and sequencing of the PCR products. These findings show evolutionary conservation of the expression of ENaC subunits in immunocytes of invertebrates to vertebrates. The observed differential expression patterns of ENaC subunits suggest that ENaC function may be regulated differentially in different types of human lymphocytes.

Engineered Protein Polymer-Gold Nanoparticle Hybrid Materials for Small Molecule Delivery

We have fabricated protein polymer-gold nanoparticle (P-GNP) nanocomposites that exhibit enhanced binding and delivery properties of the small hydrophobic molecule drug, curcumin, to the model breast cancer cell line, MCF-7. These hybrid biomaterials are constructed via in situ GNP templated-synthesis with genetically engineered histidine tags. The P-GNP nanocomposites exhibit enhanced small molecule loading, sustained release and increased uptake by MCF-7 cells. When compared to the proteins polymers alone, the P-GNPs demonstrate a greater than 7-fold increase in curcumin binding, a nearly 50% slower release profile and more than 2-fold increase in cellular uptake of curcumin. These results suggest that P-GNP nanocomposites serve as promising candidates for drug delivery vehicles.