Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases

Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors

The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets

Polycomb group (PcG) proteins are key regulators of stem-cell and cancer biology. They mainly act as repressors of differentiation and tumor-suppressor genes. One key silencing step involves the trimethylation of histone H3 on Lys27 (H3K27) by EZH2, a core component of the Polycomb Repressive Complex 2 (PRC2). The mechanism underlying the initial recruitment of mammalian PRC2 complexes is not well understood. Here, we show that NIPP1, a regulator of protein Ser/Thr phosphatase-1 (PP1), forms a complex with PP1 and PRC2 components on chromatin. The knockdown of NIPP1 or PP1 reduced the association of EZH2 with a subset of its target genes, whereas the overexpression of NIPP1 resulted in a retargeting of EZH2 from fully repressed to partially active PcG targets. However, the expression of a PP1-binding mutant of NIPP1 (NIPP1m) did not cause a redistribution of EZH2. Moreover, mapping of the chromatin binding sites with the DamID technique revealed that NIPP1 was associated with multiple PcG target genes, including the Homeobox A cluster, whereas NIPP1m showed a deficient binding at these loci. We propose that NIPP1 associates with a subset of PcG targets in a PP1-dependent manner and thereby contributes to the recruitment of the PRC2 complex

A key function for the PP1 interactor NIPP1 in cancer cell proliferation and gene expression

Polycomb Group (PcG) proteins are repressive chromatin modifiers that are important in maintaining stem cell pluripotency, establishing cell fate and cancer development. One of the key events in PcG-mediated gene silencing is the trimethylation of Lysine 27 on histone H3 (H3K27) by the methyltransferase EZH2, a core component of the Polycomb Repressive Complex 2 (PRC2) complex. NIPP1, an interactor of protein Ser/Thr phosphatase PP1, is a ubiquitously expressed nuclear protein that interacts with the two PRC2 components EZH2 and EED. Furthermore, NIPP1 associates with a subset of PcG target genes, where it is essential for the trimethylation of H3K27. In addition, the loss of NIP1 is associated with a defective proliferation and embryonic lethality.In the first part of my thesis, we demonstrated that NIPP1 is an essential regulator of EZH2 recruitment in a PP1-dependent manner. We found that the depletion of either NIPP1 or PP1 inhibits the EZH2 occupancy on NIPP1-associated PcG target genes whereas ectopically expressed NIPP1 induces a redistribution of EZH2 from fully repressed to partially active PcG target genes. Conversely, the expression of a PP1-binding mutant of NIPP1 (NIPP1-RATA) has no effect on the chromatin targeting of EZH2. Although NIPP1-RATA still interacts with EED and EZH2, we demonstrated via DamID that it is no longer associated with PcG target genes. In the second part of my thesis, we explored the role of NIPP1 and associated PP1 in cancer cell proliferation. The cellular concentration of NIPP1 needs to be strictly regulated in cancer cells since both the depletion and the stable expression of NIPP1-WT show inhibitory effects on tumor growth. Furthermore, the expression of a NIPP1 mutant that is associated with constitutively active PP1 (NIPP1-&#8710;C) abrogates tumor growth, whereas the expression NIPP1-RATA only has limited effects, indicating that NIPP1-mediated growth inhibition is dependent on PP1. Moreover, the expression of NIPP1-WT and NIPP1-&#8710;C, but not of NIPP1-RATA, induces the expression of smooth muscle-specific genes in HeLa cells and increases cell contraction upon KCl-induced depolarization. The smooth muscle-like phenotype is lost upon EZH2 depletion, indicating that NIPP1 transdifferentiates HeLa cells in an EZH2- and PP1-dependent manner.In conclusion, we have demonstrated that the NIPP1-PP1 holoenzyme is a key regulator of PRC2 chromatin targeting but also plays a role in PcG-mediated differentiation processes. In addition, we have shown that NIPP1 is strictly regulated in cancer cells, making it a potential therapeutic target. <w:latentstyles <br semihidden="false" priority="0" locked="false" <w:latentstyles role="" target="" semihidden="false" priority="0" locked="false" <w:lsdexception="" defunhidewhenused="true" deflockedstate="false" target.< therapeutic="" potential="" making="" cells,="" shown="" processes.="" differentiation="" plays="" also="" holoenzyme="" nipp1-pp1="" have="" conclusion,="" manner.in="" ezh2-="" transdifferentiates="" depletion,="" lost="" phenotype="" muscle-like="" depolarization.="" kcl-induced="" upon="" contraction="" increases="" hela="" muscle-specific="" smooth="" nipp1-rata,="" not="" but="" nipp1-?c,="" moreover,="" pp1.="" dependent="" inhibition="" growth="" nipp1-mediated="" indicating="" effects,="" limited="" only="" growth,="" abrogates="" (nipp1-?c)="" constitutively="" growth.="" tumor="" effects="" inhibitory="" show="" nipp1-wt="" stable="" both="" since="" cells="" regulated="" strictly="" be="" needs="" concentration="" cellular="" proliferation.="" explored="" second="" genes. in="" longer="" damid="" via="" eed="" still="" nipp1-rata="" although="" ezh2.="" targeting="" effect="" no="" has="" (nipp1-rata)="" mutant="" pp1-binding="" expression="" conversely,="" genes.="" active="" partially="" to="" repressed="" fully="" from="" redistribution="" induces="" ectopically="" whereas="" genes="" nipp1-associated="" occupancy="" inhibits="" pp1="" or="" either="" depletion="" found="" manner.="" pp1-dependent="" recruitment="" regulator="" demonstrated="" we="" thesis,="" my="" part="" first="" lethality.in="" embryonic="" proliferation="" defective="" associated="" nip1="" loss="" addition,="" h3k27.="" for="" essential="" it="" where="" genes,="" pcg="" subset="" associates="" nipp1="" furthermore,="" eed. ="" ezh2="" components="" prc2="" two="" with="" interacts="" nuclear="" expressed="" ubiquitously="" pp1,="" phosphatase="" thr="" ser="" protein="" interactor="" an="" nipp1,="" complex.="" (prc2)="" 2="" complex="" component="" core="" a="" ezh2,="" methyltransferase="" by="" (h3k27)="" h3="" histone="" on="" 27="" lysine="" trimethylation="" is="" silencing="" gene="" pcg-mediated="" events="" key="" the="" of="" one="" development.="" cancer="" and="" fate="" establishing="" pluripotency,="" cell="" stem="" maintaining="" in="" important="" that="" modifiers="" chromatin="" repressive="" are="" proteins="" (pcg)="" group="" polycomb=""Table of contents Dankwoord V Table of content VII List of abbreviations XI List of publications XIII Chapter I: Introduction 1 I Epigenetics 1 I.A DNA methylation 1 I.B Histone modifications 2 II Polycomb-mediated gene silencing 4 II.A The molecular composition of PcG complexes 4 II.A.1 The PRC2 complex 4 II.A.2 The PRC1 complex 8 II.B Genome-wide PcG distribution and targeting 10 II.B.1 Genome-wide mapping of the PcG binding sites 10 II.B.2 Targeting PcG complexes in Drosophila 10 II.B.3 Targeting PcG complexes in mammalian cells 11 II.C Regulation of H3K27 trimethylation 16 II.C.1 Spreading of the H3K27me3 mark 16 II.C.2 Demethylation of H3K27 16 II.C.3 PRC2-antagonizing marks 17 II.D The physiological function of PcG signaling 19 II.D.1 PcG silencing, stem cell regulation and differentiation 19 II.D.2 PcG silencing, X-inactivation and imprinting 23 II.D.3 PcG silencing and cell cycle 24 III Cancer epigenetics 26 III.A DNA methylation 26 III.A.1 Hypomethylation 26 III.A.2 Hypermethylation 27 III.B PcG silencing 28 III.B.1 EZH2 in cancer proliferation 28 III.C Epigenetic markers and therapy 30 IV NIPP1 is a multifunctional scaffold protein 32 IV.A NIPP1 structure and interactome 32 IV.B NIPP1-regulated cellular processes 33 IV.B.1 NIPP1 is involved in cellular signaling 33 IV.B.2 NIPP1 is essential player in PcG-mediated gene silencing 34 IV.B.3 NIPP1 is involved in pre-mRNA splicing 35 IV.C NIPP1 is essential for proliferation and development 35 Chapter II: Aims and scopes 37 Chapter III: The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets 39 I Abstract 39 II Introduction 40 III Material and methods 42 IV Results 46 IV.A Identification of a chromatin-associated complex of NIPP1, PP1 and PRC2 46 IV.B The loss of NIPP1 or PP1 reduces the targeting of EZH2 47 IV.C The overexpression of NIPP1 redistributes EZH2 48 IV.D The association of NIPP1 with PcG target genes depends on PP1 53 IV.E Mapping of NIPP1 chromatin binding sites 56 IV.F PP1 is not required for the assembly of the NIPP1–PRC2 complex 58 V Discussion 59 VI References 62 VII Supplementary materials and methods 65 VIII Supplementary figures 68 Chapter IV: Transdifferentiation of HeLa cells into smooth muscle-like cells by NIPP1, a regulator of PP1 and EZH2 71 I Abstract 71 II Introduction 72 III Material and Methods 74 IV Results 77 IV.A The stable ectopic expression of NIPP1 hampers cancer cell proliferation and tumor growth in a PP1-dependent manner 77 IV.B The ectopic expression of NIPP1 induces the expression of differentiation genes 81 IV.C NIPP1 transdifferentiates HeLa cells into a smooth muscle-like phenotype 85 IV.D The depletion of NIPP1 reduces cancer cell proliferation, colony formation and tumor growth 87 V Discussion 91 VI References 93 VII Supplementary material and methods 95 VIII Supplementary figures 96 Chapter V: Discussion and perspectives 101 I NIPP1 is a PP1-dependent regulator of PRC2 recruitment 101 II NIPP1 is a putative differentiation factor 103 III NIPP1 as a therapeutic target 104 Summary 107 Samenvatting 109 References 111nrpages: 138status: publishe

The motility regulator flhDC drives intracellular accumulation and tumor colonization of Salmonella

Abstract Background Salmonella have potential as anticancer therapeutic because of their innate tumor specificity. In clinical studies, this specificity has been hampered by heterogeneous responses. Understanding the mechanisms that control tumor colonization would enable the design of more robust therapeutic strains. Two mechanisms that could affect tumor colonization are intracellular accumulation and intratumoral motility. Both of these mechanisms have elements that are controlled by the master motility regulator flhDC. We hypothesized that 1) overexpressing flhDC in Salmonella increases intracellular bacterial accumulation in tumor cell masses, and 2) intracellular accumulation of Salmonella drives tumor colonization in vitro. Methods To test these hypotheses, we transformed Salmonella with genetic circuits that induce flhDC and express green fluorescent protein after intracellular invasion. The genetically modified Salmonella was perfused into an in vitro tumor-on-a-chip device. Time-lapse fluorescence microscopy was used to quantify intracellular and colonization dynamics within tumor masses. A mathematical model was used to determine how these mechanisms are related to each other. Results Overexpression of flhDC increased intracellular accumulation and tumor colonization 2.5 and 5 times more than control Salmonella, respectively (P < 0.05). Non-motile Salmonella accumulated in cancer cells 26 times less than controls (P < 0.001). Minimally invasive, ΔsipB, Salmonella colonized tumor masses 2.5 times less than controls (P < 0.05). When flhDC was selectively induced after penetration into tumor masses, Salmonella both accumulated intracellularly and colonized tumor masses 2 times more than controls (P < 0.05). Mathematical modeling of tumor colonization dynamics demonstrated that intracellular accumulation increased retention of Salmonella in tumors by effectively causing the bacteria to bind to cancer cells and preventing leakage out of the tumors. These results demonstrated that increasing intracellular bacterial density increased overall tumor colonization and that flhDC could be used to control both. Conclusions This study demonstrates a mechanistic link between motility, intracellular accumulation and tumor colonization. Based on our results, we envision that therapeutic strains of Salmonella could use inducible flhDC to drive tumor colonization. More intratumoral bacteria would enable delivery of higher therapeutic payloads into tumors and would improve treatment efficacy

Protein phosphatase PP1-NIPP1 activates mesenchymal genes in HeLa cells

The deletion of the protein phosphatase-1 (PP1) regulator known as Nuclear Inhibitor of PP1 (NIPP1) is embryonic lethal during gastrulation, hinting at a key role of PP1-NIPP1 in lineage specification. Consistent with this notion we show here that a mild, stable overexpression of NIPP1 in HeLa cells caused a massive induction of genes of the mesenchymal lineage, in particular smooth/cardiac-muscle and matrix markers. This reprogramming was associated with the formation of actin-based stress fibers and retracting filopodia, and a reduced proliferation potential. The NIPP1-induced mesenchymal transition required functional substrate and PP1-binding domains, suggesting that it involves the selective dephosphorylation of substrates of PP1-NIPP1.publisher: Elsevier articletitle: Protein phosphatase PP1-NIPP1 activates mesenchymal genes in HeLa cells journaltitle: FEBS Letters articlelink: http://dx.doi.org/10.1016/j.febslet.2015.04.017 content_type: article copyright: Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.status: publishe

The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth

The serine/threonine protein phosphatase-1 (PP1) complex is a key regulator of the cell cycle. However, the redundancy of PP1 isoforms and the lack of specific inhibitors have hampered studies on the global role of PP1 in cell cycle progression in vertebrates. Here, we show that the overexpression of nuclear inhibitor of PP1 (NIPP1; also known as PPP1R8) in HeLa cells culminated in a prometaphase arrest, associated with severe spindle-formation and chromosome-congression defects. In addition, the spindle assembly checkpoint was activated and checkpoint silencing was hampered. Eventually, most cells either died by apoptosis or formed binucleated cells. The NIPP1-induced mitotic arrest could be explained by the inhibition of PP1 that was titrated away from other mitotic PP1 interactors. Consistent with this notion, the mitotic-arrest phenotype could be rescued by the overexpression of PP1 or the inhibition of the Aurora B kinase, which acts antagonistically to PP1. Finally, we demonstrate that the overexpression of NIPP1 also hampered colony formation and tumor growth in xenograft assays in a PP1-dependent manner. Our data show that the selective inhibition of PP1 can be used to induce cancer cell death through mitotic catastrophe.status: publishe

PP1 loss impairs electrotaxis in HeLa cells.

<p><b>A.</b> Treatment of parental HeLa Tet-Off (HTO) cells with siRNA strongly depletes PP1 levels 48 h post transfection. Endogenous PP1 levels were visualized with PP1 antibodies that recognize all isoforms. <b>B.</b> Plot diagrams show that loss of PP1 impairs the ability of cells to migrate towards the cathode. Each line represents the migration trajectory of a single cell. The starting point for each cell migration track is at the origin. Cell tracks with end positions to the right appear in red (“C”, cathode) and those to the left appear in black (“A”, anode). EF-untreated cells were assayed as controls. Control siRNA cells migrate strongly towards the cathode; PP1 siRNA treated cells are unable to migrate in response to a DC EF. Scales show distance migrated in µm. <b>C.</b> PP1 depletion strongly reduces distance migrated, speed, and directedness in response to physiological DC EF. Error bars are S.E.M. <i>p</i> values for significant differences in distance, speed and directedness are shown. <b>D.</b> Localization of endogenous PP1 and distribution of filamentous-actin in control and PP1 depleted cells treated with DC EF. Endogenous PP1 levels were visualized with PP1 antibodies that recognize all isoforms (green) and polymerised actin was detected using rhodamine phalloidin (red). The nuclei have been stained with DAPI (blue). Arrows mark cells with a strong decrease in PP1 levels which correlate with defects in the formation of actin rich protrusions. Representative images are shown. Scale bar is 50 µm. <b>E.</b> Numbers of cells with filopodia were quantified by counting 100 cells. Error bars are S.E.M. <i>p</i> values for significant differences are shown. Images show a detail of cell protrusions in control siRNA and PP1 siRNA cells. Arrows mark numerous filopodia in control cells and outline areas with a major lack of filopodia at the cell edges in PP1 siRNA cells.</p

Cartoon showing the basic organization of the cervical epithelium and a mechanistic model to explain how PP1/NIPP1 may contribute to invasiveness of tumour cells.

<p>Cervical and vaginal epithelia have lumen potentials of about −25 to −50 mV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040769#pone.0040769-Boskey1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040769#pone.0040769-Szatkowski1" target="_blank">[66]</a>. Such a lumen potential would correspond to a transepithelial voltage gradients of 1.7 V/cm (170 mV/mm). In these electrophysiological conditions cervical epithelial cells would migrate towards the lumen as they turn over the epithelial lining layer (green arrow). Upregulation of NIPP1 and its recruitment to PP1 would reverse migration into the lumen, encouraging invasion of the surrounding tissue (red arrow).</p