Shannah Boens

MELK-T1, a small-molecule inhibitor of protein kinase MELK, decreases DNA-damage tolerance in proliferating cancer cells

Protein kinase MELK has oncogenic properties and is highly overexpressed in some tumors. In the present study, we show that a novel MELK inhibitor causes both the inhibition and degradation of MELK, culminating in replication stress and a senescence phenotype.

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

ABSTRACT 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. Highlighted Article: Mitosis is an established target for cancer chemotherapy. We show that the inhibition of protein phosphatase PP1 causes an arrest in mid-mitosis that often culminates in cell death.

Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits

Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, β and γ isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1β emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits.

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.

Interactor-guided dephosphorylation by protein phosphatase-1

Protein phosphatase-1 (PP1) is an essential enzyme for every eukaryotic cell and catalyzes more than half of all protein dephosphorylations at serine and threonine residues. The free catalytic subunit of PP1 shows little substrate selectivity but is tightly regulated in vivo by a large variety of structurally unrelated PP1-interacting proteins (PIPs). PIPs form highly specific dimeric or trimeric PP1 holoenzymes by acting as substrates, inhibitors, and/or substrate-specifiers. The surface of PP1 contains many binding sites for short PP1-docking motifs that are combined by PIPs to create a PP1-binding code that is universal, specific, degenerate, nonexclusive, and dynamic. These properties of the PP1-binding code can be used for the rational design of small molecules that disrupt subsets of PP1 holoenzymes and have a therapeutic potential.

The deletion of the phosphatase regulator NIPP1 causes progenitor cell expansion in the adult liver

The Ppp1r8 gene encodes NIPP1, a nuclear interactor of protein phosphatase PP1. The deletion of NIPP1 is embryonic lethal at the gastrulation stage, which has hampered its functional characterization in adult tissues. Here, we describe the effects of a conditional deletion of NIPP1 in mouse liver epithelial cells. Ppp1r8−/− livers developed a ductular reaction, that is, bile‐duct hyperplasia with associated fibrosis. The increased proliferation of biliary epithelial cells was at least partially due to an expansion of the progenitor cell compartment that was independent of liver injury. Gene‐expression analysis confirmed an upregulation of progenitor cell markers in the liver knockout livers but showed no effect on the expression of liver‐injury associated regulators of cholangiocyte differentiation markers. Consistent with an inhibitory effect of NIPP1 on progenitor cell proliferation, Ppp1r8−/− livers displayed an increased sensitivity to diet‐supplemented 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine, which also causes bile‐duct hyperplasia through progenitor cell expansion. In contrast, the liver knockouts responded normally to injuries (partial hepatectomy, single CCl4 administration) that are restored through proliferation of differentiated parenchymal cells. Our data indicate that NIPP1 does not regulate the proliferation of hepatocytes but is a suppressor of biliary epithelial cell proliferation, including progenitor cells, in the adult liver. Stem Cells 2016;34:2256–2262

Brief Report: The Deletion of the Phosphatase Regulator NIPP1 Causes Progenitor Cell Expansion in the Adult Liver

The Ppp1r8 gene encodes NIPP1, a nuclear interactor of protein phosphatase PP1. The deletion of NIPP1 is embryonic lethal at the gastrulation stage, which has hampered its functional characterization in adult tissues. Here, we describe the effects of a conditional deletion of NIPP1 in mouse liver epithelial cells. Ppp1r8−/− livers developed a ductular reaction, that is, bile‐duct hyperplasia with associated fibrosis. The increased proliferation of biliary epithelial cells was at least partially due to an expansion of the progenitor cell compartment that was independent of liver injury. Gene‐expression analysis confirmed an upregulation of progenitor cell markers in the liver knockout livers but showed no effect on the expression of liver‐injury associated regulators of cholangiocyte differentiation markers. Consistent with an inhibitory effect of NIPP1 on progenitor cell proliferation, Ppp1r8−/− livers displayed an increased sensitivity to diet‐supplemented 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine, which also causes bile‐duct hyperplasia through progenitor cell expansion. In contrast, the liver knockouts responded normally to injuries (partial hepatectomy, single CCl4 administration) that are restored through proliferation of differentiated parenchymal cells. Our data indicate that NIPP1 does not regulate the proliferation of hepatocytes but is a suppressor of biliary epithelial cell proliferation, including progenitor cells, in the adult liver. Stem Cells 2016;34:2256–2262

The protein phosphatase 1 regulator NIPP1 is essential for mammalian spermatogenesis

NIPP1 is one of the major nuclear interactors of protein phosphatase PP1. The deletion of NIPP1 in mice is early embryonic lethal, which has precluded functional studies in adult tissues. Hence, we have generated an inducible NIPP1 knockout model using a tamoxifen-inducible Cre recombinase transgene. The inactivation of the NIPP1 encoding alleles (Ppp1r8) in adult mice occurred very efficiently in testis and resulted in a gradual loss of germ cells, culminating in a Sertoli-cell only phenotype. Before the overt development of this phenotype Ppp1r8−/− testis showed a decreased proliferation and survival capacity of cells of the spermatogenic lineage. A reduced proliferation was also detected after the tamoxifen-induced removal of NIPP1 from cultured testis slices and isolated germ cells enriched for undifferentiated spermatogonia, hinting at a testis-intrinsic defect. Consistent with the observed phenotype, RNA sequencing identified changes in the transcript levels of cell-cycle and apoptosis regulating genes in NIPP1-depleted testis. We conclude that NIPP1 is essential for mammalian spermatogenesis because it is indispensable for the proliferation and survival of progenitor germ cells, including (un)differentiated spermatogonia.