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Tumor suppressor protein Lgl mediates G1 cell cycle arrest at high cell density by forming an Lgl-VprBP-DDB1 complex.

ABSTRACT: Lethal giant larvae (Lgl) is an evolutionarily conserved tumor suppressor whose loss of function causes disrupted epithelial architecture with enhanced cell proliferation and defects in cell polarity. A role for Lgl in the establishment and maintenance of cell polarity via suppression of the PAR-aPKC polarity complex is established; however, the mechanism by which Lgl regulates cell proliferation is not fully understood. Here we show that depletion of Lgl1 and Lgl2 in MDCK epithelial cells results in overproliferation and overproduction of Lgl2 causes G1 arrest. We also show that Lgl associates with the VprBP-DDB1 complex independently of the PAR-aPKC complex and prevents the VprBP-DDB1 subunits from binding to Cul4A, a central component of the CRL4 [VprBP] ubiquitin E3 ligase complex implicated in G1- to S-phase progression. Consistently, depletion of VprBP or Cul4 rescues the overproliferation of Lgl-depleted cells. In addition, the affinity between Lgl2 and the VprBP-DDB1 complex increases at high cell density. Further, aPKC-mediated phosphorylation of Lgl2 negatively regulates the interaction between Lgl2 and VprBP-DDB1 complex. These results suggest a mechanism protecting overproliferation of epithelial cells in which Lgl plays a critical role by inhibiting formation of the CRL4 [VprBP] complex, resulting in G1 arrest.

SUBMITTER: Yamashita K

PROVIDER: S-EPMC4571298 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Tumor suppressor protein Lgl mediates G1 cell cycle arrest at high cell density by forming an Lgl-VprBP-DDB1 complex.

Yamashita Kazunari K Ide Mariko M Furukawa Kana T KT Suzuki Atsushi A Hirano Hisashi H Ohno Shigeo S

Molecular biology of the cell 20150506 13

Lethal giant larvae (Lgl) is an evolutionarily conserved tumor suppressor whose loss of function causes disrupted epithelial architecture with enhanced cell proliferation and defects in cell polarity. A role for Lgl in the establishment and maintenance of cell polarity via suppression of the PAR-aPKC polarity complex is established; however, the mechanism by which Lgl regulates cell proliferation is not fully understood. Here we show that depletion of Lgl1 and Lgl2 in MDCK epithelial cells resul ...[more]

PMID: 25947136

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Project description:During the initial stages of carcinogenesis, transformation events occur in a single cell within an epithelial monolayer. However, it remains unknown what happens at the interface between normal and transformed epithelial cells during this process. In Drosophila, it has been recently shown that normal and transformed cells compete with each other for survival in an epithelial tissue; however the molecular mechanisms whereby "loser cells" undergo apoptosis are not clearly understood. Lgl (lethal giant larvae) is a tumor suppressor protein and plays a crucial role in oncogenesis in flies and mammals. Here we have examined the involvement of Lgl in cell competition and shown that a novel Lgl-binding protein is involved in Lgl-mediated cell competition. Using biochemical immunoprecipitation methods, we first identified Mahjong as a novel binding partner of Lgl in both flies and mammals. In Drosophila, Mahjong is an essential gene, but zygotic mahjong mutants (mahj(-/-)) do not have obvious patterning defects during embryonic or larval development. However, mahj(-/-) cells undergo apoptosis when surrounded by wild-type cells in the wing disc epithelium. Importantly, comparable phenomena also occur in Mahjong-knockdown mammalian cells; Mahjong-knockdown Madin-Darby canine kidney epithelial cells undergo apoptosis, only when surrounded by non-transformed cells. Similarly, apoptosis of lgl(-/-) cells is induced when they are surrounded by wild-type cells in Drosophila wing discs. Phosphorylation of the c-Jun N-terminal kinase (JNK) is increased in mahj(-/-) or lgl(-/-) mutant cells, and expression of Puckered (Puc), an inhibitor of the JNK pathway, suppresses apoptosis of these mutant cells surrounded by wild-type cells, suggesting that the JNK pathway is involved in mahj- or lgl-mediated cell competition. Finally, we have shown that overexpression of Mahj in lgl(-/-) cells strongly suppresses JNK activation and blocks apoptosis of lgl(-/-) cells in the wild-type wing disc epithelium. These data indicate that Mahjong interacts with Lgl biochemically and genetically and that Mahjong and Lgl function in the same pathway to regulate cellular competitiveness. As far as we are aware, this is the first report that cell competition can occur in a mammalian cell culture system.

Project description:BackgroundNBPF1 (Neuroblastoma Breakpoint Family, member 1) was originally identified in a neuroblastoma patient on the basis of its disruption by a chromosomal translocation t(1;17)(p36.2;q11.2). Considering this genetic defect and the frequent genomic alterations of the NBPF1 locus in several cancer types, we hypothesized that NBPF1 is a tumor suppressor. Decreased expression of NBPF1 in neuroblastoma cell lines with loss of 1p36 heterozygosity and the marked decrease of anchorage-independent clonal growth of DLD1 colorectal carcinoma cells with induced NBPF1 expression further suggest that NBPF1 functions as tumor suppressor. However, little is known about the mechanisms involved.MethodsExpression of NBPF was analyzed in human skin and human cervix by immunohistochemistry. The effects of NBPF1 on the cell cycle were evaluated by flow cytometry. We investigated by real-time quantitative RT-PCR the expression profile of a panel of genes important in cell cycle regulation. Protein levels of CDKN1A-encoded p21(CIP1/WAF1) were determined by western blotting and the importance of p53 was shown by immunofluorescence and by a loss-of-function approach. LC-MS/MS analysis was used to investigate the proteome of DLD1 colon cancer cells with induced NBPF1 expression. Possible biological interactions between the differentially regulated proteins were investigated with the Ingenuity Pathway Analysis tool.ResultsWe show that NBPF is expressed in the non-proliferative suprabasal layers of squamous stratified epithelia of human skin and cervix. Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in a p53-dependent manner. Additionally, forced expression of NBPF1 in two p53-mutant neuroblastoma cell lines also resulted in a G1 cell cycle arrest and CDKN1A upregulation. However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific effects. In addition, proteome analysis of NBPF1-overexpressing DLD1 cells identified 32 differentially expressed proteins, of which several are implicated in carcinogenesis.ConclusionsWe demonstrated that NBPF1 exerts different tumor suppressive effects, depending on the cell line analyzed, and provide new clues into the molecular mechanism of the enigmatic NBPF proteins.

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Tumor suppressor protein Lgl mediates G1 cell cycle arrest at high cell density by forming an Lgl-VprBP-DDB1 complex.

Publications

Tumor suppressor protein Lgl mediates G1 cell cycle arrest at high cell density by forming an Lgl-VprBP-DDB1 complex.

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