Plk4 is required for cytokinesis and maintenance of chromosomal stability

Abstract

Aneuploidy is a characteristic feature of established cancers and can promote tumor development. Aneuploidy may arise directly, through unequal distribution of chromosomes into daughter cells, or indirectly, through a tetraploid intermediate. The polo family kinase Plk4/Sak is required for late mitotic progression and is haploinsufficient for tumor suppression in mice. Here we show that loss of heterozygosity (LOH) occurs at the Plk4 locus in 50% of human hepatocellular carcinomas (HCC) and is present even in preneoplastic cirrhotic liver nodules. LOH at Plk4 is associated with reduced Plk4 expression in HCC tumors but not with mutations in the remaining allele. Plk4(+/-) murine embryonic fibroblasts (MEFs) at early passage show a high incidence of multinucleation, supernumerary centrosomes, and a near-tetraploid karyotype. Underlying these phenotypes is a high rate of primary cytokinesis failure, associated with aberrant actomyosin ring formation, reduced RhoA activation, and failure to localize the RhoA guanine nucleotide exchange factor Ect2 to the spindle midbody. We further show that Plk4 normally localizes to the midbody and binds to and phosphorylates Ect2 in vitro. With serial passaging Plk4(+/-) MEFs rapidly immortalize, acquiring an increasing burden of nonclonal and clonal gross chromosomal irregularities, and form tumors in vivo. Our results indicate that haploid levels of Plk4 disrupt RhoGTPase function during cytokinesis, resulting in aneuploidy and tumorigenesis, thus implicating early LOH at Plk4 as one of the drivers of human hepatocellular carcinogenesis. These findings represent an advance in our understanding of genetic predisposition to HCC, which continues to increase in incidence globally and particularly in North America.

Fig. 1.

Haploid levels of Plk4 are common in human hepatoma and drive cancer formation by murine cells. (A) Schematic representation of a region of the q arm of human chromosome 4, showing high rates of LOH at microsatellite markers closest to Plk4 in microdissected human hepatomas (n = 32 cases). All provisional, validated, and reviewed (the latter are bolded) genes are shown in black (University of California, Santa Cruz Gene Browser); none are recognized as or suggested to be tumor suppressors. (B) Decreased Plk4 mRNA levels in patients with LOH at the Plk4 locus, comparing tumor (T) in microdissected hepatoma samples with adjacent nonneoplastic liver (N) from the same patient. (C) Spontaneous immortalization of Plk4^+/− MEFs (mean ± SEM). y axis indicates the total number of live cells present on a 10-cm dish 5 days after plating 3 × 10⁵ cells. (D) Beta galactosidase staining (blue) in MEFs at passage 3 (P3) and P8 (Top), showing reduced senescence in P8 Plk4^+/− cells (n = 3 lines/genotype, *P = 0.001 vs. P8 Plk4^+/+). (E) P15 Plk4^+/− MEFs formed tumors at the site of s.c. injection into NOD-SCID mice, whereas P3 wild-type and P3 Plk4^+/− cells did not. A representative example of a high-grade tumor with spindle morphology is shown.

Fig. 2.

Plk4^+/− MEFs show multinucleation, increased centrosome number, and aneuploidy. (A) Increased incidence of multinucleation in P3 Plk4^+/− interphase MEFs (n = 400 cells per genotype; *P = 0.007). Representative photomicrographs of MEFs stained for DNA (blue) and α-tubulin (red). (B) Supernumerary centrosomes in P3 Plk4^+/− interphase MEFs (n = 200 cells per genotype, *P = 0.014). Centrosomes stain positive for centrin (green), pericentrin (red), and Plk4 (green). (C) Increasing ploidy with increasing passage (P) number in Plk4^+/− compared with Plk4^+/+ MEFs. Plk4^+/+ MEFs could not be examined past P7 because of senescence. (D) Increased ploidy in P3 Plk4^+/− MEFs is exaggerated in p53 null background. (E) Plk4^+/−p53^−/− mice show accelerated development of spontaneous lymphomas and sarcomas compared with Plk4^+/+p53^−/− mice (P < 0.0001). (F) Representative spectral karyotype analyses showing diploidy and nonclonal structural change (red arrow) in P3 Plk4^+/− MEFs and near tetraploidy and development of multiple clonal chromosomal rearrangements (green arrows), gains, and losses in late-passage Plk4^+/− MEFs; 10 spreads were evaluated per embryo.

Fig. 3.

Plk4 is required for myosin II localization and completion of cytokinesis. (A) Time-lapse DIC microscopy of mitotic P3 MEFs, showing failure of cytokinesis in a bipolar Plk4^+/− cell, resulting in multinucleation (arrows indicate nuclei, time points are in minutes). (B) Proportion of bipolar mitotic P3 Plk4^+/+ and Plk4^+/− MEFs completing and failing cytokinesis. (C) (Upper) P3 Plk4^+/+ and Plk4^+/− MEFs in late mitosis stained for F-actin (red), γ-tubulin (green), and DNA (blue). White arrows indicate cleavage furrow formation. (Lower) P3 Plk4^+/− MEF in late mitosis stained for DNA (blue) and α-tubulin (green) and/or γ-tubulin (red). (Insets) A single γ-tubulin–positive centrosome is seen at each pole. (D) Summary of mitotic phenotypes in P3 Plk4 MEFs. Plk4^+/− MEFs frequently undergo asymmetric division and ectopic cleavage furrow formation. (E) Representative immunofluorescent images of MEFs in cytokinesis showing mislocalized myosin II in Plk4^+/− cells (arrows indicate midbody location).

Fig. 4.

Plk4 is required for RhoA activation and Ect2 localization in cytokinesis. (A) Reduced levels of activated RhoA in Plk4^+/− MEFs after release from nocodazole (mean ± SEM, n = 3, *P < 0.001 vs. heterozygous). (B) Representative images of MEFs in cytokinesis. Staining for Ect2 (red), α-tubulin (green), and DNA (blue) reveals Ect2 localization to the spindle midbody in Plk4^+/+ but not Plk4^+/− cells. (C) Reciprocal coimmunoprecipitation assay on HEK293T cells shows physical interaction of Flag-Plk4 with endogenous Ect2. (D) Ratio of GTP-bound/total RhoA in unsynchronized HEK293T cells transfected with 3Flag-Plk4, with or without prior depletion of Ect2 (siEct2), showing dependence of Plk4-induced RhoA activation on Ect2 (mean ± SEM, n = 2).