Juping Yuan

Polo-like kinases and oncogenesis.

Polo-like kinases (Plks) play pivotal roles in the regulation of cell cycle progression. Plk1, the best characterized family member among mammalian Plks, strongly promotes the progression of cells through mitosis. Furthermore, Plk1 is found to be overexpressed in a variety of human tumors and its expression correlates with cellular proliferation and prognosis of tumor patients. Although all Plks share two conserved elements, the N-terminal Ser/Thr kinase domain and a highly homologues C-terminal region termed the polo-box motif, their functions diverge considerably. While Plk1 is inhibited by different checkpoint pathways, Plk2 and Plk3 are activated by the spindle checkpoint or the DNA damage checkpoint. Thus, Plk2 and Plk3 seem to inhibit oncogenic transformation. Deregulation of Plk1 activity contributes to genetic instability, which in turn leads to oncogenic transformation. In contrast, Plk2 and Plk3 are involved in checkpoint-mediated cell cycle arrest to ensure genetic stability, thereby inhibiting the accumulation of genetic defects. In this review, we shall discuss the roles of Plks in oncogenesis and Plk1 as a target for therapeutic intervention against cancer.

Inhibition of Polo-like Kinase 1 by Blocking Polo-Box Domain-Dependent Protein-Protein Interactions

The serine/threonine kinase Polo-like kinase 1 (Plk1) is overexpressed in many types of human cancers, and has been implicated as an adverse prognostic marker for cancer patients. Plk1 localizes to its intracellular anchoring sites via its polo-box domain (PBD). Here we show that Plk1 can be inhibited by small molecules which interfere with its intracellular localization by inhibiting the function of the PBD. We report the natural product thymoquinone and, especially, the synthetic thymoquinone derivative Poloxin as inhibitors of the Plk1 PBD. Both compounds inhibit the function of the Plk1 PBD in vitro, and cause Plk1 mislocalization, chromosome congression defects, mitotic arrest, and apoptosis in HeLa cells. Our data validate the Plk1 PBD as an anticancer target and provide a rationale for developing thymoquinone derivatives as anticancer drugs.

Cyclin B1 depletion inhibits proliferation and induces apoptosis in human tumor cells

Cyclin B1 is the regulatory subunit of M-phase promoting factor, and proper regulation of cyclin B1 is essential for the initiation of mitosis. Increasing evidence indicates that the deregulation of cyclin B1 is involved in neoplastic transformation, suggesting the suppression of cyclin B1 could be an attractive strategy for antiproliferative therapy. In the present work, we analysed the impact of small interfering RNAs (siRNAs) targeted to cyclin B1 on different human tumor cell lines. Cyclin B1 siRNAs reduced the protein level of cyclin B1 in HeLa, MCF-7, BT-474 and MDA-MB-435 tumor cells and efficiently reduced the kinase activity of Cdc2/cyclin B1 in HeLa cells. siRNA-treated cells were arrested in G2/M phase in all tumor cell lines tested. Proliferation of tumor cells from different origins was suppressed by 50–80% 48 h after transfection and apoptosis was increased from 5 to 40–50%. Furthermore, tumor cells showed less colony-forming ability after siRNA treatment. In contrast, primary human umbilical vein endothelial cells exhibited only a slight change in cell cycle, and neither apoptosis nor clear inhibition of proliferation was observed after cyclin B1 siRNA treatment for 48 h. These results indicate that siRNAs against cyclin B1 could become a powerful antiproliferative tool in future antitumor therapy.

Cooperative phosphorylation including the activity of polo-like kinase 1 regulates the subcellular localization of cyclin B1

The cyclin-dependent kinase 1 (Cdc2)/cyclin B1 complex performs cardinal roles for eukaryotic mitotic progression. Phosphorylation of four serine residues within cyclin B1 promotes the rapid nuclear translocation of Cdc2/cyclin B1 at the G2/M transition. Still, the role of individual phosphorylation sites and their corresponding kinases remain to be elucidated. Polo-like kinase 1 (Plk1) shows a spatial and temporal distribution which makes it a candidate kinase for the phosphorylation of cyclin B1. We could demonstrate the interaction of both proteins in mammalian cells. Plk1 phosphorylated wild-type cyclin B1 expressed in bacteria and in mammalian cells. Ser-133 within the cytoplasmic retention signal (CRS) of cyclin B1, which regulates the nuclear entry of the heterodimeric complex during prophase, is a target of Plk1. In contrast, MAPK (Erk2) and MPF phosphorylate Ser-126 and Ser-128 within the CRS. Phosphorylation of CRS by MAPK (Erk2) prior to Plk1 treatment induced enhanced phosphorylation of cyclin B1 by Plk 1 suggesting a synergistic action of both enzymes towards cyclin B1. In addition, pretreatment of cyclin B1 by MAPK (Erk2) altered the phosphorylation pattern of Plk 1. Mutation of Ser-133 to Ala decreased the phosphorylation of cyclin B1 in vivo. An immunofluorescence study revealed that a mutation of Ser-133 reduced the nuclear import rate of cyclin B1. Still, multiple serine mutations are required to prevent nuclear translocation completely indicating that orchestrated phosphorylation within the CRS triggers rapid import of cyclin B1.

Polo-like Kinase 1-mediated Phosphorylation Stabilizes Pin1 by Inhibiting Its Ubiquitination in Human Cells

The Polo-like kinase 1 (Plk1) is a key regulator of mitosis. It is reported that the human peptidyl-prolyl cis/trans-isomerase Pin1 binds to Plk1 from mitotic cell extracts in vitro. Here we demonstrate that Ser-65 in Pin1 is the major site for Plk1-specific phosphorylation, and the polo-box domain of Plk1 is required for this phosphorylation. Interestingly, the phosphorylation of Pin1 by Plk1 does not affect its isomerase activity but rather is linked to its protein stability. Pin1 is ubiquitinated in HeLa S3 cells, and substitution of Glu for Ser-65 reduces the ubiquitination of Pin1. Furthermore, inhibition of Plk1 activity by expression of a dominant negative form of Plk1 or by transfection of small interfering RNA targeted to Plk1 enhances the ubiquitination of Pin1 and subsequently reduces the amount of Pin1 in human cancer cells. Since previous reports suggested that Plk1 is a substrate of Pin1, our work adds a new dimension to this interaction of two important mitotic regulators.

Expression of p16 and lack of pRB in primary small cell lung cancer.

The retinoblastoma protein (pRB), p16, and cyclin D1 are major components of the RB pathway, which controls the G1 checkpoint of the cell cycle. Proper regulation of this pathway is crucial for normal cell proliferation. Abnormal forms of these proteins have been found in various types of malignant tumours. In the present report, immunohistochemical techniques were applied to study the expression of pRB, p16, and cyclin D1 in 161 samples of primary small cell lung cancer (SCLC) and 20 samples of non‐small cell lung cancer (NSCLC). While pRB and cyclin D1 staining was negative in 161 specimens of SCLC, expression of p16 was observed in 153 samples. In contrast to SCLC, 16 out of 20 NSCLC cases exhibited pRB expression and 15 showed cyclin D1 expression, but only very weak p16 staining was found in five samples. These observations could provide additional criteria for the distinction between SCLC and NSCLC. Furthermore, these findings, based on primary tissues, implicate different mechanisms in the tumourigenesis of SCLC and NSCLC. Copyright

Stable gene silencing of cyclin B1 in tumor cells increases susceptibility to taxol and leads to growth arrest in vivo.

Cyclin B1 is the regulatory subunit of cyclin-dependent kinase 1 (Cdk1) and is critical for the initiation of mitosis. Accumulating data indicate that the deregulation of cyclin B1 is tightly linked to neoplastic transformation. To study the phenotype and the potential preclinical relevance, we generated HeLa cell lines stably transfected with the plasmids encompassing short hairpin RNA (shRNA) targeting cyclin B1. We demonstrate that the reduction of cyclin B1 caused inhibition of proliferation by arresting cells in G2 phase and by inducing apoptosis. Cells, entering mitosis, were impaired in chromosome condensation and alignment. Importantly, HeLa cells with reduced cyclin B1 were more susceptible to the treatment of small interfering RNA targeting Polo-like kinase 1 (Plk1) and to the administration of the chemotherapeutic agent taxol. Finally, HeLa cells with reduced cyclin B1 showed inhibited tumor growth in nude mice compared to that of control cells. In summary, our data indicate that cyclin B1 is an essential molecule for tumor cell survival and aggressive proliferation, suggesting that the downregulation of cyclin B1, especially in combination with other molecular targets, might become an interesting strategy for antitumor intervention.

Down-regulation of Polo-like Kinase 1 Elevates Drug Sensitivity of Breast Cancer Cells In vitro and In vivo

Human polo-like kinase 1 (Plk1) is a key player in different stages of mitosis and modulates the spindle checkpoint at the metaphase-anaphase transition. Overexpression of Plk1 is observed in various human tumors and it is a negative prognostic factor in patients suffering from diverse cancers. We used phosphorothioate antisense oligonucleotides (ASO) targeted against Plk1, together with paclitaxel, carboplatin, and Herceptin, for the treatment of breast cancer cells to identify conditions for enhanced drug sensitivity. After transfection of the breast cancer cell lines BT-474, MCF-7, and MDA-MB-435 with Plk1-specific ASOs, paclitaxel, carboplatin, or Herceptin was added and cell proliferation, cell cycle distribution, and apoptosis were measured. Whereas the dual treatment of breast cancer cells with Plk1-specific ASOs with carboplatin or Herceptin caused only a limited antiproliferative effect in breast cancer cells, we observed synergistic effects after combination of low doses of Plk1-specific ASOs with paclitaxel, which is used in a variety of clinical anticancer regimens. Plk1-specific ASOs also acted synergistically with paclitaxel in the arrest of the cell cycle at the G(2)-M phase and in the induction of apoptosis. Interestingly, in a human xenograft experiment using MDA-MB-435 cells, the combination of Plk1 ASOs with paclitaxel led to synergistic reduction of tumor growth after 3 weeks of treatment compared with either agent alone. This study suggests that antisense inhibitors against Plk1 at well-tolerated doses may be considered as highly efficient promoters for the antineoplastic potential of taxanes, such as paclitaxel, causing synergistic effects in breast cancer cells.

Polo-Box Domain Inhibitor Poloxin Activates the Spindle Assembly Checkpoint and Inhibits Tumor Growth in Vivo

Polo-like kinase 1 (Plk1) is widely established as one of the most promising targets in oncology. Although the protein kinase domain of Plk1 is highly conserved, the polo-box domain (PBD) of Plk1 provides a much more compelling site to specifically inhibit the localization and target binding of Plk1. We recently identified, via fluorescence polarization assay, the natural product derivative, Poloxin, as the first small-molecule inhibitor specifically targeting the function of the Plk1 PBD. In this study, we characterized its mitotic phenotype and its function in vitro and in vivo. Poloxin induces centrosome fragmentation and abnormal spindle and chromosome misalignment, which activate the spindle assembly checkpoint and prolong mitosis. Notably, centrosomal fragmentation induced by Poloxin is partially attributable to dysfunctional Kizuna, a key substrate of Plk1 at centrosomes. Moreover, Poloxin strongly inhibits proliferation of a panel of cancer cells by inducing mitotic arrest, followed by a surge of apoptosis. More important, we report, for the first time to our knowledge, that the PBD inhibitor, Poloxin, significantly suppresses tumor growth of cancer cell lines in xenograft mouse models by lowering the proliferation rate and triggering apoptosis in treated tumor tissues. The data highlight that targeting the PBD by Poloxin is a powerful approach for selectively inhibiting Plk1 function in vitro and in vivo.

A Pan-Specific Inhibitor of the Polo-Box Domains of Polo-like Kinases Arrests Cancer Cells in Mitosis

Playing polo: Small‐molecule inhibitors of polo‐like kinase 1 are mostly ATP‐competitive, and thus face enormous specificity hurdles. This communication explores the concept of inhibiting Plk1 with a small‐molecule inhibitor of the protein–protein interactions required for Plk1 function.