Dominant-negative polo-like kinase 1 induces mitotic catastrophe independent of cdc25C function.

Polo-like kinase 1 (PLK1), which has been shown to have a critical role in mitosis, is one possible target for cancer therapeutic intervention. PLK1, at least in Xenopus, starts the mitotic cascade by phosphorylating and activating cdc25C phosphatase. Also, loss of PLK1 function has been shown to induce mitotic catastrophe in a HeLa cervical carcinoma cell line but not in normal Hs68 fibroblasts. We wanted to understand whether the selective mitotic catastrophe in HeLa cells could be extended to other tumor types, and, if so, whether it could be attributable to a tumor-specific loss of dependence on PLK1 for cdc25C activation. When PLK1 function was blocked through adenovirus delivery of a dominant-negative gene, we observed tumor-selective apoptosis in most tumor cell lines. In some lines, dominant-negative PLK1 induced a mitotic catastrophe similar to that published in HeLa cells (K. E. Mundt et al., Biochem. Biophys Res. Commun., 239: 377-385, 1997). Normal human mammary epithelial cells, although arrested in mitosis, appeared to escape the loss of centrosome maturation and mitotic catastrophe seen in tumor lines. Mitotic phosphorylation of cdc25C and activation of cdk1 was blocked by dominant-negative PLK1 in human mammary epithelial cells as well as in the tumor lines regardless of whether they underwent mitotic catastrophe. These data strongly argue that the mitotic catastrophe is not attributable to a lack of dependence for PLK1 in activating cdc25C.     

MLL5 maintains spindle bipolarity by preventing aberrant cytosolic aggregation of PLK1

Faithful chromosome segregation with bipolar spindle formation is critical for the maintenance of genomic stability. Perturbation of this process often leads to severe mitotic failure, contributing to tumorigenesis. MLL5 has been demonstrated to play vital roles in cell cycle progression and the maintenance of genomic stability. Here, we identify a novel interaction between MLL5 and PLK1 in the cytosol that is crucial for sustaining spindle bipolarity during mitosis. Knockdown of MLL5 caused aberrant PLK1 aggregation that led to acentrosomal microtubule-organizing center (aMTOC) formation and subsequent spindle multipolarity. Further molecular studies revealed that the polo-box domain (PBD) of PLK1 interacted with a binding motif on MLL5 (Thr887-Ser888-Thr889), and this interaction was essential for spindle bipolarity. Overexpression of wild-type MLL5 was able to rescue PLK1 mislocalization and aMTOC formation in MLL5-KD cells, whereas MLL5 mutants incapable of interacting with the PBD failed to do so. We thus propose that MLL5 preserves spindle bipolarity through maintaining cytosolic PLK1 in a nonaggregated form.     

PTEN regulates PLK1 and controls chromosomal stability during cell division

PTEN functions as a guardian of the genome through multiple mechanisms. We have previously established that PTEN maintains the structural integrity of chromosomes. In this report, we demonstrate a fundamental role of PTEN in controlling chromosome inheritance to prevent gross genomic alterations. Disruption of PTEN or depletion of PTEN protein phosphatase activity causes abnormal chromosome content, manifested by enlarged or polyploid nuclei. We further identify polo-like kinase 1 (PLK1) as a substrate of PTEN phosphatase. PTEN can physically associate with PLK1 and reduce PLK1 phosphorylation in a phosphatase-dependent manner. We show that PTEN deficiency leads to PLK1 phosphorylation and that a phospho-mimicking PLK1 mutant causes polyploidy, imitating functional deficiency of PTEN phosphatase. Inhibition of PLK1 activity or overexpression of a non-phosphorylatable PLK1 mutant reduces the polyploid cell population. These data reveal a new mechanism by which PTEN controls genomic stability during cell division.     

Liver kinase B1 regulates the centrosome via PLK1

Liver kinase B1 (LKB1) is a tumor suppressor mutationally inactivated in Peutz–Jeghers syndrome (PJS) and various sporadic cancers. Although LKB1 encodes a kinase that possesses multiple functions, no individual hypothesis posed to date has convincingly explained how loss of LKB1 contributes to carcinogenesis. In this report we demonstrated that LKB1 maintains genomic stability through the regulation of centrosome duplication. We found that LKB1 colocalized with centrosomal proteins and was situated in the mitotic spindle pole. LKB1 deficiency-induced centrosome amplification was independent of AMP-activated protein kinase (AMPK), a well-defined substrate of LKB1. Cells lacking LKB1 exhibited an increase in phosphorylated and total Polo-like kinase 1 (PLK-1), NIMA-related kinase 2 (NEK2), and ninein-like protein (NLP). Overexpression of active PLK1 (T210D) reversed the inhibition of LKB1 on centrosome amplification. In contrast, depletion of PLK1 with siRNA or suppression of PLK1 kinase activity with BTO-1 (5-Cyano-7-nitro-2-benzothiazolecarboxamide-3-oxide) abrogated LKB1 deficiency-induced centrosome amplification. We further characterized that LKB1 phosphorylated and activated AMPK-related kinase 5 (NUAK1 or ARK5) that in turn increased the phosphorylation of MYPT1, enhanced the binding between MYPT1–PP1 and PLK1, and conferred an effective dephosphorylation of PLK1. More importantly, we noted that LKB1-deficient cells exhibited multiple nuclear abnormalities, such as mitotic delay, binuclear, polylobed, grape, large, and micronuclear. Immediate depletion of LKB1 resulted in the accumulation of multiploidy cells. Expression of LKB1 is reversely correlated with the levels of PLK1 in human cancer tissues. Thus, we have uncovered a novel function of LKB1 in the maintenance of genomic stability through the regulation of centrosome mediated by PLK1.     

PLK1 Down-Regulates Parainfluenza Virus 5 Gene Expression

The paramyxoviruses are a family of negative-sense RNA viruses that includes many important human and animal pathogens. Paramyxovirus RNA synthesis requires the viral phosphoprotein (P) and the large (L) protein. Phosphorylation of P is thought to regulate viral gene expression, though direct proof remains elusive. Recently, we reported that phosphorylation of a specific residue (Ser157) of the P protein of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, correlates with decreased viral gene expression and cytokine expression in infected cells. Here, we show that: Polo-like kinase 1 (PLK1), a serine/theronine kinase that plays a critical role in regulating the cell cycle, interacts with PIV5 P through the S157 residue; PLK1 inhibition increases viral gene expression; PLK1 over-expression inhibits viral gene expression; and PLK1 directly phosphorylates P in vitro, indicating that PLK1 down-regulates viral gene expression by phosphorylating P. Furthermore, we have determined the PLK1 phosphorylation site on P and found that mutant recombinant PIV5 whose P proteins cannot either bind to or be phosphorylated by PLK1 have similar phenotypes. Increased viral gene expression in PIV5 with mutations in the PLK1 binding/phosphorylation sites correlates with increased induction of cell death and cytokine expression, suggesting that PIV5 limits its viral gene expression to avoid these host effects. It is possible that targeting PLK1 will enhance host innate immune responses, leading to a novel strategy of clearing paramyxovirus infections quickly.     

Autophosphorylation of Polo-like Kinase 4 and Its Role in Centriole Duplication

Centrosome duplication occurs once every cell cycle in a strictly controlled manner. Polo-like kinase 4 (PLK4) is a key regulator of this process whose kinase activity is essential for centriole duplication. Here, we show that PLK4 autophosphorylation of serine S305 is a consequence of kinase activation and enables the active fraction to be identified in the cell. Active PLK4 is detectable on the replicating mother centriole in G1/S, with the proportion of active kinase increasing through interphase to reach a maximum in mitosis. Activation of PLK4 at the replicating daughter centriole is delayed until G2, but a level equivalent to the replicating mother centriole is achieved in M phase. Active PLK4 is regulated by the proteasome, because either proteasome inhibition or mutation of the degron motif of PLK4 results in the accumulation of S305-phosphorylated PLK4. Autophosphorylation probably plays a role in the process of centriole duplication, because mimicking S305 phosphorylation enhances the ability of overexpressed PLK4 to induce centriole amplification. Importantly, we show that S305-phosphorylated PLK4 is specifically sequestered at the centrosome contrary to the nonphosphorylated form. These data suggest that PLK4 activity is restricted to the centrosome to prevent aberrant centriole assembly and sustained kinase activity is required for centriole duplication.     

特异小干扰RNA敲除PLK1基因的表达

为研究特异小干扰RNA（siRNA）作用于大肠癌细胞株SW480中PLK1 (Polo-like kinase 1)基因表达的mRNA对该细胞分裂生长的影响,设计了对应于PLK1基因表达mRNA不同位点的10种特异siRNA,经化学合成后,用脂质体转染SW480细胞,实时定量PCR检测PLK1基因的表达,观察不同的siRNA作用强度,并计数细胞了解相应细胞的生长情况,western-blot观察PLK1表达蛋白的变化和流式细胞计数分析细胞周期改变。发现10种siRNA均可敲除PLK1基因表达的20 %以上,其中P1、P4和P9 3组敲除mRNA达80 ％以上,这3种siRNA及其混合物对PLK1基因mRNA的作用具有相应浓度效应,在25 nmol/L时达到最佳作用效果,而且相同浓度的混合物作用效果更好（超过95%）,PLK1表达蛋白质明显降低,细胞周期在G2期受到阻碍。72 h后的各种siRNA浓度下细胞生长变化与PLK1基因的mRNA水平变化相一致。结果表明化学合成的特异siRNA对SW480细胞中PLK1基因表达具有消除作用,混合物作用更强,在细胞水平上抑制了SW480细胞的分裂生长。     

Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

MicroRNAs (miRNAs) are small noncoding RNAs that act as endogenous regulatory molecules targeting specific mRNAs for translational repression. Studies of breast cancer genomics indicate that breast cancer subtypes are distinguished and regulated by specific sets of miRNAs which affect activities such as tumor initiation, progression, and even drug response. Polo-like Kinase 1 (PLK1) is widely considered to be a proto-oncogene due to its increased expression in multiple tumor types, as well as its crucial role in regulating mitosis. Pharmacological inhibition of PLK1 can reduce tumor volume and induce tumor cell death in solid and hematologic malignancies. This prompted us to investigate how PLK1 inhibition with the target-specific inhibitor NMS-P937 would impact breast cancer cells, and how miRNAs may influence the overall response of these cells to this inhibition. We found that miR-183-5p targets PLK1 gene, effectively reducing its protein expression. Such miRNA-driven regulation of PLK1 expression sensitizes breast cancer cells to NMS-P937, resulting in synergistically increased apoptosis. We also show that the miRNA-regulated reduction of PLK1 influences the expression of apoptosis-related key proteins and possibly inducing further indirect PLK1 downmodulation through a DNMT1-p53 axis. These results suggest a potential biologically significant link between the expression of miR-183-5p and the efficacy of PLK1-specific inhibitors in breast cancer cells. Our work further elucidates how miR-183-5p regulates PLK1 gene while also enhancing NMS-P937 effect in breast cancer. Future studies assessing the role of miR-183-5p as a novel biomarker for anti-PLK1 chemotherapy agents are warranted.Subject terms: Cancer, Tumour biomarkers     

Pharmacological and functional comparison of the polo-like kinase family: insight into inhibitor and substrate specificity

PLK1 (polo-like kinase 1) is a key mitotic kinase and a therapeutic target in the treatment of proliferative diseases. Here we investigate the relative substrate specificity and pharmacological relatedness of PLK1, -2, -3, and -4 that together comprise a conserved family of Ser/Thr kinases (PLK family). We report consensus substrate sequences for PLK2, -3, and -4 and an expanded consensus sequence for PLK1, which we use to design an optimal peptide substrate, PLKtide. We report inhibitory activity for the entire PLK family across a diverse set of small-molecule ATP-competitive inhibitors including several clinical compounds. With respect to both substrate and ATP-site specificity, highest similarity is observed between PLK2 and PLK3, PLK1 is next most similar, and PLK4 is least similar. Further, we have identified and report time-dependent inhibition by two potent and selective PLK inhibitors.     

Tazarotene-induced gene 1 interacts with Polo-like kinase 2 and inhibits cell proliferation in HCT116 colorectal cancer cells

Tazarotene-induced gene 1 (TIG1) is considered to be a tumor suppressor gene that is highly expressed in normal or well-differentiated colon tissues, while downregulation of TIG1 expression occurs in poorly differentiated colorectal cancer (CRC) tissues. However, it is still unclear how TIG1 regulates the tumorigenesis of CRC. Polo-like kinases (Plks) are believed to play an important role in regulating the cell cycle. The performance of PLK2 in CRC is negatively correlated with the differentiation status of CRC tissues. Here, we found that PLK2 can induce the growth of CRC cells and that TIG1 can prevent PLK2 from promoting the proliferation of CRC cells. We also found that the expression of PLK2 in CRC cells was associated with low levels of Fbxw7 protein and increased expression of cyclin E1. When TIG1 was coexpressed with PLK2, the changes in Fbxw7/cyclin E1 levels induced by PLK2 were reversed. In contrast, silencing TIG1 promoted the proliferation of CRC, and when PLK2 was also silenced, the proliferation of CRC cells induced by TIG1 silencing was significantly inhibited. The above research results suggest that TIG1 can regulate the tumorigenesis of CRC by regulating the activity of PLK2.     

PLK1, A Potential Target for Cancer Therapy

Polo-like kinase 1 (PLK1) plays an important role in the initiation, maintenance, and completion of mitosis. Dysfunction of PLK1 may promote cancerous transformation and drive its progression. PLK1 overexpression has been found in a variety of human cancers and was associated with poor prognoses in cancers. Many studies have showed that inhibition of PLK1 could lead to death of cancer cells by interfering with multiple stages of mitosis. Thus, PLK1 is expected to be a potential target for cancer therapy. In this article, we examined PLK1’s structural characteristics, its regulatory roles in cell mitosis, PLK1 expression, and its association with survival prognoses of cancer patients in a wide variety of cancer types, PLK1 interaction networks, and PLK1 inhibitors under investigation. Finally, we discussed the key issues in the development of PLK1-targeted cancer therapy.     

PLK1 (polo like kinase 1) inhibits MTOR complex 1 and promotes autophagy

Mechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1′s and PLK1′s functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy.     

Inhibition of polo-like kinase 1 (PLK1) facilitates reactivation of gamma-herpesviruses and their elimination

Both Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and associate with certain types of tumors, such as B cell lymphomas, especially in immuno-compromised individuals including people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHV de novo infection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-infected lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Furthremore, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. We also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV lytic reactivation and cell death of EBV-infected lymphoma cells. Lastly, we noticed that PLK1 in B cells is elevated in the context of HIV infection and caused by HIV Nef protein to favor KSHV/EBV latency.     

Non-mitotic functions of polo-like kinases in cancer cells

Inhibitors of mitotic protein kinases are currently being developed as non-neurotoxic alternatives of microtubule-targeting agents (taxanes, vinca alkaloids) which provide a substantial survival benefit for patients afflicted with different types of solid tumors. Among the mitotic kinases, the cyclin-dependent kinases, the Aurora kinases, the kinesin spindle protein and Polo-like kinases (PLKs) have emerged as attractive targets of cancer therapeutics.The functions of mammalian PLK1-5 are traditionally linked to the regulation of the cell cycle and to the stress response. Especially the key role of PLK1 and PLK4 in cellular growth and proliferation, their overexpression in multiple types of human cancer and their druggability, make them appealing targets for cancer therapy. Inhibitors for PLK1 and PLK4 are currently being tested in multiple cancer trials. The clinical success of microtubule-targeting agents is attributed not solely to the induction of a mitotic arrest in cancer cells, but also to non-mitotic effects like targeting intracellular trafficking on microtubules. This raises the question whether new cancer targets like PLK1 and PLK4 regulate critical non-mitotic functions in tumor cells. In this article we summarize the important roles of PLK1-5 for the regulation of non-mitotic signaling. Due to these functions it is conceivable that inhibitors for PLK1 or PLK4 can target interphase cells, which underscores their attractive potential as cancer drug targets. Moreover, we also describe the contribution of the tumor-suppressors PLK2, PLK3 and PLK5 to cancer cell signaling outside of mitosis. These observations highlight the urgent need to develop highly specific ATP-competitive inhibitors for PLK4 and for PLK1 like the 3^rd generation PLK-inhibitor Onvansertib to prevent the inhibition of tumor-suppressor PLKs in- and outside of mitosis. The remarkable feature of PLKs to encompass a unique druggable domain, the polo-box-domain (PBD) that can be found only in PLKs offers the opportunity for the development of inhibitors that target PLKs exclusively. Beyond the development of mono-specific ATP-competitive PLK inhibitors, the PBD as drug target will support the design of new drugs that eradicate cancer cells based on the mitotic and non-mitotic function of PLK1 and PLK4.