Figure 1. PLK signaling pathway.
Polo-like kinase (PLK) is a highly conserved serine/threonine protein kinase with a highly homologous serine/threonine kinase domain at its N-terminus, which regulates PLK activity and subcellular dynamics at the C-terminus and targeted polo-box domain (PBD). There are many PLK family members, and there are four subtypes in the human body, namely PLK1, PLK2, PLK3, and PLK4, which play important roles in the regulation of various phases of the cell cycle. Among the four family members, the current research on PLK1 is the most thorough. PLK proteins regulate many key steps in the cell cycle, including the formation of bipolar spindles, chromosome segregation, late regulation of complexes, and cytokinesis. PLK is a very effective anticancer strategy as a target for inhibiting spindle formation during mitosis, thereby preventing cancer cell division.
The most thorough study in the PLK family is PLK1. The first gene of PLK was reported in 1994, which is located at 16p12, with a mRNA length of about 2.3 kb, encoding a protein with a molecular mass of about 66000. Its amino acid sequence and drosophila Polo and yeast CDC5 and other genes have greater homology, and there is a highly conserved catalytic region at the N-terminus. The C-terminal PBD consists of two conserved regions called polo boxes. PLK1 plays an important role in the normal operation of the cell cycle, centrosome maturation, and cytoplasmic separation, but it is overexpressed in most human tumor cells, and this overexpression is often accompanied by a poor prognosis. Therefore, examining the expression level of PLK1 in cells is of great significance for the early diagnosis of tumors and the prediction of tumor patients' prognosis. In addition to PLK1, the most studied is PLK4. In 1994, the scientists' cDNA library screening resulted in a gene whose encoded product is very similar to the POLO family of proteins (Snk and PLK1) and contains a highly homologous N-terminal kinase. Therefore, snk/Plk, which is a protein kinase of the same type, was later named PLK4. PLK4 is widely found in eukaryotes such as nematodes, fruit flies, and primates, but not in prokaryotes. The mouse PLK4 gene is located on chromosome 13, and the human PLK4 gene is located on chromosome 4q28, which frequently undergoes chromosomal rearrangements or deletions in human tumor cells. The PLK 4 gene in mouse encodes two protein subtypes, PLK-a and PLK-b. PLK-b is 464 amino acids in length, and the N segments are 416 amino-terminally identical, and the C-region is different. A PLK4 protein has a total length of 970 amino acids with high homology to murine PLK4-a. Unlike other PLK family proteins, PLK4 has only one polo box that can form intermolecular counterpart dimers. These dimers are like the two polo boxes of PLK1 forming an intramolecular heterodimer structure.
PLK signaling pathway
PLK1 mainly assists the functional maturation of the centrosome and the formation of the spindle in the late G2 to the early stage of M, thereby promoting the entry of cells into the M phase; and it simultaneously participates in the activation of the cell cycle regulator CDC25, which in turn contributes to cylindered dentine kinase 2 (activation of CDK2). Somatic cell studies have found that the centrosome regulates the spindle assembly at the appropriate time and position. In the G2-M transformation, the centrosome gradually matures, manifesting as γ-tubulin ring complex (γ-TuRCs) and another microtubule regulation. The factors accumulate, and the two centrosomes are separated from each other to form the poles of the spindle. This series of processes is regulated by three proteins: TPX2, a microtubule tissue protein, PLK1, and AuroraA. Antisense RNA interference experiments show that these three key enzymes may regulate centrosome maturation by the following mechanism: PLK1 is at the uppermost end of this cascade, which on the one hand attaches Aurora A to the centrosome and promotes centrosome mature and correct polarization; The other side regulates TPX2, which accumulates at the poles of the spindle and recruits tubulin to form a spindle. At the same time, TPX2 also causes Aurora A to localize to the spindle. In Hela cells, if Aurora A is completely blocked, a multi-polarized centrosome will be formed, but tubulin can still accumulate around the centrosome, and cells can enter the mitosis phase; however, if the PLK1 expression is inhibited, cells will stagnate in the G2 period. This suggests that in addition to its role in the maturation of centrosomes, PLK1 can regulate cell cycle progression through other mechanisms. It is currently known that the competing interaction between CDK1 and its antagonistic enzyme WEE1 is another key factor determining the transformation of cells from G2 to M phase. CDK1 initiates chromosome condensation and nuclear membrane process through phosphorylation, while CDC25 activates CDK1, a key enzyme that induces its migration to the nucleus; while WEE1 inhibits its activity by amino acid phosphorylation at a site on CDK1, it delays or even arrests cells into mitosis. In contrast, PLK1 catalyzes the localization of CDC25 in the nucleus, activates CDK1, and activated CDK1 phosphorylates more CDC25 by positive feedback, advancing the process of mitotic expansion loop; at the same time, PLK1 can also determine the cell by controlling the relative content of WEE1 and CDC25. The study also found that when abnormal chromosomal replication occurs in cells, such as tumor cells, CDC25 and PLK1 are overexpressed, and the activity of WEE1 is inhibited. Therefore, cancerous cells can multiply over the regulation of G2-M checkpoint and malignant proliferation. In the middle and late stages of mitosis, with the degradation of the binding protein-adhesin between the centromeres, centromere division, and sister chromatid separation, this process is affected by early mitotic inhibition 1 (Emi1) and late promotion factor (APC). In the early stage of mitosis, APC exists as an inactive precursor, and the mature promoter-promoting factor (MPF) remaining during G2-M transformation depends on cyclinB to promote phosphorylation activation by transferring ATP phosphate group to APC. And thus, it could hydrolyze adhesin. In this process, PLK1 phosphorylates Emi1 and MPF, which results in inhibition of Emi1 activity, activation of APC, activation of APC-mediated cytokine linB ubiquitination, and degradation by the corresponding protease; since MPF activity is dependent on cyclinB, the activity of MPF disappears and the nucleolus is reconstituted. The cells thus normally exit the mitosis, otherwise the mitosis will be arrested in the later stage, eventually forming multinucleated cells. Study found that PLK1 localizes to the middle of the spindle in the late stages of mitosis, suggesting that it may play an important role in cytokinesis. One of the experiments showed that in the PLK1 homologous polo gene dominant mutant of yeast S. pombe, the polo gene could not be expressed normally, which not only inhibited the formation of the spindle, but also caused the shrinkage ring and the division groove not to be in M. Late formation prevents new membrane formation. Another experiment showed that in animal cells, the overexpression of PLK1 is accompanied by the formation of multinucleated cells. Two experimental results indicate that the precise expression of PLK1 may play an important role in normal cytokinesis. In recent years, the hypothesis that PLK1 may indirectly activate the G protein and mediate the formation of the plasma membrane may be promoted by promoting the maturation of the spindle body.
The most recent research on the regulation of PLK1 is the development of related inhibitors: like most anticancer drugs targeting protein kinases, the current development of PLK1 inhibitors is mainly through the competitive binding of ATP sites to directly inhibit the catalytic activity of PLK1. Recently, scientists have discovered a series of quinazoline derivatives that can competitively bind to the core region of ATP by high-throughput screening techniques, thereby inhibiting the kinase activity of PLK1. We would like to summarize the current research progress of PLK inhibitors: a. GSK461364 is an ATP-competitive PLK1 inhibitor developed by Glaxo Smith Kline. It can form a reversible complex with PLK1 rapidly. The inhibitory effect on PLK1 is 400 times than that of PLK2 and PLK3. In vitro experiments show that GSK461364 is up to the proliferation of 120 tumor cell lines and has inhibitory activity, and in more than 83% of the tested cell lines, the IC50 is less than 50 nmol/L. A phase I clinical study (solid tumor) has been performed, showing that 20% of the subjects have developed the response, and follow-up Phase II clinical studies are ongoing. b. LFM-A13 was first recognized as a specific blocker of Btk (Bruton S tyrosine kinase), and it has recently been found that it may also be a potential small molecule inhibitor of PLK1. c. LFM-A13, a small-molecular chemical inhibitor of PLK1, acts on the PTP region of Plx1 (a homologous gene of PLK1) on the ATP binding site of the Plx1 silk/threonine kinase domain through a kinase domain. The binding of the ATP site leads to a change in the conformation of Plx1, which inhibits the activity of PLK1 kinase, thereby causing the mitosis of tumor cells to stagnate, forming a monopolar or multipolar abnormal spindle. And 10 tyrosine kinases such as ABL, BRK, BMX, c-KIT, FYN, IGF1R, PDGFR, JAK2, MET and YES did not produce inhibition. d. GW843682X is a novel ATP competitive PLK inhibitor that selectively inhibits the activity of PLK1 and PLK3 and has a broad inhibitory effect on most tumors. In lung adenocarcinoma NCI-H460 cell line, GW843682X can induce transient G2-M arrest, mitotic spindle loss, multinucleated cell formation, and ultimately lead to cancer cell apoptosis, while normal diploid fibroblasts rarely die. BI 2536 is a dihydropteryridone compound that binds to the domain of PLK1 and inhibits PLK1 activity, causing abnormalities in mitosis of tumor cells, such as arresting cells in the G2/M phase, thereby inducing apoptosis. The IC50 for BI2536 inhibition of PLK1 activity is 0.83 nmol/L, and the inhibition of PLK1 is 10000 times of other 63 protein kinases. BI 2536 can inhibit the proliferation of 32 human cancer cell lines (EC50=2~25nmol/L). Cells treated with BI 2536 were arrested in the G2-M phase, forming a monopolar spindle like the change after siRNA interference with PLK1. After injection of tumor-bearing mice (including A549 and NCI-H460 cells) twice a week by BI 2536 40~50mg/kg, polo-blocked cancer cells gradually increased, followed by many apoptosis. The description of BI 2536 can mediate mitotic arrest and apoptosis in cancer cells. RO3280 is also a novel ATP-competitive PLK1 inhibitor with strong inhibition and good selectivity for PLK1, which not only has anti-tumor activity in vitro, but also has strong anticancer effect in xenograft mouse tumor models.
As mentioned above, the PLK signaling pathway plays an important role in the development and expansion of cancer, and current studies have also demonstrated that PLK inhibitors have the potential to treat cancer.