The OTX2 Gene Induces Tumor Growth and Triggers Leptomeningeal Metastasis by Regulating the mTORC2 Signaling Pathway in Group 3 Medulloblastomas.

Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.

Plk2-mediated phosphorylation and translocalization of Nrf2 activates anti-inflammation through p53/Plk2/p21cip1 signaling in acute kidney injury.

The Plk2 is a cellular stress-responsive factor that is induced in response to oxidative stress. However, the roles of Plk2 in acute kidney injury (AKI) have not been clarified. We previously found that Plk2 is an interacting factor of Nrf2 in response to cellular stress, since Plk2 is upregulated in the Nrf2-dependent network. Here, we show that the levels of p53, Plk2, p21cip1, and chromatin-bound Nrf2 were all upregulated in kidney tissues of mice or NRK52E cells treated with either cisplatin or methotrexate. Upregulation of Plk2 by p53 led to an increase of Nrf2 in both soluble and chromatin fractions in cisplatin-treated NRK52E cells. Consistently, depletion of Plk2 suppressed the levels of Nrf2. Of note, Plk2 directly phosphorylated Nrf2 at Ser40, which facilitated its interaction with p21cip1 and translocation into the nuclei for the activation of anti-oxidative and anti-inflammatory factors in response to AKI. Together, these findings suggest that Plk2 may serve as an anti-oxidative and anti-inflammatory regulator through the phosphorylation and activation of Nrf2 to protect kidney cells from kidney toxicants and that Plk2 and Nrf2 therefore work cooperatively for the protection and survival of kidney cells from harmful stresses.

Nrf2-lncRNA controls cell fate by modulating p53-dependent Nrf2 activation as an miRNA sponge for Plk2 and p21cip1.

Long noncoding RNA (lncRNA) capable of controlling antioxidative capacity remains to be investigated. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a central molecule for cellular defense that increases antioxidative capacity. We identified a novel lncRNA named Nrf2-activating lncRNA (Nrf2-lncRNA) transcribed from an upstream region of the microRNA 122 gene (MIR122). Nrf2-lncRNA existed in the cytoplasm, suggestive of its function as a competing endogenous RNA [ceRNA, microRNA (miRNA) sponge]. Nrf2-lncRNA served as a ceRNA for polo-like kinase (Plk) 2 and cyclin-dependent kinase inhibitor 1 (p21cip1) through binding of miRNA 128 and miRNA 224, inducing Plk2/Nrf2/p21cip1 complexation for Nrf2 activation in the cells under p53-activating conditions (i.e., DNA damage and serum deprivation). Nrf2-lncRNA expression was suppressed with the initiation of apoptosis, being a rheostat for cell fate determination. Nrf2-lncRNA levels correlated with the recurrence-free postsurgery survival rate of patients with hepatocellular carcinoma. Collectively, Nrf2-lncRNA promotes Plk2 and p21cip1 translation by competing for specific miRNAs and activating Nrf2 under surviving conditions from oxidative stress, implying that Nrf2-lncRNA serves as a fine-tuning rheostat for cell fate decision.-Joo, M. S., Shin, S.-B., Kim, E. J., Koo, J. H., Yim, H., Kim, S. G. Nrf2-lncRNA controls cell fate by modulating p53-dependent Nrf2 activation as an miRNA sponge for Plk2 and p21cip1.

Combination of Inhibitors of USP7 and PLK1 has a Strong Synergism against Paclitaxel Resistance.

USP7 is a promising target for the development of cancer treatments because of its high expression and the critical functions of its substrates in carcinogenesis of several different carcinomas. Here, we demonstrated the effectiveness of targeting USP7 in advanced malignant cells showing high levels of USP7, especially in taxane-resistant cancer. USP7 knockdown effectively induced cell death in several cancer cells of lung, prostate, and cervix. Depletion of USP7 induced multiple spindle pole formation in mitosis, and, consequently, resulted in mitotic catastrophe. When USP7 was blocked in the paclitaxel-resistant lung cancer NCI-H460TXR cells, which has resistance to mitotic catastrophe, NCI-H460TXR cells underwent apoptosis effectively. Furthermore, combination treatment with the mitotic kinase PLK1 inhibitor volasertib and the USP7 inhibitor P22077 showed a strong synergism through down-regulation of MDR1/ABCB1 in paclitaxel-resistant lung cancer. Therefore, we suggest USP7 is a promising target for cancer therapy, and combination therapy with inhibitors of PLK1 and USP7 may be valuable for treating paclitaxel-resistant cancers, because of their strong synergism.

7-O-Methylwogonin from Scutellaria baicalensis Disturbs Mitotic Progression by Inhibiting Plk1 Activity in Hep3B Cells.

Polo-like kinase 1, a mitotic Ser/Thr kinase, has emerged as a molecular target for the development of anticancer drugs. In this study, we found that polo-like kinase 1 activity was inhibited by 7-O-methylwogonin and related flavones, including baicalein, dihydrobaicalein, and viscidulin II, isolated from Scutellaria baicalensis. Although dihydrobaicalein exhibited the highest polo-like kinase 1 inhibitory activity among the four compounds, it also inhibited other kinases, such as vaccinia-related kinase 2 and polo-like kinase 2. Baicalein and viscidulin II also showed low selectivity to polo-like kinase 1 since they inhibited polo-like kinase 3 and polo-like kinase 2, respectively. However, 7-O-methylwogonin exhibited selective polo-like kinase 1 inhibitory activity, as evidenced from in vitro kinase assays based on fluorescence resonance energy transfer assays and ADP-Glo kinase assays. In addition, examination of mitotic morphology and immunostaining using specific antibodies for the mitotic markers, p-histone H3 and mitotic protein monoclonal 2, in Hep3B cells showed that 7-O-methylwogonin treatment increased mitotic cell populations due to inhibition of mitotic progression as a result of polo-like kinase 1 inhibition. The pattern of 7-O-methylwogonin-induced mitotic arrest was similar to that of BI 2536, a specific polo-like kinase 1 inhibitor. Thus, it was suggested that 7-O-methylwogonin disturbed mitotic progression by inhibiting polo-like kinase 1 activity. These data suggest that 7-O-methylwogonin, a polo-like kinase 1 inhibitor, may be a useful anticancer agent because of its polo-like kinase 1 selectivity and effectiveness.

Molecular Targets of Genistein and Its Related Flavonoids to Exert Anticancer Effects.

Increased health awareness among the public has highlighted the health benefits of dietary supplements including flavonoids. As flavonoids target several critical factors to exert a variety of biological effects, studies to identify their target-specific effects have been conducted. Herein, we discuss the basic structures of flavonoids and their anticancer activities in relation to the specific biological targets acted upon by these flavonoids. Flavonoids target several signaling pathways involved in apoptosis, cell cycle arrest, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/AKT kinase, and metastasis. Polo-like kinase 1 (PLK1) has been recognized as a valuable target in cancer treatment due to the prognostic implication of PLK1 in cancer patients and its clinical relevance between the overexpression of PLK1 and the reduced survival rates of several carcinoma patients. Recent studies suggest that several flavonoids, including genistein directly inhibit PLK1 inhibitory activity. Later, we focus on the anticancer effects of genistein through inhibition of PLK1.

Sensitivity of TP53-Mutated Cancer Cells to the Phytoestrogen Genistein Is Associated With Direct Inhibition of Plk1 Activity.

Polo-like kinase 1 (Plk1), a conserved Ser/Thr mitotic kinase, has been identified as a promising target for anticancer drug development because its overexpression is correlated with malignancy. Here, we found that genistein, an isoflavone, inhibits Plk1 kinase activity directly. Previously the mitotic disturbance phenomenon induced by treatment with genistein was not fully explained by its inhibitory effect on EGFR. In kinase profiling assays, it showed selectivity relative to a panel of kinases, including EGFR. Treatment with genistein induced cell death in a concentration-dependent manner in cancer cells from diverse tissue origins, but not in non-transformed cells such as hTERT-RPE or MCF10A cells. We also observed that genistein tended to be more selective against cancer cells with mutations in the TP53 gene. TP53-depeleted LNCaP and NCI-H460 cells using shRNA targeting human TP53 were more sensitive to cell death by treatment of genistein. Furthermore, genistein induced mitotic arrest by inhibiting Plk1 activity and, consequently, led to mitotic catastrophe and apoptosis. These data suggest that genistein may be a promising anticancer drug candidate due to its inhibitory activity against Plk1 as well as EGFR and effectiveness toward cancer cells, especially those with p53-mutation. J. Cell. Physiol. 232: 2818-2828, 2017. © 2016 Wiley Periodicals, Inc.

Two nuclear export signals of Cdc6 are differentially associated with CDK-mediated phosphorylation residues for cytoplasmic translocation.

Cdc6 is cleaved at residues 442 and 290 by caspase-3 during apoptosis producing p49-tCdc6 and p32-tCdc6, respectively. While p32-tCdc6 is unable to translocate into the cytoplasm, p49-tCdc6 retains cytoplasmic translocation activity, but it has a lower efficiency than wild-type Cdc6. We hypothesized that a novel nuclear export signal (NES) sequence exists between amino acids 290 and 442. Cdc6 contains a novel NES in the region of amino acids 300-315 (NES2) that shares sequence similarity with NES1 at residues 462-476. In mutant versions of Cdc6, we replaced leucine with alanine in NES1 and NES2 and co-expressed the mutant constructs with cyclin A. We observed that the cytoplasmic translocation of these mutants was reduced in comparison to wild-type Cdc6. Moreover, the cytoplasmic translocation of a mutant in which all four leucine residues were mutated to alanine was significantly inhibited in comparison to the translocation of wild-type Cdc6. The Crm1 binding activities of Cdc6 NES mutants were consistent with the efficiency of its cytoplasmic translocation. Further studies have revealed that L468 and L470 of NES1 are required for cytoplasmic translocation of Cdc6 phosphorylated at S74, while L311 and L313 of NES2 accelerate the cytoplasmic translocation of Cdc6 phosphorylated at S54. These results suggest that the two NESs of Cdc6 work cooperatively and distinctly for the cytoplasmic translocation of Cdc6 phosphorylated at S74 and S54 by cyclin A/Cdk2.

Differential Cellular Effects of Plk1 Inhibitors Targeting the ATP-binding Domain or Polo-box Domain.

The expression of polo-like kinase 1 (Plk1) correlates with malignancy and is thus recognized as a target for cancer therapy. In addition to the development of ATP-competitive Plk1 inhibitors, the polo-box domain (PBD), a unique functional domain of PLKs, is being targeted to develop Plk1-specific inhibitors. However, the action mechanisms of these two classes of Plk1 inhibitors have not been thoroughly evaluated. Here, we evaluate the differences in cellular effects of ATP-binding domain inhibitors (BI 2536, GSK 461364) and PBD inhibitors (poloxin, thymoquinone) to determine their mechanisms of Plk1 inhibition. Our data show that BI 2536 and GSK461364 increased the population of cells in the G2/M phase compared with controls, while treatment with poloxin and thymoquinone increased cell population in the S phase as well as in G2/M, in a p53-independent manner. The population of cells staining positively for p-Histone H3 and MPM2, mitotic index, was increased by treatment with BI 2536 or GSK461364, but not by treatment with poloxin or thymoquinone. Furthermore, treatment with BI 2536 or GSK461364 resulted in activation of the BubR1 spindle checkpoint kinase, suggesting that treatment with ATP-binding domain inhibitors induces metaphase arrest. However, the administration of poloxin and thymoquinone resulted in an increase in p21(WAF1) and S arrest, indicating that PBD inhibitors also affected interphase before mitotic entry. Taken together, these data suggest that the PDB of Plk1 plays a role in S phase progression through interaction with other proteins, while its ATP-binding domain is important for regulating mitotic progression mediated by its catalytic activity involving consumption of ATP.

The tumor suppressor protein p150(Sal2) in carcinogenesis.

The transcription factor p150(Sal2), the product of the SALL2 gene, was first identified as a binding target of the oncogenic mouse polyomavirus T antigen. However, mouse polyomavirus is not the only oncogenic virus that targets p150(Sal2); the human papillomavirus E6 protein also targets this cellular protein in order to overcome p150(Sal2)-mediated growth arrest. Studies have demonstrated that p150(Sal2) recognizes GC-rich regions of the promoter and transcriptionally induces p21(Cip1/Waf1) and BAX in human ovarian epithelial cancer cells, resulting in cell growth arrest and apoptosis. Although the p150(Sal2) protein is strongly expressed in surface epithelial cells of the ovary, immunostaining experiments showed that expression of p150(Sal2) was lost in 90 % of 210 human ovarian carcinomas, supporting an important tumor suppressive role for p150(Sal2) in the human ovary. Mechanisms of silencing SALL2 in OVCA cell lines and primary tumors and possible therapeutic approaches for ovarian carcinoma are discussed in this review.

Synergistic effect of simvastatin plus NS398 on inhibition of proliferation and survival in hepatocellular carcinoma cell line.

BACKGROUND AND AIMS: NS398, a selective cyclooxygenase-2 inhibitor, and simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, both exert an anticancer effect on hepatocellular carcinoma cells, but the effect of co-administration of the two drugs remains unknown. We aimed to investigate the synergistic in vitro anticancer effect of co-administration of NS398 and simvastatin and its mechanism. METHODS: The Hep3B and Huh-7 cell lines were cultured. Cells were treated with simvastatin, NS398, or a combination. 5-bromo-2'-deoxyuridine ELISA assay, flow cytometry, Western blot analyses, and immunofluorescence assay were performed. RESULTS: In both cell lines, co-administration of simvastatin and NS398 resulted in a greater effect on proliferation and apoptosis. In Hep3B cells, co-administration of the two drugs resulted in a greater decrease in procaspase 3 and Bcl-2 and an increase in cleaved caspase 9 than that noted with monotherapy. In Huh-7 cells, co-administration of the two drugs resulted in a greater decrease in procaspase 3 and cyclin D1 and an increase in cleaved caspase 9. Expression of NF-κB and Akt were also decreased to a greater extent when the two drugs were co-administered in both cell lines. Immunofluorescence assay showed suppression of the nuclear localization of NF-κB by simvastatin or NS398. The effect was greater by co-administration. CONCLUSIONS: The co-administration of NS398 and simvastatin produced greater antiproliferative and proapoptotic effects against Hep3B cells and Huh-7 cells. Inhibition of the NF-κB and Akt pathway and activation of caspase cascade, which are considered as the major mechanism of synergistic anticancer properties, were observed in both cell lines.

Predictive value of urinary and serum biomarkers in young children with febrile urinary tract infections.

BACKGROUND: Early predictive biomarkers for the diagnosis and management of febrile urinary tract infections (UTIs) can be valuable diagnostic tools in children. METHODS: The study cohort comprised 73 pediatric patients with febrile UTIs [46 with acute pyelonephritis (APN) and 27 with lower UTIs] and 56 healthy children. Urine neutrophil gelatinase-associated lipocalin (uNGAL) and kidney injury molecule-1 (uKIM-1) levels and serum cystatin C (sCysC) levels were measured. RESULTS: The uNGAL/creatinine (Cr) and uKIM-1/Cr levels were higher in the UTI group than in the controls (P < 0.05). uNGAL/Cr and sCysC levels were higher in patients with APN than in those with lower UTIs (P < 0.05). uNGAL/Cr levels in both the APN and UTI groups decreased following the administration of antibiotics compared to those before treatment (P < 0.05). The uNGAL/Cr level was correlated with serum levels of white blood cells, C-reactive protein, CysC and with uKIM-1/Cr (P < 0.05). uKIM-1/Cr was also correlated with sCysC (P < 0.05). Receiver operating curve analyses showed good diagnostic profiles of uNGAL/Cr and uKIM-1/Cr for identifying UTIs [area under the curve (AUC) 0.9 and 0.66, respectively) and of uNGAL/Cr and sCysC for predicting APN (AUC 0.78 and 0.72, respectively). CONCLUSIONS: Our results suggest that uNGAL, uKIM-1 and sCysC levels may be useful for predicting and managing febrile UTIs in children.

Phosphorylation of Cdc6 at serine 74, but not at serine 106, drives translocation of Cdc6 to the cytoplasm.

Phosphorylation-dependent cytoplasmic translocation of human Cdc6 during S phase is sufficient to control its activity after origin firing. Export from the nucleus also serves as a mechanism for preventing re-replication in mammalian cells. Phosphorylation of the CDK consensus serine residues 54, 74, and 106 has been suggested to be involved in the cytoplasmic translocation of Cdc6. To determine the relative importance of the three phosphorylation sites, we have generated Cdc6 variants by substituting one or more of the three serine residues with alanine or aspartic acid and have assessed their cytoplasmic translocation behavior. Phosphorylation of serine 74 mainly contributes to the cytoplasmic translocation of Cdc6, while serine 54 phosphorylation provides a minor contribution. In contrast, phosphorylation at serine 106 does not affect the nuclear export of Cdc6. Comparative results were found in cells coexpressing the phosphorylation defective mutants of Cdc6 and cyclin A as well as in non-transfected cells synchronized by their release from a double thymidine block. We conclude that Cdk-mediated phosphorylation of Cdc6 at serine 74 is required for the cytoplasmic translocalization of Cdc6 during the cell cycle. Phosphorylation of Cdc6 at serine 54 plays a minor role and phosphorylation of serine 106 plays no role in the cytoplasmic localization of Cdc6. The phosphorylation of S74 in Cdc6 could be important for binding to the nuclear export protein for translocalization.

The accumulation of DNA repair defects is the molecular origin of carcinogenesis.

Genomic instability has been considered to be one of the prominent factors for carcinogenesis and the development of a number of degenerative disorders, predominantly related to the aging. The cellular machineries involved in the maintenance of genomic integrity such as DNA repair and DNA damage responses are extensively characterized by a large number of studies. The failure of proper actions of such cellular machineries may lead to the devastating effects mostly inducing cancer or premature aging, even with no acute exogenous DNA damage stimuli. In this review, we especially focus on the pathophysiological aspects of the defective DNA damage responses in carcinogenesis and premature aging. Clear understanding the causes of carcinogenesis and age-related degenerative diseases will provide novel and efficient approaches for prevention and rational treatment of cancer and premature aging.

Current clinical trials with polo-like kinase 1 inhibitors in solid tumors.

Significant advances in cancer treatment have resulted from the targeted cancer therapy by understanding the process of malignant transformation. Polo-like kinase 1 (PLK1) has been investigated as a target for cancer therapy for several years. Recently, anticancer drug candidates targeting PLK1 have been developed. To investigate the significance of PLK1 inhibitors in cancer patients, the current clinical statuses of PLK1 inhibitors including BI 2536, volasertib, and GSK461364A were analyzed. Monotherapy with BI 2536, the first human study of PLK1 inhibitors, has been terminated now, but its combinational study is still available in several solid tumors. The second-generation PLK1 inhibitor volasertib has an improved pharmacokinetic profile, safety, and efficacy, which is currently being developed under phase I/II. GSK461364 has shown a greater sensitive antitumor effect in p53-mutated cancer compared with that of p53-wild type cancer cells in a preclinical study. However, it has to be coadministered with an anticoagulator because of the high incidence of venous thrombotic emboli in clinical studies. PLK1 inhibitors showed a favorable pharmacokinetic profile, safety, and efficacy in patients with solid tumors. Further investigation with the use of PLK1 inhibitors in cancer patients who have mutated p53 or Ras and a high level of PLK1 as biomarkers is needed to consider the context and evaluation criteria of therapy.

Cell division cycle 6, a mitotic substrate of polo-like kinase 1, regulates chromosomal segregation mediated by cyclin-dependent kinase 1 and separase.

Defining the links between cell division and DNA replication is essential for understanding normal cell cycle progression and tumorigenesis. In this report we explore the effect of phosphorylation of cell division cycle 6 (Cdc6), a DNA replication initiation factor, by polo-like kinase 1 (Plk1) on the regulation of chromosomal segregation. In mitosis, the phosphorylation of Cdc6 was highly increased, in correlation with the level of Plk1, and conversely, Cdc6 is hypophosphorylated in Plk1-depleted cells, although cyclin A- and cyclin B1-dependent kinases are active. Binding between Cdc6 and Plk1 occurs through the polo-box domain of Plk1, and Cdc6 is phosphorylated by Plk1 on T37. Immunohistochemistry studies reveal that Cdc6 and Plk1 colocalize to the central spindle in anaphase. Expression of T37V mutant of Cdc6 (Cdc6-TV) induces binucleated cells and incompletely separated nuclei. Wild-type Cdc6 but not Cdc6-TV binds cyclin-dependent kinase 1 (Cdk1). Expression of wild-type Plk1 but not kinase-defective mutant promotes the binding of Cdc6 to Cdk1. Cells expressing wild-type Cdc6 display lower Cdk1 activity and higher separase activity than cells expressing Cdc6-TV. These results suggest that Plk1-mediated phosphorylation of Cdc6 promotes the interaction of Cdc6 and Cdk1, leading to the attenuation of Cdk1 activity, release of separase, and subsequent anaphase progression.

Targeting dysregulated lipid metabolism in the tumor microenvironment.

The reprogramming of lipid metabolism and its association with oncogenic signaling pathways within the tumor microenvironment (TME) have emerged as significant hallmarks of cancer. Lipid metabolism is defined as a complex set of molecular processes including lipid uptake, synthesis, transport, and degradation. The dysregulation of lipid metabolism is affected by enzymes and signaling molecules directly or indirectly involved in the lipid metabolic process. Regulation of lipid metabolizing enzymes has been shown to modulate cancer development and to avoid resistance to anticancer drugs in tumors and the TME. Because of this, understanding the metabolic reprogramming associated with oncogenic progression is important to develop strategies for cancer treatment. Recent advances provide insight into fundamental mechanisms and the connections between altered lipid metabolism and tumorigenesis. In this review, we explore alterations to lipid metabolism and the pivotal factors driving lipid metabolic reprogramming, which exacerbate cancer progression. We also shed light on the latest insights and current therapeutic approaches based on small molecular inhibitors and phytochemicals targeting lipid metabolism for cancer treatment. Further investigations are worthwhile to fully understand the underlying mechanisms and the correlation between altered lipid metabolism and carcinogenesis.

PLK1-mediated phosphorylation of ß-catenin enhances its stability and transcriptional activity for extracellular matrix remodeling in metastatic NSCLC.

Rationale: ß-catenin is a component for cell adhesion and a transcriptional coactivator in epithelial-mesenchymal transition (EMT). Previously we found that catalytically active PLK1 drives EMT in non-small cell lung cancer (NSCLC), upregulating extracellular matrix factors including TSG6, laminin γ2, and CD44. To understand the underlying mechanism and clinical significance of PLK1 and ß-catenin in NSCLC, their relationship and function in metastatic regulation were investigated. Methods: The clinical relevance between the survival rate of NSCLC patients and the expression of PLK1 and ß-catenin was analyzed by a KM plot. Immunoprecipitation, kinase assay, LC-MS/MS spectrometry, and site-directed mutagenesis were performed to reveal their interaction and phosphorylation. A lentiviral doxycycline-inducible system, Transwell-based 3D culture, tail-vein injection model, confocal microscopy, and chromatin immunoprecipitation assays were used to elucidate the function of phosphorylated ß-catenin in the EMT of NSCLC. Results: Clinical analysis revealed that the high expression of CTNNB1/PLK1 was inversely correlated with the survival rates of 1,292 NSCLC patients, especially in metastatic NSCLC. In TGF-ß-induced or active PLK1-driven EMT, ß-catenin, PLK1, TSG6, laminin γ2, and CD44 were concurrently upregulated. ß-catenin is a binding partner of PLK1 in TGF-ß-induced EMT and is phosphorylated at S311. Phosphomimetic ß-catenin promotes cell motility, invasiveness of NSCLC cells, and metastasis in a tail-vein injection mouse model. Its upregulated stability by phosphorylation enhances transcriptional activity through nuclear translocation for the expression of laminin γ2, CD44, and c-Jun, therefore enhancing PLK1 expression by AP-1. Conclusions: Our findings provide evidence for the critical role of the PLK1/ß-catenin/AP-1 axis in metastatic NSCLC, implying that ß-catenin and PLK1 may serve as a molecular target and prognostic indicator of the therapeutic response in metastatic NSCLC patients.

Invasive FoxM1 phosphorylated by PLK1 induces the polarization of tumor-associated macrophages to promote immune escape and metastasis, amplified by IFITM1.

BACKGROUND: Understanding the mechanism behind immune cell plasticity in cancer metastasis is crucial for identifying key regulators. Previously we found that mitotic factors regulate epithelial-mesenchymal transition, but how these factors convert to metastatic players in the tumor microenvironment (TME) is not fully understood. METHODS: The clinical importance of mitotic factors was analyzed by heatmap analysis, a KM plot, and immunohistochemistry in lung adenocarcinoma (LUAD) patients. Immunoprecipitation, LC-MS/MS, kinase assay, and site-directed mutagenesis were performed for the interaction and phosphorylation. A tail-vein injection mouse model, Transwell-based 3D culture, microarray analysis, coculture with monocytes, and chromatin immunoprecipitation assays were used to elucidate the function of phosphorylated FoxM1 in metastasis of TME. RESULTS: The phosphorylated FoxM1 at Ser25 by PLK1 acquires the reprogramming ability to stimulate the invasive traits in cancer and influence immune cell plasticity. This invasive form of p-FoxM1 upregulates the expression of IL1A/1B, VEGFA, and IL6 by direct activation, recruiting monocytes and promoting the polarization of M2d-like tumor-associated macrophages (TAMs). Upregulation of PD-L1 in LUAD having phosphomimetic FoxM1 facilitates immune evasion. In invasive LUAD with phosphomimetic FoxM1, IFITM1 is the most highly expressed through the activation of the STING-TBK1-IRF3 signaling, which enhances FoxM1-mediated signaling. Clinically, higher expression of FOXM1, PLK1, and IFITM1 is inversely correlated with the survival rate of advanced LUAD patients, providing a promising therapeutic strategy for the treatment of LUAD. CONCLUSION: FoxM1-based therapy would be a potential therapeutic strategy for LUAD to reduce TAM polarization, immune escape, and metastasis, since FoxM1 functions as a genetic reprogramming factor reinforcing LUAD malignancy in the TME.

DNA-binding and regulatory properties of the transcription factor and putative tumor suppressor p150(Sal2).

The product of the SALL2 protein p150(Sal2) is a multi-zinc finger transcription factor with growth arrest and proapoptotic functions that overlap those of p53. Its DNA-binding properties are unknown. We have used a modified SELEX procedure with purified p150(Sal2) and a pool of oligonucleotides of random sequence to identify those that are bound preferentially by p150(Sal2). The consensus sequence for optimal binding in vitro is GGG(T/C)GGG, placing p150(Sal2) among a large group of GC box-binding proteins including the Sp1 family of transcription factors. A triple zinc finger motif in p150(Sal2) similar to that in Sp1 is required for DNA binding. p150(Sal2) and Sp1 show evidence of co-operative binding in vitro and of interaction in vivo. p150(Sal2), a known activator of the CDK inhibitor p21(Cip1/Waf1) (p21), binds to regions of the human p21 promoter that contain variations of the consensus sequence in multiple copies. p150(Sal2) is also shown to bind to the BAX promoter with similar elements and to activate its expression following an apoptotic stimulus. These results demonstrate binding of p150(Sal2) to two natural promoters with GC elements related to the optimal binding sequence defined in vitro and whose regulation is important for suppression of tumor growth.