PBK Expression Research Articles

DEPDC1B: A novel tumor suppressor gene associated with immune infiltration in colon adenocarcinoma.

Recent research indicates a positive correlation between DEP structural domain-containing 1B (DEPDC1B) and the cell cycle in various tumors. However, the role of DEPDC1B in the infiltration of the tumor immune microenvironment (TIME) remains unexplored. We analyzed the differential expression and prognostic significance of DEPDC1B in colon adenocarcinoma (COAD) using the R package "limma" and the Gene Expression Profiling Interactive Analysis (GEPIA) website. Gene set enrichment analysis (GSEA) was employed to investigate the functions and interactions of DEPDC1B expression in COAD. Cell Counting Kit-8 (CCK-8) assays and colony formation assays were utilized to assess the proliferative function of DEPDC1B. Correlations between DEPDC1B expression and tumor-infiltrating immune cells, immune checkpoints, tumor mutational burden (TMB), and microsatellite instability (MSI) status were examined using Spearman correlation analysis and CIBERSORT. DEPDC1B was highly expressed in COAD. Elevated DEPDC1B expression was associated with lower epithelial-to-mesenchymal transition (EMT) and TNM stages, leading to a favorable prognosis. DEPDC1B mRNA was prominently expressed in COAD cell lines. CCK-8 and colony formation assays demonstrated that DEPDC1B inhibited the proliferation of COAD cells. Analysis using the CIBERSORT database and Spearman correlation revealed that DEPDC1B correlated with four types of tumor-infiltrating immune cells. Furthermore, high DEPDC1B expression was linked to the expression of PD-L1, CTLA4, SIGLEC15, PD-L2, TMB, and MSI-H. High DEPDC1B expression also indicated responsiveness to anti-PD-L1 immunotherapy. DEPDC1B inhibits the proliferation of COAD cells and positively regulates the cell cycle, showing a positive correlation with CCNB1 and PBK expression. DEPDC1B expression in COAD is associated with tumor-infiltrating immune cells, immune checkpoints, TMB, and MSI-H in the tumor immune microenvironment. This suggests that DEPDC1B may serve as a novel prognostic marker and a potential target for immunotherapy in COAD.

Development and validation of multi-omic prognostic signature of anoikis-related genes in liver hepatocellular carcinoma.

Liver hepatocellular carcinoma (LIHC) is characterized by high morbidity, rapid progression and early metastasis. Although many efforts have been made to improve the prognosis of LIHC, the situation is still dismal. Inability to initiate anoikis process is closely associated with cancer proliferation and metastasis, affecting patients' prognosis. In this study, a corresponding gene signature was constructed to comprehensively assess the prognostic value of anoikis-related genes (ARGs) in LIHC. Using TCGA-LIHC dataset, the mRNA levels of the differentially expressed ARGs in LIHC and normal tissues were compared by Student t test. And prognostic ARGs were identified through Cox regression analysis. Prognostic signature was established and then externally verified by ICGC-LIRI-JP dataset and GES14520 dataset via LASSO Cox regression model. Potential functions and mechanisms of ARGs in LIHC were evaluated by functional enrichment analyses. And the immune infiltration status in prognostic signature was analyzed by ESTIMATE algorithm and ssGSEA algorithm. Furthermore, ARGs expression in LIHC tissues was validated via qRT-PCR and IHC staining from the HPA website. A total of 97 differentially expressed ARGs were detected in LIHC tissues. Functional enrichment analysis revealed these genes were mainly involved in MAP kinase activity, apoptotic signaling pathway, anoikis and PI3K-Akt signaling pathway. Afterward, the prognostic signature consisting of BSG, ETV4, EZH2, NQO1, PLK1, PBK, and SPP1 had a moderate to high predictive accuracy and served as an independent prognostic indicator for LIHC. The prognostic signature was also applicable to patients with distinct clinical parameters in subgroup survival analysis. And it could reflect the specific immune microenvironment in LIHC, which indicated high-risk group tended to profit from ICI treatment. Moreover, qRT-PCR and IHC staining showed increasing expression of BSG, ETV4, EZH2, NQO1, PLK1, PBK and SPP1in LIHC tissues, which were consistent to the results from TCGA database. The current study developed a novel prognostic signature comprising of 7 ARGs, which could stratify the risk and effectively predict the prognosis of LIHC patients. Furthermore, it also offered a potential indicator for immunotherapy of LIHC.

Tumor-suppressive E3 ubiquitin ligase CHIP inhibits the PBK/ERK axis to repress stem cell properties and radioresistance in non-small cell lung cancer.

Recently, radioresistant cancer cells surviving radiotherapy have been suggested to show more aggressive phenotypes than parental cells, and the underlying mechanisms may be associated with cancer stem cells. This study provided novel mechanistic insights for E3 ubiquitin ligase CHIP in stem cell properties and radioresistance of non-small cell lung cancer (NSCLC). After bioinformatic prediction for key genes involved, NSCLC tissues and cells were collected to measure the expression of CHIP and PBK. E3 ubiquitin ligase CHIP was poorly expressed, while PBK was highly expressed in NSCLC tissues and cells. CHIP reduced the protein stability of PBK through the ubiquitin-protease pathway to repress the activation of ERK pathway. Based on the gain- or loss-of-function experiments, it was noted that restoration of CHIP curtailed stem cell properties and radioresistance in NSCLC, as manifested by inhibited sphere formation and cell proliferation, decreased number of CD133+CD44+ cells and expression of OCT4, SOX2, and NANOG, as well as facilitated apoptosis of NSCLC cells. Besides, in vivo animal experiments further confirmed that CHIP restrained tumorigenic ability and improved radiosensitivity of NSCLC cells by inhibiting PBK/ERK axis. Collectively, CHIP suppressed stem cell properties and radioresistance of NSCLC cells by inhibiting PBK/ERK axis, therefore offering a potential therapeutic target for enhancing efficacy of radiotherapy.

Evaluation and Comparison of PBK and E2F7 Gene Expression Between Early and Advanced Stages of Colorectal Cancer

Background and Aim PBK is a mitogen-activated protein kinase (MAPKK) among MEK1/2 and MEK7 and can phosphorylate P38, JNK, and ERK in many cellular functions. The E2F transcription factor family also belongs to a class of cellular regulators acting as oncogenes and tumor suppressors. This study aims to investigate the expression of PBK and E2F7 in the early stages of colorectal cancer (CRC) compared to advanced stages based on the experimental and TCGA (The Cancer Genome Atlas) database. Methods & Materials A total of 32 tissue samples of patients with CRC with the approval of a pathobiologist were collected according to the examination and criteria reported from different stages. After RNA extraction and cDNA synthesis, the RT-qPCR technique was used to evaluate the expression of the desired genes in the study groups. A receiver operating characteristic (ROC) curve analysis was also used to determine the ability of each of the selected genes to differentiate the two populations: stage I+II and stage III+IV. Ethical Considerations In all stages of this research, codes of ethics of research and publication were observed. Results In this study, it was shown that the expression of PBK and E2F7 significantly increased in stage I+II samples compared to stage III+IV. These data were confirmed by laboratory results and information extracted from the TCGA database. Also, based on the area under curve obtained from the ROC curves, these two genes are significantly distinguishable between stage I+II and III+IV populations in CRC. Conclusion According to the results of this study, PBK and E2F7 genes are good markers in the diagnosis of CRC.

Down-regulation of PBK inhibits proliferation of human endometrial stromal cells in thin endometrium

BackgroundThin endometrium (TE) is a challenging clinical issue in the reproductive medicine characterized by inadequate endometrial thickness, poor response to estrogen and no effective treatments currently. At present, the precise pathogenesis of thin endometria remains to be elucidated. We aimed to explore the related molecular mechanism of TE by comparing the transcriptome profiles of late-proliferative phase endometria between TE and matched controls.MethodsWe performed a bulk RNA-Seq (RNA-sequencing) of endometrial tissues in the late-proliferative phase in 7 TE and 7 matched controls for the first time. Differential gene expression analysis, gene ontology enrichment analysis and protein-protein interactions (PPIs) network analysis were performed. Immunohistochemistry was used for molecular expression and localization in endometria. Human endometrial stromal cells (HESCs) were isolated and cultured for verifying the functions of hub gene.ResultsIntegrative data mining of our RNA-seq data in endometria revealed that most genes related to cell division and cell cycle were significantly inhibited, while inflammation activation, immune response and reactive oxygen species associated genes were upregulated in TE. PBK was identified as a hub of PPIs network, and its expression level was decreased by 2.43-fold in endometria of TE patients, particularly reduced in the stromal cells, which was paralleled by the decreased expression of Ki67. In vitro experiments showed that the depletion of PBK reduced the proliferation of HESCs by 50% and increased the apoptosis of HESCs by 1 time, meanwhile PBK expression was inhibited by oxidative stress (reduced by 76.2%), hypoxia (reduced by 51.9%) and inflammatory factors (reduced by approximately 50%). These results suggested that the insufficient expression of PBK was involved in the poor endometrial thickness in TE.ConclusionsThe endometrial transcriptome in late-proliferative phase showed suppressed cell proliferation in women with thin endometria and decreased expression of PBK in human endometrial stromal cells (HESCs), to which inflammation and reactive oxygen species contributed.

Prolactin-regulated Pbk is involved in pregnancy-induced β-cell proliferation in mice.

Gestational diabetes mellitus (GDM) is a condition of diabetes with onset or first recognition in pregnancy. Its incidence is increasing, and GDM deleteriously affects both mother and the fetus during and even after pregnancy. Previous studies in mice have shown that during pregnancy, β-cell proliferation increases in the middle and late stages of pregnancy and returns to normal levels after delivery. Hormones, such as prolactin, estradiol, and progesterone as well as protein kinases, play important roles in regulating gestation-mediated β-cell proliferation; however, the regulatory relationship between them is uncertain. We previously found that protein kinase Pbk was crucial for basal proliferation of mouse islet cells. Herein we show that Pbk is upregulated during pregnancy in mice and Pbk kinase activity is required for enhanced β- cell proliferation during pregnancy. Notably, knock-in (KI) of a kinase-inactivating Pbk mutation leads to impaired glucose tolerance and reduction of β-cell proliferation and islet mass in mice during pregnancy. Prolactin upregulates the expression of Pbk, but the upregulation is diminished by knockdown of the prolactin receptor and by the inhibitors of JAK and STAT5, which mediate prolactin receptor signaling, in β-cells. Treatment of β-cells with prolactin increases STAT5 binding to the Pbk locus, as well as the recruitment of RNA polymerase II, resulting in increased Pbk transcription. These results demonstrate that Pbk is upregulated during pregnancy, at least partly by prolactin-induced and STAT5-mediated enhancement of gene transcription, and Pbk is essential for pregnancy-induced β-cell proliferation, increase in islet mass, and maintenance of normal blood glucose during pregnancy in preclinical models. These findings provide new insights into the interplay between hormones and protein kinases that ultimately prevent the development of GDM.

High expression of PDZ-binding kinase is correlated with poor prognosis and immune infiltrates in hepatocellular carcinoma

BackgroundPDZ-binding kinase (PBK) encodes a serine/threonine protein kinase related to the dual specific mitogen-activated protein kinase kinase (MAPKK) family. There is evidence that overexpression of this gene is associated with tumorigenesis. However, the role of PBK in hepatocellular carcinoma (HCC) remains unclear. Therefore, we evaluated the prognostic role of PBK and its correlation with immune infiltrates in hepatocellular carcinoma.MethodsThe expression of PBK in pan-cancers was studied by Onconmine and TIMER. The expression of PBK in HCC patients and its relationship with clinicopathological characteristics were analyzed using The Gene Expression Profiling Interactive Analysis (GEPIA), The human protein atlas database (HPA), The Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO) databases. Receiver operating characteristic (ROC) curve was used to determine the diagnostic value of PBK in HCC patients. The relationship between PBK and prognosis of HCC was performed by GEPIA and Kaplan Meier plotter web tool. The correlations between the clinical characteristics and overall survival were analyzed by Univariate Cox regression and Multivariate Cox hazards regression to identify possible prognostic factors for HCC patients. LinkedOmics was applied to investigate co-expression associated with PBK and to analyze Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The network map of PBK and related genes is constructed by GeneMANIA. Finally, TIMER and TISIDB were used to analyze the correlations between PBK and tumor-infiltrating immune cells.ResultsMultiple database analysis shows that PBK was highly expressed in many types of tumors, including hepatocellular carcinoma, and was significantly related to tumor stage (P=0.0089), age (P=0.0131), and race (P=0.0024) of HCC patients. The receiver operating characteristic (ROC) curve analysis showed that PBK had high diagnostic potential to HCC in GSE76427 (AUC=0.8799), GSE121248 (AUC=0.9224), GSE62232 (AUC=0.9975), and GSE84402 (AUC=0.9541). Multivariate Cox hazards regression showed that high expression of PBK may be an independent risk factor for overall survival in HCC patients (HR = 1.566, 95% CI=1.062–2.311, P= 0.024). The Protein–protein interaction network showed that PBK significantly interacted with LRRC47, ARAF, LGALS9B, TTK, DLG1, and other essential genes. Furthermore, enrichment analysis showed that PBK and co-expressed genes participated in many biological processes, cell composition, molecular functions, and pathways in HCC. Finally, the immune infiltration analysis by TIMER and TISIDB indicated that a significant tightly correlation between PBK and macrophages, neutrophils, as well as chemokines and receptors.ConclusionsHigh expression of PBK is significantly correlated with poor survival and immune infiltrates in hepatocellular carcinoma. Our study suggests that PBK can be used as a biomarker of poor prognosis and potential immune therapy target in hepatocellular carcinoma.

Label propagation-based semi-supervised feature selection on decoding clinical phenotypes with RNA-seq data

BackgroundClinically, behavior, cognitive, and mental functions are affected during the neurodegenerative disease progression. To date, the molecular pathogenesis of these complex disease is still unclear. With the rapid development of sequencing technologies, it is possible to delicately decode the molecular mechanisms corresponding to different clinical phenotypes at the genome-wide transcriptomic level using computational methods. Our previous studies have shown that it is difficult to distinguish disease genes from non-disease genes. Therefore, to precisely explore the molecular pathogenesis under complex clinical phenotypes, it is better to identify biomarkers corresponding to different disease stages or clinical phenotypes. So, in this study, we designed a label propagation-based semi-supervised feature selection approach (LPFS) to prioritize disease-associated genes corresponding to different disease stages or clinical phenotypes.MethodsIn this study, we pioneering put label propagation clustering and feature selection into one framework and proposed label propagation-based semi-supervised feature selection approach. LPFS prioritizes disease genes related to different disease stages or phenotypes through the alternative iteration of label propagation clustering based on sample network and feature selection with gene expression profiles. Then the GO and KEGG pathway enrichment analysis were carried as well as the gene functional analysis to explore molecular mechanisms of specific disease phenotypes, thus to decode the changes in individual behavioral and mental characteristics during neurodegenerative disease progression.ResultsLarge amounts of experiments were conducted to verify the performance of LPFS with Huntington’s gene expression data. Experimental results shown that LPFS performs better in comparison with the-state-of-art methods. GO and KEGG enrichment analysis of key gene sets shown that TGF-beta signaling pathway, cytokine-cytokine receptor interaction, immune response, and inflammatory response were gradually affected during the Huntington’s disease progression. In addition, we found that the expression of SLC4A11, ZFP474, AMBP, TOP2A, PBK, CCDC33, APSL, DLGAP5, and Al662270 changed seriously by the development of the disease.ConclusionsIn this study, we designed a label propagation-based semi-supervised feature selection model to precisely selected key genes of different disease phenotypes. We conducted experiments using the model with Huntington’s disease mice gene expression data to decode the mechanisms of it. We found many cell types, including astrocyte, microglia, and GABAergic neuron, could be involved in the pathological process.

Menin‐regulated Pbk controls high fat diet‐induced compensatory beta cell proliferation

Pancreatic beta cells undergo compensatory proliferation in the early phase of type 2 diabetes. While pathways such as FoxM1 are involved in regulating compensatory beta cell proliferation, given the lack of therapeutics effectively targeting beta cell proliferation, other targetable pathways need to be identified. Herein, we show that Pbk, a serine/threonine protein kinase, is essential for high fat diet (HFD)‐induced beta cell proliferation in vivo using a Pbk kinase deficiency knock‐in mouse model. Mechanistically, JunD recruits menin and HDAC3 complex to the Pbk promoter to reduce histone H3 acetylation, leading to epigenetic repression of Pbk expression. Moreover, menin inhibitor (MI) disrupts the menin–JunD interaction and augments Pbk transcription. Importantly, MI administration increases beta cell proliferation, ameliorating hyperglycemia, and impaired glucose tolerance (IGT) in HFD‐induced diabetic mice. Notably, Pbk is required for the MI‐induced beta cell proliferation and improvement of IGT. Together, these results demonstrate the repressive role of the menin/JunD/Pbk axis in regulating HFD‐induced compensatory beta cell proliferation and pharmacologically regulating this axis may serve as a novel strategy for type 2 diabetes therapy.

Six Novel Biomarkers for Diagnosis and Prognosis of Esophageal squamous cell carcinoma: validated by scRNA-seq and qPCR.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. ESCC has a generally poor prognosis and there is a lack of available biomarkers for diagnosis and prognosis. The aim of the study was to identify novel biomarkers for ESCC. We screened the overlapping differentially expressed genes (DEGs) acquired from six Gene Expression Omnibus (GEO) ESCC datasets and The Cancer Genome Atlas (TCGA) ESCC datasets. Subsequently, protein-protein interaction network analysis was performed to identify the key hub genes. Then, Kaplan Meier survival and receiver operating curve (ROC) analysis were utilized to clarify the diagnostic and prognostic role of these hub genes. The UALCAN database, single cell RNA sequencing (scRNA-seq) and real-time quantitative PCR (qPCR) were performed to confirm the expression levels of identified hub genes. Finally, immune infiltration analysis was conducted to investigate the role of these genes in the pathogenesis of ESCC. The results showed that PBK, KIF2C, NUF2, KIF20A, RAD51AP1, and DEPDC1 effectively distinguish ESCC tissues from normal samples, and all of them were significantly correlated with overall survival. The results of scRNA-seq and qPCR indicated that the expression levels of hub genes in ESCC were significantly higher than in normal cells or tissues. Further immune infiltration analysis showed that infiltration of dendritic cells was significantly negatively correlated with PBK, KIF2C, NUF2, RAD51AP1, and DEPDC1 expression levels. In conclusion, our results suggest that PBK, KIF2C, NUF2, KIF20A, RAD51AP1 and DEPDC1 are all potential biomarkers for ESCC diagnosis and prognosis may also be potential therapeutic targets for ESCC.

Abstract 2940: 4-Hydroxytamoxifen enhances sensitivity of estrogen receptor α-positive breast cancer to docetaxel in an estrogen and ZNF423 SNP-dependent fashion

Abstract Background: In early stage, ERα-positive breast cancer, the concurrent use of endocrine therapy and chemotherapy has not been shown to be superior to sequential use. We hypothesized that genetic biomarkers can aid in selecting patients who would benefit from chemo-endocrine therapy. Our previous genome-wide association study (GWAS) and functional studies revealed that a single nucleotide polymorphism (SNP) in ZNF423, rs9940645, determines tamoxifen response. Specifically, 4-hydroxytamoxifen (4-OH-TAM) increases the gene expression of ZNF423, a transcription factor for BRCA1, in cells that are carry the rs9940645 variant, but not the wildtype, genotype. In this study, we set out to identify additional genes regulated by ZNF423, which, upon identifying a relationship between ZNF423 and cell cycle genes, led us to investigate taxane response in a SNP- and tamoxifen-dependent fashion. Method: Gene correlation analysis using The Cancer Genome Atlas (TCGA) breast cancer dataset was used to identify genes with expression correlated with that of ZNF423. Quantitative reverse transcription PCR, chromatin immunoprecipitation, and luciferase reporter assays were used to validate the gene associations. To test ZNF423 rs9940645-specific phenotypes, we used ZR75-1 cells, which are homozygous for the variant SNP, and ZR75-1 cells that were generated to be homozygous wild type (WT) using CRISPR-Cas9 technology, in addition to Hs578T breast cells with ERα overexpression (WT) and HCC1500 cells (variant). Cell cycle was assessed by propidium iodide staining. Cytotoxicity was assessed using a colorimetric assay. Results: Mitosis-related genes VRK1 and PBK, which encode histone H3 kinases, were experimentally validated to be regulated by ZNF423. Specifically, ZNF423 knockdown resulted in decreased VRK1 and PBK expression and activity. Additionally, ZNF423 knockdown resulted in enhanced docetaxel-induced G2/M arrest and cytotoxicity which could be rescued by VRK1 or PBK overexpression. Lastly, breast cancer cells carrying the rs9940645 variant SNP genotype had increased G2-M arrest and decreased cell viability when treated with docetaxel in combination with estradiol and 4-OH-TAM. Conclusion: We identified ZNF423regulated genes involved in the G2/M phase of the cell cycle. We found that 4-OH-TAM sensitized ERα-positive breast cancer cells to docetaxel treatment in a ZNF423 SNP-dependent manner. Our findings suggest that patients with rs9940645 variant genotype may benefit from concurrent tamoxifen endocrine therapy and docetaxel chemotherapy. This would impact a substantial proportion of patients given the minor allele frequency of 0.47 for this SNP. Citation Format: Gen Wang, Sisi QIN, Jacqueline Zayas, James N. Ingle, Mohan Liu, Richard Weinshilboum, Kunwei Shen, Liewei Wang. 4-Hydroxytamoxifen enhances sensitivity of estrogen receptor α-positive breast cancer to docetaxel in an estrogen and ZNF423 SNP-dependent fashion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2940.

4-Hydroxytamoxifen enhances sensitivity of estrogen receptor α-positive breast cancer to docetaxel in an estrogen and ZNF423 SNP-dependent fashion.

In early stage, ERα-positive breast cancer, concurrent use of endocrine therapy and chemotherapy has not been shown to be superior to sequential use. We hypothesized that genetic biomarkers can aid in selecting patients who would benefit from chemo-endocrine therapy. Our previous studies revealed that ZNF423 is a transcription factor for BRCA1 and an intronic single nucleotide polymorphism (SNP) in ZNF423, rs9940645, determines tamoxifen response. Here, we identified mitosis-related genes that are regulated by ZNF423 which led us to investigate taxane response in a rs9940645 SNP- and tamoxifen-dependent fashion. The Cancer Genome Atlas (TCGA) breast cancer dataset was used to identify genes correlated with ZNF423. Quantitative reverse transcription PCR, chromatin immunoprecipitation, and luciferase reporter assays were used to validate the gene regulation. We used CRISPR/Cas9 to engineer paired ZR-75-1 cells which differ only in ZNF423 rs9940645 SNP genotype to test SNP-dependent phenotypes including cell cycle and cell viability. We validated our findings in an additional two breast cancer cell lines, Hs578T-ERα and HCC1500. Mitosis-related genes VRK1 and PBK, which encode histone H3 kinases, were experimentally validated to be regulated by ZNF423. ZNF423 knockdown decreased VRK1 and PBK expression and activity. Additionally, ZNF423 knockdown enhanced docetaxel-induced G2/M arrest and cytotoxicity through VRK1 or PBK regulation. Lastly, cells carrying the rs9940645 variant genotype had increased G2/M arrest and decreased cell viability when treated with docetaxel in combination with estradiol and 4-OH-TAM. We identified ZNF423 regulated genes involved in the G2/M phase of the cell cycle. 4-OH-TAM sensitized ERα-positive breast cancer cells to docetaxel in a ZNF423 SNP-dependent manner. Our findings suggest that patients with rs9940645 variant genotype may benefit from concurrent tamoxifen and docetaxel. This would impact a substantial proportion of patients because this SNP has a minor allele frequency of 0.47.

MicroRNA-372 enhances radiosensitivity while inhibiting cell invasion and metastasis in nasopharyngeal carcinoma through activating the PBK-dependent p53 signaling pathway.

Nasopharyngeal carcinoma (NPC) is a common cancer found in the nasopharynx, which plagues countless NPC patients. MicroRNA‐372 (miR‐372) has been reported to be involved in various tumors. Here, we explored the important role of miR‐372 in radiosensitivity, invasion, and metastasis of NPC. Microarray analysis was conducted to search the NPC‐related differentially expressed genes (DEGs) and predict the miRs regulating PBK, which suggested that miR‐372 could influence the development of NPC via PBK and the p53 signaling pathway. Importantly, miR‐372 was observed to target PBK, thus down‐regulating its expression. Then, NPC 5‐8F and C666‐1 cells were selected, and treated with ionization radiation and alteration of miR‐372 and PBK expression to explore the functional role of miR‐372 in NPC. The expression of miR‐372, PBK, Bcl‐2, p53, and Bax as well as the extent of Akt phosphorylation were measured. In addition, cell colony formation, cell cycle, proliferation, apoptosis, migration, and invasion were detected. At last, tumor growth and the effect of miR‐372 on radiosensitivity of NPC were evaluated. Besides, over‐expressed miR‐372 down‐regulated Bcl‐2 and PBK expression and the extent of Akt phosphorylation while up‐regulated the expression of p53 and Bax. Additionally, miR‐372 over‐expression and radiotherapy inhibited cell clone formation, proliferation, tumor growth, migration, invasion, and cell cycle entry, but promoted cell apoptosis. However, the restoration of PBK in NPC cells expressing miR‐372 reversed the anti‐tumor effect of miR‐372 and activation of the p53 signaling pathway. In conclusion, the study shows that up‐regulated miR‐372 promotes radiosensitivity by activating the p53 signaling pathway via inhibition of PBK.

Comprehensive analysis of tumor immune infiltration associated with endogenous competitive RNA networks in lung adenocarcinoma

Cancer immunotherapy has achieved unprecedented success in the treatment of cancer. However, different patients have different responses to immunotherapy. More and more studies have shown that tumor immune heterogeneity has an important influence on the prognosis of cancer. Therefore, understanding the clinical impact of tumor immune infiltration and the regulatory mechanism of RNA molecules is crucial for exploring the pathogenesis of lung adenocarcinoma (LUAD) and the development of immunotherapy protocols.The endogenous competitive RNA hypothesis provides new ideas for studying immune heterogeneity. Therefore, by using the method of immune genomics, this article explores the relationship between immune infiltration and prognosis of patients with lung adenocarcinoma, and found that B-cell immune infiltration highly affects the survival of patients. Through differential analysis, differential mRNAs, lncRNAs and miRNAs were extracted, and 318 differentially expressed mRNAs related to B cell immunity were screened by correlation analysis, and prognosis of patients with COX risk regression model was predicted and analyzed. Through multiple database searches, an immune-related ceRNA regulatory network was constructed, containing 3 key mRNAs, 4 miRNAs, and 50 lncRNAs. Three mRNAs and most miRNAs, lncRNAs, are significantly associated with LUAD prognosis. Bioinformatics analysis of the network showed that LINC00337 may up-regulate the expression of PBK and KIF23 through competitive binding of has-mir-373 and has-mir-519d. The competitive binding of has-mir-373 and has-mir-372 can up-regulate the expression of SLC7A11. The interaction between these RNAs may have an important regulatory role in the immune infiltration in lung adenocarcinoma, thereby affecting the patient's prognosis and immunotherapy efficacy.

Enhanced Expression of the Key Mitosis Regulator Cyclin B1 Is Mediated by PDZ-Binding Kinase in Islets of Pregnant Mice.

The proliferation of pancreatic β cells is enhanced to enable an increase in β-cell mass and to compensate for insulin resistance during pregnancy. To elucidate the mechanisms involved, we previously investigated islets from pregnant and nonpregnant mice by gene expression profiling and found that the expression of postsynaptic density-95/Discs large/zonula occludens-1 (PDZ)–binding kinase (Pbk), a member of the mitogen-activated protein kinase kinase family, is increased in pregnant mouse islets compared with control mouse islets. Among the pregnancy hormones, treatment with estradiol upregulated Pbk expression. Inhibition of Pbk expression using a small interfering RNA for Pbk reduced bromodeoxyuridine incorporation in mouse insulinoma 6 cells, which was accompanied by a decreased expression of Ccnb1, a regulatory gene involved in mitosis. Ccnb1 expression was augmented in mouse islets during pregnancy. The forced expression of Pbk using an adenovirus system in isolated mouse islets increased Ccnb1 expression, and the Pbk inhibitor HI-TOPK-032 suppressed Ccnb1 expression in islets isolated from pregnant mice. Our results suggest that Pbk contributes to the expansion of islets during pregnancy and that Ccnb1 may assist Pbk in its role in β-cell proliferation.

MiR-216b promotes cell growth and enhances chemosensitivity of colorectal cancer by suppressing PDZ-binding kinase

PDZ-binding kinase (PBK/TOPK) acts as oncogene in various cancers and correlates with drug response. However, few studies have examined the expression and roles of PBK in colonrectal cancer (CRC). In this study, we found a significant increase in the expression of PBK in CRC tissues and cell lines. While overexpression of PBK promoted cell growth and decreased the toxicity effect of oxaliplation (OXA), targeting PBK with short hairpin RNA (shRNA) or novel PBK inhibitor HI-TOPK-032 effectively suppressed tumor growth and potentiated chemosensitivity in vitro and in vivo. Furthermore, there was a significant inverse correlation between the expressions of miR-216b and PBK. Further found that miR-216b could down-regulate PBK levels by binding to the 3′ untranslated region (3′UTR) of PBK. Notably, while miR-216b decreased cell proliferation and enhanced sensitivity of CRC cells to oxaliplation, re-expression of PBK dramatically reversed these events. Collectively, our data indicated that miR-216b may function as a tumor suppressor though regulating PBK expression, which provided promising targets and possible therapeutic strategies for CRC treatment.

PSMB8 and PBK as potential gastric cancer subtype-specific biomarkers associated with prognosis.

Gastric adenocarcinoma is a common form of cancer associated with a poor prognosis. We analyzed microarray profiling data from 48 patients with gastric adenocarcinoma to characterize gastric cancer subtypes and identify biomarkers associated with prognosis. We identified two major subtypes of gastric adenocarcinoma differentially associated with overall survival (P = 0.025). Genes that were differentially expressed were identified using specific criteria (P < 0.001 and >1.5-fold); expression of 294 and 116 genes was enriched in good and poor prognosis subtypes, respectively. Genes related to translational elongation and cell cycle were upregulated in the poor prognosis group. Of these genes, upregulation of proteasome subunit beta type 8 PSMB8 and PDZ binding kinase PBK was confirmed by real-time reverse transcription-PCR analysis. PSMB8 or PBK knockdown had no effect on gastric cancer cell proliferation but suppressed cell migration and invasion, respectively. Furthermore, immunohistochemistry analysis of 385 gastric cancer patients revealed that increased nuclear expression of PSMB8 and PBK was correlated with depth of invasion, lymph node metastasis, and lower survival rates. Taken together, two gastric adenocarcinoma subtypes were predictive of prognosis. PSMB8 and PBK were predictive of gastric cancer prognosis and could be potential gastric cancer subtype-specific biomarkers.

C-Myc and E2F1 drive PBK/TOPK expression in high-grade malignant lymphomas

c-Myc has been implicated in a variety of hematologic malignancies including Burkitt's lymphoma. Targeting c-Myc driven growth pathways could be therapeutically useful but might require the identification of critical downstream proteins. Here we show that the serine–threonine kinase PBK/TOPK is frequently overexpressed in high-grade lymphomas and its expression is positively correlated to that of c-Myc and E2F1. Further we demonstrate that c-Myc regulates PBK expression through E2F1. Additionally, inhibition of c-Myc, E2F1 or PBK comparably decreased cell growth and survival. In conclusion, a c-Myc–E2F1–PBK signaling pathway operates in high-grade lymphomas and may provide a useful target for novel antineoplastic therapeutics.

PDZ-Binding Kinase (PBK/TOPK), a Serine-Threonine Kinase Known to downmodulate P53 Function, is Frequently Overexpressed In a Variety of Hematological Malignancies

AbstractAbstract 5110 Background:PBK/TOPK (PDZ-binding kinase, T-LAK-cell-originated protein kinase) is a serine-threonine kinase that was originally cloned as an IL-2 inducible gene exclusively expressed in cytotoxic T lymphocytes, and an interaction partner with the human tumor suppressor hdlg. PBK/TOPK mRNA has been found to be expressed in normal testicular and embryonic tissues, proliferating neural progenitor cells and various tumor cell lines. Numerous studies have demonstrated that PBK exerts its proproliferation function by phophorylating and thus activating its substrate proteins p38, ERK2, LGN/GPSM2, and histone H3, which are involved in cell growth, cell cycle progression and chromatin remodeling. Recently, we further reported that PBK could interact with tumor suppressor p53 via its DNA binding domain and impair its function as a transcription factor (Oncogene, 2010). These lines of evidence all point to PBK as a novel multifunctional oncoprotein. Study Aim:To investigate the frequency of PBK expression in hematological malignancies. Methods:Immunohistochemistry for PBK expression on various tissue microarrays (US Biomax) was performed. Briefly, paraffin-embedded tissue microarrays were treated with xylene and ethanol followed by a protein-blocking reagent. After incubation with 1:50 diluted PBK antibody (Cell Signaling) and HRP-labeled-secondary antibody (US Biomax), the tissue microarrays were stained with substrate DAB. The slides were scored according to the following criteria: 0 (negative), <25% cells were stained; +1, 25–50% cells were stained; +2, 51–75% cells were stained; +3, >75% cells were stained. Results:In 108 cases of diffuse large B-cell lymphoma, 50% were positive for PBK, among which 27 scored +1, 21 scored +2, and 6 scored +3; in 8 cases of Burkitt lymphoma, 7/8 (87.5%) were positive for PBK with all of them scoring +3; in 58 cases of Hodgkin lymphoma, 67% were positive for PBK with 35 scoring +1 and only 4 scoring +2; in 9 cases of plasma cell myeloma, only 2 were positive for PBK and both scored +1. Three follicular lymphomas and 4 reactive lymph nodes scored 0. Conclusions and Future Directions:PBK protein was highly expressed in a high proportion of Burkitt lymphoma. PBK was also expressed in a relatively high proportion of diffuse large B-cell lymphoma and Hodgkin lymphoma, suggesting that PBK is implicated in the pathogenesis of hematological malignancies with higher proliferation rates. PBK expression was much less frequent in more indolent hematologic neoplasms such as myeloma and follicular lymphoma. Our lab is now working on the crystal structure of PBK with the long-term goal to identify small molecular inhibitors of PBK function. These new results in combination with our earlier data suggest that inhibition of PBK function may be a rational strategy to sensitize aggressive/highly proliferative hematologic neoplasms to anti-neoplastic therapy. Disclosures:No relevant conflicts of interest to declare.

Attenuation of DNA damage checkpoint by PBK, a novel mitotic kinase, involves protein–protein interaction with tumor suppressor p53

Pathways adopted by developing cancer cells for evasion of cellular surveillance mechanism deserve attention for therapeutic exploitation as well as for better prognosis. A novel mitotic kinase, PDZ-binding kinase or PBK, which is upregulated in a variety of neoplasms including hematological malignancies, has been the focus of our attention with a goal to understand its role in malignant conversion and to examine as a possible new therapeutic target in disparate types of cancer. Earlier, we reported that PBK expression was downregulated during macrophage differentiation of HL60 promyelocytic leukemia cells, during doxorubicin-induced growth arrest in G2/M phase and that PBK was regulated by cell cycle-specific transcription factors E2F and CREB/ATF. Here, we demonstrate that HT1080 fibrosarcoma cells become adapted to doxorubicin-induced DNA damage checkpoint upon ectopic expression of a phosphomimetic mutant of PBK as indicated by the accumulation of polyploid cells. Aberrant entry into the mitotic phase by these cells is suggested by the appearance of a mitotic phase-specific marker, MPM-2. We propose that the effect is due to downregulation of p53 caused by direct physical interaction with PBK as detected by both a biochemical means as well as by yeast two-hybrid analysis. Together, our studies provide a plausible explanation for the role of PBK augmenting tumor cell growth following transient appearance in different types of progenitor cells in vivo as reported.