PDZ-binding Kinase Research Articles

PDZ-Binding Kinase, a Novel Regulator of Vascular Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.

Abstract 1221: Selective translational activation of PBK (PDZ- binding kinase) reveals a molecular vulnerability of drug-tolerant persister cells to targeted therapies in EGFR-mutant lung adenocarcinoma

Abstract In lung adenocarcinoma (LUAD), EGFR-targeted therapies often result in incomplete tumor responses followed by disease progression caused by acquired resistance. As EGFR mutations are truncal (i.e., present in every cell of a tumor), it remains unclear why a fraction of tumor cells can survive in the presence of drug treatment. It is increasingly recognized that drug-tolerant persister cells (DTPCs) can escape selective drug pressure and are important drivers of therapy failure and tumor relapse. The mechanisms underlying tumor cell adaptation and survival of DTPCs during therapy are not fully understood.Using EGFR-mutant LUAD cell lines treated with osimertinib, we unexpectedly observed that DTPCs display uncoupled mRNA and protein variations of PBK (PDZ Binding Kinase), a spindle assembly serine/threonine protein kinase. Mechanistically we discovered that although global protein synthesis is decreased upon osimertinib treatment, PBK translation is selectively maintained in DTPCs.The establishment and survival of DTPCs following EGFR inhibition is critically dependent on PBK, as genetic depletion of PBK reduces persistence to osimertinib treatment in vitro. PBK knockout leads to suppression of ERK1/2 reactivation following EGFR inhibition, and results in decreased expression of ZEB1 and Vimentin, key effectors of epithelial-to-mesenchymal transition (EMT), a hallmark of the DTPC phenotype. Consistently, combined treatment with osimertinib and OTS514 (PBK inhibitor) in vitro, reduces the DTPC fraction and decreases the enrichment of EMT-related transcriptional signatures. Similar results were obtained in LUAD patient-derived organoids, used to recapitulate the phenotype of residual disease in patients. In PDX treated with osimertinib, PBK is activated at both residual disease and acquired resistance. Concordantly, combined EGFR/PBK pharmacological inhibition was also effective in reverting osimertinib acquired resistance in vitro. Finally, in matched diagnosis and relapse samples from 7 EGFR-mutant LUAD patients treated with EGFR inhibitors, we observed increased PBK activation at the time of acquired resistance in 5 cases, including 3 EGFRT790M-positive cases, suggesting that PBK activation may co-occur with genetic resistant alterations.Our findings unveil a novel mechanism by which EGFR-mutant lung cancer DTPCs selectively translate PBK mRNA as an adaptive survival mechanism to persist during osimertinib treatment; and provide a therapeutic target of non-genetic drug resistance otherwise unlooked-for in routine transcriptomic analyses. A deeper understanding on the mRNA translational reprogramming in DTPCs is currently performed to uncover therapeutic vulnerabilities to reduce residual disease and delay or revert the emergence of acquired resistance. Citation Format: Camille Leonce, Anne-Laure Désage, Nour El-Houda Mourksi, Geneviève De Souza, Béatrice Vanbervliet, Camille Charrondière, Constance Nicq, Jérémy Néri, Aurélie Swalduz, Laurie Tonon, Eric Cumunel, Nicolas Gadot, Gabriel Ichim, Jean-Jacques Diaz, Christine Lovly, Julien Mazières, Mayeul Tabutin, Maurice Pérol, Sylvie Lantuejoul, Olivier Calvayrac, Fabien Forest, Virginie Marcel, Pierre Saintigny, Sandra Ortiz-Cuaran. Selective translational activation of PBK (PDZ- binding kinase) reveals a molecular vulnerability of drug-tolerant persister cells to targeted therapies in EGFR-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1221.

Small-molecule inhibitor HI-TOPK-032 improves NK-92MI cell infiltration into ovarian tumours.

The effectiveness of natural killer (NK) cells transferred adoptively in combating solid tumours is limited by challenges such as their difficulty in penetrating tumours from the bloodstream and maintaining viability without the support of interleukin-2 (IL-2). Genetically modified NK-92MIcells, which can release IL-2 to sustain their viability, have been identified as a promising alternative. This adaptation addresses the negative consequences of systemic IL-2 administration. The role of PSD-95/discs large/ZO-1 (PDZ)-binding kinase (PBK) in cancer development is recognized, but its effects on immunity are not fully understood. This study explores how PBK expression influences the ability of NK-92MI cells to infiltrate ovarian tumours. Elevated levels of PBK expression have been found in various cancers, including ovarian cancer (OV), with analyses showing higher PBK mRNA levels in tumour tissues compared to normal ones. Immunohistochemistry has confirmed increased PBK expression in OV tissues. Investigations into PBK's role in immune regulation reveal its association with immune cell infiltration, indicating a potentially compromised immune environment in OV with high PBK expression. The small-molecule inhibitor HI-TOPK-032, which inhibits PBK, enhances the cytotoxicity of NK-92MI cells toward OV cells. It increases the production of interferon-γ and tumour necrosis factor-α, reduces apoptosis and encourages cell proliferation. Mechanistic studies showed that contact with OV cells treated with HI-TOPK-032 upregulates CD107a on NK-92 cells. In vivo studies demonstrated that HI-TOPK-032 improves the antitumour effects of NK-92MI cells in OVCAR3Luc xenografts, extending survival without significant side effects. Safety assessments in mice confirm HI-TOPK-032's favourable safety profile, highlighting its potential as a viable antitumour therapy. These results suggest that combining NK-92MI cells with HI-TOPK-032 enhances antitumour effectiveness against OV, indicating a promising, safe and effective treatment strategy that warrants further clinical investigation.

FHND004 inhibits malignant proliferation of multiple myeloma by targeting PDZ-binding kinase in MAPK pathway.

Inhibitors of Epidermal growth factor receptor tyrosine kinase (EGFR-TKIs) are producing impressive benefits to responsive types of cancers but challenged with drug resistances. FHND drugs are newly modified small molecule inhibitors based on the third-generation EGFR-TKI AZD9291 (Osimertinib) that are mainly for targeting the mutant-selective EGFR, particularly for the non-small cell lung cancer (NSCLC). Successful applications of EGFR-TKIs to other cancers are less certain, thus the present pre-clinical study aims to explore the anticancer effect and downstream targets of FHND in multiple myeloma (MM), which is an incurable hematological malignancy and reported to be insensitive to first/second generation EGFR-TKIs (Gefitinib/Afatinib). Cell-based assays revealed that FHND004 and FHND008 significantly inhibited MM cell proliferation and promoted apoptosis. The RNA-seq identified the involvement of the MAPK signaling pathway. The protein chip screened PDZ-binding kinase (PBK) as a potential drug target. The interaction between PBK and FHND004 was verified by molecular docking and microscale thermophoresis (MST) assay with site mutation (N124/D125). Moreover, the public clinical datasets showed high expression of PBK was associated with poor clinical outcomes. PBK overexpression evidently promoted the proliferation of two MM cell lines, whereas the FHND004 treatment significantly inhibited survival of 5TMM3VT cell-derived model mice and growth of patient-derived xenograft (PDX) tumors. The mechanistic study showed that FHND004 downregulated PBK expression, thus mediating ERK1/2 phosphorylation in the MAPK pathway. Our study not only demonstrates PBK as a promising novel target of FHND004 to inhibit MM cell proliferation, but also expands the EGFR kinase-independent direction for developing anti-myeloma therapy.

The role and mechanism of PDZ binding kinase in hypobaric and hypoxic acute lung injury.

Acute lung injury (ALI) caused by hypobaric hypoxia (HH) is frequently observed in high-altitude areas, and it is one of the leading causes of death in high-altitude-related diseases due to its rapid onset and progression. However, the pathogenesis of HH-related ALI (HHALI) remains unclear, and effective treatment approaches are currently lacking. A new mouse model of HHALI developed by our laboratory was used as the study subject (Chinese patent No. ZL 2021 1 1517241 X). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the messenger RNA (mRNA) expression levels of PDZ-binding kinase (PBK), sirtuin 1 (SIRT1), and PTEN-induced kinase 1 (PINK1) in mouse lung tissue. Hematoxylin and eosin staining was used to observe the main types of damage and damaged cells in lung tissue, and the lung injury score was used for quantification. The wet-dry (W/D) ratio was used to measure lung water content. Enzyme-linked immunosorbent assay was used to detect changes in inflammatory factors and oxidative stress markers in the lungs. Western blotting verified the expression of various mitochondrial autophagy-related proteins. The 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1) method was used determined the health status of mitochondria based on changes in mitochondrial membrane potential. Transmission electron microscopy was used to directly observe the morphology of mitochondria. Multicolor immunofluorescence was used to observe the levels of mitochondrial autophagy markers. Other signaling pathways and molecular mechanisms that may play a role in epithelial cells were analyzed via through RNA sequencing. Low pressure and hypoxia caused pathological changes in mouse lung tissue, mainly ALI, leading to increased levels of inflammatory factors and intensified oxidative stress response in the lungs. Overexpression of PBK was found to alleviate HHALI, and activation of the p53 protein was shown to abrogate this therapeutic effect, while activation of SIRT1 protein reactivated this therapeutic effect. The therapeutic effect of PBK on HHALI is achieved via the activation of mitochondrial autophagy. Finally, RNA sequencing demonstrated that besides mitochondrial autophagy, PBK also exerts other functions in HHALI. Overexpression of PBK inhibits the expression of p53 and activates SIRT1-PINK1 axis mediated mitochondrial autophagy to alleviate HHALI.

Multi-Level Control of Myeloma Cell Proliferation and Genomic Instability By the PDZ Binding Kinase (PBK)

Genomic instability and cell proliferation play important roles in the development and progression of cancer. We identified a six-gene kinase signature that correlated with overall genomic instability in multiple human cancers including multiple myeloma (MM). PDZ Binding Kinase (PBK) was one of the top kinases in this signature. Inhibiting PBK downregulated E2F and FOXM1-related pathways and impacted several members of the DREAM complex, which represses cell cycle genes during quiescence. We further evaluated whether PBK affects entry into mitosis by controlling FOXM1 phosphorylation and affects genome stability. Using co-immunoprecipitation and chromatin immunoprecipitation assays, we observed that PBK binds to and phosphorylates FOXM1, leading to upregulation of FOXM1 target genes. Overexpression of PBK increased the association of LIN54 (a major component of the MuvB complex) with phosphorylated-FOXM1 and inhibited LIN54 interaction with E2F4 in the DREAM complex. Consistently, PBK knockdown or inhibitor increased the LIN54-E2F4 interaction and inhibited the LIN54 and phosphorylated-FOXM1 interaction. The inhibitor also impaired tumor growth in vitro and significantly increased the efficacy of a chemotherapeutic agent ( P = 0.0011) in a subcutaneous mouse model of MM. Overall, these data indicate that PBK phosphorylates FOXM1, then FOXM1 interacts with MuvB which is associated with the start of mitosis, dissociating it from E2F4 in the DREAM complex, thus allowing entry into mitosis and subsequent tumor growth. Given that phosphorylation of FOXM1 also activates the expression of major DNA repair genes (including RAD51, EXO1), we investigated whether PBK affects genome stability. Surprisingly, we observed that PBK also directly phosphorylates DNA repair/HR genes, such as flap structure-specific nuclease 1 (FEN1), and that PBK inhibition reduces spontaneous and chemotherapy (melphalan)-induced genomic instability ( P < 0.05). A significant upregulation of PBK has also been observed in MM patient samples at relapse (after melphalan treatment) relative to paired samples collected at diagnosis (GSE19554). To further investigate the consequences of elevated PBK in vivo, we transformed the normalized PBK expression into a Z-score and used this to divide patients (in MMRF-COMPASS) into three groups based on PBK expression (i.e., high, medium and low PBK expression groups). Patients with high PBK expression had poor overall survival as compared to those with medium and low PBK expression ( P=1.127e−13). DNA replication, mismatch repair, homologous recombination, cell cycle and Fanconi anemia were the top upregulated pathways in patients with high PBK expression. Known drivers of DNA replication, including those involved in the initiation of once-per-cell cycle replication and several DNA repair and HR genes, which are part of different genomic instability signatures (such as RAD51, RAD51C, RAD54B, RAD54L, EXO1, BRCA1, FEN1, ATAD2, CENPA, MAD2L1, TTK), were significantly overexpressed in patients with high PBK expression. Interestingly, FOXM1, which can impact both replication and genome stability was also overexpressed (Log2 Fold change of 1.8; P adj = 1.27E-86). In summary, we demonstrate that PBK drives DNA replication and genomic instability in MM cells by acting at multiple levels. Therefore, PBK inhibitors investigated here have the potential to impair growth and increase the efficacy of chemotherapeutic agents while reducing spontaneous as well as chemotherapeutic agent-induced genomic instability in MM.

PBK correlates with prognosis, immune escape and drug response in LUAD

PBK (PDZ-binding kinase) is a protein-coding gene that encodes a serine/threonine protein kinase associated with the dual-specific mitogen-activated protein kinase (MAPKK) family. Overexpression of this gene is closely linked to tumor development. In this study, we aimed to investigate the role of PBK in lung adenocarcinoma (LUAD) progression, prognosis, and immune evasion. We conducted a pan-cancer analysis of PBK to examine its expression and prognostic value. In the LUAD cohort, we analyzed PBK expression, prognosis, mutational features, and immune infiltration in groups with different PBK expression levels. We constructed a PBK-associated genomic model, integrated it into a nomogram, and compared high and low-risk subgroups. In our pan-cancer analysis, PBK was significantly upregulated, particularly in LUAD patients, and displayed poor prognosis. The high PBK expression group had many deletion mutations but still showed gene upregulation. Immune infiltration analysis indicated that PBK-triggered immune escape in the high expression group might relate to antigen presentation, dendritic cell, and CD8+ T cell infiltration. We constructed a 5-gene prognostic model and a nomogram to quantify individual survival probabilities. The PBK-associated gene prognostic model reliably predicted patient prognosis and drug response. Our findings offer new insights into PBK-induced immune escape and targeted therapy during LUAD development, providing valuable suggestions for clinical treatment approaches.

Abstract PO-061: Utilizing T cell receptor-based therapy to treat anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is considered to be among the most aggressive solid tumors in thyroid tissue, with a 6-month medium overall survival. The effectiveness of conventional treatments, such as surgery, chemotherapy, and radiation therapy, in extending the survival of patients is limited. Therefore, there is an urgent need to develop novel systemic therapeutic interventions that can induce long-lasting responses. Adoptive cell therapy has revolutionized cancer immunotherapy by successfully treating B-cell malignancies through chimeric antigen receptor-based approaches. For solid tumors, there is a need to identify a broader spectrum of tumor target antigens, and the field has now shifted towards leveraging T cell receptors (TCRs) which can recognize both intracellular and extracellular antigen-derived epitopes displayed by major histocompatibility complex (MHC) molecules. Our team has established an immunogenic epitope discovery pipeline, followed by a specialized protocol for empiric validation of candidate epitopes. We characterized immunogenic epitopes by coupling tandem mass spectrometry of MHC-eluted peptides with tumor proteome and MHC-peptide binding prediction databases. By integrating multi-omics data (genomics, transcriptomics, and immunopeptidomics) from both in-house and publicly available ATC databases, we find that ATC exhibits significant upregulation of pathways associated with microtubule-based movement, chromosome segregation, kinetochore complex components, and mitotic cytokinesis. We have identified several HLA-A2- and A24-restricted immunogenic peptides representing these pathway-associated proteins that covered more than 60% of the patient population. NDC80 kinetochore complex components (NDC80) and PDZ binding kinase (PBK) have been selected as top candidates. Peptides derived from NDC80 and PBK were subsequently utilized to isolate antigen-specific T cells from peripheral blood and identify cognate TCR. These peptide-specific T lymphocytes elicit cytotoxicity toward multiple HLA-matched ATC cell lines and tumor samples in vitro and in vivo. Furthermore, understanding the role of these genes in ATC tumorigenesis may identify novel combination strategies with adoptive cell therapy to yield synergistic effects. In conclusion, our studies contribute to the development of a new treatment paradigm for treating ATC, with the prospect of improving survival and outcome in this rare cancer patient population. Citation Format: Shao-Hsi Hung, Ying Henderson, Ke Pan, Yulun Chiu, Shailbala Singh, Yunyun Chen, Stephen Lai, Cassian Yee. Utilizing T cell receptor-based therapy to treat anaplastic thyroid cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-061.

Identification of early diagnostic biomarkers for breast cancer through bioinformatics analysis.

In the realm of clinical practice, there is currently an insufficiency of distinct biomarkers available for the detection of breast cancer. It is of utmost importance to promptly employ bioinformatics methodologies to investigate prospective biomarkers for breast cancer, with the ultimate goal of achieving early diagnosis of the disease. The initial phase of this investigation involved the identification of 2 breast cancer gene chips meeting the specified criteria within the gene expression omnibus database. Subsequently, paired data analysis was conducted on these datasets, leading to the identification of differentially expressed genes (DEGs). In addition, this study executed Gene Ontology enrichment analysis and Kyoto encyclopedia of genes and genomes pathway enrichment analysis. The subsequent stage involved the construction of a protein-protein interaction network graph using the STRING website and Cytoscape software, facilitating the calculation of Hub genes. Lastly, the UALCAN database and Kaplan-Meier survival plots were utilized to perform differential expression and survival analysis on the selected Hub genes. A total of 733 DEGs were identified from the combined analysis of 2 datasets. Among these DEGs, 441 genes were found to be downregulated, while 292 genes were upregulated. The selected DEGs underwent comprehensive analysis, including gene ontology enrichment analysis, Kyoto encyclopedia of genes and genomes pathway enrichment analysis, and establishing a protein-protein interaction network. As a result, 10 Hub genes closely associated with early diagnosis of breast cancer were identified: PDZ-binding kinase, cell cycle protein A2, cell division cycle-associated protein 8, maternal embryonic leucine zipper kinase, nucleolar and spindle-associated protein 1, BIRC5, cell cycle protein B2, hyaluronan-mediated motility receptor, mitotic arrest deficient 2-like 1, and protein regulator of cytokinesis 1. The findings of this study unveiled the significant involvement of the identified 10 Hub genes in facilitating the growth and proliferation of cancer cells, particularly cell cycle protein A2, cell division cycle-associated protein 8, maternal embryonic leucine zipper kinase, nucleolar and spindle-associated protein 1, hyaluronan-mediated motility receptor, and protein regulator of cytokinesis 1, which demonstrated a more pronounced connection with the onset and progression of breast cancer. Further analysis through differential expression and survival analysis reaffirmed their strong correlation with the incidence of breast cancer. Consequently, the investigation of these 10 pertinent Hub genes presents novel prospects for potential biomarkers and valuable insights into the early diagnosis of breast cancer.

The LIN28B-let-7-PBK pathway is essential for group 3 medulloblastoma tumor growth and survival.

Children with Group 3 medulloblastoma (G3 MB) have a very poor prognosis, and many do not survive beyond 5 years after diagnosis. A factor that may contribute to this is the lack of available targeted therapy. Expression of protein lin-28 homolog B (LIN28B), a regulator of developmental timing, is upregulated in several cancers, including G3 MB, and is associated with worse survival in this disease. Here, we investigate the role of the LIN28B pathway in G3 MB and demonstrate that the LIN28B-lethal-7 (let-7; a microRNA that is a tumor suppressor)-lymphokine-activated killer T-cell-originated protein kinase (PBK; also known as PDZ-binding kinase) axis promotes G3 MB proliferation. LIN28B knockdown in G3-MB-patient-derived cell lines leads to a significant reduction in cell viability and proliferation in vitro and in prolonged survival of mice with orthotopic tumors. The LIN28 inhibitor N-methyl-N-[3-(3-methyl-1,2,4-triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide (1632) significantly reduces G3 MB cell growth and demonstrates efficacy in reducing tumor growth in mouse xenograft models. Inhibiting PBK using HI-TOPK-032 also results in a significant reduction in G3 MB cell viability and proliferation. Together, these results highlight a critical role for the LIN28B-let-7-PBK pathway in G3 MB and provide preliminary preclinical results for drugs targeting this pathway.

PDZ-binding kinase inhibitor OTS514 suppresses the proliferation of oral squamous carcinoma cells.

PDZ-binding kinase (PBK) has been reported as a poor prognostic factor and is a promising molecular target for anticancer therapeutics. Here, we aimed to investigate the effect of specific PBK inhibitor OTS514 on the survival of OSCC cells. Four OSCC cell lines (HSC-2, HSC-3, SAS, and OSC-19) were used to examine the effect of OTS514 on cell survival and apoptosis. DNA microarray analysis was conducted to investigate the effect of OTS514 on gene expression in OSCC cells. Gene set enrichment analysis was performed to identify molecular signatures related to the antiproliferative effect of OTS514. OTS514 decreased the cell survival of OSCC cells dose-dependently, and administration of OTS514 readily suppressed the HSC-2-derived tumor growth in immunodeficient mice. Treatment with OTS514 significantly increased the number of apoptotic cells and caspase-3/7 activity. Importantly, OTS514 suppressed the expression of E2F target genes with a marked decrease in protein levels of E2F1, a transcriptional factor. Moreover, TP53 knockdown attenuated OTS514-induced apoptosis. OTS514 suppressed the proliferation of OSCC cells by downregulating the expression of E2F target genes and induced apoptosis by mediating the p53 signaling pathway. These results highlight the clinical application of PBK inhibitors in the development of molecular-targeted therapeutics against OSCC.

PDZ-binding kinase aggravates pancreatic neuroendocrine neoplasm progression by activating the AKT/mTOR pathway.

The therapeutic effects of existing drug regimens against pancreatic neuroendocrine neoplasms (pNENs) remain limited, and identifying ideal therapeutic targets is warranted. PDZ binding kinase (PBK) may play an oncogenic role in most solid tumors. However, its function in pNEN remains unclear. In this study, pNEN samples and International Cancer Genome Consortium data were used to determine the clinical significance of PBK. Cell counting and CCK8 assays were used to assess cell proliferation. Flow cytometry was used to assess drug-induced apoptosis and cell cycle arrest. An in vivo PBK-targeting experiment was performed in mice bearing pNENs. Western blotting, quantitative PCR, and immunohistochemistry were performed to assess the molecular mechanisms. PBK was significantly upregulated in pNEN tissues compared with paracancerous tissues. Additionally, PBK was a poor prognostic factor for pNEN patients. PBK was found to promote the proliferation of pNEN cells by activating the AKT/mTOR pathway. Furthermore, PBK inhibition combined with everolimus treatment had enhanced antitumour effects on pNEN via inhibiting AKT/mTOR pathway and inducing G0/G1 phase cell cycle arrest. This study highlights that PBK plays an oncogenic role in and is a promising therapeutic target for pNEN.

E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance.

SUMMARYPrevention or amelioration of declining β cell mass is a potential strategy to cure diabetes. Here, we report the pathways utilized by β cells to robustly replicate in response to acute insulin resistance induced by S961, a pharmacological insulin receptor antagonist. Interestingly, pathways that include CENP-A and the transcription factor E2F1 that are independent of insulin signaling and its substrates appeared to mediate S961-induced β cell multiplication. Consistently, pharmacological inhibition of E2F1 blocks β-cell proliferation in S961-injected mice. Serum from S961-treated mice recapitulates replication of β cells in mouse and human islets in an E2F1-dependent manner. Co-culture of islets with adipocytes isolated from S961-treated mice enables β cells to duplicate, while E2F1 inhibition limits their growth even in the presence of adipocytes. These data suggest insulin resistance-induced proliferative signals from adipocytes activate E2F1, a potential therapeutic target, to promote β cell compensation.

Identification of biomarkers related to tumorigenesis and prognosis in breast cancer.

BackgroundThe aim of the present study was to identify the central genes and prognostic index of breast cancer, and to determine the relationship between prognostic index and immune infiltration levels to provide useful information for the diagnosis and treatment of breast cancer.MethodsThe Cancer Genome Atlas breast cancer dataset and 2 microarray datasets were applied to screen overlapping differentially expressed genes (DEGs) between breast cancer tissue and normal breast tissue samples. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted through the Database for Annotation, Visualization, and Integrated Discovery. Protein-protein interaction (PPI) networks were used to screen hub genes of the overlapping DEGs. Gene Expression Profiling Interactive Analysis (GEPIA), The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN), and The Human Protein Atlas (HPA) databases were used to validate their expression. The correlation of hub genes with immune infiltration was analyzed using TISIDB software. Kaplan-Meier Plotter was used to analyze the prognosis of hub genes.ResultsTen hub genes [cyclin A2 (CCNA2), cyclin dependent kinase 1 (CDK1), centromere protein F (CENPF), kinesin family member 2C (KIF2C), kinesin family member 4A (KIF4A), maternal embryonic leucine zipper kinase (MELK), PDZ binding kinase (PBK), protein regulator of cytokinesis 1 (PRC1), DNA topoisomerase II alpha (TOP2A), and TPX2 microtubule nucleation factor (TPX2)] were selected and their overexpression in breast cancer tissue was verified. All were associated with a poor prognosis for breast cancer. CDK1, CENPF, KIF2C, KIF4A, MELK, PBK, PRC1, and TPX2 were correlated with CD4 T cells in breast cancer, while TOP2A was correlated with CD8 T cells.ConclusionsThe findings indicated that the 10 hub genes could be potential biomarkers for progression in breast cancer.

PBK drives PARP inhibitor resistance through the TRIM37/NFκB axis in ovarian cancer

Resistance to PARP inhibitors (PARPi) remains a therapeutic challenge in ovarian cancer patients. PDZ-binding kinase (PBK) participates in the chemoresistance of many malignancies. However, the role of PBK in PARPi resistance of ovarian cancer is obscure. In the current study, we demonstrated that overexpression of PBK contributed to olaparib resistance in ovarian cancer cells. Knockdown of PBK sensitized olaparib-resistant SKOV3 cells to olaparib. Inhibition of PBK using a specific inhibitor enhanced the therapeutic efficiency of olaparib. Mechanically, PBK directly interacted with TRIM37 to promote its phosphorylation and nuclear translocation. which subsequently activates the NFκB pathway. Additionally, PBK enhanced olaparib resistance of ovarian cancer by regulating the NFκB/TRIM37 axis in vitro and in vivo. In conclusion, PBK confers ovarian cancer resistance to PARPi through activating the TRIM37-mediated NFκB pathway, and targeted inhibition of PBK provided the new therapy to improve PARPi treatment outcomes for ovarian cancer patients.

Identification of PBK as a hub gene and potential therapeutic target for medulloblastoma.

Medulloblastoma (MB) is the most frequent malignant brain tumor in pediatrics. Since the current standard of care for MB consisting of surgery, cranio-spinal irradiation and chemotherapy often leads to a high morbidity rate, a number of patients suffer from long-term sequelae following treatment. Targeted therapies hold the promise of being more effective and less toxic. Therefore, the present study aimed to identify hub genes with an upregulated expression in MB and to search for potential therapeutic targets from these genes. For this purpose, gene expression profile datasets were obtained from the Gene Expression Omnibus database and processed using R 3.6.0 software to screen differentially expressed genes (DEGs) between MB samples and normal brain tissues. A total of 282 upregulated and 436 downregulated DEGs were identified. Functional enrichment analysis revealed that the upregulated DEGs were predominantly enriched in the cell cycle, DNA replication and cell division. The top 10 hub genes were identified from the protein-protein interaction network of upregulated genes, and one identified hub gene [PDZ binding kinase (PBK)] was selected for further investigation due to its possible role in the pathogenesis of MB. The aberrant expression of PBK in MB was verified in additional independent gene expression datasets. Survival analysis demonstrated that a higher expression level of PBK was significantly associated with poorer clinical outcomes in non-Wingless MBs. Furthermore, targeting PBK with its inhibitor, HI-TOPK-032, impaired the proliferation and induced the apoptosis of two MB cell lines, with the diminished phosphorylation of downstream effectors of PBK, including ERK1/2 and Akt, and the activation of caspase-3. Hence, these results suggest that PBK may be a potential prognostic biomarker and a novel candidate of targeted therapy for MB.

PDZ‐Binding Kinase Drives Pulmonary Artery Smooth Muscle Proliferation and Vascular Remodeling in Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is a progressive disease that is caused by high blood pressure in the lungs that originates from increased resistance of small pulmonary arteries (PA). A defining characteristic of PAH is vascular smooth muscle cell (VSMC) proliferation and remodeling of PA, that eventually results in right ventricular failure and death. The hyperproliferative nature of vascular cells in PAH shares many characteristics with cancer cells such as sustained proliferative signaling, changes in hypoxia signaling and cellular metabolism. Using microarray analysis, we discovered a novel gene, PDZ‐Binding Kinase (PBK) that is upregulated in hypertensive PA. PBK is a serine/threonine kinase that has important roles in mitosis and cell proliferation. Also, elevated expression of PBK is associated with numerous aggressive cancers suggesting that it may be an important contributor to PAH. Our hypothesis is that PBK has a functional role in the pathogenesis of PAH but the therapeutic utility of targeting PBK in PAH and the mechanisms by which PBK promotes pulmonary vascular remodeling have not been studied. To investigate the role of PBK in PAH, we performed immunostaining, and found that PBK was highly upregulated in pulmonary arterial smooth muscle in the lungs of two experimental PAH rat models (MCT; Su/H), in the Su/H mouse model, as well as in human PAs and lungs with PAH. Furthermore, human pulmonary arterial smooth cells (HPASMCs) overexpressing PBK showed increased proliferation, while silencing PBK or in vitro pharmacological inhibition significantly reduced HPASMC proliferation. To determine whether PBK plays an important role in PA remodeling and PAH in vivo, we next utilized PBK inhibitor (OTS‐514) to treat MCT‐induced PAH rats in a reversal treatment method and Su/H Rats and Mouse in a preventative treatment. In all of the PAH animal models, right ventricle (RV) thickness, velocity time integral (VTI, index of PA stiffness), right ventricular systolic pressure (RVSP), and the Fulton Index were significantly improved in the presence of the PBK inhibitor. To complement our pharmacological studies with a genetic approach, we have generated PBK KO rats to investigate whether genetic ablation of PBK protects against the development of PAH. Taken together, these data support the hypothesis, that PBK drives pulmonary artery smooth muscle proliferation leading to abnormal vascular remodeling and the development of PAH. Our findings advance the utility of novel therapeutic approaches targeting PBK to improve the morbidity and mortality associated with PAH.

Combined screening analysis of aberrantly methylated-differentially expressed genes and pathways in hepatocellular carcinoma.

Methylation plays an important role in hepatocellular carcinoma (HCC) by altering the expression of key genes. The aim of this study was to screen the aberrantly methylated-differentially expressed genes (DEGs) in HCC and elucidate their underlying molecular mechanism. Gene expression microarrays (GSE101685) and gene methylation microarrays (GSE44909) were selected. DEGs and differentially methylated genes (DMGs) were screened. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the Database for Annotation, Visualization, and Integrated discovery (DAVID). The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to analyze the functional protein-protein interaction (PPI) network. Molecular Complex Detection (MCODE) analysis was performed using the Cytoscape software. Hub genes were verified in The Cancer Genome Atlas (TCGA) database. A total of 80 hypomethylation-high expression genes (Hypo-HGs) were identified. Pathway enrichment analysis showed DNA replication, cell cycle, viral carcinogenesis, and the spliceosome. The top 5 hub genes were minichromosome maintenance complex component 3 (MCM3), checkpoint kinase 1 (CHEK1), kinesin family member 11 (KIF11), PDZ binding kinase (PBK), and Rac GTPase activating protein 1 (RACGAP1). In addition, 189 hypermethylation-low expression genes (Hyper-LGs) were identified. Pathway enrichment analysis indicated enrichment in metabolic pathways, drug metabolism-other enzymes, and chemical carcinogenesis. The top 5 hub genes were leukocyte immunoglobulin like receptor B2 (LILRB2), formyl peptide receptor 1 (FPR1), S100 calcium binding protein A9 (S100A9), S100 calcium binding protein A8 (S100A8), and myeloid cell nuclear differentiation antigen (MNDA). The methylation status and mRNA expression of MCM3, CHEK1, KIF11, PBK, and S100A9 were consistent in the TCGA database and significantly correlated with the prognosis of patients. Combined screening of aberrantly methylated-DEGs based on bioinformatic analysis may provide new clues for elucidating the epigenetic mechanism in HCC. Hub genes, including MCM3, CHEK1, KIF11, PBK, and S100A9, may serve as biomarkers for the precise diagnosis of HCC.

Global Phosphoproteomics Analysis of IBRS-2 Cells Infected With Senecavirus A.

Phosphorylation is a widespread posttranslational modification that regulates numerous biological processes. Viruses can alter the physiological activities of host cells to promote virus particle replication, and manipulating phosphorylation is one of the mechanisms. Senecavirus A (SVA) is the causative agent of porcine idiopathic vesicular disease. Although numerous studies on SVA have been performed, comprehensive phosphoproteomics analysis of SVA infection is lacking. The present study performed a quantitative mass spectrometry-based phosphoproteomics survey of SVA infection in Instituto Biologico-Rim Suino-2 (IBRS-2) cells. Three parallel experiments were performed, and 4,520 phosphosites were quantified on 2,084 proteins. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that many phosphorylated proteins were involved in apoptosis and spliceosome pathways, and subcellular structure localization analysis revealed that more than half were located in the nucleus. Motif analysis of proteins with differentially regulated phosphosites showed that proline, aspartic acid, and glutamic acid were the most abundant residues in the serine motif, while proline and arginine were the most abundant in the threonine motif. Forty phosphosites on 27 proteins were validated by parallel reaction monitoring (PRM) phosphoproteomics, and 30 phosphosites in 21 proteins were verified. Nine proteins with significantly altered phosphosites were further discussed, and eight [SRRM2, CDK13, DDX20, DDX21, BAD, ELAVL1, PDZ-binding kinase (PBK), and STAT3] may play a role in SVA infection. Finally, kinase activity prediction showed 10 kinases’ activity was reversed following SVA infection. It is the first phosphoproteomics analysis of SVA infection of IBRS-2 cells, and the results greatly expand our knowledge of SVA infection. The findings provide a basis for studying the interactions of other picornaviruses and their mammalian host cells.

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.