Neurotropin® ameliorates chronic pain associated with scar formation in a mouse model: A gene expression analysis of the inflammatory response.

Background: Scar formation after trauma and surgery involves an inflammatory response and can lead to the development of chronic pain. Neurotropin® (NTP) is a nonprotein extract of inflamed skin of rabbits inoculated with vaccinia virus. It has been widely used for the treatment of chronic pain. However, the in vivo effects of NTP on painful scar formation have not been determined. To investigate the molecular mechanisms underlying the effects of NTP on the inflammatory response, we evaluated gene expression in the scar tissues and dorsal root ganglions (DRGs) of mice administered NTP and control mice. Methods and results: Mice injected with saline or NTP were used as controls; other mice were subjected to surgery on the left hind paw to induce painful scar formation, and then injected with saline or NTP. Hind paw pain was evaluated by measuring the threshold for mechanical stimulation using the von Frey test. The paw withdrawal threshold gradually returned to pre-operative levels over 4 weeks post-operation; NTP-treated mice showed a significantly shortened recovery time of approximately 3 weeks, suggesting that NTP exerted an analgesic effect in this mouse model. Total RNA was extracted from the scarred hind paw tissues and DRGs were collected 1 week post-operation for a microarray analysis. Gene set enrichment analysis revealed that the expression of some gene sets related to inflammatory responses was activated or inhibited following surgery and NTP administration. Quantitative real-time reverse transcription-polymerase chain reaction analysis results for several genes were consistent with the microarray results. Conclusion: The administration of NTP to the hind paws of mice with painful scar formation following surgery diminished nociceptive pain and reduced the inflammatory response. NTP inhibited the expression of some genes involved in the response to surgery-induced inflammation. Therefore, NTP is a potential therapeutic option for painful scar associated with chronic pain.

ARK5 enhances cell survival associated with mitochondrial morphological dynamics from fusion to fission in human multiple myeloma cells.

5' adenosine monophosphate-activated protein kinase-related kinase 5 (ARK5) is involved in mitochondrial ATP production and associated with poor prognosis of multiple myeloma (MM). However, the molecular mechanisms of ARK5 in MM remain largely unknown. This study examined the pathogenic role of ARK5 in mitochondria by using genetically modified isogenic cell clones with or without ARK5 in human myeloma cell lines, KMS-11 and Sachi, which overexpress ARK5. The biallelic knockout of ARK5 (ARK5-KO) inhibited cell proliferation, colony formation, and migration with increased apoptosis. Mitochondrial fusion was enhanced in ARK5-KO cells, unlike in ARK5 wild-type (ARK5-WT) cells, which exhibited increased mitochondrial fission. Furthermore, ARK5-KO cells demonstrated a lower phosphorylated dynamin-related protein 1 at serine 616, higher protein expression of mitofusin-1 (MFN1) and MFN2, optic atrophy 1 with a lower level of ATP, and higher levels of lactate and reactive oxygen species than ARK5-WT cells. Our findings suggest that ARK5-enhanced myeloma cells can survive associated mitochondrial fission and activity. This study first revealed the relationship between ARK5 and mitochondrial morphological dynamics. Thus, our outcomes show novel aspects of mitochondrial biology of ARK5, which can afford a more advanced treatment approach for unfavorable MM expressing ARK5.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Leucine zipper protein 1 (LUZP1) regulates the constriction velocity of the contractile ring during cytokinesis.

There has been a great deal of research on cell division and its mechanisms; however, its processes still have many unknowns. To find novel proteins that regulate cell division, we performed the screening using siRNAs and/or the expression plasmid of the target genes and identified leucine zipper protein 1 (LUZP1). Recent studies have shown that LUZP1 interacts with various proteins and stabilizes the actin cytoskeleton; however, the function of LUZP1 in mitosis is not known. In this study, we found that LUZP1 colocalized with the chromosomal passenger complex (CPC) at the centromere in metaphase and at the central spindle in anaphase and that these LUZP1 localizations were regulated by CPC activity and kinesin family member 20A (KIF20A). Mass spectrometry analysis identified that LUZP1 interacted with death-associated protein kinase 3 (DAPK3), one regulator of the cleavage furrow ingression in cytokinesis. In addition, we found that LUZP1 also interacted with myosin light chain 9 (MYL9), a substrate of DAPK3, and comprehensively inhibited MYL9 phosphorylation by DAPK3. In line with a known role for MYL9 in the actin-myosin contraction, LUZP1 suppression accelerated the constriction velocity at the division plane in our time-lapse analysis. Our study indicates that LUZP1 is a novel regulator for cytokinesis that regulates the constriction velocity of the contractile ring.

Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.

CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies.

CAMK2D: a novel molecular target for BAP1-deficient malignant mesothelioma.

Malignant mesothelioma (MMe) is a rare but aggressive malignancy. Although the molecular genetics of MMe is known, including BRCA1-associated protein-1 (BAP1) gene alterations, the prognosis of MMe patients remains poor. Here, we generated BAP1 knockout (BAP1-KO) human mesothelial cell clones to develop molecular-targeted therapeutics based on genetic alterations in MMe. cDNA microarray and quantitative RT-PCR (qRT-PCR) analyses revealed high expression of a calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D) gene in the BAP1-KO cells. CAMK2D was highly expressed in 70% of the human MMe tissues (56/80) and correlated with the loss of BAP1 expression, making it a potential diagnostic and therapeutic target for BAP1-deficient MMe. We screened an anticancer drugs library using BAP1-KO cells and successfully identified a CaMKII inhibitor, KN-93, which displayed a more potent and selective antiproliferative effect against BAP1-deficient cells than cisplatin or pemetrexed. KN-93 significantly suppressed the tumor growth in mice xenografted with BAP1-deficient MMe cells. This study is the first to provide a potential molecular-targeted therapeutic approach for BAP1-deficient MMe.

Inhibition of VEGFR2 and EGFR signaling cooperatively suppresses the proliferation of oral squamous cell carcinoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently overexpressed in oral squamous cell carcinoma (OSCC), and EGFR-targeting therapeutics have been widely employed to treat patients with a variety of carcinomas including OSCC. Here, we aimed to investigate alternative signaling for OSCC survival under the disruption of EGFR signaling. METHODS: OSCC cell lines, namely HSC-3 and SAS, were utilized to investigate how EGFR disruption affects cell proliferation. Gene set enrichment analysis was performed to examine how EGFR disruption affects oncogenic signaling in OSCC cells. Disruption of KDR gene was performed using CRISPR/Cas9 techniques. A VEGFR inhibitor, vatalanib was used to research the impact of VEGFR inhibition on OSCC survival. RESULTS: EGFR disruption significantly decreased the proliferation and oncogenic signaling including Myc and PI3K-Akt, in OSCC cells. Chemical library screening assays revealed that VEGFR inhibitors continued to inhibit the proliferation of EGFR-deficient OSCC cells. In addition, CRISPR-mediated disruption of KDR/VEGFR2 retarded OSCC cell proliferation. Furthermore, combined erlotinib-vatalanib treatment exhibited a more potent anti-proliferative effect on OSCC cells, compared to either monotherapy. The combined therapy effectively suppressed the phosphorylation levels of Akt but not p44/42. CONCLUSION: VEGFR-mediated signaling would be an alternative signaling pathway for the survival of OSCC cells under the disruption of EGFR signaling. These results highlight the clinical application of VEGFR inhibitors in the development of multi-molecular-targeted therapeutics against OSCC.

Long noncoding RNA profile of the intracranial artery in patients with moyamoya disease.

OBJECTIVE: Moyamoya disease (MMD) is a rare cerebrovascular disease characterized by progressive stenosis of the internal carotid artery (ICA) and secondary formation of collateral vessels. Revascularization surgery is performed in patients with MMD to prevent stroke; however, the pathogenesis of MMD remains unknown. Recently, long noncoding RNAs (lncRNAs) have been found to play a key role in gene regulation and are implicated in various vascular diseases. However, the lncRNA expression profile in MMD lesions has not been investigated. In this study the authors aimed to determine the characteristics of lncRNA expression in MMD lesions. METHODS: The authors collected microsamples of the middle cerebral artery (MCA) from patients with MMD (n = 21) and patients with control conditions (n = 11) who underwent neurosurgical treatment. Using microarray experiments, the authors compared the profiles of lncRNA expression in the MCAs of the MMD and control patient groups and identified differentially expressed lncRNAs (fold change > 2, q < 0.05). In addition, the neighboring coding genes, whose transcription can be regulated in cis by the identified differentially expressed lncRNAs, were investigated and Gene Ontology (GO) analysis was applied to predict associated biological functions. RESULTS: The authors detected 308 differentially expressed lncRNAs (fold change > 2, q < 0.05), including 306 upregulated and 2 downregulated lncRNAs in the MCA from patients with MMD. Regarding the prediction of biological function, GO analyses with possible coding genes whose transcription was regulated in cis by the identified differentially expressed lncRNAs suggested involvement in the antibacterial humoral response, T-cell receptor signaling pathway, positive regulation of cytokine production, and branching involved in blood vessel morphogenesis. CONCLUSIONS: The profile of lncRNA expression in MMD lesions was different from that in the normal cerebral artery, and differentially expressed lncRNAs were identified. This study provides new insights into the pathophysiology of MMD.

BCL6 inhibition ameliorates resistance to ruxolitinib in CRLF2-rearranged acute lymphoblastic leukemia.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an intractable disease and most cases harbor genetic alterations that activate JAK or ABL signaling. The commonest subtype of Ph-like ALL exhibits a CRLF2 gene rearrangement that brings about JAK1/2-STAT5 pathway activation. However, JAK1/2 inhibition alone is insufficient as a treatment, so combinatorial therapies targeting multiple signals are needed. To better understand the mechanisms underlying the insufficient efficacy of JAK inhibition, we explored gene expression changes upon treatment with a JAK1/2 inhibitor (ruxolitinib) and found that elevated BCL6 expression was one such mechanism. Upregulated BCL6 suppressed the expression of TP53 along with its downstream cell cycle inhibitor p21 (CDKN2A) and pro-apoptotic molecules, such as FAS, TNFRSF10B, BID, BAX, BAK, PUMA, and NOXA, conferring cells some degree of resistance to therapy. BCL6 inhibition (with FX1) alone was able to upregulate TP53 and restore the TP53 expression that ruxolitinib had diminished. In addition, ruxolitinib and FX1 concertedly downregulated MYC. As a result, FX1 treatment alone had growth-inhibitory and apoptosis- sensitizing effects, but the combination of ruxolitinib and FX1 more potently inhibited leukemia cell growth, enhanced apoptosis sensitivity, and prolonged the survival of xenografted mice. These findings provide one mechanism for the insufficiency of JAK inhibition for the treatment of CRLF2-rearranged ALL and indicate BCL6 inhibition as a potentially helpful adjunctive therapy combined with JAK inhibition.

Clostridium butyricum-induced ω-3 fatty acid 18-HEPE elicits anti-influenza virus pneumonia effects through interferon-λ upregulation.

The precise mechanism by which butyrate-producing bacteria in the gut contribute to resistance to respiratory viral infections remains to be elucidated. Here, we describe a gut-lung axis mechanism and report that orally administered Clostridium butyricum (CB) enhances influenza virus infection resistance through upregulation of interferon (IFN)-λ in lung epithelial cells. Gut microbiome-induced ω-3 fatty acid 18-hydroxy eicosapentaenoic acid (18-HEPE) promotes IFN-λ production through the G protein-coupled receptor (GPR)120 and IFN regulatory factor (IRF)-1/-7 activations. CB promotes 18-HEPE production in the gut and enhances ω-3 fatty acid sensitivity in the lungs by promoting GPR120 expression. This study finds a gut-lung axis mechanism and provides insights into the treatments and prophylaxis for viral respiratory infections.

Correction of a CD55 mutation to quantify the efficiency of targeted knock-in via flow cytometry.

BACKGROUND: Targeted knock-in assisted by the CRISPR/Cas9 system is an advanced technology with promising applications in various research fields including medical and agricultural sciences. However, improvements in the efficiency, precision, and specificity of targeted knock-in are prerequisites to facilitate the practical application of this technology. To improve the efficiency of targeted knock-in, it is necessary to have a molecular system that allows sensitive monitoring of targeted knock-in events with simple procedures. METHODS AND RESULTS: We developed an assay, named CD55 correction assay, with which to monitor CD55 gene correction accomplished by targeted knock-in. To create the reporter clones used in this assay, we initially introduced a 7.7-kb heterozygous deletion covering CD55 exons 2-5, and then incorporated a truncating mutation within exon 4 of the remaining CD55 allele in human cell lines. The resultant reporter clones that lost the CD55 protein on the cell membrane were next transfected with Cas9 constructs along with a donor plasmid carrying wild-type CD55 exon 4. The cells were subsequently stained with fluorescence-labeled CD55 antibody and analyzed by flow cytometry to detect CD55-positive cells. These procedures allow high-throughput, quantitative detection of targeted gene correction events occurring in an endogenous human gene. CONCLUSIONS: The current study demonstrated the utility of the CD55 correction assay to sensitively quantify the efficiency of targeted knock-in. When used with the PIGA correction assay, the CD55 correction assay will help accurately determine the efficiency of targeted knock-in, precluding possible experimental biases caused by cell line-specific and locus-specific factors.

Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking.

Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700-2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.

Flow cytometry-based quantification of targeted knock-in events in human cell lines using a GPI-anchor biosynthesis gene PIGP.

Targeted knock-in supported by the CRISPR/Cas systems enables the insertion, deletion, and substitution of genome sequences exactly as designed. Although this technology is considered to have wide range of applications in life sciences, one of its prerequisites for practical use is to improve the efficiency, precision, and specificity achieved. To improve the efficiency of targeted knock-in, there first needs to be a reporter system that permits simple and accurate monitoring of targeted knock-in events. In the present study, we created such a system using the PIGP gene, an autosomal gene essential for GPI-anchor biosynthesis, as a reporter gene. We first deleted a PIGP allele using Cas9 nucleases and then incorporated a truncating mutation into the other PIGP allele in two near-diploid human cell lines. The resulting cell clones were used to monitor the correction of the PIGP mutations by detecting GPI anchors distributed over the cell membrane via flow cytometry. We confirmed the utility of these reporter clones by performing targeted knock-in in these clones via a Cas9 nickase-based strategy known as tandem paired nicking, as well as a common process using Cas9 nucleases, and evaluating the efficiencies of the achieved targeted knock-in. We also leveraged these reporter clones to test a modified procedure for tandem paired nicking and demonstrated a slight increase in the efficiency of targeted knock-in by the new procedure. These data provide evidence for the utility of our PIGP-based assay system to quantify the efficiency of targeted knock-in and thereby help improve the technology of targeted knock-in.

Transcriptome-wide analysis of intracranial artery in patients with moyamoya disease showing upregulation of immune response, and downregulation of oxidative phosphorylation and DNA repair.

OBJECTIVE: Moyamoya disease (MMD) is a rare cerebrovascular disease characterized by progressive occlusion of the internal carotid artery and the secondary formation of collateral vessels. Patients with MMD have ischemic attacks or intracranial bleeding, but the disease pathophysiology remains unknown. In this study, the authors aimed to identify a gene expression profile specific to the intracranial artery in MMD. METHODS: This was a single-center, prospectively sampled, retrospective cohort study. Microsamples of the middle cerebral artery (MCA) were collected from patients with MMD (n = 11) and from control patients (n = 9). Using microarray techniques, transcriptome-wide analysis was performed. RESULTS: Comparison of MCA gene expression between patients with MMD and control patients detected 62 and 26 genes whose expression was significantly (p < 0.001 and fold change > 2) up- or downregulated, respectively, in the MCA of MMD. Gene set enrichment analysis of genes expressed in the MCA of patients with MMD revealed positive correlations with genes involved in antigen processing and presentation, the dendritic cell pathway, cytokine pathway, and interleukin-12 pathway, and negative correlations with genes involved in oxidative phosphorylation and DNA repair. Microarray analysis was validated by quantitative polymerase chain reaction. CONCLUSIONS: Transcriptome-wide analysis showed upregulation of genes for immune responses and downregulation of genes for DNA repair and oxidative phosphorylation within the intracranial artery of patients with MMD. These findings may represent clues to the pathophysiology of MMD.

CD52 is a novel target for the treatment of FLT3-ITD-mutated myeloid leukemia.

Internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) confers poor prognosis and is found in approximately 25% of cases of acute myeloid leukemia (AML). Although FLT3 inhibitors have shown clinical benefit in patients with AML harboring FLT3-ITD, the therapeutic effect is limited. Here, to explore alternative therapeutics, we established a cellular model of monoallelic FLT3ITD/WT cells using the CRISPR-Cas9 system in a human myeloid leukemia cell line, K562. cDNA microarray analysis revealed elevated CD52 expression in K562-FLT3ITD/WT cells compared to K562-FLT3WT/WT cells, an observation that was further confirmed by quantitative real-time-PCR and flow cytometric analyses. The elevated expression of CD52 in K562-FLT3ITD/WT cells was decreased in wild-type FLT3 (FLT3-WT) knock-in K562-FLT3ITD/WT cells. In K562-FLT3ITD/WT cells, a STAT5 inhibitor, pimozide, downregulated CD52 protein expression while an AKT inhibitor, afuresertib, did not affect CD52 expression. Notably, an anti-CD52 antibody, alemtuzumab, induced significant antibody-dependent cell-mediated cytotoxicity (ADCC) in K562-FLT3ITD/WT cells compared to K562-FLT3WT/WT cells. Furthermore, alemtuzumab significantly suppressed the xenograft tumor growth of K562-FLT3ITD/WT cells in severe combined immunodeficiency (SCID) mice. Taken together, our data suggested that genetically modified FLT3-ITD knock-in human myeloid leukemia K562 cells upregulated CD52 expression via activation of STAT5, and alemtuzumab showed an antitumor effect via induction of ADCC in K562-FLT3ITD/WT cells. Our findings may allow establishment of a new therapeutic option, alemtuzumab, to treat leukemia with the FLT3-ITD mutation.

Chromosomal translocation t(11;14) and p53 deletion induced by the CRISPR/Cas9 system in normal B cell-derived iPS cells.

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain potential to transform by reprograming, and then chromosomal aberrations cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we have already established normal B cell-derived induced pluripotent stem cells (BiPSCs). Here we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgH Eµ region of either the VDJ rearranged allele or non-rearranged allele of IgH and the 5'-upsteam region of the CCND1 (two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14). These BiPSCs differentiated into hematopoietic progenitor cells (HPCs). However, unlike cord blood, those HPCs did not differentiated into B lymphocytes by co-culture with BM stromal cell. Therefore, further ingenuity is required to differentiate those BiPSCs-derived HPCs into B lymphocytes.

PBK expression predicts favorable survival in colorectal cancer patients.

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers worldwide with high morbidity and mortality rates. The discovery of small molecule anticancer reagents has significantly affected cancer therapy. However, the anticancer effects of these therapies are not sufficient to completely cure CRC. PDZ-binding kinase (PBK) was initially identified as a mitotic kinase for mitogen-activated protein kinase and is involved in cytokinesis and spermatogenesis. Aberrant expression of PBK has been reported to be closely associated with malignant phenotypes of many cancers and/or patient survival. However, the expression of PBK and its association to patient survival in CRC have not been fully elucidated. In the present study, 269 primary CRCs were evaluated immunohistochemically for PBK expression to assess its ability as a prognostic factor. CRC tumor cells variably expressed PBK (range, 0-100%; median, 32%) in the nucleus and cytoplasm. Univariate analyses identified a significant inverse correlation between PBK expression and pT stage (P<0.0001). Furthermore, patients carrying CRC with higher PBK expression showed significantly favorable survival (P=0.0094). Multivariate Cox proportional hazards regression analysis revealed high PBK expression (HR, 0.52; P=0.015) as one of the potential favorable factors for CRC patients. PBK expression showed significant correlation to Ki-67 labeling indices (ρ=0.488, P<0.0001). In vitro, the PBK inhibitor OTS514 suppressed cellular proliferation of CRC cells with PBK expression through downregulation of P-ERK and induction of apoptosis. These results suggest that PBK-targeting therapeutics may be useful for the treatment of PBK-expressing CRC patients.

PBK Enhances Cellular Proliferation With Histone H3 Phosphorylation and Suppresses Migration and Invasion With CDH1 Stabilization in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most frequent gastrointestinal malignancies with high morbidity and mortality rates. Several biological markers for the prognostication of patient outcome of CRCs are available. Recently, our group identified two favorable factors for the survival of CRC patients: PDZ-binding kinase (PBK) and phospho-histone H3 (PHH3). Both showed a significant inverse association to pT stage. The aim of this study was to uncover the mechanism through which these cellular proliferation-associated protein expressions lead to favorable clinical outcome in CRC patients. We first confirmed co-expression of PBK and PHH3 in CRC cells. Further investigation showed that aberrantly expressed PBK up-regulated the cellular proliferation of CRC cells with accumulation of PHH3. The PBK inhibitor OTS514 suppressed cellular proliferation of CRC cells through down-regulation of PHH3 and induction of apoptosis. In vitro studies revealed that PBK suppressed the migration and invasion of CRC cells with suppression of Wnt/ß-catenin signaling and CDH1 stabilization. Exogeneous PBK up-regulated the phosphorylated CDH1 at S840, S846, and S847 residues in cultured cells. Recombinant PBK directly phosphorylated HH3; however, it failed to phosphorylate CDH1 directly in vitro. The present study demonstrated the association of two markers PBK and PHH3 in CRC. We further identified one of the potential mechanisms by which higher expression of these cellular proliferation-associated proteins leads to the better survival of CRC patients, which likely involves PBK-mediated suppression of the migration and invasion of CRC cells. Our findings suggest that PBK-targeting therapeutics may be useful for the treatment of CRC patients with PBK-expressing tumors.

Identification of CD24 as a potential diagnostic and therapeutic target for malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy of the pleura that is currently incurable due to the lack of an effective early diagnostic method and specific medication. The CDKN2A (p16) and NF2 genes are both frequently mutated in MPM. To understand how these mutations contribute to MPM tumor growth, we generated NF2/p16 double-knockout (DKO) cell clones using human MeT-5A and HOMC-B1 mesothelial cell lines. Cell growth and migration activities were significantly increased in DKO compared with parental cells. cDNA microarray analysis revealed differences in global gene expression profiles between DKO and parental cells. Quantitative PCR and western blot analyses showed upregulation of CD24 concomitant with increased phosphorylation of AKT, p70S6K, and c-Jun in DKO clones. This upregulation was abrogated by exogenous expression of NF2 and p16. CD24 knockdown in DKO cells significantly decreased TGF-ß1 expression and increased expression of E-cadherin, an epithelial-mesenchymal transition marker. CD24 was highly expressed in human mesothelioma tissues (28/45 cases, 62%) and associated with the loss of NF2 and p16. Public data analysis revealed a significantly shorter survival time in MPM patients with high CD24 gene expression levels. These results strongly indicate the potential use of CD24 as a prognostic marker as well as a novel diagnostic and therapeutic target for MPM.

Identification of cisplatin-resistant factor by integration of transcriptomic and proteomic data using head and neck carcinoma cell lines.

Cisplatin is an important drug for the treatment of head and neck squamous cell carcinoma (HNSCC). Determining chemoresistant factors prior to treatment will lead to great benefits for clinicians and patients. Here, we evaluated chemoresistant factors by integrating proteomic and transcriptomic data using HNSCC cell lines to identify a more precise chemoresistant factor in HNSCC. We used four HNSCC cell lines: cisplatin-sensitive, acquired cisplatin resistance, naturally cisplatin-resistant, and acquired 5-FU resistance. Proteomic analysis was performed using iTRAQ, tandem mass spectrometry, and liquid chromatography-electrospray ionization-tandem mass spectrometry. Transcriptomic analysis was performed using microarrays. By integrating these independent data, common factors were addressed and functional analysis was performed using small interfering RNAs (siRNAs) to change the chemosensitivity. Using iTRAQ analysis, 7 proteins were identified as specific for cisplatin chemoresistance factors. Transcriptomic analysis revealed hundreds of potential candidate factors. By combining and integrating these data, S100A2 was identified as a potential cisplatin-specific chemoresistance factor. Functional analysis with siRNA revealed that the expression of S100A2 was reduced and cisplatin sensitivity recovered in the acquired and naturally cisplatin-resistant cell lines, but not in the cisplatin-sensitive cell lines. S100A2 was identified as a cisplatin-specific chemoresistance factor by integrating the transcriptomic and proteomic results obtained using HNSCC cell lines. This is a novel technique that allows for a precise identification, also known as a comprehensive analysis. Our findings indicate that these proteins could be used as biomarkers of HNSCC treatments, providing physicians with new treatment strategies for patients with HNSCC, showing chemoresistance.