Cyanine Dye Conjugation Enhances Crizotinib Localization to Intracranial Tumors, Attenuating NF-κB-Inducing Kinase Activity and Glioma Progression.

Glioblastoma (GBM) is a highly aggressive form of brain cancer with a poor prognosis and limited treatment options. The ALK and c-MET inhibitor Crizotinib has demonstrated preclinical therapeutic potential for newly diagnosed GBM, although its efficacy is limited by poor penetration of the blood brain barrier. Here, we identify Crizotinib as a novel inhibitor of nuclear factor-κB (NF-κB)-inducing kinase, which is a key regulator of GBM growth and proliferation. We further show that the conjugation of Crizotinib to a heptamethine cyanine dye, or a near-infrared dye (IR-Crizotinib), attenuated glioma cell proliferation and survival in vitro to a greater extent than unconjugated Crizotinib. Moreover, we observed increased IR-Crizotinib localization to orthotopic mouse xenograft GBM tumors, which resulted in impaired tumor growth in vivo. Overall, IR-Crizotinib exhibited improved intracranial chemotherapeutic delivery and tumor localization with concurrent inhibition of NIK and noncanonical NF-κB signaling, thereby reducing glioma growth in vitro, as well as in vivo, and increasing survival in a preclinical rodent model.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) promotes glioma cell invasion through induction of NF-κB-inducing kinase (NIK) and noncanonical NF-κB signaling.

BACKGROUND: High-grade gliomas are one of the most invasive and therapy-resistant cancers. We have recently shown that noncanonical NF-κB/RelB signaling is a potent driver of tumorigenesis and invasion in the aggressive, mesenchymal subtype of glioma. However, the relevant signals that induce activation of noncanonical NF-κB signaling in glioma and its function relative to the canonical NF-κB pathway remain elusive. METHODS: The ability of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to regulate NF-κB signaling and promote tumor progression was investigated in both established and primary high-grade glioma tumor lines using a three-dimensional (3-D) collagen invasion assay. The roles of specific NF-κB proteins in regulating glioma cell invasion and expression of Matrix Metalloproteinase 9 (MMP9) in response to TWEAK were evaluated using shRNA-mediated loss-of-function studies. The ability of NF-κB-inducing kinase (NIK) to promote glioma growth in vivo was investigated using an orthotopic xenograft mouse model. RESULTS: In glioma cells that display elevated noncanonical NF-κB signaling, loss of RelB attenuates invasion without affecting RelA expression or phosphorylation and RelB is sufficient to promote invasion in the absence of RelA. The cytokine TWEAK preferentially activates the noncanonical NF-κB pathway through induction of p100 processing to p52 and nuclear accumulation of both RelB and p52 without activating the canonical NF-κB pathway. Moreover, TWEAK, but not TNFα, significantly increases NIK mRNA levels. TWEAK also promotes noncanonical NFκB-dependent MMP9 expression and glioma cell invasion. Finally, expression of NIK is sufficient to increase gliomagenesis in vivo. CONCLUSIONS: Our data establish a key role for NIK and noncanonical NF-κB in mediating TWEAK-induced, MMP-dependent glioma cell invasion. The findings also demonstrate that TWEAK induces noncanonical NF-κB signaling and signal-specific regulation of NIK mRNA expression. Together, these studies reveal the important role of noncanonical NF-κB signaling in regulating glioma invasiveness and highlight the therapeutic potential of targeting activation of NIK in this deadly disease.

NF-κB-inducing kinase maintains mitochondrial efficiency and systemic metabolic homeostasis.

NF-κB-inducing kinase (NIK) is an essential upstream inducer of noncanonical NF-κB signaling and a critical regulator of immunity and inflammation. Our recent work has demonstrated that NIK regulates mitochondrial respiration and adaptive metabolic responses in cancer and innate immune cells. However, it is not clear whether NIK also has roles in regulating systemic metabolism. In this study, we demonstrate that NIK has local and systemic effects on developmental and metabolic processes. Our findings show that NIK-deficient mice exhibit reduced adiposity, as well as elevated energy expenditure both basally, and under the stress of a high-fat diet. Moreover, we identify NF-κB-independent and -dependent functions for NIK in white adipose tissue metabolism and development. Specifically, we found that in an NF-κB-independent manner NIK is required for maintaining mitochondrial fitness, as NIK-deficient adipocytes have impaired mitochondrial membrane potential and spare respiratory capacity. In addition to mitochondrial exhaustion, NIK-deficient adipocytes and ex vivo adipose tissue exhibit a compensatory upregulation of glycolysis to meet bioenergetic demands. Finally, while NIK regulation of mitochondrial metabolism in preadipocytes is NF-κB-independent, we demonstrate that NIK has a complementary role in adipocyte differentiation that requires activation of RelB and the noncanonical NF-κB pathway. Collectively, these data demonstrate that NIK has critical roles in local and systemic development and metabolism. Our findings establish NIK as an important regulator of organelle, cell, and systemic metabolic homeostasis, suggesting that metabolic dysfunction may be an important and unappreciated component of immune disorders and inflammatory diseases arising from NIK deficiency.

Transcriptional induction of NF-κB-inducing kinase by E2F4/5 facilitates collective invasion of GBM cells.

The prognosis of high-grade gliomas, such as glioblastoma multiforme (GBM), is extremely poor due to the highly invasive nature of these aggressive cancers. Previous work has demonstrated that TNF-weak like factor (TWEAK) induction of the noncanonical NF-κB pathway promotes the invasiveness of GBM cells in an NF-κB-inducing kinase (NIK)-dependent manner. While NIK activity is predominantly regulated at the posttranslational level, we show here that NIK (MAP3K14) is upregulated at the transcriptional level in invading cell populations, with the highest NIK expression observed in the most invasive cells. GBM cells with high induction of NIK gene expression demonstrate characteristics of collective invasion, facilitating invasion of neighboring cells. Furthermore, we demonstrate that the E2F transcription factors E2F4 and E2F5 directly regulate NIK transcription and are required to promote GBM cell invasion in response to TWEAK. Overall, our findings demonstrate that transcriptional induction of NIK facilitates collective cell migration and invasion, thereby promoting GBM pathogenesis.

NIK promotes metabolic adaptation of glioblastoma cells to bioenergetic stress.

Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and ß (IKKα/ß) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK-/- cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.

Comparison of postoperative voice outcomes after postauricular facelift robotic hemithyroidectomy and conventional transcervical hemithyroidectomy.

BACKGROUND: The purpose of this study was to determine whether postauricular robotic and conventional hemithyroidectomy result in significantly different voice outcomes. METHODS: We prospectively compared the voice handicap index (VHI)-10 and acoustic parameters of a postauricular facelift robotic group and a conventional group preoperatively, 1 week, 1 month, and 6 months after surgery. RESULTS: Forty-two patients in the postauricular group and 68 patients in the conventional group completed the VHI-10 questionnaire and acoustic analysis. The postoperative VHI-10 scores were not significantly different between the two groups. In female patients, the highest frequency was higher and the frequency range was wider in the postauricular group compared to the conventional group postoperatively until 1 month after surgery. CONCLUSION: Postauricular facelift robotic thyroidectomy has advantages over conventional thyroidectomy in terms of postoperative voice pitch.

NIK/MAP3K14 Regulates Mitochondrial Dynamics and Trafficking to Promote Cell Invasion.

Although the role of NF-κB-inducing kinase (NIK) in immunity is well established, its relevance in cancer is just emerging. Here we describe novel functions for NIK in regulating mitochondrial dynamics and motility to promote cell invasion. We show that NIK is localized to mitochondria in cancer cell lines, ex vivo tumor tissue, and mouse embryonic fibroblasts (MEFs). NIK promotes mitochondrial fission, velocity, and directional migration, resulting in subcellular distribution of mitochondria to the periphery of migrating cells. Moreover, NIK is required for recruitment of Drp1 to mitochondria, forms a complex with Drp1, and regulates Drp1 phosphorylation at Ser-616 and dephosphorylation at Ser-637. Consistent with a role for NIK in regulating mitochondrial dynamics, we demonstrate that Drp1 is required for NIK-dependent, cytokine-induced invasion. Importantly, using MEFs, we demonstrate that the established downstream mediators of NIK signaling, IκB kinase α/ß (IKKα/ß) and NF-κB, are not required for NIK to regulate cell invasion, Drp1 mitochondrial localization, or mitochondrial fission. Our results establish a new paradigm for IKK-independent NIK signaling and significantly expand the current dogma that NIK is predominantly cytosolic and exclusively regulates NF-κB activity. Overall, these findings highlight the importance of NIK in tumor pathogenesis and invite new therapeutic strategies that attenuate mitochondrial dysfunction through inhibition of NIK and Drp1.

DNA methylation affects meiotic trans-sensing, not meiotic silencing, in Neurospora.

During the early stages of meiosis in Neurospora, the symmetry of homologous chromosomal regions is carefully evaluated by actively trans-sensing their identity. If a DNA region cannot be detected on the opposite homologous chromosome, then this lack of "sensing" activates meiotic silencing, a post-transcriptional gene silencing-like mechanism that silences all genes in the genome with homology to the loop of unpaired DNA, whether they are paired or unpaired. In this work, we genetically dissected the meiotic trans-sensing step from meiotic silencing by demonstrating that DNA methylation affects sensing without interfering with silencing. We also determined that DNA sequence is an important parameter considered during meiotic trans-sensing. Altogether, these observations assign a previously undescribed role for DNA methylation in meiosis and, on the basis of studies in other systems, we speculate the existence of an intimate connection among meiotic trans-sensing, meiotic silencing, and meiotic recombination.

An argonaute-like protein is required for meiotic silencing.

We demonstrate the involvement of suppressor of meiotic silencing-2 (sms-2(+)), a Neurospora gene coding for an Argonaute-like protein, in meiotic silencing and normal sexual development.

Properties of unpaired DNA required for efficient silencing in Neurospora crassa.

The presence of unpaired copies of a gene during meiosis triggers silencing of all copies of the gene in the diploid ascus cell of Neurospora. This phenomenon is called meiotic silencing and on the basis of genetic studies appears to be a post-transcriptional gene silencing (PTGS) mechanism. Previously, meiotic silencing was defined to be induced by the presence of a DNA region lacking an identical segment in the homologous chromosome. However, the determinants of unpaired DNA remained a mystery. Using the Ascospore maturation-1 (Asm-1) gene, we defined what needs to be "unpaired" to silence a gene. For efficient silencing, an unpaired region of DNA needs to be of a sufficient size and contain homology to the reporter transcript. The greater the size of the loop and the larger the homology to the reporter transcript, the better the resulting meiotic silencing is. Conversely, regions not containing homology to the transcript, e.g., intergenic regions, did not silence the reporter. Surprisingly, unpaired fragments lacking a canonical promoter silenced the reporter. Additionally, we detected the unpairing-dependent loss of a transcript during meiotic silencing. Our observations further support a PTGS mechanism for meiotic silencing and offer insight into the evolutionary consequences resulting from this novel meiotic checkpoint.

Effects of oral prednisolone on recovery after tonsillectomy.

OBJECTIVES/HYPOTHESIS: To evaluate the effect of oral prednisolone on recovery from tonsillectomy. STUDY DESIGN: Prospective, randomized, controlled trial of 198 consecutive patients, aged 4 years and older, with no previous or known contraindications to steroid therapy. METHODS: All 198 patients scheduled for elective tonsillectomy with or without adenoidectomy from April 2013 to April 2014 were included. The participants were then randomly assigned to receive a postoperative course of prednisolone 0.25 mg/kg/d or no prednisolone over 7 days. During the first postoperative day, pain, type of diet (none, fluid, soft, normal), type of activity (none, bed rest, quiet, restricted, normal), presence of nausea and vomiting, postoperative bleeding rate, and sleep disturbance were assessed using questionnaires. All patients were followed up on days 7 and 14 by endoscopic photographic examination of both tonsillar fossa and by completion of questionnaires. RESULTS: No statistically significant differences in pain, diet, activity, rate of minor bleeding, nausea/vomiting, fever, or sleep disturbance were observed between the groups on day 1. On day 7, however, in pediatric patients, differences in pain (P = .001), diet (P = .001), activity (P = .004), mean area of re-epithelialization (P = .000), fever (P = .04), and sleep disturbance (P = .04) were observed. On day 14, differences in the mean area of re-epithelialization (P = .000, .001) remained in both pediatric and adult patients. CONCLUSIONS: Oral prednisolone may be beneficial during recovery from tonsillectomy without causing any serious complications.

Increased cyclooxygenase-2 expression in human squamous cell carcinomas of the head and neck and inhibition of proliferation by nonsteroidal anti-inflammatory drugs.

BACKGROUND: Cyclooxygenase-2 (COX-2) has been found to be up-regulated in several types of human cancers and its role in the carcinogenic process has been proposed The aim of this study was to examine the expression of COX-2 in human squamous cell carcinoma of the head and neck (SCCHN) and to find out the effects of COX-2 inhibitors on the growth of cultured cells. MATERIALS AND METHODS: We investigated the effect of indomethacin and NS-398 at various concentrations on the growth of SCCHN cell lines using cell proliferation assay, cell cycle analysis and quantification of apoptosis. RESULTS: Immunostaining revealed a significantly increased COX-2 expression in tumor tissues compared with normal controls (p<0.05). Western blotting analysis using a COX-2 antibody, indicated that seven SCCHN cell lines tested constitutively expressed COX-2 protein. Treatment of head and neck cancer cells with NS-398 (10-200 microM) or indomethacin (50-1000 microM) for 72 hours showed a significant dose-dependent inhibition of cell growth (p<0.01) and a significant increase in the number of cells in the G0/G1-phases of the cell cycle with a concomitant reduction at the S-phase in a dose-dependent manner (p<0.05). NS-398 was more effective in cell cycle arrest and growth inhibition than indomethacin (p<0.05) and induced significant apoptosis in two out of three SCCHN cell lines tested at the concentration of 100 microM. CONCLUSION: Our study showed that COX-2 could be a participant in carcinogenesis of SCCHN and that COX-2 inhibitors would be a potential tool for the treatment and prevention of SCCHN.

A cytosine methyltransferase homologue is essential for sexual development in Aspergillus nidulans.

BACKGROUND: The genome defense processes RIP (repeat-induced point mutation) in the filamentous fungus Neurospora crassa, and MIP (methylation induced premeiotically) in the fungus Ascobolus immersus depend on proteins with DNA methyltransferase (DMT) domains. Nevertheless, these proteins, RID and Masc1, respectively, have not been demonstrated to have DMT activity. We discovered a close homologue in Aspergillus nidulans, a fungus thought to have no methylation and no genome defense system comparable to RIP or MIP. PRINCIPAL FINDINGS: We report the cloning and characterization of the DNA methyltransferase homologue A (dmtA) gene from Aspergillus nidulans. We found that the dmtA locus encodes both a sense (dmtA) and an anti-sense transcript (tmdA). Both transcripts are expressed in vegetative, conidial and sexual tissues. We determined that dmtA, but not tmdA, is required for early sexual development and formation of viable ascospores. We also tested if DNA methylation accumulated in any of the dmtA/tmdA mutants we constructed, and found that in both asexual and sexual tissues, these mutants, just like wild-type strains, appear devoid of DNA methylation. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that a DMT homologue closely related to proteins implicated in RIP and MIP has an essential developmental function in a fungus that appears to lack both DNA methylation and RIP or MIP. It remains formally possible that DmtA is a bona fide DMT, responsible for trace, undetected DNA methylation that is restricted to a few cells or transient but our work supports the idea that the DMT domain present in the RID/Masc1/DmtA family has a previously undescribed function.

The Anabaena sp. strain PCC 7120 asr1734 gene encodes a negative regulator of heterocyst development.

The novel asr1734 gene of Anabaena (Nostoc) sp. strain PCC 7120 inhibited heterocyst development when present in extra copies. Overexpression of asr1734 inhibited heterocyst development in several strains including the wild type and two strains that form multiple contiguous heterocysts (Mch phenotype): a PatS null mutant and a hetR(R223W) mutant. Overexpression of asr1734 also caused increased nblA messenger RNA levels, and increased loss of autofluorescence in vegetative cells throughout filaments after nitrogen or sulphur depletion. Unlike the wild type, an asr1734 knockout mutant formed 5% heterocysts after a nitrogen shift from ammonium to nitrate, and formed 15% heterocysts and a weak Mch phenotype after step-down to medium lacking combined nitrogen. After nitrogen step-down, the asr1734 mutant had elevated levels of ntcA messenger RNA. A green fluorescent protein reporter driven by the asr1734 promoter, P(asr1734)-gfp, was expressed specifically in differentiating proheterocysts and heterocysts after nitrogen step-down. Strains overexpressing asr1734 and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show normal patterned upregulation 24 h after nitrogen step-down even though hetR expression was upregulated at 6 h. Apparent orthologues of asr1734 are found only in two other filamentous nitrogen-fixing cyanobacteria, Anabaena variabilis and Nostoc punctiforme.

Construction of strains for rapid homokaryon purification after integration of constructs at the histidine-3 ( his-3) locus of Neurospora crassa.

We report the construction of histidine-3 (his-3) strains of Neurospora crassa containing the hygromycin B phosphotransferase gene of Escherichia coli (hph(+)) fused in-frame to the herpes simplex virus thymidine kinase gene (tk(+); Lupton et al. 1991), integrated at the his-3 locus. We also report the construction of two ampicillin-resistant and two kanamycin-resistant his-3 gene-replacement vector plasmids. The combined use of these strains and plasmids for his-3-targeted gene integration allows for the rapid identification of homokaryotic transformants containing the expected gene replacement event.