Autophagy is considered as a double-edged sword. It can prolong the survival of cancer cells and enhance its resistance to apoptosis, and paradoxically, defective autophagy has been linked to increased tumorigenesis, but the mechanism behind this phenomenon is unclear. In this study, we demonstrated that decreased phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) was correlated with increased autophagy through the Akt/mTOR and Erk signaling pathways in human head and neck squamous cell carcinoma (HNSCC). We also showed that blockage of STAT3 by NSC74859 could markedly induce apoptotic cell death and autophagy. Meanwhile, increased autophagy inhibited apoptosis. The pharmacological or genetic inhibition of autophagy and STAT3 further sensitized HNSCC cells to apoptosis. Furthermore, evidence from xenograft model proved that suppressed STAT3 activity combined with inhibition of autophagy promoted tumor regression better than either treatment alone. Taken together, this present study demonstrated that autophagy alleviates apoptotic cell death in HNSCC, and combination of inhibition of STAT3 by NSC74859 and autophagy might be a promising new therapeutic strategy for HNSCC.

The Ca(2+)-activated Cl(-) channel is considered a key constituent of odor transduction. Odorant binding to a specific receptor in the cilia of olfactory sensory neurons (OSNs) triggers a cAMP cascade that mediates the opening of a cationic cyclic nucleotide-gated channel (CNG), allowing Ca(2+) influx. Ca(2+) ions activate Cl(-) channels, generating a significant Cl(-) efflux, with a large contribution to the receptor potential. The Anoctamin 2 channel (ANO2) is a major constituent of the Cl(-) conductance, but its knock-out has no impairment of behavior and only slightly reduces field potential odorant responses of the olfactory epithelium. Likely, an additional Ca(2+)-activated Cl(-) channel of unknown molecular identity is also involved. In addition to ANO2, we detected two members of the ClCa family of Ca(2+)-activated Cl(-) channels in the rat olfactory epithelium, ClCa4l and ClCa2. These channels, also expressed in the central nervous system, may correspond to odorant transduction channels. Whole Sprague Dawley olfactory epithelium nested RT-PCR and single OSNs established that the mRNAs of both channels are expressed in OSNs. Real time RT-PCR and full length sequencing of amplified ClCa expressed in rat olfactory epithelium indicated that ClCa4l is the most abundant. Immunoblotting with an antibody recognizing both channels revealed immunoreactivity in the ciliary membrane. Immunochemistry of olfactory epithelium and OSNs confirmed their ciliary presence in a subset of olfactory sensory neurons. The evidence suggests that ClCa4l and ClCa2 might play a role in odorant transduction in rat olfactory cilia.

Rap1 signaling is important for migration, differentiation, axonal growth, and during neuronal polarity. Rap1 can be activated by external stimuli, which in turn regulates specific guanine nucleotide exchange factors such as C3G, among others. Cdk5 functions are also important to neuronal migration and differentiation. Since we found that pharmacological inhibition of Cdk5 by using roscovitine reduced Rap1 protein levels in COS-7 cells and also C3G contains three putative phosphorylation sites for Cdk5, we examined whether the Cdk5-dependent phosphorylation of C3G could affect Rap1 expression and activity. We co-transfected C3G and tet-OFF system for p35 over-expression, an activator of Cdk5 activity into COS-7 cells, and then we evaluated phosphorylation in serine residues in C3G by immunoprecipitation and Western blot. We found that p35 over-expression increased C3G-serine-phosphorylation while inhibition of p35 expression by tetracycline or inhibition of Cdk5 activity with roscovitine decreased it. Interestingly, we found that MG-132, a proteasome inhibitor, rescue Rap1 protein levels in the presence of roscovitine. Besides, C3G-serine-phosphorylation and Rap1 protein levels were reduced in brain from Cdk5(-/-) as compared with the Cdk5(+/+) brain. Finally, we found that p35 over-expression increased Rap1 activity while inhibition of p35 expression by tetracycline or roscovitine decreased Rap1 activity. These results suggest that Cdk5-mediated serine-phosphorylation of C3G may control Rap1 stability and activity, and this may potentially impact various neuronal functions such as migration, differentiation, and polarity.

Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. In this study, we show that Cdk5 phosphorylation of p27(Kip1) at Thr187 is crucial to neural differentiation because 1) neurogenesis is specifically suppressed by transfection of p27(Kip1) siRNA into Cdk5(+/+) NSCs; 2) reduced neuronal differentiation in Cdk5(-/-) compared with Cdk5(+/+) NSCs; 3) Cdk5(+/+) NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; and 4) transfection of mutant p27(Kip1) (p27/187A) into Cdk5(+/+) NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27(Kip1) at theThr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27(Kip1), a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth, and migration.

Mutations in the doublecortin (DCX) gene in human or targeted disruption of the cdk5 gene in mouse lead to similar cortical lamination defects in the developing brain. Here we show that Dcx is phosphorylated by Cdk5. Dcx phosphorylation is developmentally regulated and corresponds to the timing of expression of p35, the major activating subunit for Cdk5. Mass spectrometry and Western blot analysis indicate phosphorylation at Dcx residue Ser297. Phosphorylation of Dcx lowers its affinity to microtubules in vitro, reduces its effect on polymerization, and displaces it from microtubules in cultured neurons. Mutation of Ser297 blocks the effect of Dcx on migration in a fashion similar to pharmacological inhibition of Cdk5 activity. These results suggest that Dcx phosphorylation by Cdk5 regulates its actions on migration through an effect on microtubules.

Neuronal migration disorders are often identified in patients with epilepsy refractory to medical treatment. The prolonged or repeated seizures are known to cause neuronal death; however, the mechanism underlying seizure-induced neuronal death remains to be elucidated. An essential role of cyclin-dependent kinase 5 (Cdk5) in brain development has been demonstrated in Cdk5(-/-) mice, which show neuronal migration defects and perinatal lethality. Here, we show the consequences of Cdk5 deficiency in the postnatal brain by generating Cdk5 conditional knockout mice, in which Cdk5is selectively eliminated from neurons in the developing forebrain. The conditional mutant mice were viable, but exhibited complex neurological deficits including seizures, tremors, and growth retardation. The forebrain not only showed disruption of layering, but also neurodegenerative changes accompanied by neuronal loss and microglial activation. The neurodegenerative changes progressed with age and were accompanied by up-regulation of the neuronal tissue-type plasminogen activator, a serine protease known to mediate microglial activation. Thus age-dependent neurodegeneration in the Cdk5 conditional knockout mouse brain invoked a massive inflammatory reaction. These findings indicate an important role of Cdk5 in inflammation, and also provide a mouse model to examine the possible involvement of inflammation in the pathogenesis of progressive cognitive decline in patients with neuronal migration disorders.

Epithelial-mesenchymal transition (EMT) is associated with metastasis formation, generation and maintenance of cancer stem cells (CSCs). However, the regulatory mechanisms of CSCs have not been clarified. This study aims to investigate the role of TNF receptor-associated factor 6 (TRAF6) on EMT and CSC regulation in squamous cell carcinoma of head and neck (SCCHN). We found TRAF6 was overexpressed in human SCCHN tissues, and high TRAF6 expression was associated with lymphatic metastasis and resulted in poor prognosis in patients with SCCHN. In addition, elevated TRAF6 expression was observed in several HNSCC cell lines, and wound healing and transwell assay results showed that TRAF6 knockdown inhibited the migration and invasion ability of the SCCHN cells. Moreover, the expression of Vimentin, Slug and N-cadherin was down-regulated and that of E-cadherin was elevated after TRAF6 knockdown but decreased by transforming growth factor beta 1 (TGF-β1) and CAL27 similar to mesenchymal cells formed after TGF-β1 induction. In addition, the expression levels of CD44, ALDH1, KLF4 and SOX2 were inhibited after TRAF6 knockdown, and the anchor-dependent colony formation number and sphere number were remarkably reduced. Flow cytometry showed TRAF6 knockdown reduced ALDH1-positive cancer stem cells. We also demonstrated that TRAF6 is closely associated with EMT process and cancer stem cells using a Tgfbr1/Pten 2cKO mice SCCHN model and human SCCHN tissue microarray. Our findings indicate that TRAF6 plays a role in EMT phenotypes, the generation and maintenance of CSCs in SCCHN, suggesting that TRAF6 is a potential therapeutic target for SCCHN.

Alzheimer's disease (AD) is characterized by extracellular senile plaques, intracellular neurofibrillary tangles, and neuronal death. Aggregated amyloid-β (Aβ) induces inflammation and oxidative stress, which have pivotal roles in the pathogenesis of AD. Hepcidin is a key regulator of systemic iron homeostasis. Recently, an anti-inflammatory response to hepcidin was reported in macrophages. Under the hypothesis that hepcidin mediates anti-inflammatory response in the brain, in this study, we evaluated the putative anti-inflammatory role of hepcidin on Aβ-activated astrocytes and microglia. Primary culture of astrocytes and microglia were treated with Aβ, with or without hepcidin, and cytokine levels were then evaluated. In addition, the toxicity of Aβ-treated astrocyte- or microglia-conditioned media was tested on neurons, evaluating cellular death and oxidative stress generation. Finally, mice were injected in the right lateral ventricle with Aβ, with or without hepcidin, and hippocampus glial activation and oxidative stress were evaluated. Pre-treatment with hepcidin reduced the expression and secretion of TNF-α and IL-6 in astrocytes and microglia treated with Aβ. Hepcidin also reduced neurotoxicity and oxidative damage triggered by conditioned media obtained from astrocytes and microglia treated with Aβ. Stereotaxic intracerebral injection of hepcidin reduced glial activation and oxidative damage triggered by Aβ injection in mice. Overall, these results are consistent with the hypothesis that in astrocytes and microglia hepcidin down-regulates the inflammatory and pro-oxidant processes induced by Aβ, thus protecting neighboring neurons. This is a newly described property of hepcidin in the central nervous system, which may be relevant for the development of strategies to prevent the neurodegenerative process associated with AD.

Immature myeloid cells including myeloid-derived suppressor cells (MDSCs) and tumour-associated macrophages (TAMs) promote tumour growth and metastasis by facilitating tumour transformation and angiogenesis, as well as by suppressing antitumour effector immune responses. Therefore, strategies designed to reduce MDSCs and TAMs accumulation and their activities are potentially valuable therapeutic goals. In this study, we show that negative immune checkpoint molecule B7-H3 is significantly overexpressed in human head and neck squamous cell carcinoma (HNSCC) specimen as compared with normal oral mucosa. Using immunocompetent transgenic HNSCC models, we observed that targeting inhibition of B7-H3 reduced tumour size. Flow cytometry analysis revealed that targeting inhibition of B7-H3 increases antitumour immune response by decreasing immunosuppressive cells and promoting cytotoxic T cell activation in both tumour microenvironment and macroenvironment. Our study provides direct in vivo evidence for a rationale for B7-H3 blockade as a future therapeutic strategy to treat patients with HNSCC.

Partial epithelial mesenchymal transition (p-EMT) was found to play a potential role in the initial stage of metastasis in human head and neck squamous cell carcinoma (HNSCC). Some long noncoding RNAs (lncRNAs) have been reported to function as promoters or inhibitors of cancer metastasis. This study aimed to identify p-EMT-related lncRNAs in HNSCC.

With the global population aging and life expectancy increasing, dementia has turned a priority in the health care system. In Chile, dementia is one of the most important causes of disability in the elderly and the most rapidly growing cause of death in the last 20 years. Cognitive complaint is considered a predictor for cognitive and functional decline, incident mild cognitive impairment, and incident dementia. The GERO cohort is the Chilean core clinical project of the Geroscience Center for Brain Health and Metabolism (GERO). The objective of the GERO cohort is to analyze the rate of functional decline and progression to clinical dementia and their associated risk factors in a community-dwelling elderly with subjective cognitive complaint, through a population-based study. We also aim to undertake clinical research on brain ageing and dementia disorders, to create data and biobanks with the appropriate infrastructure to conduct other studies and facilitate to the national and international scientific community access to the data and samples for research.

Oncolytic viruses (OVs) encoding various transgenes are being evaluated for cancer immunotherapy. Diverse factors such as cytokines, immune checkpoint inhibitors, tumor-associated antigens, and T cell engagers have been exploited as transgenes. These modifications are primarily aimed to reverse the immunosuppressive tumor microenvironment. By contrast, antiviral restriction factors that inhibit the replication of OVs and result in suboptimal oncolytic activity have received far less attention. Here, we report that guanylate-binding protein 1 (GBP1) is potently induced during HSV-1 infection and restricts HSV-1 replication. Mechanistically, GBP1 remodels cytoskeletal organization to impede nuclear entry of HSV-1 genome. Previous studies have established that IpaH9.8, a bacterial E3 ubiquitin ligase, targets GBPs for proteasomal degradation. We therefore engineered an oncolytic HSV-1 to express IpaH9.8 and found that the modified OV effectively antagonized GBP1, replicated to a higher titer in vitro and showed superior antitumor activity in vivo. Our study features a strategy for improving the replication of OVs via targeting a restriction factor and achieving promising therapeutic efficacy.

Dysregulated central-energy metabolism is a hallmark of brain aging. Supplying enough energy for neurotransmission relies on the neuron-astrocyte metabolic network. To identify genes contributing to age-associated brain functional decline, we formulated an approach to analyze the metabolic network by integrating flux, network structure and transcriptomic databases of neurotransmission and aging. Our findings support that during brain aging: (1) The astrocyte undergoes a metabolic switch from aerobic glycolysis to oxidative phosphorylation, decreasing lactate supply to the neuron, while the neuron suffers intrinsic energetic deficit by downregulation of Krebs cycle genes, including mdh1 and mdh2 (Malate-Aspartate Shuttle); (2) Branched-chain amino acid degradation genes were downregulated, identifying dld as a central regulator; (3) Ketone body synthesis increases in the neuron, while the astrocyte increases their utilization, in line with neuronal energy deficit in favor of astrocytes. We identified candidates for preclinical studies targeting energy metabolism to prevent age-associated cognitive decline.

Human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2) and transmembrane and immunoglobulin domain containing 2 (TMIGD2) are new immune checkpoint molecules of the B7:CD28 family; however, little research has been performed on these immune checkpoint molecules. In this study, we used oral squamous cells carcinoma (OSCC) tissue microarrays and immunohistochemistry methods to investigate the expression patterns of HHLA2 and TMIGD2 in OSCC. After comparing the HHLA2 and TMIGD2 expression levels in OSCC, dysplasia, and mucosa, we found increased HHLA2 expression in OSCC and dysplasia, while the TMIGD2 expression was decreased in OSCC and dysplasia. Using the Kaplan-Meier method and log-rank test, we found that higher HHLA2 or TMIGD2 expression levels in OSCC indicate poor prognosis. Furthermore, two-tailed Pearson's statistical analysis revealed that the HHLA2 expression levels in OSCC, dysplasia, and mucosa were positively correlated with the T cell immunoglobulin and mucin-domain containing-3 (TIM3), lymphocyte-activation gene 3 (LAG3), B7 homolog 3 protein (B7-H3), B7 homolog 4 protein (B7H4), and V-domain Ig suppressor of T cell activation (VISTA) levels, while the TMIGD2 expression levels in OSCC, dysplasia, and mucosa were inversely correlated with the TIM3, LAG3, and B7H3 levels. Our current study demonstrates that HHLA2 may serve as an immune target for OSCC therapy and that the TMIGD2 expression level in OSCC could forecast patient prognosis.

Late endosomal/lysosomal adaptor and MAPK and mTOR activator 5 (LAMTOR5) is a novel oncoprotein associated with several human malignancies, but its clinical role in head and neck squamous cell carcinoma (HNSCC) remains unclear. The present study aims to investigate the clinical and pathological significance of LAMTOR5 in HNSCC. We utilized immunohistochemical staining of human tissue microarrays (210 primary HNSCC, 42 normal oral mucosae, 69 oral epithelial dysplasia, and 68 metastasis lymph nodes) to explore the clinical and pathological significance of LAMTOR5 in HNSCC. Additionally, expression level of LAMTOR5 in immunoreactivity of Pten conditional knock out (Pten cKO) mice HNSCC was also assessed. We found LAMTOR5 was overexpressed in human and Pten cKO mice HNSCC, and its expression was significantly associated with patients' overall survival, lymph node metastasis and lymph node grade. Furthermore, LAMTOR5 expression was significantly correlated with the expression of p-AktSer473, p-S6Ser235/236, immune checkpoints (PD-L1, Galectin 9, VISTA and B7-H4) and macrophage markers (CD68 and CD163). In Pten cKO mice HNSCC, it was also significantly correlated with VISTA and F4/80. Consequently, we consider that high expression of LAMTOR5 might be a poor prognostic indicator and correlated with the immunosuppression of tumor microenvironment.

Cyclin-dependent kinase 5 (Cdk5) is essential for brain development and function, and its deregulated expression is implicated in some of neurodegenerative diseases. We reported earlier that the forebrain-specific Cdk5 conditional knockout (cKO) mice displayed an early lethality associated with neuroinflammation, increased expression of the neuronal tissue-type plasminogen activator (tPA), and neuronal migration defects.

Human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease characterized by selective loss of axons and myelin in the corticospinal tracts. This central axonopathy may originate from the impairment of anterograde axoplasmic transport. Previous work showed tau hyperphosphorylation at T(181) in cerebrospinal fluid of HAM/TSP patients. Similar hyperphosphorylation occurs in SH-SY5Y cells incubated with supernatant from MT-2 cells (HTLV-I-infected lymphocytes secreting viral proteins, including Tax) that produce neurite shortening. Tau phosphorylation at T(181) is attributable to glycogen synthase kinase 3-β (GSK3-β) and cyclin-dependent kinase 5 (CDK5) activation. Here we investigate whether neurite retraction in the SH-SY5Y model associates with concurrent changes in other tau hyperphosphorylable residues. Threonine 181 turned out to be the only tau hyperphosphorylated residue. We also evaluate the role of GSK3-β and CDK5 in this process by using specific kinase inhibitors (LiCl, TDZD-8, and roscovitine). Changes in both GSK3-β active and inactive forms were followed by measuring the regulatory phosphorylable sites (S(9) and Y(216) , inactivating and activating phosphorylation, respectively) together with changes in β-catenin protein levels. Our results showed that LiCl and TDZD-8 were unable to prevent MT-2 supernatant-mediated neurite retraction and also that neither Y(216) nor S(9) phosphorylations were changed in GSK3-β. Thus, GSK3-β seems not to play a role in T(181) hyperphosphorylation. On the other hand, the CDK5 involvement in tau phosphorylation was confirmed by both the increase in its enzymatic activity and the absence of MT-2 neurite retraction in the presence of roscovitine or CDK5 siRNA transfection.

Tumor-associated macrophages (TAMs) play an important role in the progression and prognostication of numerous cancers. However, the role and clinical significance of TAM markers in oral squamous cell carcinoma (OSCC) has not been elucidated. The present study was designed to investigate the correlation between the expression of TAM markers and pathological features in OSCC by tissue microarray. Tissue microarrays containing 16 normal oral mucosa, 6 oral epithelial dysplasia, and 43 OSCC specimens were studied by immunohistochemistry. We observed that the protein expression of the TAM markers CD68 and CD163 as well as the cancer stem cell (CSC) markers ALDH1, CD44, and SOX2 increased successively from the normal oral mucosa to OSCC. The expressions of CD68 and CD163 were significantly associated with lymph node status, and SOX2 was significantly correlated with pathological grade and lymph node status, whereas ALDH1 was correlated with tumor stage. Furthermore, CD68 was significantly correlated with CD163, SOX2, and ALDH1 (P < 0.05). Kaplan-Meier analysis revealed that OSCC patients overexpressing CD163 had significantly worse overall survival (P < 0.05). TAM markers are associated with cancer stem cell marker and OSCC overall survival, suggesting their potential prognostic value in OSCC.

Myeloid-derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) play key roles in the tumor immune suppressive network and tumor progression. However, precise roles of programmed death-1 (PD-1) in immunological functions of MDSCs and TAMs in head and neck squamous cell carcinoma (HNSCC) have not been clearly elucidated. In the present study, we show that PD-1 and PD-L1 levels were significantly higher in human HNSCC specimen than in normal oral mucosa. MDSCs and TAMs were characterized in mice and human HNSCC specimen, correlated well with PD-1 and PD-L1 expression. αPD-1 treatment was well tolerated and significantly reduced tumor growth in the HNSCC mouse model along with significant reduction in MDSCs and TAMs in immune organs and tumors. Molecular analysis suggests a reduction in the CD47/SIRPα pathway by PD-1 blockade, which regulates MDSCs, TAMs, dendritic cell as well as effector T cells. Hence, these data identify that PD-1/PD-L1 axis is significantly increased in human and mouse HNSCC. Adoptive αPD-1 immunotherapy may provide a novel therapeutic approach to modulate the micro- and macro-environment in HNSCC.

In the present study, we explored the expression and correlation of survivin with HIF-1α, TGF-β1 and TFE3 in adenoid cystic carcinoma (AdCC). The expression of survivin, HIF-1α, TGF-β1 and TFE3 was assessed by immunohistochemical staining of a tissue microarray containing tissue samples of normal salivary gland (NSG), pleomorphic adenoma (PA) and AdCC. Correlation analysis of these proteins revealed that increased survivin expression was associated with the overexpression of HIF-1α (P<0.001, r = 0.5599), TGF-β1 (P<0.001, r = 0.6616) and TFE3 (P<0.001, r = 0.7747). The expression of survivin, HIF-1α, TGF-β1 and TFE3 was not correlated with the pathological type of human AdCC (P>0.05). Selective inhibition of survivin by YM155 and siRNA significantly reduced human SACC-83 cell proliferation, with the corresponding decrease in expression of HIF-1α, TGF-β1 and TFE3. The data indicate that the overexpression of survivin in AdCC is related to HIF-1α, TGF-β1 and TFE3. We hypothesize from these findings that the inhibition of survivin may be a novel strategy for neoadjuvant chemotherapeutic and radiosensitive treatment of AdCC.