Ceramide-1-phosphate is a regulator of Golgi structure and is co-opted by the obligate intracellular bacterial pathogen Anaplasma phagocytophilum.

Many intracellular pathogens structurally disrupt the Golgi apparatus as an evolutionarily conserved promicrobial strategy. Yet, the host factors and signaling processes involved are often poorly understood, particularly for Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We found that A. phagocytophilum elevated cellular levels of the bioactive sphingolipid, ceramide-1-phosphate (C1P), to promote Golgi fragmentation that enables bacterial proliferation, conversion from its non-infectious to infectious form, and productive infection. A. phagocytophilum poorly infected mice deficient in ceramide kinase, the Golgi-localized enzyme responsible for C1P biosynthesis. C1P regulated Golgi morphology via activation of a PKCα/Cdc42/JNK signaling axis that culminates in phosphorylation of Golgi structural proteins, GRASP55 and GRASP65. siRNA-mediated depletion of Cdc42 blocked A. phagocytophilum from altering Golgi morphology, which impaired anterograde trafficking of trans-Golgi vesicles into and maturation of the pathogen-occupied vacuole. Cells overexpressing phosphorylation-resistant versions of GRASP55 and GRASP65 presented with suppressed C1P- and A. phagocytophilum-induced Golgi fragmentation and poorly supported infection by the bacterium. By studying A. phagocytophilum, we identify C1P as a regulator of Golgi structure and a host factor that is relevant to disease progression associated with Golgi fragmentation.IMPORTANCECeramide-1-phosphate (C1P), a bioactive sphingolipid that regulates diverse processes vital to mammalian physiology, is linked to disease states such as cancer, inflammation, and wound healing. By studying the obligate intracellular bacterium Anaplasma phagocytophilum, we discovered that C1P is a major regulator of Golgi morphology. A. phagocytophilum elevated C1P levels to induce signaling events that promote Golgi fragmentation and increase vesicular traffic into the pathogen-occupied vacuole that the bacterium parasitizes. As several intracellular microbial pathogens destabilize the Golgi to drive their infection cycles and changes in Golgi morphology is also linked to cancer and neurodegenerative disorder progression, this study identifies C1P as a potential broad-spectrum therapeutic target for infectious and non-infectious diseases.

Skewing cPLA2α activity toward oxoeicosanoid production promotes neutrophil N2 polarization, wound healing, and the response to sepsis.

Uncontrolled inflammation is linked to poor outcomes in sepsis and wound healing, both of which proceed through distinct inflammatory and resolution phases. Eicosanoids are a class of bioactive lipids that recruit neutrophils and other innate immune cells. The interaction of ceramide 1-phosphate (C1P) with the eicosanoid biosynthetic enzyme cytosolic phospholipase A2 (cPLA2) reduces the production of a subtype of eicosanoids called oxoeicosanoids. We investigated the effect of shifting the balance in eicosanoid biosynthesis on neutrophil polarization and function. Knockin mice expressing a cPLA2 mutant lacking the C1P binding site (cPLA2αKI/KI mice) showed enhanced and sustained neutrophil infiltration into wounds and the peritoneum during the inflammatory phase of wound healing and sepsis, respectively. The mice exhibited improved wound healing and reduced susceptibility to sepsis, which was associated with an increase in anti-inflammatory N2-type neutrophils demonstrating proresolution behaviors and a decrease in proinflammatory N1-type neutrophils. The N2 polarization of cPLA2αKI/KI neutrophils resulted from increased oxoeicosanoid biosynthesis and autocrine signaling through the oxoeicosanoid receptor OXER1 and partially depended on OXER1-dependent inhibition of the pentose phosphate pathway (PPP). Thus, C1P binding to cPLA2α suppresses neutrophil N2 polarization, thereby impairing wound healing and the response to sepsis.

Neointima abating and endothelium preserving - An adventitia-localized nanoformulation to inhibit the epigenetic writer DOT1L.

Open vascular reconstructions such as bypass are common treatments for cardiovascular disease. Unfortunately, neointimal hyperplasia (IH) follows, leading to treatment failure for which there is no approved therapy. Here we combined the strengths of tailoring nanoplatforms for open vascular reconstructions and targeting new epigenetic mechanisms. We produced adhesive nanoparticles (ahNP) that could be pen-brushed and immobilized on the adventitia to sustainably release pinometostat, an inhibitor drug selective to the epigenetic writer DOT1L that catalyzes histone-3 lysine-79 dimethylation (H3K79me2). This treatment not only reduced IH by 76.8% in injured arteries mimicking open reconstructions in obese Zucker rats with human-like diseases but also avoided the shortcoming of endothelial impairment in IH management. In mechanistic studies, chromatin immunoprecipitation (ChIP) sequencing revealed co-enrichment of the histone mark H3K27ac(acetyl) and its reader BRD4 at the gene of aurora kinase B (AURKB), where H3K79me2 was also enriched as indicated by ChIP-qPCR. Accordingly, DOT1L co-immunoprecipitated with H3K27ac. Furthermore, the known IH driver BRD4 governed the expression of DOT1L which controlled AURKB's protein level, revealing a BRD4- > DOT1L- > AURKB axis. Consistently, AURKB-selective inhibition reduced IH. Thus, this study presents a prototype nanoformulation suited for open vascular reconstructions, and the new insights into chromatin modulators may aid future translational advances.

miR579-3p is an inhibitory modulator of neointimal hyperplasia and transcription factors c-MYB and KLF4.

Neointimal hyperplasia (IH) is a common vascular pathology that typically manifests in in-stent restenosis and bypass vein graft failure. Smooth muscle cell (SMC) phenotypic switching is central to IH, both regulated by some microRNAs, yet the role of miR579-3p, a scarcely studied microRNA, is not known. Unbiased bioinformatic analysis suggested that miR579-3p was repressed in human primary SMCs treated with different pro-IH cytokines. Moreover, miR579-3p was software-predicted to target both c-MYB and KLF4 - two master transcription factors known to promote SMC phenotypic switching. Interestingly, treating injured rat carotid arteries via local infusion of miR579-3p-expressing lentivirus reduced IH 14 days after injury. In cultured human SMCs, transfection with miR579-3p inhibited SMC phenotypic switching, as indicated by decreased proliferation/migration and increased SMC contractile proteins. miR579-3p transfection downregulated c-MYB and KLF4, and luciferase assays indicated miR579-3p's targeting of the 3'UTRs of the c-MYB and KLF4 mRNAs. In vivo, immunohistochemistry showed that treatment of injured rat arteries with the miR579-3p lentivirus reduced c-MYB and KLF4 and increased SMC contractile proteins. Thus, this study identifies miR579-3p as a previously unrecognized small-RNA inhibitor of IH and SMC phenotypic switch involving its targeting of c-MYB and KLF4. Further studies on miR579-3p may provide an opportunity for translation to develop IH-mitigating new therapeutics.

Angioplasty induces epigenomic remodeling in injured arteries.

Neointimal hyperplasia/proliferation (IH) is the primary etiology of vascular stenosis. Epigenomic studies concerning IH have been largely confined to in vitro models, and IH-underlying epigenetic mechanisms remain poorly understood. This study integrates information from in vivo epigenomic mapping, conditional knockout, gene transfer and pharmacology in rodent models of IH. The data from injured (IH-prone) rat arteries revealed a surge of genome-wide occupancy by histone-3 lysine-27 trimethylation (H3K27me3), a gene-repression mark. This was unexpected in the traditional view of prevailing post-injury gene activation rather than repression. Further analysis illustrated a shift of H3K27me3 enrichment to anti-proliferative genes, from pro-proliferative genes where gene-activation mark H3K27ac(acetylation) accumulated instead. H3K27ac and its reader BRD4 (bromodomain protein) co-enriched at Ezh2; conditional BRD4 knockout in injured mouse arteries reduced H3K27me3 and its writer EZH2, which positively regulated another pro-IH chromatin modulator UHRF1. Thus, results uncover injury-induced loci-specific H3K27me3 redistribution in the epigenomic landscape entailing BRD4→EZH2→UHRF1 hierarchical regulations. Given that these players are pharmaceutical targets, further research may help improve treatments of IH.

Repositioning Fenofibrate to Reactivate p53 and Reprogram the Tumor-Immune Microenvironment in HPV+ Head and Neck Squamous Cell Carcinoma.

Human papillomavirus-associated head and neck squamous cell carcinoma (HPV+ HNSCC) is recognized as a distinct disease with unique etiology and clinical features. Current standard of care therapeutic modalities are identical for HPV+ and HPV- HNSCC and thus, there remains an opportunity to develop innovative pharmacologic approaches to exploit the inherent vulnerabilities of HPV+ HNSCC. In this study, using an inducible HPVE6E7 knockdown system, we found that HPV+ HNSCC cells are addicted to HPVE6E7, such that loss of these viral oncogenes impaired tumorigenicity in vitro and in vivo. A number of druggable pathways, including PPAR and Wnt, were modulated in response to HPVE6E7 loss. Fenofibrate showed significant anti-proliferative effects in a panel of HPV+ cancer cell lines. Additionally, fenofibrate impaired tumor growth as monotherapy and potentiated the activity of cisplatin in a pre-clinical HPV+ animal model. Systemic fenofibrate treatment induced p53 protein accumulation, and surprisingly, re-programmed the tumor-immune microenvironment to drive immune cell infiltration. Since fenofibrate is FDA-approved with a favorable long-term safety record, repositioning of this drug, as a single agent or in combination with cisplatin or checkpoint blockade, for the HPV+ HNSCC setting should be prioritized.

miR548ai antagonism attenuates exosome-induced endothelial cell dysfunction.

Endothelial cell (EC) and smooth muscle cell (SMC) are major cell types adjacent in the vascular wall. Recent progress indicates that their communication is crucial for vascular homeostasis and pathogenesis. In particular, dysfunctional (proliferative) SMCs through exosomes can induce EC dysfunction (impaired growth). The current study suggests that miR548ai, a rarely known microRNA, may provide a molecular target for protection against SMC/exosome-induced EC dysfunction. We performed microarray profiling of microRNAs of dysfunctional human primary aortic SMCs induced by different cytokines (PDGF-BB, TGFß1, TNFα, IL1ß). Among the microRNAs commonly upregulated by these cytokines, miR548ai showed the most robust changes, as also validated through quantitative PCR. This cytokine-induced miR548ai upregulation was recapitulated in the qPCR determination of SMC-derived exosomal microRNAs. Consistent with SMC-to-EC communication, the exosomes extracted from cytokine-stimulated SMCs impaired human EC proliferation and migration. Of particular interest, this SMC exosomal impingement on ECs was countered by transfection of miR548ai inhibitor microRNA into ECs. Furthermore, the miR548ai inhibitor transfected into SMCs attenuated SMC dysfunction/proliferation. Thus, these results identify miR548ai as a novel target; namely, miR548ai inhibitor mitigates EC dysfunction induced by exosomes derived from dysfunctional SMCs. This new knowledge may aid the future development of microRNA-based treatment of vascular disorders.

An adventitial painting modality of local drug delivery to abate intimal hyperplasia.

A major medical problem is the persistent lack of approved therapeutic methods to prevent postoperative intimal hyperplasia (IH) which leads to high-rate failure of open vascular reconstructions such as bypass grafting. Hydrogel has been widely used in preclinical trials for perivascular drug administration to mitigate postoperative IH. However, bulky hydrogel is potentially pro-inflammatory, posing a significant hurdle to clinical translation. Here we developed a new modality of directly "painting" drug-loaded unimolecular micelles (UM) to the adventitia thus obviating the need for a hydrogel. To render tissue adhesion, we generated amine-reactive unimolecular micelles with N-hydroxysuccinimide ester (UM-NHS) terminal groups to form stable amide bonds with the adventitia. To test periadventitial application, we either soaked balloon-injured rat carotid arteries in crosslinked UM-NHS (Mode-1) or non-crosslinked UM-NHS (Mode-2), or painted the vessel surface with non-crosslinked UM-NHS (Mode-3). The UM-NHS were loaded with or without a model drug (rapamycin) known to be IH inhibitory. We found that Mode-1 produced a marked IH-mitigating drug effect but also caused severe tissue damage. Mode-2 resulted in lower tissue toxicity yet less drug effect on IH. However, the painting method, Mode-3, demonstrated a pronounced therapeutic effect (75% inhibition of IH) without obvious toxicity. In summary, we present a simple painting modality of periadventitial local drug delivery using tissue-adhesive UM. Given the robust IH-abating efficacy and low tissue toxicity, this prototype merits further development towards an effective anti-stenosis therapy suitable for open vascular reconstructions.

Smad2 inhibition of MET transcription potentiates human vascular smooth muscle cell apoptosis.

Background: Vascular smooth muscle cell (SMC) apoptosis is involved in major cardiovascular diseases. Smad2 is a transcription factor implicated in aortic aneurysm. The molecular mediators of Smad2-driven SMC apoptosis are not well defined. Here we have identified a Smad2-directed mechanism involving MET and FAS, both encoding cell membrane signaling receptors. Methods and results: Guided by microarray analysis in human primary aortic SMCs, loss/gain-of-function (siRNA/overexpression) indicated that Smad2 negatively and positively regulated, respectively, the gene expression of Met which was identified herein as anti-apoptotic and that of Fas, a known pro-apoptotic factor. While co-immunoprecipitation suggested a physical association of Smad2 with p53, chromatin immunoprecipitation followed by quantitative PCR revealed their co-occupancy in the same region of the MET promoter. Activating p53 with nutlin3a further potentiated the suppression of MET promoter-dependent luciferase activity and the exacerbation of SMC apoptosis that were caused by Smad2 overexpression. These results indicated that Smad2 in SMCs repressed the transcription of MET by cooperating with p53, and that Smad2 also activated FAS, a target gene of its transcription factor activity. Conclusions: Our study suggests a pro-apoptotic mechanism in human SMCs, whereby Smad2 negatively and positively regulates MET and FAS, genes encoding anti-apoptotic and pro-apoptotic factors, respectively.

BRD2 regulation of sigma-2 receptor upon cholesterol deprivation.

The sigma-2 receptor (S2R) has long been pharmacologically targeted for antipsychotic treatment and tumor imaging. Only recently was it known for its coding gene and for its role implicated in cholesterol homeostasis. Here, we have investigated the transcriptional control of S2R by the Bromo/ExtraTerminal epigenetic reader family (BETs, including BRD2, 3, and 4) upon cholesterol perturbation. Cholesterol deprivation was induced in ARPE19 cells using a blocker of lysosomal cholesterol export. This condition up-regulated S2R mRNA and protein, and also SREBP2 but not SREBP1, both transcription factors key to cholesterol/fatty acid metabolism. Silencing BRD2 but not BRD3 or BRD4 (though widely deemed a master regulator) averted S2R up-regulation that was induced by cholesterol deprivation. Silencing SREBP2 but not SREBP1 diminished S2R expression. Furthermore, endogenous BRD2 co-immunoprecipitated with the transcription-active N-terminal half of SREBP2, and chromatin immunoprecipitation-qPCR signified co-occupancy of BRD2, H3K27ac (histone acetylation), and SREBP2Nterm at the S2R gene promoter. In summary, this study reveals a previously unrecognized BRD2/SREBP2 cooperative regulation of S2R transcription, thus shedding new light on signaling in response to cholesterol deprivation.

Smad3 Regulates Neuropilin 2 Transcription by Binding to its 5' Untranslated Region.

Background Vascular smooth muscle cell phenotypic change and consequential intimal hyperplasia (IH) cause arterial stenosis and posttreatment restenosis. Smad3 is a master transcription factor, yet its underlying functional mechanisms in this disease context are not well defined. Methods and Results In cultured smooth muscle cells, Smad3 silencing and overexpression respectively reduced and increased the mRNA and protein of NRP2 (neuropilin 2), a recently reported pro-IH signaling factor. Smad3 silencing attenuated pro-IH smooth muscle cell phenotypes including proliferation, migration, and dedifferentiation (reduced smooth muscle α-actin). While increased Smad3 enhanced these phenotypes, NRP2 silencing abolished this enhancement. Interestingly, the 5' untranslated region but not the promoter of NRP2 was indispensable for Smad3-enhanced transcriptional activity (luciferase assay); both chromatin immunoprecipitation and electrophoretic mobility shift assay showed predominant Smad3 binding in the +51 to +78 bp region of NRP2's 5' untranslated region. In vivo, Smad3 haploinsufficiency reduced NRP2 (immunostaining) and IH (by 47%) in wire-injured mouse femoral arteries. Conclusions Smad3 controls NRP2 expression by occupying its 5' untranslated region in promoting smooth muscle cell phenotypic change in vitro. This and in vivo results shed new light on the long-debated role of Smad3 in IH.

p53 functional states are associated with distinct aldehyde dehydrogenase transcriptomic signatures.

p53 and aldehyde dehydrogenase (ALDH) have been implicated in key tumorigenesis processes including cancer initiating cell (CIC) maintenance; however, the relationship between these two mediators remains poorly defined. In this study, ALDH isoform expression diversity was revealed in CICs with disparate p53 functional states: gain of function, high risk p53 mutation (p53HRmut) and wildtype p53 (p53WT) inactivated by the human papillomavirus 16 (HPV16) E6 oncogene. Interrogation of head and neck squamous cell carcinoma (HNSCC) cell lines and patient tumors showed that HPV16+/p53WT cases have higher ALDH variance score (AVS), a measure of tumor ALDH isoform expression diversity, compared to HPV-/p53HRmut cases (p = 0.03). AVS and several individual ALDH isoforms were associated with prognosis in HPV16+/p53WT HNSCC but not in HPV-/p53HRmut HNSCC. Knockdown of the dominant ALDH isoform in high AVS HNSCC depleted the CIC pool in vitro and in vivo. Our results demonstrate that p53 functional states are associated with distinct ALDH isoform transcriptomic signatures. Moreover, tumor ALDH profiling may provide insight on which ALDH isoform to target in high AVS HNSCC tumors to deplete the CIC population.

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation.

Vascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

TriCurin, a novel formulation of curcumin, epicatechin gallate, and resveratrol, inhibits the tumorigenicity of human papillomavirus-positive head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600,000 new cases diagnosed in the last year. The incidence of human papillomavirus-positive head and neck squamous cell carcinoma (HPV-positive HNSCC) has rapidly increased over the past 30 years prompting the suggestion that an epidemic may be on the horizon. Therefore, there is a clinical need to develop alternate therapeutic strategies to manage the growing number of HPV-positive HNSCC patients. TriCurin is a composition of three food-derived polyphenols in unique stoichiometric proportions consisting of curcumin from the spice turmeric, resveratrol from red grapes, and epicatechin gallate from green tea. Cell viability, clonogenic survival, and tumorsphere formation were inhibited and significant apoptosis was induced by TriCurin in UMSCC47 and UPCI:SCC090 HPV-positive HNSCC cells. Moreover, TriCurin decreased HPV16E6 and HPV16E7 and increased p53 levels. In a pre-clinical animal model of HPV-positive HNSCC, intra-tumoral injection of TriCurin significantly inhibited tumor growth by 85.5% compared to vehicle group (P < 0.05, n = 7). Our results demonstrate that TriCurin is a potent anti-tumor agent for HPV-positive HNSCC. Further development of TriCurin as a novel anti-cancer therapeutic to manage the HPV-positive HNSCC population is warranted.

Immunotherapy for head and neck cancer: the future of treatment?

INTRODUCTION: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with >500,000 cases diagnosed each year. HNSCC patients often present to the clinic with advanced disease and are managed with a multi-disciplinary approach consisting of surgery, chemotherapy, and/or radiation. Morbidity and quality of life issues are major challenges in this patient population due to the debilitating effects of standard of care treatment paradigms. There is a critical need for new therapeutic approaches to manage HNSCC with better anti-tumor activities and toxicity profiles. Immunotherapy has gained traction as a precision medicine initiative to manage solid malignancies. Areas covered: The authors review current knowledge of immune escape mechanisms and discuss key immunotherapies in HNSCC with an emphasis on clinical trials data. Expert opinion: The excitement over the potential of immunotherapy to manage solid malignancies, including HNSCC is high and warranted based on the impressive clinical data accrued to date. Research in immunity and immune modulation in cancer has been invigorated and offers the potential to reveal novel vulnerabilities that may be exploitable pharmacologically. The evolution of immunotherapy will continue and move toward rational combinations with other immunotherapies or molecularly-targeted agents in the first-line, adjuvant, and recurrent/metastatic settings in HNSCC.

miR-124 Regulates the Epithelial-Restricted with Serine Box/Epidermal Growth Factor Receptor Signaling Axis in Head and Neck Squamous Cell Carcinoma.

Epithelial-restricted with serine box (ESX), a member of the ETS transcription factor family, is elevated and regulates EGFR in head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanisms that contribute to ESX dysregulation remain to be elucidated. In this study, in silico analysis of the 3'-untranslated region (UTR) of ESX predicted two miR-124-binding sites. Delivery of miR-124 inhibited the 3'UTR ESX-driven reporter activity by 50% (P < 0.05) confirming ESX as a direct target of miR-124. Loss of miR-124 was found to be a frequent event in HNSCC. miR-124 expression was significantly depleted in the primary tumor compared with matched normal tissue in 100% (12/12) of HNSCC patients; relative mean miR-124 expression of 0.01197 and 0.00118 (P < 0.001, n = 12) in matched normal adjacent tissue and primary HNSCC tumor, respectively. Overexpression of miR-124 decreased ESX and EGFR levels in miR-124(low)/ESX(high)/EGFR(high) SCC15 HNSCC cells and reduced cell invasion, migration, proliferation, and colony formation. SCC15 cells with miR-124 restoration were less tumorigenic in vivo than miR-control SCC15 cells (70% inhibition, P < 0.01). Restoration of miR-124 in SCC15 cells enhanced the antiproliferative efficacy of the EGFR/Her2 tyrosine kinase inhibitors. Furthermore, recapitulation of EGFR in miR-124-overexpressing SCC15 cells was sufficient to completely block the antiproliferative effects of lapatinib and afatinib. Taken together, our work provides intriguing evidence that miR-124 is a novel therapeutic approach to reduce ESX/EGFR, and may be a tractable strategy to enhance the response rate of HNSCC patients to current anti-EGFR/Her2 therapies.

Are all cancer stem cells created equal?

Numerous solid malignancies have been reported to contain cancer stem cells (CSCs). Distinct functional characteristics have been attributed to CSCs, and thus it is widely believed that these unique cells may have genetic and phenotypic homogeneity. Recent exciting but limited evidence, however, contradicts this tenet and supports the intriguing concept of genetic and phenotypic diversity in the CSC population. We propose that CSC heterogeneity at the inter- and intrapatient levels may be due to the cell of origin, to environmental cues, and/or to human papillomavirus infection. Additional insight into CSC heterogeneity is needed to identify actionable targets for optimal eradication of the diverse CSC subpopulations within a tumor.

Elevated intrinsic cancer stem cell population in human papillomavirus-associated head and neck squamous cell carcinoma.

BACKGROUND: Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC), particularly the development of oropharyngeal squamous cell carcinoma (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies, and it is postulated that they are responsible for disease recurrence and/or progression. Because the prognoses of patients with HPV16-positive and HPV-negative HNSCC are distinct, the authors sought to determine whether differences in the number of CSCs could account for this clinical observation. METHODS: CSC populations in HPV16-positive and HPV-negative HNSCC were assessed using a proprietary assay based on expression of the enzyme aldehyde dehydrogenase (ALDH), an in vitro tumorsphere formation assay, and an in vivo limiting cell dilution in nonobese diabetic/severe combined immunodeficiency mice. A high-density tissue microarray was stained with ALDH1, a CSC marker, to determine the association between CSCs and HPV16-positive/HPV-negative OPSCC. RESULTS: HPV16-positive HNSCC had a greater intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 has been identified as a major mechanism for the elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using tumors from patients with HPV16-positive and HPV-negative OPSCC indicated that the CSC frequency was 62.5-fold greater in an HPV16-positive OPSCC tumor than in an HPV-negative OPSCC tumor. Primary tumors from patients with HPV16-positive OPSCC were associated with elevated tumor ALDH1 staining, further extending the association between HPV16 and CSCs. CONCLUSIONS: The current data and the clinical observation that patients with HPV16-positive HNSCC respond more favorably to current treatment paradigms than patients with HPV-negative HNSCC support the suggestion that CSC phenotype is not homogeneous. Therefore, the reliance on the CSC number may be insufficient to accurately assess the potential of a particular tumor for disease recurrence and/or progression.

Source, distribution, and health risk assessment of polycyclic aromatic hydrocarbons in urban street dust from Tianjin, China.

To better assess and understand potential health risk of urban residents exposed to urban street dust, the total concentration, sources, and distribution of 16 polycyclic aromatic hydrocarbons (PAHs) in 87 urban street dust samples from Tianjin as a Chinese megacity that has undergone rapid urbanization were investigated. In the meantime, potential sources of PAHs were identified using the principal component analysis (PCA), and the risk of residents' exposure to PAHs via urban street dust was calculated using the Incremental Lifetime Cancer Risk (ILCR) model. The results showed that the total PAHs (∑PAHs) in urban street dust from Tianjin ranged from 538 µg kg(-1) to 34.3 mg kg(-1), averaging 7.99 mg kg(-1). According to PCA, the two to three- and four to six-ring PAHs contributed 10.3 and 89.7 % of ∑PAHs, respectively. The ratio of the sum of major combustion specific compounds (ΣCOMB) / ∑PAHs varied from 0.57 to 0.79, averaging 0.64. The ratio of Ant/(Ant + Phe) varied from 0.05 to 0.41, averaging 0.10; Fla/(Fla + Pyr) from 0.40 to 0.68, averaging 0.60; BaA/(BaA + Chry) from 0.29 to 0.51, averaging 0.38; and IcdP/(IcdP + BghiP) from 0.07 to 0.37, averaging 0.22. The biomass combustion, coal combustion, and traffic emission were the main sources of PAHs in urban street dust with the similar proportion. According to the ILCR model, the total cancer risk for children and adults was up to 2.55 × 10(-5) and 9.33 × 10(-5), respectively.