技术资料

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties,yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME,we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway,possibly by modulating availability of hyaluronic acid (HA),the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships,and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions,respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction,whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo,targeting CD44 decreased PI3K-4EBP1-SOX2 signaling,tumor growth,and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally,a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes,respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44,TAM,and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery. View Publication

BACKGROUND Taking into consideration a recent surge of a lung injury condition associated with electronic cigarette use,we devised an in vitro model of sub-chronic exposure of human bronchial epithelial cells (HBECs) in air-liquid interface,to determine deterioration of epithelial cell barrier from sub-chronic exposure to cigarette smoke (CS),e-cigarette aerosol (EC),and tobacco waterpipe exposures (TW). METHODS Products analyzed include commercially available e-liquid,with 0{\%} or 1.2{\%} concentration of nicotine,tobacco blend (shisha),and reference-grade cigarette (3R4F). In one set of experiments,HBECs were exposed to EC (0 and 1.2{\%}),CS or control air for 10 days using 1 cigarette/day. In the second set of experiments,exposure of pseudostratified primary epithelial tissue to TW or control air exposure was performed 1-h/day,every other day,until 3 exposures were performed. After 16-18 h of last exposure,we investigated barrier function/structural integrity of the epithelial monolayer with fluorescein isothiocyanate-dextran flux assay (FITC-Dextran),measurements of trans-electrical epithelial resistance (TEER),assessment of the percentage of moving cilia,cilia beat frequency (CBF),cell motion,and quantification of E-cadherin gene expression by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS When compared to air control,CS increased fluorescence (FITC-Dextran assay) by 5.6 times,whereby CS and EC (1.2{\%}) reduced TEER to 49 and 60{\%} respectively. CS and EC (1.2{\%}) exposure reduced CBF to 62 and 59{\%},and cilia moving to 47 and 52{\%},respectively,when compared to control air. CS and EC (1.2{\%}) increased cell velocity compared to air control by 2.5 and 2.6 times,respectively. The expression of E-cadherin reduced to 39{\%} of control air levels by CS exposure shows an insight into a plausible molecular mechanism. Altogether,EC (0{\%}) and TW exposures resulted in more moderate decreases in epithelial integrity,while EC (1.2{\%}) substantially decreased airway epithelial barrier function comparable with CS exposure. CONCLUSIONS The results support a toxic effect of sub-chronic exposure to EC (1.2{\%}) as evident by disruption of the bronchial epithelial cell barrier integrity,whereas further research is needed to address the molecular mechanism of this observation as well as TW and EC (0{\%}) toxicity in chronic exposures. View Publication

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO,Ac-DEVD-CHO,Ac-YVAD-CHO,t-butoxycarbonyl-IETD-CHO,and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes,with dissociation constants ranging from 75 pM to {\textgreater}10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor,with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki {\textless} 20 nM) and selective inhibitor of Group I caspases (caspase-1,-4,and -5) and most Group III caspases (caspase-8,-9,and -10),suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response. View Publication

View Publication

PURPOSE A major clinical problem in the treatment of breast cancer is the inherent and acquired resistance to antiestrogen therapy. In this study,we sought to determine whether antiprogestin treatment,used as a monotherapy or in combination with antiestrogen therapy,induced growth arrest and active cell death in antiestrogen-resistant breast cancer cells. EXPERIMENTAL DESIGN MCF-7 sublines were established from independent clonal isolations performed in the absence of drug selection and tested for their response to the antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 (fulvestrant),and the antiprogestin mifepristone (MIF). The cytostatic (growth arrest) effects of the hormones were assessed with proliferation assays,cell counting,flow cytometry,and a determination of the phosphorylation status of the retinoblastoma protein. The cytotoxic (apoptotic) effects were analyzed by assessing increases in caspase activity and cleavage of poly(ADP-ribose) polymerase. RESULTS All of the clonally derived MCF-7 sublines expressed estrogen receptor and progesterone receptor but showed a wide range of antiestrogen sensitivity,including resistance to physiological levels of 4-OHT. Importantly,all of the clones were sensitive to the antiprogestin MIF,whether used as a monotherapy or in combination with 4-OHT. MIF induced retinoblastoma activation,G(1) arrest,and apoptosis preceded by caspase activation. CONCLUSIONS We demonstrate that: (a) estrogen receptor(+)progesterone receptor(+),4-OHT-resistant clonal variants can be isolated from an MCF-7 cell line in the absence of antiestrogen selection; and (b) MIF and MIF plus 4-OHT combination therapy induces growth arrest and active cell death of the antiestrogen-resistant breast cancer cells. These preclinical findings show potential for a combined hormonal regimen of an antiestrogen and an antiprogestin to combat the emergence of antiestrogen-resistant breast cancer cells and,ultimately,improve the therapeutic index of antiestrogen therapy. View Publication

Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2),a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2-CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently,myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca,but not Na,currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw,a model of postoperative pain. These effects are compared with those of tat-CBD3-the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex,superior suppression of calcium influx,and better antinociceptive potential. View Publication

Increased extracellular sodium activates Th17 cells,which provide protection from bacterial and fungal infections. Whilst high salt diets have been shown to worsen autoimmune disease,the immunological consequences of clinical salt depletion are unknown. Here,we investigate immunity in patients with inherited salt-losing tubulopathies (SLT). Forty-seven genotyped SLT patients (with Bartter,Gitelman or EAST Syndromes) are recruited. Clinical features of dysregulated immunity are recorded with a standardised questionnaire and immunological investigations of IL-17 responsiveness undertaken. The effects of altering extracellular ionic concentrations on immune responses are then assessed. Patients are hypokalaemic and hypomagnesaemic,with reduced interstitial sodium stores determined by 23Na-magnetic resonance imaging. SLT patients report increased mucosal infections and allergic disease compared to age-matched controls. Aligned with their clinical phenotype,SLT patients have an increased ratio of Th2:Th17 cells. SLT Th17 and Tc17 polarisation is reduced in vitro,yet STAT1 and STAT3 phosphorylation and calcium flux following T cell activation are unaffected. In control cells,the addition of extracellular sodium (+40 mM),potassium (+2 mM),or magnesium (+1 mM) reduces Th2:Th17 ratio and augments Th17 polarisation. Our results thus show that the ionic environment typical in SLT impairs IL-17 immunity,but the intracellular pathways that mediate salt-driven Th17 polarisation are intact and in vitro IL-17 responses can be reinvigorated by increasing extracellular sodium concentration. Whether better correction of extracellular ions can rescue the immunophenotype in vivo in SLT patients remains unknown. View Publication

Eltrombopag is a first-in-class,orally bioavailable,small-molecule,nonpeptide agonist of the thrombopoietin receptor (TpoR),which is being developed as a treatment for thrombocytopenia of various etiologies. In vitro studies have demonstrated that the activity of eltrombopag is dependent on expression of TpoR,which activates the signaling transducers and activators of transcription (STAT) and mitogen-activated protein kinase signal transduction pathways. The objective of this preclinical study is to determine if eltrombopag interacts selectively with the TpoR to facilitate megakaryocyte differentiation in platelets. Functional thrombopoietic activity was demonstrated by the proliferation and differentiation of primary human CD34(+) bone marrow cells into CD41(+) megakaryocytes. Measurements in platelets in several species indicated that eltrombopag specifically activates only the human and chimpanzee STAT pathways. The in vivo activity of eltrombopag was demonstrated by an increase of up to 100{\%} in platelet numbers when administered orally (10 mg/kg per day for 5 days) to chimpanzees. In conclusion,eltrombopag interacts selectively with the TpoR without competing with Tpo,leading to the increased proliferation and differentiation of human bone marrow progenitor cells into megakaryocytes and increased platelet production. These results suggest that eltrombopag and Tpo may be able to act additively to increase platelet production. View Publication

Many tumors become addicted to autophagy for survival,suggesting inhibition of autophagy as a potential broadly applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation,how ULK1 promotes autophagy remains poorly understood. Here,we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered 15 phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells,regulating autophagy and cell survival. SBI-0206965 greatly synergized with mechanistic target of rapamycin (mTOR) inhibitors to kill tumor cells,providing a strong rationale for their combined use in the clinic. View Publication

Although regenerative medicine products are at the forefront of scientific research,technological innovation,and clinical translation,their reproducibility and large-scale production are compromised by automation,monitoring,and standardization issues. To overcome these limitations,new technologies at software (e.g.,algorithms and artificial intelligence models,combined with imaging software and machine learning techniques) and hardware (e.g.,automated liquid handling,automated cell expansion bioreactor systems,automated colony-forming unit counting and characterization units,and scalable cell culture plates) level are under intense investigation. Automation,monitoring and standardization should be considered at the early stages of the developmental cycle of cell products to deliver more robust and effective therapies and treatment plans to the bedside,reducing healthcare expenditure and improving services and patient care. View Publication

Autophagy is a cellular catabolic process responsible for the degradation of cytoplasmic constituents,including organelles and long-lived proteins,that helps maintain cellular homeostasis and protect against various cellular stresses. Verteporfin is a benzoporphyrin derivative used clinically in photodynamic therapy to treat macular degeneration. Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light. We report that verteporfin directly targets and modifies p62,a scaffold and adaptor protein that binds both polyubiquitinated proteins destined for degradation and LC3 on autophagosomal membranes. Western blotting experiments revealed that exposure of cells or purified p62 to verteporfin causes the formation of covalently crosslinked p62 oligomers by a mechanism involving low-level singlet oxygen production. Rose bengal,a singlet oxygen producer structurally unrelated to verteporfin,also produced crosslinked p62 oligomers and inhibited autophagosome formation. Co-immunoprecipitation experiments demonstrated that crosslinked p62 oligomers retain their ability to bind to LC3 but show defective binding to polyubiquitinated proteins. Mutations in the p62 PB1 domain that abolish self-oligomerization also abolished crosslinked oligomer formation. Interestingly,small amounts of crosslinked p62 oligomers were detected in untreated cells,and other groups noted the accumulation of p62 forms with reduced SDS-PAGE mobility in cellular and animal models of oxidative stress and aging. These data indicate that p62 is particularly susceptible to oxidative crosslinking and lead us to propose a model whereby oxidized crosslinked p62 oligomers generated rapidly by drugs like verteporfin or over time during the aging process interfere with autophagy. View Publication

We used synthetic lethal high-throughput screening to interrogate 23,550 compounds for their ability to kill engineered tumorigenic cells but not their isogenic normal cell counterparts. We identified known and novel compounds with genotype-selective activity,including doxorubicin,daunorubicin,mitoxantrone,camptothecin,sangivamycin,echinomycin,bouvardin,NSC146109,and a novel compound that we named erastin. These compounds have increased activity in the presence of hTERT,the SV40 large and small T oncoproteins,the human papillomavirus type 16 (HPV) E6 and E7 oncoproteins,and oncogenic HRAS. We found that overexpressing hTERT and either E7 or LT increased expression of topoisomerase 2alpha and that overexpressing RAS(V12) and ST both increased expression of topoisomerase 1 and sensitized cells to a nonapoptotic cell death process initiated by erastin. View Publication