A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.

Accumulating evidence implicates heterogeneity within cancer cell populations in the response to stressful exposures, including drug treatments. While modeling the acute response to various anticancer agents in drug-sensitive human tumor cell lines, we consistently detected a small subpopulation of reversibly "drug-tolerant" cells. These cells demonstrate >100-fold reduced drug sensitivity and maintain viability via engagement of IGF-1 receptor signaling and an altered chromatin state that requires the histone demethylase RBP2/KDM5A/Jarid1A. This drug-tolerant phenotype is transiently acquired and relinquished at low frequency by individual cells within the population, implicating the dynamic regulation of phenotypic heterogeneity in drug tolerance. The drug-tolerant subpopulation can be selectively ablated by treatment with IGF-1 receptor inhibitors or chromatin-modifying agents, potentially yielding a therapeutic opportunity. Together, these findings suggest that cancer cell populations employ a dynamic survival strategy in which individual cells transiently assume a reversibly drug-tolerant state to protect the population from eradication by potentially lethal exposures.

Clinical efficacy comparison of low-temperature plasma radiofrequency ablation and Nd:YAG laser in treating recurrent acquired nasolacrimal duct obstruction.

To evaluate the effectiveness and safety of low-temperature plasma radiofrequency ablation (coblation) and Nd:YAG laser in treating recurrent nasolacrimal duct obstruction. A prospective study was performed on patients who agreed to be treated with coblation or Nd:YAG laser for recurrent nasolacrimal duct obstruction after failed lacrimal Nd:YAG laser combined with silicone intubation. The visual analogue scale (VAS) pain grade was assessed at baseline, immediately, and 3 and 7 days after surgery. The degree of watering, lacrimal passage irrigation, and complications were also evaluated 1 week and 1, 3, and 6 months after surgery. Ninety-five patients who met the criteria for recurrent nasolacrimal duct obstruction from February 2018 to February 2019 were included in this study, with 46 patients receiving coblation and 49 patients Nd:YAG laser. The intraoperative and postoperative (3 days after surgery) VAS pain grades of the patients who received coblation were significantly lower than those who received Nd:YAG laser (P < 0.001). The number of patients in the coblation group who achieved complete clinical relief (no epiphora symptoms with fluent lacrimal passage irrigation) was significantly larger than that in the lacrimal Nd:YAG laser group at 1, 3, and 6 months after surgery (P = 0.033, P = 0.006, P = 0.003, respectively). During the follow-up period, there were no unexpected complications in either group. Compared with Nd:YAG laser, coblation performed well in alleviating pain and maintaining sustained disease relief and may therefore be an alternative to conventional laser or dacryocystorhinostomy surgery in the management of recurrent nasolacrimal duct obstruction.

Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis.

The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.

Field-induced lifetime enhancement of photorefractive gratings in a Mn:Fe:KTN crystal.

We report the lifetime enhancement of light-induced refractive index grating by applying a bias field during writing. In comparison with the lifetime of about 10 hours of the photorefractive grating prepared without a bias field, the lifetime of the grating with a 4 kV/cm bias field can be prolonged to 7.5 years, which is obtained from the dynamic behavior of grating visualized and monitored with digital holographic microscopy. The higher the bias field is applied, the longer the dark decay time of grating can be achieved. The enhanced lifetime of phase grating is attributed to polar nanoregions oriented by external field. This effect is of great significance for electro-holographic device applications.

Small-molecule probe for IBD risk variant GPR65 I231L alters cytokine signaling networks through positive allosteric modulation.

The proton-sensing heterotrimeric guanine nucleotide-binding protein-coupled receptor GPR65 is expressed in immune cells and regulates tissue homeostasis in response to decreased extracellular pH, which occurs in the context of inflammation and tumorigenesis. Genome-wide association studies linked GPR65 to several autoimmune and inflammatory diseases such as multiple sclerosis and inflammatory bowel disease (IBD). The loss-of-function GPR65 I231L IBD risk variant alters cellular metabolism, impairs protective tissue functions, and increases proinflammatory cytokine production. Hypothesizing that a small molecule designed to potentiate GPR65 at subphysiological pH could decrease inflammatory responses, we found positive allosteric modulators of GPR65 that engage and activate both human and mouse orthologs of the receptor. We observed that the chemical probe BRD5075 alters cytokine and chemokine programs in dendritic cells, establishing that immune signaling can be modulated by targeting GPR65. Our investigation offers improved chemical probes to further interrogate the biology of human GPR65 and its clinically relevant genetic variants.

Constitutively active autophagy in macrophages dampens inflammation through metabolic and post-transcriptional regulation of cytokine production.

Autophagy is an essential cellular process that is deeply integrated with innate immune signaling; however, studies that examine the impact of autophagic modulation in the context of inflammatory conditions are lacking. Here, using mice with a constitutively active variant of the autophagy gene Beclin1, we show that increased autophagy dampens cytokine production during a model of macrophage activation syndrome and in adherent-invasive Escherichia coli (AIEC) infection. Moreover, loss of functional autophagy through conditional deletion of Beclin1 in myeloid cells significantly enhances innate immunity in these contexts. We further analyzed primary macrophages from these animals with a combination of transcriptomics and proteomics to identify mechanistic targets downstream of autophagy. Our study reveals glutamine/glutathione metabolism and the RNF128/TBK1 axis as independent regulators of inflammation. Altogether, our work highlights increased autophagic flux as a potential approach to reduce inflammation and defines independent mechanistic cascades involved in this control.

Genetic vulnerability to Crohn's disease reveals a spatially resolved epithelial restitution program.

Effective tissue repair requires coordinated intercellular communication to sense damage, remodel the tissue, and restore function. Here, we dissected the healing response in the intestinal mucosa by mapping intercellular communication at single-cell resolution and integrating with spatial transcriptomics. We demonstrated that a risk variant for Crohn's disease, hepatocyte growth factor activator (HGFAC) Arg509His (R509H), disrupted a damage-sensing pathway connecting the coagulation cascade to growth factors that drive the differentiation of wound-associated epithelial (WAE) cells and production of a localized retinoic acid (RA) gradient to promote fibroblast-mediated tissue remodeling. Specifically, we showed that HGFAC R509H was activated by thrombin protease activity but exhibited impaired proteolytic activation of the growth factor macrophage-stimulating protein (MSP). In Hgfac R509H mice, reduced MSP activation in response to wounding of the colon resulted in impaired WAE cell induction and delayed healing. Through integration of single-cell transcriptomics and spatial transcriptomics, we demonstrated that WAE cells generated RA in a spatially restricted region of the wound site and that mucosal fibroblasts responded to this signal by producing extracellular matrix and growth factors. We further dissected this WAE cell-fibroblast signaling circuit in vitro using a genetically tractable organoid coculture model. Collectively, these studies exploited a genetic perturbation associated with human disease to disrupt a fundamental biological process and then reconstructed a spatially resolved mechanistic model of tissue healing.

Single-cell transcriptomic profiles reveal changes associated with BCG-induced trained immunity and protective effects in circulating monocytes.

Bacillus Calmette-Guérin (BCG) vaccine is one of the most widely used vaccines worldwide. In addition to protection against tuberculosis, BCG confers a degree of non-specific protection against other infections by enhancing secondary immune responses to heterologous pathogens, termed "trained immunity." To better understand BCG-induced immune reprogramming, we perform single-cell transcriptomic measurements before and after BCG vaccination using secondary immune stimulation with bacterial lipopolysaccharide (LPS). We find that BCG reduces systemic inflammation and identify 75 genes with altered LPS responses, including inflammatory mediators such as CCL3 and CCL4 that have a heightened response. Co-expression analysis reveals that gene modules containing these cytokines lose coordination after BCG. Other modules exhibit increased coordination, including several humanin nuclear isoforms that we confirm induce trained immunity in vitro. Our results link in vivo BCG administration to single-cell transcriptomic changes, validated in human genetics experiments, and highlight genes that are putatively responsible for non-specific protective effects of BCG.

Growth of Ga-doped ZnS nanowires constructed by self-assembled hexagonal platelets with excellent photocatalytic properties.

A new process for making single crystalline undoped and Ga-doped ZnS nanowires with simple evaporation and condensation procedures on Si and GaN is introduced. The process does not need additional catalysts or precursors. The growth mechanism is studied using transmission electron microscopy (TEM), x-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. TEM images show that the undoped ZnS nanowires exhibit an ordinary straight morphology, whereas the Ga-doped nanowires are composed of aligned hexagonal platelets, connected in the center into nanowires to maximize surface area. The Ga 2p3 and S 2p peaks in the XPS results confirm the presence of Ga doping in the form of Ga-S bonding. Raman spectra show that the ZnS LO peak is red-shifted from 349 to 347 cm(-1), indicative of a tensile stress caused by the Ga dopants. The growth mechanism and photocatalytic activity of the Ga-doped ZnS nanowires are discussed. We also demonstrate the excellent photocatalytic activity of Ga-doped ZnS nanowires as compared to those of undoped ZnS nanowires and Ga-doped ZnS nanosheets.

The retinoblastoma protein is required for Ras-induced oncogenic transformation.

Most human cancers involve either mutational activation of the Ras oncogenic pathway and/or inactivation of the retinoblastoma tumor suppressor (RB) pathway. Paradoxically, tumors that harbor Ras mutations almost invariably retain expression of a wild-type pRB protein. We explain this phenomenon by demonstrating that Ras-induced oncogenic transformation surprisingly depends on functional pRB protein. Cells lacking pRB are less susceptible to the oncogenic actions of H-RasV12 than wild-type cells and activated Ras has an inhibitory effect on the proliferation of pRB-deficient human tumor cells. In addition, depletion of pRB from Ras-transformed murine cells or human tumor cells that harbor Ras pathway mutations inhibits their proliferation and anchorage-independent growth. In sharp contrast to pRB-/- 3T3 cells, fibroblasts deficient in other pRB family members (p107 and p130) are more susceptible to Ras-mediated transformation than wild-type 3T3 cells. Moreover, loss of pRB in tumor cells harboring a Ras mutation results in increased expression of p107, and overexpression of p107 but not pRB strongly inhibits proliferation of these tumor cells. Together, these findings suggest that pRB and p107 have distinct roles in Ras-mediated transformation and suggest a novel tumor-suppressive role for p107 in the context of activated Ras.

Bioinformatics-based discovery and characterization of an AKT-selective inhibitor 9-chloro-2-methylellipticinium acetate (CMEP) in breast cancer cells.

AKT is a promising target for anticancer drug development. In this work, a bioinformatics approach was applied to search for AKT inhibitors based on the correlation analysis between phospho-Ser473 AKT expression level and the antiproliferative data of NCI small molecule compounds against NCI 60 cancer cell lines, the candidate compounds were then subject to AKT kinase assay. The possible effects of potent compound on PI3K/AKT, PDK1, and MAPK, its antiproliferative and apoptosis-inducing effects on breast cancer cells which have high-levels of AKT activation were assessed by Western blot analysis, cell viability assay, and apoptosis assay. One compound, CMEP (NSC632855, 9-chloro-2-methylellipticinium acetate) was identified with all three correlation algorithm, Pearson's, Sperman's, and Kendall's, showing a high-ranked correlation coefficient. CMEP inhibits only AKT, but does not inhibit PI3K, PDK1, or MAPK. CMEP also inhibits heregulin-induced AKT activation, does not inhibit heregulin-induced MAPK activation in MCF-7 breast cancer cells. Increased concentrations of ATP reverse the AKT inhibitory effect of CMEP. CMEP inhibits growth and induces apoptosis in breast cancer cells which have high-levels of AKT activation and lack functional PTEN; however, CMEP only shows a minimal activity in NIH3T3 cells which do not have AKT activation. In conclusion, a lead compound CMEP, as an AKT selective inhibitor has been identified started with a bioinformatics-based approach. CMEP inhibits growth and induces apoptosis in cancer cells which have high-levels of AKT activation and lack PTEN or harbor PTEN mutation.

Interaction and stabilization of X-linked inhibitor of apoptosis by Raf-1 protein kinase.

Raf-1 serine/threonine protein kinase plays an important role in cell growth, differentiation and cell survival. Recent reports using c-raf-1 gene-knockouts have observed MEK/ERK independent functions of Raf-1 in cell survival and protection from apoptosis. Raf-1 has also been shown to be involved in counteracting specific apoptotic pathways by restraining caspase activation, although the precise mechanism is unknown. XIAP is a potent inhibitor of apoptosis that blocks both the mitochondria and death receptor mediated pathways of apoptosis by directly binding to and inhibiting the initiator and effector caspases. In our efforts to understand the mechanism by which Raf-1 inhibits caspase activation, we discovered a novel interaction between Raf-1 and XIAP. In this study, we describe the physical interaction between Raf-1 and XIAP in vitro and in vivo in mammalian cells. We also demonstrate that Raf-1 phosphorylates XIAP in vitro and in vivo. Additionally, Raf-1 prevents XIAP degradation in response to different apoptotic triggers. Our studies identify XIAP as a new substrate of Raf-1 and provide potentially important insight into mechanisms underlying Raf-1 effects on cell survival.

Gene expression profile by inhibiting Raf-1 protein kinase in breast cancer cells.

Raf-1 protein serine-threonine kinase plays an important role in cell growth, proliferation, and cell survival. Previously, we and others have demonstrated that antisense raf oligonucleotide-mediated inhibition of Raf-1 expression leads to tumor growth arrest, radiosensitization and chemosensitization in vivo. Raf-1 inhibition is also associated with apoptotic cell death. In this study, we inhibited Raf-1 using an antisense raf oligonucleotide (AS-raf-ODN) to identify downstream targets of Raf-1 using microarray gene expression analysis. Treatment of MDA-MB-231 breast cancer cells with 250 nM AS-raf-ODN led to significant inhibition of Raf-1 protein (75.2 +/- 9.6%) and c-raf-1 mRNA levels (86.2 +/- 3.3%) as compared to untreated control cells. The lipofectin control or mismatch oligonucleotide had no effect on Raf-1 expression. To determine the changes in gene expression profiles that were due to inhibition of Raf-1, we simultaneously compared the gene expression patterns in AS-raf-ODN treated cells with untreated control cells and cells treated with lipofectin alone or MM-ODN. A total of 17 genes (4 upregulated and 13 down-regulated) including c-raf-1 were identified that were altered after AS-raf-ODN treatment. Functional clustering analysis revealed genes involved in apoptosis (Bcl-XL), cell adhesion (paxillin, plectin, Rho GDIalpha, CCL5), metabolism (GM2A, SLC16A3, PYGB), signal transduction (protein kinase C nu), and transcriptional regulation (HMGA1), and membrane-associated genes (GNAS, SLC16A3). Real-time PCR, Northern analysis and Western analysis confirmed the microarray findings. Our study provides insight into Raf-1 related signaling pathways and a model system to identify potential target genes.

Macrocyclization in the design of Grb2 SH2 domain-binding ligands exhibiting high potency in whole-cell systems.

While most SH2 domains bind phosphotyrosyl (pTyr) containing peptides in extended fashion, the growth factor receptor-bound protein 2 (Grb2) SH2 domain preferentially binds ligands in bend conformations. Accordingly, incorporation of bend-inducing functionality into synthetic ligands could potentially enhance their affinity for this SH2 domain. A macrocyclic tripeptide mimetic that contains a simplified pTyr surrogate lacking an alpha-nitrogen has recently been shown to exhibit high Grb2 SH2 domain-binding affinity in extracellular ELISA-based assays. However, the same compound is largely ineffective in whole-cell assays. It is known that acidic functionality originating from the alpha-nitrogen of pTyr residues or from the alpha-position of P0 pTyr mimetics not only increases binding affinity of peptides to Grb2 SH2 domains in extracellular assays but also enhances potency in cell-based systems. Such functionality is absent from the previously reported macrocycle. Therefore, the current study was undertaken to examine the effects of introducing carboxylic functionality at the pTyr mimetic alpha-position of macrocyclic ligands. It was found that such a modification not only enhanced Grb2 SH2 domain binding in extracellular assays but also conferred high efficacy in whole-cell systems. The most potent compound of the current study exhibited an IC(50) value of 0.002 microM in an extracellular ELISA-based assay, and in MDA-MB-453 cells, it both inhibited the association of Grb2 with p185(erbB-2) and exhibited antimitogenic effects with submicromolar IC50 values.

Molecular mechanism of gossypol-induced cell growth inhibition and cell death of HT-29 human colon carcinoma cells.

Gossypol, a male contraceptive drug, has been demonstrated to have antiproliferative and antimetastatic effects on many kinds of cancer cells in vitro. HT-29 human carcinoma cell line is one of the most susceptible cell lines to gossypol-induced cell death. Here, it is shown that treatment of HT-29 cells with gossypol not only induces cell cycle arrest on the G0/G1 phase, but also induces apoptosis. With a serial of Western blot analysis, it is revealed that gossypol-induced cell cycle arrest is involved in P21 up-regulation and cyclin D1 down-regulation; gossypol-induced apoptosis triggers down-regulation of anti-apoptosis Bcl-2 members: Bcl-X(L), Bag-1 and Mcl-1, up-regulation of pro-apoptosis Bcl-2 member Bak, activation of caspase-3, -6, -7, -8, and -9, up-regulation of Apaf-1, release of cytochrome c (cyto-c) from mitochondria, and activation of both DFF45 and PARP. Taken together, gossypol-induced cell death initiates extensive alterations of cell cycle and apoptosis proteins. Gossypol-induced apoptosis of HT-29 cells is through first the mitochondrial pathway, then the death receptor pathway, and the mitochondria pathway is, at least in part, involved in cyto-c release.

Development of a phosphatase-stable phosphotyrosyl mimetic suitably protected for the synthesis of high-affinity Grb2 SH2 domain-binding ligands.

Synthesis of (2R)-2-carboxymethyl-3-(4-(phosphonomethyl)phenyl) proprionic acid (5) in tert-butyl-protected form (6) and its use for the preparation of a Grb2 SH2 domain-directed tripeptide (8a) is reported. In extracellular ELISA-based assays, 8a exhibits potent Grb2 SH2 domain binding affinity (IC(50)=8 nM). Against cultures of MDA-MB-453 breast cancer cells, which over-express erbB-2 tyrosine kinase, 8a is also antimitogenic at concentrations equivalent to those required to inhibit intracellular association of Grb2 protein with phosphorylated p185(erbB-2) protein (IC(50)=8 microM). Analogue 6 may be useful for the preparation of a variety of phosphatase-stable SH2 domain-directed ligands.