CX-5461 Treatment Leads to Cytosolic DNA-Mediated STING Activation in Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the deadliest of the gynecologic malignancies, with an overall survival rate of <30%. Recent research has suggested that targeting RNA polymerase I (POL I) with small-molecule inhibitors may be a viable therapeutic approach to combating EOC, even when chemoresistance is present. CX-5461 is one of the most promising POL I inhibitors currently being investigated, and previous reports have shown that CX-5461 treatment induces DNA damage response (DDR) through ATM/ATR kinase. Investigation into downstream effects of CX-5461 led us to uncovering a previously unreported phenotype. Treatment with CX-5461 induces a rapid accumulation of cytosolic DNA. This accumulation leads to transcriptional upregulation of 'STimulator of Interferon Genes' (STING) in the same time frame, phosphorylation of IRF3, and activation of type I interferon response both in vitro and in vivo. This activation is mediated and dependent on cyclic GMP-AMP synthase (cGAS). Here, we show THAT CX-5461 leads to an accumulation of cytosolic dsDNA and thereby activates the cGAS-STING-TBK1-IRF3 innate immune pathway, which induces type I IFN. CX-5461 treatment-mediated immune activation may be a powerful mechanism of action to exploit, leading to novel drug combinations with a chance of increasing immunotherapy efficacy, possibly with some cancer specificity limiting deleterious toxicities.

Aurora B Kinase Promotes CHIP-Dependent Degradation of HIF1α in Prostate Cancer Cells.

Hypoxia is a major factor in tumor progression and resistance to therapies, which involves elevated levels of the transcription factor HIF1α. Here, we report that prostate tumor xenografts express high levels of HIF1α and show greatly enhanced growth in response to knockdown of the E3 ligase CHIP (C-terminus of Hsp70-interacting protein). In multiple human prostate cancer cell lines under hypoxia, taxol treatment induces the degradation of HIF1α, and this response is abrogated by knockdown of CHIP, but not by E3 ligase VHL or RACK1. HIF1α degradation is accompanied by loss of function, evidenced by reduced expression of HIF1α-dependent genes. CHIP-dependent HIF1α degradation also occurs in cells arrested in mitosis by nocodazole instead of taxol. Mitotic kinase Aurora B activity is required for taxol-induced HIF1α degradation. Purified Aurora B directly phosphorylates HIF1α at multiple sites, and these modifications enhance its polyubiquitination by CHIP in a purified reconstituted system. Our results show how activation of Aurora B promotes CHIP-dependent degradation of HIF1α in prostate cancer cells. This new knowledge may affect the use of mitotic kinase inhibitors and open new approaches for treatment of hypoxic prostate tumors.

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance.

Lung cancer resists radiotherapy, making it one of the deadliest forms of cancer. Here, we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically, ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor, p21 protein, is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene (CDKN1A) in CHIP knockdown cells. Conversely, overexpression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone HSP70. These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity, thus suggesting a novel strategy for the treatment of lung cancer.Implications: The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. Mol Cancer Res; 15(6); 651-9. ©2017 AACR.

Essential role of class II phosphatidylinositol-3-kinase-C2α in sphingosine 1-phosphate receptor-1-mediated signaling and migration in endothelial cells.

The phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes, including cell proliferation, migration, and vesicular trafficking, through catalyzing 3'-phosphorylation of phosphoinositides. In contrast to class I PI3Ks, including p110α and p110ß, functional roles of class II PI3Ks, comprising PI3K-C2α, PI3K-C2ß, and PI3K-C2γ, are little understood. The lysophospholipid mediator sphingosine 1-phosphate (S1P) plays the important roles in regulating vascular functions, including vascular formation and barrier integrity, via the G-protein-coupled receptors S1P(1-3). We studied the roles of PI3K-C2α in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK, and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P(1) in ECs. Knockdown of either PI3K-C2α or class I p110ß markedly inhibited S1P-induced migration, lamellipodium formation, and tube formation, whereas that of p110α or Vps34 did not. Only p110ß was necessary for S1P-iduced Akt activation, but both PI3K-C2α and p110ß were required for Rac1 activation. FRET imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns 3-phosphate (PtdIns(3)P)-enriched endosomes. Knockdown of PI3K-C2α but not p110ß markedly reduced PtdIns(3)P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2α but not p110ß suppressed S1P-induced S1P(1) internalization into PtdIns(3)P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P(1) internalization, Rac1 activation, migration, and tube formation. These observations indicate that PI3K-C2α plays the crucial role in S1P(1) internalization into the intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in ECs.

Endothelial PI3K-C2α, a class II PI3K, has an essential role in angiogenesis and vascular barrier function.

The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles; however, its functional role is not well understood. Global or endothelial-cell-specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in endothelial cells resulted in a decrease in the number of PI3-phosphate-enriched endosomes, impaired endosomal trafficking, defective delivery of VE-cadherin to endothelial cell junctions and defective junction assembly. PI3K-C2α knockdown also impaired endothelial cell signaling, including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. Together, the effects of PI3K-C2α knockdown led to defective endothelial cell migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deficiency in vivo suppressed postischemic and tumor angiogenesis and diminished vascular barrier function with a greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α has a crucial role in vascular formation and barrier integrity and represents a new therapeutic target for vascular disease.

Functional analysis of VopF activity required for colonization in Vibrio cholerae.

Vibrio cholerae, a Gram-negative facultative pathogen, is the etiologic agent for the diarrheal disease cholera. We previously characterized a clinical isolate, AM-19226, that translocates a type III secretion system (T3SS) effector protein with actin-nucleating activity, VopF, into the host cells. From comparative genomic studies, we identified a divergent T3SS island in additional isolates which possess a VopF homolog, VopN. Unlike the VopF-mediated protrusion formation, VopN localizes to stress fiber in host cells similarly to VopL, which is present in the pandemic strain of Vibrio parahaemolyticus. Chimera and yeast two-hybrid studies indicated that the amino-terminal regions of VopF and VopN proteins interact with distinct host cell factors. We determined that AM-19226-infected cells are arrested at S phase of the cell cycle and that VopF/VopN are antiapoptotic factors. To understand how VopF may contribute to the pathogenesis of AM-19226, we examined the effect of VopF in an in vitro polarized-epithelial model and an in vivo adult rabbit diarrheal model. Within the T3SS pathogenicity island is VopE, a homolog of YopE from Yersinia, which has been shown to loosen tight junctions. In polarized intestinal epithelia, VopF and VopE compromised the integrity of tight junctions by inducing cortical actin depolymerization and aberrant localization of the tight-junction protein ZO-1. An assay for pathogenicity in the adult rabbit diarrhea model suggested that these effectors are involved in eliciting the diarrheal response in infected rabbits.

Genetic characterization of hepatitis B virus in peripheral blood leukocytes: evidence for selection and compartmentalization of viral variants with the immune escape G145R mutation.

The compartmentalization of viral variants in distinct host tissues is a frequent event in many viral infections. Although hepatitis B virus (HBV) classically is considered hepatotropic, it has strong lymphotropic properties as well. However, unlike other viruses, molecular evolutionary studies to characterize HBV variants in compartments other than hepatocytes or sera have not been performed. The present work attempted to characterize HBV sequences from the peripheral blood leukocytes (PBL) of a large set of subjects, using advanced molecular biology and computational methods. The results of this study revealed the exclusive compartmentalization of HBV subgenotype Ae/A2-specific sequences with a potent immune escape G145R mutation in the PBL of the majority of the subjects. Interestingly, entirely different HBV genotypes/subgenotypes (C, D, or Aa/A1) were found to predominate in the sera of the same study populations. These results suggest that subgenotype Ae/A2 is selectively archived in the PBL, and the high prevalence of G145R indicates high immune pressure and high evolutionary rates of HBV DNA in the PBL. The results are analogous to available literature on the compartmentalization of other viruses. The present work thus provides evidence in favor of the compartment-specific abundance, evolution, and emergence of the potent immune escape mutant. These findings have important implications in the field of HBV molecular epidemiology, transmission, transfusion medicine, organ transplantation, and vaccination strategies.

Frequency and distribution of hepatitis B virus genotypes among eastern Indian voluntary blood donors: Association with precore and basal core promoter mutations.

AIM: To screen hepatitis B virus (HBV) genotypes and associated basal core promoter (BCP; T1762/A1764) and precore (PreC; A1896) mutations among the HBV surface antigen (HBsAg) positive voluntary blood donors in eastern India. METHODS: HBV genotypes, BCP and PreC mutations of 141 HBsAg positive voluntary blood donors were determined by the restriction fragment length polymorphism (RFLP) method and a phylogenetic tree was constructed from surface (S) gene region sequences of representative HBsAg positive donors to confirm the results. RESULTS: HBV/D was the most predominant (79, 56.0%) genotype followed by HBV/C (33, 23.4%) and HBV/A (29, 20.6%). HBV/C infected blood donors are mostly young (18-25 years). The occurrence of BCP mutation was found to be significantly higher in HBV/C (24, 72.7%) than in HBV/A (7, 24.1%, P < 0.001) and HBV/D (17, 21.5%, P < 0.001), whereas PreC mutation was more frequent in HBV/D (28, 35.4%) than in HBV/C (9, 27.3%). However, the simultaneous presence of BCP and PreC mutations was more common in HBV/C (8/33, 24.2%), followed by HBV/D (6/79, 7.6%). CONCLUSION: In addition to HBV/D and HBV/A, a significant proportion of HBV/C (23.4%) was also present among the voluntary blood donors from eastern India, most frequently in the 18-25 year age group. BCP mutation was more common in HBV/C infected donors.

Acquisition of classical CTX prophage from Vibrio cholerae O141 by El Tor strains aided by lytic phages and chitin-induced competence.

The El Tor biotype of Vibrio cholerae O1, causing the current seventh pandemic of cholera, has replaced the classical biotype, which caused the sixth pandemic. The CTX prophages encoding cholera toxin in the two biotypes have distinct repressor (rstR) genes. Recently, new variants of El Tor strains that carry the classical type (CTX(class)) prophage have emerged. These "hybrid" strains apparently originate through lateral gene transfer and recombination events. To explore possible donors of the CTX(class) prophage and its mode of transfer, we tested environmental V. cholerae isolates for the presence of CTX(class) prophage and mobility of the phage genome. Of the 272 environmental V. cholerae isolates tested, 6 were found to carry the CTX(class) prophage; all of these belonged to the O141 serogroup. These O141 strains were unable to produce infectious CTX(class) phage or to transmit the prophage to recipient strains in the mouse model of infection; however, the CTX(class) prophage was acquired by El Tor strains when cultured with the O141 strains in microcosms composed of filtered environmental water, a chitin substrate, and a V. cholerae O141-specific bacteriophage. The CTX(class) prophage either coexisted with or replaced the resident CTX(ET) prophage, resulting in El Tor strains with CTX genotypes similar to those of the naturally occurring hybrid strains. Our results support a model involving phages and natural chitin substrate in the emergence of new variants of pathogenic V. cholerae. Furthermore, the O141 strains apparently represent an alternative reservoir of the CTX(class) phage genome, because the classical V. cholerae O1 strains are possibly extinct.

Distribution of genes for virulence and ecological fitness among diverse Vibrio cholerae population in a cholera endemic area: tracking the evolution of pathogenic strains.

The pathogenic strains of Vibrio cholerae that cause acute enteric infections in humans are derived from environmental nonpathogenic strains. To track the evolution of pathogenic V. cholerae and identify potential precursors of new pathogenic strains, we analyzed 324 environmental or clinical V. cholerae isolates for the presence of diverse genes involved in virulence or ecological fitness. Of 251 environmental non-O1, non-O139 strains tested, 10 (3.9%) carried the toxin coregulated pilus (TCP) pathogenicity island encoding TCPs, and the CTX prophage encoding cholera toxin, whereas another 10 isolates carried the TCP island alone, and were susceptible to transduction with CTX phage. Most V. cholerae O1 and O139 strains carried these two major virulence determinants, as well as the Vibrio seventh pandemic islands (VSP-1 and VSP-2), whereas 23 (9.1%) non-O1, non-O139 strains carried several VSP island genes, but none carried a complete VSP island. Conversely, 30 (11.9%) non-O1, non-O139 strains carried type III secretion system (TTSS) genes, but none of 63 V. cholerae O1 or O139 strains tested were positive for TTSS. Thus, the distribution of major virulence genes in the non-O1, non-O139 serogroups of V. cholerae is largely different from that of the O1 or O139 serogroups. However, the prevalence of putative accessory virulence genes (mshA, hlyA, and RTX) was similar in all strains, with the mshA being most prevalent (98.8%) followed by RTX genes (96.2%) and hlyA (94.6%), supporting more recent assumptions that these genes imparts increased environmental fitness. Since all pathogenic strains retain these genes, the epidemiological success of the strains presumably depends on their environmental persistence in addition to the ability to produce major virulence factors. Potential precursors of new pathogenic strains would thus require to assemble a combination of genes for both ecological fitness and virulence to attain epidemiological predominance.

Diagnostic evaluation of childhood cervical lymphadenopathy by fine needle aspiration cytology.

Lymphadenopathy is an age old affliction of mankind. It is one of the very common presentations in clinical practice. The present study was carried out to evaluate the merits and demerits of fine needle aspiration cytology (FNAC) as a diagnostic procedure in childhood lymphadenopathy in comparison to open surgical biopsy. Altogether 70 children with lymphadenopathy in the age group of up to 12 years were selected for FNAC. Only 38 could be motivated for open surgical biopsy. Out of 38 cases, FNAC was consistent with histopathology in 33 cases, thus giving a diagnostic accuracy or percentage of agreement-86.8%. The diagnostic accuracy of FNAC, sensitivity, specificity, positive predictive values of FNAC in the present study were fairly high ranging from 80%-100%. FNAC gave false positive diagnosis in 13.2% cases and false negative result in 13.2% cases.

Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment.

The factors that enhance the waterborne spread of bacterial epidemics and sustain the epidemic strain in nature are unclear. Although the epidemic diarrheal disease cholera is known to be transmitted by water contaminated with pathogenic Vibrio cholerae, routine isolation of pathogenic strains from aquatic environments is challenging. Here, we show that conditionally viable environmental cells (CVEC) of pathogenic V. cholerae that resist cultivation by conventional techniques exist in surface water as aggregates (biofilms) of partially dormant cells. Such CVEC can be recovered as fully virulent bacteria by inoculating the water into rabbit intestines. Furthermore, when V. cholerae shed in stools of cholera patients are inoculated in environmental water samples in the laboratory, the cells exhibit characteristics similar to CVEC, suggesting that CVEC are the infectious form of V. cholerae in water and that CVEC in nature may have been derived from human cholera stools. We also observed that stools from cholera patients contain a heterogeneous mixture of biofilm-like aggregates and free-swimming planktonic cells of V. cholerae. Estimation of the relative infectivity of these different forms of V. cholerae cells suggested that the enhanced infectivity of V. cholerae shed in human stools is largely due to the presence of clumps of cells that disperse in vivo, providing a high dose of the pathogen. The results of this study support a model of cholera transmission in which in vivo-formed biofilms contribute to enhanced infectivity and environmental persistence of pathogenic V. cholerae.

An improved technique for isolation of environmental Vibrio cholerae with epidemic potential: monitoring the emergence of a multiple-antibiotic-resistant epidemic strain in Bangladesh.

Predicting cholera epidemics through monitoring the environment for the presence of pathogenic Vibrio cholerae is complicated by the presence in water of a large number of mostly nonpathogenic V. cholerae strains. V. cholerae strains causing recent cholera epidemics in Bangladesh carry the sulfamethoxazole-trimethoprim (SXT) element, which encodes resistance to several antibiotics. Here, we show that the use of a culture medium containing streptomycin, sulfamethoxazole, and trimethoprim (the antibiotic selection technique [AST]) can significantly enhance the isolation of environmental V. cholerae O1 with epidemic potential (P<.001). The AST was also used to monitor the recent emergence and spread of a new multiple-antibiotic-resistant strain of V. cholerae in Bangladesh. The results of this study support the hypothesis that pre-epidemic amplification of pathogenic V. cholerae occurs in the human host and leads to the start of an epidemic cycle dominated by a single clone of V. cholerae that spreads rapidly through environmental waters.