Mechanistic Rationale to Target PTEN-Deficient Tumor Cells with Inhibitors of the DNA Damage Response Kinase ATM

Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response (DDR). ATM loss of function can produce a synthetic lethal phenotype in combination with tumor-associated mutations in FA/BRCA pathway components. In this study, we took an siRNA screening strategy to identify other tumor suppressors that, when inhibited, similarly sensitized cells to ATM inhibition. In this manner, we determined that PTEN and ATM were synthetically lethal when jointly inhibited. PTEN-deficient cells exhibited elevated levels of reactive oxygen species, increased endogenous DNA damage, and constitutive ATM activation. ATM inhibition caused catastrophic DNA damage, mitotic cell cycle arrest, and apoptosis specifically in PTEN-deficient cells in comparison with wild-type cells. Antioxidants abrogated the increase in DNA damage and ATM activation in PTEN-deficient cells, suggesting a requirement for oxidative DNA damage in the mechanism of cell death. Lastly, the ATM inhibitor KU-60019 was specifically toxic to PTEN mutant cancer cells in tumor xenografts and reversible by reintroduction of wild-type PTEN. Together, our results offer a mechanistic rationale for clinical evaluation of ATM inhibitors in PTEN-deficient tumors.

The significance of combining VEGFA, FLT1, and KDR expressions in colon cancer patient prognosis and predicting response to bevacizumab

Targeting angiogenesis through inhibition of the vascular endothelial growth factor (VEGF) pathway has been successful in the treatment of late stage colorectal cancer. However, not all patients benefit from inhibition of VEGF. Ras status is a powerful biomarker for response to anti-epidermal growth factor receptor therapy; however, an appropriate biomarker for response to anti-VEGF therapy is yet to be identified. VEGF and its receptors, FLT1 and KDR, play a crucial role in colon cancer progression; individually, these factors have been shown to be prognostic in colon cancer; however, expression of none of these factors alone was predictive of tumor response to anti-VEGF therapy. In the present study, we analyzed the expression levels of VEGFA, FLT1, and KDR in two independent colon cancer datasets and found that high expression levels of all three factors afforded a very poor prognosis. The observation was further confirmed in another independent colon cancer dataset, wherein high levels of expression of this three-gene signature was predictive of poor prognosis in patients with proficient mismatch repair a wild-type KRas status, or mutant p53 status. Most importantly, this signature also predicted tumor response to bevacizumab, an antibody targeting VEGFA, in a cohort of bevacizumab-treated patients. Since bevacizumab has been proven to be an important drug in the treatment of advanced stage colon cancer, our results suggest that the three-gene signature approach is valuable in terms of its prognostic value, and that it should be further evaluated in a prospective clinical trial to investigate its predictive value to anti-VEGF treatment.

A Secretory Leukocyte Protease Inhibitor Variant with Improved Activity against Lung Infection

Secretory leukocyte protease inhibitor (SLPI) is an important respiratory tract host defense protein, which is proteolytically inactivated by excessive neutrophil elastase (NE) during chronic Pseudomonas infection in the cystic fibrosis (CF) lung. We generated two putative NE-resistant variants of SLPI by site-directed mutagenesis, SLPI-A16G and SLPI-S15G-A16G, with a view to improving SLPI’s proteolytic stability. Both variants showed enhanced resistance to degradation in the presence of excess NE as well as CF patient sputum compared with SLPI-wild type (SLPI-WT). The ability of both variants to bind bacterial lipopolysaccharides and interact with nuclear factor-κB DNA binding sites was also preserved. Finally, we demonstrate increased anti-inflammatory activity of the SLPI-A16G protein compared with SLPI-WT in a murine model of pulmonary Pseudomonas infection. This study demonstrates the increased stability of these SLPI variants compared with SLPI-WT and their therapeutic potential as a putative anti-inflammatory treatment for CF lung disease.

Infrequent mutations of Archipelago (hAGO, hCDC4, Fbw7) in primary ovarian cancer

Objective: Archipelago (AGO, also known as hCdc4, Fbw7, or Sel-10) is an F-box containing component of the SCF complex implicated in the ubiquitination and proteolysis of cyclin E and c-Myc, and found to be mutated in 16% of endometrial carcinomas. We have previously reported somatic mutations in AGO in 3/10 ovarian cancer cell lines, but the frequency of such mutations in primary ovarian cancer is unknown.

Methods: The coding sequence of AGO was analyzed in 95 primary sporadic ovarian tumors and 16 cases of familial ovarian cancer, and correlated with levels of cyclin E and c-Myc protein expression. Constructs encoding mutations in AGO were transfected into an AGO-null cell line to directly test their ability to regulate cyclin E and c-Myc levels.

Results: Mutations were present in only 2 of 95 sporadic cases: a premature stop within the WD domain (471 Ter) and a missense change near the F-box (S245T). Both primary tumor specimens containing these mutations showed high levels of cyclin E and c-Myc, but reconstitution of an AGO-null cell line with constructs encoding these mutations showed 471 Ter to be inactive in regulating endogenous cyclin E and c-Myc levels, while the S245T mutant was indistinguishable from wild-type. No germ-line mutations were found in familial cases of ovarian cancer.

Conclusion: Somatic AGO mutations are infrequent in primary ovarian cancers and are unlikely to contribute to familial ovarian cancer. Reconstitution experiments, rather than measuring tumor levels of cyclin E and c-Myc, provide an effective approach to determine the functional significance of AGO mutations identified in human cancers.

Selective induction of apoptosis by the pyrrolo-1,5-benzoxazepine 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) in Leukemia cells occurs via the c-Jun NH2-terminal kinase-dependent phosphorylation and inactivation of Bcl-2 and Bcl-XL

Overexpression of the Bcl-2 proto-oncogene in tumor cells confers resistance against chemotherapeutic drugs. In this study, we describe how the novel pyrrolo-1,5-benzoxazepine compound 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) selectively induces apoptosis in Bcl-2-overexpressing cancer cells, whereas it shows no cytotoxic effect on normal peripheral blood mononuclear cells. PBOX-6 overcomes Bcl-2-mediated resistance to apoptosis in chronic myelogenous leukemia (CML) K562 cells by the time- and dose-dependent phosphorylation and inactivation of antiapoptotic Bcl-2 family members Bcl-2 and Bcl-XL. PBOX-6 also induces Bcl-2 phosphorylation and apoptosis in wild-type T leukemia CEM cells and cells overexpressing Bcl-2. This is in contrast to chemotherapeutic agents such as etoposide, actinomycin D, and ultraviolet irradiation, whereby overexpression of Bcl-2 confers resistance against apoptosis. In addition, PBOX-6 induces Bcl-2 phosphorylation and apoptosis in wild-type Jurkat acute lymphoblastic leukemia cells and cells overexpressing Bcl-2. However, Jurkat cells containing a Bcl-2 triple mutant, whereby the principal Bcl-2 phosphorylation sites are mutated to alanine, demonstrate resistance against Bcl-2 phosphorylation and apoptosis. PBOX-6 also induces the early and transient activation of c-Jun NH2-terminal kinase (JNK) in CEM cells. Inhibition of JNK activity prevents Bcl-2 phosphorylation and apoptosis, implicating JNK in the upstream signaling pathway leading to Bcl-2 phosphorylation. Collectively, these findings identify Bcl-2 phosphorylation and inactivation as a critical step in the apoptotic pathway induced by PBOX-6 and highlight its potential as an effective antileukemic agent.

Prevalence of the prothrombin G20210A mutation in the Irish populations:use of a novel polymerase chain reaction approach

The prothrombin G20210A polymorphism is associated with a threefold-increased risk of venous thrombosis. There is considerable variation in the reported prevalence of this polymorphism within normal populations, ranging from 0 to 6.5%. The prevalence within the Irish population has not been determined. A restriction fragment length polymorphism (RFLP)-based assay is commonly used for the detection of the prothrombin 20210A allele. This assay does not include a control restriction digest fragment and, consequently, failure of the enzyme activity or lack of addition of enzyme to the sample cannot be distinguished from wild-type prothrombin. We developed a RFLP-based assay, which incorporates an invariant digest site, resulting in the generation of a control digest fragment. Furthermore, we developed a nested polymerase chain reaction (PCR) method for the amplification and digestion of poor-quality or low-concentration DNA. In the Irish population studied, five of 385 (1.29%) were heterozygous and one patient was homozygous for the prothrombin 20210A polymorphism. This is the first reported data on an Irish or Celtic population and suggests that the allele frequency is similar to Anglo-Saxon populations. The nested PCR method successfully amplified and digested 100/100 (100%) of the archived samples; none of these samples could be analyzed by the standard single-round PCR method. In conclusion, nested PCR should be considered in the analysis of archived samples. Single-round PCR is appropriate for recently collected samples; however, an invariant control digest site should be incorporated in RFLP-based assays to validate the integrity of the digestion enzyme and limit the risk of false-negative results.

The TGF-β-inducible miR-23a cluster attenuates IFN-γ levels and antigen-specific cytotoxicity in human CD8+ T cells

Cytokine secretion and degranulation represent key components of CD8(+) T-cell cytotoxicity. While transcriptional blockade of IFN-γ and inhibition of degranulation by TGF-β are well established, we wondered whether TGF-β could also induce immune-regulatory miRNAs in human CD8(+) T cells. We used miRNA microarrays and high-throughput sequencing in combination with qRT-PCR and found that TGF-β promotes expression of the miR-23a cluster in human CD8(+) T cells. Likewise, TGF-β up-regulated expression of the cluster in CD8(+) T cells from wild-type mice, but not in cells from mice with tissue-specific expression of a dominant-negative TGF-β type II receptor. Reporter gene assays including site mutations confirmed that miR-23a specifically targets the 3'UTR of CD107a/LAMP1 mRNA, whereas the further miRNAs expressed in this cluster-namely, miR-27a and -24-target the 3'UTR of IFN-γ mRNA. Upon modulation of the miR-23a cluster by the respective miRNA antagomirs and mimics, we observed significant changes in IFN-γ expression, but only slight effects on CD107a/LAMP1 expression. Still, overexpression of the cluster attenuated the cytotoxic activity of antigen-specific CD8(+) T cells. These functional data thus reveal that the miR-23a cluster not only is induced by TGF-β, but also exerts a suppressive effect on CD8(+) T-cell effector functions, even in the absence of TGF-β signaling.

Glucocorticoid receptor and Klf4 co-regulate anti-inflammatory genes in keratinocytes

The glucocorticoid (GC) receptor (GR) and Kruppel-like factor Klf4 are transcription factors that play major roles in skin homeostasis. However, whether these transcription factors cooperate in binding genomic regulatory regions in epidermal keratinocytes was not known. Here, we show that in dexamethasone-treated keratinocytes GR and Klf4 are recruited to genomic regions containing adjacent GR and KLF binding motifs to control transcription of the anti-inflammatory genes Tsc22d3 and Zfp36. GR- and Klf4 loss of function experiments showed total GR but partial Klf4 requirement for full gene induction in response to dexamethasone. In wild type keratinocytes induced to differentiate, GR and Klf4 protein expression increased concomitant with Tsc22d3 and Zfp36 up-regulation. In contrast, GR-deficient cells failed to differentiate or fully induce Klf4, Tsc22d3 and Zfp36 correlating with increased expression of the epithelium-specific Trp63, a known transcriptional repressor of Klf4. The identified transcriptional cooperation between GR and Klf4 may determine cell-type specific regulation and have implications for developing therapies for skin diseases.

ER stress-mediated autophagy promotes Myc-dependent transformation and tumor growth

The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/-) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc-induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.

Tumor necrosis factor receptor expression and signaling in renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC), a tubular epithelial cell (TEC) malignancy, frequently secretes tumor necrosis factor (TNF). TNF signals via two distinct receptors (TNFRs). TNFR1, expressed in normal kidney primarily on endothelial cells, activates apoptotic signaling kinase 1 and nuclear factor-kappaB (NF-kappaB) and induces cell death, whereas TNFR2, inducibly expressed on endothelial cells and on TECs by injury, activates endothelial/epithelial tyrosine kinase (Etk), which trans-activates vascular endothelial growth factor receptor 2 (VEGFR2) to promote cell proliferation. We investigated TNFR expression in clinical samples and function in short-term organ cultures of ccRCC tissue treated with wild-type TNF or specific muteins selective for TNFR1 (R1-TNF) or TNFR2 (R2-TNF). There is a significant increase in TNFR2 but not TNFR1 expression on malignant TECs that correlates with increasing malignant grade. In ccRCC organ cultures, R1-TNF increases TNFR1, activates apoptotic signaling kinase and NF-kappaB, and promotes apoptosis in malignant TECs. R2-TNF increases TNFR2, activates NF-kappaB, Etk, and VEGFR2 and increases entry into the cell cycle. Wild-type TNF induces both sets of responses. R2-TNF actions are blocked by pretreatment with a VEGFR2 kinase inhibitor. We conclude that TNF, acting through TNFR2, is an autocrine growth factor for ccRCC acting via Etk-VEGFR2 cross-talk, insights that may provide a more effective therapeutic approach to this disease.

Clathrin is spindle-associated but not essential for mitosis

BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

METHODOLOGY/PRINCIPAL FINDINGS: Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

CONCLUSIONS/SIGNIFICANCE: We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.

Inhibition of endosome fusion by wortmannin persists in the presence of activated Rab5

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5(Q79L) or of a xanthosine triphosphate-binding mutant, rab5(D136N), in the presence of the nonhydrolysable analogue XTPgammaS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPgammaS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5(Q79L), beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPgammaS.

Synapsin- and actin-dependent frequency enhancement in mouse hippocampal mossy fiber synapses

The synapsin proteins have different roles in excitatory and inhibitory synaptic terminals. We demonstrate a differential role between types of excitatory terminals. Structural and functional aspects of the hippocampal mossy fiber (MF) synapses were studied in wild-type (WT) mice and in synapsin double-knockout mice (DKO). A severe reduction in the number of synaptic vesicles situated more than 100 nm away from the presynaptic membrane active zone was found in the synapsin DKO animals. The ultrastructural level gave concomitant reduction in F-actin immunoreactivity observed at the periactive endocytic zone of the MF terminals. Frequency facilitation was normal in synapsin DKO mice at low firing rates (approximately 0.1 Hz) but was impaired at firing rates within the physiological range (approximately 2 Hz). Synapses made by associational/commissural fibers showed comparatively small frequency facilitation at the same frequencies. Synapsin-dependent facilitation in MF synapses of WT mice was attenuated by blocking F-actin polymerization with cytochalasin B in hippocampal slices. Synapsin III, selectively seen in MF synapses, is enriched specifically in the area adjacent to the synaptic cleft. This may underlie the ability of synapsin III to promote synaptic depression, contributing to the reduced frequency facilitation observed in the absence of synapsins I and II.

Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes

To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

Innate Lymphoid Cells are the Predominant Source of Interleukin-17A During the Early Pathogenesis of Acute Respiratory Distress Syndrome

Rationale: IL-17A is purported to help drive early pathogenesis in acute respiratory distress syndrome (ARDS) by enhancing neutrophil recruitment. Whilst IL-17A is the archetypal cytokine of T helper (Th)17 cells, it is produced by a number of lymphocytes, the source during ARDS being unknown.

Objectives: To identify the cellular source and the role of IL17A in the early phase of lung injury

Methods: Lung injury was induced in WT (C57BL/6) and IL-17 KO mice with aerosolised LPS (100 µg) or Pseudomonas aeruginosa infection. Detailed phenotyping of the cells expressing RORγt, the transcriptional regulator of IL-17 production, in the mouse lung at 24 hours was carried out by flow cytometry.

Measurement and Main Results: A 100-fold reduction in neutrophil infiltration was observed in the lungs of the IL-17A KO compared to wild type (WT) mice. The majority of RORγt+ cells in the mouse lung were the recently identified type 3 innate lymphoid cells (ILC3). Detailed characterisation revealed these pulmonary ILC3s (pILC3s) to be discrete from those described in the gut. The critical role of these cells was verified by inducing injury in Rag2 KO mice which lack T cells but retain ILCs. No amelioration of pathology was observed in the Rag2 KO mice.

Conclusions: IL-17 is rapidly produced during lung injury and significantly contributes to early immunopathogenesis. This is orchestrated largely by a distinct population of pILC3 cells. Modulation of pILC3s’ activity may potentiate early control of the inflammatory dysregulation seen in ARDS, opening up new therapeutic targets.