Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer

Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.

Respiratory Infections Cause the Release of Extracellular Vesicles: Implications in Exacerbation of Asthma/COPD

Background: Infection-related exacerbations of respiratory diseases are a major health concern; thus understanding the mechanisms driving them is of paramount importance. Despite distinct inflammatory profiles and pathological differences, asthma and COPD share a common clinical facet: raised airway ATP levels. Furthermore, evidence is growing to suggest that infective agents can cause the release of extracellular vesicle (EVs) in vitro and in bodily fluids. ATP can evoke the P2X7/caspase 1 dependent release of IL-1β/IL-18 from EVs; these cytokines are associated with neutrophilia and are increased during exacerbations. Thus we hypothesized that respiratory infections causes the release of EVs in the airway and that the raised ATP levels, present in respiratory disease, triggers the release of IL-1β/IL-18, neutrophilia and subsequent disease exacerbations.

Methods: To begin to test this hypothesis we utilised human cell-based assays, ex vivo murine BALF, in vivo pre-clinical models and human samples to test this hypothesis.

Results: Data showed that in a murine model of COPD, known to have increased airway ATP levels, infective challenge causes exacerbated inflammation. Using cell-based systems, murine models and samples collected from challenged healthy subjects, we showed that infection can trigger the release of EVs. When exposed to ATP the EVs release IL-1b/IL-18 via a P2X₇/caspase-dependent mechanism. Furthermore ATP challenge can cause a P2X₇ dependent increase in LPS-driven neutrophilia.

Conclusions: This preliminary data suggests a possible mechanism for how infections could exacerbate respiratory diseases and may highlight a possible signalling pathway for drug discovery efforts in this area.

Fibroblast growth factor receptor 4 (FGFR4): a targetable regulator of drug resistance in colorectal cancer

The discovery of underlying mechanisms of drug resistance, and the development of novel agents to target these pathways, is a priority for patients with advanced colorectal cancer (CRC). We previously undertook a systems biology approach to design a functional genomic screen and identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of drug resistance. The aim of this study was to examine the role of FGFR4 in drug resistance using RNAi and the small-molecule inhibitor BGJ398 (Novartis). We found that FGFR4 is highly expressed at the RNA and protein levels in colon cancer tumour tissue compared with normal colonic mucosa and other tumours. Silencing of FGFR4 reduced cell viability in a panel of colon cancer cell lines and increased caspase-dependent apoptosis. A synergistic interaction was also observed between FGFR4 silencing and 5-fluorouracil (5-FU) and oxaliplatin chemotherapy in colon cancer cell lines. Mechanistically, FGFR4 silencing decreased activity of the pro-survival STAT3 transcription factor and expression of the anti-apoptotic protein c-FLIP. Furthermore, silencing of STAT3 resulted in downregulation of c-FLIP protein expression, suggesting that FGFR4 may regulate c-FLIP expression via STAT3. A similar phenotype and downstream pathway changes were observed following FGFR4 silencing in cell lines resistant to 5-FU, oxaliplatin and SN38 and upon exposure of parental cells to the FGFR small-molecule inhibitor BGJ398. Our results indicate that FGFR4 is a targetable regulator of chemo-resistance in CRC, and hence inhibiting FGFR4 in combination with 5-FU and oxaliplatin is a potential therapeutic strategy for this disease.

Efficient Drug Delivery and Induction of Apoptosis in Colorectal Tumors Using a Death Receptor 5-Targeted Nanomedicine

Death Receptor 5 (DR5) is a pro-apoptotic cell-surface receptor that is a potential therapeutic target in cancer. Despite the potency of DR5-targeting agents in preclinical models, the translation of these effects into the clinic remains disappointing. Herein, we report an alternative approach to exploiting DR5 tumor expression using antibody-targeted, chemotherapy-loaded nanoparticles. We describe the development of an optimized polymer-based nanotherapeutic incorporating both a functionalized polyethylene glycol (PEG) layer and targeting antibodies to limit premature phagocytic clearance whilst enabling targeting of DR5-expressing tumor cells. Using the HCT116 colorectal cancer model, we show that following binding to DR5, the nanoparticles activate caspase 8, enhancing the anti-tumor activity of the camptothecin payload both in vitro and in vivo. Importantly, the combination of nanoparticle-induced DR5 clustering with camptothecin delivery overcomes resistance to DR5-induced apoptosis caused by loss of BAX or overexpression of anti-apoptotic FLIP. This novel approach may improve the clinical activity of DR5-targeted therapeutics while increasing tumor-specific delivery of systemically toxic chemotherapeutics.Molecular Therapy (2014); doi:10.1038/mt.2014.137.

HPV16 activates the AIM2 inflammasome in keratinocytes

Human papillomaviruses (HPV) are double-stranded DNA viruses, which selectively infect keratinocytes in stratified epithelia. After an initial infection, many patients clear HPV. In some patients, however, HPV persist, and dysfunctional innate immune responses to HPV infection could be involved in the ineffective clearing of these viruses. In this study, the mechanisms of HPV-induced immune responses in keratinocytes were investigated. Binding of viral DNA leads to AIM2 inflammasome activation and IL-1β release, while IFI16 activation results in IFN-β release. Using immunohistochemistry, AIM2 and IFI16-two recently identified sensors for cytosolic DNA-were also detected in HPV positive skin lesions. CISH stainings further confirmed the presence of cytosolic HPV16 DNA in biopsy samples. Moreover, active IL-1β and cleaved caspase-1 were detected in HPV infected skin, suggesting inflammasome activation by viral DNA. In subsequent functional studies, HPV16 DNA triggered IL-1β and IL-18 release via the AIM2 inflammasome in normal human keratinocytes. Although HPV DNA did not induce IFN-β in keratinocytes, IFN-β secretion was observed when AIM2 was blocked. Meanwhile, blocking of IFI16 increased HPV16 DNA-induced IL-1β, but not IL-18, secretion. These findings suggest crosstalk between IFI16 and AIM2 in the immune response to HPV DNA. In sum, novel aspects concerning HPV-induced innate immune responses were identified. Eventually, understanding the mechanisms of HPV-induced inflammasome activation could lead to the development of novel strategies for the prevention and treatment of HPV infections.

Endoplasmic reticulum targeting in Ewing’s sarcoma by the alkylphospholipid analog edelfosine

Ewing's sarcoma (ES) is the second most common bone cancer in children and young people. Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is the prototype of a family of synthetic antitumor compounds, collectively known as alkylphospholipid analogs (APLs). We have found that APLs ranked edelfosine>perifosine>erucylphosphocholine>miltefosine for their capacity to promote apoptosis in ES cells. Edelfosine accumulated in the endoplasmic reticulum (ER) and triggered an ER stress response that eventually led to caspase-dependent apoptosis in ES cells. This apoptotic response involved mitochondrial-mediated processes, with cytochrome c release, caspase-9 activation and generation of reactive oxygen species. Edelfosine-induced apoptosis was also dependent on sustained c-Jun NH2-terminal kinase activation. Oral administration of edelfosine showed a potent in vivo antitumor activity in an ES xenograft animal model. Histochemical staining gave evidence for ER stress response and apoptosis in the ES tumors isolated from edelfosine-treated mice. Edelfosine showed a preferential action on ES tumor cells as compared to non-transformed osteoblasts, and appeared to be well suited for combination therapy regimens. These results demonstrate in vitro and in vivo antitumor activity of edelfosine against ES cells that is mediated by caspase activation and ER stress, and provide the proof of concept for a putative edelfosine-and ER stress-mediated approach for ES treatment.

Interactions of a non-thermal atmospheric pressure plasma effluent with PC-3 prostate cancer cells

The effect of a radio-frequency driven, microscale non thermal atmospheric pressure plasma jet operated in helium with vol. 0.3% molecular oxygen gas admixture, on PC-3 prostate cancer cells has been investigated. The viability of cells exposed to the plasma was found to decrease with increasing plasma exposure time, with apoptosis through caspase and PARP cleavage being observed. High concentrations of nitrite and nitrate were detected in growth media exposed to the plasma and were found to increase in a time dependent manner post exposure. This indicates a slow release of reactive nitrogen species into the growth media, which is likely to influence cellular response to plasma exposure.

HDAC Inhibition Overcomes Acute Resistance to MEK Inhibition in BRAF-Mutant Colorectal Cancer by Downregulation of c-FLIPL

Purpose: Activating mutations in the BRAF oncogene are found in 8% to 15% of colorectal cancer patients and have been associated with poor survival. In contrast with BRAF-mutant (MT) melanoma, inhibition of the MAPK pathway is ineffective in the majority of BRAFMT colorectal cancer patients. Therefore, identification of novel therapies for BRAFMT colorectal cancer is urgently needed.

Experimental Design: BRAFMT and wild-type (WT) colorectal cancer models were assessed in vitro and in vivo. Small-molecule inhibitors of MEK1/2, MET, and HDAC were used, overexpression and siRNA approaches were applied, and cell death was assessed by flow cytometry, Western blotting, cell viability, and caspase activity assays.

Results: Increased c-MET-STAT3 signaling was identified as a novel adaptive resistance mechanism to MEK inhibitors (MEKi) in BRAFMT colorectal cancer models in vitro and in vivo. Moreover, MEKi treatment resulted in acute increases in transcription of the endogenous caspase-8 inhibitor c-FLIPL in BRAFMT cells, but not in BRAFWT cells, and inhibition of STAT3 activity abrogated MEKi-induced c-FLIPL expression. In addition, treatment with c-FLIP–specific siRNA or HDAC inhibitors abrogated MEKi-induced upregulation of c-FLIPL expression and resulted in significant increases in MEKi-induced cell death in BRAFMT colorectal cancer cells. Notably, combined HDAC inhibitor/MEKi treatment resulted in dramatically attenuated tumor growth in BRAFMT xenografts.

Conclusions: Our findings indicate that c-MET/STAT3-dependent upregulation of c-FLIPL expression is an important escape mechanism following MEKi treatment in BRAFMT colorectal cancer. Thus, combinations of MEKi with inhibitors of c-MET or c-FLIP (e.g., HDAC inhibitors) could be potential novel treatment strategies for BRAFMT colorectal cancer.

Inflammatory and cytotoxic effects of acrolein, nicotine, acetylaldehyde and cigarette smoke extract on human nasal epithelial cells

Background: Cigarette smoke induces a pro-inflammatory response in airway epithelial cells but it is not clear which of the various chemicals contained within cigarette smoke (CS) should be regarded as predominantly responsible for these effects. We hypothesised that acrolein, nicotine and acetylaldehyde, important chemicals contained within volatile cigarette smoke in terms of inducing inflammation and causing addiction, have immunomodulatory effects in primary nasal epithelial cell cultures (PNECs).

Methods: PNECs from 19 healthy subjects were grown in submerged cultures and were incubated with acrolein, nicotine or acetylaldehyde prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide (PA LPS). Experiments were repeated using cigarette smoke extract (CSE) for comparison. IL-8 was measured by ELISA, activation of NF-κB by ELISA and Western blotting, and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method.

Results: CSE was pro-inflammatory after a 24 h exposure and 42% of cells were apoptotic or necrotic after this exposure time. Acrolein was pro-inflammatory for the PNEC cultures (30 μM exposure for 4 h inducing a 2.0 fold increase in IL-8 release) and also increased IL-8 release after stimulation with PA LPS. In contrast, nicotine had anti-inflammatory properties (0.6 fold IL-8 release after 50 μM exposure to nicotine for 24 h), and acetylaldehyde was without effect. Acrolein and nicotine had cellular stimulatory and anti-inflammatory effects respectively, as determined by NF-κB activation. Both chemicals increased levels of cleaved caspase 3 and induced cell death.

Conclusions: Acrolein is pro-inflammatory and nicotine anti-inflammatory in PNEC cultures. CSE induces cell death predominantly by apoptotic mechanisms. 

Protein expression differences between lung adenocarcinoma and squamous cell carcinoma with brain metastasis

AIM: We investigated tissue biomarkers in non-small cell lung cancer (NSCLC) to find indicators of brain metastasis and peritumoral brain edema.

PATIENTS AND METHODS: Fifty-two cases were studied out of which 26 had corresponding brain metastatic tissue. Clinicopathological characteristics of tumors were correlated with biomarkers of cell adhesion, cell growth, cell cycle and apoptosis regulation that were previously immunohistochemically studied but never analyzed separately according to histological subgroups, gender and smoking history.

RESULTS: Increased collagen XVII in adenocarcinoma (ADC) and increased caspase-9, CD44v6, and decreased cellular apoptosis susceptibility protein (CAS) and Ki-67 in squamous cell carcinoma (SCC) correlated significantly with brain metastasis. Increased β-catenin, E-cadherin and decreased caspase-9 expression in primary SCC, and decreased CD44v6 expression in brain metastatic SCC tissues showed a significant correlation with the extent of peritumoral brain edema. Positive correlation between smoking and biomarker expression could be observed in metastatic ADCs with p16 and caspase-8, while-negative correlation was found in SCC without brain metastasis with caspase-3, and in SCC with brain metastasis with p27.

CONCLUSION: Our results highlight the importance of separate analysis of biomarker expression in histological subtypes of NSCLC.

The Ability of Secretory Leukocyte Protease Inhibitor to Inhibit Apoptosis in Monocytes Is Independent of Its Antiprotease Activity

Secretory Leukocyte Protease Inhibitor (SLPI) is a serine protease inhibitor produced by epithelial and myeloid cells with anti-inflammatory properties. Research has shown that SLPI exerts its anti-inflammatory activity by directly binding to NF-κB DNA binding sites and, in so doing, prevents binding and subsequent transcription of proinflammatory gene expression. In the current study, we demonstrate that SLPI can inhibit TNF-α-induced apoptosis in U937 cells and peripheral blood monocytes. Specifically, SLPI inhibits TNF-α-induced caspase-3 activation and DNA degradation associated with apoptosis. We go on to show that this ability of SLPI to inhibit apoptosis is not dependent on its antiprotease activity as antiprotease deficient variants of SLPI can also inhibit TNF-α-induced apoptosis. This reduction in monocyte apoptosis may preserve monocyte function during inflammation resolution and promote infection clearance at mucosal sites.

A selective antagonist of histamine H₄ receptors prevents antigen-induced airway inflammation and bronchoconstriction in guinea pigs:involvement of lipocortin-1

BACKGROUND AND PURPOSE: Among the pathogenic mechanisms of asthma, a role for oxidative/nitrosative stress has been well documented. Recent evidence suggests that histamine H₄ receptors play a modulatory role in allergic inflammation. Here we report the effects of compound JNJ 7777120 (JNJ), a selective H4 receptor antagonist, on antigen-induced airway inflammation, paying special attention to its effects on lipocortin-1 (LC-1/annexin-A1), a 37 kDA anti-inflammatory protein that plays a key role in the production of inflammatory mediators.

EXPERIMENTAL APPROACH: Ovalbumin (OA)-sensitized guinea pigs placed in a respiratory chamber were challenged with antigen. JNJ (5, 7.5 and 10 mg.kg⁻¹) was given i.p. for 4 days before antigen challenge. Respiratory parameters were recorded. Bronchoalveolar lavage (BAL) fluid was collected and lung specimens taken for further analyses 1 h after antigen challenge. In BAL fluid, levels of LC-1, PGD2 , LTB4 and TNF-α were measured. In lung tissue samples, myeloperoxidase, caspase-3 and Mn-superoxide dismutase activities and 8-hydroxy-2-deoxyguanosine levels were measured.

KEY RESULTS: OA challenge decreased LC-1 levels in BAL fluid, induced cough, dyspnoea and bronchoconstriction and increased PGD2 , LTB4 and TNF-α levels in lung tissue. Treatment with JNJ dose-dependently increased levels of LC-1, reduced respiratory abnormalities and lowered levels of PGD2 , LTB4 and TNF-α in BAL fluid.

CONCLUSIONS AND IMPLICATIONS: Antigen-induced asthma-like reactions in guinea pigs decreased levels of LC-1 and increased TNF-α and eicosanoid production. JNJ pretreatment reduced allergic asthmatic responses and airway inflammation, an effect associated with LC-1 up-regulation.

BubR1 is required for the mitotic block induced by combretastatin-A4 and a novel cis-restricted ß-lactam analogue in human cancer cells

BubR1 is a well-defined guardian of the mitotic spindle, initiating mitotic arrest in response to the lack of tension and/or chromosome alignment across the mitotic plate. However, the role of BubR1 in combretastatin-induced cell death remains unknown. In this study, we describe the effects of combretastatin A-4 (CA-4) and a synthetic cis-restricted 3,4-diaryl-2-azetidinone (ß-lactam) analogue (CA-432) on the modulation and phosphorylation of BubR1 in human cervical cancer-derived cells. We demonstrate that CA-4 and CA-432 depolymerise the microtubular network of human cervical carcinoma-derived cells. Both compounds induced the disassembly of the microtubules and the loss of microtubule tension led to the early phosphorylation of BubR1 and the late cleavage of BubR1. The phosphorylation of BubR1 correlated with the onset of G2M cell cycle arrest whilst the cleavage of BubR1 coincided with apoptosis induced by the combretastatins. The combretastatin-induced apoptosis and the BubR1 cleavage were caspase-dependent. In vitro enzyme digests demonstrated that combretastatin-activated BubR1 is a substrate for caspase-3. Gene silencing of BubR1 with small interfering RNA severely compromised combretastatin-induced G2M cell cycle arrest with a corresponding increase in the formation of polyploid cells in both cervical and breast cancer-derived cells. In summary, BubR1 is required to maintain the G2M arrest and limit the formation of polyploid cells in response to continued combretastatin exposure. Moreover, substitution of the ethylene bridge with 3,4-diaryl-2-azetidinone did not alter the tubulin depolymerising properties or the subsequent mitotic spindle checkpoint response to CA-4 in human cancer cells.

The vascular targeting agent combretastatin-A4 and a novel cis-Restricted {beta}-Lactam Analogue, CA-432, induce apoptosis in human chronic myeloid leukemia cells and ex vivo patient samples including those displaying multidrug resistance

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected β-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead β-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G(2)M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-x(L) preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.

Dual targeting of tumour cells and host endothelial cells by novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines

PURPOSE: Some members of a novel series of pyrrolo-1,5-benzoxazepines (PBOXs) are microtubule-targeting agents capable of inducing apoptosis in a variety of human cancerous cells, hence, they are currently being developed as potential anti-cancer agents. The purpose of this study was to first characterise the activities of a novel PBOX analogue, PBOX-16 and then investigate the anti-angiogenic potential of both PBOX-16 and its prototype PBOX-6.

METHODS: The effects of PBOX-6 and -16 on cancerous cells (chronic myeloid leukaemia K562 cells and ovarian carcinoma A2780 cells) and primary cultured human umbilical vein endothelial cells (HUVECs) were examined by assessing cell proliferation, microtubular organisation, DNA analysis of cell cycle progression and caspase-3/7 activity. Their anti-angiogenic properties were then investigated by examining their ability to interfere with HUVEC differentiation into capillary-like structures and vascular endothelial growth factor (VEGF)-stimulated HUVEC migration.

RESULTS: PBOX-6 and -16 inhibited proliferation of K562, A2780 and HUVEC cells in a concentration-dependent manner. PBOX-16, confirmed as a novel depolymerising agent, was approximately tenfold more potent than PBOX-6. Inhibition of cell proliferation was mediated by G(2)/M arrest followed by varying degrees of apoptosis depending on the cell type; endothelial cells underwent less apoptosis than either of the cancer cell lines. In addition to the antitumourigenic properties, we also describe a novel antiangiogenic function for PBOXs: treatment with PBOXs inhibited the spontaneous differentiation of HUVECs into capillary-like structures when grown on a basement membrane matrix preparation (Matrigel™) and also significantly reduced VEGF-stimulated HUVEC migration.

CONCLUSION: Dual targeting of both the tumour cells and the host endothelial cells by PBOX compounds might enhance the anti-cancer efficacy of these drugs.