Epidermal Growth Factor Removal or Tyrphostin AG1478 Treatment Reduces Goblet Cells & Mucus Secretion of Epithelial Cells from Asthmatic Children Using the Air-Liquid Interface Model

RATIONALE: Epithelial remodelling in asthma is characterised by goblet cell hyperplasia and mucus hypersecretion for which no therapies exist. Differentiated bronchial air-liquid interface cultures from asthmatic children display high goblet cell numbers. Epidermal growth factor and its receptor have been implicated in goblet cell hyperplasia.

OBJECTIVES: We hypothesised that EGF removal or tyrphostin AG1478 treatment of differentiating air-liquid interface cultures from asthmatic children would result in a reduction of epithelial goblet cells and mucus secretion.

METHODS: In Aim 1 primary bronchial epithelial cells from non-asthmatic (n = 5) and asthmatic (n = 5) children were differentiated under EGF-positive (10ng/ml EGF) and EGF-negative culture conditions for 28 days. In Aim 2, cultures from a further group of asthmatic children (n = 5) were grown under tyrphostin AG1478, a tyrosine kinase inhibitor, conditions. All cultures were analysed for epithelial resistance, markers of differentiation using immunocytochemistry, ELISA for MUC5AC mucin secretion and qPCR for MUC5AC mRNA.

RESULTS: In cultures from asthmatic children the goblet cell number was reduced in the EGF negative group (p = 0.01). Tyrphostin AG1478 treatment of cultures from asthmatic children had significant reductions in goblet cells at 0.2μg/ml (p = 0.03) and 2μg/ml (p = 0.003) as well as mucus secretion at 2μg/ml (p = 0.04).

CONCLUSIONS: We have shown in this preliminary study that through EGF removal and tyrphostin AG1478 treatment the goblet cell number and mucus hypersecretion in differentiating air-liquid interface cultures from asthmatic children is significantly reduced. This further highlights the epidermal growth factor receptor as a potential therapeutic target to inhibit goblet cell hyperplasia and mucus hypersecretion in asthma.

Sequential treatment with flavopiridol synergistically enhances pyrrolo-1,5-benzoxazepine-induced apoptosis in human chronic myeloid leukaemia cells including those resistant to imatinib treatment

The Bcr-Abl kinase inhibitor, imatinib mesylate, is the front line treatment for chronic myeloid leukaemia (CML), but the emergence of imatinib resistance has led to the search for alternative drug treatments and the examination of combination therapies to overcome imatinib resistance. The pro-apoptotic PBOX compounds are a recently developed novel series of microtubule targeting agents (MTAs) that depolymerise tubulin. Recent data demonstrating enhanced MTA-induced tumour cell apoptosis upon combination with the cyclin dependent kinase (CDK)-1 inhibitor flavopiridol prompted us to examine whether this compound could similarly enhance the effect of the PBOX compounds. We thus characterised the apoptotic and cell cycle events associated with combination therapy of the PBOX compounds and flavopiridol and results showed a sequence dependent, synergistic enhancement of apoptosis in CML cells including those expressing the imatinib-resistant T315I mutant. Flavopiridol reduced the number of polyploid cells formed in response to PBOX treatment but only to a small extent, suggesting that inhibition of endoreplication was unlikely to play a major role in the mechanism by which flavopiridol synergistically enhanced PBOX-induced apoptosis. The addition of flavopiridol following PBOX-6 treatment did however result in an accelerated exit from the G2/M transition accompanied by an enhanced downregulation and deactivation of the CDK1/cyclin B1 complex and an enhanced degradation of the inhibitor of apoptosis protein (IAP) survivin. In conclusion, results from this study highlight the potential of these novel series of PBOX compounds, alone or in sequential combination with flavopiridol, as an effective therapy against CML.

CD38 expression level and pattern of expression remains a reliable and robust marker of progressive disease in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) follows a variable clinical course which is difficult to predict at diagnosis. We assessed somatic mutation (SHM) status, CD38 and ZAP-70 expression in 87 patients (49 male, 38 female) with stage A CLL and known cytogenetic profile to compare their role in predicting disease progression, which was assessed by the treatment free interval (TFI) from diagnosis. Sixty (69%) patients were SHM+, 24 (28%) were CD38+ and ten (12%) were ZAP-70+. The median TFI for: (i) SHM + versus SHM- patients was 124 versus 26 months; hazard ratio (HR) = 3.6 [95% confidence interval (CI) = 1.8 - 7.3; P = 0.001]: (ii) CD38- versus CD38+ patients was 120 versus 34 months; HR = 2.4 (95% CI = 1.4 - 5.3; P = 0.02); and (iii) ZAP70- versus ZAP70+ was 120 versus 16 months; HR = 3.4 (95% CI = 1.4 - 8.7; P = 0.01). SHM status and CD38 retained prognostic significance on multivariate analysis whereas ZAP-70 did not. We conclude that ZAP-70 analysis does not provide additional prognostic information in this group of patients.

Pyrrolo-1,5-benzoxazepines induce apoptosis in chronic myelogenous leukemia (CML) cells by bypassing the apoptotic suppressor bcr-abl

Expression of the transforming oncogene bcr-abl in chronic myelogenous leukemia (CML) cells is reported to confer resistance against apoptosis induced by many chemotherapeutic agents such as etoposide, ara-C, and staurosporine. In the present study some members of a series of novel pyrrolo-1,5-benzoxazepines potently induce apoptosis, as shown by cell shrinkage, chromatin condensation, DNA fragmentation, and poly(ADP-ribose) polymerase (PARP) cleavage, in three CML cell lines, K562, KYO.1, and LAMA 84. Induction of apoptosis by a representative member of this series, PBOX-6, was not accompanied by either the down-regulation of Bcr-Abl or by the attenuation of its protein tyrosine kinase activity up to 24 h after treatment, when approximately 50% of the cells had undergone apoptosis. These results suggest that down-regulation of Bcr-Abl is not part of the upstream apoptotic death program activated by PBOX-6. By characterizing the mechanism in which this novel agent executes apoptosis, this study has revealed that PBOX-6 caused activation of caspase 3-like proteases in only two of the three CML cell lines. In addition, inhibition of caspase 3-like protease activity using the inhibitor z-DEVD-fmk blocked caspase 3-like protease activity but did not prevent the induction of apoptosis, suggesting that caspase 3-like proteases are not essential in the mechanism by which PBOX-6 induces apoptosis in CML cells. In conclusion, this study demonstrates that PBOX-6 can bypass Bcr-Abl-mediated suppression of apoptosis, suggesting an important potential use of these compounds in the treatment of CML.

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.

Prognostic significance of combining VEGFA, FLT1 and KDR mRNA expression in lung cancer

Angiogenesis is important in cancer progression. Promising results in clinical trials have indicated that targeting vascular epidermal growth factor (VEGF) signaling may prolong lung cancer patient survival. In particular, various studies have implicated VEGFA as a potential prognostic marker in lung cancer, although prognostication using the expression of VEGF receptors (VEGFRs), such as fms-related tyrosine kinase 1 (FLT1; also known as VEGFR1) and kinase insert domain receptor (KDR; also known as VEGFR2), has produced varied results in different lung cancer studies. The present study aimed to investigate the prognostic significance of these three factors, alone or in combination. mRNA expression data were extracted from four independent lung cancer cohorts totaling 583 patients, and the association between mRNA expression and survival was investigated by performing statistical analyses. When VEGFA, FLT1 and KDR expression were considered alone, only VEGFA demonstrated a significant association with patient survival consistently across all four datasets (P<0.05). Patients with a high expression of VEGFA and one of the two receptors were associated with significantly worse survival than patients expressing low levels of VEGFA and the particular receptor (P<0.05). Notably, patients with a high level expression of all three genes in their tumor specimens were associated with a significantly shorter survival time compared with patients exhibiting a low level expression of one, two or all three genes (P<0.05). The results indicate that a high level of VEGFA expression and its receptors may be required for cancer progression. Therefore, these three factors should be considered together as a prognostic indicator for lung cancer patients.

Epirubicin, oxaliplatin, and capecitabine with or without panitumumab for patients with previously untreated advanced oesophagogastric cancer (REAL3): a randomised, open-label phase 3 trial.

BACKGROUND: EGFR overexpression occurs in 27-55% of oesophagogastric adenocarcinomas, and correlates with poor prognosis. We aimed to assess addition of the anti-EGFR antibody panitumumab to epirubicin, oxaliplatin, and capecitabine (EOC) in patients with advanced oesophagogastric adenocarcinoma. METHODS: In this randomised, open-label phase 3 trial (REAL3), we enrolled patients with untreated, metastatic, or locally advanced oesophagogastric adenocarcinoma at 63 centres (tertiary referral centres, teaching hospitals, and district general hospitals) in the UK. Eligible patients were randomly allocated (1:1) to receive up to eight 21-day cycles of open-label EOC (epirubicin 50 mg/m(2) and oxaliplatin 130 mg/m(2) on day 1 and capecitabine 1250 mg/m(2) per day on days 1-21) or modified-dose EOC plus panitumumab (mEOC+P; epirubicin 50 mg/m(2) and oxaliplatin 100 mg/m(2) on day 1, capecitabine 1000 mg/m(2) per day on days 1-21, and panitumumab 9 mg/kg on day 1). Randomisation was blocked and stratified for centre region, extent of disease, and performance status. The primary endpoint was overall survival in the intention-to-treat population. We assessed safety in all patients who received at least one dose of study drug. After a preplanned independent data monitoring committee review in October, 2011, trial recruitment was halted and panitumumab withdrawn. Data for patients on treatment were censored at this timepoint. This study is registered with ClinicalTrials.gov, number NCT00824785. FINDINGS: Between June 2, 2008, and Oct 17, 2011, we enrolled 553 eligible patients. Median overall survival in 275 patients allocated EOC was 11.3 months (95% CI 9.6-13.0) compared with 8.8 months (7.7-9.8) in 278 patients allocated mEOC+P (hazard ratio [HR] 1.37, 95% CI 1.07-1.76; p=0.013). mEOC+P was associated with increased incidence of grade 3-4 diarrhoea (48 [17%] of 276 patients allocated mEOC+P vs 29 [11%] of 266 patients allocated EOC), rash (29 [11%] vs two [1%]), mucositis (14 [5%] vs none), and hypomagnesaemia (13 [5%] vs none) but reduced incidence of haematological toxicity (grade ≥ 3 neutropenia 35 [13%] vs 74 [28%]). INTERPRETATION: Addition of panitumumab to EOC chemotherapy does not increase overall survival and cannot be recommended for use in an unselected population with advanced oesophagogastric adenocarcinoma. FUNDING: Amgen, UK National Institute for Health Research Biomedical Research Centre.

Personalized cancer medicine: molecular diagnostics, predictive biomarkers, and drug resistance.

The progressive elucidation of the molecular pathogenesis of cancer has fueled the rational development of targeted drugs for patient populations stratified by genetic characteristics. Here we discuss general challenges relating to molecular diagnostics and describe predictive biomarkers for personalized cancer medicine. We also highlight resistance mechanisms for epidermal growth factor receptor (EGFR) kinase inhibitors in lung cancer. We envisage a future requiring the use of longitudinal genome sequencing and other omics technologies alongside combinatorial treatment to overcome cellular and molecular heterogeneity and prevent resistance caused by clonal evolution.

Combinations of ZAP-70, CD38 and IGHV mutational status as predictors of time to first treatment in CLL.

ZAP-70, CD38 and IGHV mutations have all been reported to have prognostic impact in chronic lymphocytic leukemia (CLL), both individually and in paired combinations. We aimed to determine whether the combination of all three factors provided more refined prognostic information concerning the treatment-free interval (TFI) from diagnosis. ZAP-70, CD38 and IGHV mutations were evaluated in 142 patients. Combining all three factors, the ZAP-70-/CD38-/Mutated group showed the longest median TFI (62 months, n = 37), ZAP-70+/CD38+/Unmutated cases the shortest (11 months, n = 37) and cases discordant for > or = 1 factor, an intermediate TFI (27 months, n = 68) (p = 0.006). Analysis of discordant cases revealed values that were otherwise masked when measuring single prognostic factors. The presence or absence of cytogenetic abnormalities did not explain the variability among discordant cases. Simultaneous analysis of ZAP-70, CD38 and IGHV mutations in CLL provides more discriminatory prediction of TFI than any factor alone.

Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling pathways in vivo

Attaching and effacing (A/E) lesions and actin polymerization, the hallmark of enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) infections, are dependent on the effector Tir. Phosphorylation of Tir(EPEC/CR) Y474/1 leads to recruitment of Nck and neural Wiskott-Aldrich syndrome protein (N-WASP) and strong actin polymerization in cultured cells. Tir(EPEC/CR) also contains an Asn-Pro-Tyr (NPY(454/1)) motif, which triggers weak actin polymerization. In EHEC the NPY(458) actin polymerization pathway is amplified by TccP/EspF(U), which is recruited to Tir via IRSp53 and/or insulin receptor tyrosine kinase substrate (IRTKS). Here we used C. rodentium to investigate the different Tir signalling pathways in vivo. Following infection with wild-type C. rodentium IRTKS, but not IRSp53, was recruited to the bacterial attachment sites. Similar results were seen after infection of human ileal explants with EHEC. Mutating Y471 or Y451 in Tir(CR) abolished recruitment of Nck and IRTKS respectively, but did not affect recruitment of N-WASP or A/E lesion formation. This suggests that despite their crucial role in actin polymerization in cultured cells the Tir:Nck and Tir:IRTKS pathways are not essential for N-WASP recruitment or A/E lesion formation in vivo. Importantly, wild-type C. rodentium out-competed the tir tyrosine mutants during mixed infections. These results uncouple the Tir:Nck and Tir:IRTKS pathways from A/E lesion formation in vivo but assign them an important in vivo role.

Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1- Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti- phospho-Tyr9 antibodies showed that the level of Tyr9 phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr9, distinct from Tyr373/376, is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr9 phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.

5-aryl-pyrazolo[3,4-b]pyridazines: Potent inhibitors of glycogen synthase kinase-3 (GSK-3)

Introduction of a nitrogen atom into the 6-position of a series of pyrazolo[3,4-b]pyridines led to a dramatic improvement in the potency of GSK-3 inhibition. Rationalisation of the binding mode suggested participation of a putative structural water molecule, which was subsequently confirmed by X-ray crystallography. (C) 2003 Elsevier Science Ltd. All rights reserved.

3-anilino-4-arylmaleimides: Potent and selective inhibitors of glycogen synthase kinase-3 (GSK-3)

Potent 3-anilino-4-arylmaleimide glycogen synthase kinase-3 (GSK-3) inhibitors have been prepared using automated array methodology. A number of these are highly selective, having little inhibitory potency against more than 20 other protein kinases. (C) 2001 Elsevier Science Ltd. All rights reserved.

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription

Background: Glycogen synthase kinase-3 (GSK-8) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3.