Search

The development of drug resistance in lung cancer is a major clinical challenge, leading to a 5-year survival rate of only 18%. Therefore, unravelling the mechanisms of drug resistance and developing novel therapeutic strategies is of crucial importance. This study systematically explores the novel biomarkers of drug resistance using a lung cancer model (DLKP) with a series of drug-resistant variants. In-depth label-free quantitative mass spectrometry-based proteomics and gene ontology analysis shows that parental DLKP cells significantly differ from drug-resistant variants, and the cellular proteome changes even among the drug-resistant subpopulations. Overall, ABC transporter proteins and lipid metabolism were determined to play a significant role in the formation of drug resistance in DKLP cells. A series of membrane-related proteins such as HMOX1, TMB1, EPHX2 and NEU1 were identified to be correlated with levels of drug resistance in the DLKP subpopulations. The study also showed enrichment in biological processes and molecular functions such as drug metabolism, cellular response to the drug and drug binding. In gene ontology analysis, 18 proteins were determined to be positively or negatively correlated with resistance levels. Overall, 34 proteins which potentially have a therapeutic and diagnostic value were identified.

The multi-drug combination regime, FOLFIRINOX, is a standard of care chemotherapeutic therapy for pancreatic cancer patients. However, systematic evaluation of potential pharmacodynamic interactions among multi-drug therapy has not been reported previously. Here, pharmacodynamic interactions of the FOLFIRINOX agents (5-fluorouracil (5-FU), oxaliplatin (Oxa) and SN-38, the active metabolite of irinotecan) were assessed across a panel of primary and established pancreatic cancer cells. Inhibition of cell proliferation was quantified for each drug, alone and in combination, to obtain quantitative, drug-specific interaction parameters and assess the nature of drug interactions. The experimental data were analysed assuming Bliss independent interactions, and nonlinear regression model fitting was conducted in SAS. Estimates of the drug interaction term, psi (ψ), revealed that the Oxa/SN-38 combination appeared synergistic in PANC-1 (ψ = 0.6, 95% CI = 0.4, 0.9) and modestly synergistic, close to additive, in MIAPaCa-2 (ψ = 0.8, 95% CI = 0.6, 1.0) in 2D assays. The triple combination was strongly synergistic in MIAPaCa-2 (ψ = 0.2, 95% CI = 0.1, 0.3) and modestly synergistic/borderline additive in PANC-1 2D (ψ = 0.8, 95% CI = 0.6, 1.0). The triple combination showed antagonistic interactions in the primary PIN-127 and 3D PANC-1 model (ψ 1). Quantitative pharmacodynamic interactions have not been described for the FOLFIRINOX regimen; this analysis suggests a complex interplay among the three chemotherapeutic agents. Extension of this pharmacodynamic analysis approach to clinical/translational studies of the FOLFIRINOX combination could reveal additional pharmacodynamic interactions and guide further refinement of this regimen to achieve optimal clinical responses.

Objectives A limited repertoire of good pancreatic ductal adenocarcinoma (PDAC) models is one of the main barriers in developing effective new PDAC treatments. We aimed to characterize 6 commonly used PDAC cell lines and compare them with PDAC patient tumor samples using proteomics. Methods Proteomic methods were used to generate an extensive catalog of proteins from 10 PDAC surgical specimens, 9 biopsies of adjacent normal tissue, and 6 PDAC cell lines. Protein lists were interrogated to determine what extent the proteome of the cell lines reflects the proteome of primary pancreatic tumors. Results We identified 7973 proteins from the cell lines, 5680 proteins from the tumor tissues, and 4943 proteins from the adjacent normal tissues. We identified 324 proteins unique to the cell lines, some of which may play a role in survival of cells in culture. Conversely, a list of 63 proteins expressed only in the patient samples, whose expression is lost in culture, may place limitations on the degree to which these model systems reflect tumor biology in vivo. Conclusions Our work offers a catalog of proteins detected in each of the PDAC cell lines, providing a useful guide for researchers seeking model systems for PDAC functional studies.

The Caco-2 cell line is composed of a heterogeneous mix of cells; isolation of individual clonal populations from this mix allows for specific mechanisms and phenotypes to be further explored. Previously we exposed Caco-2 cells to inorganic copper sulphate or organic copper proteinate to generate resistant variant populations. Here we describe the isolation and characterisation of clonal subpopulations from these resistant variants to organic (clone Or1, Or2, Or3) or inorganic (clone In1 and In2) copper. The clones show considerable homogeneity in response to Cu-induced toxicity and heterogeneity in morphology with variations in level of cross-resistance to other metals and doxorubicin. Population growth was reduced for Cu-resistant clones In2 and Or3 in selective pressure relative to parental Caco-2 cells. Gene expression analysis identified 4026 total (2102 unique and 1924 shared) differentially expressed genes including those involved in the MAP Kinase and Rap1 signalling pathways, and in the focal adhesion and ECM-receptor contact pathways. Gene expression changes common to all clones included upregulation of ANXA13 and GPx2. Our analysis additionally identified differential expression of multiple genes specific to copper proteinate exposure (including overexpressed UPK1B) in isolated clones Or1, Or2 and Or3 and CuSO4 exposure (including decreased AIFM2 expression) in isolated clones In1 and In2. The adaptive transcriptional responses established in this study indicate a cohort of genes, which may be involved in copper resistance regulation and chronic copper exposure.

Pancreatic cancer is usually advanced and drug resistant at diagnosis. A potential therapeutic approach outlined here uses nanoparticle (NP)-based drug carriers, which have unique properties that enhance intra-tumor drug exposure and reduce systemic toxicity of encapsulated drugs. Here we report that patients whose pancreatic cancers express elevated levels of Death Receptor 5 (DR5) and its downstream regulators/effectors FLIP, Caspase-8, and FADD had particularly poor prognoses. To take advantage of elevated expression of this pathway, we designed drug-loaded NPs with a surface-conjugated αDR5 antibody (AMG 655). Binding and clustering of the DR5 is a prerequisite for efficient apoptosis initiation, and the αDR5-NPs were indeed found to activate apoptosis in multiple pancreatic cancer models, whereas the free antibody did not. The extent of apoptosis induced by αDR5-NPs was enhanced by down-regulating FLIP, a key modulator of death receptor- mediated activation of caspase-8. Moreover, the DNA topoisomerase-1 inhibitor camptothecin (CPT) down-regulated FLIP in pancreatic cancer models and enhanced apoptosis induced by αDR5-NPs. CPT-loaded αDR5-NPs significantly increased apoptosis and decreased cell viability in vitro in a caspase-8- and FADD-dependent manner consistent with their expected mechanism-of-action. Importantly, CPT-loaded αDR5-NPs markedly reduced tumor growth rates in vivo in established pan- creatic tumor models, inducing regressions in one model. These proof-of-concept studies indicate that αDR5-NPs loaded with agents that downregulate or inhibit FLIP are promising candidate agents for the treatment of pancreatic cancer.

Cell line engineering using microRNAs represents a desirable route for improving the efficiency of recombinant protein production by CHO cells. In this study we generated stable CHO DP12 cells expressing a miR-7 sponge transcript which sequesters miR-7 from its endogenous targets. Depletion of miR-7 results in a 65% increase in cell growth and >3-fold increase in yield of secreted IgG protein. Quantitative labelfree LC-MS/MS proteomic profiling was carried out to identify the targets of miR-7 and understand the functional drivers of the improved CHO cell phenotypes. Subcellular enrichment and total proteome analysis identified more than 3000 proteins per fraction resulting in over 5000 unique proteins identified per timepoint analysed. Early stage culture analysis identified 117 proteins overexpressed in miR-7 depleted cells. A subset of these proteins are involved in the Akt pathway which could be the underlying route for cell density improvement and may be exploited more specifically in the future. Late stage culture identified 160 proteins overexpressed in miR-7 depleted cells with some of these involved in ribosome biogenesis which may be causing the increased productivity through improved translational efficiency. This is the first in-depth proteomic profiling of the IgG producing CHO DP12 cell line stably depleted of miR-7. SIGNIFICANCE: Chinese hamster ovary (CHO) cells are the mammalian cell expression system of choice for production of recombinant therapeutic proteins. There is much research ongoing to characterise CHO cell factories through the application of systems biology approaches that will enable a fundamental understanding of CHO cell physiology, and as a result, a better knowledge and understanding of recombinant protein production. This study profiles the proteomic effects of microRNA-7 depletion on the IgG producing CHO DP12 cell line. This is one of the very few studies that attempts to identify the functioning proteins driving improved CHO cell phenotypes resulting from microRNA manipulation. Using subcellular enrichment and total proteome analysis we identified over 5000 unique proteins in miR-7 depleted CHO cells. This work has identified a cohort of proteins involved in the Akt pathway and ribosome biogenesis. These proteins may drive improved CHO cell phenotypes and are of great interest for future work.

Copper is an essential dietary micronutrient in humans for proper cell function; however, in excess, it is toxic. The human cell line Caco-2 is popular as an in vitro model for intestinal absorption and toxicology. This study investigated the response of exponentially growing Caco-2 cells to prolonged copper exposure (120 h). An unexpected non-monotonic dose-response profile was observed in Caco-2 cells. Exposure to media supplemented with 3.125 μM CuSO4 resulted in decreased cell yield vs. untreated. However, toxicity was progressively reduced from 90% at 3.125 μM to 60% at 25 μM. This effect was documented between 48 and 120 h continuous exposure (p

Objectives We used miRNA and proteomic profiling to understand intracellular pathways that contribute to high and low specific productivity (Qp) phenotypes in CHO clonally derived cell lines (CDCLs) from the same cell line generation project. Results Differentially expressed (DE) miRNAs were identified which are predicted to target several proteins associated with protein folding. MiR-200a was found to have a number of predicted targets associated with the unfolded protein response (UPR) which were shown to have decreased expression in high Qp CDCLs and have no detected change at the mRNA level. MiR-200a overexpression in a CHO CDCL was found to increase recombinant protein titer by 1.2 fold and Qp by 1.8 fold. Conclusion These results may suggest a role for miR-200a in post-transcriptional regulation of the UPR, presenting miR-200a as a potential target for engineering industrially attractive CHO cell phenotypes.

Chinese hamster ovary (CHO) cells are one of the most commonly used host cell lines used for the production human therapeutic proteins. Much research over the past two decades has focussed on improving the growth, titre and cell specific productivity of CHO cells and in turn lowering the costs associated with production of recombinant proteins. CHO cell engineering has become of particular interest in recent years following the publication of the CHO cell genome and the availability of data relating to the proteome, transcriptome and metabolome of CHO cells. However, data relating to the cellular post-translational modification (PTMs) which can affect the functionality of CHO cellular proteins has only begun to be presented in recent years. PTMs are important to many cellular processes and can further alter proteins by increasing the complexity of proteins and their interactions. In this review, we describe the research presented from CHO cells to date related on three of the most important PTMs; glycosylation, phosphorylation and ubiquitination.

Pancreatic ductal adenocarcinoma (PDAC), which represents approximately 80% of all pancreatic cancers, is a highly aggressive malignant disease and one of the most lethal among all cancers. Overall, the 5-year survival rate among all pancreatic cancer patients is less than 9%; these rates have shown little change over the past 30 years. A more comprehensive understanding of the molecular mechanisms underlying this complex disease is crucial to the development of new diagnostic tools for early detection and disease monitoring, as well as to identify new and more effective therapeutics to improve patient outcomes.We summarize recent advances in proteomic strategies and mass spectrometry to identify new biomarkers for early detection and monitoring of disease progression, predict response to therapy, and to identify novel proteins that have the potential to be 'druggable' therapeutic targets. An overview of proteomic studies that have been conducted to further our mechanistic understanding of metastasis and chemotherapy resistance in PDAC disease progression will also be discussed.The results from these PDAC proteomic studies on a variety of PDAC sample types (e.g., blood, tissue, cell lines, exosomes, etc.) provide great promise of having a significant clinical impact and improving patient outcomes.

We previously described a non-monotonic dose response curve at low copper concentrations where 3.125 μM CuSO4 (the early inflection point) was more toxic than 25 μM CuSO4 in Caco-2 cells. We employed global proteomics to investigate this observation. The altered expression levels of a small number of proteins displaying a temporal response may provide the best indication of the underlying mechanism; more well-known copper response proteins including the metal binding metallothioneins (MT1X, MT1F, MT2A) and antioxidant response proteins including Heme oxygenase were upregulated to a similar level in both copper concentrations and so are less likely to underpin this phenomenon.The temporal response proteins include Granulins, AN1-type zinc finger protein 2A (ZFAND2A), and the heat shock proteins (HSPA6 and HSPA1B). Granulins were decreased after 4 h only in 25 μM CuSO4 but from 24 h, were decreased in both copper concentrations to a similar level. Induction of ZFAND2A and increases in HSPA6 and HSPA1B were observed at 24 h only in 25 μM CuSO4 but were present at 48 h in both copper conditions. The early expression of ZFAND2A, HSPs, and higher levels of α-crystallin B (CRYAB) correlated with lower levels of misfolded proteins in 25 μM CuSO4 compared to 3.125 μM CuSO4 at 48 h. These results suggest that 3.125 μM CuSO4 at early time points was unable to activate the plethora of stress responses invoked by the higher copper concentration, paradoxically exposing the Caco-2 cells to higher levels of misfolded proteins and greater proteotoxic stress.

We report a series of copper(II) artificial metallo-nucleases (AMNs) and demonstrate their DNA damaging properties and in-vitro cytotoxicity against human-derived pancreatic cancer cells. The compounds combine a tris-chelating polypyridyl ligand, di-(2-pycolyl)amine (DPA), and a DNA intercalating phenanthrene unit. Their general formula is Cu-DPA-N,N' (where N,N'=1,10-phenanthroline (Phen), dipyridoquinoxaline (DPQ) or dipyridophenazine (DPPZ)). Characterisation was achieved by X-ray crystallography and continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE) and Davies electron-nuclear double resonance (ENDOR) spectroscopies. The presence of the DPA ligand enhances solution stability and facilitates enhanced DNA recognition with apparent binding constants (Kapp) rising from 105 to 107 m−1 with increasing extent of planar phenanthrene. Cu-DPA-DPPZ, the complex with greatest DNA binding and intercalation effects, recognises the minor groove of guanine–cytosine (G-C) rich sequences. Oxidative DNA damage also occurs in the minor groove and can be inhibited by superoxide and hydroxyl radical trapping agents. The complexes, particularly Cu-DPA-DPPZ, display promising anticancer activity against human pancreatic tumour cells with in-vitro results surpassing the clinical platinum(II) drug oxaliplatin.

Homeostasis of metal micronutrients such as copper is tightly regulated to ensure deficiency does not occur while restricting damage resulting from excess accumulation. Using LC-MS the effect on the proteome of intestinal Caco-2 cells of exposure to the chelator triethylenetetramine (TETA) was investigated. Continuous exposure of TETA at 25 μM to Caco-2 cells caused decreased cell yields and morphological changes. These effects were reversed when cells were no longer exposed to TETA. Quantitative proteomic analysis identified 957 mostly low-fold differentially expressed proteins, 41 of these returned towards control Caco-2 expression following recovery. Proteins exhibiting this “reciprocal” behaviour included upregulated deoxyhypusine hydroxylase (DOHH, 15.69- fold), a protein essential for eIF-5A factor hypsuination, a post translational modification responsible for eIF-5A maturation, which in turn is responsible for translation elongation. Exposure to TETA also resulted in 87 proteins, the expression of which was stable and remained differentially expressed following recovery. This study helps to elucidate the stable and transient proteomic effects of TETA exposure in intestinal cells.

Pancreatic cancer remains among the most lethal cancers worldwide, with poor early detection rates and poor survival rates. Patient-derived xenograft (PDX) models have increasingly been used in preclinical and clinical research of solid cancers to fulfil unmet need. Fresh tumour samples from human pancreatic adenocarcinoma patients were implanted in severe combined immunodeficiency (SCID) mice. Samples from 78% of treatment-na&iuml, ve pancreatic ductal adenocarcinoma patients grew as PDX tumours and were confirmed by histopathology. Frozen samples from F1 PDX tumours could be later successfully passaged in SCID mice to F2 PDX tumours. The human origin of the PDX was confirmed using human-specific antibodies, however, the stromal component was replaced by murine cells. Cell lines were successfully developed from three PDX tumours. RNA was extracted from eight PDX tumours and where possible, corresponding primary tumour (T) and adjacent normal tissues (N). mRNA profiles of tumour vs. F1 PDX and normal vs. tumour were compared by Affymetrix microarray analysis. Differential gene expression showed over 5000 genes changed across the N vs. T and T vs. PDX samples. Gene ontology analysis of a subset of genes demonstrated genes upregulated in normal vs. tumour vs. PDX were linked with cell cycle, cycles cell process and mitotic cell cycle. Amongst the mRNA candidates elevated in the PDX and tumour vs. normal were SERPINB5, FERMT1, AGR2, SLC6A14 and TOP2A. These genes have been associated with growth, proliferation, invasion and metastasis in pancreatic cancer previously. Cumulatively, this demonstrates the applicability of PDX models and transcriptomic array to identify genes associated with growth and proliferation of pancreatic cancer.

AIM To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. METHODS Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward’s clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. RESULTS Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. CONCLUSION This first study providing “tri-omics” analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression.

Background: Discovery and validation of new antibody tractable targets is critical for the development of new antibody therapeutics to address unmet needs in oncology. Methods: A highly invasive clonal variant of the MDA-MB-435S cell line was used to generate monoclonal antibodies (MAbs), which were screened for anti-invasive activity against aggressive cancer cells in vitro. The molecular target of selected inhibitory MAb 9E1 was identified using immunoprecipitation/liquid chromatography-tandem mass spectrometry. The potential anti-tumour effects of MAb 9E1 were investigated in vitro together with immunohistochemical analysis of the 9E1 target antigen in normal and cancer tissues. Results: MAb 9E1 significantly decreases invasion in pancreatic, lung squamous and breast cancer cells and silencing of its target antigen, which was revealed as AnxA6, leads to markedly reduced invasive capacity of pancreatic and lung squamous cancer in vitro. IHC using MAb 9E1 revealed that AnxA6 exhibits a high prevalence of membrane immunoreactivity across aggressive tumour types with restricted expression observed in the majority of normal tissues. In pancreatic ductal adenocarcinoma, high AnxA6 IHC score correlated with the presence of tumour budding at the invasive front of tumours (P=0.082), the presence of perineural invasion (P=

Background Identification of mechanisms promoting neutrophil trafficking to the lungs of patients with cystic fibrosis (CF) is a challenge for next generation therapeutics. Cholesterol, a structural component of neutrophil plasma membranes influences cell adhesion, a key step in transmigration. The effect of chronic inflammation on neutrophil membrane cholesterol content in patients with CF (PWCF) remains unclear. To address this we examined neutrophils of PWCF to evaluate the cause and consequence of altered membrane cholesterol and identified the effects of lung transplantation and ion channel potentiator therapy on the cellular mechanisms responsible for perturbed membrane cholesterol and increased cell adhesion. Methodology PWCF homozygous for the ΔF508 mutation or heterozygous for the G551D mutation were recruited (n = 48). Membrane protein expression was investigated by mass spectrometry. The effect of lung transplantation or ivacaftor therapy was assessed by ELISAs, and calcium fluorometric and μ-calpain assays. Findings Membranes of CF neutrophils contain less cholesterol, yet increased integrin CD11b expression, and respond to inflammatory induced endoplasmic reticulum (ER) stress by activating μ-calpain. In vivo and in vitro, increased μ-calpain activity resulted in proteolysis of the membrane cholesterol trafficking protein caveolin-1. The critical role of caveolin-1 for adequate membrane cholesterol content was confirmed in caveolin-1 knock-out mice. Lung transplant therapy or treatment of PWCF with ivacaftor, reduced levels of circulating inflammatory mediators and actuated increased caveolin-1 and membrane cholesterol, with concurrent normalized neutrophil adhesion. Interpretation Results demonstrate an auxiliary benefit of lung transplant and potentiator therapy, evident by a reduction in circulating inflammation and controlled neutrophil adhesion., Highlights • This study explored neutrophil adhesion in cystic fibrosis. • Altered membrane cholesterol lead to increased adhesion. • Circulating inflammatory mediators caused increased calpain activity and reduced membrane cholesterol content. In patients with cystic fibrosis (CF), chronic inflammation in the circulation, in part originating from the pulmonary compartment, leads to decreased membrane cholesterol in circulating neutrophils, resulting in increased cell adhesion. The mechanism of action involves proteolytic down-regulation of the cholesterol trafficking protein caveolin-1. The overall effect of lung transplant therapy, or CFTR potentiator treatment, was to significantly diminish the circulating inflammatory burden thereby permitting caveolin-1 expression, with concomitant decreased CF cell adhesion and significant clinical improvement.

Studies in hepatic systems identify multiple factors involved in the generation of copper resistance. As the intestine is the route of exposure to dietary copper, we wanted to understand how intestinal cells overcome the toxic effects of high copper and what mechanisms of resistance develop. Using the intestinal cell line Caco-2, resistance was developed by serial subculture in 50 μM copper in inorganic (CuSO4) or organic (Cu proteinate) forms. Caco-2 variants exhibited resistance to copper and retained the non-monotonic dose response while displaying stable phenotypes following repeated subculture in the absence of copper. Phenotypic changes on exposure to copper in parental Caco-2 cells included significantly increased total protein yield, ROS, SOD, metallothionein expression, GSH and total glutathione. These phenotypic changes were not replicated in resistant variants on a per cell basis. Quantitative label-free LC-MS/MS proteomic analysis identified 1113 differentially expressed proteins (DEPs) between parental Caco-2 and resistant cells. With some exceptions, most of the DEPs were overexpressed to a low level around 2-fold suggesting resistance was supported by multiple small changes in protein expression. These variants may be a useful tool in studying the toxicity of stress responses in further Cu-related studies.

Cancer research relies on key values such as creativity, collaboration, research integrity and resource sharing. A positive research environment which fosters these key values is becoming a decisive factor for some funders and research institutions. To create a supportive research culture in laboratories, the training and mentoring of young scientists is important. However, the fast-paced and fierce competition for funding and jobs can present a challenge to the younger generation of scientists who depend on the guidance and mentorship of scientific leaders. The annual Nature Awards for Mentoring in Science have been created to bring attention to one of the most essential but least recognized skills in scientific leadership. Thus far, 35 scientists from across the world, who are working in a range of disciplines, have been recognized by this award for their outstanding scientific mentorship. In this Viewpoint, we have asked three recipients of this award who work in fields associated with cancer research about their views on good mentoring, and how a revised approach to mentorship can help to achieve a positive research culture and contribute to scientific discovery.

Recent success in demonstrating the translation of circular RNA open reading frames or circular mRNA, may offer a new avenue for improving recombinant protein production from cell and cell-free expression platforms. Initiation and termination are two rate limiting steps of translation. Circular RNA as a class of RNA is defined by covalent joining of terminal ends to give a closed loop structure. By encoding a gene lacking a stop codon on a circular RNA molecule an infinite open reading frame is generated permitting continuously translating circular mRNA (CTC mRNA). CTC mRNAs have shown promise in enhancing the production of multimeric polyproteins in bacterial cell-free expression systems. Problems arise when homogenous, functional post-translationally modified protein is required. To produce post-translationally modified, secreted protein from an CTC mRNA we investigated co-translational cleavage of nascent polypeptide chains by incorporating a 2 A “self-cleavage” peptide motif. Using a model recombinant human glycoprotein Erythropoietin (EPO) we demonstrate for the first time the ability to produce secreted protein from an infinite circular mRNA in live mammalian cells. Both cell-specific and volumetric productivity were improved by using circular mRNAs. This study pioneers the potential of recombinant protein production from CTC mRNA in mammalian cells.

Copper is an essential trace element micronutrient in human and animal nutrition. Trace amounts present even in ultrapure water, serum and other cell culture medium components are sufficient to support the health requirements of most cell types in culture. Analysis of a variety of different types of basal media from a number of different suppliers revealed large fluctuations in the levels of copper, and also of other micronutrients including zinc, iron, selenium and cobalt. Investigations on proliferating Caco-2 cells revealed reductions in growth with increasing copper concentrations within the range seen in the commercial media and changes in expression of apoptosis- and autophagy-related proteins were noted. Even at concentrations of 1 μM CuSO4 where there was no significant change in cell growth, there was a significant decrease in procaspase-3 expression. These results stress the importance of batch testing of basal media when undertaking trace metal research since the baseline levels may vary. Batch variation of serum is well established but our results suggest that batch variation of the media may also be important.

microRNA engineering of CHO cells has already proved successful in enhancing various industrially relevant phenotypes and producing various recombinant products. A single miRNA's ability to interact with multiple mRNA targets allows their regulatory capacity to extend to processes such as cellular metabolism. Various metabolic states have previously been associated with particular CHO cell phenotypes such as glycolytic or oxidative metabolism accommodating growth and productivity, respectively. miR-23 has previously been demonstrated to play a role in glutamate metabolism resulting in enhanced oxidative phosphorylation through the TCA cycle. Re-programming cellular bioenergetics through miR-23 could tip the balance, forcing mammalian production cells to be more productive by favoring metabolic channelling into oxidative metabolism. CHO clones depleted of miR-23 using a miR-sponge decoy demonstrated an average ∼three-fold enhanced specific productivity with no impact on cell growth. Using a cell respirometer, mitochondrial activity was found to be enhanced by ∼30% at Complex I and II of the electron transport system. Additionally, label-free proteomic analysis uncovered various potential novel targets of miR-23 including LE1 and IDH1, both implicated in oxidative metabolism and mitochondrial activity. These results demonstrate miRNA-based engineering as a route to re-programming cellular metabolism resulting in increased productivity, without affecting growth.

Lung cancer is the second most common type of cancer in the world and is the most common cause of cancer-related death in both men and women. Research into causes, prevention and treatment of lung cancer is ongoing and much progress has been made recently in these areas, however survival rates have not significantly improved. Therefore, it is essential to develop biomarkers for early diagnosis of lung cancer, prediction of metastasis and evaluation of treatment efficiency, as well as using these molecules to provide some understanding about tumour biology and translate highly promising findings in basic science research to clinical application. In this investigation, two-dimensional difference gel electrophoresis and mass spectrometry were initially used to analyse conditioned media from a panel of lung cancer and normal bronchial epithelial cell lines. Significant proteins were identified with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), pyruvate kinase M2 isoform (PKM2), Hsc-70 interacting protein and lactate dehydrogenase A (LDHA) selected for analysis in serum from healthy individuals and lung cancer patients. hnRNPA2B1, PKM2 and LDHA were found to be statistically significant in all comparisons. Tissue analysis and knockdown of hnRNPA2B1 using siRNA subsequently demonstrated both the overexpression and potential role for this molecule in lung tumorigenesis. The data presented highlights a number of in vitro derived candidate biomarkers subsequently verified in patient samples and also provides some insight into their roles in the complex intracellular mechanisms associated with tumour progression.

Genetic engineering of mammalian cells is of interest as a means to boost bio-therapeutic protein yield. X-linked inhibitor of apoptosis (XIAP) overexpression has previously been shown to enhance CHO cell growth and prolong culture longevity while additionally boosting productivity. The authors confirmed this across a range of recombinant products (SEAP, EPO, and IgG). However, stable overexpression of an engineering transgene competes for the cells translational machinery potentially compromising product titre. MicroRNAs are attractive genetic engineering candidates given their non-coding nature and ability to regulate multiple genes simultaneously, thereby relieving the translational burden associated with stable overexpression of a protein-encoding gene. The large number of potential targets of a single miRNA, falsely predicted in silico, presents difficulties in identifying those that could be useful engineering tools. The authors explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH protocol. A biotin-tagged antisense DNA oligonucleotide for XIAP mRNA is designed and used to pull down and capture bound miRNAs. Two miRNAs are chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both results in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increases XIAP protein levels. A 3'UTR reporter assay confirms miR-124-3p to be a bona fide regulator of XIAP in CHO-K1 cells. This method demonstrates a useful approach to finding miRNA candidates for CHO cell engineering without competing for the cellular translational machinery.

Background Docetaxel based therapy is one of the first line chemotherapeutic agents for the treatment of metastatic castrate-resistant prostate cancer. However, one of the major obstacles in the treatment of these patients is docetaxel-resistance. Defining the mechanisms of resistance so as to inform subsequent treatment options and combinations represents a challenge for clinicians and scientists. Previous work by our group has shown complex changes in pro and anti-apoptotic proteins in the development of resistance to docetaxel. Targeting these changes individually does not significantly impact on the resistant phenotype but understanding the central signalling pathways and transcription factors (TFs) which control these could represent a more appropriate therapeutic targeting approach. Methods Using a number of docetaxel-resistant sublines of PC-3 cells, we have undertaken a transcriptomic analysis by expression microarray using the Affymetrix Human Gene 1.0 ST Array and in conjunction with bioinformatic analyses undertook to predict dysregulated TFs in docetaxel resistant prostate cancer. The clinical significance of this prediction was ascertained by performing immunohistochemical (IHC) analysis of an identified TF (SRF) in the metastatic sites from men who died of advanced CRPC. Investigation of the functional role of SRF was examined by manipulating SRF using SiRNA in a docetaxel-resistant PC-3 cell line model. Results The transcription factors identified include serum response factor (SRF), nuclear factor kappa-B (NFκB), heat shock factor protein 1 (HSF1), testicular receptor 2 & 4 (TR2 &4), vitamin-D and retinoid x receptor (VDR-RXR) and oestrogen-receptor 1 (ESR1), which are predicted to be responsible for the differential gene expression observed in docetaxel-resistance. IHC analysis to quantify nuclear expression of the identified TF SRF correlates with both survival from date of bone metastasis (p = 0.003), survival from androgen independence (p = 0.00002), and overall survival from prostate cancer (p = 0.0044). Functional knockdown of SRF by siRNA demonstrated a reversal of apoptotic resistance to docetaxel treatment in the docetaxel-resistant PC-3 cell line model. Conclusions Our results suggest that SRF could aid in treatment stratification of prostate cancer, and may also represent a therapeutic target in the treatment of men afflicted with advanced prostate cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3100-4) contains supplementary material, which is available to authorized users.

Identification of protein targets that play a role in breast cancer invasion may help to understand the rapid progression of cancer and may lead to the development of new biomarkers for the disease. In this study, we compared two highly invasive and two poorly invasive breast cancer cell lines using comparative label-free LC-MS profiling in order to identify differentially expressed proteins that may be linked to the invasive phenotype in vitro. Forty-five proteins were found to be upregulated, and 34 proteins, downregulated. UV excision repair protein RAD23 homologue B (RAD23B) was found among the downregulated proteins in highly invasive breast cancer cell lines. In poorly invasive breast cancer cell lines, siRNA-mediated downregulation of RAD23B subsequently led to an increase in invasion and adhesion in vitro. Immunohistochemistry analysis of 164 specimens of invasive breast cancer showed that having a high percentage (>80%) of RAD23B positive nuclei was significantly associated with histopathological grades 1 and 2 breast cancer and with low mitotic activity. In addition, a high staining intensity for RAD23B in the cytoplasm was significantly associated with histopathological grade 3 breast cancer. This study suggests a potential role of RAD23B in breast cancer progression and may further imply a tumor suppressor role of nuclear RAD23B in breast cancer.

Development of more effective therapeutic strategies for cancers of high unmet need requires the continued discovery of disease-specific protein targets for therapeutic antibody targeting. In order to identify novel proteins associated with cancer cell invasion/metastasis, we present here an alternative to antibody targeting of cell surface proteins with an established role in invasion; our functional antibody screening approach involves the isolation and selection of MAbs that are primarily screened for their ability to inhibit tumour invasion. A clonal population of the Mia PaCa-2, a pancreatic ductal adenocarcinoma (PDAC) cell line, which displays a highly invasive phenotype, was used to generate MAbs with the objective of identifying membrane targets directly involved in cancer invasion. Selected MAb 7B7 can significantly reduce invasion in a dose-responsive manner in Mia PaCa-2 clone 3 and DLKP-M squamous lung carcinoma cells. Using immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis, the target antigen of anti-invasive antibody, 7B7, was determined to be the heterodimeric Ku antigen, Ku70/80, a core protein composed of the Ku70 and Ku80 subunits which is involved in non-homologous end-joining (NHEJ) DNA repair. RNA interference-mediated knockdown of Ku70 and Ku80 resulted in a marked decrease in the invasive capacity of Mia PaCa-2 clone 3 and DLKP-M cells, indicating that Ku70/Ku80 is functionally involved in pancreatic and lung cancer invasion. Immunohistochemical analysis demonstrated Ku70/Ku80 immunoreactivity in 37 PDAC tumours, indicating that this heterodimer is highly expressed in this aggressive cancer type. This study demonstrates that a functional MAb screening approach coupled with immunoprecipitation/proteomic analyses can be successfully applied to identify functional anti-invasive MAbs and potential novel targets for therapeutic antibody targeting.

Uveal melanoma is the most common primary intraocular malignancy in adults, with an incidence of 5-7 per million per year. It is associated with the development of metastasis in about 50% of cases, and 40% of patients with uveal melanoma die of metastatic disease despite successful treatment of the primary tumour. The survival rates at 5, 10 and 15 years are 65%, 50% and 45% respectively. Unlike progress made in many other areas of cancer, uveal melanoma is still poorly understood and survival rates have remained similar over the past 25 years. Recently, advances made in molecular genetics have improved our understanding of this disease and stratification of patients into low risk and high risk for developing metastasis. However, only a limited number of studies have been performed using proteomic methods. This review will give an overview of various proteomic technologies currently employed in life sciences research, and discuss proteomic studies of uveal melanoma.

Conditioned medium (CM) from clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with differing invasive abilities, were examined for their effect on in vitro invasion. Conditioned medium from Clone #3 (CM#3) strongly promoted invasion, while CM from Clone #8 (CM#8) inhibited invasion in vitro. 2D DIGE followed by MALDI–TOF MS analysis of CM#3 and CM#8 identified 41 proteins which were differentially regulated; 27 proteins were down-regulated and 14 proteins up-regulated in the invasion-promoting CM#3 when compared to CM#8. Western blotting analysis confirmed the down-regulated expression of gelsolin and the up-regulation of aldehyde dehydrogenase 1A1 in CM#3. Down-regulation of aldehyde dehydrogenase 1A1 in Clone #3 CM and gelsolin levels in Clone #8 CM by siRNA transfection revealed an important involvement of these proteins in promoting and inhibiting invasion in these pancreatic cancer cell lines. Naomi Walsh⁎,1, Paul Dowling1, Norma O'Donovan, Michael Henry, Paula Meleady, Martin Clynes

The majority of patients afflicted with Duchenne muscular dystrophy develop cardiomyopathic complications, warranting large-scale proteomic studies of global cardiac changes for the identification of new protein markers of dystrophinopathy. The aged heart from the X-linked dystrophic mdx mouse has been shown to exhibit distinct pathological aspects of cardiomyopathy. In order to establish age-related alterations in the proteome of dystrophin-deficient hearts, cardiomyopathic tissue from young versus aged mdx mice was examined by label-free LC-MS/MS. Significant age-dependent alterations were established for 67 proteins, of which 28 proteins were shown to exhibit a lower abundance and 39 proteins were found to be increased in their expression levels. Drastic changes were demonstrated for 17 proteins, including increases in Ig chains and transferrin, and drastic decreases in laminin, nidogen and annexin. An immunblotting survey of young and old wild-type versus mdx hearts confirmed these proteomic findings and illustrated the effects of natural aging versus dystrophin deficiency. These proteome-wide alterations suggest a disintegration of the basal lamina structure and cytoskeletal network in dystrophin-deficient cardiac fibres, increased levels of antibodies in a potential autoimmune reaction of the degenerating heart, compensatory binding of excess iron and a general perturbation of metabolic pathways in dystrophy-associated cardiomyopathy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers; despite a low incidence rate it is the fourth leading cause of cancer-related death in the world. Improvement of the diagnosis, prognosis and treatment remains the main focus of pancreatic cancer research. Rapid developments in proteomic technologies has improved our understanding of the pancreatic cancer proteome. Here, the authors summarise the recent proteomic strategies undertaken in the search for: novel biomarkers for early diagnosis, pancreatic cancer-specific proteins which may be used for novel targeted therapies and proteins which may be useful for monitoring disease progression post-therapy. Recent advances and findings discussed here provide great promise of having a significant clinical impact and improving the outcome of patients with this malignancy.

Aspergillus fumigatus is an opportunistic fungal pathogen. Siderophore biosynthesis and iron acquisition are essential for virulence. Yet, limited data exist with respect to the adaptive nature of the fungal microsomal proteome under iron-limiting growth conditions, as encountered during host infection. Here, we demonstrate that under siderophore biosynthetic conditions--significantly elevated fusarinine C (FSC) and triacetylfusarinine C (TAFC) production (p0.0001), extensive microsomal proteome remodelling occurs. Specifically, a four-fold enrichment of transmembrane-containing proteins was observed with respect to whole cell lysates following ultracentrifugation-based microsomal extraction. Comparative label-free proteomic analysis of microsomal extracts, isolated following iron-replete and -deplete growth, identified 710 unique proteins. Scatterplot analysis (MaxQuant) demonstrated high correlation amongst biological replicates from each growth condition (Pearson correlation0.96 within groups; biological replicates (n=4)). Quantitative and qualitative comparison revealed 231 proteins with a significant change in abundance between the iron-replete and iron-deplete conditions (p0.05, fold change ≥ 2), with 96 proteins showing increased abundance and 135 with decreased abundance following iron limitation, including predicted siderophore transporters. Fluorescently labelled FSC was only sequestered following A. fumigatus growth under iron-limiting conditions. Interestingly, human sera exhibited significantly increased reactivity (p0.0001) against microsomal protein extracts obtained following iron-deplete growth.The opportunistic fungal pathogen Aspergillus fumigatus must acquire iron to facilitate growth and pathogenicity. Iron-chelating non-ribosomal peptides, termed siderophores, mediate iron uptake via membrane-localised transporter proteins. Here we demonstrate for the first time that growth of A. fumigatus under iron-deplete conditions, concomitant with siderophore biosynthesis, leads to an extensive remodelling of the microsomal proteome which includes significantly altered levels of 231 constituent proteins (96 increased and 135 decreased in abundance), many of which have not previously been localised to the microsome. We also demonstrate the first synthesis of a fluorescent version of fusarinine C, an extracellular A. fumigatus siderophore, and its uptake and localization under iron-restricted conditions. This infers the use of an A. fumigatus siderophore as a 'Trojan horse' to potentiate the efficacy of anti-fungal drugs. Finally, in addition to revealing the Aspergillus-specific IgG reactivity in normal human sera against microsomal proteins, there appears to be a significantly increased reactivity against microsomal proteins obtained following iron-restricted growth. We hypothesise that iron-limiting environment in humans, which has evolved to nutritionally limit pathogen growth in vivo, may also alter the fungal microsomal proteome.

High-grade gliomas (HGG), are the most common aggressive brain tumours in adults. Inhibitors targeting growth factor signalling pathways in glioma have shown a low clinical response rate. To accurately evaluate response to targeted therapies further in vitro studies are necessary. Growth factor pathway expression using epidermal growth factor receptor (EGFR), mutant EGFR (EGFRvIII), platelet derived growth factor receptor (PDGFR), C-Kit and C-Abl together with phosphatase and tensin homolog (PTEN) expression and downstream activation of AKT and phosphorylated ribosomal protein S6 (P70S6K) was analysed in 26 primary glioma cultures treated with the tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib and imatinib. Response to TKIs was assessed using 50% inhibitory concentrations (IC 50 ). Response for each culture was compared with the EGFR/PDGFR immunocytochemical pathway profile using hierarchical cluster analysis (HCA) and principal component analysis (PCA). Erlotinib response was not strongly associated with high expression of the growth factor pathway components. PTEN expression did not correlate with response to any of the three TKIs. Increased EGFR expression was associated with gefitinib response; increased PDGFR-α expression was associated with imatinib response. The results of this in vitro study suggest gefitinib and imatinib may have therapeutic potential in HGG tumours with a corresponding growth factor receptor expression profile.

Three clonal subpopulations of DLKP, a poorly differentiated squamous lung carcinoma cell line, display striking differences in ability to survive in suspension (anoikis resistance). DLKP-SQ is anoikis resistant (7.5% anoikis at 24 h). In contrast, DLKP-M and DLKP-I are sensitive to anoikis (49.2% and 42.6% respectively). DLKP-I shows increased apoptosis consistently over all time points tested while DLKP-M appear to slow down metabolically and perhaps delays onset of anoikis by undergoing autophagy. Expression microarray analysis identified pronounced differential expression of Olfactomedin 3 (OLFM3) between the clones. High expression of OLFM3 was confirmed at the RNA level by qRT-PCR in DLKP-SQ and at the protein level by Western blotting (within the cell and secreted). Little or no OLFM3 was detected in the other two clones (DLKPM and DLKP-I). Following siRNA knockdown of OLFM3 in DLKP-SQ, anoikis was increased 2.8- fold to 21% which was intermediate between the anoikis levels in DLKP-SQ and DLKP-M or DLKP-I. This knockdown correlated with increased apoptosis in suspension but not in attached culture conditions. Addition of recombinant OLFM3 reduced anoikis in DLKP-I. This is the first instance of OLFM3 being linked with anoikis resistance in a human cancer cell line.

Early detection, clinical management and disease recurrence monitoring are critical areas in cancer treatment in which specific biomarker panels are likely to be very important in each of these key areas. We have previously demonstrated that levels of alpha-2-heremans-schmid-glycoprotein (AHSG), complement component C3 (C3), clusterin (CLI), haptoglobin (HP) and serum amyloid A (SAA) are significantly altered in serum from patients with squamous cell carcinoma of the lung. Here, we report the abundance levels for these proteins in serum samples from patients with advanced breast cancer, colorectal cancer (CRC) and lung cancer compared to healthy controls (age and gender matched) using commercially available enzyme-linked immunosorbent assay kits. Logistic regression (LR) models were fitted to the resulting data, and the classification ability of the proteins was evaluated using receiver-operating characteristic curve and leave-one-out cross-validation (LOOCV). The most accurate individual candidate biomarkers were C3 for breast cancer [area under the curve (AUC) 5 0.89, LOOCV 5 73%], CLI for CRC (AUC 5 0.98, LOOCV 5 90%), HP for small cell lung carcinoma (AUC 5 0.97, LOOCV 5 88%), C3 for lung adenocarcinoma (AUC 5 0.94, LOOCV 5 89%) and HP for squamous cell carcinoma of the lung (AUC 5 0.94, LOOCV 5 87%). The best dual combination of biomarkers using LR analysis were found to be AHSG 1 C3 (AUC 5 0.91, LOOCV 5 83%) for breast cancer, CLI 1 HP (AUC 5 0.98, LOOCV 5 92%) for CRC, C3 1 SAA (AUC 5 0.97, LOOCV 5 91%) for small cell lung carcinoma and HP 1 SAA for both adenocarcinoma (AUC 5 0.98, LOOCV 5 96%) and squamous cell carcinoma of the lung (AUC 5 0.98, LOOCV 5 84%). The high AUC values reported here indicated that these candidate biomarkers have the potential to discriminate accurately between control and cancer groups both individually and in combination with other proteins.

Melanoma-associated antigen (MAGE) family members are generally described as tumor-specific antigens. An association between MAGE-D4B and breast cancer has yet to be reported and the functional role of the encoded protein has never been established. We performed microarray analysis of 104 invasive breast tumors and matched non-cancerous breast biopsies. qPCR was used for validation in an independent biobank. To investigate the biological relevance of MAGE-D4B in breast tumorigenesis, its phenotypic effects were assessed in vitro. Overall, MAGE-D4B was detected in 43% of tumors while undetected in normal breast tissue. MAGE-D4B was found to correlate with tumor progression and to be an independent prognostic marker for poor outcome in term of relapse-free and overall survival, with potential predictive relevance in relation to response to chemotherapy. RNA interference-mediated knockdown of MAGE-D4B significantly hampered the invasive properties of Hs578T cells by affecting anchorage-independent growth, adhesion, migration and invasion affecting anchorage-independent growth, adhesion, migration and invasion and by modulating expression of invasion-suppressor gene E-cadherin.

BACKGROUND: Although trastuzumab has improved the prognosis for HER-2 positive breast cancer, not all HER-2 positive breast tumours respond to trastuzumab treatment and those that initially respond frequently develop resistance. Insulin-like growth factor-1 receptor (IGF1R) signalling has been previously implicated in trastuzumab resistance. We tested IGF1R inhibition to determine if dual targeting of HER-2 and IGF1R improves response in cell line models of acquired trastuzumab resistance. METHODS: HER-2, IGF1R, phospho-HER-2, and phospho-IGF1R levels were measured by ELISA in parental and trastuzumab-resistant SKBR3 and BT474 cells. IGF1R signalling was targeted in these cells using siRNA and the tyrosine kinase inhibitor NVP-AEW541. RESULTS: IGF1R levels were significantly increased in the trastuzumab resistant model, SKBR3/Tr, compared to the parental SKBR3 cell line. In both the SKBR3/Tr and BT474/Tr cell lines, inhibition of IGF1R expression with siRNA or inhibition of tyrosine kinase activity by NVP-AEW541 significantly increased response to trastuzumab. The dual targeting approach also improved response in the parental SKBR3 cells but not in the BT474 parental cells. CONCLUSIONS: Our results confirm that IGF1R inhibition improves response to trastuzumab in resistant HER-2 positive breast cancer cells, and also suggest that dual targeting of IGF1R and HER-2 may improve response in some trastuzumab-sensitive cells.

The efficient production of recombinant proteins by Chinese Hamster Ovary (CHO) cells in modern bioprocesses is often augmented by the use of proliferation control strategies. The most common method is to shift the culture temperature from 37 °C to 28-33 °C though genetic approaches to achieving the same effect are also of interest. In this work we used qRT-PCR-based expression profiling using TLDA™ cards to identify miRNAs displaying differential expression 24h after temperature-shift (TS) from 37 °C to 31 °C. Six miRNAs were found to be significantly up-regulated (mir-219, mir-518d, mir-126, mir-30e, mir-489 and mir-345) and four down-regulated (mir-7, mir-320, mir-101 and mir-199). Furthermore, qRT-PCR analysis of miR-7 expression over a 6 day batch culture, with and without TS, demonstrated decreased expression over time in both cultures but to a significantly greater extent in cells shifted to a lower culture temperature. Unexpectedly, when miR-7 levels were increased transiently by transfection with miR-7 mimic in CHO-K1 cells, cell proliferation at 37 °C was effectively blocked over a 96 h culture period. On the other hand, transient inhibition of endogenous miR-7 levels using antagonists had no impact on cell growth. The exogenous overexpression of miR-7 also resulted in increased normalised (per cell) production at 37 °C, though the yield was lower than cells grown at reduced temperature. This is the first report demonstrating a functional impact of specific miRNA disregulation on CHO cell behavior in batch culture and provides some evidence of the potential which these molecules may have in terms of engineering targets in CHO production clones. Finally, we report the cloning and sequencing of the hamster-specific cgr-miR-7.

Hereditary deficiency of the protein α-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor–mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT–IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositol-anchored (GPI-anchored) Fc receptor FcγRIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of FcγRIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased FcγRIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency.

The ultimate objective of clinical proteomics is the successful discovery, validation and translation of biomarkers, together with new therapeutic targets into medical practices. New highly developed technologies in proteomics and their use in understanding tumor biology have significant clinical potential in the diagnosis, prognosis and treatment of disease. Areas such as MS, new labeling technologies and advancements in bioinformatics systems are now used to successfully detect disease-associated biomarkers together with therapeutic targets in complex biological specimens, including biofluids, cell lysates and tissue biopsies. Recent improvements in sample preparation (specifically focused on fractionation and enrichment) are enabling the analysis of low-abundance proteins together with many types of post-translational modifications. Targeted proteomic diagnostics will play a significant role in the development of personalized molecular medicine, a process that will be vital in modernizing healthcare structures.

Chemotherapy drug resistance is a major obstacle in the treatment of cancer. It can result from an increase in levels of cellular drug efflux pumps, such as P-glycoprotein (P-gp). Lapatinib, a growth factor receptor tyrosine kinase inhibitor, is currently in clinical trials for treatment of breast cancer. We examined the impact of co-incubation of chemotherapy drugs in combination with lapatinib in P-gp over-expressing drug resistant cells. Unexpectedly, lapatinib treatment, at clinically relevant concentrations, increased levels of the P-gp drug transporter in a dose- and time-responsive manner. Conversely, exposure to the epidermal growth factor (EGF), an endogenous growth factor receptor ligand, resulted in a decrease in P-gp expression. Despite the lapatinib-induced alteration in P-gp expression, use of accumulation, efflux and toxicity assays demonstrated that the induced alteration in P-gp expression by lapatinib had little direct impact on drug resistance.

A highly sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed to quantify cellular levels of the tyrosine kinase inhibitors (TKIs) dasatinib (Sprycel) and lapatinib (Tykerb, Tyverb). Cellular samples were extracted with a tert-butyl methyl ether:acetonitrile (3:1, v/v):1 M ammonium formate pH 3.5 (8:1, v/v) mixture. Separation was achieved on a Hyperclone BDS C18 (150 mm x 2.0 mm 3 microm) column with isocratic elution using a mobile phase of acetonitirile-10 mM ammonium formate, pH 4 (54:46, v/v), at a flow rate of 0.2 mL/min. The TKIs were quantified using a triple quadrupole mass spectrometer which was operated in multi-reaction-monitoring mode employing positive electrospray ionisation. The limit of detection and limit of quantification for lapatinib was determined to be 15 and 31 pg on column, respectively. The limit of detection and quantification for dasatinib was 3 and 15 pg on column, respectively. The method allowed for sensitive and accurate determination of cellular levels of dasatinib and lapatinib. In addition, we examined the potential for this method to be utilised to quantitate other TKIs, using gefitinib, erlotinib, imatinib and sorafenib as examples. In principle, these agents were also quantifiable by this method, however, no drug specific validation studies were undertaken with these TKIs. The data indicates that in the cancer cell-line model, DLKP, significantly more lapatinib accumulates in cells in comparison to dasatinib. Additionally, over-expression of the membrane protein drug transporter, P-glycoprotein (P-gp) a common cancer drug resistance mechanism, greatly reduces the cellular accumulation of dasatinib but not of lapatinib.

Purpose: p53 is inactivated in many human malignancies through missense mutations or overexpression of the human homologue of Mdm2 (Hdm2), an E3 ubiquitin ligase that ubiquitinates p53, thereby promoting its proteasomal degradation. The cis-imidazoline nutlin-3 can disrupt the p53-Hdm2 interaction and activate p53, inducing apoptosis in vitro in many malignancies, including multiple myeloma (MM). Experimental Design: We hypothesized that suppression of Hdm2-mediated p53 ubiquitination may augment sequelae of p53 accumulation caused by proteasomal inhibition. We compared the response of MM cells versus several epithelial cancer models to the proteasome inhibitor bortezomib in combination with nutlin-3. Results: The combination of sublethal concentrations of bortezomib plus nutlin-3 induced additive cytotoxicity against bortezomib-sensitive MM cell lines. Importantly, however, in breast, prostate, colon, and thyroid (papillary, follicular, anaplastic, and medullary) carcinoma cell lines, this combination triggered synergistic cytotoxicity, and increased expression of p53, p21, Hdm2, Bax, Noxa, PUMA, and cleavage of caspase-3 and poly ADP ribose polymerase. Coculture with bone marrow stromal cells attenuated MM cell sensitivity to nutlin-3 monotherapy and was associated with evidence of suppression of p53 activity in MM cells, whereas combined bortezomib-nutlin-3 treatment maintained cytotoxicity even in the presence of bone marrow stromal cells. Conclusions: This differential response of MM versus epithelial carcinomas to combination of nutlin-3 with bortezomib sheds new light on the role of p53 in bortezomib-induced apoptosis. Concurrent Hdm2 inhibition with bortezomib may extend the spectrum of bortezomib applications to malignancies with currently limited sensitivity to single-agent bortezomib or, in the future, to MM patients with decreased clinical responsiveness to bortezomib-based therapy. (Clin Cancer Res 2009;15(23):7153–60)

No proven targeted therapy is currently available for the treatment of triple-negative breast cancer (TNBC). Epidermal growth factor receptor (EGFR) is frequently overexpressed in TNBC. We studied the activity of EGFR antagonists alone, and in combination with chemotherapy, in TNBC cell lines.EGFR and phosphorylated EGFR were measured by enzyme-linked immunosorbent assay. Sensitivity to EGFR inhibitors alone and in combination with chemotherapy was assessed. Effects of gefitinib on EGFR signalling and cell cycle were also examined.EGFR was overexpressed in the TNBC compared with the human epidermal growth factor receptor 2 (HER-2)-positive cell lines. Phosphorylation of EGFR was detected in the TNBC cells in response to epidermal growth factor stimulation and was blocked by gefitinib treatment. However, the TNBC cell lines were less sensitive to EGFR inhibition than the HER-2-positive cell lines. Response to gefitinib was associated with reduced phosphorylation of both mitogen activated protein kinase (MAPK) and Akt and induction of G(1) arrest. Gefitinib enhanced response to both carboplatin and docetaxel in the TNBC cells, and the triple combination of gefitinib, carboplatin and docetaxel was synergistic.Although the TNBC cells are less sensitive to EGFR inhibition than the HER-2-positive cell lines, gefitinib enhanced response to chemotherapy. Gefitinib combined with carboplatin and docetaxel warrants further investigation in TNBC.

Alterations in protein expression associated with adriamycin resistance in a panel of variants derived from the poorly differentiated squamous cell lung carcinoma DLKP were investigated using 2-D DIGE. Of the 80 proteins identified as being differentially expressed, 32 correlated to adriamycin resistance. Twenty-four proteins showed positive correlations with drug resistance, 11 correlated directly with increase in the resistance (including NDPK, RPA2, CCT2, HSP70 and Annexin A1) while 13 proteins (including HNRP K and H1, aldehyde dehydrogenase (ALDH), stomatin and CCT3) increased to a similar level in all drug-resistant variants. Fewer proteins showed an inverse correlation with resistance; two (protein disulphide isomerase (PDI) and HSP70 variant 1) displayed a similar decrease in all variants and six (including prohibitin (PHB) and EIF5A) correlated inversely with resistance. Three proteins (EEF1D, Actin G1 and Annexin 1) correlated with the invasive status of these variants. Some expected targets of adriamycin action showed correlation with resistance including RPA2 (critical for DNA damage repair), while others proteins involved in protection from ROS production (such as GST, peroxiredoxins and thioredoxins) did not. The proteomic analysis revealed a large number of changes in protein expression that may contribute to a more apoptosis-resistant state. Many of these changes could provide novel targets for overcoming resistance.

Pulse selections on a chemotherapy naive squamous lung carcinoma cell line, SKMES-1, with clinically relevant concentrations of taxanes (taxol or taxotere) resulted in the development of a stable taxotere-resistant variant, SKMES-1-Taxotere and an unstable taxol-resistant variant, SKMES-1-Taxol. Both variants exhibited increased invasiveness in vitro. The unstable nature of SKMES-1-Taxol facilitated looking at factors involved in loss of taxol resistance and increased invasion. The taxotere and taxol-resistant cell lines were 5.9-fold and 12.5-fold resistant to taxotere and taxol respectively. Alterations in expression of/or point mutations in tubulin, the main target of taxanes, is a major mechanism or resistance. However, alterations in expression of beta tubulin were not consistent in the taxane-selected variants. Cross-resistance to adriamycin, vincristine and etoposide (VP-16) was consistent with overexpression of P-glycoprotein (P-gp). However, P-gp alone is not sufficient to confer the complete multiple drug resistance phenotype as all cell lines exhibited cross-resistance to 5-Fluorouracil (5-FU) and more than one mechanism has been linked to taxane resistance. There was no correlation between the fall of taxol resistance in SKMES-1-Taxol and P-gp expression indicating the loss in resistance to be independent of P-gp expression. Furthermore, resistance to the other drugs was not unstable in SKMES-1-Taxol suggesting some parallel mechanisms of resistance. Two-dimensional electrophoresis coupled with matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify alterations in expression of specific proteins associated with taxane resistance. A large number of differentially regulated proteins were identified in the resistant and invasive variants affecting cellular processes including stress response, protein turnover and cytoskeleton proteins.

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant protein biopharmaceuticals. The purpose of this study was to investigate differences in the proteome of CHO DUKX cells expressing recombinant human bone morphogenetic protein-2 (rhBMP-2) (G5 cells) compared to cells also expressing soluble exogenous paired basic amino acid cleaving enzyme soluble paired basic amino acid cleaving enzyme (PACEsol) (3C9 cells), which has been previously found to improve the post-translational processing of the mature rhBMP-2 dimer. PACEsol co-expression was also associated with a significant increase (almost four-fold) in cellular productivity of rhBMP-2 protein. Differential proteomic expression profiling using 2-D DIGE and MALDI-TOF MS was performed to compare 3C9 and G5 cells, and revealed a list of 60 proteins that showed differential expression (up/downregulated), with a variety of different cellular functions. A substantial number of these altered proteins were found to have chaperone activity, involved with protein folding, assembly and secretion, as well as a number of proteins involved in protein translation. These results support the use of proteomic profiling as a valuable tool towards understanding the biology of bioprocess cultures.

The cytochrome P450 CYP1B1 is consistently overexpressed in tumour cells as compared to their normal counterparts, but its precise role in drug resistance is yet to be defined. It has been reported that transfection of CYP1B1 results in increased resistance to docetaxel in V79 cells (McFadyen et al, 2001). In this study, we analysed changes in expression of CYP1B1 mRNA associated with pulse selection of MCF-7 cells with docetaxel. Docetaxel-selected MCF-7 cells (MCF-7 Txt), which showed increased resistance to this drug as compared to parental MCF-7 cells, showed a noteworthy increase in CYP1B1 mRNA expression, paralleled by increased ethoxyresorufin-O-deethylase (EROD) activity levels. This effect was not observed in cisplatin- or adriamycin-selected MCF-7 cells, or in docetaxel-selected colon, lung or pancreatic carcinoma cells. Short-term treatment with docetaxel induced CYP1B1 mRNA expression in MDA 453 and BT-20 breast carcinoma cells, but not in MCF-7 cells. Transfection of MCF-7 Txt cells with CYP1B1 siRNA did not significantly affect docetaxel-induced toxicity, but it decreased cell survival in the absence of drug. Preincubation of docetaxel with recombinant CYP1B1 did not affect drug toxicity in A549 cells. These results suggest that CYP1B1 does not directly inactivate docetaxel, but rather might promote cell survival in MCF-7 Txt cells, providing an explanation for its association with drug resistance.

Background: Detecting extracellular nucleic acids in the serum/plasma of cancer patients may help in cancer diagnosis. We investigated whether extracellular mRNAs are reproducibly detectable in conditioned medium (CM) from insulin-producing cell cultures and if their presence and amounts are indicative of cell number and/or function.Methods: We isolated mRNA from medium conditioned by the culture of several insulin-producing cell types: MIN6(L) (glucose-responsive), MIN6(H) (glucose-nonresponsive), and MIN6 B1 murine beta cells and monkey kidney fibroblast cells engineered to produce human preproinsulin (PPI) (Vero-PPI). We used reverse transcription–PCR analyses to evaluate the occurrence of several mRNAs and investigated whether the presence and amounts of the various extracellular mRNAs are associated with cell mass and/or function.Results: Reproducible amplification of mRNAs encoded by Pdx1, Npy, Egr1, Pld1, Chgb, Ins1, Ins2, and Actb from MIN6(L), MIN6(H), and MIN6 B1 cells and their CM suggests that beta cells transcribe and release these mRNAs into their culture environment. Similarly, PPI mRNA was detected in samples of Vero-PPI cells and CM. The amounts of some mRNAs reflected the numbers and functional status (i.e., glucose responsiveness vs nonresponsiveness) of the cells conditioning the medium. Although Pax4 mRNA was detected in the MIN6 B1 cell line, the fact that this transcript was not amplifiable from the corresponding CM suggested that mRNA release was selective.Conclusion: mRNAs may be secreted from insulin-producing cells, are reproducibly detected in the extracellular environment, and may have potential as extracellular biomarkers for assessing beta cell mass and function.

The complex role of drug metabolism-related enzymes and their possible influence in cancer development, treatment and outcome has not yet been completely elucidated. There is evidence that these enzymes can activate certain environmental procarcinogens to more toxic derivatives and thus a role has been proposed for them in carcinogenesis. The fact that they can also inactivate a number of chemotherapeutic drugs has raised the possibility of these enzymes influencing the sensitivity of tumour cells to anticancer agents. In this report, we analyse the expression of drug metabolism-related genes within a whole genome microarray study of 104 breast cancer and 17 normal breast specimens. Kaplan-Meier survival curves, Chi-squared, and Cox Regression analyses were used to identify associations between expression of gene transcripts and patients' clinicopathological and survival data. Our results show that several of these genes are significantly expressed in both normal and tumour tissue; in many cases, expression is altered in the tumour specimens as compared to normal breast tissue. Moreover, expression of ARNT2 and GST A1 was correlated with prognosis. Kaplan-Meier analysis showed expression of ARNT2 mRNA to correlate significantly with favourable disease outcome for patients, in terms of both their disease-free survival (P = 0.0094) and overall survival (P = 0.0018) times from diagnosis, while detection of GST A1 mRNA correlated with shortened disease-free survival (P = 0.0131) and overall survival (P = 0.0028). Multivariate analysis indicated GST A1 expression to be an independent prognostic factor for overall survival (P = 0.045). Our results suggest a possible use of ARNT2 and GST A1 as prognostic breast cancer biomarkers.