Paul Dowling

Analysis of the saliva proteome from patients with head and neck squamous cell carcinoma reveals differences in abundance levels of proteins associated with tumour progression and metastasis

The objective of this study was to identify differentially expressed proteins in saliva from HNSCC patients compared to a control group. Saliva samples from eight individuals with non-malignant conditions of the head and neck region were employed as a control group and compared to saliva from eight patients with HNSCC using 2D DIGE analysis and subsequent mass spectrometry identification of candidate proteins. Beta fibrin (+2.77-fold), S100 calcium binding protein (+5.35-fold), transferrin (+3.37-fold), immunoglobulin heavy chain constant region gamma (+3.28) and cofilin-1 (+6.42) were all found to be significantly increased in the saliva from HNSCC samples compared to the control group whereas transthyretin (-2.92-fold) was significantly decreased. The increased abundance of one of the proteins identified (S100 calcium binding protein) was confirmed by immunoblot analysis. Many of these proteins are involved in tumour progression, metastasis and angiogenesis. The proximity of saliva to the developing tumour is undoubtedly a major factor in facilitating detection of these proteins and such a strategy may lead to the development of a panel of biomarkers useful for therapeutic monitoring and for early detection of HNSCC.

Conditioned media from cell lines: A complementary model to clinical specimens for the discovery of disease-specific biomarkers†

In the strictest sense, the cell secretome (conditioned media) refers to the collection of proteins that contain a signal peptide and are processed via the endoplasmic reticulum and Golgi apparatus through the classical secretion pathway. More generally, the secretome also encompasses proteins shed from the cell surface and intracellular proteins released through non‐classical secretion pathway or exosomes. These secreted proteins include numerous enzymes, growth factors, cytokines and hormones or other soluble mediators. They are fundamental in the processes of cell growth, differentiation, invasion and angiogenesis by regulating cell‐to‐cell and cell‐to‐extracellular matrix interactions. The main aim of this review is to provide a synopsis of findings from the analysis of the secretome taking diabetes, cancer and neurodegenerative diseases as examples. We will also discuss the preparation of conditioned media and on the main proteomic‐based methodological approaches that have been developed for the study of secreted/shed proteins.

RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation

We previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered. In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours. Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

Drastic reduction of sarcalumenin in Dp427 (dystrophin of 427 kDa)-deficient fibres indicates that abnormal calcium handling plays a key role in muscular dystrophy.

Although the primary abnormality in dystrophin is the underlying cause for mdx (X-chromosome-linked muscular dystrophy), abnormal Ca2+ handling after sarcolemmal microrupturing appears to be the pathophysiological mechanism leading to muscle weakness. To develop novel pharmacological strategies for eliminating Ca2+-dependent proteolysis, it is crucial to determine the fate of Ca2+-handling proteins in dystrophin-deficient fibres. In the present study, we show that a key luminal Ca2+-binding protein SAR (sarcalumenin) is affected in mdx skeletal-muscle fibres. One- and two-dimensional immunoblot analyses revealed the relative expression of the 160 kDa SR (sarcoplasmic reticulum) protein to be approx. 70% lower in mdx fibres when compared with normal skeletal muscles. This drastic reduction in SAR was confirmed by immunofluorescence microscopy. Patchy internal labelling of SAR in dystrophic fibres suggests an abnormal formation of SAR domains. Differential co-immunoprecipitation experiments and chemical cross-linking demonstrated a tight linkage between SAR and the SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1) isoform of the SR Ca2+-ATPase. However, the relative expression of the fast Ca2+ pump was not decreased in dystrophic membrane preparations. This implies that the reduction in SAR and calsequestrin-like proteins plays a central role in the previously reported impairment of Ca2+ buffering in the dystrophic SR [Culligan, Banville, Dowling and Ohlendieck (2002) J. Appl. Physiol. 92, 435-445]. Impaired Ca2+ shuttling between the Ca2+-uptake SERCA units and calsequestrin clusters via SAR, as well as an overall decreased luminal ion-binding capacity, might indirectly amplify the Ca2+-leak-channel-induced increase in cytosolic Ca2+ levels. This confirms the idea that abnormal Ca2+ cycling is involved in Ca2+-induced myonecrosis. Hence, manipulating disturbed Ca2+ handling might represent new modes of abolishing proteolytic degradation in muscular dystrophy.

2-D DIGE analysis of the mitochondrial proteome from human skeletal muscle reveals time course-dependent remodelling in response to 14 consecutive days of endurance exercise training.

Adaptation of skeletal muscle to repeated bouts of endurance exercise increases aerobic capacity and improves mitochondrial function. However, the adaptation of human skeletal muscle mitochondrial proteome to short‐term endurance exercise training has not been investigated. Eight sedentary males cycled for 60 min at 80% of peak oxygen consumption (VO2peak) each day for 14 consecutive days, resulting in an increase in VO2peak of 17.5±3.8% (p<0.01). Mitochondria‐enriched protein fractions from skeletal muscle biopsies taken from m. vastus lateralis at baseline, and on the morning following the 7th and 14th training sessions were subjected to 2‐D DIGE analysis with subsequent MS followed by database interrogation to identify the proteins of interest. Thirty‐one protein spots were differentially expressed after either 7 or 14 days of training (ANOVA, p<0.05). These proteins included subunits of the electron transport chain, enzymes of the tricarboxylic acid cycle, phosphotransfer enzymes, and regulatory factors in mitochondrial protein synthesis, oxygen transport, and antioxidant capacity. Several proteins demonstrated a time course‐dependent induction during training. Our results illustrate the phenomenon of skeletal muscle plasticity with the extensive remodelling of the mitochondrial proteome occurring after just 7 days of exercise training suggestive of enhanced capacity for adenosine triphosphate generation at a cellular level.

Brain dystrophin-glycoprotein complex: Persistent expression of beta-dystroglycan, impaired oligomerization of Dp71 and up-regulation of utrophins in animal models of muscular dystrophy

BackgroundAside from muscle, brain is also a major expression site for dystrophin, the protein whose abnormal expression is responsible for Duchenne muscular dystrophy. Cognitive impairments are frequently associated with this genetic disease, we therefore studied the fate of brain and skeletal muscle dystrophins and dystroglycans in dystrophic animal models.ResultsAll dystrophin-associated glycoproteins investigated were reduced in dystrophic muscle fibres. In Dp427-deficient mdx brain and Dp71-deficient mdx-3cv brain, the expression of α-dystroglycan and laminin was reduced, utrophin isoforms were up-regulated and β-dystroglycan was not affected. Immunofluorescence localization of β-dystroglycan in comparison with glial, endothelial and neuronal cell markers revealed co-localization of von Willebrand factor with β-dystroglycan. Its expression at the endothelial-glial interface was preserved in dystrophin isoform-deficient brain from mdx and mdx-3cv mice. In addition, chemical crosslinking revealed that the Dp71 isoform exists in mdx brain predominantly as a monomer.ConclusionsThis suggests an association of β-dystroglycan with membranes at the vascular-glial interface in the forebrain. In contrast to dystrophic skeletal muscle fibres, dystrophin deficiency does not trigger a reduction of all dystroglycans in the brain, and utrophins may partially compensate for the lack of brain dystrophins. Abnormal oligomerization of the dystrophin isoform Dp71 might be involved in the pathophysiological mechanisms underlying abnormal brain functions.

Identification of pancreatic cancer invasion-related proteins by proteomic analysis

BackgroundMarkers of pancreatic cancer invasion were investigated in two clonal populations of the cell line, MiaPaCa-2, Clone #3 (high invasion) and Clone #8 (low invasion) using proteomic profiling of an in vitro model of pancreatic cancer.Materials and methodsUsing 2D-DIGE followed by MALDI-TOF MS, two clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with high and low invasive capacities were incubated on matrigel 24 hours prior to analysis to stimulate cell-ECM contact and mimic in vivo interaction with the basement membrane.ResultsSixty proteins were identified as being differentially expressed (> 1.2 fold change and p ≤ 0.05) between Clone #3 and Clone #8. Proteins found to have higher abundance levels in the highly invasive Clone #3 compared to the low invasive Clone #8 include members of the chaperone activity proteins and cytoskeleton constituents whereas metabolism-associated and catalytic proteins had lower abundance levels. Differential protein expression levels of ALDH1A1, VIM, STIP1 and KRT18 and GAPDH were confirmed by immunoblot. Using RNAi technology, STIP1 knockdown significantly reduced invasion and proliferation of the highly invasive Clone #3. Knockdown of another target, VIM by siRNA in Clone #3 cells also resulted in decreased invasion abilities of Clone #3. Elevated expression of STIP1 was observed in pancreatic tumour tissue compared to normal pancreas, whereas ALDH1A1 stained at lower levels in pancreatic tumours, as detected by immunohistochemistry.ConclusionIdentification of targets which play a role in the highly invasive phenotype of pancreatic cancer may help to understand the biological behaviour, the rapid progression of this cancer and may be of importance in the development of new therapeutic strategies for pancreatic cancer.

Bone Disease in Multiple Myeloma: Pathophysiology and Management

Myeloma bone disease (MBD) is a devastating complication of multiple myeloma (MM). More than 80% of MM patients suffer from destructive bony lesions, leading to pain, fractures, mobility issues, and neurological deficits. MBD is not only a main cause of disability and morbidity in MM patients but also increases the cost of management. Bone destruction and lack of bone formation are main factors in the development of MBD. Some novel factors are found to be involved in the pathogenesis of MBD, eg, receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) system (RANKL/OPG), Wingless (Wnt), dickkopf-1 (Wnt/DKK1) pathway. The addition of novel agents in the treatment of MM, use of bisphosphonates and other supportive modalities such as radiotherapy, vertebroplasty/kyphoplasty, and surgical interventions, all have significant roles in the treatment of MBD. This review provides an overview on the pathophysiology and management of MBD.

Drug resistance in cancer - searching for mechanisms, markers and therapeutic agents

Treatment resistance, whether inherent or acquired, is a major problem reducing the activity of conventional and newer, molecularly targeted, cancer drugs. A more complex picture of the causes and contributions of specific forms of resistance is now emerging through application of pharmacological, proteomic and gene expression technologies and we have entered an exciting time where new molecular research tools are being applied not only to characterise the causes of such resistance, but to identify rational new treatments and treatment combinations that are being rapidly translated to clinical evaluations with increasing success. This review outlines many of the contributing causes of resistance to established cytotoxics and to the new breed of molecularly targeted agents, both monoclonal antibodies and small molecules, and the research methods being used to wage war on resistant cancer.

Circulating miRNAs miR-34a and miR-150 associated with colorectal cancer progression

BackgroundScreening for the early detection of colorectal cancer is important to improve patient survival. The aim of this study was to investigate the potential of circulating cell-free miRNAs as biomarkers of CRC, and their efficiency at delineating patients with polyps and benign adenomas from normal and cancer patient groups.MethodsThe expression of 667 miRNAs was assessed in a discovery set of 48 plasma samples comprising normal, polyp, adenoma, and early and advanced cancer samples. Three miRNAs (miR-34a, miR-150, and miR-923) were further examined in a validation cohort of 97 subjects divided into the same five groups, and in an independent public dataset of 40 CRC samples and paired normal tissues.ResultsHigh levels of circulating miR-34a and low miR-150 levels distinguished groups of patients with polyps from those with advanced cancer (AUC = 0.904), and low circulating miR-150 levels separated patients with adenomas from those with advanced cancer (AUC = 0.875). In addition, the altered expression of miR-34a and miR-150 in an independent public dataset of forty CRC samples and paired normal tissues was confirmed.ConclusionWe identified two circulating miRNAs capable of distinguishing patient groups with different diseases of the colon from each other, and patients with advanced cancer from benign disease groups.