Automated annotation and visualisation of high-resolution spatial proteomic mass spectrometry imaging data using HIT-MAP.

Spatial proteomics has the potential to significantly advance our understanding of biology, physiology and medicine. Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a powerful tool in the spatial proteomics field, enabling direct detection and registration of protein abundance and distribution across tissues. MALDI-MSI preserves spatial distribution and histology allowing unbiased analysis of complex, heterogeneous tissues. However, MALDI-MSI faces the challenge of simultaneous peptide quantification and identification. To overcome this, we develop and validate HIT-MAP (High-resolution Informatics Toolbox in MALDI-MSI Proteomics), an open-source bioinformatics workflow using peptide mass fingerprint analysis and a dual scoring system to computationally assign peptide and protein annotations to high mass resolution MSI datasets and generate customisable spatial distribution maps. HIT-MAP will be a valuable resource for the spatial proteomics community for analysing newly generated and retrospective datasets, enabling robust peptide and protein annotation and visualisation in a wide array of normal and disease contexts.

'MCC' protein interacts with E-cadherin and ß-catenin strengthening cell-cell adhesion of HCT116 colon cancer cells.

E-cadherin and ß-catenin are key proteins that are essential in the formation of the epithelial cell layer in the colon but their regulatory pathways that are disrupted in cancer metastasis are not completely understood. Mutated in colorectal cancer (MCC) is a tumour suppressor gene that is silenced by promoter methylation in colorectal cancer and particularly in patients with increased lymph node metastasis. Here, we show that MCC methylation is found in 45% of colon and 24% of rectal cancers and is associated with proximal colon, poorly differentiated, circumferential and mucinous tumours as well as increasing T stage and larger tumour size. Knockdown of MCC in HCT116 colon cancer cells caused a reduction in E-cadherin protein level, which is a hallmark of epithelial-mesenchymal transition in cancer, and consequently diminished the E-cadherin/ß-catenin complex. MCC knockdown disrupted cell-cell adhesive strength and integrity in the dispase and transepithelial electrical resistance assays, enhanced hepatocyte growth factor-induced cell scatter and increased tumour cell invasiveness in an organotypic assay. The Src/Abl inhibitor dasatinib, a candidate anti-invasive drug, abrogated the invasive properties induced by MCC deficiency. Mechanistically, we establish that MCC interacts with the E-cadherin/ß-catenin complex. These data provide a significant advance in the current understanding of cell-cell adhesion in colon cancer cells.

SerpinB2 regulates stromal remodelling and local invasion in pancreatic cancer.

Pancreatic cancer has a devastating prognosis, with an overall 5-year survival rate of ~8%, restricted treatment options and characteristic molecular heterogeneity. SerpinB2 expression, particularly in the stromal compartment, is associated with reduced metastasis and prolonged survival in pancreatic ductal adenocarcinoma (PDAC) and our genomic analysis revealed that SERPINB2 is frequently deleted in PDAC. We show that SerpinB2 is required by stromal cells for normal collagen remodelling in vitro, regulating fibroblast interaction and engagement with collagen in the contracting matrix. In a pancreatic cancer allograft model, co-injection of PDAC cancer cells and SerpinB2-/- mouse embryonic fibroblasts (MEFs) resulted in increased tumour growth, aberrant remodelling of the extracellular matrix (ECM) and increased local invasion from the primary tumour. These tumours also displayed elevated proteolytic activity of the primary biochemical target of SerpinB2-urokinase plasminogen activator (uPA). In a large cohort of patients with resected PDAC, we show that increasing uPA mRNA expression was significantly associated with poorer survival following pancreatectomy. This study establishes a novel role for SerpinB2 in the stromal compartment in PDAC invasion through regulation of stromal remodelling and highlights the SerpinB2/uPA axis for further investigation as a potential therapeutic target in pancreatic cancer.

The Δ133p53 isoform and its mouse analogue Δ122p53 promote invasion and metastasis involving pro-inflammatory molecules interleukin-6 and CCL2.

A number of naturally occurring isoforms of the tumour suppressor protein p53 have been discovered, which appear to have differing roles in tumour prevention or promotion. We are investigating the tumour-promoting activities of the Δ133p53 isoform using our mouse model of Δ133p53 (Δ122p53). Here, we report that tumours from Δ122p53 homozygous mice show evidence of invasion and metastasis and that Δ122p53 promotes migration though a 3-dimensional collagen matrix. We also show that Δ122p53 and Δ133p53 promote cell migration in scratch wound and Transwell assays, similar to the 'gain-of-function' phenotypes seen with mutant p53. Using the well-defined B16 mouse melanoma metastatic model, we show that Δ122p53 leads to faster generation of lung metastases. The increased migratory phenotypes are dependent on secreted factors, including the cytokine interleukin-6 and the chemokine CCL2. We propose that Δ122p53 (and Δ133p53) acts in a similar manner to 'gain-of-function' mutant p53 proteins to promote migration, invasion and metastasis, which may contribute to poor survival in patients with Δ133p53-expressing tumours.

Functions of TAp63 and p53 in restraining the development of metastatic cancer.

Many tumours harbour mutations in the p53 tumour-suppressor gene that result in the expression of a mutant p53 protein. This mutant p53 protein has, in most cases, lost wild-type transcriptional activity and can also acquire novel functions in promoting invasion and metastasis. One of the mechanisms underlying these novel functions involves the ability of the mutant p53 to interfere with other transcription factors, including the p53 family protein TAp63. To investigate whether simultaneous depletion of both p53 and TAp63 can recapitulate the effect of mutant p53 expression in vivo, we used a mouse model of pancreatic cancer in which the expression of mutant p53 resulted in the rapid appearance of primary tumours and metastases. As shown previously, loss of one allele of wild-type (WT) p53 accelerated tumour development. A change of one WT p53 allele into mutant p53 did not further accelerate tumour development, but did promote the formation of metastasis. By contrast, loss of TAp63 did not significantly accelerate tumour development or metastasis. However, simultaneous depletion of p53 and TAp63 led to both rapid tumour development and metastatic potential, although the incidence of metastases remained lower than that seen in mutant p53-expressing tumours. TAp63/p53-null cells derived from these mice also showed an enhanced ability to scatter and invade in tissue culture as was observed in mutant p53 cells. These data suggest that depletion of TAp63 in a p53-null tumour can promote metastasis and recapitulate-to some extent-the consequences of mutant p53 expression.

Mutant p53 enhances MET trafficking and signalling to drive cell scattering and invasion.

Tumour-derived mutant p53 proteins promote invasion, in part, by enhancing Rab coupling protein (RCP)-dependent receptor recycling. Here we identified MET as an RCP-binding protein and showed that mutant p53 promoted MET recycling. Mutant p53-expressing cells were more sensitive to hepatocyte growth factor, the ligand for MET, leading to enhanced MET signalling, invasion and cell scattering that was dependent on both MET and RCP. In cells expressing the p53 family member TAp63, inhibition of TAp63 also lead to cell scattering and MET-dependent invasion. However, in cells that express very low levels of TAp63, the ability of mutant p53 to promote MET-dependent cell scattering was independent of TAp63. Taken together, our data show that mutant p53 can enhance MET signalling to promote cell scattering and invasion through both TAp63-dependent and -independent mechanisms. MET has a predominant role in metastatic progression and the identification of mechanisms through which mutations in p53 can drive MET signalling may help to identify and direct therapy.

Mtss1 regulates epidermal growth factor signaling in head and neck squamous carcinoma cells.

Mtss1 is located within chromosomal region 8q23-24, which is one of the three most commonly amplified regions in head and neck squamous cell carcinoma (HNSCC). Mtss1 is lost in metastatic cells, but confusingly is commonly overexpressed in primary tumors. Here we address possible reasons why Mtss1 is positively selected for in primary tumors. We find that Mtss1 enhances the localization of the epidermal growth factor (EGF) receptor to the plasma membrane, prolonging EGF signaling and resulting in enhanced proliferation in HNSCC. Depletion of Mtss1 results in decreased EGF receptor levels and decreased phosphorylation of Erk1/2 and Akt. However, when cells are at high density and adherent to each other, analogous to conditions in a solid tumor, Mtss1 does not confer any growth advantage, either in basal conditions or following EGF stimulation. This could indicate why Mtss1 might be lost in metastases, but preserved in early primary tumors. This is supported by an organotypic assay showing that Mtss1-expressing cells display a less proliferative more epithelial-like morphology on top of a collagen matrix. Furthermore, xenograft tumors expressing Mtss1 initially grow more rapidly, but later show less proliferation and more differentiation. Mtss1 positively modulates EGF signaling at low cell densities to promote proliferation and, therefore, may be beneficial for the early stages of primary HNSCC tumor growth. However, at high cell densities, Mtss1 impacts negatively on EGF signaling and this suggests why it inhibits metastasis.

Annexin A6 inhibits Ras signalling in breast cancer cells.

Overexpression of epidermal growth factor receptor (EGFR) is associated with enhanced activation of wild-type (hyperactive) Ras in breast cancer. Little is known about the regulation of Ras inactivation and GTPase-activating proteins (GAPs), such as p120GAP, in cells with hyperactive Ras. Recently, we showed that in EGFR-overexpressing A431 cells, which lack endogenous Annexin A6 (AnxA6), ectopic expression of AnxA6 stimulates membrane recruitment of p120GAP to modulate Ras signalling. We now demonstrate that, AnxA6 is downregulated in a number of EGFR-overexpressing and estrogen receptor (ER)-negative breast cancer cells. In these cells, AnxA6 overexpression promotes Ca(2+)- and EGF-inducible membrane targeting of p120GAP. In ER-negative MDA-MB-436 cells, overexpression of p120GAP, but not CAPRI or a p120GAP mutant lacking the AnxA6-binding domain inhibits Ras/MAPK activity. AnxA6 knockdown in MDA-MB-436 increases Ras activity and cell proliferation in anchorage-independent growth assays. Furthermore, AnxA6 co-immunoprecipitates with H-Ras in a Ca(2+)- and EGF-inducible manner and fluorescence resonance energy transfer (FRET) microscopy confirmed that AnxA6 is in close proximity of active (G12V), but not inactive (S17N) H-Ras. Thus, association of AnxA6 with H-Ras-containing protein complexes may contribute to regulate p120GAP/Ras assembly in EGFR-overexpressing and ER-negative breast cancer cells.

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.

Cryptosporidium parvum in environmental samples in the Sligo area, Republic of Ireland: a preliminary report.

To detect Cryptosporidium in environmental specimens in the Republic of Ireland, grab samples of river water were prepared by calcium carbonate flocculation, and marine mussel tissue homogenated prior to testing with a fluorescently labelled monoclonal antibody and fluorescence microscopy. The parasite was detected in both river waters and marine mussels (Mytilus edulis). Filter feeders such as Mytilus edulis may be of value as biological monitors for the presence of cryptosporidial oocysts in sea water. The presence of Cryptosporidium in river and marine waters and, in particular, contaminating mussels used for human consumption, has obvious health implications.