Tumor-derived CXCL8 signaling augments stroma-derived CCL2-promoted proliferation and CXCL12-mediated invasion of PTEN-deficient prostate cancer cells

Impaired PTEN function is a genetic hallmark of aggressive prostate cancers (CaP) and is associated with increased CXCL8 expression and signaling. The current aim was to further characterize biological responses and mechanisms underpinning CXCL8-promoted progression of PTEN-depleted prostate cancer, focusing on characterizing the potential interplay between CXCL8 and other disease-promoting chemokines resident within the prostate tumor microenvironment. Autocrine CXCL8-stimulation (i) increased expression of CXCR1 and CXCR2 in PTEN-deficient CaP cells suggesting a self-potentiating signaling axis and (ii) induced expression of CXCR4 and CCR2 in PTEN-wild-type and PTEN-depleted CaP cells. In contrast, paracrine CXCL8 signaling induced expression and secretion of the chemokines CCL2 and CXCL12 from prostate stromal WPMY-1 fibroblasts and monocytic macrophage-like THP-1 cells. In vitro studies demonstrated functional co-operation of tumor-derived CXCL8 with stromal-derived chemokines. CXCL12-induced migration of PC3 cells and CCL2-induced proliferation of prostate cancer cells were dependent upon intrinsic CXCL8 signaling within the prostate cancer cells. For example, in co-culture experiments, CXCL12/CXCR4 signaling but not CCL2/CCR2 signaling supported fibroblast-mediated migration of PC3 cells while CXCL12/CXCR4 and CCL2/CCR2 signaling underpinned monocyte-enhanced migration of PC3 cells. Combined inhibition of both CXCL8 and CXCL12 signaling was more effective in inhibiting fibroblast-promoted cell motility while repression of CXCL8 attenuated CCL2-promoted proliferation of prostate cancer cells. We conclude that tumor-derived CXCL8 signaling from PTEN-deficient tumor cells increases the sensitivity and responsiveness of CaP cells to stromal chemokines by concurrently upregulating receptor expression in cancer cells and inducing stromal chemokine synthesis. Combined chemokine targeting may be required to inhibit their multi-faceted actions in promoting the invasion and proliferation of aggressive CaP.

p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling

Using microarray information from oro-pharyngeal data sets and results from primary human foreskin keratinocytes (HFK) expressing Human Papilloma Virus (HPV)-16 E6/E7 proteins, we show that p63 expression regulates signalling molecules which initiate cell migration such as Src and focal adhesion kinase (FAK) and induce invasion in 3D-organotypic rafts; a phenotype that can be reversed by depletion of p63. Knockdown of Src or FAK in the invasive cells restored focal adhesion protein paxillin at cell periphery and impaired the cell migration. In addition, specific inhibition of FAK (PF573228) or Src (dasatinib) activities mitigated invasion and attenuated the expression/activity of matrix metalloproteinase 14 (MMP14), a pivotal MMP in the MMP activation cascade. Expression of constitutively active Src in non-invasive HFK expressing E6/E7 proteins upregulated the activity of c-Jun and MMP14, and induced invasion in rafts. Depletion of Src, FAK or AKT in the invasive cells normalised the expression/activity of c-Jun and MMP14, thus implicating the Src-FAK/AKT/AP-1 signalling in MMP14-mediated extra-cellular matrix remodelling. Up-regulation of Src, AP-1, MMP14 and p63 expression was confirmed in oro-pharyngeal cancer. Since p63 transcriptionally regulated expression of many of the genes in this signalling pathway, it suggests that it has a central role in cancer progression.

HPV16 Down-Regulates the Insulin-Like Growth Factor Binding Protein 2 to Promote Epithelial Invasion in Organotypic Cultures

Cervical cancer is a multi-stage disease caused by human papillomaviruses (HPV) infection of cervical epithelial cells, but the mechanisms regulating disease progression are not clearly defined. Using 3-dimensional organotypic cultures, we demonstrate that HPV16 E6 and E7 proteins alter the secretome of primary human keratinocytes resulting in local epithelial invasion. Mechanistically, absence of the IGF-binding protein 2 (IGFBP2) caused increases in IGFI/II signalling and through crosstalk with KGF/FGFR2b/AKT, cell invasion. Repression of IGFBP2 is mediated by histone deacetylation at the IGFBP2 promoter and was reversed by treatment with histone deacetylase (HDAC) inhibitors. Our in vitro findings were confirmed in 50 invasive cancers and 79 cervical intra-epithelial neoplastic lesions caused by HPV16 infection, where IGFBP2 levels were reduced with increasing disease severity. In summary, the loss of IGFBP2 is associated with progression of premalignant disease, and sensitises cells to pro-invasive IGF signalling, and together with stromal derived factors promotes epithelial invasion.

The paradox of invasion: Reeves’ muntjac deer invade the British Isles from a limited number of founding females

High levels of genetic diversity and high propagule pressure are favoured by conservation biologists as the basis for successful reintroductions and ensuring the persistence of populations. However, invasion ecologists recognize the ‘paradox of invasion’, as successful species introductions may often be characterized by limited numbers of individuals and associated genetic bottlenecks. In the present study, we used a combination of high-resolution nuclear and mitochondrial genetic markers to investigate the invasion history of Reeves' muntjac deer in the British Isles. This invasion has caused severe economic and ecological damage, with secondary spread currently a concern throughout Europe and potentially globally. Microsatellite analysis based on eight loci grouped all 176 introduced individuals studied from across the species' range in the UK into one genetic cluster, and seven mitochondrial D-loop haplotypes were recovered, two of which were present at very low frequency and were related to more common haplotypes. Our results indicate that the entire invasion can be traced to a single founding event involving a low number of females. These findings highlight the fact that even small releases of species may, if ignored, result in irreversible and costly invasion, regardless of initial genetic diversity or continual genetic influx.

Slug-dependent upregulation of L1CAM is responsible for the increased invasion potential of pancreatic cancer cells following long-term 5-FU treatment

BACKGROUND: Pancreatic adenocarcinoma is a lethal disease with 5-year survival of less than 5%. 5-fluorouracil (5-FU) is a principal first-line therapy, but treatment only extends survival modestly and is seldom curative. Drug resistance and disease recurrence is typical and there is a pressing need to overcome this. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc 03.27 cell line by long-term exposure to increasing doses of 5-FU.

RESULTS: 5-FU-resistant cell lines exhibited increased expression of markers associated with multidrug resistance explaining their reduced sensitivity to 5-FU. In addition, 5-FU-resistant cell lines showed alterations typical for an epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, and a significant upregulation of L1CAM was seen on the RNA and protein level. In pancreatic cancer, expression of L1CAM is associated with a chemoresistant and migratory phenotype. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with antibodies, we show that the increased invasiveness observed in the chemoresistant cells functionally depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we demonstrate that in the chemoresistant cell lines, L1CAM expression depends on Slug rather than β-catenin.

CONCLUSION: Our findings establish Slug-induced L1CAM expression as a mediator of a chemoresistant and migratory phenotype in pancreatic adenocarcinoma cells.

MT1-MMP regulates urothelial cell invasion via transcriptional regulation of Dickkopf-3

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a zinc-binding endopeptidase, which plays a crucial role in tumour growth, invasion and metastasis. We have shown previously that MT1-MMP has higher expression levels in the human urothelial cell carcinoma (UCC) tissue. We show here that siRNA against MT1-MMP blocks invasion in UCC cell lines. Invasion is also blocked by broad-spectrum protease and MMP inhibitors including tissue inhibitor of metalloproteinase-1 and -2. Membrane type-1-MMP can also regulate transcription. We have used expression arrays to identify genes that are differentially transcribed when siRNA is used to suppress MT1-MMP expression. Upon MT1-MMP knockdown, Dickkopf-3 (DKK3) expression was highly upregulated. The stability of DKK3 mRNA was unaffected under these conditions, suggesting transcriptional regulation of DKK3 by MT1-MMP. Dickkopf-3 has been previously shown to inhibit invasion. We confirm that the overexpression of DKK3 leads to decreased invasive potential as well as delayed wound healing. We show for the first time that the effects of MT1-MMP on cell invasion are mediated in part through changes in DKK3 gene transcription.

Forecasting invasions: resource use by mussels informs invasion patterns along the South African coast

Invasive species are often more able to rapidly and efficiently utilise resources than natives, and comparing per capita resource use at different resource densities among invaders and trophically analogous natives could allow for reliable predictions of invasiveness. In South Africa, invasion by the Mediterranean mussel Mytilus galloprovincialis has transformed wave-exposed shores, negatively affecting native mussel species. Currently, South Africa is experiencing a second mussel invasion with the recent detection of the South American Semimytilus algosus. We tested per capita uptake of an algal resource by invading M. galloprovincialis, S. algosus, and the native Aulacomya atra at different algal concentrations and temperatures, representing the west and south coasts of South Africa, to examine whether their per capita resource use could be a predictor of their spread and subsequent invasiveness. Regardless of temperature, M. galloprovincialis was the most efficient consumer, significantly reducing algal cells compared to the other species when the resource was presented in both low and high starting densities. Furthermore, these findings aligned with a greater biomass of M. galloprovincialis on the shore in comparison with the other species. Resource use by the new invader S. algosus was dependent on the density of resource and, although this species was efficient at low algal concentrations at cooler temperatures, this pattern broke down at higher algal densities. This was once more reflected in lower biomass in surveys of this species along the cool west coast. We therefore forecast that S. algosus will be become established along the south coast; however, we also predict that M. galloprovincialis will maintain dominance on these shores.

EphA2 Expression Is a Key Driver of Migration and Invasion and a Poor Prognostic Marker in Colorectal Cancer

PURPOSE: EphA2, a member of the Eph receptor tyrosine kinases family, is an important regulator of tumor initiation, neovascularization, and metastasis in a wide range of epithelial and mesenchymal cancers; however, its role in colorectal cancer recurrence and progression is unclear.

EXPERIMENTAL DESIGN: EphA2 expression was determined by immunohistochemistry in stage II/III colorectal tumors (N = 338), and findings correlated with clinical outcome. The correlation between EphA2 expression and stem cell markers CD44 and Lgr5 was examined. The role of EphA2 in migration/invasion was assessed using a panel of KRAS wild-type (WT) and mutant (MT) parental and invasive colorectal cancer cell line models.

RESULTS: Colorectal tumors displayed significantly higher expression levels of EphA2 compared with matched normal tissue, which positively correlated with high CD44 and Lgr5 expression levels. Moreover, high EphA2 mRNA and protein expression were found to be associated with poor overall survival in stage II/III colorectal cancer tissues, in both univariate and multivariate analyses. Preclinically, we found that EphA2 was highly expressed in KRASMT colorectal cancer cells and that EphA2 levels are regulated by the KRAS-driven MAPK and RalGDS-RalA pathways. Moreover, EphA2 levels were elevated in several invasive daughter cell lines, and downregulation of EphA2 using RNAi or recombinant EFNA1 suppressed migration and invasion of KRASMT colorectal cancer cells.

CONCLUSIONS: These data show that EpHA2 is a poor prognostic marker in stage II/III colorectal cancer, which may be due to its ability to promote cell migration and invasion, providing support for the further investigation of EphA2 as a novel prognostic biomarker and therapeutic target. Clin Cancer Res; 22(1); 230-42. ©2015 AACR.

Polybius’ ‘previous landslide’: Proof that Hannibal’s invasion route crossed the col de la traversette

Both Polybius and Livy described a landslide/landslip that blocked the Punic Army’s exfiltration from a high col on the water divide in the Western Alps. The landslide, more aptly termed rockfall, has been a source of contention amongst classicists for centuries despite the fact that only two cols—Clapier and Traversette—exhibit rockfall debris on the lee side of the Alps. While the Clapier rockfall is too small and too young to have provided blockage, the Traversette debris is nearly as Polybius described it when he retraced the invasion route some 60 years after the event. His ‘two-tier’ description of the deposit, a doublet of younger and older rock rubble, including measurements of width and volume are close to modern measurements and prove that he knew, in advance, the route Hannibal had followed. It would take a practiced eye to correctly identify the stratigraphic complexity inherent in the Traversette Rockfall. Here we present weathering ratios, soil stratigraphic, mineral, chemical and microbiological evidence in support of Polybius’ observations as a considerable background database for future geoarchaeological exploration.

Population model of the recent subtidal invasion of Sargassum muticum in southern Portugal

Dissertação de Mestrado, Biologia Marinha, Especialização em Ecologia e Conservação, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2007

Cadherin-5: a biomarker for metastatic breast cancer with optimum efficacy in oestrogen receptor-positive breast cancers with vascular invasion

Background: A glycoproteomic study has previously shown cadherin-5 (CDH5) to be a serological marker of metastatic breast cancer when both protein levels and glycosylation status were assessed. In this study we aimed to further validate the utility of CDH5 as a biomarker for breast cancer progression. Methods: A nested case–control study of serum samples from breast cancer patients, of which n=52 had developed a distant metastatic recurrence within 5 years post-diagnosis and n=60 had remained recurrence-free. ELISAs were used to quantify patient serum CDH5 levels and assess glycosylation by Helix pomatia agglutinin (HPA) binding. Clinicopathological, treatment and lifestyle factors associated with metastasis and elevated biomarker levels were identified. Results: Elevated CDH5 levels (P=0.028) and ratios of CDH5:HPA binding (P=0.007) distinguished patients with metastatic disease from those that remained metastasis-free. Multivariate analysis showed that the association between CDH5:HPA ratio and the formation of distant metastases was driven by patients with oestrogen receptor (ER+) positive cancer with vascular invasion (VI+). Conclusions: CDH5 levels and the CDH5 glycosylation represent biomarker tests that distinguish patients with metastatic breast cancer from those that remain metastasis-free. The test reached optimal sensitivity and specificity in ER-positive cancers with vascular invasion.

Using life strategies to explore the vulnerability of ecosystem services to invasion by alien plants

Invasive plants can have different effects of ecosystem functioning and on the provision of ecosystem services, from strongly deleterious impacts to positive effects. The nature and intensity of such effects will depend on the service and ecosystem being considered, but also on features of life strategies of invaders that influence their invasiveness as well as their influence of key processes of receiving ecosystems. To address the combined effect of these various factors we developed a robust and efficient methodological framework that allows to identify areas of possible conflict between ecosystem services and alien invasive plants, considering interactions between landscape invasibility and species invasiveness. Our framework combines the statistical robustness of multi-model inference, efficient techniques to map ecosystem services, and life strategies as a functional link between invasion, functional changes and potential provision of services by invaded ecosystems. The framework was applied to a test region in Portugal, for which we could successfully predict the current patterns of plant invasion, of ecosystem service provision, and finally of probable conflict (expressing concern for negative impacts, and value for positive impacts on services) between alien species richness (total and per plant life strategy) and the potential provision of selected services. Potential conflicts were identified for all combinations of plant strategy and ecosystem service, with an emphasis for those concerning conflicts with carbon sequestration, water regulation and wood production. Lower levels of conflict were obtained between invasive plant strategies and the habitat for biodiversity supporting service. The added value of the proposed framework in the context of landscape management and planning is discussed in perspective of anticipation of conflicts, mitigation of negative impacts, and potentiation of positive effects of plant invasions on ecosystems and their services.

Inhibition of cell migration and invasion mediated by the TAT-RasGAP317-326 peptide requires the DLC1 tumor suppressor.

TAT-RasGAP317-326, a peptide corresponding to the 317-326 sequence of p120 RasGAP coupled with a cell-permeable TAT-derived peptide, sensitizes the death response of various tumor cells to several anticancer treatments. We now report that this peptide is also able to increase cell adherence, prevent cell migration and inhibit matrix invasion. This is accompanied by a marked modification of the actin cytoskeleton and focal adhesion redistribution. Interestingly, integrins and the small Rho GTP-binding protein, which are well-characterized proteins modulating actin fibers, adhesion and migration, do not appear to be required for the pro-adhesive properties of TAT-RasGAP317-326. In contrast, deleted in liver cancer-1, a tumor suppressor protein, the expression of which is often deregulated in cancer cells, was found to be required for TAT-RasGAP317-326 to promote cell adherence and inhibit migration. These results show that TAT-RasGAP317-326, besides its ability to favor tumor cell death, hampers cell migration and invasion.