Regulation of cancer metastasis by microRNAs

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that have been found highly conserved among species. MiRNAs are able to negatively regulate gene expression through base pairing of 3’ UTRs of their target genes. Therefore, miRNAs have been shown to play an important role in regulating various cellular activities. Over the past decade, substantial evidences have been obtained to show that miRNAs are aberrantly expressed in human malignancies and could act as “OncomiRs” or “Tumor suppressor miRs”. In recent years, increasing number of studies have demonstrated the involvement of miRNAs in cancer metastasis. Many studies have shown that microRNAs could directly target genes playing a central role in epithelia-mesenchymal-transition (EMT), a cellular transformation process that allows cancer cells to acquire motility and invasiveness. EMT is considered an essential step driving the early phase of cancer metastasis. This review will summarize the recent findings and characterization of miRNAs that are involved in the regulation of EMT, migration, invasion and metastasis of cancer cells. Lastly, we will discuss potential use of miRNAs as diagnostic and prognostic biomarkers as well as therapeutic targets for cancer.     

Identification of Aedes albopictus larval index thresholds in the transmission of dengue in Guangzhou,China

Entomological indices have been used to quantitatively express vector density, but the threshold of larval indices of Aedes albopictus in dengue epidemics is still undefined. We conducted a case‐control study to identify the thresholds of Aedes albopictus larval indices in dengue epidemics. Two unit levels of analysis were used: district and street. The discriminative power of the indices was assessed by receiver operating characteristic (ROC) curves. The association between the entomologic indices and dengue transmission was further explored by a logistic regression model. At the district level, there was no significant difference in the Breteau index (BI) between districts that reported cases and those did not (t=0.164, p>0.05), but the Container index (CI) did show a significant difference (t=2.028, p<0.01). The AUC (Area Under the Curve) of BI, CI, and prediction value were 0.540, 0.630, and 0.533, respectively. Predicting at the street level, the AUC of BI, CI, and prediction values were 0.684, 0.660, and 0.685, respectively, and 0.861, 0.827, and 0.867 for outbreaks. BI=5.1, CI=5.4, or prediction value =0.491were suggested to control the epidemic efficiently with the fewest resources, where BI=4.0, CI=5.1, or PRE =0.483 were suggested to achieve effectiveness.     

Propagule pressure governs establishment of an invasive herb

The success of plant invasions may be limited by the availability of propagules and/or of suitable microsites, with microsite availability being affected by, for example, disturbance and interspecific competition. A mechanistic understanding of the contributions of propagule pressure and microsite limitation to plant invasions is therefore required to minimise future invasions. Here, we investigated the relative roles of propagule pressure, the availability of microsites, and their interaction on the establishment of an invasive herb, Lupinus polyphyllus, in two geographic regions representing different climate and growth conditions in Finland (a more productive southern region and a harsher central region). We carried out a field experiment in 14 L. polyphyllus populations, in which we manipulated both propagule pressure and disturbance. In a complementary greenhouse experiment, we manipulated propagule pressure and interspecific competition. Seedling establishment of L. polyphyllus was higher in the more productive southern region than in the harsher central region. The number of L. polyphyllus seedlings increased with increasing propagule pressure regardless of disturbance or interspecific competition. However, the number of L. polyphyllus seedlings per sown seed (relative establishment) tended to decrease with increasing propagule pressure, indicating that the positive effect of propagule pressure on early invasion is partially counteracted by density-dependent mortality at high seed densities. Our results highlight the dominant role of propagule pressure over disturbance and interspecific competition in the establishment of L. polyphyllus, suggesting that the early stage of invasion is limited by the availability of propagules rather than the availability of suitable microsites.     

Elucidating pathways of Toxoplasma gondii invasion in the gastrointestinal tract: involvement of the tight junction protein occludin

Toxoplasma gondii is an obligate intracellular parasite infecting one third of the world's population. The small intestine is the parasite's primary route of infection, although the pathway of epithelium transmigration remains unclear. Using an in vitro invasion assay and live imaging we showed that T. gondii (RH) tachyzoites infect and transmigrate between adjacent intestinal epithelial cells in polarized monolayers without altering barrier integrity, despite eliciting the production of specific inflammatory mediators and chemokines. During invasion, T. gondii co-localized with occludin. Reducing the levels of endogenous cellular occludin with specific small interfering RNAs significantly reduced the ability of T. gondii to penetrate between and infect epithelial cells. Furthermore, an in vitro invasion and binding assays using recombinant occludin fragments established the capacity of the parasite to bind occludin and in particular to the extracellular loops of the protein. These findings provide evidence for occludin playing a role in the invasion of T. gondii in small intestinal epithelial cells.     

Syndecan-1 and -4 differentially regulate oncogenic K-ras dependent cell invasion into collagen through α2β1 integrin and MT1-MMP

Syndecans function as co-receptors for integrins on different matrixes. Recently, syndecan-1 has been shown to be important for α2β1 integrin-mediated adhesion to collagen in tumor cells by regulating cell adhesion and migration on two-dimensional collagen. However, the function of syndecans in supporting α2β1 integrin interactions with three-dimensional (3D) collagen is less well studied. Using loss-of-function and overexpression experiments we show that in 3D collagen syndecan-4 supports α2β1-mediated collagen matrix contraction. Cell invasion through type I collagen containing 3D extracellular matrix (ECM) is driven by α2β1 integrin and membrane type-1 matrix metalloproteinase (MT1-MMP). Here we show that mutational activation of K-ras correlates with increased expression of α2β1 integrin, MT1-MMP, syndecan-1, and syndecan-4. While K-ras-induced α2β1 integrin and MT1-MMP are positive regulators of invasion, silencing and overexpression of syndecans demonstrate that these proteins inhibit cell invasion into collagen. Taken together, these data demonstrate the existence of a complex interplay between integrin α2β1, MT1-MMP, and syndecans in the invasion of K-ras mutant cells in 3D collagen that may represent a mechanism by which tumor cells become more invasive and metastatic.     

The Pyk2 FERM regulates Pyk2 complex formation and phosphorylation

The focal adhesion kinase Pyk2 integrates signals from cell adhesion receptors, growth factor receptors, and G-protein-coupled receptors leading to the activation of intracellular signaling pathways that regulate cellular phenotypes. The intrinsic mechanism for the activation of Pyk2 activity remains to be fully defined. Previously, we reported that mutations in the N-terminal FERM domain result in loss of Pyk2 activity and expression of the FERM domain as an autonomous fragment inhibits Pyk2 activity. In the present study, we sought to determine the mechanism that underlies these effects. Utilizing differentially epitope-tagged Pyk2 constructs, we observed that Pyk2 forms oligomeric complexes in cells and that complex formation correlates positively with tyrosine phosphorylation. Similarly, when expressed as an autonomous fragment, the Pyk2 FERM domain formed a complex with other Pyk2 FERM domains but not the FAK FERM domain. When co-expressed with full-length Pyk2, the autonomously expressed Pyk2 FERM domain formed a complex with full-length Pyk2 preventing the formation of Pyk2 oligomers and resulting in reduced Pyk2 phosphorylation. Deletion of the FERM domain from Pyk2 enhanced Pyk2 complex formation and phosphorylation. Together, these data indicate that the Pyk2 FERM domain is involved in the regulation of Pyk2 activity by acting to regulate the formation of Pyk2 oligomers that are critical for Pyk2 activity.     

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1^nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.     

Actin cytoskeleton disruption by ExoY and its effects on Pseudomonas aeruginosa invasion

Three of the Type III-secreted effectors of Pseudomonas aeruginosa (ExoS, ExoT, and ExoY) each alter mammalian cell morphology in culture without causing a loss of cell viability. For ExoS and ExoT this property involves RhoGAP activity, and leads to actin cytoskeleton disruption and a reduced capacity for internalizing bacteria. ExoY does not possess RhoGAP activity. Instead, cell rounding depends upon its adenylate cyclase catalytic region. Since anti-phagocytic activities of ExoS and ExoT are associated with cell rounding and cytoskeleton disruption, we hypothesized that ExoY would also inhibit P. aeruginosa invasion of epithelial cells coinciding with adenylate cyclase-mediated cytoskeleton disruption. The results showed actin disruption of epithelial cells at 2 h post-infection associated with both adenylate cyclase-active ExoY and its catalytic mutant form ExoYK81M, and which coincided with inhibition of bacterial invasion (76% inhibition by ExoY, and 37% by ExoYK81M). Surprisingly, at 4h post-infection, neither form of ExoY inhibited invasion despite extensive actin disruption. These data suggest that ExoY, like ExoS and ExoT, contains more than one active domain affecting mammalian cell function. The data also suggest that cytoskeleton disruption does not necessarily predict invasion inhibitory activity, supporting the recently proposed model that P. aeruginosa internalization can proceed through more than one pathway.