Rho GTPase signaling in cancer progression and dissemination.

Rho GTPases are a family of small G proteins that regulate a wide array of cellular processes related to their key roles controlling the cytoskeleton. Cancer is a multistep disease caused by the accumulation of genetic mutations and epigenetic alterations, from the initial stages of cancer development when cells in normal tissues undergo transformation, to the acquisition of invasive and metastatic traits, responsible for a large number of cancer related deaths. In this review, we discuss the role of Rho GTPase signaling in cancer in every step of disease progression. Rho GTPases contribute to tumor initiation and progression, by regulating proliferation and apoptosis, but also metabolism, senescence, and cancer cell stemness. Rho GTPases play a major role in cell migration and in the metastatic process. They are also involved in interactions with the tumor microenvironment and regulate inflammation, contributing to cancer progression. After years of intensive research, we highlight the importance of relevant models in the Rho GTPase field, and we reflect on the therapeutic opportunities arising for cancer patients.

Extracellular matrix anisotropy is determined by TFAP2C-dependent regulation of cell collisions.

The isotropic or anisotropic organization of biological extracellular matrices has important consequences for tissue function. We study emergent anisotropy using fibroblasts that generate varying degrees of matrix alignment from uniform starting conditions. This reveals that the early migratory paths of fibroblasts are correlated with subsequent matrix organization. Combined experimentation and adaptation of Vicsek modelling demonstrates that the reorientation of cells relative to each other following collision plays a role in generating matrix anisotropy. We term this behaviour 'cell collision guidance'. The transcription factor TFAP2C regulates cell collision guidance in part by controlling the expression of RND3. RND3 localizes to cell-cell collision zones where it downregulates actomyosin activity. Cell collision guidance fails without this mechanism in place, leading to isotropic matrix generation. The cross-referencing of alignment and TFAP2C gene expression signatures against existing datasets enables the identification and validation of several classes of pharmacological agents that disrupt matrix anisotropy.

Controls on Iron Reduction and Biomineralization over Broad Environmental Conditions as Suggested by the Firmicutes Orenia metallireducens Strain Z6.

Microbial iron reduction is a ubiquitous biogeochemical process driven by diverse microorganisms in a variety of environments. However, it is often difficult to separate the biological from the geochemical controls on bioreduction of Fe(III) oxides. Here, we investigated the primary driving factor(s) that mediate secondary iron mineral formation over a broad range of environmental conditions using a single dissimilatory iron reducer, Orenia metallireducens strain Z6. A total of 17 distinct geochemical conditions were tested with differing pH (6.5-8.5), temperature (22-50 °C), salinity (2-20% NaCl), anions (phosphate and sulfate), electron shuttle (anthraquinone-2,6-disulfonate), and Fe(III) oxide mineralogy (ferrihydrite, lepidocrocite, goethite, hematite, and magnetite). The observed rates and extent of iron reduction differed significantly with kint between 0.186 and 1.702 mmol L-1 day-1 and Fe(II) production ranging from 6.3% to 83.7% of the initial Fe(III). Using X-ray absorption and scattering techniques (EXAFS and XRD), we identified and assessed the relationship between secondary minerals and the specific environmental conditions. It was inferred that the observed bifurcation of the mineralization pathways may be mediated by differing extents of Fe(II) sorption on the remaining Fe(III) minerals. These results expand our understanding of the controls on biomineralization during microbial iron reduction and aid the development of practical applications.

Formation of a statewide community pharmacy practice-based research network: Pharmacist opinions on research participation and engagement.

OBJECTIVE: To gather the insights and opinions of pharmacist stakeholders to inform the creation of a community pharmacy practice-based research network (PBRN) in Pennsylvania. DESIGN: A stakeholder advisory board of pharmacists, patients, and researchers was established to guide this research. This was a qualitative study using a semistructured interview guide. SETTING AND PARTICIPANTS: Community pharmacists from the Pennsylvania Pharmacist Care Network. OUTCOME MEASURES: Themes were identified that describe pharmacist insights and opinions on research participation and preferences for engagement in the PBRN. RESULTS: A total of 16 pharmacists participated in the study. The pharmacists believed that participating in research would help demonstrate their value and commitment to improving patients' health. Enhancing patient-pharmacist relationships and driving innovation were additional benefits that were reported. The pharmacists believed that they could effectively leverage their relationships with patients to engage them in research opportunities. The pharmacists reported that they would like to share research ideas and successful research practices with other members of the PBRN. CONCLUSION: Gathering pharmacists' opinions on participating in research was an important step in developing a community pharmacy PBRN that meets stakeholder needs. The results of this study can help others who seek to form community pharmacy PBRNs that facilitate stakeholder-driven research.

Cutting edge: Dexamethasone potentiates the responses of both regulatory T cells and B-1 cells to antigen immunization in the ApoE(-/-) mouse model of atherosclerosis.

The immunosuppressant dexamethasone was shown to preferentially deplete CD4+ effector T cells while sparing regulatory T cells (Tregs) in vivo. In the current study, we show that it also preferentially depletes B-2 cells while sparing B-1 cells. In the ApoE(-/-) mouse model of atherosclerosis, in which both Tregs and B-1 cells are thought to play an atheroprotective role, we show that HSP60-targeted immunization in the presence of dexamethasone raises Ag-reactive Tregs and B-1 cells concomitantly and reduces the severity of atherosclerosis. These results indicate that dexamethasone is an adjuvant that potentiates both the Treg and B-1 responses to immunogens. This study shows that B-1 cells with a specificity for a disease-relevant Ag can be raised in vivo by immunization.

Organelle adaptations in response to mechanical forces during tumour dissemination.

Cell migration plays a pivotal role in various biological processes including cancer dissemination and successful metastasis, where the role of mechanical signals is increasingly acknowledged. This review focuses on the intricate mechanisms through which cancer cells modulate their migratory strategies via organelle adaptations in response to the extracellular matrix (ECM). Specifically, the nucleus and mitochondria emerge as pivotal mediators in this process. These organelles serve as sensors, translating mechanical stimuli into rapid metabolic alterations that sustain cell migration. Importantly, prolonged exposure to such stimuli can induce transcriptional or epigenetic changes, ultimately enhancing metastatic traits. Deciphering the intricate interplay between ECM properties and organelle adaptations not only advances our understanding of cytoskeletal dynamics but also holds promise for the development of innovative anti-metastatic therapeutic strategies.

AMPK is a mechano-metabolic sensor linking cell adhesion and mitochondrial dynamics to Myosin-dependent cell migration.

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton.

CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.

Protocol to drive human monocyte-to-macrophage polarization in vitro using tumor conditioned media.

Tumor-associated macrophages (TAMs) are key contributors to antitumor immunity. Here, we present a protocol to drive human monocyte-macrophage differentiation using tumor-derived conditioned media, followed by phenotypic and functional characterization of TAMs in vitro. We describe CD14+ cell isolation from healthy human blood, and detail the procedure to induce macrophage polarization. Finally, we outline morphological assessment of macrophages, and validation of their functional behaviors with a tumor cell killing assay. This translatable-based approach can be applied to different cancer cell types. For complete details on the use and execution of this protocol, please refer to Georgouli et al. (2019).

Amoeboid migration in health and disease: Immune responses versus cancer dissemination.

Cell migration is crucial for efficient immune responses and is aberrantly used by cancer cells during metastatic dissemination. Amoeboid migrating cells use myosin II-powered blebs to propel themselves, and change morphology and direction. Immune cells use amoeboid strategies to respond rapidly to infection or tissue damage, which require quick passage through several barriers, including blood, lymph and interstitial tissues, with complex and varied environments. Amoeboid migration is also used by metastatic cancer cells to aid their migration, dissemination and survival, whereby key mechanisms are hijacked from professionally motile immune cells. We explore important parallels observed between amoeboid immune and cancer cells. We also consider key distinctions that separate the lifespan, state and fate of these cell types as they migrate and/or fulfil their function. Finally, we reflect on unexplored areas of research that would enhance our understanding of how tumour cells use immune cell strategies during metastasis, and how to target these processes.

Podoplanin drives dedifferentiation and amoeboid invasion of melanoma.

Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilize amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by Rho GTPase signalling. Migrastatic drugs targeting actin polymerization and contractility are therefore a promising treatment option for metastatic melanoma. To predict amoeboid invasion and metastatic potential, biomarkers functionally linked to contractility pathways are needed. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts and is overexpressed in many cancers. We show that podoplanin enhances amoeboid invasion in melanoma. Podoplanin expression in murine melanoma drives rounded cell morphology, increasing motility, and invasion in vivo. Podoplanin expression is increased in a subset of dedifferentiated human melanoma, and in vitro is sufficient to upregulate melanoma-associated marker Pou3f2/Brn2. Together, our data define podoplanin as a functional biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target.

A novel aspochalasin with HIV-1 integrase inhibitory activity from Aspergillus flavipes.

A novel aspochalasin, aspochalasin L (1), was isolated from the fermentation broth of a soil-derived fungal culture identified as Aspergillus flavipes (Deuteromycota). Structure elucidation of 1 was accomplished by detailed spectroscopic data analyses and by comparison with related cytochalasins. Aspochalasin L demonstrated activity against HIV integrase with an IC50 of 71.7microM.

The effect of ICER on screening methods involving CRE-mediated reporter gene expression.

We describe a mechanism whereby increasing levels of cAMP in Chinese hamster ovary (CHO) and other cell lines lead to a significant repression in cAMP response element (CRE)-mediated luciferase reporter gene expression. This effect was shown to be mediated by a modulatory factor located downstream of cyclic AMP (cAMP), which displayed the temporal regulation pattern of an immediate early gene. The expression of this inducible cAMP early repressor (ICER) was shown to be coincident with the time and concentration dependency of the repression of CRE-mediated luciferase gene expression on the treatment of CHO cells with forskolin. Furthermore, this phenomenon was also observed in JEG and GH3 cell lines (both previously reported to express ICER), but not in COS-7 cells, which do not express ICER. These studies suggest that, in certain cell lines, expression of ICER can be induced at pharmacologically elevated cAMP levels, leading to a potent inhibition of CRE-mediated gene expression. We therefore conclude that screening methodologies employing such CRE-linked reporter genes (particularly in high-throughput screening assays) may produce false functional responses in certain cell lines. Moreover, such effects are likely to be exacerbated in screening assays in which receptors either are overexpressed or high concentrations of potent cAMP-elevating compounds are used.