Cancer metastases: challenges and opportunities.

Cancer is a complex disease that is dictated by both cancer cell-intrinsic and cell-extrinsic processes. Adenosine is an ancient extracellular signalling molecule that can regulate almost all aspects of tissue function. As such, several studies have recently highlighted a crucial role for adenosine signalling in regulating the various aspects of cell-intrinsic and cell-extrinsic processes of cancer development. This Review critically discusses the role of adenosine and its receptors in regulating the complex interplay among immune, inflammatory, endothelial and cancer cells during the course of neoplastic disease.

Immunity, inflammation and cancer: a leading role for adenosine

Rho GTPase signalling in cell migration.

Breast cancer is now the most frequently diagnosed cancer and leading cause of cancer death in women worldwide. Strategies targeting the primary tumour have markedly improved, but systemic treatments to prevent metastasis are less effective; metastatic disease remains the underlying cause of death in the majority of patients with breast cancer who succumb to their disease. The long latency period between initial treatment and eventual recurrence in some patients suggests that a tumour may both alter and respond to the host systemic environment to facilitate and sustain disease progression. Results from studies in animal models suggest that specific subtypes of breast cancer may direct metastasis through recruitment and activation of haematopoietic cells. In this review, we focus on data implicating breast cancer as a systemic disease.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/joim.12084

Breast cancer as a systemic disease: a view of metastasis

The goal of this study was to understand if exosomes derived from high-metastatic cells may influence the behavior of less aggressive cancer cells and the properties of the endothelium. We found that metastatic colon cancer cells are able to transfer their amoeboid phenotype to isogenic primary cancer cells through exosomes, and that this morphological transition is associated with the acquisition of a more aggressive behavior. Moreover, exosomes from the metastatic line (SW620Exos) exhibited higher ability to cause endothelial hyperpermeability than exosomes from the non metastatic line (SW480Exos). SWATH-based quantitative proteomic analysis highlighted that SW620Exos are significantly enriched in cytoskeletal-associated proteins including proteins activating the RhoA/ROCK pathway, known to induce amoeboid properties and destabilization of endothelial junctions. In particular, thrombin was identified as a key mediator of the effects induced by SW620Exos in target cells, in which we also found a significant increase of RhoA activity. Overall, our results demonstrate that in a heterogeneous context exosomes released by aggressive sub-clones can contribute to accelerate tumor progression by spreading malignant properties that affect both the tumor cell plasticity and the endothelial cell behavior.

https://iris.unipa.it/bitstream/10447/237658/1/Scientific%20Reports%202017.pdf

Exosomes from metastatic cancer cells transfer amoeboid phenotype to non-metastatic cells and increase endothelial permeability: their emerging role in tumor heterogeneity

Molecular networks that regulate cancer metastasis.

An epithelial-mesenchymal transition (EMT) underlies malignant tumor progression and metastatic spread by enabling cancer cells to depart from the primary tumor, invade surrounding tissue, and disseminate to distant organs. EMT also enriches for cancer stem cells (CSC) and increases the capacity of cancer cells to initiate and propagate tumors upon transplantation into immune-deficient mice, a major hallmark of CSCs. However, the molecular mechanisms promoting the tumorigenicity of cancer cells undergoing an EMT and of CSCs have remained widely elusive. We here report that EMT confers efficient tumorigenicity to murine breast cancer cells by the upregulated expression of the proangiogenic factor VEGF-A and by increased tumor angiogenesis. On the basis of these data, we propose a novel interpretation of the features of CSCs with EMT-induced, VEGF-A-mediated angiogenesis as the connecting mechanism between cancer cell stemness and tumor initiation.

https://cancerres.aacrjournals.org/content/canres/74/5/1566.full.pdf

VEGF-Mediated Angiogenesis Links EMT-Induced Cancer Stemness to Tumor Initiation

Rheumatoid arthritis (RA) is a progressive chronic inflammatory autoimmune disease characterized by synovial hyperplasia, articular inflammation and excessive joint and bone erosion. Activated RA synovial fibroblasts (SF) are key players of cartilage erosion by attaching and invading into cartilage. The kindlin family of focal adhesion proteins consists of three members, whose function is to activate integrins and thereby enable focal adhesion. Integrins play a central role in regulating cell-matrix and cell-cell adhesion. The effects of integrin activation are cell adhesion and migration, which are known to be pathologically increased in RASF. It is well described that dysfunction of integrins and kindlins and their co-factor talin-1 are involved in the pathogenesis of certain diseases characterized by inflammation and malignant transformation. However, the role of kindlins and talin-1 in RA, and specifically RASF, remain unclear.To investigate the compartmental and cellular localisation of kindlin-1, -2, -3 and talin-1 in RA synovial tissue and RASF, and the impact of pro-inflammatory activation on their expression on mRNA and protein level.Synovial fibroblasts were isolated from synovial tissue after joint replacement surgery (RA, osteoarthritis (OA)) or synovectomy of hand joints (non-arthritic controls). RASF were stimulated with 10 ng/ml IL-1β for 6, 17, 24 and 48 h. In a second approach, RASF were repetitively stimulated three times with 0.05 ng/ml IL-1β every 24 h. RNA was isolated, reverse-transcribed and qPCR performed for kindlin-1, -2, -3 and talin-1. Whole RNA sequencing was performed for the repetitive stimulation approach and evaluated using matrix combining pairwise contrasts (DESeq2 and DESeq) normalized counts and thresholds for significance of differential expression per contrast. Immunocytochemistry on RASF seeded on chamber slides was performed. Immunohistochemistry on synovial tissue was performed on 5 µm acetone-fixed cryosections. Biological replicates and Kruskal-Wallis-test with Dunn’s multiple comparison were used for statistics.In RA synovial tissues, kindlin-2 and talin-1 protein were detectable adjacent to blood vessels, in the synovial lining layer and in the sublining. Kindlin-2 and talin-1 could be detected in cultured RASF with a consistent signal of the cell body without a nuclear signal. Kindlin-1 and -3 were neither detectable in synovial tissue nor in synovial fibroblasts. Relative mRNA levels (arbitrary units) of kindlins and talin-1 in RASF differed, with the strongest signal for talin-1 (663 ± 186), followed by kindlin-2 (88 ± 23), kindlin-1 (2.15 ± 2.06) and kindlin-3 (0.12 ± 0.08). In comparison to unstimulated controls, stimulation of RASF with IL-1β led to a temporary downregulation of kindlin-2 (-4.1-fold, p=0.0456) and talin-1 (-1.6-fold) after 17 h and returned to the baseline levels after 48 h. This time-dependent effect could also be observed by immunocytochemistry of IL-1β-stimulated RASF. Kindlin-1 mRNA expression was significantly upregulated after 6 h (2.9-fold, p=0.0178) of stimulation and downregulated after 17 h (-1.5-fold, p<0.0001), 24 h (-0.7-fold, p=0.0004) and 48 h (-1.2-fold, p<0.0001) compared to 6 h. After repetitive activation of RASF, kindlins and talin-1 were constantly regulated compared to the first stimulation (kindlin-1: 1.03-fold; kindlin-2: -1.06-fold; talin-1: 1.00-fold). In contrast, IL-6 showed an inflammatory adjustment to IL-1β leading to lower induction of IL-6 (49.1%) after repetitive IL-1β exposure.Focal adhesion proteins kindlin-2 and talin-1, factors involved in integrin activation of cells, are present on RA synovial tissue and in RASF. As a time-dependent regulation of kindlin-2 and talin-1 after stimulation was observed, this effect may lead to a temporary and repetitive reproducible activation of RASF and activation of integrins, subsequently contributing to altered adhesion and migration of RASF under inflammatory arthritic conditions.NIL.NIL.None Declared.

Pos1035 focal adhesion proteins kindlin-1, -2 and talin-1 are regulated in il-1β-stimulated rheumatoid arthritis synovial fibroblasts

Increased neovascularization in the inflamed synovium of patients with rheumatoid arthritis (RA) is a pathological feature in the pathogenesis of RA. Key players in RA progression are chronically activated RA synovial fibroblasts (RASF), which secrete proinflammatory factors (e.g. IL-6), proangiogenic VEGF, IL-11 and matrix metalloproteases (MMP) promoting inflammation, synovial angiogenesis, and invasion of cartilage and bone by degrading the consecutive matrix, resulting in the release of the anti-angiogenic collagen IV fragment canstatin. Canstatin may further activate RASF leading to altered expression of vascular markers such as angiopoietin-2 (ANGPT2) and neovascularization.Analysis of repetitively stimulated RASF- and canstatin-mediated effects on vessel formation in the tube formation assay, in the SCID-mouse model for RA and in synovial tissue of RA patients with respect to ANGPT2 expression and RASF-endothelial cell (EC) interactions.2D tube formation assay was performed using HUVEC seeded on Matrigel. 15% Calcein AM-stained RASF were added. RASF/HUVEC were treated with 0.5µg/ml canstatin. HUVEC were pre-treated with 0.2µg/ml canstatin. RASF were repetitively stimulated three times with 0.05ng/µl IL-1ß every 24h starting at day 2. Tube thickness and the area covered by the formed cellular network were measured. RNA was extracted from tubes and ANGPT2 expression was analysed by qPCR. IL-6 and IL-11 in supernatants were measured by ELISA. Healthy cartilage was subcutaneously co-implanted with RASF into SCID mice. Contralaterally, healthy cartilage without RASF was implanted. Vessel formation was evaluated after 3-45 days. RA and osteoarthritis (OA) synovial tissue was stained for ANGPT2 and CD31.RASF or HUVEC stimulated once showed a significant IL-6 increase compared to unstimulated controls. After subsequent repetitive stimulation of RASF or HUVEC, a significant decrease in IL-6 (1st stimulation: 5076±1730pg/ml vs. 3rd: 1890±758pg/ml, p<0.0001/ 1st: 488±186pg/ml vs. 3rd: 382±109pg/ml, p=0.041) and IL-11 in RASF (1st: 451±205pg/ml vs. 3rd: 69±51pg/ml, p<0.0001) compared to the first stimulation was observed. Repetitive stimulation of HUVEC+RASF resulted in a significant IL-6 increase for each subsequent stimulation (1st vs. 3rd: p=0.02). RASF significantly reduced tube thickness (22.9µm (SD=6.3) vs. 16.6µm (SD=2.2), p=0.014) and the network area (p<0.0001) and significantly increased ANGPT2 2-fold compared to HUVEC alone. RASF stimulated only once further reduced the network area (p=0.038), while repetitive stimulation significantly attenuated the proinflammatory effect (IL-6, p=0.029). Stimulation of pre-treated HUVEC and unstimulated RASF with canstatin led to disturbed tube formation with reduced tube thickness from 22.9 to 16.9µm (SD=4.4, p=0.011). Co-culture of RASF with pre-treated HUVEC with canstatin further increased the RASF-mediated effect by reducing tube thickness (22.9 to 14.6µm (SD=1.4), p<0.001), with a significant 1.6-fold decrease of ANGPT2. In human RA synovium, ANGPT2 was also found to be significantly upregulated in vessels compared to OA tissue. In SCID mice, RASF-mediated altered vessel formation started at day 3. Helix-like vessels were detectable at early time points of vessel formation in implants with RASF (d3-9), e.g. at day 3, 60% of vessels were helix-like.RASF induced helix-like vessels in the SCID mouse model suggest an influence on vessel formation. This could be confirmed in vitro as well as on molecular level inducing the vascular marker ANGPT2 in newly formed tubes. ANGPT2-upregulation in vessels was also observed in human RA synovium. Repetitive stimulation of RASF resulted in an inflammatory adjustment to IL-1ß, leading to a significant reduction of IL-6 and improved neovascularization in vitro. The anti-angiogenic canstatin could in part enhance the RASF-mediated effect and was not able to restore RASF-altered tube formation in vitro.NIL.NIL.None Declared.

/pdf/op0209-impact-of-chronic-inflammation-and-collagen-iv-1wyc46mf.pdf

Op0209 impact of chronic inflammation and collagen iv fragment canstatin on rheumatoid arthritis synovial fibroblasts and endothelial cell interactions in vitro and in vivo

Chantal Heck

Papers

Molecular networks that regulate cancer metastasis.

VEGF-Mediated Angiogenesis Links EMT-Induced Cancer Stemness to Tumor Initiation

Pos1035 focal adhesion proteins kindlin-1, -2 and talin-1 are regulated in il-1β-stimulated rheumatoid arthritis synovial fibroblasts

Op0209 impact of chronic inflammation and collagen iv fragment canstatin on rheumatoid arthritis synovial fibroblasts and endothelial cell interactions in vitro and in vivo