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Atomic force microscopy and graph analysis to study the P-cadherin/SFK mechanotransduction signalling in breast cancer cells
Publication . Ribeiro, A. S.; Carvalho, Filomena Almeida; Figueiredo, J.; Carvalho, R.; Mestre, T.; Monteiro, J.; Guedes, Ana Filipa; Fonseca, M.; Sanches, J.; Seruca, R.; Santos, Nuno C.; Paredes, J.
Physical forces mediated by cell–cell adhesion molecules, as cadherins, play a crucial role in preserving normal tissue architecture. Accordingly, altered cadherins’ expression has been documented as a common event during cancer progression. However, in most studies, no data exist linking protumorigenic signaling and variations in the mechanical balance mediated by adhesive forces. In breast cancer, P-cadherin overexpression increases in vivo tumorigenic ability, as well as in vitro cell invasion, by activating Src family kinase (SFK) signalling. However, it is not known how P-cadherin and SFK activation impact cell–cell biomechanical properties. In the present work, using atomic force microscopy (AFM) images, cell stiffness and cell–cell adhesion measurements, and undirected graph analysis based on microscopic images, we have demonstrated that P-cadherin overexpression promotes significant alterations in cell’s morphology, by decreasing cellular height and increasing its area. It also affects biomechanical properties, by decreasing cell–cell adhesion and cell stiffness. Furthermore, cellular network analysis showed alterations in intercellular organization, which is associated with cell–cell adhesion dysfunction, destabilization of an E-cadherin/p120ctn membrane complex and increased cell invasion.
Remarkably, inhibition of SFK signaling, using dasatinib, reverted the pathogenic P-cadherin induced effects by increasing cell’s height, cell–cell adhesion and cell stiffness, and generating more compact epithelial aggregates, as quantified by intercellular network analysis. In conclusion, P-cadherin/SFK signalling induces topological, morphological and biomechanical cell–cell alterations, which are associated with more invasive breast cancer cells. These effects could be further reverted by dasatinib treatment, demonstrating the applicability of AFM and cell network diagrams for measuring the epithelial biomechanical properties and structural organization.
Pathogenic Rickettsia in ticks of spur-thighed tortoise (Testudo graeca) sold in a Qatar live animal market
Publication . Barradas, Patricia F.; Mesquita, Joao R.; Lima, Clara; Cardoso, Luis; Alho, Ana Margarida; Ferreira, Paula; Amorim, Irina; de Sousa, Rita; Gartner, Fatima
The dissemination of vector arthropods harbouring zoonotic pathogens through
the uncontrolled transboundary trade of exotic and pet animals poses an important
threat to Public Health. In the present report, we describe the introduction of
pathogenic Rickettsia africae and R. aeschlimanni in ticks removed from imported tortoises
in Qatar. A total of 21 ticks were collected from pet spur‐thighed tortoises
(Testudo graeca) from Doha, May 2018, and studied for species identification and
characterization of Rickettsia spp. Morphological and molecular analysis of ticks allowed
their identification as Hyalomma aegyptium. Molecular analysis of partial ompA
and gltA genes showed that Rickettsia sequences found on these ticks clustered with
sequences classified as R. aeschilimanii and R. africae. Since pre‐adult stages of H. aegyptium
also feed on humans, this tick species may play a role in the transmission of
R. aeschilimanii and R. africae. We alert for the introduction of non‐native pets as vehicles
for tick importation, known vectors for animal and human pathogenic agents.
Importation of exotic species into non‐autochthonous countries deserves strict control
to enforce robust surveillance and mitigate potential exotic diseases epidemics.
SRC inhibition prevents P-cadherin mediated signaling and function in basallike breast cancer cells
Publication . Ribeiro, Ana Sofia; Nobre, Ana Rita; Mendes, Nuno; Almeida, João; Vieira, André Filipe; Sousa, Bárbara; Carvalho, Filomena Almeida; Monteiro, Joana; Polónia, António; Fonseca, Martina; Sanches, João Miguel; Santos, Nuno C.; Seruca, Raquel; Paredes, Joana
BACKGROUND: Basal-like breast cancer (BLBC) is a poor prognosis subgroup of triple-negative carcinomas that still lack specific target therapies and accurate biomarkers for treatment selection. P-cadherin is frequently overexpressed in these tumors, promoting cell invasion, stem cell activity and tumorigenesis by the activation of Src-Family kinase (SRC) signaling. Therefore, our aim was to evaluate if the treatment of BLBC cells with dasatinib, the FDA approved SRC inhibitor, would impact on P-cadherin induced tumor aggressive behavior.
METHODS: P-cadherin and SRC expression was evaluated in a series of invasive Breast Cancer and contingency tables and chi-square tests were performed. Cell-cell adhesion measurements were performed by Atomic Force Microscopy, where frequency histograms and Gaussian curves were applied. 2D and 3D cell migration and invasion, proteases secretion and self-renew potential were evaluated in vitro. Student's t-tests were used to determine statistically significant differences. The cadherin/catenin complex interactions were evaluated by in situ proximity-ligation assay, and statistically significant results were determined by using Mann-Whitney test with a Bonferroni correction. In vivo xenograft mouse models were used to evaluate the impact of dasatinib on tumor growth and survival. ANOVA test was used to evaluate the differences in tumor size, considering a confidence interval of 95%. Survival curves were estimated by the Kaplan-Meier's method, using the log-rank test to assess significant differences for mice overall survival.
RESULTS: Our data demonstrated that P-cadherin overexpression is significantly associated with SRC activation in breast cancer cells, which was also validated in a large series of primary tumor samples. SRC activity suppression with dasatinib significantly prevented the in vitro functional effects of P-cadherin overexpressing cells, as well as their in vivo tumorigenic and metastatic ability, by increasing mice overall survival. Mechanistically, SRC inhibition affects P-cadherin downstream signaling, rescues the E-cadherin/p120-catenin complex to the cell membrane, recovering cell-cell adhesion function.
CONCLUSIONS: In conclusion our findings show that targeting P-cadherin/SRC signaling and functional activity may open novel therapeutic opportunities for highly aggressive and poor prognostic basal-like breast cancer.
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Fundação para a Ciência e a Tecnologia
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PEst-C/SAU/LA0003/2013