Glioblastomas are aggressive brain tumors for which effective therapy is still lacking, resulting in dismal survival rates. These tumors display significant phenotypic plasticity, harboring diverse cell populations ranging from tumor core cells to dispersed, highly invasive cells. Neuron navigator 3 (NAV3), a microtubule-associated protein affecting microtubule growth and dynamics, is downregulated in various cancers, including glioblastoma, and has thus been considered a tumor suppressor. In this study, we challenge this designation and unveil distinct expression patterns of NAV3 across different invasion phenotypes. Using glioblastoma cell lines and patient-derived glioma stem-like cell cultures, we disclose an upregulation of NAV3 in invading glioblastoma cells, contrasting with its lower expression in cells residing in tumor spheroid cores. Furthermore, we establish an association between low and high NAV3 expression and the amoeboid and mesenchymal invasive phenotype, respectively, and demonstrate that overexpression of NAV3 directly stimulates glioblastoma invasive behavior in both 2D and 3D environments. Consistently, we observed increased NAV3 expression in cells migrating along blood vessels in mouse xenografts. Overall, our results shed light on the role of NAV3 in glioblastoma invasion, providing insights into this lethal aspect of glioblastoma behavior.

• MeSH
• fenotyp * MeSH
• glioblastom * patologie   genetika   metabolismus   MeSH
• invazivní růst nádoru * genetika   MeSH
• lidé MeSH
• membránové proteiny MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory mozku * patologie   genetika   metabolismus   MeSH
• pohyb buněk genetika   fyziologie   MeSH
• proteiny nervové tkáně metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Cancer is still one of the leading causes of death, with an estimated 19.3 million new cases every year. Our paper presents the tumor-suppressing effect of Taenia crassiceps and Mesocestoides corti on B16F10 melanoma, the intraperitoneal application of which followed the experimental infection with these tapeworms, resulting in varying degrees of effectiveness in two strains of mice. In the case of M. corti-infected ICR mice, a strong tumor growth suppression occurred, which was accompanied by a significant reduction in the formation of distant metastases in the liver and lung. Tapeworm-infected C57BL/6J mice also showed a suppression of tumor growth and, in addition, the overall survival of infected C57BL/6J mice was significantly improved. Experiments with potential cross-reaction of melanoma and tapeworm antigens with respective specific antibodies, restimulation of spleen T cells, or the direct effect of tapeworm excretory-secretory products on melanoma cells in vitro could not explain the phenomenon. However, infections with T. crassiceps and M. corti increased the number of leukocytes possibly involved in anti-tumor immunity in the peritoneal cavity of both ICR and C57BL/6J mice. This study unveils the complex interplay between tapeworm infections, immune responses, and melanoma progression, emphasizing the need for further exploration of the mechanisms driving observed tumor-suppressive effects.

• MeSH
• Cestoda * MeSH
• cestodózy * komplikace   patologie   MeSH
• melanom * komplikace   MeSH
• Mesocestoides * fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši inbrední ICR MeSH
• myši MeSH
• Taenia * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork.

• MeSH
• blízká infračervená spektroskopie metody   MeSH
• fibrosarkom patologie   MeSH
• invazivní růst nádoru MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• nádorové buňky kultivované MeSH
• nádory prsu patologie   MeSH
• počítačové zpracování obrazu MeSH
• podozomy patologie   MeSH
• zobrazování trojrozměrné metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Src kinase plays an important role in a multitude of fundamental cellular processes and is often found deregulated in tumors. Active Src adopts an open conformation, whereas inactive Src is characterized by a very compact structure stabilized by inhibitory intramolecular interactions. Taking advantage of this spatial regulation, we constructed a fluorescence resonance energy transfer (FRET)-based Src biosensor and analyzed conformational changes of Src following Src activation and the spatiotemporal dynamics of Src activity in cells. We found that activatory mutations either in regulatory or kinase domains induce opening of the Src structure. Surprisingly, we discovered that Src inhibitors differ in their effect on the Src structure, some counterintuitively inducing an open conformation. Finally, we analyzed the dynamics of Src activity in focal adhesions by FRET imaging and found that Src is rapidly activated during focal adhesion assembly, and its activity remains steady and high throughout the life cycle of focal adhesion and decreases during focal adhesion disassembly.

• MeSH
• biosenzitivní techniky metody   MeSH
• fokální adheze metabolismus   MeSH
• FRAP MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mutageneze MeSH
• rezonanční přenos fluorescenční energie MeSH
• skupina kinas odvozených od src-genu antagonisté a inhibitory   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Observation and analysis of cancer cell behaviour in 3D environment is essential for full understanding of the mechanisms of cancer cell invasion. However, label-free imaging of live cells in 3D conditions is optically more challenging than in 2D. Quantitative phase imaging provided by coherence controlled holographic microscopy produces images with enhanced information compared to ordinary light microscopy and, due to inherent coherence gate effect, enables observation of live cancer cells' activity even in scattering milieu such as the 3D collagen matrix. Exploiting the dynamic phase differences method, we for the first time describe dynamics of differences in cell mass distribution in 3D migrating mesenchymal and amoeboid cancer cells, and also demonstrate that certain features are shared by both invasion modes. We found that amoeboid fibrosarcoma cells' membrane blebbing is enhanced upon constriction and is also occasionally present in mesenchymally invading cells around constricted nuclei. Further, we demonstrate that both leading protrusions and leading pseudopods of invading fibrosarcoma cells are defined by higher cell mass density. In addition, we directly document bundling of collagen fibres by protrusions of mesenchymal fibrosarcoma cells. Thus, such a non-invasive microscopy offers a novel insight into cellular events during 3D invasion.

• MeSH
• buněčná membrána metabolismus   MeSH
• buněčné kultury metody   MeSH
• fibrosarkom diagnostické zobrazování   patologie   MeSH
• holografie přístrojové vybavení   metody   MeSH
• intravitální mikroskopie přístrojové vybavení   metody   MeSH
• invazivní růst nádoru diagnostické zobrazování   patologie   MeSH
• kolagen metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• pohyb buněk * MeSH
• pseudopodia metabolismus   MeSH
• zobrazování trojrozměrné přístrojové vybavení   metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The tyrosine kinase Src acts as a key regulator of cell motility by phosphorylating multiple protein substrates that control cytoskeletal and adhesion dynamics. In an earlier phosphotyrosine proteomics study, we identified a novel Rho-GTPase activating protein, now known as ARHGAP42, as a likely biologically relevant Src substrate. ARHGAP42 is a member of a family of RhoGAPs distinguished by tandem BAR-PH domains lying N-terminal to the GAP domain. Like other family members, ARHGAP42 acts preferentially as a GAP for RhoA. We show that Src principally phosphorylates ARHGAP42 on tyrosine 376 (Tyr-376) in the short linker between the BAR-PH and GAP domains. The expression of ARHGAP42 variants in mammalian cells was used to elucidate its regulation. We found that the BAR domain is inhibitory toward the GAP activity of ARHGAP42, such that BAR domain deletion resulted in decreased active GTP-bound RhoA and increased cell motility. With the BAR domain intact, ARHGAP42 GAP activity could be activated by phosphorylation of Tyr-376 to promote motile cell behavior. Thus, phosphorylation of ARHGAP42 Tyr-376 is revealed as a novel regulatory event by which Src can affect actin dynamics through RhoA inhibition.

• MeSH
• fokální adheze metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• myši MeSH
• pohyb buněk fyziologie   MeSH
• proteiny aktivující GTPasu genetika   metabolismus   MeSH
• rho proteiny vázající GTP antagonisté a inhibitory   metabolismus   MeSH
• rhoA protein vázající GTP antagonisté a inhibitory   metabolismus   MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• tyrosin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Although there is extensive evidence for the amoeboid invasiveness of cancer cells in vitro, much less is known about the role of amoeboid invasiveness in metastasis and the importance of Rho/ROCK/MLC signaling in this process. RESULTS: We analyzed the dependence of amoeboid invasiveness of rat and chicken sarcoma cells and the metastatic activity of chicken cells on individual elements of the Rho/ROCK/MLC pathway. In both animal models, inhibition of Rho, ROCK or MLC resulted in greatly decreased cell invasiveness in vitro, while inhibition of extracellular proteases using a broad spectrum inhibitor did not have a significant effect. The inhibition of both Rho activity and MLC phosphorylation by dominant negative mutants led to a decreased capability of chicken sarcoma cells to metastasize. Moreover, the overexpression of RhoA in non-metastatic chicken cells resulted in the rescue of both invasiveness and metastatic capability. Rho and ROCK, unlike MLC, appeared to be directly involved in the maintenance of the amoeboid phenotype, as their inhibition resulted in the amoeboid-mesenchymal transition in analyzed cell lines. CONCLUSION: Taken together, these results suggest that protease-independent invasion controlled by elements of the Rho/ROCK/MLC pathway can be frequently exploited by metastatic sarcoma cells.

• MeSH
• invazivní růst nádoru MeSH
• kinázy asociované s rho metabolismus   MeSH
• krysa rodu rattus MeSH
• kur domácí MeSH
• lehké řetězce myosinu metabolismus   MeSH
• nádorové buněčné linie MeSH
• pohyb buněk MeSH
• rho proteiny vázající GTP metabolismus   MeSH
• sarkom metabolismus   patologie   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Focal adhesions are specific types of cellular adhesion structures through which both mechanical force and regulatory signals are transmitted. Recently, the existence of focal adhesions in 3D environment has been questioned. Using a unique life-like model of dermis-based matrix we analysed the presence of focal adhesions in a complex 3D environment. Although the dermis-based matrix constitutes a 3D environment, the interface of cell-to-matrix contacts on thick bundled fibres within this matrix resembles 2D conditions. We call this a quasi-2D situation. We suggest that the quasi-2D interface of cell-to-matrix contacts constituted in the dermis-based matrix is much closer to in tissue conditions than the meshed structure of mostly uniform thin fibres in the gel-based matrices. In agreement with our assumption, we found that the cell adhesion structures are formed by cells that invade the dermis-based matrix and that these structures are of similar size as focal adhesions formed on fibronectin-coated coverslips (2D). In both 2D situation and the dermis-based matrix, we observed comparable vinculin dynamics in focal adhesions and comparable enlargement of the focal adhesions in response to a MEK inhibitor. We conclude that focal adhesions that are formed in the 3D environment are similar in size and dynamics as those seen in the 2D setting.

• MeSH
• buněčné kultury * MeSH
• butadieny farmakologie   MeSH
• fokální adheze účinky léků   metabolismus   ultrastruktura   MeSH
• FRAP MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy kinas antagonisté a inhibitory   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nitrily farmakologie   MeSH
• škára účinky léků   metabolismus   MeSH
• Sus scrofa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes and plays an important role in invasiveness of Src-transformed cells. A novel phosphorylation site on CAS, Tyr-12 (Y12) within the ligand-binding hydrophobic pocket of the CAS SH3 domain, was identified and found to be enriched in Src-transformed cells and invasive human carcinoma cells. To study the biological significance of CAS Y12 phosphorylation, phosphomimicking Y12E and nonphosphorylatable Y12F mutants of CAS were studied. The phosphomimicking mutation decreased interaction of the CAS SH3 domain with focal adhesion kinase (FAK) and PTP-PEST and reduced tyrosine phosphorylation of FAK. Live-cell imaging showed that green fluorescent protein-tagged CAS Y12E mutant is, in contrast to wild-type or Y12F CAS, excluded from focal adhesions but retains its localization to podosome-type adhesions. Expression of CAS-Y12F in cas-/- mouse embryonic fibroblasts resulted in hyperphosphorylation of the CAS substrate domain, and this was associated with slower turnover of focal adhesions and decreased cell migration. Moreover, expression of CAS Y12F in Src-transformed cells greatly decreased invasiveness when compared to wild-type CAS expression. These findings reveal an important role of CAS Y12 phosphorylation in the regulation of focal adhesion assembly, cell migration, and invasiveness of Src-transformed cells.

• MeSH
• fokální adheze metabolismus   MeSH
• fokální adhezní tyrosinkinasy metabolismus   MeSH
• fosforylace MeSH
• invazivní růst nádoru MeSH
• lidé MeSH
• molekuly buněčné adheze metabolismus   MeSH
• mutace MeSH
• myši MeSH
• nádorová transformace buněk MeSH
• nádorové buněčné linie MeSH
• pohyb buněk MeSH
• signální transdukce MeSH
• src homologní domény MeSH
• substrátový protein asociovaný s Crk chemie   genetika   metabolismus   MeSH
• transformované buněčné linie MeSH
• tyrosin metabolismus   MeSH
• tyrosinfosfatasa nereceptorového typu 12 metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Pharmacological inhibition of protein kinases that are responsible for the phosphorylation of the carboxy-terminal domain (CTD) of RNA Pol II during transcription by 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) leads to severe inhibition of mRNA synthesis and activates p53. Transcription of the p53 effectors that are induced under these conditions, such as p21 or PUMA, must bypass the requirement for CTD phosphorylation by the positive elongation factor P-TEFb. Here, we have downregulated SNW1/SKIP, a splicing factor and a transcriptional co-regulator, which was found to interact with P-TEFb and synergistically affect Tat-dependent transcription elongation of HIV 1. Using the colon cancer derived cell line HCT116, we have found that both doxorubicin- and DRB-induced expression of p21 or PUMA is insensitive to SNW1 downregulation by siRNA. This suggests that transcription of stress response genes, unlike, e.g., the SNW1-sensitive mitosis-specific genes, can proceed uncoupled from regulators that normally function under physiological conditions.

• MeSH
• buněčné jádro metabolismus   MeSH
• down regulace genetika   MeSH
• fyziologický stres genetika   MeSH
• HCT116 buňky MeSH
• HeLa buňky MeSH
• koaktivátory jaderných receptorů genetika   metabolismus   MeSH
• lidé MeSH
• lidské chromozomy metabolismus   MeSH
• mitóza MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH