Bio-nano interactions have been extensively explored in nanomedicine to develop selective delivery strategies and reduce systemic toxicity. To enhance the delivery of nanocarriers to cancer cells and improve the therapeutic efficiency, different nanomaterials have been developed. However, the limited clinical translation of nanoparticle-based therapies, largely due to issues associated with poor targeting, requires a deeper understanding of the biological phenomena underlying cell-nanoparticle interactions. In this context, we investigate the molecular and cellular mechanobiology parameters that control such interactions. We demonstrate that the pharmacological inhibition or the genetic ablation of the key mechanosensitive component of the Hippo pathway, i.e., yes-associated protein, enhances nanoparticle internalization by 1.5-fold. Importantly, this phenomenon occurs independently of nanoparticle properties, such as size, or cell properties such as surface area and stiffness. Our study reveals that the internalization of nanoparticles in target cells can be controlled by modulating cell mechanosensing pathways, potentially enhancing nanotherapy specificity.

• Klíčová slova
• bio−nano interactions, mechanobiology, mechanotransduction, nanoparticles,
• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• buněčný převod mechanických signálů účinky léků   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nanočástice * chemie   MeSH
• nanomedicína MeSH
• signální dráha Hippo MeSH
• signální proteiny YAP MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• signální proteiny YAP MeSH
• YAP1 protein, human MeSH Prohlížeč

Trophoblastic cell surface antigen 2 (TROP2) is a membrane glycoprotein overexpressed in many solid tumors with a poor prognosis, including intestinal neoplasms. In our study, we show that TROP2 is expressed in preneoplastic lesions, and its expression is maintained in most colorectal cancers (CRC). High TROP2 positivity correlated with lymph node metastases and poor tumor differentiation and was a negative prognostic factor. To investigate the role of TROP2 in intestinal tumors, we analyzed two mouse models with conditional disruption of the adenomatous polyposis coli (Apc) tumor-suppressor gene, human adenocarcinoma samples, patient-derived organoids, and TROP2-deficient tumor cells. We found that Trop2 is produced early after Apc inactivation and its expression is associated with the transcription of genes involved in epithelial-mesenchymal transition, the regulation of migration, invasiveness, and extracellular matrix remodeling. A functionally similar group of genes was also enriched in TROP2-positive cells from human CRC samples. To decipher the driving mechanism of TROP2 expression, we analyzed its promoter. In human cells, this promoter was activated by β-catenin and additionally by the Yes1-associated transcriptional regulator (YAP). The regulation of TROP2 expression by active YAP was verified by YAP knockdown in CRC cells. Our results suggest a possible link between aberrantly activated Wnt/β-catenin signaling, YAP, and TROP2 expression.

• Klíčová slova
• APC, EMT, TACSTD2, WNT/β-catenin signaling, colorectal cancer, expression profiling, organoids,
• Publikační typ
• časopisecké články MeSH

The Hippo pathway effector, Yes-associated protein (YAP), is a transcriptional coactivator implicated in cholangiocarcinoma (CCA) pathogenesis. YAP is known to be regulated by a serine/threonine kinase relay module (MST1/2-LATS1/2) culminating in phosphorylation of YAP at Serine 127 and cytoplasmic sequestration. However, YAP also undergoes tyrosine phosphorylation, and the role of tyrosine phosphorylation in YAP regulation remains unclear. Herein, YAP regulation by tyrosine phosphorylation was examined in human and mouse CCA cells, as well as patient-derived xenograft (PDX) models. YAP was phosphorylated on tyrosine 357 (Y357) in CCA cell lines and PDX models. SRC family kinase (SFK) inhibition with dasatinib resulted in loss of YAPY357 phosphorylation, promoted its translocation from the nucleus to the cytoplasm, and reduced YAP target gene expression, including cell lines expressing a LATS1/2-resistant YAP mutant in which all serine residues were mutated to alanine. Consistent with these observations, precluding YAPY357 phosphorylation by site-directed mutagenesis (YAPY357F) excluded YAP from the nucleus. Targeted siRNA experiments identified LCK as the SFK that most potently mediated YAPY357 phosphorylation. Likewise, inducible CRISPR/Cas9-targeted LCK deletion decreased YAPY357 phosphorylation and its nuclear localization. The importance of LCK in CCA biology was demonstrated by clinical observations suggesting LCK expression levels were associated with early tumor recurrence following resection of CCA. Finally, dasatinib displayed therapeutic efficacy in PDX models. Mol Cancer Res; 16(10); 1556-67. ©2018 AACR.

• MeSH
• adaptorové proteiny signální transdukční genetika   MeSH
• buněčné jádro účinky léků   MeSH
• cholangiokarcinom farmakoterapie   genetika   patologie   MeSH
• cytoplazma účinky léků   MeSH
• dasatinib aplikace a dávkování   MeSH
• fosfoproteiny genetika   MeSH
• fosforylace účinky léků   MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• proliferace buněk účinky léků   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• signální proteiny YAP MeSH
• signální transdukce účinky léků   MeSH
• skupina kinas odvozených od src-genu antagonisté a inhibitory   genetika   MeSH
• transkripční faktory MeSH
• tyrosin genetika   MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty genetika   MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• dasatinib MeSH
• fosfoproteiny MeSH
• LATS1 protein, human MeSH Prohlížeč
• LCK protein, human MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• signální proteiny YAP MeSH
• skupina kinas odvozených od src-genu MeSH
• transkripční faktory MeSH
• tyrosin MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty MeSH
• YAP1 protein, human MeSH Prohlížeč

In this review, we address aspects of Wnt, R-Spondin (RSPO) and Hippo signalling, in both healthy and transformed intestinal epithelium. In intestinal stem cells (ISCs), the Wnt pathway is essential for intestinal crypt formation and renewal, whereas RSPO-mediated signalling mainly affects ISC numbers. In human colorectal cancer (CRC), aberrant Wnt signalling is the driving mechanism initiating this type of neoplasia. The signalling role of the RSPO-binding transmembrane proteins, the leucine-rich-repeat-containing G-protein-coupled receptors (LGRs), is possibly more pleiotropic and not only limited to the enhancement of Wnt signalling. There is growing evidence for multiple crosstalk between Hippo and Wnt/β-catenin signalling. In the ON state, Hippo signalling results in serine/threonine phosphorylation of Yes-associated protein (YAP1) and tafazzin (TAZ), promoting formation of the β-catenin destruction complex. In contrast, YAP1 or TAZ dephosphorylation (and YAP1 methylation) results in β-catenin destruction complex deactivation and β-catenin nuclear localization. In the Hippo OFF state, YAP1 and TAZ are engaged with the nuclear β-catenin and participate in the β-catenin-dependent transcription program. Interestingly, YAP1/TAZ are dispensable for intestinal homeostasis; however, upon Wnt pathway hyperactivation, the proteins together with TEA domain (TEAD) transcription factors drive the transcriptional program essential for intestinal cell transformation. In addition, in many CRC cells, YAP1 phosphorylation by YES proto-oncogene 1 tyrosine kinase (YES1) leads to the formation of a transcriptional complex that includes YAP1, β-catenin and T-box 5 (TBX5) DNA-binding protein. YAP1/β-catenin/T-box 5-mediated transcription is necessary for CRC cell proliferation and survival. Interestingly, dishevelled (DVL) appears to be an important mediator involved in both Wnt and Hippo (YAP1/TAZ) signalling and some of the DVL functions were assigned to the nuclear DVL pool. Wnt ligands can trigger alternative signalling that directly involves some of the Hippo pathway components such as YAP1, TAZ and TEADs. By upregulating Wnt pathway agonists, the alternative Wnt signalling can inhibit the canonical Wnt pathway activity.

• Klíčová slova
• Hippo pathway, LGR, R-Spondins, Wnt/β-catenin signalling, YAP1/TAZ, colorectal cancer,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The Wnt pathway plays a crucial role in self-renewal and differentiation of cells in the adult gut. In the present study, we revealed the functional consequences of inhibition of canonical Wnt signaling in the intestinal epithelium. The study was based on generation of a novel transgenic mouse strain enabling inducible expression of an N-terminally truncated variant of nuclear Wnt effector T cell factor 4 (TCF4). The TCF4 variant acting as a dominant negative (dn) version of wild-type (wt) TCF4 protein decreased transcription of β-catenin-TCF4-responsive genes. Interestingly, suppression of Wnt/β-catenin signaling affected asymmetric division of intestinal stem cells (ISCs) rather than proliferation. ISCs expressing the transgene underwent several rounds of division but lost their clonogenic potential and migrated out of the crypt. Expression profiling of crypt cells revealed that besides ISC-specific markers, the dnTCF4 production downregulated expression levels of epithelial genes produced in other crypt cells including markers of Paneth cells. Additionally, in Apc conditional knockout mice, dnTCF activation efficiently suppressed growth of Apc-deficient tumors. In summary, the generated mouse strain represents a convenient tool to study cell-autonomous inhibition of β-catenin-Tcf-mediated transcription.

• Klíčová slova
• Cre/loxP, TCF/LEF transcription factors, Wnt pathway, gene targeting, gut, β-catenin,
• MeSH
• beta-katenin metabolismus   MeSH
• buněčná diferenciace MeSH
• buněčné dělení MeSH
• genetická transkripce MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• myši transgenní MeSH
• myši MeSH
• proliferace buněk MeSH
• signální dráha Wnt * MeSH
• střevní sliznice cytologie   metabolismus   MeSH
• tenké střevo cytologie   metabolismus   MeSH
• transkripční faktor 4 MeSH
• transkripční faktory BHLH-Zip chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-katenin MeSH
• Tcf4 protein, mouse MeSH Prohlížeč
• transkripční faktor 4 MeSH
• transkripční faktory BHLH-Zip MeSH