The cell surface glycoprotein Trop-2 is commonly overexpressed in carcinomas and represents an exceptional antigen for targeted therapy. Here, we provide evidence that surface Trop-2 expression is functionally connected with an epithelial phenotype in breast and prostate cell lines and in patient tumor samples. We further show that Trop-2 expression is suppressed epigenetically or through the action of epithelial-to-mesenchymal transition transcription factors and that deregulation of Trop-2 expression is linked with cancer progression and poor patient prognosis. Moreover, our data suggest that the cancer plasticity-driven intratumoral heterogeneity in Trop-2 expression may significantly contribute to response and resistance to therapies targeting Trop-2-expressing cells.

• MeSH
• antigeny nádorové genetika   metabolismus   MeSH
• CD antigeny biosyntéza   MeSH
• epitelo-mezenchymální tranzice fyziologie   MeSH
• epitelové buňky metabolismus   MeSH
• kadheriny biosyntéza   MeSH
• karcinom patologie   MeSH
• lidé MeSH
• metylace DNA genetika   MeSH
• molekuly buněčné adheze genetika   metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty mortalita   patologie   MeSH
• nádory prsu mortalita   patologie   MeSH
• progrese nemoci MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

SIGNIFICANCE: Machine learning is increasingly being applied to the classification of microscopic data. In order to detect some complex and dynamic cellular processes, time-resolved live-cell imaging might be necessary. Incorporating the temporal information into the classification process may allow for a better and more specific classification. AIM: We propose a methodology for cell classification based on the time-lapse quantitative phase images (QPIs) gained by digital holographic microscopy (DHM) with the goal of increasing performance of classification of dynamic cellular processes. APPROACH: The methodology was demonstrated by studying epithelial-mesenchymal transition (EMT) which entails major and distinct time-dependent morphological changes. The time-lapse QPIs of EMT were obtained over a 48-h period and specific novel features representing the dynamic cell behavior were extracted. The two distinct end-state phenotypes were classified by several supervised machine learning algorithms and the results were compared with the classification performed on single-time-point images. RESULTS: In comparison to the single-time-point approach, our data suggest the incorporation of temporal information into the classification of cell phenotypes during EMT improves performance by nearly 9% in terms of accuracy, and further indicate the potential of DHM to monitor cellular morphological changes. CONCLUSIONS: Proposed approach based on the time-lapse images gained by DHM could improve the monitoring of live cell behavior in an automated fashion and could be further developed into a tool for high-throughput automated analysis of unique cell behavior.

• MeSH
• algoritmy MeSH
• časosběrné zobrazování MeSH
• epitelo-mezenchymální tranzice * MeSH
• holografie * MeSH
• strojové učení MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

TGFβ has roles in inflammation, wound healing, epithelial to mesenchymal transition (EMT), and cancer stem cell states, and acts as a tumor suppressor gene for squamous cell carcinoma (SCC). SCCs are also characterized by high levels of ΔNp63, which induces epithelial cell phenotypes and maintains squamous stem cells. Previous studies indicate a complex interplay between ΔNp63 and TGFβ signaling, with contradictory effects reported. We investigated the effects of TGFβ on p63 isoform proteins and mRNAs in non-malignant squamous and SCC cells, and the role of either canonical or non-canonical TGFβ signaling pathways. TGFβ selectively increased ΔNp63 protein levels in non-malignant keratinocytes in association with SMAD3 activation and was prevented by TGFβ receptor inhibition, indicating activation of canonical TGFβ pathway signaling. TP63 isoform mRNAs showed discordance from protein levels, with an initial increase in both TAP63 and ΔNP63 mRNAs followed by a decrease at later times. These data demonstrate complex and heterogeneous effects of TGFβ in squamous cells that depend on the extent of canonical TGFβ pathway aberrations. The interplay between TGFβ and p63 is likely to influence the magnitude of EMT states in SCC, with clinical implications for tumor progression and response to therapy.

• MeSH
• epitelo-mezenchymální tranzice * genetika   MeSH
• epitelové buňky metabolismus   MeSH
• lidé MeSH
• protein - isoformy genetika   metabolismus   MeSH
• spinocelulární karcinom * genetika   metabolismus   MeSH
• transformující růstový faktor beta MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Benzimidazole anthelmintics flubendazole and mebendazole are microtubule-targeting drugs that showed considerable anti-cancer activity in different preclinical models. In this study, the effects of flubendazole and mebendazole on proliferation, migration and cadherin switching were studied in a panel of oral cell lines in vitro. Both compounds reduced the viability of the PE/CA-PJ15 and H376 oral squamous carcinoma cells and of the premalignant oral keratinocytes DOK with the IC50 values in the range of 0.19-0.26 μM. Normal oral keratinocytes and normal gingival fibroblasts were less sensitive to the treatment. Flubendazole and mebendazole also reduced the migration of the PE/CA-PJ15 cell in concentrations that had no anti-migratory effects on the normal gingival fibroblasts. Levels of the focal adhesion kinase FAK, Rho-A and Rac1 GTPases and the Rho guanine nucleotide exchange factor GEF-H1 were decreased in both PE/CA-PJ15 cells and gingival fibroblasts following treatment. Both drugs also interfered with cadherin switching in the model of TGF-β-induced epithelial to mesenchymal transition (EMT) in the DOK cell line. Levels of N-cadherin were reduced in the TGF-β induced cells co-treated with flubendazol and mebendazole in very low concentration (50 nM). These results suggest direct effects of both benzimidazoles on selected processes of EMT in oral cell lines such as cadherin switching as well as cellular migration.

• MeSH
• buněčné linie MeSH
• cdc42 protein vázající GTP metabolismus   MeSH
• epitelo-mezenchymální tranzice účinky léků   MeSH
• fokální adhezní kinasa 1 metabolismus   MeSH
• kadheriny metabolismus   MeSH
• lidé MeSH
• mebendazol analogy a deriváty   farmakologie   MeSH
• nádory úst metabolismus   patologie   MeSH
• pohyb buněk účinky léků   MeSH
• proliferace buněk účinky léků   MeSH
• rhoA protein vázající GTP metabolismus   MeSH
• spinocelulární karcinom metabolismus   patologie   MeSH
• transformující růstový faktor beta farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Lipopolysaccharide (LPS)-induced inflammation of lung tissues triggers irreversible alterations in the lung parenchyma, leading to fibrosis and pulmonary dysfunction. While the molecular and cellular responses of immune and connective tissue cells in the lungs are well characterized, the specific epithelial response remains unclear due to the lack of representative cell models. Recently, we introduced human embryonic stem cell-derived expandable lung epithelial (ELEP) cells as a novel model for studying lung injury and regeneration. METHODS: ELEPs were derived from the CCTL 14 human embryonic stem cell line through activin A-mediated endoderm specification, followed by further induction toward pulmonary epithelium using FGF2 and EGF. ELEPs exhibit a high proliferation rate and express key structural and molecular markers of alveolar progenitors, such as NKX2-1. The effects of Escherichia coli LPS serotype O55:B5 on the phenotype and molecular signaling of ELEPs were analyzed using viability and migration assays, mRNA and protein levels were determined by qRT-PCR, western blotting, and immunofluorescent microscopy. RESULTS: We demonstrated that purified LPS induces features of a hybrid epithelial-to-mesenchymal transition in pluripotent stem cell-derived ELEPs, triggers the unfolded protein response, and upregulates intracellular β-catenin level through retention of E-cadherin within the endoplasmic reticulum. CONCLUSIONS: Human embryonic stem cell-derived ELEPs provide a biologically relevant, non-cancerous lung cell model to investigate molecular responses to inflammatory stimuli and address epithelial plasticity. This approach offers novel insights into the fine molecular processes underlying lung injury and repair.

• MeSH
• buněčné linie MeSH
• CD antigeny metabolismus   MeSH
• endoplazmatické retikulum * metabolismus   účinky léků   MeSH
• epitelo-mezenchymální tranzice * účinky léků   MeSH
• epitelové buňky * účinky léků   metabolismus   cytologie   MeSH
• kadheriny * metabolismus   MeSH
• lidé MeSH
• lidské embryonální kmenové buňky * cytologie   MeSH
• lipopolysacharidy * farmakologie   MeSH
• plíce * cytologie   MeSH
• tyreoidální jaderný faktor 1 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cancer-associated fibroblasts are bioactive elements influencing the biological properties of malignant tumors. Their origin from different cell types has been established, and the possibility of their formation by epithelial-to-mesenchymal transition from cancer cells is under debate. This study shows that human cancer cells grafted to nu/nu mice induced formation of tumor stroma with the presence of typical smooth muscle actin-containing cancer-associated fibroblasts. These cells seem to be of the host origin because they are not recognized by an antibody specific for human vimentin, as was also verified in vitro. These results suggest that cancer-associated stromal fibroblasts are not formed by epithelial-to-mesenchymal transition from cancer cells.

• MeSH
• adenokarcinom metabolismus   patologie   MeSH
• buněčný rodokmen * MeSH
• buňky HT-29 MeSH
• buňky stromatu metabolismus   patologie   MeSH
• časové faktory MeSH
• epitelo-mezenchymální tranzice * MeSH
• fibroblasty metabolismus   patologie   MeSH
• heterografty MeSH
• kolorektální nádory metabolismus   patologie   MeSH
• lidé MeSH
• myši nahé MeSH
• nádorové biomarkery metabolismus   MeSH
• nádory hlavy a krku metabolismus   patologie   MeSH
• nádory hltanu metabolismus   patologie   MeSH
• spinocelulární karcinom metabolismus   patologie   MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Východiska: Triple-negativní karcinomy prsu (TNBC) jsou heterogenní skupinou nádorů s převážně agresivním chováním a špatnou prognózou. V souvislosti s jejich agresivním chováním a chemorezistencí vůči léčbě se do popředí dostal koncept epitelo-mezenchymové tranzice (EMT). Proteiny CD9 a CD29 jsou spojeny s EMT a mohou hrát roli v progresi TNBC. Naším cílem bylo prozkoumat asociaci těchto markerů s metastázami do lymfatických uzlin, gradingem tumoru, proliferační aktivitou a přežitím pacientů. Pacienti a metody: Náš soubor tvořilo 66 pacientek s TNBC bez neoadjuvantní terapie ve věku 26–81 let. Patologické stadium nádoru se pohybovalo od pT1b do pT3 a histologický stupeň od II do III podle systému Bloom-Richardson. Imunohistochemické hodnocení exprese CD9, CD29, E-cadherinu, vimentinu, androgenového receptoru a Ki-67 bylo provedeno semikvantitativně pomocí H-skóre. Exprese proteinů byla statisticky hodnocena ve vztahu ke klinicko-patologickým parametrům a přežití pacientů. Výsledky: Pozorovali jsme nižší expresi CD9 v metastázách lymfatických uzlin ve srovnání s primárním nádorem (p = 0,021). Exprese CD29 v primárním nádoru byla signifikantně nižší u pacientů s metastázami v lymfatických uzlinách ve srovnání s pacienty bez diseminace (p = 0,03). Ani exprese CD9 ani CD29 proteinu nebyla spojena s přežitím specifickým pro karcinom prsu (BCSS). Nižší exprese E-cadherinu na periferii primárního tumoru byla spojena s horším BCSS (p = 0,038). Pro grading ani přítomnost metastáz v lymfatických uzlinách nebyl nalezen signifikantní vztah s BCSS. Nižší exprese E-cadherinu na periferii byla také spojena s vyšší hladinou Ki67 (Rs −0,26) a vimentinu (Rs −0,33). Závěr: Snížená exprese proteinů CD9 a CD29 byla spojena s růstem metastáz v lymfatických uzlinách, avšak jejich souvislost s přežitím nebyla prokázána. Nižší exprese E-cadherinu na periferii primárního nádoru byla spojena s vysokou proliferací a špatným nádorově specifickým přežitím.

Background: Triple-negative breast carcinomas (TNBC) are a heterogeneous group of tumors with mostly aggressive behaviour and poor prognosis. In association with their aggressive behavior and chemoresistance to treatment, the concept of epithelial-mesenchymal transition (EMT) has come to the fore. CD9 and CD29 proteins are associated with EMT and may play a role in TNBC progression. Our aim was to investigate association of these markers with the lymph node metastasis, tumor grade, proliferative activity, and patient survival. Patients and methods: Our cohort consisted of 66 TNBC patients without neoadjuvant therapy, aged 26–81 years. The pathological tumor stages ranged from pT1b to pT3 and histological grades ranged from II to III, according to the Bloom-Richardson system. Immunohistochemical evaluation of CD9, CD29, E-cadherin, vimentin, androgen receptor and Ki-67 expression was performed semiquantitatively using the H-score. Expression of the proteins was statistically evaluated in relation to the clinicopathological parameters and survival of the patients. Results: We observed lower expression of CD9 in lymph node metastases compared to the primary tumor (P = 0.021). The CD29 expression in primary tumor was significantly lower in patients with lymph node metastases compared to patients without cancer dissemination (P = 0.03). Neither CD9 nor CD29 protein expression was associated with breast cancer-specific survival (BCSS). Lower expression of E-cadherin at the periphery of the primary tumor was associated with worse BCSS (P = 0.038). Neither grade nor the presence of lymph node metastases reached significant association with the BCSS. Lower expression of E-cadherin at the periphery was also associated with higher Ki67 (Rs −0.26) and vimentin (Rs −0.33). Conclusion: Decreased protein expression of CD9 and CD29 were associated with lymph node metastasis growth, however, their association with survival was not proved. Lower expression of E-cadherin at the periphery of the primary tumor was associated with high proliferation and poor breast cancer-specific survival.

• MeSH
• antigeny CD29 analýza   MeSH
• antigeny CD9 analýza   MeSH
• epitelo-mezenchymální tranzice imunologie   MeSH
• imunohistochemie * metody   MeSH
• kadheriny analýza   MeSH
• kohortové studie MeSH
• lidé MeSH
• lymfatické metastázy diagnostické zobrazování   imunologie   MeSH
• prognóza MeSH
• triple-negativní karcinom prsu * diagnostické zobrazování   imunologie   sekundární   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (EpCAM) identification of fibroblasts from breast and prostate tumor tissues is advised. © 2017 International Society for Advancement of Cytometry.

• MeSH
• adhezní molekula epiteliálních buněk metabolismus   MeSH
• antigeny CD31 metabolismus   MeSH
• antigeny CD45 metabolismus   MeSH
• biologické markery metabolismus   MeSH
• buňky PC-3 MeSH
• epitelo-mezenchymální tranzice fyziologie   MeSH
• epitelové buňky metabolismus   MeSH
• fibroblasty metabolismus   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty metabolismus   MeSH
• nádory prsu metabolismus   MeSH
• serinové endopeptidasy metabolismus   MeSH
• transformující růstový faktor beta1 metabolismus   MeSH
• želatinasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cystic fibrosis (CF) is a monogenetic disease resulting from mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene encoding an anion channel. Recent evidence indicates that CFTR plays a role in other cellular processes, namely in development, cellular differentiation and wound healing. Accordingly, CFTR has been proposed to function as a tumour suppressor in a wide range of cancers. Along these lines, CF was recently suggested to be associated with epithelial-mesenchymal transition (EMT), a latent developmental process, which can be re-activated in fibrosis and cancer. However, it is unknown whether EMT is indeed active in CF and if EMT is triggered by dysfunctional CFTR itself or a consequence of secondary complications of CF. In this study, we investigated the occurrence of EMT in airways native tissue, primary cells and cell lines expressing mutant CFTR through the expression of epithelial and mesenchymal markers as well as EMT-associated transcription factors. Transepithelial electrical resistance, proliferation and regeneration rates, and cell resistance to TGF-β1induced EMT were also measured. CF tissues/cells expressing mutant CFTR displayed several signs of active EMT, namely: destructured epithelial proteins, defective cell junctions, increased levels of mesenchymal markers and EMT-associated transcription factors, hyper-proliferation and impaired wound healing. Importantly, we found evidence that the mutant CFTR triggered EMT was mediated by EMT-associated transcription factor TWIST1. Further, our data show that CF cells are over-sensitive to EMT but the CF EMT phenotype can be reversed by CFTR modulator drugs. Altogether, these results identify for the first time that EMT is intrinsically triggered by the absence of functional CFTR through a TWIST1 dependent mechanism and indicate that CFTR plays a direct role in EMT protection. This mechanistic link is a plausible explanation for the high incidence of fibrosis and cancer in CF, as well as for the role of CFTR as tumour suppressor protein.

• MeSH
• cystická fibróza metabolismus   patologie   MeSH
• epitelo-mezenchymální tranzice MeSH
• HEK293 buňky MeSH
• jaderné proteiny metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• onkogeny genetika   MeSH
• protein CFTR metabolismus   MeSH
• transkripční faktor Twist metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVE: We recently determined that the ectopic over-expression of miR-429 and other members of the miR-200 family of microRNAs in ovarian cancer (OC) mesenchymal-like cell lines induces mesenchymal-to-epithelial transition (MET) with a concomitant increase in sensitivity to platinum drugs. We sought to determine if metastasizing OC cells isolated from an OC patient could also be induced by miR-429 to undergo MET and become sensitized to established first-line platinum-based therapies. METHODS: We established and characterized a new primary cell line (OCI-984) from free-floating OC cells isolated from the ascites fluid of an advanced stage OC patient. miR-429 was ectopically over-expressed in these cells. RESULTS: The over-expression of miR-429 in OCI-984 cells induced morphological, functional and molecular changes consistent with MET and a concomitant significant increase in the sensitivity of the converted cells to cisplatin. CONCLUSIONS: Our findings indicate that the miR-200 family of microRNAs, and miR-429 in particular, play a critical role in the functioning of OC metastasizing cells and that targeted delivery of miR-429, and perhaps other miR-200 family members, in combination with platinum-based chemotherapies may be an effective strategy in reducing OC metastasis and tumor recurrence.

• MeSH
• cisplatina farmakologie   MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• lidé MeSH
• mikro RNA biosyntéza   MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie * MeSH
• nádory vaječníků farmakoterapie   genetika   patologie   MeSH
• protinádorové látky farmakologie   MeSH
• screeningové testy protinádorových léčiv MeSH
• senioři MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH