Breast cancer is the most frequently diagnosed cancer in women worldwide. Although dramatically increased survival rates of early diagnosed cases have been observed, late diagnosed patients and metastatic cancer may still be considered fatal. The present study's main focus was on cancer‐associated fibroblasts (CAFs) which is an active component of the tumor microenvironment (TME) regulating the breast cancer ecosystem. Transcriptomic profiling and analysis of CAFs isolated from breast cancer skin metastasis, cutaneous basal cell carcinoma, and squamous cell carcinoma unravelled major gene candidates such as IL6, VEGFA and MFGE8 that induced co‐expression of keratins‐8/‐14 in the EM‐G3 cell line derived from infiltrating ductal breast carcinoma. Western blot analysis of selected keratins (keratin‐8, ‐14, ‐18, ‐19) and epithelial‐mesenchymal transition‐associated markers (SLUG, SNAIL, ZEB1, E‐/N‐cadherin, vimentin) revealed specific responses pointing to certain heterogeneity of the studied CAF populations. Experimental in vitro treatment using neutralizing antibodies against IL-6, VEGF‐A and MFGE8 attenuated the modulatory effect of CAFs on EM‐G3 cells. The present study provided novel data in characterizing and understanding the interactions between CAFs and EM‐G3 cells in vitro. CAFs of different origins support the pro‐inflammatory microenvironment and influence the biology of breast cancer cells. This observation potentially holds significant interest for the development of novel, clinically relevant approaches targeting the TME in breast cancer. Furthermore, its implications extend beyond breast cancer and have the potential to impact a wide range of other cancer types.
- MeSH
- antigeny povrchové MeSH
- fibroblasty asociované s nádorem * metabolismus MeSH
- fibroblasty metabolismus MeSH
- keratiny genetika metabolismus MeSH
- lidé MeSH
- maligní melanom kůže MeSH
- MFC-7 buňky MeSH
- mléčné bílkoviny genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorové mikroprostředí genetika MeSH
- nádory prsu * farmakoterapie genetika metabolismus MeSH
- prognóza MeSH
- transkriptom MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND/AIM: Expression profiling was performed to delineate and characterize the impact of malignancy by comparing tissues from three sites of head and neck cancer of each patient, also determining interindividual variability. MATERIALS AND METHODS: Genome-wide analysis was carried out covering the expression of 25,832 genes with quantification for each site of seven patients with tonsillar or oropharyngeal squamous cell carcinoma. Immunohistochemical analysis was performed for adhesion/growth-regulatory galectins, three pro-inflammatory chemo- and cytokines and keratins. RESULTS: Up- and down-regulation was found for 281 (tumor vs. normal) and 276 genes (transition zone vs. normal), respectively. The profile of the transition zone had its own features, with similarity to the tumor. Galectins were affected in a network manner, with differential regulation and interindividual variability between patients, also true for keratins and the chemo- and cytokines. CONCLUSION: These results underline special features at each site of specimen origin as well as the importance of analyzing galectins as a network and of defining the expression status of the individual patient prior to reaching clinically relevant conclusions.
- MeSH
- cytokiny genetika MeSH
- epitel metabolismus MeSH
- galektiny genetika metabolismus MeSH
- genom lidský MeSH
- keratiny genetika metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádory orofaryngu genetika metabolismus MeSH
- senioři MeSH
- spinocelulární karcinom genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Keratin-associated proteins (KRTAPs) play a critical role in cross-linking the keratin intermediate filaments to build a hair shaft. The genetic polymorphisms of the bovine KRTAP7-1 gene were investigated for the first time in this study. The complete coding sequence of the KRTAP7-1 gene in 108 domestic yak, taurine and zebu cattle from China and Indonesia were successfully amplified using polymerase chain reaction and then directly sequenced. Only two single-nucleotide polymorphisms (one nonsynonymous at c.7C/G and another synonymous at c.21C/T) and three haplotypes (BOVIN-KRTAP7-1*A, B and C) were identified in the complete coding sequence of the bovine KRTAP7-1 gene among all animals. There was no polymorphism across three Chinese indigenous yak breeds and one Indonesian zebu cattle population, all sharing the BOVINKRTAP71*A haplotype. The four taurine cattle populations also had BOVIN-KRTAP7-1*A as the most common haplotype with a frequency of 0.80. The frequency of novel haplotype BOVIN-KRTAP7-1*B was only 0.07 present in one heterozygous animal in each of the four taurine cattle populations, while BOVINKRTAP7- 1*C was only found in a Simmental and a local Chinese Yellow cattle population with frequencies of 0.17 and 0.36, respectively. The monomorphic yak KRTAP7-1 gene in particular, and highly conserved bovine, sheep and goat KRTAP7-1 genes in general, demonstrated its unique intrinsic structural property (e.g., > 21% high glycine content) and primary functional importance in supporting the mechanical strength and shape of hair.
Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).
- MeSH
- biopsie MeSH
- imunohistochemie MeSH
- keratiny biosyntéza genetika MeSH
- kultivační média MeSH
- kultivované buňky MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae metabolismus ultrastruktura MeSH
- nemoci rohovky genetika patologie chirurgie MeSH
- regulace genové exprese * MeSH
- RNA genetika MeSH
- rohovkový epitel metabolismus ultrastruktura MeSH
- senioři MeSH
- transmisní elektronová mikroskopie MeSH
- transplantace rohovky * MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Triple-negative breast cancers (TNBC) comprise a heterogeneous subgroup of tumors with a generally poor prognosis. Subclassification of TNBC based on genomic analyses shows that basal-like TNBCs, specifically the basal A or BL2 subtype, are characterized by the expression of ΔNp63, a transcription factor that has been attributed a variety of roles in the regulation of proliferation, differentiation, and cell survival. To investigate the role(s) of p63 in basal-like breast cancers, we used HCC1806 cells that are classified as basal A/BL2. We show that these cells endogenously express p63, mainly as the ΔNp63α isoform. TP63 gene knockout by CRISPR resulted in viable cells that proliferate more slowly and adhere less tightly, with an increased rate of migration. Analysis of adhesion-related gene expression revealed a complex set of alterations in p63-depleted cells, with both increased and decreased adhesion molecules and adhesion substrates compared to parental cells expressing p63. Examination of the phenotype of these cells indicated that endogenous p63 is required to suppress the expression of luminal markers and maintain the basal epithelial phenotype, with increased levels of both CK8 and CK18 and a reduction in N-cadherin levels in cells lacking p63. On the other hand, the level of CK5 was not decreased and ER was not increased, indicating that p63 loss is insufficient to induce full luminal-type differentiation. Taken together, these data demonstrate that p63 exerts multiple pro-oncogenic effects on cell differentiation, proliferation and adhesion in basal-like breast cancers.
- MeSH
- buněčná adheze MeSH
- buněčná diferenciace MeSH
- CD antigeny biosyntéza genetika MeSH
- CRISPR-Cas systémy MeSH
- epitelové buňky metabolismus patologie MeSH
- fenotyp MeSH
- genový knockout MeSH
- kadheriny biosyntéza genetika MeSH
- karcinom patologie MeSH
- keratiny biosyntéza genetika MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny fyziologie MeSH
- nádorové supresorové proteiny nedostatek fyziologie MeSH
- proliferace buněk MeSH
- protein - isoformy fyziologie MeSH
- regulace genové exprese u nádorů MeSH
- transkripční faktory nedostatek fyziologie MeSH
- triple-negativní karcinom prsu patologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Tumor biomarkers increasingly provide information for predicting outcomes with chemotherapeutic regimens (personalized medicine). Topo2A is a DNA helicase targeted by anthracyclines, cytotoxic therapeutics used in both adjuvant and palliative treatments of breast cancer. TOP2A gene amplification/deletion is implicated in response to anthracycline-based chemotherapy. We describe an approach for analyzing formalin-fixed, paraffin-embedded breast tumors on tissue microarrays with TOP2A fluorescence in situ hybridization coupled with cytokeratin immunofluorescence to target tumor cells. Stained tissue from patient specimens was imaged and analyzed using Metafer/Metacyte (Metasystems, Waltham, MA, USA), including customized image classifiers. TOP2A/CEN17 ratios of 2.0 or greater (amplified) and 0.8 or less (deleted) were observed for 10.0% and 6.1% of the patients, respectively. Patient outcomes for adjuvant chemotherapy (cyclophosphamide-epirubicin-fluorouracil, cyclophosphamide-methotrexate-fluorouracil, no chemotherapy) were evaluated. No statistical significance was achieved for clinical end points regarding TOP2A status in anthracycline-treated patients. However, patients with TOP2A aberrations receiving methotrexate-based therapy exhibited a significant decrease in 5-year distant disease-free survival and breast cancer-specific overall survival, especially for patients with TOP2A deletions (disease-free survival: hazard ratio, 5.31 [P = .001], and breast cancer-specific overall survival: hazard ratio, 6.45 [P < .001]). No significant differences were seen in patients included in the no-chemotherapy group. Topo2A protein levels were assessed by immunohistochemistry with no correlative statistical relevance to immunofluorescence/fluorescence in situ hybridization-based prognosis for cyclophosphamide-epirubicin-fluorouracil or cyclophosphamide-methotrexate-fluorouracil groups. Interestingly, aberrant (under)expressing patients in the no-chemotherapy group exhibited better 5-year distant disease-free survival (hazard ratio, 0.39; P = .004), trending toward more favorable breast cancer-specific overall survival (hazard ratio, 0.61; P = .11). Our results indicate a strategy by which fluorescence in situ hybridization scoring targeted to cytokeratin-positive tumor cells may provide a tool for added precision and efficiency in TOP2A evaluation from tumor tissue. Copyright 2012 Elsevier Inc. All rights reserved.
- MeSH
- adjuvantní chemoterapie MeSH
- antigeny nádorové * genetika MeSH
- cyklofosfamid terapeutické užití MeSH
- DNA vazebné proteiny * genetika MeSH
- DNA-topoisomerasy typu II * genetika MeSH
- dospělí MeSH
- fluorouracil terapeutické užití MeSH
- hybridizace in situ fluorescenční MeSH
- keratiny * genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- methotrexát terapeutické užití MeSH
- nádory prsu * farmakoterapie genetika patologie MeSH
- přežití po terapii bez příznaků nemoci MeSH
- protokoly antitumorózní kombinované chemoterapie terapeutické užití MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- ženské pohlaví MeSH
The aim of this study was to detect a spectrum of cytokeratins (CK) present in the adult human cornea, limbus and perilimbal conjunctiva. Cryosections from seven corneo-scleral discs were fixed, and indirect immunofluorescent staining was performed using antibodies directed against CK1-CK10 and CK13-CK20. The percentage of positive cells was calculated in the epithelium of the cornea, limbus and perilimbal conjunctiva. Quantitative real time RT-PCR (qRT-PCR) was used to detect CK6 and CK18 expression in the corneal and conjunctival epithelium. The most intense staining present throughout the cornea was observed for CK3, CK5 and CK14; CK19 was found at the corneal periphery only. CK4 and CK10/13 revealed mild to moderate positivity mostly in the superficial layers of the cornea. The suprabasal cell layers of all examined areas showed a strong positivity for CK16. A heterogeneous staining pattern with a centrifugal decrease in the signal was observed for CK8 and CK18. CK5/6, CK14 and CK19 were present in the limbus, where a positive signal for CK3 was observed in the suprabasal and superficial cells only. In contrast to the cornea, CK15 appeared in the basal and suprabasal layers of the limbus. The perilimbal conjunctiva showed strong immunostaining for CK10/13, CK14 and CK19. A moderate signal for CK7 was detected in the superficial layers of the conjunctiva. qRT-PCR confirmed CK6 and CK18 expression in the corneal and conjunctival epithelium. The detailed characterization of the corneal, limbal and perilimbal conjunctival epithelium under normal circumstances may be useful for characterizing the changes occurring under pathological conditions.
- MeSH
- dospělí MeSH
- fluorescenční mikroskopie MeSH
- fluorescenční protilátková technika nepřímá MeSH
- glyceraldehyd-3-fosfátdehydrogenasy metabolismus MeSH
- imunohistochemie MeSH
- keratiny biosyntéza genetika metabolismus MeSH
- konjunktiva metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- RNA biosyntéza genetika MeSH
- rohovka metabolismus MeSH
- rohovkový epitel metabolismus MeSH
- senioři MeSH
- skléra metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Přehledový článek shrnuje nejnovější poznatky z oblasti hepatopatologie jež byly předneseny na Prague Hepatology Meeting 2010. V oblasti nealkoholické steatohepatitidy hraje v současné době důležitou roli střevní mikroflóra a permeabilita zažívacího traktu, které významným způsobem ovlivňují riziko vzniku jaterní cirhózy. U nádorů jater se pozornost zaměřuje na molekulární diagnostiku, umožňující např. u hepatocelulárního adenomu rozlišit několik podtypů s různým rizikem maligního zvratu. Experimentální hepatologie se v současnosti zabývá zejména rolí epigenetických faktorů při vzniku alkoholového postižení jater. Virové hepatitidě C je věnována pozornost z hlediska mechanizmů vzniku rezistence k terapii. Stále důležitým tématem zůstává problematika patogeneze jaterní fibrózy a problematika role keratinů v patogenezi hepatopatií.
This review summarizes the highlights in hepatopathology presented during the Prague Hepatology Meeting 2010. Gut flora (microbiome) and intestinal permeability seems to play an important role during progression into liver cirrhosis in patients with non-alcoholic steatohepatitis. Molecular diagnosis allows the distinction of several types of hepatocellular adenoma with different risks of malignant progression. Experimental hepatology is currently focused on the role of epigenetics during pathogenesis of alcoholic liver disease. The most studied topic in viral hepatitis C is the mechanisms of resistance to antiviral treatment. Pathogenesis of liver fibrosis and the role of keratins in the pathology of liver disease are areas of scientific interest as well.
- Klíčová slova
- fibróza jater, hepatitis C, alkoholová hepatopatie, nealkoholická steatohepatitida,
- MeSH
- alkoholická steatóza jater diagnóza MeSH
- hepatitida C diagnóza etiologie terapie MeSH
- hepatitida diagnóza etiologie klasifikace MeSH
- jaterní cirhóza diagnóza etiologie MeSH
- keratiny genetika imunologie izolace a purifikace MeSH
- kongresy jako téma MeSH
- lidé MeSH
- metabolický syndrom diagnóza etiologie komplikace MeSH
- nádory jater diagnóza etiologie MeSH
- rizikové faktory MeSH
- střevní mikroflóra MeSH
- ztučnělá játra diagnóza etiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Cieľ: Cieľom práce bolo zistiť incidenciu regionálnych okultných mikrometastáz semisériovým rezaním a imunohistochemickým vyšetrením na cytokeratíny u pacientov so skvamocelulárnym karcinómom hlavy a krku. Materiál a metódy: Lymfatické uzliny z krčného disekátu u 18 pacientov so skvamocelulárnym karcinómom hlavy a krku, ktorí absolvovali primárnu chirurgickú liečbu, boli semisériovo rezané v 200 μm intervaloch s farbením na hematoxylin-eosin a imunohistochemickým vyšetrením na dôkaz epitelových markerov (cytokeratínový koktail AE1/3). Výsledky: Z 18 pacientov, 28 krčných disekcií, sa získalo a vyšetrilo 600 lymfatických uzlín. Incidencia okultných mikrometastáz bola 22,2% na pacienta a 14,3% na stranu krku. Incidencia mikrometastáz na lymfatickú uzlinu bola 1%. K zmene z pN0 na pN+ došlo u štyroch pacientov, 28,6 %. Záver: Incidencia okultných mikrometastáz pri karcinómoch hlavy a krku detekovaná sériovými rezmi s doplneným imunohistochemickým vyšetrením na cytokeratíny je vyššia ako je diagnostikovaná rutínnym histopatologickým vyšetrením. Touto technikou by sa mohli presnejšie identifikovať pacienti na pooperačnú adjuvantnú liečbu.
Objectives: The aim of the study was to determine incidence of occult micrometastases by semiserial sectioning and immunohistochemistry with cytokeratin in patients with head and neck squamous cell carcinoma with clinically negative neck. Material and methods: Eighteen patients with squamous cell carcinoma of the head and neck who primary underwent surgical treatment of the primary tumor and elective selective neck dissection were included in the study. Lymph nodes of neck dissection specimen were evaluated by semiserial sectioning at 200 μm interval with hematoxylin-eosin staining and immunohistochemical stain method using pan-cytokeratin AE1/3. Results: Of 18 patients, 28 neck dissections were harvested 600 lymph nodes. The incidence of occult micrometastases was 22.2% per patient, 14.3% per neck and 1% per lymph node. In 4 patients was pN upgrading from pN0 to pN+, 28.6%. Conclusion: The incidence of occult micrometastases in head and neck cancer patients detected by semiserial sectioning with cytokeratin immunohistochemistry is higher than conventional pathologic techniques. Use of this technique should more accurately identify patients for postoperative adjuvant treatment.
- MeSH
- biopsie sentinelové lymfatické uzliny metody trendy MeSH
- diagnostické techniky molekulární metody MeSH
- imunohistochemie metody trendy MeSH
- keratiny analýza genetika MeSH
- krční disekce MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfatické metastázy diagnóza patologie prevence a kontrola MeSH
- lymfatické uzliny chirurgie metabolismus patologie MeSH
- nádorové biomarkery analýza metabolismus MeSH
- nádorové buňky kultivované MeSH
- nádory hlavy a krku chirurgie metabolismus patologie MeSH
- následné studie MeSH
- senioři MeSH
- spinocelulární karcinom diagnóza chirurgie sekundární MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
BACKGROUND: Carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) are established tumour markers and the CEA pre-operative levels in serum have prognostic value. The aim of this study was to verify the usefulness of the estimation of CEA and CK20 gene expressions in tissues of colorectal cancers and their liver metastases. PATIENTS AND METHODS: Two hundred and twenty-two specimens of colorectal cancers, their liver metastases, other liver tumours and control tissues were analysed by reverse transcription combined with real-time PCR. RESULTS: The expressions of CEA and CK20 were significantly higher in tumours than in controls; there were differences between tumour types and no relationship to staging or clinical development was found. CK20 expression was inversely dependent on grading. The CEA expression in tumours did not correlate with the CEA levels in serum, but did correlate with serum tissue-specific polypeptide antigen (TPS). CONCLUSION: The measurement of CEA and CK20 gene expressions in tumours did not supply any new prognostic information, but raised the question of the mechanism releasing CEA into the blood.
- MeSH
- adenokarcinom genetika imunologie patologie sekundární MeSH
- dospělí MeSH
- exprese genu MeSH
- financování organizované MeSH
- karcinoembryonální antigen biosyntéza genetika krev MeSH
- keratin-20 MeSH
- keratiny biosyntéza genetika krev MeSH
- kolorektální nádory genetika imunologie krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- messenger RNA biosyntéza genetika MeSH
- nádorové biomarkery krev MeSH
- nádory jater genetika imunologie sekundární MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- staging nádorů MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři nad 80 let MeSH
- senioři MeSH
