Keratin 19 interacts with GSK3ß to regulate its nuclear accumulation and degradation of cyclin D3.

Cyclin D3 regulates the G1/S transition and is frequently overexpressed in several cancer types including breast cancer, where it promotes tumor progression. Here we show that a cytoskeletal protein keratin 19 (K19) physically interacts with a serine/threonine kinase GSK3ß and prevents GSK3ß-dependent degradation of cyclin D3. The absence of K19 allowed active GSK3ß to accumulate in the nucleus and degrade cyclin D3. Specifically, the head (H) domain of K19 was required to sustain inhibitory phosphorylation of GSK3ß Ser9, prevent nuclear accumulation of GSK3ß, and maintain cyclin D3 levels and cell proliferation. K19 was found to interact with GSK3ß and K19-GSK3ß interaction was mapped out to require Ser10 and Ser35 residues on the H domain of K19. Unlike wildtype K19, S10A and S35A mutants failed to maintain total and nuclear cyclin D3 levels and induce cell proliferation. Finally, we show that the K19-GSK3ß-cyclin D3 pathway affected sensitivity of cells toward inhibitors to cyclin-dependent kinase 4 and 6 (CDK4/6). Overall, these findings establish a role for K19 in the regulation of GSK3ß-cyclin D3 pathway and demonstrate a potential strategy for overcoming resistance to CDK4/6 inhibitors.

Prognostic Role for CYFRA 21-1 in Patients With Advanced-stage NSCLC Treated With Bevacizumab Plus Chemotherapy.

AIM: To investigate potential associations between selected oncomarkers [carcinoembryonic antigen (CEA), C-terminus of cytokeratin 19 (CYFRA 21-1, CYFRA), and squamous cell carcinoma antigen (SCC)] and outcomes in patients with NSCLC treated with bevacizumab plus chemotherapy. PATIENTS AND METHODS: We retrospectively analysed 105 patients with NSCLC from the Czech TULUNG registry treated at University Hospital in Pilsen with bevacizumab plus chemotherapy. Response to therapy was tested by Fisher's exact test. Survival statistics were evaluated using the Kaplan-Meier method and Cox analysis. RESULTS: Only normal values of CYFRA (not CEA or SCC) were associated with significantly better overall and progression-free survival in univariate analysis. We also observed a trend for a better disease control rate in patients with normal levels of CYFRA. In a multivariate Cox model, only CYFRA was associated with significantly better overall but not progression-free survival. CONCLUSION: In our retrospective study, we point out the possibility of using CYFRA as a prognostic marker in patients with NSCLC treated with chemotherapy plus bevacizumab.

Serum markers of pulmonary epithelial damage in systemic sclerosis-associated interstitial lung disease and disease progression.

BACKGROUND AND OBJECTIVE: The course of systemic sclerosis-associated interstitial lung disease (SSc-ILD) is highly variable, and accurate prognostic markers are needed. KL-6 is a mucin-like glycoprotein (MUC1) expressed by type II pneumocytes, while CYFRA 21-1 is expressed by alveolar and bronchiolar epithelial cells. Both are released into the blood from cell injury. METHODS: Serum KL-6 and CYFRA 21-1 levels were measured in a retrospective (n = 189) and a prospective (n = 118) cohort of SSc patients. Genotyping of MUC1 rs4072037 was performed. Linear mixed-effect models were used to evaluate the relationship with change in lung function parameters over time, while association with survival was evaluated with Cox proportional hazard analysis. RESULTS: In both cohorts, KL-6 and CYFRA 21-1 were highest in patients with lung involvement, and in patients with extensive rather than limited ILD. KL-6 was higher in patients carrying the MUC1 rs4072037 G allele in both cohorts. In patients with SSc-ILD, serum KL-6, but not CYFRA 21-1, was significantly associated with DLCO decline in both cohorts (P = 0.001 and P = 0.004, respectively), and with FVC decline in the retrospective cohort (P = 0.005), but not the prospective cohort. When combining the cohorts and subgrouping by severity (median CPI = 45.97), KL-6 remained predictive of decline in DLCO in both milder (P = 0.007) and more severe disease (P = 0.02) on multivariable analysis correcting for age, gender, ethnicity, smoking history and MUC1 allele carriage. CONCLUSION: Our results suggest serum KL-6 predicts decline in lung function in SSc, suggesting its clinical utility in risk stratification for progressive SSc-ILD.

Comparison of the Opn-CreER and Ck19-CreER Drivers in Bile Ducts of Normal and Injured Mouse Livers.

Inducible cyclization recombinase (Cre) transgenic mouse strains are powerful tools for cell lineage tracing and tissue-specific knockout experiments. However, low efficiency or leaky expression can be important pitfalls. Here, we compared the efficiency and specificity of two commonly used cholangiocyte-specific Cre drivers, the Opn-iCreERT2 and Ck19-CreERT drivers, using a tdTomato reporter strain. We found that Opn-iCreERT2 triggered recombination of the tdTomato reporter in 99.9% of all cholangiocytes while Ck19-CreERT only had 32% recombination efficiency after tamoxifen injection. In the absence of tamoxifen, recombination was also induced in 2% of cholangiocytes for the Opn-iCreERT2 driver and in 13% for the Ck19-CreERT driver. For both drivers, Cre recombination was highly specific for cholangiocytes since recombination was rare in other liver cell types. Toxic liver injury ectopically activated Opn-iCreERT2 but not Ck19-CreERT expression in hepatocytes. However, ectopic recombination in hepatocytes could be avoided by applying a three-day long wash-out period between tamoxifen treatment and toxin injection. Therefore, the Opn-iCreERT2 driver is best suited for the generation of mutant bile ducts, while the Ck19-CreERT driver has near absolute specificity for bile duct cells and is therefore favorable for lineage tracing experiments.

Keratin 19: a key role player in the invasion of human hepatocellular carcinomas.

OBJECTIVE: Keratin (K)19, a biliary/hepatic progenitor cell (HPC) marker, is expressed in a subset of hepatocellular carcinomas (HCC) with poor prognosis. The underlying mechanisms driving this phenotype of K19-positive HCC remain elusive. DESIGN: Clinicopathological value of K19 was compared with EpCAM, and α-fetoprotein, in a Caucasian cohort of 242 consecutive patients (167 surgical specimens, 75 needle biopsies) with different underlying aetiologies. Using microarrays and microRNA profiling the molecular phenotype of K19-positive HCCs was identified. Clinical primary HCC samples were submitted to in vitro invasion assays and to side population analysis. HCC cell lines were transfected with synthetic siRNAs against KRT19 and submitted to invasion and cytotoxicity assays. RESULTS: In the cohort of surgical specimens, K19 expression showed the strongest correlation with increased tumour size (p<0.01), decreased tumour differentiation (p<0.001), metastasis (p<0.05) and microvascular invasion (p<0.001). The prognostic value of K19 was also confirmed in a set of 75 needle biopsies. Profiling showed that K19-positive HCCs highly express invasion-related/metastasis-related markers (eg, VASP, TACSTD2, LAMB1, LAMC2, PDGFRA), biliary/HPC markers (eg, CD133, GSTP1, NOTCH2, JAG1) and members of the miRNA family 200 (eg, miR-141, miR-200c). In vitro, primary human K19-positive tumour cells showed increased invasiveness, and reside in the chemoresistant side population. Functionally, K19/KRT19 knockdown results in reduced invasion, loss of invadopodia formation and decreased resistance to doxorubicin, 5-fluorouracil and sorafenib. CONCLUSIONS: Giving the distinct invasive properties, the different molecular profile and the poor prognostic outcome, K19-positive HCCs should be considered as a seperate entity of HCCs.

Influences of desmin and keratin 19 on passive biomechanical properties of mouse skeletal muscle.

In skeletal muscle fibers, forces must be transmitted between the plasma membrane and the intracellular contractile lattice, and within this lattice between adjacent myofibrils. Based on their prevalence, biomechanical properties and localization, desmin and keratin intermediate filaments (IFs) are likely to participate in structural connectivity and force transmission. We examined the passive load-bearing response of single fibers from the extensor digitorum longus (EDL) muscles of young (3 months) and aged (10 months) wild-type, desmin-null, K19-null, and desmin/K19 double-null mice. Though fibers are more compliant in all mutant genotypes compared to wild-type, the structural response of each genotype is distinct, suggesting multiple mechanisms by which desmin and keratin influence the biomechanical properties of myofibers. This work provides additional insight into the influences of IFs on structure-function relationships in skeletal muscle. It may also have implications for understanding the progression of desminopathies and other IF-related myopathies.

Cytokeratin 10 and cytokeratin 19: predictive markers for poor prognosis in hepatocellular carcinoma patients after curative resection.

PURPOSE: Cytokeratin 10 (CK10) was found to be expressed differently in human hepatocellular carcinoma (HCC) cell lines with different metastatic potentials in our previous research. The aim of this study was to assess the value of CK10 alone or in combination with cytokeratin 19 (CK19) in predicting tumor recurrence after curative resection in HCC patients. EXPERIMENTAL DESIGN: CK10 expression in stepwise metastatic HCC cell lines and tumor tissues from 50 HCC patients was investigated using immunofluorescence assay, quantitative real-time reverse transcription-PCR, and Western blot analyses. Tumor tissue microarrays of 300 HCC patients who underwent curative resection between 1997 and 2000 were used to detect the expressions of CK10 and CK19. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. RESULTS: CK10 was overexpressed in the high metastatic HCC cell line and in tumor tissues of recurrent patients. Both univariate and multivariate analyses revealed that CK10 was a significant predictor for overall survival (OS) and disease-free survival, and that CK19 was a significant predictor for OS. CK10 expression was correlated with poor prognosis regardless of alpha-fetoprotein, tumor-node-metastasis stage, and vascular invasion. The 7-year OS and disease-free survival rates in CK10+ and/or CK19+ patients were 30.0% and 37.6%, respectively, which were significantly lower than that of CK10-/CK19- patients (56.1% and 60.0%, respectively; P < 0.001). CONCLUSION: CK10 is associated with HCC invasiveness. CK10 alone, or in combination with CK19, can be a novel predictor for poor prognosis of HCC patients after curative resection.

SgIGSF is a novel biliary-epithelial cell adhesion molecule mediating duct/ductule development.

UNLABELLED: Spermatogenic immunoglobulin superfamily (SgIGSF) is an intercellular adhesion molecule of the nectin-like family. While screening its tissue distribution, we found that it was expressed in fetal liver but not adult liver. In the present study, we examined which cells in developing and regenerating liver express SgIGSF via immunohistochemistry and Western blot analysis. In developing mouse liver, SgIGSF expression was transiently upregulated at perinatal ages and was restricted to the lateral membrane of biliary epithelial cells (BECs). In regenerating rat livers from the 2-acetylaminofluorene/partial hepatectomy model, SgIGSF was detected exclusively in oval cells that aligned in ductal and trabecular patterns by the second week posthepatectomy. In human livers, fetal and newborn bile ducts and cirrhotic bile ductules were clearly positive for SgIGSF, whereas disease-free adult bile ducts were negative. To investigate the role of SgIGSF in bile duct/ductule formation, we used an in vitro model in which rat hepatocyte aggregates embedded in collagen gels containing insulin and epidermal growth factor extend epithelial sheets and processes in the first week and form ductules within a month. The process and ductular cells were continuously positive for SgIGSF and cytokeratin 19, a BEC marker. When the aggregate culture was started in the presence of a function-blocking anti-SgIGSF antibody, the number of epithelial processes per aggregate was reduced by 80%. CONCLUSION: We propose that SgIGSF is a novel and functional BEC adhesion molecule that is expressed for a limited time during active bile duct/ductule formation.

[Bootstrapping algorithm approach reveals inherent regulatory pattern in 550 invasive breast cancer cases: CK5/6/CK14 and CK8/18/CK19 builds an antagonistic set]. / Implementierung eines Bootstrap-Algorithmus zur Evaluation Zytokeratin-abhängiger Regulationsmuster an 550 invasiven Mammakarzinomen.

All the preliminary observations on a lot of marker sets defining different stages in the tumor development are building a framework of work hypothesis which can be verified in characterising large pools of histological uniform rated paraffin probes. We developed a bootstrapping algorithm based on correlation measures to uncover regulatory patterns of immunohistochemical characterized tissue arrays with 550 invasive breast cancer cases. The algorithm is implemented in 'S' a computer language used to model mathematical solutions. Focussing on the Cytokeratins versus a set of prominent markers in breast cancer differentiation it will be obvious that markers which are known to appear in early (progenitor) forms conform to CK5/6 and CK14 while others associated with late stages conform to CK8/18 and CK19. Markers examined are among others EGFR, EMA, erb-B2, Vimentin, p53, ER and PR. The developed approach is an elegant and complete procedure to reveal the real regulatory patterns which are enclosed in a certain experimental design. The statistical significance of the results calculated by our algorithm is generally high and in the presented experimental design smaller than 0.6 * 10E-6.