Downregulation of cytokeratin 18 induces cellular partial EMT and stemness through increasing EpCAM expression in breast cancer.

Induction of epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) characteristics underlie the development of metastasis, chemoresistance, and tumor recurrence in breast cancer. Downregulation of cytokeratin 18 (CK18) is a critical molecular event of EMT; however, its importance in the induction of EMT and CSC features has not been defined to date. This study aimed to investigate the biological significance and underlying molecular mechanisms of CK18 in inducing EMT phenotype and stemness properties of breast cancer cells. Three breast cancer cell lines (i.e., non-metastatic MCF-7, highly metastatic MDA-MB-231, and mitoxantrone (MX)-selected resistant MCF-7/MX cells) and two CK18-knockdown stable cell clones (MCF-7-shCK18-7D and 3C) were used to determine the association between CK18 and EMT and stemness. CK18 expression was extremely low in highly metastatic, resistant, and transforming growth factor (TGF)-ß1/tumor necrosis factor (TNF)-α-treated breast cancer cells with mesenchymal phenotype and increased expression of CSC markers. Depletion of CK18 promoted partial EMT and the acquisition of stemness properties in breast cancer MCF-7 cells. Mechanistically, CK18 interference in MCF-7 cells activated the Wnt/ß-catenin signaling, resulting in the up-regulation of epithelial cell adhesion molecule (EpCAM). Consistently, the stemness properties and metastasis can be attenuated by further knockdown of EpCAM in CK18-depleted cells. In conclusion, downregulation of CK18 promotes partial EMT and enhances breast cancer stemness by increasing EpCAM expression partly via the Wnt/ß-catenin pathway. These findings indicate that CK18 may serve as a potential treatment target for advanced breast cancer.

The Rho-guanine nucleotide exchange factor Solo decelerates collective cell migration by modulating the Rho-ROCK pathway and keratin networks.

Collective cell migration plays crucial roles in tissue remodeling, wound healing, and cancer cell invasion. However, its underlying mechanism remains unknown. Previously, we showed that the RhoA-targeting guanine nucleotide exchange factor Solo (ARHGEF40) is required for tensile force-induced RhoA activation and proper organization of keratin-8/keratin-18 (K8/K18) networks. Here, we demonstrate that Solo knockdown significantly increases the rate at which Madin-Darby canine kidney cells collectively migrate on collagen gels. However, it has no apparent effect on the migratory speed of solitary cultured cells. Therefore, Solo decelerates collective cell migration. Moreover, Solo localized to the anteroposterior regions of cell-cell contact sites in collectively migrating cells and was required for the local accumulation of K8/K18 filaments in the forward areas of the cells. Partial Rho-associated protein kinase (ROCK) inhibition or K18 or plakoglobin knockdown also increased collective cell migration velocity. These results suggest that Solo acts as a brake for collective cell migration by generating pullback force at cell-cell contact sites via the RhoA-ROCK pathway. It may also promote the formation of desmosomal cell-cell junctions related to K8/K18 filaments and plakoglobin.

Tumor necrosis factor-α confers cardioprotection through ectopic expression of keratins K8 and K18.

Tumor necrosis factor-α (TNF-α), one of the major stress-induced proinflammatory cytokines, is upregulated in the heart after tissue injury, and its sustained expression can contribute to the development of heart failure. Whether TNF-α also exerts cytoprotective effects in heart failure is not known. Here we provide evidence for a cardioprotective function of TNF-α in a genetic heart failure model, desmin-deficient mice. The cardioprotective effects of TNF-α are a consequence of nuclear factor-κB (NF-κB)-mediated ectopic expression in cardiomyocytes of keratin 8 (K8) and keratin 18 (K18), two epithelial-specific intermediate filament proteins. In cardiomyocytes, K8 and K18 (K8/K18) formed an alternative cytoskeletal network that localized mainly at intercalated discs (IDs) and conferred cardioprotection by maintaining normal ID structure and mitochondrial integrity and function. Ectopic induction of K8/K18 expression in cardiomyocytes also occurred in other genetic and experimental models of heart failure. Loss of the K8/K18 network resulted in a maladaptive cardiac phenotype following transverse aortic constriction. In human failing myocardium, where TNF-α expression is upregulated, K8/K18 were also ectopically expressed and localized primarily at IDs, which did not contain detectable amounts of desmin. Thus, TNF-α- and NF-κB-mediated formation of an alternative, stress-induced intermediate filament cytoskeleton has cardioprotective function in mice and potentially in humans.

Keratin 8/18 regulation of glucose metabolism in normal versus cancerous hepatic cells through differential modulation of hexokinase status and insulin signaling.

As differentiated cells, hepatocytes primarily metabolize glucose for ATP production through oxidative phosphorylation of glycolytic pyruvate, whereas proliferative hepatocellular carcinoma (HCC) cells undergo a metabolic shift to aerobic glycolysis despite oxygen availability. Keratins, the intermediate filament (IF) proteins of epithelial cells, are expressed as pairs in a lineage/differentiation manner. Hepatocyte and HCC (hepatoma) cell IFs are made solely of keratins 8/18 (K8/K18), thus providing models of choice to address K8/K18 IF functions in normal and cancerous epithelial cells. Here, we demonstrate distinctive increases in glucose uptake, glucose-6-phosphate formation, lactate release, and glycogen formation in K8/K18 IF-lacking hepatocytes and/or hepatoma cells versus their respective IF-containing counterparts. We also show that the K8/K18-dependent glucose uptake/G6P formation is linked to alterations in hexokinase I/II/IV content and localization at mitochondria, with little effect on GLUT1 status. In addition, we find that the insulin-stimulated glycogen formation in normal hepatocytes involves the main PI-3 kinase-dependent signaling pathway and that the K8/K18 IF loss makes them more efficient glycogen producers. In comparison, the higher insulin-dependent glycogen formation in K8/K18 IF-lacking hepatoma cells is associated with a signaling occurring through a mTOR-dependent pathway, along with an augmentation in cell proliferative activity. Together, the results uncover a key K8/K18 regulation of glucose metabolism in normal and cancerous hepatic cells through differential modulations of mitochondrial HK status and insulin-mediated signaling.

Caspase-cleaved cytokeratin-18 and tumour regression in gastro-oesophageal adenocarcinomas treated with neoadjuvant chemotherapy.

AIM: To examine cytokeratin-18 (CK-18) and caspase-cleaved CK-18 expression in tumours and correlate with clinicopathological outcomes including tumour regression grade (TRG) response. METHODS: Formalin-fixed human gastro-oesophageal cancers were constructed into tissue microarrays. The first set consisted of 122 gastric/gastro-oesophageal cancer cases not exposed to neoadjuvant chemotherapy and the second set consisted of 97 gastric/gastro-oesophageal cancer cases exposed to pre-operative platinum-based chemotherapy. Expression of CK-18 and caspase-cleaved CK-18 was investigated using immunohistochemistry. RESULTS: CK18 was commonly expressed in gastro-oesophageal tumours (92.6%). Fifty-six point seven percent of tumours previously exposed to neoadjuvant chemotherapy were positive for caspase-cleaved CK-18 expression compared to only 24.6% of tumours not previously exposed to neoadjuvant chemotherapy (P = 0.009). In patients who received neoadjuvant chemotherapy, caspase-cleaved cytokeratin-18 expression correlated with favourable TRG response (TRG 1, 2 or 3, P = 0.043). CONCLUSION: This is the largest study to date of CK-18 and caspase-cleaved CK-18 expression in gastro-oesophageal tumours. We provide the first evidence that caspase-cleaved CK-18 predicts tumour regression with neoadjuvant chemotherapy.

Cytokeratin 18 expression inhibits cytokine-induced death of cervical cancer cells.

OBJECTIVES: In cervical cancer, increased cytokeratin 18 (CK18) filament expression is associated with disease progression. However, it may also provide resistance to cytokine-induced apoptosis. The present study tested whether CK18 expression influences susceptibility to cytokine-induced apoptosis. METHODS: The cervical cancer cell lines C-4II (high CK18 expression), ME-180 (low CK18 expression), and 2 subtypes of HeLa cells containing or lacking CK18 expression (CK18+ and CK18- cells, respectively) were exposed to vehicle (control), Fas ligand (FasL) (50 ng/mL), or tumor necrosis factor α (TNF-α; 10 ng/mL) without/with cycloheximide (CHX; 2.5 µg/mL) to test the hypothesis that diminished CK18 expression increases susceptibility to cytokine-induced apoptosis. RESULTS: Flow cytometric analysis of cell death via TUNEL staining revealed that cytokine-induced apoptosis was 2-fold greater in ME-180 cells than C-4II cells in response to FasL+CHX or TNF-α+CHX (P < 0.05). Similarly, there was a higher incidence of FasL-induced apoptosis in CK18- HeLa cells (23% and 91% apoptotic for FasL and FasL+CHX, respectively) than CK18+ HeLa cells (1% and 11%, respectively; P < 0.05). Surprisingly, TNF-α had no effect on either CK18+ or CK18- HeLa cells (P > 0.05). Caspase 3 activity was greater in CK18- HeLa cells than in CK18+ HeLa cells at 8 and 18 hours after FasL treatment (P < 0.05), an effect abrogated by the caspase 8 inhibitor IETD-fmk (P < 0.05). CONCLUSIONS: Cervical cancer cells with diminished CK18 expression are more susceptible to cytokine-induced apoptosis, particularly in response to FasL treatment. These observations suggest that relative CK18 expression is an important factor when considering therapeutic strategies to enhance immune cell-mediated death of cervical cancer cells.

Crosstalk between angiogenesis, cytokeratin-18, and insulin resistance in the progression of non-alcoholic steatohepatitis.

AIM: To elucidate the possible crosstalk between angiogenesis, cytokeratin-18 (CK-18), and insulin resistance (IR) especially in patients with non-alcoholic steatohepatitis (NASH). METHODS: Twenty-eight patients with NASH and 11 with simple fatty liver disease (FL) were enrolled in this study and underwent clinicopathological examination. The measures of angiogenesis, CK-18, and IR employed were CD34-immunopositive vessels, CK-18-immunopositive cells, and homeostasis model assessment of IR (HOMA-IR), respectively. The correlations of these factors with NASH were elucidated. RESULTS: Significant development of hepatic neovascularization was observed only in NASH, whereas almost no neovascularization could be observed in FL and healthy liver. The degree of angiogenesis was almost parallel to liver fibrosis development, and both parameters were positively correlated. Similarly, CK-18 expression and HOMA-R were significantly increased in NASH as compared with FL and healthy liver. Furthermore, CK-18 and HOMA-IR were also positively correlated with the degree of neovascularization. CONCLUSION: These results indicate that the crosstalk between angiogenesis, CK-18, and IR may play an important role in the onset and progression of NASH.

Proteomic analysis to identify cytokeratin 18 as a novel biomarker of nasopharyngeal carcinoma.

PURPOSE: In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. METHODS: Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cytokeratin 18 in the above two tissues as well as 4 NPC cell lines was determined by Western blotting. Immunohistochemistry was also performed to detect the expression of cytokeratin 18 in 62 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. siRNA and in vitro cell invasion assay were used to check the correlation between the expression of cytokeratin 18 and invasive ability of NPC. RESULTS: Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cytokeratin 18 in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Significant cytokeratin 18 down-regulation was observed in NPC versus NNET (P = 0.000), whereas significant cytokeratin 18 up-regulation was observed in lymph node metastasis versus primary NPC (P = 0.001). In addition, cytokeratin 18 down-regulation was significantly correlated with poor histological differentiation (P = 0.000), whereas cytokeratin 18 up-regulation was significantly correlated with advanced clinical stage (P = 0.019), recurrence (P = 0.000), and regional lymph node metastasis (P = 0.001), and distant metastasis (P = 0.000). And down-regulated cytokeratin 18 expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Furthermore, survival curves showed that patients with cytokeratin 18 up-regulation had a poor prognosis (P = 0.000). Univariate analysis (Cox's proportional hazards model) showed that WHO histologic type (P = 0.025), lymph node metastasis (P = 0.007), distant metastasis (P = 0.005), recurrence (P = 0.000), and cytokeratin 18 (P = 0.000) were significantly associated with the prognosis of NPC. Multivariate analysis confirmed that lymph node metastasis (P = 0.012), distant metastasis (P = 0.009), recurrence (P = 0.006), and cytokeratin 18 (P = 0.001) were independent prognostic indicators. CONCLUSIONS: The data suggest that cytokeratin 18 is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.

Possible relation between histone 3 and cytokeratin 18 in human hepatocellular carcinoma.

BACKGROUND: Previously we found some low molecular weight proteins identified as histone in hepatocelluar carcinoma. Our objective was to clarify whether the coimmunoprecipitation of histone and cytokeratin 18 was an artifact or not. MATERIALS AND METHODS: Histone 3 and cytokeratin 18 were investigated in three cases of human hepatocellular carcinoma and one case of normal liver tissue. Nuclei of the tissues were isolated; the proteins inside the nuclei were analyzed by Western blot. RESULTS: The results revealed histone was co-immunoprecipitated with cytokeratin 18 in hepatocellular carcinoma. It was speculated that modulation of the cytoskeleton in human hepatocellular carcinoma might disturb the organization of the nucleoskeleton. The unstable nucleoskeleton might further cause instability and fragility of nuclei, thus possibly exposing the histone and co-immunoprecipitating it with cytokeratin 18. CONCLUSION: The evidence might indicate that expression of histone 3 was highly related to modulation of cytokeratin 18 and might play an important role in tumorigenesis of hepatocellular carcinoma.

Keratins let liver live: Mutations predispose to liver disease and crosslinking generates Mallory-Denk bodies.

Keratin polypeptides 8 and 18 (K8/K18) are the cytoskeletal intermediate filament proteins of hepatocytes while K8/K18/K19 are the keratins of hepatobiliary ductal cells. Hepatocyte K8/K18 are highly abundant and behave as stress proteins with injury-inducible expression. Human association studies show that K8/K18 germline heterozygous mutations predispose to end-stage liver disease of multiple etiologies ( approximately 3 fold increased risk), and to liver disease progression in patients with chronic hepatitis C infection. These findings are supported by extensive transgenic mouse and ex vivo primary hepatocyte culture studies showing that K8 or K18 mutations predispose the liver to acute or subacute injury and promote apoptosis and fibrosis. Mutation-associated predisposition to liver injury is likely related to mechanical and nonmechanical keratin functions including maintenance of cell integrity, protection from apoptosis and oxidative injury, serving as a phosphate sponge, regulation of mitochondrial organization/function and protein targeting. These functions are altered by mutation-induced changes in keratin phosphorylation, solubility and filament organization/reorganization. Keratins are also the major constituents of Mallory-Denk bodies (MDBs). A toxin-induced K8>K18 ratio, and keratin crosslinking by transglutaminase-2 play essential roles in MDB formation. Furthermore, intracellular or cell-released K18 fragments, generated by caspase-mediated proteolysis during apoptosis serve as markers of liver injury. Therefore, K8 and K18 are cytoprotective stress proteins that play a central role in guarding hepatocytes from apoptosis. Keratin involvement in liver disease is multi-faceted and includes modulating disease progression upon mutation, formation of MDBs in response to unique forms of injury, and serving as markers of epithelial cell death.

Keratin 8 modulation of desmoplakin deposition at desmosomes in hepatocytes.

Keratins, the intermediate filament proteins of epithelial cells, connect to desmosomes, the cell-cell adhesion structures at the surface membrane. The building elements of desmosomes include desmoglein and desmocollin, which provide the actual cell adhesive properties, and desmoplakins, which anchor the keratin intermediate filaments to desmosomes. In the work reported here, we address the role of keratin 8 in modulating desmoplakin deposition at surface membrane in mouse hepatocytes. The experimental approach is based on the use of keratin 8- and keratin 18-null mouse hepatocytes as cell models. In wild-type mouse hepatocytes, desmoplakin is aligned with desmoglein and keratin 8 at the surface membrane. In keratin 8-null hepatocytes, the intermediate filament loss leads to alterations in desmoplakin distribution at the surface membrane, but not of desmoglein. Intriguingly, a significant proportion of keratin 18-null hepatocytes express keratin 8 at the surface membrane, associated with a proper desmoplakin alignment with desmoglein at desmosomes. A Triton treatment of the monolayer reveals that most of the desmoplakin present in either wild-type, keratin 8- or keratin 18-null hepatocytes is insoluble. Deletion analysis of keratin 8 further suggests that the recovery of desmoplakin alignment requires the keratin 8 rod domain. In addition, similarly to other works revealing a key role of desmoplakin phosphorylation on its interaction with intermediate filaments, we find that the phosphorylation status of the keratin 8 head domain affects desmoplakin distribution at desmosomes. Together, the data indicate that a proper alignment/deposition of desmoplakin with keratins and desmoglein in hepatocytes requires keratin 8, through a reciprocal phosphoserine-dependent process.

[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.