Mario Mikula

The transcription factor ZEB1 (δEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity

Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, we identified transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell–cell adhesion, including the cell polarity genes Crumbs3, HUGL2 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promotor activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour–host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. Our data show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression.

Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene

The Raf kinases play a key role in relaying signals elicited by mitogens or oncogenes. Here, we report that c‐raf‐1−/− embryos are growth retarded and die at midgestation with anomalies in the placenta and in the fetal liver. Although hepatoblast proliferation does not appear to be impaired, c‐raf‐1−/− fetal livers are hypocellular and contain numerous apoptotic cells. Similarly, the poor proliferation of Raf‐1−/− fibroblasts and hematopoietic cells cultivated in vitro is due to an increase in the apoptotic index of these cultures rather than to a cell cycle defect. Furthermore, Raf‐1‐ deficient fibroblasts are more sensitive than wild‐ type cells to specific apoptotic stimuli, such as actinomycin D or Fas activation, but not to tumor necrosis factor‐α. MEK/ERK activation is normal in Raf‐1‐deficient cells and embryos, and is probably mediated by B‐Raf. These results indicate that the essential function of Raf‐1 is to counteract apoptosis rather than to promote proliferation, and that effectors distinct from the MEK/ERK cascade must mediate the anti‐apoptotic function of Raf‐1.

A crucial function of PDGF in TGF-β-mediated cancer progression of hepatocytes

Polarized hepatocytes expressing hyperactive Ha-Ras adopt an invasive and metastatic phenotype in cooperation with transforming growth factor (TGF)-β. This dramatic increase in malignancy is displayed by an epithelial to mesenchymal transition (EMT), which mimics the TGF-β-mediated progression of human hepatocellular carcinomas. In culture, hepatocellular EMT occurs highly synchronously, facilitating the analysis of molecular events underlying the various stages of this process. Here, we show that in response to TGF-β, phosphorylated Smads rapidly translocated into the nucleus and activated transcription of target genes such as E-cadherin repressors of the Snail superfamily, causing loss of cell adhesion. Within the TGF-β superfamily of cytokines, TGF-β1, -β2 and -β3 were specific for the induction of hepatocellular EMT. Expression profiling of EMT kinetics revealed 78 up- and 235 downregulated genes, which preferentially modulate metabolic activities, extracellular matrix composition, transcriptional activities and cell survival. Independent of the genetic background, platelet-derived growth factor (PDGF)-A ligand and both PDGF receptor subunits were highly elevated, together with autocrine secretion of bioactive PDGF. Interference with PDGF signalling by employing hepatocytes expressing the dominant-negative PDGF-α receptor revealed decreased TGF-β-induced migration in vitro and efficient suppression of tumour growth in vivo. In conclusion, these results provide evidence for a crucial role of PDGF in TGF-β-mediated tumour progression of hepatocytes and suggest PDGF as a target for therapeutic intervention in liver cancer.

β-Catenin and TGFβ signalling cooperate to maintain a mesenchymal phenotype after FosER-induced epithelial to mesenchymal transition

Several signalling pathways contribute to the regulation of epithelial to mesenchymal transition (EMT), either during developmentally regulated processes or in cancer progression and metastasis. Induction of EMT in fully polarized mouse mammary epithelial cells (EpH4) by an inducible c-fos estrogen receptor (FosER) oncoprotein involves loss of E-cadherin expression, nuclear translocation of β-catenin, and autocrine production of TGFβ. Reporter assays demonstrate that both β-catenin/LEF–TCF- and TGFβ–Smad-dependent signalling activities are upregulated, probably coregulating mesenchymal-specific gene expression during EMT. Stable expression of E-cadherin in mesenchymal FosER cells decreased β-catenin activity and reduced cell proliferation. However, these cells still exhibited a defect in epithelial polarization and expressed E-cadherin/β-catenin complexes in the entire plasma membrane. On the other hand, inhibition of TGFβ–Smad signalling in mesenchymal FosER cells induced flat, cobblestone-like clusters of cells, which relocalized β-catenin to the plasma membrane but still lacked detectable E-cadherin. Interestingly, inhibition of TGFβ signalling in the E-cadherin-expressing mesenchymal FosER cells caused their reversion to a polarized epithelial phenotype, in which E-cadherin, β-catenin, and ZO-1 were localized at their correct lateral plasma membrane domains. These results demonstrate that loss of E-cadherin can contribute to increased LEF/TCF-β-catenin signalling, which in turn cooperates with autocrine TGFβ signalling to maintain an undifferentiated mesenchymal phenotype.

PDGF essentially links TGF- β signaling to nuclear β -catenin accumulation in hepatocellular carcinoma progression

The cooperation of Ras – extracellular signal-regulated kinase/mitogen-activated protein kinase and transforming growth factor (TGF)-β signaling provokes an epithelial to mesenchymal transition (EMT) of differentiated p19ARF null hepatocytes, which is accompanied by a shift in malignancy and gain of metastatic properties. Upon EMT, TGF-β induces the secretion and autocrine regulation of platelet-derived growth factor (PDGF) by upregulation of PDGF-A and both PDGF receptors. Here, we demonstrate by loss-of-function analyses that PDGF provides adhesive and migratory properties in vitro as well as proliferative stimuli during tumor formation. PDGF signaling resulted in the activation of phosphatidylinositol-3 kinase, and furthermore associated with nuclear β-catenin accumulation upon EMT. Hepatocytes expressing constitutively active β-catenin or its negative regulator Axin were employed to study the impact of nuclear β-catenin. Unexpectedly, active β-catenin failed to accelerate proliferation during tumor formation, but in contrast, correlated with growth arrest. Nuclear localization of β-catenin was accompanied by strong expression of the Cdk inhibitor p16INK4A and the concomitant induction of the β-catenin target genes cyclin D1 and c-myc. In addition, active β-catenin revealed protection of malignant hepatocytes against anoikis, which provides a prerequisite for the dissemination of carcinoma. From these data, we conclude that TGF-β acts tumor progressive by induction of PDGF signaling and subsequent activation of β-catenin, which endows a subpopulation of neoplastic hepatocytes with features of cancer stem cells.

Down-regulation of sprouty2 in non-small cell lung cancer contributes to tumor malignancy via extracellular signal-regulated kinase pathway-dependent and -independent mechanisms

Sprouty (Spry) proteins function as inhibitors of receptor tyrosine kinase signaling mainly by interfering with the Ras/Raf/mitogen-activated protein kinase cascade, a pathway known to be frequently deregulated in human non–small cell lung cancer (NSCLC). In this study, we show a consistently lowered Spry2 expression in NSCLC when compared with the corresponding normal lung epithelium. Based on these findings, we investigated the influence of Spry2 expression on the malignant phenotype of NSCLC cells. Ectopic expression of Spry2 antagonized mitogen-activated protein kinase activity and inhibited cell migration in cell lines homozygous for K-Ras wild type, whereas in NSCLC cells expressing mutated K-Ras, Spry2 failed to diminish extracellular signal-regulated kinase (ERK) phosphorylation. Nonetheless, Spry2 significantly reduced cell proliferation in all investigated cell lines and blocked tumor formation in mice. Accordingly, a Spry2 mutant unable to inhibit ERK phosphorylation reduced cell proliferation significantly but less pronounced compared with the wild-type protein. Therefore, we conclude that Spry2 interferes with ERK phosphorylation and another yet unidentified pathway. Our results suggest that Spry2 plays a role as tumor suppressor in NSCLC by antagonizing receptor tyrosine kinase–induced signaling at different levels, indicating feasibility for the usage of Spry in targeted gene therapy of NSCLC. (Mol Cancer Res 2007;5(5):509–20)

The transcription factor ZEB1 (δEF1) represses Plakophilin 3 during human cancer progression

Plakophilin 3 (PKP3) belongs to the p120ctn family of armadillo‐related proteins predominantly functioning in desmosome formation. Here we report that PKP3 is transcriptionally repressed by the E‐cadherin repressor ZEB1 in metastatic cancer cells. ZEB1 physically associates with two conserved E‐box elements in the PKP3 promoter and partially represses the activity of corresponding human and mouse PKP3 promoter fragments in reporter gene assays. In human tumours ZEB1 is upregulated in invasive cancer cells at the tumour–host interface, which is accompanied by downregulation of PKP3 expression levels. Hence, the transcriptional repression of PKP3 by ZEB1 contributes to ZEB1‐mediated disintegration of intercellular adhesion and epithelial to mesenchymal transition.

Activated hepatic stellate cells induce tumor progression of neoplastic hepatocytes in a TGF‐β dependent fashion

The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra‐ and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells. For both tumorigenesis and hepatic fibrogenesis, transforming growth factor (TGF)‐β signaling executes key roles and therefore is considered as a hallmark of these pathological events. By employing cellular transplantation we show that the interaction of neoplastic MIM‐R hepatocytes with the tumor microenvironment, containing either activated hepatic stellate cells (M1‐4HSCs) or myofibroblasts derived thereof (M‐HTs), induces progression in malignancy. Cotransplantation of MIM‐R hepatocytes with M‐HTs yielded strongest MIM‐R generated tumor formation accompanied by nuclear localization of Smad2/3 as well as of β‐catenin. Genetic interference with TGF‐β signaling by gain of antagonistic Smad7 in MIM‐R hepatocytes diminished epithelial dedifferentiation and tumor progression upon interaction with M1‐4HSCs or M‐HTs. Further analysis showed that tumors harboring disrupted Smad signaling are devoid of nuclear β‐catenin accumulation, indicating a crosstalk between TGF‐β and β‐catenin signaling. Together, these data demonstrate that activated HSCs and myofibroblasts directly govern hepatocarcinogenesis in a TGF‐β dependent fashion by inducing autocrine TGF‐β signaling and nuclear β‐catenin accumulation in neoplastic hepatocytes. These results indicate that intervention with TGF‐β signaling is highly promising in liver cancer therapy. J. Cell. Physiol. 209: 560–567, 2006.

The proto-oncoprotein c-Fos negatively regulates hepatocellular tumorigenesis

Hepatocytes adopt an invasive and metastatic phenotype caused by the cooperation of transforming growth factor (TGF)-β and oncogenic Ha-Ras. In the initial phase of this process, c-Fos is rapidly induced by TGF-β, but then decreases to undetectable levels. Here, we investigated the functional implications of c-Fos activation and its contribution to hepatocellular tumorigenesis. By employing conditional c-Fos expression, we observed that continuous activation of c-Fos and consequently AP-1 activity leads to depolarization of differentiated murine epithelial hepatocytes. Most remarkably, this change in morphology was associated with inhibition of proliferation and induction of cell death. Coexpression of antiapoptotic Bcl-XL or scavenging of reactive oxygen species was sufficient to prevent the c-Fos-mediated phenotype. In contrast, the cooperation of c-Fos with oncogenic Ha-Ras or a Ras mutant selectively activating the MAPK pathway even enhanced c-Fos-induced effects. Showing the negative role in hepatocellular tumorigenesis, c-Fos repressed oncogenic Ras-driven anchorage-independent growth in vitro and strongly suppressed tumour formation in vivo. Taken together, we demonstrate that c-Fos modulates plasticity of epithelial hepatocytes and acts tumour suppressive in neoplastic hepatocytes by stimulating cell cycle inhibition and cell death.

Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans

Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, differentiation or apoptosis in tumour cells and are, therefore, promising anti‐cancer reagents. However, the specific HDAC isoforms that mediate these effects are not yet identified. To explore the role of HDAC1 in tumourigenesis and tumour proliferation, we established an experimental teratoma model using wild‐type and HDAC1‐deficient embryonic stem cells. HDAC1‐deficient teratomas showed no significant difference in size compared with wild‐type teratomas. Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation. Epithelial structures showed reduced differentiation as monitored by Oct3/4 expression and changed E‐cadherin localization and displayed up‐regulated expression of SNAIL1, a regulator of epithelial cell plasticity. Increased levels of the transcriptional regulator SNAIL1 are crucial for enhanced proliferation and reduced differentiation of HDAC1‐deficient teratoma. Importantly, the analysis of human teratomas revealed a similar link between loss of HDAC1 and enhanced tumour malignancy. These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.