<p>PDF file - 643KB, Figure S1: Reactivation of Slit2 expression in Panc1 cells by 5-AZA-dC suggests silencing of Slit2 gene due to promoter hypermethylation. Figure S2: Slit2 inhibits directed migration of pancreatic tumor cells. Figure S3: Re-expression of Slit2 does not affect random migration of MiaPaCaTR-Slit2 Cells. Figure S4: Doxycycline treatment results in induction of Slit2 protein in MiaPaCaTR-Slit2 tumors in vivo. Figure S5: Inducible re-expression of Slit2 inhibits invasion, metastasis and angiogenesis in size-matched orthotopic pancreatic tumors. Figure S6: Stable knock down of Robo1 in pancreatic tumor cells promotes metastasis, but does not affect angiogenesis of orthotopic pancreatic tumors. Figure S7: Functional inhibition of Slit2 via Robo1-Fc stimulates angiogenesis in DANGRobo1-KD tumors subjected to Robo1 receptor knock down. Figure S8: Forced expression of Slit2 inhibits metastasis and invasion in the orthotopic Panc02 syngeneic tumor model. Figure S9: Time-lapse microscopy reveals directional navigation of tumor cells along Neuritis.</p>
The potential antiproliferative effects of interferon–α (IFN–α) in the treatment of hepatocellular carcinoma (HCC) are controversial, and the growth inhibitory mechanisms remain poorly understood. Therefore, the current study was designed to delineate the molecular mechanisms responsible for direct antiproliferative actions of IFN–α in HCC cells. IFN–α receptor expression and signal transduction were examined by RT–PCR, immunoprecipitation, Western analysis, and transient transactivation assays. Effects of IFN–α on cell growth and cell–cycle distribution were evaluated based on cell numbers and flow cytometry. Composition and activity of cyclin–dependent kinase complexes were determined by immunoblotting and histone–H1–kinase assays. Expression of IFN–α receptors was found in all 3 HCC cell lines. IFN–α binding initiated phosphorylation of Jak1 and Tyk2 kinases leading to Stat1/Stat2 activation, nuclear translocation, and transactivation of an ISRE–luciferase reporter gene construct. IFN–α treatment resulted in a time– and dose–dependent reduction of proliferation. Cell cycle analysis of G1–synchronized, IFN–α–treated HCC cells revealed a substantial delay in S–phase progression but no alteration of G 1 /S–phase transition or evidence of apoptotic cell death. Reflecting the time course of S–phase accumulation, cell cycle–dependent induction of Cyclin A and Cyclin B was impaired, resulting in reduced activity of Cdk2 and Cdc2 kinases. Furthermore, Cdc25C was selectively down–regulated. IFN–α treatment inhibits growth of HCC cells by specifically delaying S–phase progression, most likely because of inhibition of Cyclin A induction, resulting in decreased activity of the associated Cdk2 and Cdc2 kinases.
Pancreatic ductal adenocarcinoma (PDAC) metastasizes by neural, vascular, and local invasion routes, which limit patient survival. In nerves and vessels, SLIT2 and its ROBO receptors constitute repellent guidance cues that also direct epithelial branching. Thus, the SLIT2-ROBO system may represent a key pinch point to regulate PDAC spread. In this study, we examined the hypothesis that escaping from repellent SLIT2-ROBO signaling is essential to enable PDAC cells to appropriate their local stromal infrastructure for dissemination. Through immunohistochemical analysis, we detected SLIT2 receptors ROBO1 and ROBO4 on epithelia, nerves, and vessels in healthy pancreas and PDAC specimens, respectively. SLIT2 mRNA expression was reduced in PDAC compared with nontransformed pancreatic tissues and cell lines, suggesting a reduction in SLIT2-ROBO pathway activity in PDAC. In support of this interpretation, restoring the SLIT2 expression in SLIT2-deficient PDAC cells inhibited their bidirectional chemoattraction with neural cells, and more specifically, impaired unidirectional PDAC cell navigation along outgrowing neurites in models of neural invasion. Restoring autocrine/paracrine SLIT2 signaling was also sufficient to inhibit the directed motility of PDAC cells, but not their random movement. Conversely, RNA interference-mediated silencing of ROBO1 stimulated the motility of SLIT2-competent PDAC cells. Furthermore, culture supernatants from SLIT2-competent PDAC cells impaired migration of endothelial cells (human umbilical vein endothelial cells), whereas an N-terminal SLIT2 cleavage fragment stimulated such migration. In vivo investigations of pancreatic tumors with restored SLIT2 expression demonstrated reduced invasion, metastasis, and vascularization, with opposing effects produced by ROBO1 silencing in tumor cells or sequestration of endogenous SLIT2. Analysis of clinical specimens of PDAC showed that those with low SLIT2 mRNA expression exhibited a higher incidence and a higher fraction of tumor-infiltrated lymph nodes. Taken together, our findings argue that disrupting SLIT2-ROBO signaling in PDAC may enhance metastasis and predispose PDAC cells to neural invasion.
Einleitung: Slit2 und seine Robo-Rezeptoren sind axon guidance Faktoren mit repulsiver Wirkung auf die Navigation von Nerven und Gefäßen. Aufgrund der neuronalen Differenzierung von NETs könnte somit das Slit2-Robo axon guidance Liganden-Rezeptor System eine entsprechend konservierte Funktion bei der Regulation der Motilität und der Interaktion von Tumorzellen mit Kompartimenten des Tumormikromilieus spielen.
Einleitung: Der Transkriptionsfaktor HIF-1a wird im Verlauf der Pathogenese des humanen Magenkarzinoms überexprimiert. Die funktionelle Inhibition von HIF-1a hemmt das Wachstum experimenteller Magenkarzinome durch eine erhöhte Apoptoserate der neoplastischen Zellen, wobei die zugrundeliegenden molekularen Mechanismen unbekannt sind.
ABSTRACT We have analyzed human pancreatic cancer cells to explore the growth regulatory function of protein kinase C (PKC)α. PKCα subcellular redistribution, activation kinetics and downregulation were examined in detail and correlated to immediate and delayed effects on cell-cycle regulatory pathways. TPA treatment resulted in transient PKCα activation accompanied by translocation of the enzyme into membrane and nuclear compartments, and was followed by subsequent downregulation. TPA-induced inhibition of DNA synthesis was prevented by a PKC-antagonist and was reproduced by microinjection of recombinant PKCα, indicating that activation of this isoenzyme was required and sufficient for growth inhibitory effects. PKCα activation arrested cells in the G1 phase of the cell cycle as a consequence of selective inhibition of cyclin dependent kinase (CDK)2 activity with concomitant hypophosphorylation of Rb. The inhibition of CDK2 activity resulted from induction of p21cip1 cyclin-dependent kinase inhibitors. Levels of p21cip1 remained elevated and CDK2 activity repressed in spite of PKCα downregulation, indicating that downstream effectors of PKCα are the primary determinants for the duration of PKC-mediated growth inhibition. The PKCα-induced block in cell proliferation persisted even though cells were kept in the presence of growth factors, suggesting that induction of PKCα results in a permanent withdrawal of pancreatic cancer cells from the cell cycle.
