Martina Welzel

Galectin-1 interacts with the {alpha}5{beta}1 fibronectin receptor to restrict carcinoma cell growth via induction of p21 and p27.

Surface binding of galectin family members has the potential to link distinct glycan structures to growth regulation. Therefore, we addressed the antiproliferative potential of galectin-1 (Gal-1) in a panel of carcinoma cell lines. We discovered growth inhibition by Gal-1 in epithelial tumor cell lines from different origins and provide evidence that this effect requires functional interaction with the α5β1 integrin. Antiproliferative effects result from inhibition of the Ras-MEK-ERK pathway and consecutive transcriptional induction of p27. We have further identified two Sp1-binding sites in the p27 promoter as crucial for Gal-1 responsiveness. Inhibition of the Ras-MEK-ERK cascade by Gal-1 increased Sp1 transactivation and DNA binding due to reduced threonine phosphorylation of Sp1. Furthermore, Gal-1 induced p21 transcription and selectively increased p27 protein stability. Gal-1-mediated accumulation of p27 and p21 inhibited cyclin-dependent kinase 2 activity and ultimately resulted in G1 cell cycle arrest and growth inhibition. These data define a novel mechanism whereby Gal-1 regulates epithelial tumor cell homeostasis via carbohydrate-dependent interaction with the α5β1 integrin.

Tumor suppressor p16INK4a: Downregulation of galectin-3, an endogenous competitor of the pro-anoikis effector galectin-1, in a pancreatic carcinoma model

The tumor suppressor p16INK4a has functions beyond cell‐cycle control via cyclin‐dependent kinases. A coordinated remodeling of N‐ and O‐glycosylation, and an increase in the presentation of the endogenous lectin galectin‐1 sensing these changes on the surface of p16INK4a‐expressing pancreatic carcinoma cells (Capan‐1), lead to potent pro‐anoikis signals. We show that the p16INK4a‐dependent impact on growth‐regulatory lectins is not limited to galectin‐1, but also concerns galectin‐3. By monitoring its expression in relation to p16INK4a status, as well as running anoikis assays with galectin‐3 and cell transfectants with up‐ or downregulated lectin expression, a negative correlation between anoikis and the presence of this lectin was established. Nuclear run‐off and northern blotting experiments revealed an effect of the presence of p16INK4a on steady‐state levels of galectin‐3‐specific mRNA that differed from decreasing the transcriptional rate. On the cell surface, galectin‐3 interferes with galectin‐1, which initiates signaling toward its pro‐anoikis activity via caspase‐8 activation. The detected opposite effects of p16INK4a at the levels of growth‐regulatory galectins‐1 and ‐3 shift the status markedly towards the galectin‐1‐dependent pro‐anoikis activity. A previously undescribed orchestrated fine‐tuning of this effector system by a tumor suppressor is discovered.

Hypoxia-Inducible Factor 1α Mediates Anoikis Resistance via Suppression of α5 Integrin

The transcription factor hypoxia-inducible factor 1 (HIF-1) alpha is abundantly expressed in the majority of human carcinomas and their metastases. HIF-1alpha controls central metastasis-associated pathways such as glycolysis, angiogenesis, and invasion. Functional inhibition of HIF-1alpha leads to impaired metastasis formation in murine tumor models. However, the precise molecular mechanisms underlying the metastasis-promoting role of HIF-1alpha have not been fully characterized. The ability of transformed epithelial cells to initiate the metastatic cascade relies on their ability to escape anoikis, a default program of apoptosis induction following loss of integrin anchoring to the extracellular matrix. Therefore, we addressed the function of HIF-1alpha in anoikis resistance and anchorage-independent growth. Inhibition of HIF-1alpha via RNA interference resulted in up-regulation of alpha5 integrin on the cell surface of human gastric cancer cells, whereas other integrins remained unaffected. Integrin alpha5 induction occurred at the level of transcription and was dependent on elevated intracellular superoxide in HIF-1alpha-knockdown cells. HIF-1alpha-deficient cells displayed significantly increased anoikis susceptibility due to up-regulated alpha5 integrin. Finally, colony formation in soft agar was shown to be dependent on HIF-1alpha as HIF-1alpha-deficient cells displayed a 70% reduction in anchorage-independent proliferation. Results obtained by RNA interference could be entirely confirmed by application of the pharmacologic HIF-1alpha-inhibitor 2-methoxyestradiol. Hence, our data argue for a pivotal role for HIF-1alpha in anoikis control via suppression of alpha5 integrin. HIF-1alpha-inhibiting drugs might therefore offer an innovative strategy for antimetastatic cancer therapy.

Axon Guidance Factor SLIT2 Inhibits Neural Invasion and Metastasis in Pancreatic Cancer

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.

The oral multitarget tumour growth inhibitor, ZK 304709, inhibits growth of pancreatic neuroendocrine tumours in an orthotopic mouse model

Background and aims: Current systemic therapies for neuroendocrine tumours (NETs) do not provide sufficient control of tumour growth. However, efficient evaluation of novel drugs is hindered by the lack of a suitable preclinical animal model. Here an orthotopic mouse model of pancreatic NET is established and used to study the action of ZK 304709, a first in class, oral multitarget tumour growth inhibitor. ZK 304709 is an inhibitor of cyclin-dependent kinases (Cdks) 1, 2, 4, 7 and 9, vascular endothelial growth factor receptor-type kinases (VEGF-RTKs) 1–3 and platelet-derived growth factor receptor-type kinase β (PDGF-RTKß). Methods: BON and QGP-1 human NET cells were used to study proliferation, survival and cell cycle distribution in vitro. For induction of orthotopic NETs, BON cells were injected into the pancreas of NMRInu/nu mice. Primary tumour growth and metastatic spread were recorded after 9 weeks, and apoptosis, microvessel density and lymphatic vessel density were determined. Results: ZK 304709 dose-dependently suppressed proliferation and colony formation of NET cells. Direct effects on NET cells were consistent with Cdk inhibition and involved G2 cell cycle arrest and apoptosis induction, which was associated with reduced expression of MCL1 (myeloid cell leukaemia sequence 1), survivin and hypoxia-inducible factor 1α (HIF1α). Apoptosis similarly occurred in vivo in ZK 304709-treated orthotopic BON tumours, resulting in a 80% reduction of primary tumour growth. In contrast, treatment with lanreotide or 5-fluorouracil and streptozotocin failed to inhibit tumour gowth. ZK 304709 also reduced tumour microvessel density, implicating antiangiogenic mechanisms. Conclusion: BON orthotopic tumours provide an informative model for preclinical drug evaluation in NETs. In this model, ZK 304709 achieved efficacious tumour growth control via induction of apoptosis and inhibition of tumour-induced angiogenesis.