Katharina Detjen

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.

Tumour suppressor p16INK4a – anoikis-favouring decrease in N/O-glycan/cell surface sialylation by down-regulation of enzymes in sialic acid biosynthesis in tandem in a pancreatic carcinoma model

Tumour suppressor p16INK4a is known to exert cell‐cycle control via cyclin‐dependent kinases. An emerging aspect of its functionality is the orchestrated modulation of N/O‐glycosylation and galectin expression to induce anoikis in human Capan‐1 pancreatic carcinoma cells. Using chemoselective N/O‐glycan enrichment technology (glycoblotting) and product characterization, we first verified a substantial decrease in sialylation. Tests combining genetic (i.e. transfection with α2,6‐sialyltransferase‐specific cDNA) or metabolic (i.e. medium supplementation with N‐acetylmannosamine to track down a bottleneck in sialic acid biosynthesis) engineering with cytofluorometric analysis of lectin binding indicated a role of limited substrate availability, especially for α2,6‐sialylation, which switches off reactivity for anoikis‐triggering homodimeric galectin‐1. Quantitative MS analysis of protein level changes confirmed an enhanced galectin‐1 presence along with an influence on glycosyltransferases (β1,4‐galactosyltransferase‐IV, α2,3‐sialyltransferase‐I) and detected p16INK4a‐dependent down‐regulation of two enzymes in the biosynthesis pathway for sialic acid [i.e. the bifunctional UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (GNE) and N‐acetylneuraminic acid 9‐phosphate synthase] (P < 0.001). By contrast, quantitative assessment for the presence of nuclear CMP‐N‐acetylneuraminic acid synthase (which is responsible for providing the donor for enzymatic sialylation that also acts as feedback inhibitor of the epimerase activity of GNE) revealed a trend for an increase. Partial restoration of sialylation in GNE‐transfected cells supports the implied role of sialic acid availability for the glycophenotype. Fittingly, the extent of anoikis was reduced in double‐transfected (p16INK4a/GNE) cells. Thus, a second means of modulating cell reactivity to the growth effector galectin‐1 is established in addition to the common route of altering α2,6‐sialyltransferase expression: regulating enzymes of the pathway for sialic acid biosynthesis.

Hypoxia-Inducible Factor 1α Determines Gastric Cancer Chemosensitivity via Modulation of p53 and NF-κB

Background Reduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1α has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1αs role in determining chemosensitivity focussing on responsible molecular pathways. Methodology and Principal Findings RNA interference was applied to inactivate HIF-1α or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-κB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1α in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1α-competent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF-1α markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1α-competent cells was identified as the molecular mechanism of HIF-1α-mediated inhibition of p53. Furthermore, loss of HIF-1α abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-κB and expression of anti-apoptotic NF-κB target genes. Accordingly, reconstitution of the NF-κB subunit p65 reversed the increased chemosensitivity of HIF-1α-deficient cells. Conclusion and Significance In summary, we identified HIF-1α as a potent regulator of p53 and NF-κB activity under conditions of genotoxic stress. We conclude that p53 mutations in human tumors hold the potential to confound the efficacy of HIF-1-inhibitors in cancer therapy.

Interferon-α: Regulatory Effects on Cell Cycle and Angiogenesis

In the current study, we investigated the effects of interferon-α (IFN-α) on proliferation and angiogenesis in neuroendocrine tumor disease. Using a panel of human neuroendocrine tumor cell lines, we confirmed functionally active IFN-α signaling by STAT activation and nuclear translocation as well as transactivation. IFN-α results in anchorage-dependent and -independent growth inhibition due to a delayed progression from S-phase to G2 phase of the cell cycle. This was due to substantial reduction in cellular cyclin B levels resulting in the inhibition of Cdc2 kinase activity. In parallel to growth inhibition, we observed a profound inhibition of VEGF gene transcription by IFN-α in human neuroendocrine tumor cells due to an Sp1/Sp3-dependent inhibition of VEGF promoter activity. Treatment of neuroendocrine tumors with IFN-α in nude mice resulted in growth inhibition and inhibition of angiogenesis. Furthermore, treatment of neuroendocrine tumor patients with IFN-α resulted in decreased VEGF expression as well as tumor angiogenesis in liver metastases. In summary, IFN-α acts via direct antiproliferative effects as well as inhibition of tumor angiogenesis mediated by suppression of VEGF gene expression in neuroendocrine tumor disease.

Angiopoietin-2 Promotes Disease Progression of Neuroendocrine Tumors

Purpose: Inhibition of angiogenesis represents a promising therapeutic strategy in neuroendocrine tumors. Angiopoietin-2 (Ang-2), a ligand of the endothelial tyrosine kinase Tie-2, is emerging as a key regulator of vascular remodeling during tumorangiogenesis. We therefore addressed the expression and biological significance of Ang-2 in human neuroendocrine tumors. Experimental Design: Surgical specimens and serum from neuroendocrine tumor patients were used to determine Ang-2 expression by in situ hybridization or ELISA (circulating Ang-2). Ang-2 biological effects were evaluated following stable transfection into BON human pancreatic neuroendocrine tumor cells. BON clones were grown as orthotopic xenografts in nude mice to determine tumor growth and abdominal metastatic spread. Further analyses included microvessel density, lymphatic vessel density, and nodal invasion. Results: Specimens from pancreatic neuroendocrine tumors and nontransformed pancreatic tissue revealed uniform expression of Ang-2 mRNA in endothelial cells. In contrast, epithelial expression of Ang-2 mRNA occurred exclusively in neuroendocrine tumors. Overexpression of Ang-2 in BON orthotopic xenografts did not affect primary tumor growth, although successful Ang-2 induction was confirmed from elevated serum levels. However, increased microvessel density and enhanced lymphatic metastasis were evident in Ang-2–expressing tumors, indicating a functional role of Ang-2 in experimental neuroendocrine tumors. Consistent with this notion, circulating Ang-2 was significantly elevated in neuroendocrine tumor patients compared with healthy controls. Circulating Ang-2 furthermore correlated with metastatic versus localized disease. The highest Ang-2 concentrations occurred in patients with liver metastasis, and concentrations ≥75th percentile predicted shorter survival (P = 0.0003). Conclusion: Induction of Ang-2 in neuroendocrine tumors represents a clinically relevant pathomechanism of disease progression and constitutes an adverse prognostic marker. Clin Cancer Res; 16(2); 420–9.

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.

Loss of UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase (GNE) induces apoptotic processes in pancreatic carcinoma cells

Early invasive growth and metastasis are features of pancreatic cancer that rely on its resistance to anoikis, an apoptosis program activated on loss of matrix anchorage. How anoikis is regulated is unclear. UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylman‐nosamine‐kinase (GNE) was silenced, or p16 was over‐expressed, in human pancreatic carcinoma cells. Gene expression profiling, enzymatic assays, Western blotting, and cell cycle analysis were conducted. Silencing of GNE, the key enzyme of sialic acid biosynthesis, sensitizes pancreatic cancer cells to anoikis. Accordingly, we observed a loss of GNE enzyme activity in cells, which became anoikis susceptible after transfection with the tumor suppressor p16. Similarly, studies of another cell line with low GNE activity revealed strong anoikis susceptibility, confirming the association of low GNE activity and anoikis susceptibility. Gene expression profiling demonstrated that the loss of GNE triggered the transcriptional activation of the ATF4‐ATF3‐CHOP pathway, leading to apoptosis in the framework of the unfolded protein response. In silico analysis showed that GNE up‐regulation occurred predominantly in pancreatic cancer but also in other malignancies. Delineation of GNE‐dependent signaling pathways may provide targets that control anchorage dependence and/or restore drug efficacy, which is of utmost relevance for the treatment of pancreatic cancer.—Kemmner, W., Kessel, P., Sanchez‐Ruderisch, H., Möller, H., Hinderlich, S., Schlag, P. M., Detjen, K. Loss of UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (GNE) induces apoptotic processes in pancreatic carcinoma cells. FASEB J. 26, 938–946 (2012). www.fasebj.org

Tumor suppressor p16INK4a controls oncogenic K-Ras function in human pancreatic cancer cells.

Pancreatic cancer is characterized by oncogenic activation of K‐Ras and inactivation of the cell cycle inhibitor p16INK4a. We previously demonstrated that reintroduction of p16INK4a reversed anoikis resistance and clonogenicity of human pancreatic cancer cells, properties commonly attributed to the transforming potential of oncogenic K‐Ras. Therefore, we aimed to determine the role of Ras after p16INK4a re‐expression. Here, we show that restitution of p16INK4a in pancreatic cancer cell lines elicits a profound suppression of K‐Ras activity. A more detailed analysis in p16INK4a reconstituted Capan‐1 cells indicated selective reduction of both K‐Ras activity and protein stability. Re‐expression of K‐Ras in p16INK4a restituted Capan‐1 cells reversed the anoikis‐sensitive phenotype and increased colony formation, indicating that K‐Ras suppression was required for p16INK4a‐mediated reversion of the transformed phenotype. Inducible expression of p16INK4a in DanG cells confirmed inhibition of K‐Ras activity as well as an increase in anoikis susceptibility. Thus, our results delineate a novel functional interaction with defined biological consequences for the two most frequent alterations observed in pancreatic cancer. (Cancer Sci 2012; 103: 169–175)

The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models

Background/Aims The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110α inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines. Methods Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA. Results BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately. Conclusion Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus.

Abstract 5106: Circulating MACC1 transcripts are elevated in pancreatic cancer patient blood and correlate to locally advanced disease.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnPancreatic cancer is a highly aggressive malignancy with a 5-year survival of < 5%. Late diagnosis, lack of prognostic markers and poor response to radiation and chemotherapies all contribute to the dismal prognosis. Surgery represents the sole curative treatment option. However, success is limited by invasive growth and metastatic spread, which frequently precede the onset of clinical symptoms and hence diagnosis.nnRecently, we identified the novel gene metastasis-associated in colon cancer-1 (MACC1). MACC1 is a prognostic indicator for metastasis formation linked to metastasis-free survival of colorectal cancer patients. Circulating MACC1 transcripts in colorectal cancer patient plasma predict metastasis and survival. Correlations of MACC1 expression levels with tumor progression, recurrence and metastasis have meanwhile been reported for several cancers, e.g. gastric, lung and hepatocellular cancer. Here, we addressed the role of MACC1 in pancreatic cancer.nnImmunohistochemical analysis of MACC1 in cryosections of human pancreatic cancer revealed strong reactivity of tumour duct epithelial cells, but not in the desmoplastic stroma. In contrast, weak immunoreactivity was present in non transformed pancreatic tissues, suggesting an induction of MACC1 in pancreatic malignancy. We furthermore analyzed blood levels of circulating MACC1 transcripts in pancreatic cancer patients. Circulating MACC1 transcript levels were elevated in pancreatic cancer patients, when compared to healthy volunteers. MACC1 transcript levels furthermore correlated to peripancreatic infiltration, but not to tumour size, tumor grading or angioinvasion.nnThus, MACC1 expression is induced in pancreatic cancer, which is reflected by increased circulating transcript levels in blood as well as high protein levels in the primary tumors. The correlation of MACC1 transcript levels with peripancreatic infiltration suggests a distinct function of MACC1 in pancreatic cancer cell biology, which translates into distinct clinical features of disease progression. Circulating MACC1 might therefore help to personalize treatment decisions in situations such as (neo-)adjuvant chemotherapy.nnCitation Format: Ulrike S. Stein, Pia Herrmann, Christian Fischer, Peter M. Schlag, Katharina Detjen. Circulating MACC1 transcripts are elevated in pancreatic cancer patient blood and correlate to locally advanced disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5106. doi:10.1158/1538-7445.AM2013-5106