Metastatic Esophageal Carcinoma Cells Exhibit Reduced Adhesion Strength and Enhanced Thermogenesis.

Despite continuous improvements in multimodal therapeutic strategies, esophageal carcinoma maintains a high mortality rate. Metastases are a major life-limiting component; however, very little is known about why some tumors have high metastatic potential and others not. In this study, we investigated thermogenic activity and adhesion strength of primary tumor cells and corresponding metastatic cell lines derived from two patients with metastatic adenocarcinoma of the esophagus. We hypothesized that the increased metastatic potential of the metastatic cell lines correlates with higher thermogenic activity and decreased adhesion strength. Our data show that patient-derived metastatic esophageal tumor cells have a higher thermogenic profile as well as a decreased adhesion strength compared to their corresponding primary tumor cells. Using two paired esophageal carcinoma cell lines of primary tumor and lymph nodes makes the data unique. Both higher specific thermogenesis profile and decreased adhesion strength are associated with a higher metastatic potential. They are in congruence with the clinical patient presentation. Understanding these functional, biophysical properties of patient derived esophageal carcinoma cell lines will enable us to gain further insight into the mechanisms of metastatic potential of primary tumors and metastases. Microcalorimetric evaluation will furthermore allow for rapid assessment of new treatment options for primary tumor and metastases aimed at decreasing the metastatic potential.

CAIX furthers tumour progression in the hypoxic tumour microenvironment of esophageal carcinoma and is a possible therapeutic target.

The hypoxic tumour microenvironment of solid tumours represents an important starting point for modulating progression and metastatic spread. Carbonic anhydrase IX (CAIX) is a known HIF-1α-dependent key player in maintaining cell pH conditions under hypoxia. We show that CAIX is strongly expressed in esophageal carcinoma tissues. We hypothesize that a moderate CAIX expression facilitates metastases and thereby worsens prognosis. Selective inhibition of CAIX by specific CAIX inhibitors and a CAIX knockdown effectively inhibit proliferation and migration in vitro. In the orthotopic esophageal carcinoma model, the humanized HER2 antibody trastuzumab down-regulates CAIX, possibly through CAIX's linkage with HER2 in the hypoxic microenvironment. Our results show CAIX to be an essential part of the tumour microenvironment and a possible master regulator of tumour progression. This makes CAIX a highly effective and feasible therapeutic target for selective cancer treatment.

Influence of hypoxia-dependent factors on the progression of neuroblastoma.

PURPOSE: Several oxygen-dependent factors, e.g., CAIX (carbonic anhydrase IX) or phosphoglycerate kinase 1 (PGK1) interacting with the CXCR4/SDF1 axis (chemokine receptor 4/stromal cell derived factor 1) have been shown to be involved in processes of tumour pathology including tumourigenicity, tumour cell dissemination and poor survival in several solid tumour entities. The aim of the current study was to evaluate the influence of the hypoxia-inducible factors CAIX and PGK1 on progression of neuroblastoma and to evaluate the clinical relevance of possible therapeutic approaches. METHODS: Expression of hypoxia-dependent factors PGK1 and CAIX was examined in neuroblastoma specimen, was correlated with clinical parameters, and was studied in neuroblastoma cells. The impact of these hypoxic factors was evaluated by proliferation assays under targeted therapy. RESULTS: Expression of hypoxia-dependent factors was found in 50 % of neuroblastoma specimen. In neuroblastoma cells, CAIX and PGK1 expression is up regulated under hypoxia and correlates with response to targeted anti-proliferative treatment. The negative impact on survival, although significant for both CAIX and PGk1, appears to be stronger for CAIX. CONCLUSIONS: Our results show that the hypoxic factors in the tumour`s microenvironment further the progression of tumour disease. This strengthens the perspectives for additive novel therapeutic approaches targeting hypoxia-dependent factors in this childhood disease.

Carbonic anhydrase IX correlates with survival and is a potential therapeutic target for neuroblastoma.

Carbonic anhydrase IX (CAIX) is involved in pathological processes including tumorgenicity, metastases and poor survival in solid tumors. Twenty-two neuroblastoma samples of patients who were surgically treated at the University Medical Center Hamburg-Eppendorf were evaluated immunohistochemically for expression of CAIX. Results were correlated with clinical parameters and outcome. Neuroblastoma Kelly and SH-EP-Tet-21/N cells were examined for CAIX expression and inhibited with specific inhibitors, FC5-207A and FC8-325A. 32% of neuroblastoma tumors expressed CAIX. This was significantly associated with poorer survival. Kelly and SH-EP-Tet-21/N cells showed a major increase of CAIX RNA under hypoxic conditions. Proliferation of Kelly cells was significantly decreased by CAIX inhibitors, FC5-207A and FC8-325A, while proliferation of SH-EP-Tet-21/N cells was only significantly affected by FC8-325A. CAIX is a potent biomarker that predicts survival in neuroblastoma patients. CAIX-targeted therapy in neuroblastoma cell lines is highly effective and strengthens the potential of CAIX as a clinical therapeutic target in a selected patient collective.

COSMC knockdown mediated aberrant O-glycosylation promotes oncogenic properties in pancreatic cancer.

BACKGROUND: Human pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies in the world and despite great efforts in research types of treatment remain limited. A frequently detected alteration in PDACs is a truncated O-linked N-acetylgalactosamine (GalNAc) glycosylation with expression of the Tn antigen. Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations. Tn antigen is a tumor associated glycan detected in 75-90 % of PDACs and up to 67 % in its precursor lesions. Since the role of Tn antigen expression in PDAC is insufficiently understood we analyzed the impact of COSMC mediated Tn antigen expression in two human PDAC cell lines on cellular oncogenic properties. METHODS: Forced expression of Tn antigen on O-glycosylated proteins in pancreatic cancer cells was induced by lentiviral-mediated knockdown of the COSMC chaperone, which prevented O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins. Altered O-GalNAc glycosylation was analyzed in human pancreatic cancer cell lines Panc-1 and L3.6pl using Western and Far-Western blot as well as immunocytochemical techniques. To assess the biological implications of COSMC function on oncogenic properties, cell viability assays, scratch assays combined with live cell imaging, migration and apoptosis assays were performed. Lectin based glycoprotein enrichment with subsequent mass spectrometric analysis identified new cancer O-GalNAc modified proteins. Expression of Tn antigen bearing Nucleolin in patient derived PDAC tumor specimens was evaluated and correlated with clinicopathological data. RESULTS: Tn antigen expression was induced on various O-GalNAc glycoproteins in COSMC deficient cell lines compared to the control. Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells. Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines. Interestingly, immunohistochemical staining and co-localization studies of patient derived PDACs revealed poor survival for patients with strong co-localization of Tn antigen and Nucleolin (p = 0.037). CONCLUSION: This study substantiates the influence of altered O-glycan (Tn/STn) expression on oncogenic properties in pancreatic cancer and identifies O-GalNAc modified Nucleolin as novel prognostic marker.

ErbB2 signaling activates the Hedgehog pathway via PI3K-Akt in human esophageal adenocarcinoma: identification of novel targets for concerted therapy concepts.

The Hedgehog pathway plays an important role in the pathogenesis of several tumor types, including esophageal cancer. In our study, we show an expression of the ligand Indian hedgehog (Ihh) and its downstream mediator Gli-1 in primary resected adenocarcinoma tissue by immunohistochemistry and quantitative PCR in fifty percent of the cases, while matching healthy esophagus mucosa was negative for both proteins. Moreover, a functionally important regulation of Gli-1 by ErbB2-PI3K-mTORC signaling as well as a Gli-1-dependent regulation of Ihh in the ErbB2 amplified esophageal adenocarcinoma cell line OE19 was observed. Treatment of OE19 cells with the Her2 antibody trastuzumab, the PI3K-mTORC1 inhibitor NVP BEZ235 (BEZ235) or the knockdown of Akt1 resulted in a downregulation of Gli-1 and Ihh as well as in a reduction of viable OE19 cells in vitro. Interestingly, the Hedgehog receptor Smo, which acts upstream of Gli-1, was not expressed in OE19 cells and in the majority of primary human esophageal adenocarcinoma, suggesting a non-canonical upregulation of Gli-1 expression by the ErbB2-PI3K axis. To translate our findings into a therapeutic concept, we targeted ErbB2-PI3K-mTORC1 by trastuzumab and BEZ235, combining both compounds with the Gli-1/2 inhibitor GANT61. The triple combination led to significantly stronger reduction of tumor cell viability than cisplatinum or each biological alone. Therefore, concomitant blockage of the ErbB2-PI3K pathway and the Hedgehog downstream mediator Gli-1 may provide a new therapeutic strategy for esophageal cancer.

Esophageal carcinoma cell line with high EGFR polysomy is responsive to gefitinib.

PURPOSE: It has previously been shown that gefitinib-treated patients with epidermal growth factor receptor (EGFR) gene amplification or high polysomy had a statistically significant improvement in response, time to progression, and survival in non-small cell lung cancer (NSCLC). Only few studies utilizing anti-EGFR treatment in advanced esophageal adenocarcinomas have been performed and the results have been heterogeneous. The aim of this study was to evaluate EGFR-targeted therapy with gefitinib in esophageal adenocarcinoma with a high EGFR polysomy. METHODS: Novel esophageal cell lines PT6216 and LN6216c were established from primary tumor and lymph node metastasis of a patient with highly aggressive and metastatic adenocarcinoma. Pathological examination including tumor differentiation and prognostic marker analysis, immunohistochemical EGFR expression analysis, EGFR fluorescence in situ hybridization, and mutation analysis were performed. Response of novel cell lines to gefitinib treatment was evaluated by cell proliferation and vitality assays. Fifty-four esophageal adenocarcinoma specimens were evaluated for EGFR gene copy gain. RESULTS: The primary tumor cell line PT6216 and the lymph node cell line LN6216c show a homogenously high polysomy for EGFR determined by FISH analysis. Cell proliferation and vitality are highly sensitive to the tyrosine kinase inhibitor gefitinib compared to esophageal control cells without a high polysomy for EGFR. High polysomy for EGFR was found in 35 % of patients. CONCLUSION: We show for the first time a significant treatment response to the EGFR tyrosine kinase inhibitor gefitinib in esophageal tumor cells with a high polysomy for EGFR, suggesting a future role of anti-EGFR therapy for esophageal adenocarcinoma patients with a high EGFR polysomy.

PGK1 as predictor of CXCR4 expression, bone marrow metastases and survival in neuroblastoma.

BACKGROUND AND AIM: A close relationship between phosphoglycerate kinase 1 (PGK1) and the CXCR4/SDF1 axis (chemokine receptor 4/stromal cell derived factor 1) has been shown for several cancers. However, the role of PGK1 has not been investigated for neuroblastoma, and PGK1 might be a therapeutic target for this tumor entity. The aim of the current study was to evaluate the role of PGK1 expression in neuroblastoma patients, to determine the impact of PGK1 expression levels on survival, and to correlate PGK1 expression with CXCR4 expression and bone marrow dissemination. MATERIALS AND METHODS: Samples from 22 patients with neuroblastoma that were surgically treated at the University Medical Center Hamburg-Eppendorf were evaluated for expression of PGK1 and CXCR4 using immunohistochemistry. Results were correlated with clinical parameters, metastases and outcome of patients. Immunocytochemistry, proliferation and expression analysis of CXCR4 and PGK1 were performed in neuroblastoma cell lines. RESULTS: PGK1 is expressed in neuroblastoma cells. PGK1 expression is significantly positively correlated with CXCR4 expression and tumor dissemination to the bone marrow. Moreover the expression of PGK1 is significantly associated with a negative impact on survival in patients with neuroblastoma. PGK1 is downregulated by inhibition of CXCR4 in neuroblastoma cells. CONCLUSION: PGK1 appears to play an important role for neuroblastoma, predicting survival and tumor dissemination. Further in vivo studies outstanding, it is a candidate target for novel therapeutic strategies.

Effective inhibition of metastases and primary tumor growth with CTCE-9908 in esophageal cancer.

BACKGROUND: In spite of multimodular treatment, the therapeutic options for esophageal carcinoma are limited, and metastases remain the leading cause of tumor-related mortality. Expression of the chemokine receptor CXCR4 significantly correlates with poor survival rates in patients with esophageal carcinoma and is associated with lymph node and bone marrow metastases. The aim of this study was to evaluate the effect of the CXCR4 antagonist CTCE-9908 on metastatic homing and primary tumor growth in vitro and in vivo in an orthotopic xenograft model of esophageal cancer. MATERIALS AND METHODS: OE19 cells were examined for stromal cell-derived factor 1 alpha-mediated migration under CTCE-9908 treatment. The CTCE-9908 treatment was further evaluated in an in vitro proliferation assay and orthotopic esophageal model, accompanied by magnetic resonance imaging. Tumor and metastases were immunohistochemically examined for CXCR4 expression. RESULTS: CTCE-9908 has an inhibitory effect on stromal cell-derived factor 1 alpha-mediated migration and proliferation of OE19 cells. Treatment with CTCE-9908 in the orthotopic esophageal model leads to a reduction of metastatic spread and primary tumor growth. This was confirmed by magnetic resonsance imaging. Treatment with CTCE-9908 results in altered CXCR4 expression pattern exhibiting a high degree of variability. CONCLUSION: CTCE-9908 effectively inhibits OE19 cell migration and proliferation in vitro, reduces metastases to lung, liver, and lymph nodes in vivo, and moreover leads to tumor growth reduction in an orthotopic model of esophageal carcinoma.

Involvement of CXCR4 chemokine receptor in metastastic HER2-positive esophageal cancer.

A functional linkage of the structurally unrelated receptors HER2 and CXCR4 has been suggested for breast cancer but has not been evaluated for esophageal carcinoma. The inhibition of HER2 leads to a reduction of primary tumor growth and metastases in an orthotopic model of esophageal carcinoma. The chemokine receptor CXCR4 has been implicated in metastatic dissemination of various tumors and correlates with poor survival in esophageal carcinoma. The aim of this study was to investigate a correlation between the expression levels of HER2 and CXCR4 and to evaluate the involvement of CXCR4-expression in HER2-positive esophageal carcinoma. The effects of HER2-inhibition with trastuzumab and of CXCR4-inhibition with AMD3100 on primary tumor growth, metastatic homing, and receptor expression were evaluated in vitro and in an orthotopic model of metastatic esophageal carcinoma using MRI for imaging. The clinical relevance of HER2- and CXCR4-expression was examined in esophageal carcinoma patients. A significant correlation of HER2- and CXCR4-expression in primary tumor and metastases exists in the orthotopic model. Trastuzumab and AMD3100 treatment led to a significant reduction of primary tumor growth, metastases and micrometastases. HER2-expression was significantly elevated under AMD3100 treatment in the primary tumor and particularly in the metastases. The positive correlation between HER2- and CXCR4-expression was validated in esophageal cancer patients. The correlation of CXCR4- and HER2-expression and the elevation of HER2-expression and reduction of metastases through CXCR4-inhibition suggest a possible functional linkage and a role in tumor dissemination in HER2-positive esophageal carcinoma.

CXCR4/SDF-1α-mediated chemotaxis in an in vivo model of metastatic esophageal carcinoma.

BACKGROUND/AIM: The chemokine receptor CXCR4 and its ligand (stromal cell-derived factor-1alpha; SDF-1α) play an important role in tumor cell chemotaxis and metastatic homing of esophageal carcinoma. Several methods are available to examine tumor cell migration in vitro. However, in vivo chemotaxis is subject to complex tumor-host interactions. The aim of this study was to establish an in vivo model of chemotaxis for esophageal carcinoma that allows the examination of tumor cell migration and metastatic homing in the complex microenvironment. MATERIALS AND METHODS: CXCR4 expression of OE19 adenocarcinoma cells was determined by immunostaining in an orthotopic esophageal model. SDF-1α-mediated migration of cells was examined in vitro. An in vivo model of chemotaxis and metastasis was established by subcutaneous injection of OE19 cells into NMRI/nu mice and by daily stimulation with SDF-1α. RESULTS: CXCR4 is expressed in the primary tumor and in the metastatic tissue. CXCR4-positive OE19 cells are susceptible to SDF-1α-mediated migration. The novel in vivo model leads to developement of metastases in liver, lung, peritoneum and retroperitoneum after stimulation with SDF-1α but not with PBS, and revealed an SDF-1α dose-dependent migratory effect. CONCLUSION: As metastasis is still the leading cause of tumor-related death, it is essential to investigate the complex tumor-host interactions involved in metastatic homing. We established an in vivo model of chemotaxis and metastasis for esophageal carcinoma, which allows investigation and inhibition of CXCR4/SDF-1α-mediated cell survival and proliferation, chemotaxis and homing, adhesion, and tumor angiogenesis.

WT1-dependent sulfatase expression maintains the normal glomerular filtration barrier.

Paracrine signaling between podocytes and glomerular endothelial cells through vascular endothelial growth factor A (VEGFA) maintains a functional glomerular filtration barrier. Heparan sulfate proteoglycans (HSPGs), located on the cell surface or in the extracellular matrix, bind signaling molecules such as VEGFA and affect their local concentrations, but whether modulation of these moieties promotes normal crosstalk between podocytes and endothelial cells is unknown. Here, we found that the transcription factor Wilms' Tumor 1 (WT1) modulates VEGFA and FGF2 signaling by increasing the expression of the 6-O-endosulfatases Sulf1 and Sulf2, which remodel the heparan sulfate 6-O-sulfation pattern in the extracellular matrix. Mice deficient in both Sulf1 and Sulf2 developed age-dependent proteinuria as a result of ultrastructural abnormalities in podocytes and endothelial cells, a phenotype similar to that observed in children with WT1 mutations and in Wt1(+/-) mice. These kidney defects associated with a decreased distribution of VEGFA in the glomerular basement membrane and on endothelial cells. Collectively, these data suggest that WT1-dependent sulfatase expression plays a critical role in maintaining the glomerular filtration barrier by modulating the bioavailability of growth factors, thereby promoting normal crosstalk between podocytes and endothelial cells.