Helmut Schaider

Low-Level Monocyte Chemoattractant Protein-1 Stimulation of Monocytes Leads to Tumor Formation in Nontumorigenic Melanoma Cells

Tumors commonly produce chemokines for recruitment of host cells, but the biological significance of tumor-infiltrating inflammatory cells, such as monocytes/macrophages, for disease outcome is not clear. Here, we show that all of 30 melanoma cell lines secreted monocyte chemoattractant protein-1 (MCP-1), whereas normal melanocytes did not. When low MCP-1-producing melanoma cells from a biologically early, nontumorigenic stage were transduced to overexpress the MCP-1 gene, tumor formation depended on the level of chemokine secretion and monocyte infiltration; low-level MCP-1 secretion with modest monocyte infiltration resulted in tumor formation, whereas high secretion was associated with massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction within a few days after injection into mice. Tumor growth stimulated by monocytes/macrophages was due to increased angiogenesis. Vessel formation in vitro was inhibited with mAbs against TNF-α, which, when secreted by cocultures of melanoma cells with human monocytes, induced endothelial cells under collagen gels to form branching, tubular structures. These studies demonstrate that the biological effects of tumor-derived MCP-1 are biphasic, depending on the level of secretion. This correlates with the degree of monocytic cell infiltration, which results in increased tumor vascularization and TNF-α production.

Downregulation of E-cadherin and Desmoglein 1 by autocrine hepatocyte growth factor during melanoma development

During melanoma development, transformed cells evade keratinocyte-mediated control by downregulating cell adhesion molecules. This study investigated the regulation of cell adhesion by hepatocyte growth factor (HGF) in melanoma. Melanocytes and two melanoma lines, WM164 and WM35, expressed normal level E-cadherin and Desmoglein 1, whereas most melanomas (18 out of 20) expressed no E-cadherin and significantly reduced Desmoglein 1. Overexpression of dominant negative E-cadherin and Desmoglein in melanocytes demonstrated that both molecules contribute to adhesion between melanocytes and keratinocytes. In contrast to melanocytes, most melanomas expressed HGF. All melanocytic cells expressed the HGF receptor c-Met, and autocrine HGF caused constitutive activation of c-Met, MAPK and PI3K. When autocrine activation was induced with HGF-expressing adenovirus, E-cadherin and Desmoglein 1 were decreased in melanocytes, WM164 and WM35. MAPK inhibitor PD98059 and PI3K inhibitor wortmannin partially blocked the downregulation, suggesting that both pathways are involved in this process. c-Met was coimmunoprecipitated with E-cadherin, Desmoglein 1 and Plakoglobin, suggesting that they form a complex (es) that acts to regulate intercellular adhesion. Together, the results indicate that autocrine HGF decouples melanomas from keratinocytes by downregulating E-cadherin and Desmoglein 1, therefore frees melanoma cells from the control by keratinocytes and allows dissemination of the tumor mass.

Nongenital cancers metastatic to the ovary

Abstract We review our experience with 82 patients with nongenital cancers metastatic to the ovary. All patients were referred for evaluation of an ovarian mass. The patients had primary carcinoma of the breast ( n = 28), colon ( n = 23), stomach ( n = 22), pancreas ( n = 7), or gallbladder ( n = 2). The overall actuarial 5-year survival rate was 10%. Five-year survival in patients with metastatic colon cancer was significantly higher (23%) than that in patients with metastatic cancer of the breast, stomach, gallbladder, or pancreas, all of whom died within 58 months ( P p = 0.003). Five-year survival in patients with disease limited to the pelvis was significantly higher than that in those with abdominal spread (22% vs 6%; P 2 cm in diameter was 18% or 4%, respectively ( P = 0.002). This pattern applied mainly to differences in patients with primary cancer of the breast or colon ( P

In search of a novel target - phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy.

This study was performed in the aim to identify potential targets for the development of novel therapy to treat cancer with poor outcome or treatment efficacy. We show that the negatively charged phospholipid phosphatidylserine (PS) is exposed in the outer leaflet of their plasma membrane not only in tumor cell lines, but also in metastases and primary cultures thereof, which contrasts with a lack of PS exposure by differentiated non-tumorigenic counterparts. Studied tumor cell lines were derived from non-tumorigenic and malignant melanomas, prostate- and renal cancer, glioblastoma and a rhabdomyosarcoma. Importantly, also metastases of melanoma expose PS and there is a correlation between malignancy of melanoma cell lines from different stages of tumor progression and PS exposure. The PS exposure we found was neither of apoptotic nor of experimental artificial origin. Finally potentially malignant and non-malignant cells could be differentiated by sorting of a primary cell culture derived from a glioblastoma based on PS exposure, which has so far not been possible within one culture due to lack of a specific marker. Our data provide clear evidence that PS could serve as uniform marker of tumor cells and metastases as well as a target for novel therapeutic approaches based on e.g. PS-specific host defense derived peptides.

Differential response of primary and metastatic melanomas to neutrophils attracted by IL‐8

IL‐8 is a strong chemoattractant for neutrophils, and it is constitutively produced by many tumors, including human melanomas. To determine the biologic importance of IL‐8 for melanoma cells from primary and metastatic lesions, we transduced selected cell lines constitutively producing low levels of IL‐8 with IL‐8 cDNA using a replication‐deficient adenoviral vector. Nontumorigenic SBcl2 primary melanoma cells formed tumors when transduced with increasing plaque‐forming units of IL‐8 per cell. However, at high IL‐8 transduction levels (100 ng/ml/105 cells in 48 hr), tumor growth was impaired due to massive neutrophil infiltration. A similar biphasic response was observed in WM115 primary melanomas, which are tumorigenic but not metastatic. Depletion of neutrophils with an antibody that blocks the accumulation of granulocytes at the site of inflammation enabled transduced primary melanomas secreting high levels of IL‐8 to survive and grow. In contrast, highly tumorigenic and metastatic 451Lu cells showed marked increases in tumor growth and number of metastatic foci in the lungs depending on the expression levels of IL‐8. Cytotoxicity assays with isolated neutrophils confirmed the preferential killing of primary over metastatic melanoma cells. SBcl2 cells stimulated by IL‐8 to form tumors in immunodeficient mice were induced to produce VEGF, suggesting that the angiogenic response is enhanced due to increased growth factor production. Our results demonstrate that nontumorigenic primary melanomas depend on IL‐8 stimulation in vivo for growth and that tumor growth depends on the level of neutrophil infiltration. Metastatic melanomas proliferate in vivo independently of infiltrating neutrophils.

Interleukin-8 overexpression is present in pyoderma gangrenosum ulcers and leads to ulcer formation in human skin xenografts.

Interleukin-8 (IL-8) is a potent chemotactic polypeptide for neutrophils. However, the role of this cytokine during inflammation remains unclear. Skin specimens from patients with pyoderma gangrenosum demonstrated IL-8 overexpression in skin ulcers, which suggests a role for IL-8 in the development of the disease. We therefore constructed a recombinant adenovirus expressing the complementary deoxyribonucleic acid encoding human IL-8 (IL-8/Ad5) that induces a 2000-fold increase in IL-8 expression of infected human fibroblasts in vitro. Human skin engrafted to severe combined immunodeficiency mice and then injected with the recombinant virus demonstrated erythema, an intense perivascular infiltration of neutrophils, and extravasation of erythrocytes after 8 hours. By 12 hours after injection, neutrophils had accumulated beneath the epidermis, which then necrotized, and one or more ulcers that remained for approximately 2 weeks were observed. Clinically and histologically, the ulcers resembled pyoderma gangrenosum. These clinical and experimental findings suggest an etiologic role of IL-8 in the pathogenesis of pyoderma gangrenosum.

Tumoural CXCL16 expression is a novel prognostic marker of longer survival times in renal cell cancer patients

The aim of our study was to analyse the expression of CXCL16, ADAM10 and CXCR6 in renal cell carcinoma (RCC) tissue and to correlate the expression pattern with clinicopathologic data, including patient survival. Furthermore, we investigated CXCL16, ADAM10 and CXCR6 expressions by FACS, immunofluorescence and ELISA analysis in renal carcinoma cell lines. Our immunohistochemical analysis on tissue microarray of renal cancer samples of 104 patients revealed that ADAM10 correlated significantly with tumour stage, pathological nodal status, M status and lymphangiosis carcinomatosa. CXCL16, CXCR6 and ADAM10 were significantly increased in papillary carcinomas. Importantly, high levels of CXCL16 expression in renal cancer tissue correlated with better survival of patients, and CXCL16 correlated inversely to the tumour stage. In addition, inhibition of CXCL16 induced the migration of renal cancer cells assuming an anti-migratory function of transmembrane CXCL16. Taken together, our data demonstrate that downregulation of CXCL16 plays an important role in renal cancer development and progression, and that CXCL16 in RCC is an independent prognostic marker for better patient survival.

Transcriptional Activation of ZEB1 by Slug Leads to Cooperative Regulation of the Epithelial–Mesenchymal Transition-Like Phenotype in Melanoma

The E-box-binding zinc finger transcription factors Slug and ZEB1 are important repressors of E-cadherin, contributing to epithelial-mesenchymal transition (EMT) in primary epithelial cancers. Activator or repressor status of EMT transcription factors defines consequences for tumorigenesis. We show that changes in expression levels of Slug in melanoma cell lines lead to concomitant alterations of ZEB1 expression. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays identified Slug as a direct transcriptional activator at E-boxes of the ZEB1 promoter. Transcriptional activation of ZEB1 was demonstrated to be specific for Slug, as EMT regulators Snail and Twist failed to influence ZEB1 expression. Slug and ZEB1 cooperatively repressed E-cadherin expression resulting in decreased adhesion to human keratinocytes, but promoted migration of melanoma cells. Our results show that the transcriptional activity of ZEB1 is increased by Slug, suggesting a hierarchical organized expression of EMT transcription factors through directed activation, triggering an EMT-like process in melanoma.

A stress-induced early innate response causes multidrug tolerance in melanoma

Acquired drug resistance constitutes a major challenge for effective cancer therapies with melanoma being no exception. The dynamics leading to permanent resistance are poorly understood but are important to design better treatments. Here we show that drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multidrug resistance, termed induced drug-tolerant cells (IDTCs). Transition into the IDTC state seems to be an inherent stress reaction for survival toward unfavorable environmental conditions or drug exposure. The response comprises chromatin remodeling, activation of signaling cascades and markers implicated in cancer stemness with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced aldehyde dehydrogenase (ALDH) activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug-resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance.

Distinct signatures of B-cell homeostatic and activation-dependent chemokine receptors in the development and progression of extragastric MALT lymphomas†‡

Chemokine receptors mediate migration and activation of lymphocytes through binding of their ligands. Recent studies have revealed important contributions of chemokine receptors to the development, progression, and dissemination of haematopoietic neoplasms. Because the chemokine receptor expression profile in extragastric MALT lymphoma is unknown, we performed a comprehensive study on tissue samples of parotid glands, parotid glands affected by Sjögren syndrome, extragastric MALT lymphoma, and extranodal diffuse large B‐cell lymphoma (eDLBCL) originating from MALT lymphoma (transformed MALT lymphoma). By investigating the expression of 19 chemokine receptors by real‐time PCR using a semi‐quantitative approach and of four chemokine receptors (CCR1, CCR5, CXCR6, and XCR1) by immunohistochemistry, we show that the chemokine receptor expression profiles of extragastric MALT lymphomas differ substantially from those of extranodal DBLCL, with lower expression of CCR1, CCR8, and CXCR3, and the absence of expression of CX3CR1 and XCR1 in eDLBCL. Expression of CCR6, CCR7, CXCR3, CXCR4, and CXCR5, responsible for B‐cell homing to secondary lymphoid tissue, was detected in both B‐cell malignancies. Expression of CCR4 was just detected in trisomy 3‐positive MALT lymphoma cases. Comparing gastric with extragastric MALT lymphomas, up‐regulation of CXCR1 and CXCR2 accompanied by down‐regulation of CCR8 and CX3CR1 and loss of XCR1 expression in extragastric MALT lymphomas appear to be key determinants for the site of origin of MALT lymphomagenesis. Our results support a model of stepwise progression of extragastric MALT lymphoma from a non‐neoplastic event to Sjögren syndrome, to MALT lymphoma, and finally to overt eDLBCL, guided by differentially expressed B‐cell homeostatic and activation‐dependent chemokine receptors and their ligands. Copyright