Tumor cell integrin ß4 and tumor stroma E-/P-selectin cooperatively regulate tumor growth in vivo.

BACKGROUND: The immunological composition of the tumor microenvironment has a decisive influence on the biological course of cancer and is therefore of profound clinical relevance. In this study, we analyzed the cooperative effects of integrin ß4 (ITGB4) on tumor cells and E-/P-selectin on endothelial cells within the tumor stroma for regulating tumor growth by shaping the local and systemic immune environment. METHODS: We used several preclinical mouse models for different solid human cancer types (xenograft and syngeneic) to explore the role of ITGB4 (shRNA-mediated knockdown in tumor cells) and E-/P-selectins (knockout in mice) for tumor growth; effects on apoptosis, proliferation and intratumoral signaling pathways were determined by histological and biochemical methods and 3D in vitro experiments; changes in the intratumoral and systemic immune cell composition were determined by flow cytometry and immunohistochemistry; chemokine levels and their attracting potential were measured by ELISA and 3D invasion assays. RESULTS: We observed a very robust synergism between ITGB4 and E-/P-selectin for the regulation of tumor growth, accompanied by an increased recruitment of CD11b+ Gr-1Hi cells with low granularity (i.e., myeloid-derived suppressor cells, MDSCs) specifically into ITGB4-depleted tumors. ITGB4-depleted tumors undergo apoptosis and actively attract MDSCs, well-known to promote tumor growth in several cancers, via increased secretion of different chemokines. MDSC trafficking into tumors crucially depends on E-/P-selectin expression. Analyses of clinical samples confirmed an inverse relationship between ITGB4 expression in tumors and number of tumor-infiltrating leukocytes. CONCLUSIONS: These findings suggest a distinct vulnerability of ITGB4Lo tumors for MDSC-directed immunotherapies.

The role of the desmosomal protein desmocollin 2 in tumour progression in triple negative breast cancer patients.

BACKGROUND: The disruption of epithelial features represents a critical step during breast cancer spread. In this context, the dysregulation of desmosomal proteins has been associated with malignant progression and metastasis formation. Curiously, both tumour suppressive and pro-metastatic roles have been attributed to desmosomal structures in different cancer entities. In the present study, we describe the pro-metastatic role of the desmosomal protein desmocollin 2 (DSC2) in breast cancer. METHODS: We analysed the prognostic role of DSC2 at mRNA and protein level using microarray data, western blot analysis and immunohistochemistry. Functional consequences of DSC2 overexpression and DSC2 knock down were investigated in the triple negative breast cancer (TNBC) cell line MDA-MB-231 and its brain-seeking subline MDA-MB-231-BR, respectively in vitro and in vivo. RESULTS: We found a significantly higher DSC2 expression in the more aggressive molecular subtypes HER2-positive and TNBC than in luminal breast cancers, as well as a significant correlation between increased DSC2 expression and a shorter disease-free-also in multivariate analysis-and overall survival. Additionally, a significant association between DSC2 expression in the primary tumour and an increased frequency of cerebral and lung metastasis could be observed. In vitro, ectopic DSC2 expression or DSC2 down-regulation in MDA-MB-231 and MDA-MB-231-BR led to a significant tumour cell aggregation increase and decrease, respectively. Furthermore, tumour cells displaying higher DSC2 levels showed increased chemoresistance in 3D structures, but not 2D monolayer structures, suggesting the importance of cell aggregation as a means for reduced drug diffusion. In an in vivo brain dissemination xenograft mouse model, reduced expression of DSC2 in the brain-seeking TNBC cells led to a decreased amount of circulating tumour cells/clusters and, in turn, to fewer and smaller brain metastatic lesions. CONCLUSION: We conclude that high DSC2 expression in primary TNBC is associated with a poorer prognosis, firstly by increasing tumour cell aggregation, secondly by reducing the diffusion and effectiveness of chemotherapeutic agents, and, lastly, by promoting the circulation and survival of tumour cell clusters, each of which facilitates distant organ colonisation.

VEGF-C serum level is associated with response to bevacizumab maintenance therapy in primary ovarian cancer patients.

OBJECTIVE: At present, maintenance therapy with the antiangiogenic agent bevacizumab or with PARP-inhibitors represent two options for BRCA-wildtype ovarian cancer patients, after platinum-based first line chemotherapy. The identification of molecular markers to predict patient response to different maintenance therapies remains a major challenge. In the present study we analyzed the predictive potential of vascular endothelial growth factor C (VEGF-C) to identify ovarian cancer patients that might benefit from an antiangiogenic therapy. METHODS: 101 patients with primary epithelial ovarian cancer were analyzed for serum levels of VEGF-A,-C and CA-125 by ELISA. Serum levels were compared between patients with low pT-stage (pT1a-pT2c n = 11), healthy individuals (n = 27) and patients with higher pT-stage (> = pT3 n = 90). Adjusted ROC curves and an adjusted logistic regression model were carried out to evaluate the potential impact of VEGF-A and -C, as well as CA-125 serum level concentration on bevacizumab-therapy response, under consideration of covariates such as FIGO, pM, pN and residual tumor after surgery. RESULTS: A patient which has in comparison twice the VEGF-C concentration in serum, has a significant increased chance of response to bevacizumab by a factor of 2.79. Further, only VEGF-C serum levels were significantly higher in the group of patients with lower pT-stage compared to healthy individuals, whereas VEGF-A or CA-125 serum levels could not discriminate between healthy individuals and patients with ovarian cancer at low pT-stages. CONCLUSION: VEGF-C serum level might serve as as a biomarker to evaluate treatment response under bevacizumab.

Fra-2 overexpression upregulates pro-metastatic cell-adhesion molecules, promotes pulmonary metastasis, and reduces survival in a spontaneous xenograft model of human breast cancer.

PURPOSE: The transcription factor Fra-2 affects the invasive potential of breast cancer cells by dysregulating adhesion molecules in vitro. Previous results suggested that it upregulates the expression of E- and P-selectin ligands. Such selectin ligands are important members of the leukocyte adhesion cascade, which govern the adhesion and transmigration of cancer cells into the stroma of the host organ of metastasis. As so far, no in vivo data are available, this study was designed to elucidate the role of Fra-2 expression in a spontaneous breast cancer metastasis xenograft model. METHODS: The effect of Fra-2 overexpression in two stable Fra-2 overexpressing clones of the human breast cancer cell line MDA MB231 on survival and metastatic load was studied after subcutaneous injection into scid and E- and P-selectin-deficient scid mice. RESULTS: Fra-2 overexpression leads to a significantly shorter overall survival and a higher amount of spontaneous lung metastases not only in scid mice, but also in E- and P-deficient mice, indicating that it regulates not only selectin ligands, but also selectin-independent adhesion processes. CONCLUSION: Thus, Fra-2 expression influences the metastatic potential of breast cancer cells by changing the expression of adhesion molecules, resulting in increased adherence to endothelial cells in a breast cancer xenograft model.

TGFB-induced factor homeobox 1 (TGIF) expression in breast cancer.

BACKGROUND: Breast cancer (BC) is the most frequent female cancer and preferentially metastasizes to bone. The transcription factor TGFB-induced factor homeobox 1 (TGIF) is involved in bone metabolism. However, it is not yet known whether TGIF is associated with BC bone metastasis or patient outcome and thus of potential interest. METHODS: TGIF expression was analyzed by immunohistochemistry in 1197 formalin-fixed, paraffin-embedded tissue samples from BC patients treated in the GAIN (German Adjuvant Intergroup Node-Positive) study with two adjuvant dose-dense schedules of chemotherapy with or without bisphosphonate ibandronate. TGIF expression was categorized into negative/low and moderate/strong staining. Endpoints were disease-free survival (DFS), overall survival (OS) and time to primary bone metastasis as first site of relapse (TTPBM). RESULTS: We found associations of higher TGIF protein expression with smaller tumor size (p = 0.015), well differentiated phenotype (p < 0.001) and estrogen receptor (ER)-positive BC (p < 0.001). Patients with higher TGIF expression levels showed a significantly longer disease-free (DFS: HR 0.75 [95%CI 0.59-0.95], log-rank p = 0.019) and overall survival (OS: HR 0.69 [95%CI 0.50-0.94], log-rank p = 0.019), but no association with TTPBM (HR 0.77 [95%CI 0.51-1.16]; p = 0.213). Univariate analysis in molecular subgroups emphasized that elevated TGIF expression was prognostic for both DFS and OS in ER-positive BC patients (DFS: HR 0.68 [95%CI 0.51-0.91]; log-rank p = 0.009, interaction p = 0.130; OS: HR 0.60 [95%CI 0.41-0.88], log-rank p = 0.008, interaction p = 0.107) and in the HER2-negative subgroup (DFS:HR 0.67 [95%CI 0.50-0.88], log-rank p = 0.004, interaction p = 0.034; OS: HR 0.57 [95%CI 0.40-0.81], log-rank p = 0.002, interaction p = 0.015). CONCLUSIONS: Our results suggest that moderate to high TGIF expression is a common feature of breast cancer cells and that this is not associated with bone metastases as first site of relapse. However, a reduced expression is linked to tumor progression, especially in HER2-negative breast cancer. TRIAL REGISTRATION: This clinical trial has been registered with ClinicalTrials.gov ; registration number: NCT00196872 .

Mechanisms of Tumor-Lymphatic Interactions in Invasive Breast and Prostate Carcinoma.

During the last few years, diverse studies have shown that tumors can actively interact with the lymphatic system and promote metastases development. In order to examine the molecular mechanisms involved in this interaction, we co-cultured tumor and lymphatic endothelial cells (LEC) and subsequently analyzed the molecular alterations of LECs. Therefore, LECs were co-cultivated with either a highly or weakly metastatic breast cancer cell line using contact (mixture) and non-contact (transwell) co-cultures. mRNA profiles from LECs were subsequently analyzed for genes specifically induced by highly metastatic tumor cells ("metastatic specific"). Among the up-regulated "metastatic specific" genes, we found candidates involved in cell cycle, cell adhesion and motility (BST2, E-selectin, and HMMR), cytokines (CCL7, CXCL6, CXCL1, and CSF2) and factors of the complement system (C1R, C3, and CFB). Among the down-regulated genes, we detected the hyaluronan receptor STAB2, angiogenic factor apelin receptor (APLNR), and the glycosylation enzyme MAN1A1. In an additional prostate cancer co-culture model, we could confirm a "metastatic specific" upregulation of E-selectin and CCL7 in LECs after interaction with the prostate cancer cell lines LNCAP (highly metastatic) and DU145 (weakly metastatic). These data allowed us to identify a set of genes regulated in LECs during in vitro communication with cancer cells, which might subsequently facilitate lymphatic metastasis.

Clinical relevance of H-RAS, K-RAS, and N-RAS mRNA expression in primary breast cancer patients.

PURPOSE: The RAS family comprises three proto-oncogenes (H-RAS, K-RAS, and N-RAS) and is among the most widely studied of oncogenes. The present study aimed at investigating the clinical relevance of mRNA levels of the three isoforms in a large group of breast cancer patients with a long-term follow-up. METHODS: 198 previously untreated patients were enrolled in the study. mRNA levels of K-RAS, H-RAS, and N-RAS were measured using microarray (Affymetrix HG-U133A). RESULTS: Elevated H-RAS levels were found significantly more frequently in patients with larger (p = 0.021) and ER-positive tumors (p = 0.048), while elevated K-RAS levels were associated with nodal positivity (p = 0.001) and HER2-positivity (p = 0.010). Patients with high N-RAS mRNA levels were more likely to be diagnosed with triple-negativity (p < 0.001) and higher grading (p = 0.001). Patients with high K-RAS levels were more likely to show an elevated H-RAS (p = 0.003). After a median follow-up of 183 months, patients with high N-RAS expression had significantly reduced overall survival (OS) compared with patients with low N-RAS (mean: 146.9 vs. 211.0 months; median 169.3 vs. not reached; p = 0.009). In patients with non-metastatic disease at the time of tissue sampling, mean disease-free survival (DFS) was 150.1 months for patients with high N-RAS versus 227.7 months with low N-RAS; median DFS was not reached (p = 0.004). The expression of H-RAS and K-RAS was not associated with DFS/OS. In the multivariable analysis, distant metastasis, HER2 positivity, and elevated N-RAS mRNA levels independently predicted reduced OS, while nodal status, HER2 status, and N-RAS predicted reduced DFS. CONCLUSIONS: Elevated N-RAS mRNA levels predict impaired clinical outcome; hypothetically, further exploration of the RAS signaling pathway might enable identifying potential targeted treatment strategies. The association between high N-RAS levels and the most aggressive among breast cancer subtypes, the triple-negative phenotype, for which targeted approaches are still lacking, underlines the need to further investigate the RAS family.

Prognostic relevance of the Golgi mannosidase MAN1A1 in ovarian cancer: impact of N-glycosylation on tumour cell aggregation.

BACKGROUND: Maturation of complex N-glycans involves the action of Golgi mannosidases and plays a major role in cancer progression. We recently showed a favourable prognostic role of α-mannosidase MAN1A1 in breast cancer mainly caused by alteration of certain adhesion molecules. METHODS: We analysed the protein expression of MAN1A1 in ovarian cancer (n = 204) using western blot and studied the impact of MAN1A1 itself and of MAN1A1-related glycosylation on the prognostic relevance of two adhesion molecules. Functional consequences of mannosidase inhibition using kifunensine and MAN1A1 knock out were investigated in ovarian cancer cells in vitro. RESULTS: Patients with high MAN1A1 expression in tumours showed significantly shorter RFS than those with low-MAN1A1 levels. Moreover, high MAN1A1 expression correlated significantly with advanced stage, lymph node involvement and distant metastasis. Further, the glycosylated adhesion molecule ALCAM reveals a significant adverse prognostic effect only in the presence of high MAN1A1 expression. In spheroid-formation assays, mannosidase inhibition and especially MAN1A1 knock out led to strong reduction of tumour cell aggregation. CONCLUSIONS: Our study demonstrates the unfavourable prognostic role of MAN1A1 in ovarian cancer, probably caused by an altered ability of spheroid formation, and the strong influence of this glycosylation enzyme on the prognostic impact of ALCAM.

Prognostic Impact of CEACAM1 in Node-Negative Ovarian Cancer Patients.

The underlying mechanisms of ovarian cancer (OvCa) dissemination are still poorly understood, and novel molecular markers for this cancer type are urgently needed. In search of adhesion molecules with prognostic relevance in OvCa, we compared tumors with good outcome (alive > 3 years) and those with poor outcome (dead < 2 years) within data from The Cancer Genome Atlas (TCGA). The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) turned out as the only gene with differential expression in these groups. In order to further investigation on its role in OvCa, we analyzed CEACAM1 mRNA levels extracted from TCGA microarray data (n = 517) as well as CEACAM1 protein expression by Western blot analysis in a cohort of 242 tumor samples. Further, CEACAM1 localization in tumour tissue was evaluated by immunohistochemistry and CEACAM1 splice variants by RT-PCR in representative tumours. In Kaplan-Meier analysis, high CEACAM1 mRNA levels significantly correlated with longer survival (p = 0.008). By Western blot analysis in the second cohort, similar associations of high CEACAM1 protein levels with longer recurrence-free survival (RFS, p = 0.035) and overall survival (OAS, p = 0.004) were observed. In multivariate Cox regression analysis including clinical prognostic parameters, CEACAM1 mRNA or protein expression turned out as independent prognostic markers. Stratified survival analysis showed that high CEACAM1 protein expression was prognostic in node-negative tumors (p = 0.045 and p = 0.0002 for DFS and OAS) but lost prognostic significance in node-positive carcinomas. Similarly, high CEACAM1 mRNA expression did not show prognostic relevance in tumors with lymphatic invasion (L1) but was associated with longer survival in cases without lymphovascular involvement. Further analysis showed a predominance of 4S and 4L isoforms and mostly membraneous CEACAM1 localization in ovarian tumours. Our results suggest that CEACAM1 might be an independent favorable prognostic marker in OvCa, especially in the subgroup of patients with solely intraperitoneal metastasis.

Prognostic role of the sialyltransferase ST6GAL1 in ovarian cancer.

Aberrant sialylation of glycoproteins has been detected in many tumors, and upregulation of the beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6GAL1) has been implicated with tumor aggressiveness and chemoresistance in experimental models. In our present study, we aimed to study the prognostic or predictive role of ST6GAL1 in ovarian carcinoma, using two independent ovarian cancer cohorts. ST6GAL1 mRNA levels were retrieved from a publicly available database (n = 517), and ST6GAL1 protein levels were analyzed by western blot analysis in a cohort of 204 ovarian tumor samples. The results were correlated with clinical and histological tumor parameters and follow-up information. High ST6GAL1 mRNA levels significantly correlated with lymphovascular invasion and shorter survival, whereas high ST6GAL1 protein expression was associated with advanced stage, distant metastasis and shorter recurrence-free intervals. In both cohorts the prognostic role was most pronounced in tumors without macroscopically visible residual tumor after surgery. In these cases, ST6GAL1 expression levels might help to identify cases with a higher risk of chemoresistance and metastatic relapse that might require an adapted therapeutic regime.

Elevated serum RAS p21 is an independent prognostic factor in metastatic breast cancer.

BACKGROUND: An important component of the RAS signalling pathway, the RAS p21 oncogene, is frequently hyperactivated in breast cancer. Its expression in tumor tissue has been linked to poor clinical outcome. This study was designed to evaluate the clinical relevance of RAS p21 levels in peripheral blood in a large cohort of metastatic breast cancer patients. METHODS: Two hundred fifty-one patients with metastatic breast cancer were enrolled in this prospective, multicentre, open-label, non-randomized study. Blood samples were collected before start of first-line or later-line treatment. RAS p21 was determined using a sandwich-type ELISA immunoassay. For the determination of the cutoff, blood samples from age-matched healthy controls were analyzed. A value above 452 pg/ml was regarded as elevated (mean + 2 x SD). In the univariate survival analysis, two other cutoffs were considered as well (50th and 75th percentile of patients, i.e. 229 pg/ml and 320 pg/ml). Circulating tumor cells (CTCs) were detected using the CellSearch system. RESULTS: 29 of 251 (12%) patients had RAS p21 levels above the cut-off level of 452 pg/ml. Clinical-pathological parameters, such as hormone receptor and HER2 status, line of therapy and CTC status, did not correlate with RAS p21 levels. Elevated RAS p21 was significantly associated with shorter progression-free and overall survival in the univariate analysis (median PFS: 3.9 months [95%-CI: 1.8-6.0] for patients with elevated RAS p21 levels versus 8.5 months [95%-CI: 7.4-9.5] with non-elevated levels [p = 0.01]; median OS: 7.1 months [95%-CI: 0.3-14.2] versus not reached [p = 0.002], respectively). When RAS p21 cutoffs other than 452 pg/ml were considered, elevated RAS p21 was significantly associated with OS but not with PFS. Classical clinical-pathological factors were included into a multivariate Cox regression analysis. In addition, factors previously shown to influence survival in a univariate analysis, such as serum HER2, CAIX and TIMP1, were included as well. In the multivariate analysis, RAS p21, presence of ≥5 CTCs per 7.5 ml blood, higher grading and higher line of therapy remained independent predictors of shorter OS. CONCLUSIONS: Metastatic breast cancer patients with elevated levels of circulating RAS p21 have significantly worse clinical outcome. Hypothetically, these patients might benefit from therapeutic strategies targeting RAS pathway. TRIAL REGISTRATION: Current Controlled Trials ISRCTN59722891 (DETECT); trial registration date: April, 17th 2010; the trial was registered retrospectively.

The Predictive Significance of Metastasis-Associated in Colon Cancer-1 (MACC1) in Primary Breast Cancer.

BACKGROUND: Metastasis-Associated in Colon Cancer-1(MACC1) was first identified as a transcriptional activator of the HGF/MET pathway. Deregulation of HGF/MET signaling is reported as a prognostic marker for tumorigenesis, early stage invasion, and metastasis which is associated with poor clinical outcome in breast cancer patients. The aim of the present study was to further investigate the prognostic or predictive value of MACC1 expression in breast cancer. MATERIALS AND METHODS: We analyzed the MACC1 expression in 105 primary breast cancer samples by Western-Blot analysis and immunohistochemistry. RESULTS: A significant correlation of high MACC1 expression with shorter disease-free survival was found within the group of lymph-node-negative patients. Additionally, an association of high MACC1 expression and shorter disease-free survival was observed within estrogen receptor positive tumors and patients without adjuvant chemotherapy. CONCLUSION: Our results support a biologic role and potentially open the perspective for the use of MACC1 as a prognostic marker for treatment decision in breast cancer patients.

Reduced mannosidase MAN1A1 expression leads to aberrant N-glycosylation and impaired survival in breast cancer.

BACKGROUND: Alterations in protein glycosylation have been related to malignant transformation and tumour progression. We recently showed that low mRNA levels of Golgi alpha-mannosidase MAN1A1 correlate with poor prognosis in breast cancer patients. METHODS: We analysed the role of MAN1A1 on a protein level using western blot analysis (n=105) and studied the impact of MAN1A1-related glycosylation on the prognostic relevance of adhesion molecules involved in breast cancer using microarray data (n=194). Functional consequences of mannosidase inhibition using the inhibitor kifunensine or MAN1A1 silencing were investigated in breast cancer cells in vitro. RESULTS: Patients with low/moderate MAN1A1 expression in tumours showed significantly shorter disease-free intervals than those with high MAN1A1 levels (P=0.005). Moreover, low MAN1A1 expression correlated significantly with nodal status, grading and brain metastasis. At an mRNA level, membrane proteins ALCAM and CD24 were only significantly prognostic in tumours with high MAN1A1 expression. In vitro, reduced MAN1A1 expression or mannosidase inhibition led to a significantly increased adhesion of breast cancer cells to endothelial cells. CONCLUSIONS: Our study demonstrates the prognostic role of MAN1A1 in breast cancer by affecting the adhesive properties of tumour cells and the strong influence of this glycosylation enzyme on the prognostic impact of some adhesion proteins.

Loss of BRCA1 promotor hypermethylation in recurrent high-grade ovarian cancer.

BACKGROUND: Approximately 20-25% of ovarian cancers are attributable to germline or somatic BRCA1/2 mutations, resulting in defects in the homologous recombination pathway. Inactivation of these genes can also be mediated by epigenetic changes, e.g., hypermethylation of CpG islands in the promoter regions. In such homologous recombination deficient tumors, platinum based chemotherapy is in general effective, however, loss of hypermethylation might lead to refractory disease. The aim of this study was to evaluate the stability of BRCA1 promoter hypermethylation in recurrent disease after platinum based chemotherapy. METHODS: Tumor tissue from 76 patients with primary and 48 patients with platinum-sensitive recurrent high-grade ovarian cancer was collected. In a subgroup of 12 patients, 'paired' tumor tissue from primary and recurrent surgery was available. BRCA1 promoter methylation status was assessed using methylation specific polymerase chain reaction and was verified by Sanger Sequencing. RESULTS: 73.7% (56/76) of primary and 20.8% (10/48) of recurrent tumors displayed BRCA1 promoter hypermethylation. BRCA1 promoter methylation status was not associated with progression-free- or overall survival. In the paired subgroup 83.3% (10/12) of the primary vs. 16.7% (2/12) of the recurrent tumors showed hypermethylation. In eight patients loss of BRCA1 hypermethylation was observed, whereas two patients had stable methylation status. CONCLUSIONS: Loss of BRCA1 promoter methylation may be a mechanism to restore BRCA1 function in recurrent disease. However, currently the clinical significance is still unclear and should be evaluated in prospective clinical trials.

Selectin-independent adhesion during ovarian cancer metastasis.

PURPOSE: Ovarian cancer (OvCa) progression mainly takes place by intraperitoneal spread. Adhesion of tumor cells to the mesothelial cells which form the inner surface of the peritoneum is a crucial step in this process. Cancer cells use in principle different molecules of the leukocyte adhesion cascade to facilitate adhesion. This cascade is initiated by selectin-ligand interactions followed by integrin - extracellular matrix protein interactions. Here we address the question whether all tumor cells predominantly employ selectin-dependent leukocyte-like adhesion cascade (SDAC) or whether they use integrin mediated adhesion for OvCa progression as well. METHODS: A comparative transcriptomic analysis of the human OvCa cell lines OVCAR8 and SKOV3 was performed. Intraperitoneal xenograft model of OVCAR8 cells was used to determine whether there is a correlation between SDAC gene expression and the metastatic potential of the control cells and the cells overexpressing c-Fos. Transcriptomic analysis of OVCAR8 and SKOV3 samples was performed using microarrays. RESULTS: One-third of the protein-coding genes involved in SDAC exhibited lower expression levels in OVCAR8 than in SKOV3 cells. In contrast to SKOV3 cells, c-Fos overexpression in OVCAR8 cells did not significantly influence the expression of SDAC genes. Intraperitoneal xenograft model of OVCAR8 cells unexpectedly demonstrated that the aggressiveness of OVCAR8 tumors was not depended on the c-Fos expression level and was comparable to that of SKOV3 control tumors. Gene expression analysis of tumors suggests that SKOV3-derived tumor progression was mainly depended on SDAC. Progression of OVCAR8 tumors relied on other cell adhesion molecules that do not interact with selectins. CONCLUSIONS: High expression of c-Fos in ovarian cancer cells is not always associated with reduced metastatic potential. Low expression level of SDAC genes may not ensure low OvCa metastatic potential hence alternative adhesion mechanisms involving laminin-integrin interactions exist as well.

VEGF-C expression attributes the risk for lymphatic metastases to ovarian cancer patients.

BACKGROUND: Peritoneal dissemination and retroperitoneal lymph node involvement are main routes for progression of epithelial ovarian cancer (EOC). Vascular endothelial growth factor (VEGF) mediated angiogenesis has been identified as an important mechanism promoting tumour progression. METHODS: Tumour tissue of 100 patients with EOC was analysed for protein expression of vascular endothelial growth factor (VEGF)-A, -C, -D by Western Blot analysis. Expression patterns in patients with 'extensive intraperitoneal' metastases (pT3c pN1 and pT3b-pT3c pN0, n=80) were compared to patients with 'predominantly retroperitoneal' metastases (pT1a-pT3b, pN1, n=20). Overall and progression-free survival was analysed by Kaplan-Meier method. RESULTS: While no significant differences in expression levels among the different modes of metastases were noted for VEGF-A and -D, VEGF-C expression was significantly higher in the group of predominantly retroperitoneal metastases compared to the group with extensive intraperitoneal metastases. Patients with high VEGF-C expression had a significantly worse overall survival compared to patients with low expression levels. CONCLUSIONS: Retroperitoneal tumour progression in EOC patients is associated with high VEGF-C expression. VEGF-C may serve as a molecular marker to identify patients with assumed high risk for lymphatic metastases, who might benefit from specific treatment strategies.

Role of protein glycosylation in cancer metastasis.

Although altered glycosylation has been detected in human cancer cells decades ago, only investigations in the last years have enormously increased our knowledge about the details of protein glycosylation and its role in tumour progression. Many proteins, which are heavily glycosylated, i.e. adhesion proteins or proteases, play an important role in cancer metastasis that represents the crucial and frequently life-threatening step in progression of most tumour types. Compared to normal tissue, tumour cells often show altered glycosylation patters with appearance of new tumour-specific antigens. In this review, we give an overview about the role of glycosylation in tumour metastasis, describing recent results about O-glycans, N-glycans and glycosaminoglycans. We show that glycan structures, glycosylated proteins and glycosylation enzymes have influence on different steps of the metastatic process, including epithelial-mesenchymal transition (EMT), migration, invasion/intravasation and extravasation of tumour cells. Regarding the important role of cancer metastasis for patients survival, further knowledge about the consequences of altered glycosylation patterns in tumour cells is needed which might eventually lead to the development of novel therapeutic approaches.

Role of HYAL1 expression in primary breast cancer in the formation of brain metastases.

BACKGROUND: The incidence of brain metastases in breast cancer patients has increased in the last years. However, the knowledge about tumor cell invasion in the brain is still very limited. Based on our recent study on cDNA microarray data of breast cancer patients, we hypothesized that two enzymes involved in the hyaluronan metabolism, namely, hyaluronan synthase 2 (HAS2) and hyaluronidase 1 (HYAL1) are associated with brain metastases formation. METHODS: Protein expression levels of hyaluronan, HAS2, and HYAL1 were analyzed in primary breast cancer, and metastatic tissue samples from different localizations (brain, bone, skin, liver, and lung) were included in four different cohorts by immunohistochemistry. Correlations of expression levels with clinical and pathological parameters were performed within the individual cohorts. RESULTS: Higher HYAL1 expression was detected among primary tumors from patients with subsequent brain metastases compared with those without brain metastases (p = 0.011). Interestingly, brain metastatic tissue showed a significantly reduced HYAL1 expression compared with the corresponding primary tumor (p = 0.003). HYAL1 expression in brain metastases was also significantly lower than in skin, liver, and lung metastases. Further, hyaluronan staining in brain metastases was mainly located on the surface of the tumor cells, whereas in all other metastatic sites hyaluronan was only detected in the extracellular matrix. We could not show an association of HAS2 with the formation of brain metastases. CONCLUSIONS: In conclusion, our results suggest that the enzyme HYAL1 plays a role in tumor dissemination and brain-specific colonization, rather than in subsequent metastatic out-growth.

Circulating Cell-Free miR-373, miR-200a, miR-200b and miR-200c in Patients with Epithelial Ovarian Cancer.

In the present study, we investigated whether circulating cell-free microRNAs serve as potential biomarkers in epithelial ovarian cancer (EOC) patients. Circulating miR-373, miR-200a, miR-200b and miR-200c were quantified in a cohort of 60 EOC patients, 20 patients with benign ovarian diseases and 32 healthy women using quantitative TaqMan MicroRNA assays. The serum concentrations of cell-free miR-373, miR-200a, miR-200b and miR-200c were significantly higher in EOC patients than in healthy women (p = 0.0001). With a sensitivity of 83 % and a specificity of 100 %, the combination of miR-200a, miR-200b and miR-200c could differ between malignant and benign ovarian tumors (p = 0.0001). Elevated levels of these cell-free microRNAs could be detected in FIGO I-II and FIGO III-IV stages, grading G1-2 and G3 and lymph node-negative and -positive EOC. In conclusion, the increased serum levels of this microRNA panel have diagnostic value for distinguishing healthy controls and benign tumors from EOC.