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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Carbonic anhydrase IX (CAIX) is a membranous expressed metalloenzyme involved in pH homeostasis and cell adhesion. The protein is overexpressed in a variety of tumors and potentially associated with ...negative outcome. This study was designed to investigate the prognostic role of CAIX in serum and tumor tissue of patients with primary cervical cancer.
Tumor samples of 221 consecutive patients with primary cervical cancer who underwent surgery between 1993 and 2008 were analyzed for CAIX expression by immunohistochemistry. Additionally, preoperative serum CAIX concentrations were determined by ELISA in a subset of patients. Correlation with intratumoral CAIX expression as well as clinicopathological factors and outcome was analyzed.
CAIX expression was observed in 81.9% of the tumor specimens; 62.0% showed a moderate or strong staining intensity. Moderate/strong expression was associated with squamous histology (p=0.024), advanced tumor stage (p=0.001), greater invasion depth (p=0.025), undifferentiated tumor grade (p<0.001) and high preoperative SCC-Ag values (p=0.042). Furthermore patients with moderate/strong intratumoral CAIX expression had a higher number of metastatic lymph nodes compared to those with none/weak intratumoral expression levels (p=0.047) and there was a non-significant association between high intratumoral CAIX expression and shorter survival (p=0.118). Preoperative serum concentrations of CAIX ranged between 23 and 499 pg/mL and did not correlate with intratumoral expression or other clinicopathological variables.
CAIX is associated with advanced tumor stages and lymph node metastases in cervical cancer, potentially representing a new target in this disease. In contrast to other epithelial cancers we could not observe a correlation between serum CAIX and its intratumoral expression.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Despite the high number of previous studies, the role of p53 alterations in prostate cancer is not clearly defined. To address the role of p53 alterations in prostate cancer biology, a total of 2514 ...cancers treated by radical prostatectomy were successfully analyzed by immunohistochemistry in a tissue microarray format. Overall a low rate of p53-positive tumors was found (2.5%). A significant underestimation of p53-positive cases was excluded by subsequent large section analyses and direct sequencing of the p53 gene in subsets of our patients. Large section analysis of 23 cases considered negative on the tissue microarray yielded only one weakly p53-positive tumor. Only 4 out of 64 (6.4%) high-grade tumors, that were considered negative for p53 by immunohistochemistry, presented exon 5-8 mutations. These data suggest a high sensitivity of our immunohistochemistry approach and confirm the overall low frequency of p53 alterations in clinically localized prostate cancer. A positive p53 immunostaining was strongly associated with presence of exon 5-8 mutations (P<0.0001), advanced pT-stage (P<0.0001), high Gleason grade (P<0.0001), positive surgical margins (P=0.03) and early biochemical tumor recurrence (P<0.0001). A higher rate of positive p53 immunostaining was detected in late-stage diseases including metastatic prostate cancer (P=0.0152) and hormone-refractory tumors (P=0.0003). Moreover, p53 expression was identified as an independent predictor of biochemical tumor recurrence in the subgroup of low- and intermediate-grade cancers. In summary, the results of this study show that p53 mutations characterize a small biologically aggressive subgroup of prostate cancers with a high risk of progression after prostatectomy. The rate of p53 alterations increases with prostate cancer progression.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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.
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.
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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. Keywords: Breast cancer, TGFB-induced factor homeobox 1, TGIF, Bone metastases
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Exosomes are membrane vesicles that mediate intercellular communication by transporting their molecular cargo from cell to cell. We investigated whether serum levels of exosomal miR-373, miR-200a, ...miR-200b and miR-200c and circulating exosomes have diagnostic and prognostic relevance in a cohort of 163 epithelial ovarian cancer (EOC) patients using TaqMan MicroRNA assays and ELISA. The serum concentrations of exosomal miR-373 (p = 0.0001), miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.028) were significantly higher in EOC patients than healthy women. The levels of miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.019) could distinguish between malignant and benign ovarian tumors. While the levels of miR-373 and miR-200a were increased in all FIGO/lymph node stages (p = 0.0001), the levels of miR-200b and miR-200c were higher in patients with FIGO stage III-IV (p = 0.0001, p = 0.008, respectively) including lymph node metastasis (p = 0.0001, p = 0.004, respectively) than FIGO stages I-II. The increased levels of miR-200b and miR-200c were also associated with CA125 values (p = 0.0001, p = 0.0001, respectively) and a shorter overall survival (p = 0.007, p = 0.017, respectively). The levels of exosomes were excessively elevated in EOC patients (p = 0.0001). In all three cohorts, they were positively associated with the serum levels of exosomal miR-373 (p = 0.004), miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.008). In conclusion, the increased levels of exosomal miR-200b and miR-200c mainly observed in advanced EOC suggest that these microRNAs may be involved in tumor progression. The high concentrations of exosomes in EOC patients imply an excessive, active exosomal secretion in EOC.
Purpose
The intercellular adhesion molecule (ICAM)-1 is expressed on many cell types including endothelial cells and different cancer cell entities. Experimental data strongly indicate that ICAM-1 ...can activate intracellular signalling pathways in cancer cells leading to enhanced cell motility, invasion and metastasis. Yet, little is known about the role of ICAM-1 expression during malignant progression in breast cancer patients.
Methods
We investigated ICAM-1 protein and mRNA expression in two partly overlapping cohorts of breast cancer patients. ICAM-1 protein was detected by Western blot analysis in 104 cases and verified by immunohistochemistry. Additionally, ICAM-1 mRNA microarray data from 169 tumours were analysed.
Results
With both methods, high ICAM-1 expression was significantly associated with a poorly differentiated phenotype, a negative estrogen receptor (ER) status and positive lymph node involvement. In addition, there was a significant prognostic impact of ICAM-1 protein overexpression on recurrence-free survival (HR = 2.82,
P
= 0.023), which was most pronounced in ER-negative tumours. ICAM-1 mRNA overexpression was associated with high urokinase plasminogen activator (uPA) and uPA-inhibitor protein 1 (PAI 1) protein and mRNA levels as well as high Ki67 protein and vascular endothelial growth factor (VEGF) mRNA expression.
Conclusions
In our group of patients, ICAM-1 expression was associated with a more aggressive tumour phenotype. Because of its association with malignant progression, ICAM-1 might represent a new target in the treatment of breast cancer patients.
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EMUNI, NUK, SBMB, SBNM, UL, UPUK
Angiogenesis appears to play an important role in ovarian cancer. Vascular endothelial growth factor (VEGF) has recently been implicated as a therapeutic target in ovarian cancer. The tissue ...inhibitor of metalloproteinase 1 (TIMP-1) is involved in tissue invasion and angiogenesis. The application of serum TIMP-1 and VEGF to monitor primary therapy and predict clinical outcome of patients with ovarian cancer is unclear.
Patients with epithelial ovarian cancer who presented for primary surgery were included in this study. A total of 148 serum samples from 37 patients were analyzed. Samples were prospectively collected at 4 predefined time points: 1. before radical debulking surgery, 2. after surgery and before platinum/taxane based chemotherapy, 3. during chemotherapy, 4. after chemotherapy. Serum VEGF-165 and TIMP-1 as well as CA-125 were quantified by ELISA or ECLIA and correlation with response and long-term clinical outcome was analyzed.
Serum levels of all markers changed substantially during first-line therapy. High CA-125 (p = 0.002), TIMP-1 (p = 0.007) and VEGF-165 (p = 0.02) after chemotherapy were associated with reduced overall survival. In addition, elevated CA-125 (p < 0.001) and VEGF-165 (p = 0.006) at this time point predicted poor progression-free survival. TIMP-1 and VEGF-165 were closely correlated at all time-points during therapy.
TIMP-1 and VEGF serum levels changed significantly during first-line therapy of ovarian cancer patients and predicted prognosis. These findings support the role of angiogenesis in ovarian cancer progression and the use of antiangiogenic therapy.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Approximately 20-25% of ovarian cancers are attributable to germline or somatic
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
promoter hypermethylation in recurrent disease after platinum based chemotherapy.
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.
promoter methylation status was assessed using methylation specific polymerase chain reaction and was verified by Sanger Sequencing.
73.7% (56/76) of primary and 20.8% (10/48) of recurrent tumors displayed
promoter hypermethylation.
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
hypermethylation was observed, whereas two patients had stable methylation status.
Loss of
promoter methylation may be a mechanism to restore
function in recurrent disease. However, currently the clinical significance is still unclear and should be evaluated in prospective clinical trials.
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