In this study, we compared the blood serum levels of circulating cell-free and exosomal microRNAs, and their involvement in the molecular subtypes of breast cancer patients. Our analyses on cell-free miR-101, miR-372 and miR-373 were performed in preoperative blood serum of 168 patients with invasive breast cancer, 19 patients with benign breast diseases and 28 healthy women. MicroRNAs were additionally quantified in exosomes of 50 cancer patients and 12 healthy women from the same cohort. Relative concentrations were measured by quantitative TaqMan MicroRNA assays and correlated to clinicopathological risk factors. The concentrations of cell-free miR-101 (p=0.013) and miR-373 (p=0.024) were significantly different between patients with breast cancer and benign tumors. A prevalence of miR-101, miR-372 and miR-373 were found in exosomes. The levels of circulating exosomal (but not cell-free) miR-373 were higher in triple negative than luminal carcinomas (p=0.027). Also, estrogen-negative (p=0.021) and progesterone-negative (p=0.01) tumors displayed higher concentrations of exosomal miR-373 than patients with hormone-receptor positive tumors. Overexpression of miR-373 by transfection of MCF-7 cells showed downregulated protein expression of the estrogen receptor, and inhibition of apoptosis induced by camptothecin. Our data indicate that serum levels of exosomal miR-373 are linked to triple negative and more aggressive breast carcinomas.
Breast cancer brain metastases (BCBM) are detected with increasing incidence. In order to detect potential genes involved in BCBM, we first screened for genes down-regulated by methylation in cell lines with site-specific metastatic ability. The expression of five genes, CADM1, SPARC, RECK, TNFAIP3 and CXCL14, which were also found down-regulated in gene expression profiling analyses of BCBM tissue samples, was verified by qRT-PCR in a larger patient cohort. CADM1 was chosen for further down-stream analyses. A higher incidence of CADM1 methylation, correlating with lower expression levels, was found in BCBM as compared to primary BC. Loss of CADM1 protein expression was detected most commonly among BCBM samples as well as among primary tumors with subsequent brain relapse. The prognostic role of CADM1 expression was finally verified in four large independent breast cancer cohorts (n=2136). Loss of CADM1 protein expression was associated with disease stage, lymph node status, and tumor size in primary BC. Furthermore, all analyses revealed a significant association between loss of CADM1 and shorter survival. In multivariate analyses, survival was significantly shorter among patients with CADM1-negative tumors. Loss of CADM1 expression is an independent prognostic factor especially associated with the development of brain metastases in breast cancer patients.
For better lung cancer diagnosis and therapy, early detection markers of tumor dissemination are urgently needed, as most lung cancers do not show symptoms until extensive metastasis formation has already taken place. Our previous studies showed that in non-small cell lung cancer (NSCLC) early tumor dissemination is associated with a loss of chromosome 4q12-q32 and the presence of disseminated tumor cells (DTC) in the bone marrow. In order to identify the potential target gene in this region, a screen for methylation-dependent expression was performed. Lung cancer cell lines showing a loss of 4q as well as a normal bronchial epithelial cell line as control were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) followed by expression profiling. Seven genes within the 4q target region, which have been associated with a positive DTC status before were found to be regulated by hypermethylation. QRT-PCR in an independent sample set identified HERC5 as a potential target gene. Quantitative methylation analysis of these lung tissue samples revealed that HERC5 promoter hypermethylation was significantly associated with positive DTC status (p?=?0.020) and occurrence of brain metastases (p?=?0.015). In addition, hypermethylation of the HERC5 promoter in NSCLC was identified as a predictor for poor survival for Stage I adenocarcinoma patients (p?=?0.022) and also for poor overall survival in metastatic lung cancer patients (p?=?0.028). In conclusion, HERC5 may function as a prognostic marker and is associated with tumor dissemination in lung cancer.
Cancer cells can divert metabolites into anabolic pathways to support their rapid proliferation and to accumulate the cellular building blocks required for tumour growth. However, the specific bioenergetic profile of invasive and metastatic cancer cells is unknown. Here we report that migratory/invasive cancer cells specifically favour mitochondrial respiration and increased ATP production. Invasive cancer cells use the transcription coactivator peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1A, also known as PGC-1?) to enhance oxidative phosphorylation, mitochondrial biogenesis and the oxygen consumption rate. Clinical analysis of human invasive breast cancers revealed a strong correlation between PGC-1? expression in invasive cancer cells and the formation of distant metastases. Silencing of PGC-1? in cancer cells suspended their invasive potential and attenuated metastasis without affecting proliferation, primary tumour growth or the epithelial-to-mesenchymal program. Inherent genetics of cancer cells can determine the transcriptome framework associated with invasion and metastasis, and mitochondrial biogenesis and respiration induced by PGC-1? are also essential for functional motility of cancer cells and metastasis.
Current standard systemic therapies for treating breast cancer patients with brain metastases are inefficient. Targeted therapies against human epidermal growth factor receptors are of clinical interest because of their alteration in a subset of breast cancers (BCs). We analyzed copy number, mutation status, and protein expression of epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2), phosphatase and tensin homologue (PTEN), and PI3K catalytic subunit (PIK3CA) in 110 ductal carcinoma in situ, primary tumor, and metastatic BC samples. Alterations in EGFR, HER2, and PTEN, alone or in combination, were found in a significantly larger fraction of breast cancer brain metastases tumor tissue compared with samples from primary tumors with good prognosis, bone relapse, or other distant metastases (all P < 0.05). Primary tumor patients with a subsequent brain relapse showed almost equally high frequencies of especially EGFR and PTEN alteration as the breast cancer brain metastases patients. PIK3CA was not associated with an increased risk of brain metastases. Genetic alterations in both EGFR and PTEN were especially common in triple-negative breast cancer patients and rarely were seen among HER2-positive patients. In conclusion, we identified two independent high-risk primary BC subgroups for developing brain metastases, represented by genetic alterations in either HER2 or EGFR/PTEN-driven pathways. In contrast, none of these pathways was associated with an increased risk of bone metastasis. These findings highlight the importance of both pathways as possible targets in the treatment of brain metastases in breast cancer.
The progress of non-small cell lung cancer (NSCLC) is dependent on sufficient angiogenesis. Thrombin induced activation of proteinase-activated receptor 1 (PAR-1) on platelets leads to platelet secretion and aggregation. This influences cell survival, apoptosis and angiogenesis by the release of VEGF and Endostatin (ES), a potent angiogenesis inhibitor. Interleukin-8 (IL-8) induces tumor angiogenesis independent of the VEGF pathway through the chemokine C-X-C motif receptor 2 (CXCR-2). Our purpose was to evaluate germline polymorphisms of these potential therapy targets as prognostic markers for disease free survival (DFS) and overall survival (OS) in surgically treated NSCLC patients. In total 209 Caucasian patients, treated between 1996 and 2011, were included in this study. Genomic DNA was extracted from peripheral blood leucocytes. Genotyping of CXCR-2 +1208 C > T and +785 C > T, PAR-1 -506 Ins/del and -14 Ivs A > T and ES +4349 G > A was performed by TaqMan(®) genotyping assays or by polymerase chain reaction (PCR) followed by capillary electrophoresis. Chi-square test, Kaplan-Meier estimator and cox regression hazard model were used to assess the prognostic value of selected polymorphisms. The PAR-1 -14 Ivs A/A genotype was associated with advanced tumor stages (p = 0.024) and, in univariate analysis, with shorter median OS in squamous cell lung carcinoma (SqCC, p = 0.035). The CXCR-2 + 1208T/T genotype was associated with aggressive tumor biology (p = 0.038), and shorter DFS and OS (p = 0.018, p = 0.021) in NSCLC and especially in SqCC a negative predictor for DFS and OS (p = 0.045, p = 0.041). Genotyping of the CXCR-2 +1208 C >T polymorphism could be a useful tool to identify high-risk SqCC subgroups.
The activated leukocyte cell adhesion molecule (ALCAM) is overexpressed in many mammary tumors, but controversial results about its role and prognostic impact in breast cancer have been reported. Therefore, we evaluated the biologic effects of ALCAM expression in two breast cancer cell lines and a larger cohort of mammary carcinomas. By stable transfections, MCF7 cells with ALCAM overexpression and MDA-MB231 cells with reduced ALCAM levels were generated and analyzed in functional assays and cDNA microarrays. In addition, an immunohistochemical study on 347 patients with breast cancer with long-term follow-up and analysis of disseminated tumor cells (DTCs) was performed. In both cell lines, high ALCAM expression was associated with reduced cell motility. In addition, ALCAM silencing in MDA-MB231 cells resulted in lower invasive potential, whereas high ALCAM expression was associated with increased apoptosis in both cell lines. Among genes which were differentially expressed in clones with altered ALCAM expression, there was an overlap of 15 genes between both cell lines, among them cathepsin D, keratin 7, gelsolin, and ets2 whose deregulation was validated by western blot analysis. In MDA-MB231 cells, we observed a correlation with VEGF expression which was validated by enzyme-linked immuno sorbent assay (ELISA). Our IHC results on primary breast carcinomas showed that ALCAM expression was associated with an estrogen receptor-positive phenotype. In addition, strong ALCAM immunostaining correlated with nodal involvement and the presence of tumor cells in bone marrow. By Kaplan-Meier analysis, strong ALCAM expression in ductal carcinomas correlated with shorter recurrence-free intervals (P=0.048) and overall survival (OAS, P=0.003). Our results indicate that the biologic role of ALCAM in breast cancer is complex, but overexpression might be relevant for outcome in ductal carcinomas.
Lung cancer is the most common cancer worldwide. Polymorphisms in genes associated with carcinogen metabolism may modulate risk of disease. Glutathione S-transferase pi (GSTP1) detoxifies polycyclic aromatic hydrocarbons found in cigarette smoke and is the most highly expressed glutathione S-transferase in lung tissue. A polymorphism in the GSTP1 gene, an A-to-G transition in exon 5 (Ile105Val, 313A --> 313G), results in lower activity among individuals who carry the valine allele. The authors present a meta- and a pooled analysis of case-control studies that examined the association between this polymorphism in GSTP1 and lung cancer risk (27 studies, 8,322 cases and 8,844 controls and 15 studies, 4,282 cases and 5,032 controls, respectively). Overall, the meta-analysis found no significant association between lung cancer risk and the GSTP1 exon 5 polymorphism. In the pooled analysis, there was an overall association (odds ratio = 1.11, 95% confidence interval: 1.03, 1.21) between lung cancer and carriage of the GSTP1 Val/Val or Ile/Val genotype compared with those carrying the Ile/Ile genotype. Increased risk varied by histologic type in Asians. There appears to be evidence for interaction between amount of smoking, the GSTP1 exon 5 polymorphism, and risk of lung cancer in whites.
Bone marrow is a common homing organ for early disseminated tumor cells (DTC) and their presence can predict the subsequent occurrence of overt metastasis and survival in lung cancer. It is still unclear whether the shedding of DTC from the primary tumor is a random process or a selective release driven by a specific genomic pattern.
Asbestos causes DNA damage and the fibers, together with tobacco smoke, have a synergistic effect on lung cancer risk. We recently identified 18 chromosomal regions that showed differences in DNA copy number between the lung tumors of asbestos-exposed and nonexposed patients. One of the previously identified asbestos-associated chromosomal regions at 9q was further analyzed for allelic imbalance and DNA copy number alterations (CNA) in the lung tumors of asbestos-exposed and nonexposed patients. In addition, the ploidy level of the tumors was studied.
The occurrence of brain metastases among breast cancer patients is currently rising with approximately 20-25% incidence rates, underlining the importance of the identification of new therapeutic and prognostic markers. We have previously screened for new markers for brain metastasis by array CGH. We found that loss of 11p15 is common among these patients. In this study, we investigated the clinical significance of loss of 11p15 in primary breast cancer (BC) and breast cancer brain metastases (BCBM). 11p15 aberration patterns were assessed by allelic imbalance (AI) analysis in primary BC (n = 78), BCBM (n = 21) and metastases from other distant sites (n = 6) using six different markers. AI at 11p15 was significantly associated with BCBM (p = 0.002). Interestingly, a subgroup of primary BC with a later relapse to the brain had almost equally high AI rates as the BCBM cases. In primary BC, AI was statistically significantly associated with high grade, negative hormone receptor status, and triple-negative (TNBC) tumors. Gene expression profiling identified PRKCDBP in the 11p15 region to be significantly downregulated in both BCBM and primary BC with brain relapse compared to primary tumors without relapse or bone metastasis (fdr<0.05). qRT-PCR confirmed these results and methylation was shown to be a common way to silence this gene. In conclusion, we found loss at 11p15 to be a marker for TNBC primary tumors and BCBM and PRKCDBP to be a potential target gene in this locus.
We have previously demonstrated an association between genomic alterations in 19p13, 2p16, and 9q33.1 and asbestos exposure in patients lung tumours. This study detected allelic imbalance (AI) in these regions in asbestos-exposed lung cancer (LC) patients histologically normal pulmonary epithelium. We extended the analyses of tumour tissue to cover a large LC patient cohort and studied DNA copy number alteration (CNA) and AI in 19p13, 2p16, and 9q33.1 for the first time in combination. We found both CNA and AI in ?2/3 of the regions to be significantly and dose-dependently (P < 0.001) associated with pulmonary asbestos fibre count. Twenty percent of the exposed patients LC showed CNA in ?2/3 of the regions, whereas none of the non-exposed patients LC showed CNA in more than one region. AI was evident in 89% of the exposed and in only 26% of the non-exposed patients LC. The genomic alterations in 19p13, 2p16, and 9q33.1 in compilation identified asbestos-exposed patients lung tumours better than each of the regions alone. These alterations form the basis for the development of a combinatorial molecular assay that could be used to identify asbestos-related LC.
With the improvement of therapeutic options for the treatment of breast cancer, the development of brain metastases has become a major limitation to life expectancy in many patients. Therefore, our aim was to identify molecular markers associated with the development of brain metastases in breast cancer.
VEGFR-2 gene displays several functional germline polymorphisms with impact on VEGFR-2 mediated angiogenesis. Our purpose was to evaluate VEGFR-2 polymorphisms as prognostic markers for tumor recurrence and overall survival (OS) in non-small-cell lung cancer (NSCLC).
Gene copy number profiles of primary lung tumors were screened for high-level amplifications. We detected 22 high-level amplifications in various loci, including 14q13. This locus is known to harbor the adenocarcinoma (AC) lineage-specific target gene NKX2-1, which is not expressed in squamous cell carcinoma (SCC). As the 14q amplification was also found in SCC, we investigated whether or not FOXA1 might be the corresponding target gene for SCC. Focusing on these two target genes, we assessed gene amplifications and protein expression of NKX2-1 and FOXA1 in primary lung tumors (n = 554) and brain metastases (n = 68). Primary AC (n = 194) showed positive protein expression of NKX2-1 in 58.2% of the samples compared with 4.2% of primary SCC samples (n = 212). Positive staining for FOXA1 was seen in 34.7% of the SCC samples, which was comparable with 39.6% in the AC samples. For brain metastases, FOXA1 expression was slightly higher in the SCC samples (55.6%) compared with the non-matched primary SCC tumor samples (43.4%), whereas NKX2-1 expression was comparable in both primary tumors and brain metastases. Positive FOXA1 and NKX2-1 expression was associated with a gain or amplification in 34.6% and 28.6% of cases, respectively. The expression of NKX2-1 was associated with early stage and grade among the AC cases. In contrast, FOXA1 expression in SCC was associated with distant metastases as well as an unfavorable survival rate (P = 0.039). These results suggest that both FOXA1 and NKX2-1 may act as lineage-specific target genes within the 14q amplicon with opposite functions in lung cancer.
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