[Show abstract][Hide abstract] ABSTRACT: Background: Metabolic and transcriptomic differences between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) compartments, particularly in the context of obesity, may play a role in colorectal carcinogenesis. We investigated the differential functions of their metabolic compositions.
Objectives: Biochemical differences between adipose tissues (VAT vs. SAT) in patients with colorectal carcinoma (CRC) were investigated by using mass spectrometry metabolomics and gene expression profiling. Metabolite compositions were compared between VAT, SAT, and serum metabolites. The relation between patients’ tumor stage and metabolic profiles was assessed.
Design: Presurgery blood and paired VAT and SAT samples during tumor surgery were obtained from 59 CRC patients (tumor stages I–IV) of the ColoCare cohort. Gas chromatography time-of-flight mass spectrometry and liquid chromatography quadrupole time-of-flight mass spectrometry were used to measure 1065 metabolites in adipose tissue (333 identified compounds) and 1810 metabolites in serum (467 identified compounds). Adipose tissue gene expression was measured by using Illumina’s HumanHT-12 Expression BeadChips.
Results: Compared with SAT, VAT displayed elevated markers of inflammatory lipid metabolism, free arachidonic acid, phospholipases (PLA2G10), and prostaglandin synthesis–related enzymes (PTGDS/PTGS2). Plasmalogen concentrations were lower in VAT compared with SAT, which was supported by a lower FAR1 gene expression, the rate-limiting enzyme for ether-lipid synthesis in VAT. Serum sphingomyelin concentrations were inversely correlated (P = 0.0001) with SAT adipose triglycerides. Logistic regression identified lipids in patients’ adipose tissues, which were associated with CRC tumor stage.
Conclusions: As one of the first studies, we comprehensively assessed differences in metabolic, lipidomic, and transcriptomic profiles between paired human VAT and SAT and their association with CRC tumor stage. We identified markers of inflammation in VAT, which supports prior evidence regarding the role of visceral adiposity and cancer. This trial was registered at clinicaltrials.gov as NCT02328677.
American Journal of Clinical Nutrition 07/2015; 102(2). DOI:10.3945/ajcn.114.103804 · 6.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Local recurrence of rectal cancer after curative surgery predicts patients' prognosis. The correlation between the exact anatomic location of tumour recurrence and patients' survival is still under debate. Thus, this study aimed to investigate the impact of the exact location of recurrent rectal cancer on post-operative morbidity and survival.
This is a retrospective study including 90 patients with locally recurrent rectal cancer. The location of tumour recurrence was classified into intraluminal and extraluminal recurrence. Univariate and multivariable Cox regression analyses were used to determine the impact on post-operative morbidity and survival.
Patients' survival with intraluminal recurrence was significantly longer compared to patients with extraluminal recurrence (p = 0.027). Curative resection was associated with prolonged survival in univariate and multivariable analyses (p = 0.0001) and was more often achieved in patients with intraluminal recurrence (p = 0.024). Survival of curative resected patients with intraluminal recurrence was significantly longer compared to curatively resected patients with extraluminal recurrence (p = 0.0001). The rate of post-operative morbidity between intraluminal and extraluminal recurrence was not statistically different (p = 0.59).
Based on the present investigation, intraluminal recurrence is associated with superior outcome. Post-operative morbidity did not differ significantly between both groups.
Journal of Gastrointestinal Surgery 03/2015; 19(6). DOI:10.1007/s11605-015-2804-5 · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lynch syndrome is caused by germline mutations of DNA mismatch repair (MMR) genes, most frequently MLH1 and MSH2. Recently, MMR-deficient crypt foci (MMR-DCF) have been identified as a novel lesion which occurs at high frequency in the intestinal mucosa from Lynch syndrome mutation carriers, but very rarely progress to cancer. To shed light on molecular alterations and clinical associations of MMR-DCF, we systematically searched the intestinal mucosa from Lynch syndrome patients for MMR-DCF by immunohistochemistry. The identified lesions were characterised for alterations in microsatellite-bearing genes with proven or suspected role in malignant transformation. We demonstrate that the prevalence of MMR-DCF (mean 0.84 MMR-DCF per 1 cm2 mucosa in the colorectum of Lynch syndrome patients) was significantly associated with patients' age, but not with patients' gender. No MMR-DCF were detectable in the mucosa of patients with sporadic MSI-H colorectal cancer (n = 12). Microsatellite instability of at least one tested marker was detected in 89% of the MMR-DCF examined, indicating an immediate onset of microsatellite instability after MMR gene inactivation. Coding microsatellite mutations were most frequent in the genes HT001 (ASTE1) with 33%, followed by AIM2 (17%) and BAX (10%). Though MMR deficiency alone appears to be insufficient for malignant transformation, it leads to measurable microsatellite instability even in single MMR-deficient crypts. Our data indicate for the first time that the frequency of MMR-DCF increases with patients' age. Similar patterns of coding microsatellite instability in MMR-DCF and MMR-deficient cancers suggest that certain combinations of coding microsatellite mutations, including mutations of the HT001, AIM2 and BAX gene, may contribute to the progression of MMR-deficient lesions into MMR-deficient cancers.
PLoS ONE 03/2015; 10(3):e0121980. DOI:10.1371/journal.pone.0121980 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Signal transducer and activator of transcription proteins (STATs) are crucial regulators of cell growth and differentiation; however, their specific prognostic impact in human colon cancer has only been studied to limited extent. We aimed to assess the prognostic significance of specific STAT expression patterns in colon carcinoma.
Protein expression patterns of activated STAT1, STAT3, STAT4, and STAT5 in human colon carcinoma tissue and corresponding healthy mucosa (n = 104) were assessed using multiplex bead-based immunoassay technologies. Expression patterns were correlated with clinical and survival data. Immunohistochemistry was performed to assess spatial expression of STAT3 and STAT5.
STAT3 was underexpressed whereas STAT4 and STAT5 were overexpressed in colon carcinoma tissue. Primary tumors from patients with distant metastases (M1) displayed significantly increased expression of STAT1 and STAT3 but decreased expression of STAT4 and STAT5. Increased tumor expression of STAT1 or STAT3 was associated with impaired patient survival, whereas increased expression of STAT4 or STAT5 correlated with improved survival. Multivariate analysis identified an increased STAT3/STAT5 expressional ratio as an adverse prognostic marker in colon cancer patients.
The tumor progression-associated transcription factors STAT3, STAT4, and STAT5 are differently expressed in colon carcinoma tissue and colon mucosa. Moreover, the STAT3/STAT5 expression ratio is an independent prognostic marker in colon cancer patients.
Annals of Surgical Oncology 03/2015; DOI:10.1245/s10434-015-4485-4 · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxygen is essential for metazoans to generate energy. Upon oxygen deprivation adaptive and protective pathways are induced, mediated by hypoxia-inducible factors (HIFs) and prolyl hydroxylase domain-containing enzymes (PHDs). Both play a pivotal role in various conditions associated with prolonged ischemia and inflammation, and are promising targets for therapeutic intervention. This review focuses on aspects of therapeutic PHD modulation in surgically relevant disease conditions such as hepatic and intestinal disorders, wound healing, innate immune responses, and tumorigenesis, and discusses the therapeutic potential and challenges of PHD inhibition in surgical patients.
[Show abstract][Hide abstract] ABSTRACT: Pancreatic cancer consists of a heterogenous bulk of tumor cells and stroma cells which contribute to tumor progression by releasing angiogenic factors. Those factors can be detected as circulating serum factors. We performed a compartment-specific analysis of tumor-derived and stroma-derived angiogenic factors to identify biomarkers and molecular targets for the treatment of pancreatic cancer. Kryo-frozen tissue from primary ductal adenocarcinomas (n = 51) was laser-microdissected to isolate tumor and stroma tissue. Expression of 17 angiogenic factors (angiopoietin-2, follistatin, GCSF, HGF, interleukin-8, leptin, PDGF-BB, PECAM-1, VEGF, matrix metalloproteinase -1, -2, -3, -7, -9, -10, -12, and -13) was analyzed using a multiplex elisa assay for tissue-derived proteins and corresponding serum.
Our study reveals a compartment-specific expression profile for several angiogenic factors and matrix metalloproteinases. ROC analysis of corresponding serum samples reveals MMP-7 and MMP-12 as strong classifiers for the diagnosis of patients with pancreatic cancer vs. healthy control donors. High expression of tumor-derived PDGF-BB and MMP-1 correlates with prolonged survival in univariate and multivariate analysis. In conclusion, a distinct expression patterns for angiogenic cytokines and MMPs in pancreatic cancer and surrounding stroma may implicate them as novel targets for cancer treatment. Tumor-derived PDGF-BB and MMP-1 are significant and independent prognostic markers for poor survival.
[Show abstract][Hide abstract] ABSTRACT: Background:
Whether anastomotic leakage (AL) has a negative impact on survival remains a matter of debate. This study aimed to assess the impact of AL on the overall and disease-free survival of patients undergoing curative resection of stages 1-3 rectal cancer using propensity-scoring methods.
In a single-center study, 570 patients undergoing curative resection of stages 1-3 rectal cancer between January 2002 and December 2011 were assessed. The mean follow-up period was 4.7 ± 2.9 years. Patients who did and did not experience AL were compared using Cox regression and propensity score analyses.
Overall, 51 patients (8.9 %) experienced an AL. The characteristics of the patients were highly biased concerning AL (propensity score, 0.16 ± 0.12 vs. 0.09 ± 0.07; P < 0.001). Anastomotic leakage was uniformly associated with a significantly increased risk of mortality in unadjusted analysis [hazard ratio (HR) 2.30; 95 % confidence interval (CI) 1.40-3.76; P = 0.003], multivariable Cox regression (HR 2.27; 95 % CI 1.33-3.88; P = 0.005), and propensity score-adjusted Cox regression (HR 2.07; 95 % CI 1.21-3.55; P = 0.014). Similarly, disease-free survival was significantly impaired in patients who experienced AL according to unadjusted analysis (HR 1.88; 95 % CI 1.19-2.95; P = 0.011), multivariable Cox regression (HR 1.90; 95 % CI 1.17-3.09; P = 0.014), and propensity score-adjusted Cox regression (HR 2.31; 95 % CI 1.40-3.80; P = 0.002).
This is the first propensity score-based analysis providing evidence that oncologic outcome may be impaired after curative rectal cancer resection in patients with AL.
[Show abstract][Hide abstract] ABSTRACT: Humoral immune responses against tumor antigens are studied as indirect markers of antigen exposure and in cancer vaccine studies. An increasing number of tumor antigens potentially translated from mutant genes is identified by advances in genomic sequencing. They represent an interesting source for yet unknown immunogenic epitopes. We here describe a multiplex method using the Luminex technology allowing for the detection of antibodies against multiple in silico-predicted linear neo-antigens in large sets of sera. The approach included 32 synthetic biotinylated peptides comprising a predicted set of frameshift mutation-induced neo-antigens. The antigens were fused to a FLAG epitope to ensure monitoring antigen binding to avidin-linked microspheres in the absence of monoclonal antibodies. Analytical specificity of measured serum antibody reactivity was proven by the detection of immune responses in immunized rabbits and a colorectal cancer patient vaccinated with peptides included in the assay. The measured antibody responses were comparable to peptide ELISA, and inter-assay reproducibility of the multiplex approach was excellent (R
2 > 0.98) for 20 sera tested against all antigens. Our methodic approach represents a valuable platform to monitor antibody responses against predicted antigens. It may be used in individualized cancer vaccine studies, thereby extending the relevance beyond the model system in the presented approach.
Cancer Immunology and Immunotherapy 08/2014; 63(12). DOI:10.1007/s00262-014-1595-y · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract
BACKGROUND & AIMS:
Hypoxia inducible factor (HIF) prolyl hydroxylase inhibitors are protective in mouse models of inflammatory bowel disease (IBD). Here, we investigated the therapeutic target(s) and mechanism(s) involved.
The effect of genetic deletion of individual HIF-prolyl hydroxylase (PHD) enzymes on the development of dextran sulphate sodium (DSS)-induced colitis was examined in mice.
PHD1(-/-), but not PHD2(+/-) or PHD3(-/-), mice were less susceptible to the development of colitis than wild-type controls as determined by weight loss, disease activity, colon histology, neutrophil infiltration, and cytokine expression. Reduced susceptibility of PHD1(-/-) mice to colitis was associated with increased density of colonic epithelial cells relative to wild-type controls, which was because of decreased levels of apoptosis that resulted in enhanced epithelial barrier function. Furthermore, with the use of cultured epithelial cells it was confirmed that hydroxylase inhibition reversed DSS-induced apoptosis and barrier dysfunction. Finally, PHD1 levels were increased with disease severity in intestinal tissue from patients with IBD and in colonic tissues from DSS-treated mice.
These results imply a role for PHD1 as a positive regulator of intestinal epithelial cell apoptosis in the inflamed colon. Genetic loss of PHD1 is protective against colitis through decreased epithelial cell apoptosis and consequent enhancement of intestinal epithelial barrier function. Thus, targeted PHD1 inhibition may represent a new therapeutic approach in IBD.
[Show abstract][Hide abstract] ABSTRACT: Tumors from colorectal cancer (CRC) are generally immunogenic and commonly infiltrated with T lymphocytes. However, the details of the adaptive immune reaction to these tumors are poorly understood. We have accrued both colon tumor samples and adjacent healthy mucosal samples from 15 CRC patients to study lymphocytes infiltrating these tissues. We apply a method for detailed sequencing of T-cell receptor (TCR) sequences from tumor-infiltrating lymphocytes (TILs) in CRC tumors at high throughput to probe T-cell clones in comparison with the TCRs from adjacent healthy mucosal tissue. In parallel, we captured TIL counts using standard immunohistochemistry. The variation in diversity of the TIL repertoire was far wider than the variation of T-cell clones in the healthy mucosa, and the oligoclonality was higher on average in the tumors. However, the diversity of the T-cell repertoire in both CRC tumors and healthy mucosa was on average 100-fold lower than in peripheral blood. Using the TCR sequences to identify and track clones between mucosal and tumor samples, we determined that the immune response in the tumor is different than in the adjacent mucosal tissue, and the number of shared clones is not dependent on distance between the samples. Together, these data imply that CRC tumors induce a specific adaptive immune response, but that this response differs widely in strength and breadth between patients.
Cancer Immunology and Immunotherapy 06/2013; 62(9). DOI:10.1007/s00262-013-1446-2 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Liver regeneration after partial hepatectomy (PH) occurs in conditions of reduced oxygen supply. HIF prolyl hydroxylase enzymes (PHD1, PHD2, and PHD3) are oxygen sensors involved in adaptive response to hypoxia. Specific functions of these PHD enzymes in liver regeneration have, however, remained enigmatic. Here, we investigated the significance of PHD1 in liver regeneration following hepatectomy.
Liver regeneration was studied in PHD1-deficient (PHD1(-/-)) and wild type (WT) mice subjected to 80% hepatectomy. For in vitro analyses, hepatocytes were isolated from PHD1(-/-) and WT livers. Cell cycle progression was studied via FACS-based analysis of nuclear DNA profile. Transcription factor binding assays, qRT-PCR, and immunoblotting were applied to study the relevance of PHD1 downstream effectors during liver regeneration.
Liver regeneration was significantly enhanced in PHD1(-/-) mice compared to WT littermates. This effect was due to enhanced proliferation rather than to hypertrophy of liver cells. Cell cycle progression was significantly enhanced, and transcriptional activity of the cell cycle regulator c-Myc was increased in PHD1-deficient hepatocytes. These changes coincided with increased expression of cyclin D2, a cell cycle-promoting c-Myc target, and decreased expression of the cell cycle-delaying c-Myc target p21.
Loss of PHD1 enhances liver regeneration by boosting hepatocyte proliferation in a c-Myc-dependent fashion. PHD1 might, therefore, represent a potential target to facilitate liver regeneration after surgical resection.
Langenbeck s Archives of Surgery 09/2012; 397(8). DOI:10.1007/s00423-012-0998-5 · 2.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypoxia and HIFs (HIF-1α and HIF-2α) modulate innate immune responses in the setting of systemic inflammatory responses and sepsis. The HIF prolyl hydroxylase enzymes PHD1, PHD2 and PHD3 regulate the mammalian adaptive response to hypoxia; however, their significance in the innate immune response has not been elucidated. We demonstrate in this study that deficiency of PHD3 (PHD3(-/-)) specifically shortens the survival of mice subjected to various models of abdominal sepsis because of an overwhelming innate immune response, leading to premature organ dysfunction. By contrast, this phenotype was absent in mice deficient for PHD1 (PHD1(-/-)) or PHD2 (PHD2(+/-)). In vivo, plasma levels of proinflammatory cytokines were enhanced, and recruitment of macrophages to internal organs was increased in septic PHD3-deficient mice. Reciprocal bone marrow transplantation in sublethally irradiated mice revealed that enhanced susceptibility of PHD3-deficient mice to sepsis-related lethality was specifically caused by loss of PHD3 in myeloid cells. Several in vitro assays revealed enhanced cytokine production, migration, phagocytic capacity, and proinflammatory activation of PHD3-deficient macrophages. Increased proinflammatory activity of PHD3-deficient macrophages occurred concomitantly with enhanced HIF-1α protein stabilization and increased NF-κB activity, and interference with the expression of HIF-1α or the canonical NF-κB pathway blunted their proinflammatory phenotype. It is concluded that impairment of PHD3 enzyme function aggravates the clinical course of abdominal sepsis via HIF-1α- and NF-κB-mediated enhancement of the innate immune response.
The Journal of Immunology 07/2012; 189(4):1955-65. DOI:10.4049/jimmunol.1103471 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Since mammalian cells rely on the availability of oxygen, they have devised mechanisms to sense environmental oxygen tension, and to efficiently counteract oxygen deprivation (hypoxia). These adaptive responses to hypoxia are essentially mediated by hypoxia inducible transcription factors (HIFs). Three HIF prolyl hydroxylase enzymes (PHD1, PHD2 and PHD3) function as oxygen sensing enzymes, which regulate the activity of HIFs in normoxic and hypoxic conditions. Many of the compensatory functions exerted by the PHD-HIF system are of immediate surgical relevance since they regulate the biological response of ischemic tissues following ligation of blood vessels, of oxygen-deprived inflamed tissues, and of tumors outgrowing their vascular supply. PURPOSE: Here, we outline specific functions of PHD enzymes in surgically relevant pathological conditions, and discuss how these functions might be exploited in order to support the treatment of surgically relevant diseases.
Langenbeck s Archives of Surgery 03/2012; 397(4):603-10. DOI:10.1007/s00423-012-0930-z · 2.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epithelial-to-mesenchymal transition (EMT) plays a pivotal role in tumor invasion and dissemination. EMT occurs predominantly at the tumor edge where it is induced by cytokines, the extracellular matrix environment, or hypoxia. In the tumor cell, it is further mediated by several transcription factors and microRNAs. The aim of this study was to explore the expression of EMT-associated genes at the invasive front in colorectal cancer and to evaluate their prognostic significance.
We evaluated the expression of 13 EMT-associated genes at the invasion front of 30 colorectal liver metastases by quantitative real-time PCR. Immunostaining against zinc finger E-box-binding homeobox 2 (ZEB2) was carried out on 175 primary colorectal cancer specimens and 30 colorectal liver metastases and correlated to clinical and histopathologic data. DLD-1 cells were transfected with siRNA and subjected to migration and invasion assays.
Gene expression analysis and immunohistochemistry showed an upregulation of ZEB2 at the invasion front in primary colorectal cancer and liver metastases. Overexpression of ZEB2 at the invasion front correlated significantly with tumor stage in primary colorectal cancer. Moreover, univariate and multivariate analysis revealed overexpression of ZEB2 at the invasion front as an independent prognostic marker for cancer-specific survival. Downregulation of ZEB2 by siRNA decreased the migration and invasion capacity of DLD-1 cells in vitro.
Overexpression of ZEB2 at the invasion front correlates with tumor progression and predicts cancer-specific survival in primary colorectal cancer. Therefore, ZEB2 may be interesting as biomarker and potential target for treatment of colorectal cancer.
Clinical Cancer Research 12/2011; 17(24):7654-63. DOI:10.1158/1078-0432.CCR-10-2816 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The regulation of neutrophil lifespan by induction of apoptosis is critical for maintaining an effective host response and preventing excessive inflammation. The hypoxia-inducible factor (HIF) oxygen-sensing pathway has a major effect on the susceptibility of neutrophils to apoptosis, with a marked delay in cell death observed under hypoxic conditions. HIF expression and transcriptional activity are regulated by the oxygen-sensitive prolyl hydroxylases (PHD1-3), but the role of PHDs in neutrophil survival is unclear. We examined PHD expression in human neutrophils and found that PHD3 was strongly induced in response to hypoxia and inflammatory stimuli in vitro and in vivo. Using neutrophils from mice deficient in Phd3, we demonstrated a unique role for Phd3 in prolonging neutrophil survival during hypoxia, distinct from other hypoxia-associated changes in neutrophil function and metabolic activity. Moreover, this selective defect in neutrophil survival occurred in the presence of preserved HIF transcriptional activity but was associated with upregulation of the proapoptotic mediator Siva1 and loss of its binding target Bcl-xL. In vivo, using an acute lung injury model, we observed increased levels of neutrophil apoptosis and clearance in Phd3-deficient mice compared with WT controls. We also observed reduced neutrophilic inflammation in an acute mouse model of colitis. These data support what we believe to be a novel function for PHD3 in regulating neutrophil survival in hypoxia and may enable the development of new therapeutics for inflammatory disease.
The Journal of clinical investigation 03/2011; 121(3):1053-63. DOI:10.1172/JCI43273 · 13.22 Impact Factor