Thomas A. Werner

Prognostic and Clinicopathological Significance of Survivin in Colorectal Cancer: A Meta-Analysis

Survivin/BIRC5 is a potentially interesting prognostic marker and therapeutic target in colorectal cancer (CRC). However, the available data on survivin expression in CRC are heterogeneous. Thus, to clarify the prognostic relevance of survivin in patients with CRC and its association with clinicopathological parameters we performed a meta-analysis. We screened PubMed and EMBASE for those studies that investigated the prognostic value of survivin and its association with clinicopathological parameters in CRC. Data from eligible studies were extracted and included into the meta-analyses using a random effects model. Electronical literature search identified 15 studies including 1934 patients with CRC mostly detecting survivin by immunohistochemistry (IHC). Pooled hazard ratios of 11 studies that performed survival analysis revealed a positive correlation between survivin expression and poor prognosis (HR 1.93; 95% CI: 1.55–2.42; P<0.00001; I2 = 23%). Subgroup analyses with respect to the detection method, HR estimation, global quality score and the country of origin in which the study was conducted supported the stability of this observation. In addition, meta-analyses revealed a significant association between expression of survivin and the presence of lymph node metastases (OR: 0.37; 95% CI: 0.19–0.75; I2 = 61%) or blood vessel invasion (OR: 0.50; 95% CI: 0.28–0.90; I2 = 0%). Expression of survivin indicates poor prognosis and a pro-metastatic phenotype and may be useful in identifying a subgroup of patients that could benefit from a targeted therapy against survivin in CRC.

Survivin and XIAP: two valuable biomarkers in medullary thyroid carcinoma

Background:Medullary thyroid carcinoma (MTC) accounts for ∼5% of all thyroid malignancies. To date, surgery is the first-line therapy with curative intention. However, for advanced MTC, conventional chemotherapeutic agents do not provide convincing results. Therefore, the identification of biomarkers that can be antagonised by small-molecule therapeutics may lead to novel encouraging treatment options.Methods:Seventy-nine patients with surgically resected and histologically confirmed MTC were included in this study. Tissue microarrays were constructed to assess the relationship between inhibitor of apoptosis proteins (IAPs) survivin or XIAP expression levels and clinicopathological variables as well as overall survival.Results:High survivin or XIAP expression was associated with an advanced T-stage and metastatic disease. Whereas tissue expression levels of survivin correlated with serum calcitonin levels, XIAP was overexpressed in the subgroup of patients with sporadic MTC. Both IAPs were negatively associated with patient survival in the multivariate Cox regressions analysis (survivin: hazard ratio (HR) 1.62; 95% confidence interval (CI): 1.21–2.16; P=0.001; XIAP: HR 1.78; 95% CI: 1.16–2.72; P=0.008).Conclusions:Survivin and XIAP demonstrate distinct expression patterns in MTCs, which are associated with advanced disease and poor prognosis. We thus provide first evidence that both IAPs might serve as viable targets in patients with MTC.

Role of microRNAs Located on Chromosome Arm 10q in Malignant Gliomas.

Deletions of chromosome arm 10q are found in most glioblastomas and subsets of lower grade gliomas. Mutations in the PTEN gene at 10q23.3 are restricted to less than half of the 10q‐deleted gliomas, suggesting additional glioma‐associated tumor suppressors on 10q. We investigated 64 astrocytic gliomas of different malignancy grades for aberrant expression of 16 microRNAs (miRNAs) on 10q. Thereby, we identified four miRNAs (miR‐107, miR‐146b‐5p, miR‐346, miR‐1287‐5p) whose expression was frequently down‐regulated in anaplastic astrocytomas and/or glioblastomas. DNA methylation analyses revealed 5′‐CpG site hypermethylation of miR‐346 in more than two‐thirds of primary glioblastomas, while aberrant 5′‐CpG site methylation of miR‐146b‐5p was frequent in IDH1‐mutant astrocytomas and secondary glioblastomas. Overexpression of either of the four miRNAs in glioma cell lines reduced cell proliferation and/or increased caspase‐3/7 activity. Expression analyses of miRNA overexpressing glioma cells and 3′‐untranslated region luciferase reporter gene assays revealed evidence that these miRNAs post‐transcriptionally regulate expression of glioma‐relevant genes, including CDK6 (miR‐107), EGFR (miR‐146b‐5p, miR‐1287‐5p), TERT and SEMA6A (miR‐346), all of which are overexpressed in malignant gliomas in situ. In summary, we show that the 10q‐located miRNAs miR‐107, miR‐146b‐5p, miR‐346 and miR‐1287‐5p are frequently down‐regulated in malignant gliomas and thereby may support overexpression of important glioma growth‐promoting genes.

Preclinical assesement of survivin and XIAP as prognostic biomarkers and therapeutic targets in gastroenteropancreatic neuroendocrine neoplasia

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) represent a rare and heterogenous tumor entity. Importantly, the highly proliferative subgroup of neuroendocrine carcinoma (GEP-NEC) is characterized by high resistance to conventional chemotherapy. Consequently, there is an urgent need to identify novel therapeutic targets, especially for GEP-NEC. Thus, we focused on Inhibitor of apoptosis protein (IAP) family members survivin and XIAP that orchestrate inhibition of apoptosis, induce resistance against chemotherapeutics and facilitate tumor metastasis. Copy number gains (CNGs) could be detected by microarray comparative genomic hybridization for survivin and XIAP in 60 % and 26.7 % of all GEP-NENs, respectively. Immunohistochemical staining of tissue specimens from 77 consecutive patients with GEP-NEN demonstrated increased survivin protein expression levels in tissue specimens of highly proliferative GEP-NEC or GEP-NEN located in the stomach and colon. In contrast, XIAP overexpression was associated with advanced tumor stages. Knockdown of survivin and XIAP markedly reduced cell proliferation and tumor growth. In vitro, YM155 induced apoptotic cell death accompanied by a reduction in cell proliferation and inhibited GEP-NEC xenograft growth. Taken together, our data provide evidence for a biological relevance of these IAPs in GEP-NEN and support a potential role of survivin as therapeutic target especially in the subgroup of aggressive GEP-NEC.

Encapsulation status of papillary thyroid microcarcinomas is associated with the risk of lymph node metastases and tumor multifocality.

The management of papillary microcarcinoma (PMC) of the thyroid is controversial, especially after partial thyroid resection for benign thyroid disease. In order to detect prognostic factors for PMC, we analyzed 116 patients with PMC for encapsulation status and lymph node metastases. Between 10/1992 and 12/2010, 116 patients with PMC have been operated in our department (87 females, 29 males, median age 49 years). Eighty per cent of PMCs were diagnosed postoperatively. Seventy-six patients (66%) received a more extended resection with either thyroidectomy, near total thyroidectomy, or Dunhill operation either primarily or after completion operation, whereas 40 patients (34%) had only partial resection. Fifty patients (43%) received radioiodine (RIA) ablation. Lymph node metastases were found in 21 patients (18%). Univariate analysis showed four risk factors to be significantly associated with the risk of lymph node metastasis (p<0.05): male gender, younger age, age group<50 years and nonencapsulation of the tumor. Multivariate analysis demonstrated statistical significance for gender and tumor capsulation status. The tumor capsulation status also correlated with tumor multifocality. Our data show that the risk of lymph node metastases is significantly higher in partially or nonencapsulated PMC than in encapsulated specimens. We therefore suggest that the WHO classification should be extended to a compulsory notification of the encapsulation status in PMC.

IAPs cause resistance to TRAIL-dependent apoptosis in follicular thyroid cancer

Follicular thyroid cancers (FTC) excellent long-term prognosis is mainly dependent on postoperative radioactive iodine (RAI) treatment. However, once the tumour becomes refractory, the 10-year disease-specific survival rate drops below 10%. The aim of our study was to evaluate the prognostic and biological role of the TRAIL system in FTC and to elucidate the influence of small-molecule-mediated antagonisation of inhibitor of apoptosis proteins (IAPs) on TRAIL sensitivity in vitro Tissue microarrays were constructed from forty-four patients with histologically confirmed FTC. Expression levels of TRAIL and its receptors were correlated with clinicopathological data and overall as well as recurrence-free survival. Non-iodine-retaining FTC cell lines TT2609-bib2 and FTC133 were treated with recombinant human TRAIL alone and in combination with Smac mimetics GDC-0152 or Birinapant. TRAIL-R2/DR5 as well as TRAIL-R3/DcR1 and TRAIL-R4/DcR2 were significantly higher expressed in advanced tumour stages. Both decoy receptors were negatively associated with recurrence-free and overall survival. TRAIL-R4/DcR2 additionally proved to be an independent negative prognostic marker in FTC (HR = 1.446, 95% CI: 1.144-1.826; P < 0.001). In vitro, the co-incubation of Birinapant or GDC-0152 with rh-TRAIL-sensitised FTC cell lines for TRAIL-induced apoptosis, through degradation of cIAP1/2. The TRAIL system plays an important role in FTC tumour biology. Its decoy receptors are associated with poor prognosis as well as earlier recurrence. The specific degradation of cIAP1/2 sensitises FTC cells to TRAIL-induced apoptosis and might highlight a new point of attack in patients with RAI refractory disease.

Survivin and XIAP – two potential biological targets in follicular thyroid carcinoma

Follicular thyroid carcinoma’s (FTC) overall good prognosis deteriorates if the tumour fails to retain radioactive iodine. Therefore, new druggable targets are in high demand for this subset of patients. Here, we investigated the prognostic and biological role of survivin and XIAP in FTC. Survivin and XIAP expression was investigated in 44 FTC and corresponding non-neoplastic thyroid specimens using tissue microarrays. Inhibition of both inhibitor of apoptosis proteins (IAP) was induced by shRNAs or specific small molecule antagonists and functional changes were investigated in vitro and in vivo. Survivin and XIAP were solely expressed in FTC tissue. Survivin expression correlated with an advanced tumour stage and recurrent disease. In addition, survivin proved to be an independent negative prognostic marker. Survivin or XIAP knockdown caused a significant reduction in cell viability and proliferation, activated caspase3/7 and was associated with a reduced tumour growth in vivo. IAP-targeting compounds induced a decrease of cell viability, proliferation and cell cycle activity accompanied by an increase in apoptosis. Additionally, YM155 a small molecule inhibitor of survivin expression significantly inhibited tumour growth in vivo. Both IAPs demonstrate significant functional implications in the oncogenesis of FTCs and thus prove to be viable targets in patients with advanced FTC.

CXCR4/CXCR7/CXCL12 axis promotes an invasive phenotype in medullary thyroid carcinoma

Background:Medullary thyroid carcinoma (MTC) is a rare and challenging endocrine malignancy. Once spread, the therapeutic options are limited and the outcome poor. For these patients, the identification of new druggable biological markers is of great importance. Here, we investigated the prognostic and biological role of the C-X-C chemokine receptors type 4 and 7 (CXCR4/7) in MTC.Methods:Eighty-six MTC and corresponding non-neoplastic thyroid specimens were immunohistochemically stained for CXCR4/7 using tissue microarray technology and expression levels correlated with clinicopathological variables. Medullary thyroid carcinoma cell line TT was treated with recombinant human SDF1α/CXCL12 (rh-SDF1α) and CXCR4 antagonists AMD3100 and WZ811. Changes in cell cycle activation, tumour cell invasiveness as well as changes in mRNA expression levels of genes associated with epithelial–mesenchymal transition (EMT) were investigated.Results:High CXCR4 expression was associated with large tumour size and metastatic disease. CXCR4 antagonists significantly reduced tumour cell invasiveness, while the treatment with rh-SDF1α stimulated invasive growth, caused cell cycle activation and induced EMT.Conclusions:The CXCR4/CXCR7/CXCL12 axis plays an important role in MTC. We provide first evidence that the chemokine receptors might serve as potential therapeutic targets in patients with advanced MTC and offer new valuable insight into the underlying molecular machinery of metastatic MTC.

Clinicopathological and functional implications of the inhibitor of apoptosis proteins survivin and XIAP in esophageal cancer

Based on their overexpression and important roles in progression and therapy-resistance in malignant diseases, the inhibitor of apoptosis protein family (IAP) members, survivin and X-linked inhibitor of apoptosis protein (XIAP), represent attractive candidates for targeted therapy. The present study investigated the prognostic and biological relevance of survivin and XIAP in esophageal squamous-cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Survivin and XIAP expression was analyzed by immunohistochemistry using tissue microarrays containing 120 ESCC and 90 EAC samples as well as the corresponding non-neoplastic esophageal mucosa samples. IAP expression levels were then correlated to clinicopathological parameters and overall survival to identify any associations. In addition, esophageal cancer cell lines were treated with the survivin inhibitor YM155, and the XIAP inhibitors Birinapant and GDC-0152 in vitro. Survivin and XIAP expression were significantly increased in EAC and ESCC when compared with tumor-adjacent mucosa. In patients with ESCC XIAP expression was associated with female gender and advanced tumor stages, and nuclear survivin expression was associated with poor grading. High XIAP expression was identified as an independent negative prognostic marker in ESCC. By contrast, XIAP inhibitors did not affect cancer cell viability in vitro, and the small molecule survivin inhibitor YM155 significantly reduced cell viability and proliferation in esophageal cancer cell lines. Western blot analysis revealed a dose dependent decrease of survivin accompanied by an increased poly (adenosine diphosphate-ribose) polymerase cleavage following YM155 treatment. These findings underline the potential role of survivin and XIAP in the oncogenesis of esophageal cancer and provide a rationale for future clinical studies investigating the therapeutic efficacy of IAP directed therapies in patients with esophageal cancer.

Establishment and characterization of a novel cell line derived from a small cell neuroendocrine carcinoma of the anal canal

Background: Gastroenteropancreatic neuroendocrine carcinomas (GEP-NECs) are biologically aggressive tumors, associated with a very poor survival. Due to their rarity, our knowledge on GEP-NEC biology is very limited. The aim of this study was to establish a GEP-NEC cell line model that might contribute to a better understanding of this rare malignant disease to further develop novel therapeutic approaches in preclinical studies. Methods: Small cell neuroendocrine cancer cell line NEC-DUE3 was derived from a lymph node metastasis of a neuroendocrine carcinoma (NEC) located at the anal canal. Morphological characteristics and the expression of neuroendocrine markers were comprehensively investigated. For genetic profiling, NEC-DUE3 cells were analyzed by DNA fingerprinting. Chromosomal aberrations were mapped by array comparative genomic hybridization. NEC-DUE3 cell tumorigenicity was evaluated in vivo and the sensitivity to chemotherapeutic agents was assessed in vitro. Results: NEC-DUE3 cells were characterized by the expression of molecular markers that are commonly observed in GEP-NECs, were sensitive to treatment with cisplatin, and able to form tumors in immunodeficient mice. Conclusion: We established and characterized the first small cell GEP-NEC cell line that may serve as a valuable tool to create a better understanding of the biology of these rare tumors and to develop novel treatment strategies.