Christian Röder

TRAIL promotes metastasis of human pancreatic ductal adenocarcinoma.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention for its potential use in tumor therapy, as some recombinant variants of this ligand induce apoptosis in tumor cells without harming most normal cells. Here, we show that TRAIL strongly induces the expression of the proinflammatory cytokines interleukin-8 and monocyte chemoattractant protein 1 and enhances the invasion of apoptosis-resistant pancreatic ductal adenocarcinoma cells in vitro by upregulation of the urokinase-type plasminogen activator expression. Most importantly, we also demonstrate for the first time that TRAIL treatment results in strongly increased distant metastasis of pancreatic tumors in vivo. We orthotopically transplanted human pancreatic ductal adenocarcinoma cells to the pancreata of severe combined immunodeficiency mice and observed a dramatic increase in metastatic spread including a sixfold increase in the volume and fourfold increase in the number of liver metastases upon TRAIL treatment. Our results point to the necessity to carefully evaluate in vivo side effects of TRAIL and to select therapy conditions that not only enhance apoptosis induction but in addition prevent proinvasive and proinflammatory non-apoptotic TRAIL signaling.

CD95 and TRAIL receptor-mediated activation of protein kinase C and NF-kappaB contributes to apoptosis resistance in ductal pancreatic adenocarcinoma cells.

The molecular alterations in tumour cells leading to resistance towards apoptosis induced by CD95 and TRAIL-receptors are not fully understood. We report here that the stimulation of the CD95- and TRAIL-resistant human pancreatic adenocarcinoma cell line PancTuI with an agonistic anti-CD95 antibody or TRAIL resulted in activation of protein kinase C and NF-κB. Inhibition of protein kinase C by Gö6983 sensitized these cells to apoptotic challenges and strongly diminished activation of NF-κB by anti-CD95 and TRAIL. Similarly, inhibition of NF-κB by MG132 or by transient transfection with a dominant negative mutant of IκBα restored the responsiveness of PancTuI cells to both death ligands. In the CD95 and TRAIL-sensitive cell line Colo357 the induction of protein kinase C and NF-κB following activation of CD95 and TRAIL-R was very moderate compared with PancTuI cells. However, pre-incubation of these cells with PMA strongly reduced their apoptotic response to anti-CD95 and TRAIL. Taken together, we show that activation of protein kinase C operates directly in a death receptor-dependent manner in PancTuI cells and protect pancreatic tumour cells from anti-CD95 and TRAIL-mediated apoptosis by preventing the loss ΔΨm and Cytochrome c release as well as by induction of NF-κB.

The detection of disseminated tumor cells in bone marrow from colorectal-cancer patients by a cytokeratin-20-specific nested reverse-transcriptase-polymerase-chain reaction is related to the stage of disease.

The dissemination of cancer cells is a prerequisite in the development of micrometastases and solid metastases. Our previous examinations of these cells were based on immunocytological staining of tumor‐associated antigens and cytokeratins. We have now developed a highly sensitive and specific detection method based on a nested reverse‐transcriptase‐polymerase‐chain reaction (RT‐PCR) of cytokeratin‐20 (CK‐20) mRNA. Using this method, we examined the bone marrow of 57 patients with colorectal cancer and detected increasing numbers of CK‐20‐positive samples, depending on the UICC stage. Some 35% of all bone‐marrow samples tested positive for CK‐20: none were found in colorectal cancer stage I, 24% were in stage II, 31% in stage III and 71% in stage IV. Investigation of bone‐marrow specimens of patients with pancreatic cancer showed that 4 out of 11 patients were positive for CK‐20 mRNA. To confirm that sample positivity for CK‐20 expression was due to disseminated tumor cells, we examined bone marrow from a control group (n = 16) without apparent carcinoma. In this group, 15 out of 16 donors were CK‐20‐negative, while one donor with familial adenomatous polyposis showed a CK‐20‐specific signal.

Increased proteasome subunit protein expression and proteasome activity in colon cancer relate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2)

An elevated proteasome activity contributes to tumorigenesis, particularly by providing cancer cells with antiapoptotic protection and efficient clearance from irregular proteins. Still, the underlying mechanisms are poorly known. In this study, we report that in colon cancer patients, higher proteasome activity was detected in tumoral tissue compared with surrounding normal tissue, and also that increased levels of proteasomal subunit proteins, such as S5a/PSMD4 and α-5/PSMA5, could be detected. Colon tumors showed higher nuclear levels of nuclear factor E2-related factor 2 (Nrf2), a transcription factor supposed to be involved in the control of proteasomal subunit protein expression. The induction or overexpression of Nrf2 led to stronger S5a and α-5 expression in the human colon cancer cell lines, Colo320 and Lovo, as well as in NCM460 colonocytes along with higher proteasome activity. The small interfering RNA (siRNA)-mediated Nrf2 knockdown decreased S5a and α-5 expression and reduced proteasome activity. Additionally, Nrf2-dependent S5a and α-5 expression conferred protection from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, an effect preceded by an increased nuclear factor (NF)-κB activation and higher expression of antiapoptotic NF-κB target genes. These findings point to an important role of Nrf2 in the gain of proteasome activity, thereby contributing to colorectal carcinogenesis. Nrf2 may therefore serve as a potential target in anticancer therapy.

PKC|[mu]| prevents CD95-mediated apoptosis and enhances proliferation in pancreatic tumour cells

Loss of growth control and a marked resistance to apoptosis are considered major mechanisms driving tumour progression. Protein kinases C (PKC) have been shown to be important in the regulation of proliferation and apoptosis. In this report, we investigated the role of the PKC-like kinase PKCμ in the control of these processes in pancreatic adenocarcinoma cells. We demonstrate that in these cells, PKCμ expression strongly correlates with resistance to CD95-induced apoptosis. Inhibition of PKCμ with Goe6983 sensitized resistant cells to CD95-induced apoptosis. In CD95-sensitive Colo357 cells, forced overexpression of PKCμ strongly reduced CD95-mediated apoptosis, an effect that could be reversed by pretreatment with Goe6983. In addition, PKCμ overexpression led to a strongly enhanced cell growth and to a significant increase of telomerase activity. In an attempt to identify the signalling pathways affected by PKCμ, we identified the antiapoptotic proteins c-FLIPL and survivin to be strongly upregulated in PKCμ overexpressing cells. Immunohistochemical analysis of pancreatic tumour tissue of 48 patients and 10 normal pancreatic tissues revealed marked overexpression of PKCμ in tumours. In conclusion, we showed that PKCμ controls proliferative, as well as anti-apoptotic, signalling pathways and therefore plays an important role in acquiring the malignant phenotype of pancreatic tumours.

CD95 and TRAF2 promote invasiveness of pancreatic cancer cells

Pancreatic adenocarcinoma represents a tumor type with extremely poor prognosis. High apoptosis resistance and a strong invasive and early metastatic potential contribute to its highly malignant phenotype. Here we identified the death receptor adaptor molecule TRAF2 as a key player in pancreatic cancer pathophysiology. Using immunohistochemistry and Western blot analysis we found TRAF2 overexpressed in 34 of 36 pancreatic tumor samples as well as in pancreatic tumor cell lines resistant to CD95‐mediated apoptosis. The high TRAF2 protein level was not related to chromosomal changes, as monitored by FISH analysis. Instead, the NF‐κB‐and MEK‐signaling pathways were involved. Introduction of a TRAF2 expression vector in CD95‐sensitive Colo357 cells resulted in (i) resistance to CD95‐induced apoptosis; (ii) increased constitutive NF‐κB and AP‐1 activity; and (iii) higher basal secretion of matrix metalloproteinases (MMPs), urokinase‐type plasminogen activator (uPA), and IL‐8, leading to increased invasiveness. High apoptosis resistance and uPA secretion could be reverted by TRAF2‐specific siRNA. Stimulation of TRAF2‐overexpressing cells with CD95 ligand led to induction of NF‐κB and AP‐1, enhanced IL‐8‐ and uPA‐secretion, and a further increased invasiveness. Thus, TRAF2 overexpression does not only block apoptosis induction by CD95 but also converts this death receptor into a mediator of invasiveness.

TRAIL signaling is mediated by DR4 in pancreatic tumor cells despite the expression of functional DR5

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and agonistic anti-DR4/TRAIL-R1 and anti-DR5/TRAIL-R2 antibodies are currently under clinical investigation for treatment of different malignancies. TRAIL activates DR4 and DR5 and thereby triggers apoptotic and non-apoptotic signaling pathways, but possible different roles of DR4 or DR5 in these responses has poorly been addressed so far. In the present work, we analyzed cell viability, DISC formation as well as IL-8 and NF–κB activation side by side in responses to TRAIL and agonistic antibodies against DR4 (mapatumumab) and against DR5 (lexatumumab) in pancreatic ductal adenocarcinoma cells. We found that all three reagents are able to activate cell death and pro-inflammatory signaling. Death-inducing signaling complex (DISC) analysis revealed that mapatumumab and lexatumumab induce formation of homocomplexes of either DR4 or DR5, whereas TRAIL additionally stimulated the formation of heterocomplexes of both receptors. Notably, blocking of receptors using DR4- and DR5-specific Fab fragments indicated that TRAIL exerted its function predominantly via DR4. Interestingly, inhibition of PKC by Goe6983 enabled DR5 to trigger apoptotic signaling in response to TRAIL and also strongly enhanced lexatumumab-mediated cell death. Our results suggest the existence of mechanisms that silence DR5 for TRAIL- but not for agonistic-antibody treatment.

Mucin 7 and Cytokeratin 20 as New Diagnostic Urinary Markers for Bladder Tumor

PURPOSE We determine the sensitivity and specificity of cytokeratin 20 (CK-20) and mucin 7 (MUC7) gene expression in voided urine samples taken from patients with bladder tumor and from control groups to investigate putative, noninvasive urinary markers for bladder tumor detection and monitoring. MATERIALS AND METHODS Voided urine samples were collected from 50 patients with histologically proven bladder neoplasms (pTaN0M0G1-3 in 19 and pTisN0M0G3-pT4pN1M1G3 in 31), 20 patients with urolithiasis, 20 patients with urinary tract infection, 20 patients with other urological neoplasms and 20 healthy volunteers. Total RNA was extracted from exfoliated cells collected from 200 ml. voided urine. All RNA samples were investigated by a specific CK-20 and MUC7 nested reverse transcriptase polymerase chain reaction. RESULTS The overall sensitivity of CK-20 gene expression in voided urine samples for the detection of bladder neoplasms was 78%. In contrast, voided urine samples from control patients and healthy volunteers showed a high rate of false-positive CK-20 detection resulting in a low specificity of 36%. The overall sensitivity of the MUC7 test for all bladder tumor cases was 66%. The sensitivity for papillary urothelial neoplasms (pTaN0M0G1-3) was 42% whereas analysis of the carcinoma in situ and invasive bladder cancer group (pTisN0M0G3-pT4pN1M1G3) yielded a sensitivity of 81%. The overall specificity of the MUC7 nested reverse transcriptase polymerase chain reaction method in the control groups was 80%. CONCLUSIONS A high positive CK-20 detection rate was found not only in voided urine samples from patients with bladder tumor, but also in urine specimens from control groups. Therefore, CK-20 is not a reliable urinary tumor marker for bladder neoplasms. In contrast to CK-20, analysis of MUC7 demonstrated a high sensitivity and high specificity for carcinoma in situ and invasive bladder cancer, thus fulfilling the criteria of a urinary tumor marker.

Nuclear Death Receptor TRAIL-R2 Inhibits Maturation of Let-7 and Promotes Proliferation of Pancreatic and Other Tumor Cells

BACKGROUND & AIMS Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. METHODS Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. RESULTS TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. CONCLUSIONS Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.

Cytokeratin–20 Reverse–Transcriptase Polymerase Chain Reaction as a New Tool for the Detection of Circulating Tumor Cells in Peripheral Blood and Bone Marrow of Bladder Cancer Patients

Objectives: Systemic progression is the prevalent form of bladder tumor recurrence after radical cystectomy. The ability to detect circulating tumor cells in peripheral blood or bone marrow could be of prognostic value for the disease with the consequence of early adjuvant chemotherapy. We established a sensitive and specific method using a double cytokeratin–20 (CK–20) reverse–transcriptase polymerase chain reaction (RT–PCR) to detect circulating bladder cancer cells in venous blood and bone marrow Material and Methods: The sensitivity of the detection method was determined by a serial dilution of bladder cancer cells from the cell line HT1376 in whole blood. Bone marrow from 20 bladder cancer patients was drawn prior to radical cystectomy and CK–20 cDNA was amplified by RT–PCR. Additionally, pre– and postoperative venous blood samples from 11 of these patients with bone marrow aspirates and 9 patients undergoing only transurethral resection of the bladder as well as blood samples of 25 healthy volunteers were investigated by CK–20 RT–PCR. Results: The detection limit of the method was 2 bladder cancer cells/ml whole blood containing one million peripheral blood mononuclear cells. The positive detection rate in bone marrow was 7 of 20 (35%) for bladder cancer patients of all stages. However, investigation of the preoperatively collected venous blood samples from 20 patients revealed onyl 2 positive findings, belonging to advanced tumor stages pT4pN0M0 and pT3pN2M0. In contrast, CK–20 was detected in 3 of 20 postoperatively collected venous blood samples from patients with low tumor stages (pTaNXM0 and pT1NXM0) as well as from 1 patient with pelvic lymph node metastases (pT3apN2M0). All venous blood samples of the control group (n = 25) were negative for CK–20. Conclusion: The detection of circulating bladder tumor cells in venous blood and bone marrow by the CK–20 RT–PCR is a promising approach that could improve risk assessment and the identification of bladder cancer patients who would benefit from adjuvant chemotherapy.