Henning Wege

Presence of EpCAM-positive circulating tumor cells as biomarker for systemic disease strongly correlates to survival in patients with hepatocellular carcinoma

Current imaging technologies do not sufficiently detect micrometastasis and therefore do not allow adequate stratification of patients with hepatocellular carcinoma (HCC) for curative or systemic therapy. In HCC, presence of stem cell‐like, epithelial cell adhesion molecule (EpCAM)‐positive cells correlates with tumor aggressiveness and formation of metastasis. Therefore, we investigated the prognostic relevance of EpCAM‐positive circulating tumor cells (CTCs) in patients with HCC. Blood from 78 patients (19 patients in the control cohort and 59 patients with HCC) was tested for CTCs with the CellSearch™ system. Correlation analysis to overall survival (OS), the Barcelona Clinic Liver Cancer (BCLC) staging system, macroscopic and microscopic vascular invasion and alpha‐fetoprotein (AFP) levels were performed. We detected ≥1 CTC in 18/59 HCC patients and in 1/19 patients with cirrhosis or benign hepatic tumor (p = 0.026). OS was significantly shorter (460 vs. 746 days) in the CTC‐positive cohort (p = 0.017). Comparing BCLC stages, significant differences in CTC detection rates were also observed: BCLC stages A 1/9, B 6/31 and C 11/19 (p = 0.006). Ten of 18 patients with macroscopic and 10/16 patients with microscopic vascular invasion exhibited positive findings in CTC testing (p = 0.004 and p = 0.006). Furthermore, CTC results correlated to AFP (cutoff > 400 ng/mL) levels (p = 0.050). Our study demonstrates frequent presence of EpCAM‐positive CTC in patients with intermediate or advanced HCC and its prognostic value for OS with possible implications for future treatment stratification.

Functional p53 is required for effective execution of telomerase inhibition in BCR-ABL-positive CML cells.

OBJECTIVE In chronic myeloid leukemia (CML), increased cellular turnover of hematopoietic cells driven by the oncogene BCR-ABL leads to accelerated telomere shortening despite increased telomerase activity. It has been postulated that shortened telomeres, particularly in the context of increased telomerase activity, might facilitate accumulation of genetic aberrations and, consequently, disease progression from chronic phase to accelerated phase and blast crisis. Therefore, inhibition of telomerase might be a promising approach in CML therapy. MATERIAL AND METHODS To investigate the therapeutic potential of telomerase inhibition in this model disorder, we used a small molecule telomerase inhibitor, BIBR1532 as well as expression of a dominant-negative mutant of hTERT (DNhTERT-IRES-GFP) in the p53-negative CML blast crisis cell line K562 and characterized the effects in long-term culture. Furthermore, we expressed an inducible p53 construct (vector pBabe-p53ER(tam)) via retroviral transduction in cells with critically short telomeres and in cells with a normal telomere length to explain the role of the tumor suppressor in response to critical telomere shortening in BCR-ABL-positive cells. RESULTS BIBR1532-treated bulk cultures did not show altered growth kinetics despite significant telomere shortening to a critical length of approximately 5 kb. In comparison, DNhTERT-expressing clones either lost telomere length, leading to a significant but transient slow down in proliferation but eventually all escaped senescence/crisis (group I) or, alternatively, remained virtually unaffected despite measurable telomerase inhibition (group II). Further analyses of group I clones revealed impaired DNA damage response and an accumulation of dicentric chromosomes. However, upon restoration of p53 in telomerase-negative K562 clones with critically short telomeres, immediate reinduction of apoptosis and complete eradication of cells was observed, whereas vector control cells continued to escape from crisis. CONCLUSIONS These results suggest that the success of strategies aimed at telomerase inhibition in CML is highly dependent on the presence of functional p53 and should be explored preferentially in chronic phase CML.

Telomeres and telomerase in chronic myeloid leukaemia: impact for pathogenesis, disease progression and targeted therapy.

Telomeres are specialized structures localized at the end of human chromosomes. Due to the end replication problem, each cell division results in a loss of telomeric repeats in normal somatic cells. In germ line and stem cells, the multicomponent enzyme telomerase maintains the length of telomere repeats. However, elevated telomerase activity has also been reported in the majority of solid tumours as well as in acute and chronic leukaemia. Chronic myeloid leukaemia (CML) serves as a model disease to study telomere biology in clonal myeloproliferative disorders. In CML, telomere shortening correlates with disease stage, duration of chronic phase (CP), prognosis measured by the Hasford risk score and the response to disease‐modifying therapeutics such as the tyrosine kinase inhibitor Imatinib. In addition, telomerase activity (TA) is already increased in CP CML and further upregulated with disease progression to accelerated phase and blast crisis (BC). Furthermore, a correlation of TA with increased genetic instability as well as a shorter survival of the patients has been reported. Here, we review the current state of knowledge of the role of telomere and telomerase biology in CML and discuss the possible impact of novel treatment approaches. Copyright

Telomerase activation in liver regeneration and hepatocarcinogenesis: Dr. Jekyll or Mr. Hyde?

The liver has a remarkable capability to restore its functional capacity following liver injury. According to the current paradigm, differentiated and usually quiescent hepatocytes are the primary cell type responsible for liver repair. As reserve compartment, bipotent hepatic progenitor cells are activated, especially if extensive loss or damage of hepatocytes with impaired replication occurs, e.g. in cirrhotic liver tissue. Recently, animal studies have suggested that liver regeneration following partial hepatectomy is associated with telomerase activation. Telomerase, a ribonucleoprotein with reverse transcriptase activity, plays a pivotal role in maintaining telomere length and chromosomal stability in proliferating cells. In cells lacking telomerase activity, replication-associated telomere shortening limits the replicative lifespan. Therefore, in the context of liver regeneration, telomerase activation might be a cellular mechanism to confer an extended lifespan to replicating hepatocytes and hepatic progenitor cells. On the other hand, high levels of telomerase activity are a hallmark of cancer, including hepatocellular carcinoma. Moreover, recent data indicate that telomerase activation may be an early event in hepatocarcinogenesis. At present, it is unclear, whether telomerase activation preserves the non-malignant phenotype and replicative longevity of liver cells or constitutes an early alteration obligatory for an unlimited proliferation and malignant transformation.

Inhibition of experimental HCC growth in mice by use of the kinase inhibitor DMAT

The multi-kinase-inhibitor Sorafenib has been shown to prolong survival of patients suffering from hepatocellular carcinoma (HCC). We investigated effects of the serine/threonine kinase inhibitor 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) on experimental HCC growth, and identified mechanisms and target kinases of DMAT. Our results show that DMAT application in vivo reduced tumor growth in a xenotransplant model by interference with tumor cell proliferation. Biochemical parameters and histology following DMAT administration revealed no alterations in liver tissue. Similar to Sorafenib, DMAT interfered with NFκB activation and Wnt-signaling. Of the kinases inhibited by DMAT at almost equimolar IC50, CK2 and PIM-3 were found to be over-expressed or more active in hepatoma cells and human HCC tissue. Knockdown of PIM-3 or CK2 by shRNA revealed that both kinases are important for hepatoma cell proliferation and survival. In conclusion, DMAT reduces HCC growth by interference with NFκB- and Wnt-signaling. PIM-3 and CK2 seem to be important target kinases. Inhibition of these kinases by application of inhibitors, e.g., DMAT, might represent a promising therapeutic approach in future HCC therapy.

Different Soluble Forms of the Interleukin-6 Family Signal Transducer gp130 Fine-tune the Blockade of Interleukin-6 Trans-signaling.

Soluble forms of the IL-6 receptor (sIL-6R) bind to the cytokine IL-6 with similar affinity as the membrane-bound IL-6R. IL-6·sIL-6R complexes initiate IL-6 trans-signaling via activation of the ubiquitously expressed membrane-bound β-receptor glycoprotein 130 (gp130). Inhibition of IL-6 trans-signaling has been shown to be favorable in numerous inflammatory diseases. Furthermore, different soluble forms of gp130 (sgp130) exist that, together with the sIL-6R, are thought to form a buffer for IL-6 in the blood. However, a functional role for the different sgp130 forms has not been described to date. Here we demonstrate that the metalloproteases ADAM10 and ADAM17 can produce sgp130 by ectodomain shedding of gp130, even though this mechanism only accounts for a minor proportion of sgp130 in the circulation. We further show that full-length sgp130 and the shorter forms sgp130-rheumatoid arthritis-associated peptide (RAPS) and sgp130-E10 are differentially expressed in a cell type- specific manner. Remarkably, full-length sgp130 is expressed by monocytes, but this expression is completely lost during differentiation into macrophages in vitro. Using genetically engineered murine pre-B cells that secrete different forms of sgp130, we found that these secreted sgp130 proteins are able to prevent trans-signaling-driven cell proliferation of the secreting cells, whereas conditioned supernatant from these cells failed to block IL-6 trans-signaling in other cells. Thus, our data suggest that the different sgp130 forms are released from cells into their immediate surroundings and appear to form cell-associated gradients to modulate their own susceptibility for IL-6 trans-signaling.

Forced Activation of β-Catenin Signaling Supports the Transformation of hTERT-Immortalized Human Fetal Hepatocytes

Hepatocarcinogenesis is a multistep process driving the progressive transformation of normal liver cells into highly malignant derivatives. Unlimited proliferation and telomere maintenance have been recognized as prerequisites for the development of liver cancer. Moreover, recent studies identified illegitimate β-catenin signaling as relevant hit in a considerable subset of patients. To further investigate the currently not well-understood malignant evolution driven by telomerase and β-catenin, we monitored cytogenetic and phenotypic alterations in untransformed telomerase-immortalized human fetal hepatocytes following forced activation of β-catenin signaling. As expected, constitutive activation of β-catenin signaling significantly enhanced proliferation with decreasing serum dependence. Previously intact contact inhibition was almost completely eliminated. Interestingly, after several passages in cell culture, immortalized clones with dominant-positive β-catenin signaling acquired additional chromosomal aberrations, in particular translocations, anchorage-independent growth capabilities, and formed tumors in athymic nude mice. In further support for the driving role of β-catenin during hepatocarcinogenesis, improved colony growth in soft agar and accelerated tumor formation was also confirmed in Huh7 cells following stable expression of the constitutively active S33Y β-catenin mutant. Telomerase inhibition showed that short-term expansion of transformed clones was not telomerase dependent. Finally, cancer pathway profiling in derived tumors revealed upregulation of characteristic genes associated with invasion and angiogenesis. In conclusion, illegitimate activation of β-catenin signaling enhances the transformation from immortalization to malignant growth in human fetal hepatocytes. Our data functionally confirm a permissive role for β-catenin signaling in the initial phase of hepatocarcinogenesis. Mol Cancer Res; 9(9); 1222–31. ©2011 AACR.

Regeneration in pig livers by compensatory hyperplasia induces high levels of telomerase activity.

BackgroundSeveral highly proliferative human cells transiently activate telomerase, a ribonucleoprotein with reverse transcriptase activity, to counterbalance replication-associated telomere erosion and to increase stress resistance. Quiescent human hepatocytes exhibit very low or undetectable levels of telomerase activity. However, hepatocytes display a remarkable proliferative capability following liver injury. To investigate whether liver regeneration by compensatory hyperplasia is associated with telomerase activation, we measured telomerase activity in pig livers after 70 to 80% partial hepatectomy using a fully quantitative real-time telomeric repeat amplification protocol. In contrast to commonly studied inbred laboratory mouse strains, telomere length and telomerase activity in porcine tissues are comparable to humans.ResultsFollowing partial hepatectomy, histology revealed mitotic hepatocytes as marker for compensatory hyperplasia. As expected, there was no induction of inflammation. Telomerase activity increased significantly showing the highest levels (5-fold upregulation) in pigs treated with partial hepatectomy and hepatic decompression. Moreover, telomerase activity significantly correlated to the number of mitotic hepatocytes.ConclusionOur data demonstrate telomerase activation in liver regeneration by compensatory hyperplasia in a large animal model with telomere biology comparable to humans. Telomerase activation may constitute a mechanism to protect proliferating liver cells against telomere shortening and oxidative stress.

Transduction of fetal mice with a feline lentiviral vector induces liver tumors which exhibit an E2F activation signature.

Lentiviral vectors are widely used in basic research and clinical applications for gene transfer and long-term expression; however, safety issues have not yet been completely resolved. In this study, we characterized hepatocarcinomas that developed in mice 1 year after in utero administration of a feline-derived lentiviral vector. Mapped viral integration sites differed among tumors and did not coincide with the regions of chromosomal aberrations. Furthermore, gene expression profiling revealed that no known cancer-associated genes were deregulated in the vicinity of viral integrations. Nevertheless, five of the six tumors exhibited highly significant upregulation of E2F target genes, of which a majority are associated with oncogenesis, DNA damage response, and chromosomal instability. We further show in vivo and in vitro that E2F activation occurs early on following transduction of both fetal mice and cultured human hepatocytes. On the basis of the similarities in E2F target gene expression patterns among tumors and the lack of evidence implicating insertional mutagenesis, we propose that transduction of fetal mice with a feline lentiviral vector induces E2F-mediated major cellular processes that drive hepatocytes toward uncontrolled proliferation culminating in tumorigenesis.

New foe treated with old guns – supportive role of steroids in the treatment of acute severe hepatitis E

BackgroundAutochthonous hepatitis E has been observed with growing incidence in industrialized countries. Hepatitis E virus infection causes an acute hepatitis with spontaneous resolution in the majority of cases. However, in individual cases, hepatitis E may lead to life-threatening acute liver failure. In this report, we describe a case of acute liver injury caused by an autochthonous hepatitis E that resolved under steroid treatment. To our knowledge, this is the first case report describing supportive steroid monotherapy for acute liver injury due to hepatitis E.Case presentationA 63-year-old Caucasian male presented with acute liver injury of unknown origin. After excluding the most prevalent causes of acute liver injury, liver histology revealed signs of immune-mediated toxic or drug-induced liver injury. Therefore, immunosuppressive treatment with prednisolone was started. After initialization of steroid treatment, polymerase chain reaction analyses of peripheral blood and liver tissue revealed an acute hepatitis E virus infection (genotype 3). Under sustained steroid treatment, acute liver injury improved and hepatitis E infection resolved.ConclusionSteroid treatment might be an option to prevent progress of life-threatening liver failure and liver transplantation in patients with hepatitis E-induced acute liver injury and high-grade inflammation.