Karl Quint

Clinical significance of histone deacetylases 1, 2, 3, and 7: HDAC2 is an independent predictor of survival in HCC

Histone deacetylases (HDAC) are responsible for the transcriptional control of genes through chromatin remodeling and control tumor suppressor genes. In several tumors, their expression has been linked to clinicopathological factors and patient survival. This study investigates HDACs 1, 2, 3, and 7 expressions in hepatocellular carcinoma (HCC) and their correlation with clinical data and patient survival. Tissue microarrays of 170 surgically resected primary HCCs and adjacent uninvolved tissue were evaluated immunohistochemically for the expression of HDACs 1, 2, 3, 7, and Ki-67 and were analyzed with respect to clinicopathological data and patient survival. HDACs 1, 2, 3, and Ki-67 were expressed significantly higher in cancer cells compared to normal tissue (HDAC1: p = 0.034, HDACs 2 and 3 and Ki-67: p < 0.001), while HDAC7 expression did not differ between HCC and non-cancerous liver tissue. In tumor tissue HDACs 1–3 expression levels showed high concordance with each other, Ki-67 and tumor grade (p < 0.001). High HDAC2 expression was associated with poor survival in low-grade and early-stage tumors (p < 0.05). The expression of the HDACs 1, 2, and 3 (but not HDAC7) isoenzymes correlates with clinicopathological factors, and HDAC2 expression has an impact on patient survival.

The pan-deacetylase inhibitor panobinostat inhibits growth of hepatocellular carcinoma models by alternative pathways of apoptosis.

Inhibition of deacetylases represents a new treatment option for human cancer diseases. We applied the novel and potent pan-deacetylase inhibitor panobinostat (LBH589) to human hepatocellular carcinoma models and investigated by which pathways tumor cell survival is influenced. HepG2 (p53wt) and Hep3B (p53null) responded to panobinostat treatment with a reduction of cell proliferation and a significant increase in apoptotic cell death at low micromolar concentrations. Apoptosis was neither mediated by the extrinsic nor the intrinsic pathway but quantitative RT-PCR showed an upregulation of CHOP, a marker of the unfolded protein response and endoplasmic reticulum stress with subsequent activation of caspase 12. Dependent on the p53 status, a transcriptional upregulation of p21cip1/waf1, an increased phosphorylation of H2AX, and an activation of the MAPK pathway were observed. In a subcutaneous xenograft model, daily i.p. injections of 10 mg/kg panobinostat lead to a significant growth delay with prolonged overall survival, mediated by reduced tumor cell proliferation, increased apoptosis and reduced angiogenesis in tumor xenografts. Panobinostat increased the acetylation of histones H3 and H4. Panobinostat is a well tolerated new treatment option for HCC that activates alternative pathways of apoptosis, also in p53-deficient tumors.

The Expression Pattern of PDX-1, SHH, Patched and Gli-1 Is Associated with Pathological and Clinical Features in Human Pancreatic Cancer

Background and Aims: Pancreatic cancer cells have been shown to possess stem-cell-like properties, especially by reactivating embryonic transcription factors involved in tissue differentiation. We therefore investigated if and to what extent developmental genes of the human pancreas are expressed in pancreatic ductal adenocarcinomas and precursor lesions, pancreatic intraepithelial neoplasia (PanIN), and if this correlates or predicts response to treatment and overall survival. Material and Methods: Invasive ductal adenocarcinomas of the pancreas [UICC pT3pN0 (n = 13) vs. pT3pN1 (n = 25)] and tumors after neoadjuvant chemotherapy [5-fluorouracil (FU)/folic-acid and gemcitabine; UICC ypN0 (n = 7) vs. ypN1 (n = 6)] resected between 1997 and 2003 were characterized histochemically and immunohistochemically [pancreas duodenum homeobox 1 (PDX-1), Sonic hedgehog protein (SHH), Patched (Ptc) and Gli-1]. Gene distribution was compared with morphological patterns of the pancreatic carcinoma and PanIN as well as with peritumorous reactions of normal pancreas. Results: The overall expression of PDX-1, SHH, Ptc and Gli-1 was low, but showed a distinctive and topographic linkage inside pancreatic carcinomas as well as inside PanINs. Additionally, a topographic and significant association of these markers with nodal status (PDX-1, Ptc, Gli-1), tumor size (PDX-1, Gli-1) and R status (PDX-1) was found. After stratification with the strongest outcome predictor, grading, survival analysis revealed that Ptc expression in grade 2 and PDX-1 expression in grade 3 carcinomas are independent survival factors. Conclusions: Markers of pancreas development are reexpressed in invasive ductal adenocarcinomas and their expression is essentially associated with general clinical and pathological features such as survival or nodal status.

AKT inhibition by triciribine alone or as combination therapy for growth control of gastroenteropancreatic neuroendocrine tumors.

Up-regulation of phosphatidylinositol-3-kinase (PI3K)-AKT signaling facilitates tumor cell growth and inhibits cell demise. The AKT-pathway also plays an important role in cytostatic therapy resistance and response to hypoxia and angiogenesis. Using real-time cell proliferation assay we examined the potency of triciribine in three distinct neuroendocrine gastrointestinal tumor cell lines. Also we investigated triciribines induction of apoptosis and effects on a broad range of cancer-associated gene products. Furthermore, we characterized the role of PTEN as a possible predictor of sensitivity to triciribine in GEP-NETs. We also looked for additive anti-neoplastic effects of triciribine when combined with conventional cytostatic drugs or other targeted drugs, affecting different molecules of the PI3K-AKT-pathway and we assessed the potency of triciribine to inhibit tumor growth in vivo, by using the chick chorioallantoic membrane assay. Treatment of insulinoma (CM) or gut neuroendocrine tumor cells (STC-1) with triciribine significantly reduced tumor cell growth by 59% and 65%, respectively. By contrast, the highly expressing PTEN carcinoid cell line BON did not respond, even at higher doses. Combinations of triciribine with classic cytostatic drugs as well as drugs targeting other molecules of the PI3K-AKT-pathway led to synergistic anti-proliferative effects. Additional in vivo-evaluations confirmed the anti-neoplastic potency of triciribine. Thus, our data show that inhibition the AKT-pathway potently reduces the growth of GEP-NET cells alone or in combination therapies. AKT inhibition may provide a rationale for future evaluations.

Influence of stress and health-behaviour on miRNA expression.

Psychological stress is correlated with and may even cause DNA damage, which contributes to the etiology of various diseases. Recent studies point to the role of micro-RNA (miRNA), small molecules that regulate gene expression, in health and disease. This study investigated the relationship between transient stress and two cancer-related miRNAs, and determined whether health-behaviour moderated these relationships. Using a pre-post design, 37 German students completed measures on health-behaviour and perceived stress, the latter after a study break (low stress) and after an exam (high stress). On both occasions, students underwent blood tests to determine the expression of let-7b and miR-21, two miRNAs recently found to be related to cancer. The students reported significantly higher stress after the exam than in the study break period. The levels of let-7b and miR-21 expression significantly declined from low- to high-stress periods. Importantly, baseline health-behaviour interacted with time in relation to miR-21, such that the expression of this marker decreased only in students with inadequate health-behaviour, while it did not change in students with adequate health-behaviour. This is the first study showing that brief academic stress can alter the expression of two cancer-related miRNA molecules, and that health-behaviour may moderate these effects for miR-21.

Airtightness of lung parenchyma without a closing suture after atypical resection using the Nd:YAG Laser LIMAX® 120

OBJECTIVES Lung metastases can be non-anatomically resected with a Nd:YAG Laser. It is recommended that the resected lung surface be sealed by slowly resorbable sutures. However, the lung tissue may be restricted by the sutures once it is re-ventilated. Thus, it was analysed whether the lung parenchyma is airtight after laser resection without suturing the defect. METHODS The pulmonary artery of unimpaired paracardial lung lobes of freshly slaughtered pigs (mean weight 46 g) was cannulated and rinsed out via a hypotonic saline-heparin solution (5000 IE) until the perfusate was clear of body fluid. The lobular bronchus was connected to an airtight ventilation tube (Fa. VYGON 520 3.5 oral tube) and ventilated pressure-controlled (PEEP + 5 cm H₂O, P₁ = 20 cm H₂O, frequency = 10/min) via a respirator. All lobes were perfused with Ringer solution at 42°C at normothermia and normotonia. In group 1 (n = 8), an atypical peripheral parenchymal resection (average resected surface: 2 × 2 cm(2)) and in group 2 (n = 8), a deep atypical parenchymal resection (average resected surface: 4 × 4 cm(2)) were performed with the Nd:YAG Laser LIMAX 120 (output power at 100 watts). After post-resection ventilation of 15 min, the resection surface was tested for airtightness and burst pressure. RESULTS All group 1 lobes tested airtight under pressure-controlled ventilation. The mean burst pressure was 34.4 mbar (SD ± 3.2 mbar). Six lobes of group 2 were also completely airtight. The remaining two lobes, however, revealed a serious parenchymal leak (score 3). This was caused by the cross-opening of a segmental bronchus, although the surrounding lung parenchyma was also airtight. The mean burst pressure of these lobes was 31.7 mbar (SD ± 4.08 mbar). There was no significant difference between the two groups (P = 0.12). CONCLUSIONS Peripheral lung defects after Nd:YAG Laser resection might not be sutured, since the laser-induced vaporization of the lung parenchyma seems to be initially airtight. These experimental data warrant confirmation in a controlled clinical study.

RETRACTED: Nef from SIVmac239 decreases proliferation and migration of adenoid-cystic carcinoma cells and inhibits angiogenesis

The HIV/SIV accessory protein Nef is known to down-modulate cell surface receptors that are required for virus entry such as CD4, CCR5 and CXCR4 to block lethal viral superinfection of the infected cell. The chemokine receptor CXCR4 also plays an important role in promoting cell proliferation, metastasis and tumor angiogenesis. Therefore it was of interest to evaluate if Nef can down-regulate CXCR4 in tumor cells since this could affect these critical prognostic parameters. The CXCR4-expressing cell line ACC3 that was derived from a salivary gland adenoid cystic carcinoma (ACC) of the head and neck was transfected with Nef from SIV(mac239) and cell surface expression of the receptor was monitored by FACS analysis. Real time proliferation of cells was measured with the xCELLigence system (Roche, Mannheim, Germany). Cell migration was detected by an in vitro scratch assay. Similarly, COS-7 cells were co-transfected with CXCR4 and Nef and were treated as described for ACC3. In vitro tube formation was deployed to assess the effect of Nef on angiogenesis. siRNA was used for CXCR4 knockdown. Cell surface down-modulation of endogenous CXCR4 could be observed in ACC3 cells after Nef-transfection as well as in COS-7 cells after co-transfection of CXCR4 and Nef. Proliferation as well as migration of Nef-transfected ACC3 tumor cells appeared significantly reduced. In vitro tube formation was significantly lowered after Nef-transfection or CXCR4 knockdown with siRNA. SIV-Nef could serve as an interesting tool to study the biologic behavior of CXCR4-expressing tumors such as ACC. Deploying SIV-Nef thereby could help in the discovery of new therapeutic approaches for the treatment of ACC and other CXCR4-expressing tumors.

Exogenous hepatitis B virus envelope proteins induce endoplasmic reticulum stress: involvement of cannabinoid axis in liver cancer cells

HBV represents the most common chronic viral infection and major cause of hepatocellular carcinoma (HCC), although its exact role in liver tumorigenesis is unclear. Massive storage of the small (SHBs), middle (MHBs) and large surface (LHBs) HBV envelope proteins leads to cell stress and sustained inflammatory responses. Cannabinoid (CB) system is involved in the pathogenesis of liver diseases, stimulating acute and chronic inflammation, liver damage and fibrogenesis; it triggers endoplasmic reticulum (ER) stress response. The aim of our work was to investigate the activation of ER stress pathway after ectopic HBV envelope proteins expression, in liver cancer cells, and the role exerted by CB receptors. PCR, immunofluorescence and western blotting showed that exogenous LHBs and MHBs induce a clear ER stress response in Huh-7 cells expressing CB1 receptor. Up-regulation of the chaperone BiP/GRP78 (Binding Immunoglobulin Protein/Glucose-Regulated Protein 78) and of the transcription factor CHOP/GADD153 (C/EBP Homologous Protein/Growth Arrest and DNA Damage inducible gene 153), phosphorylation of PERK (PKR-like ER Kinase) and eIF2α (Eukaryotic Initiation Factor 2α) and splicing of XBP1 (X-box binding protein 1) was observed. CB1−/− HepG2 cells did not show any ER stress activation. Inhibition of CB1 receptor counteracted BiP expression in transfected Huh-7 and in HBV+ PLC/PRF/5 cells; whereas no effect was observed in HBV− HLF cells. These results suggest that HBV envelope proteins are able to induce the ER stress pathway. CB1 expression is directly correlated with ER stress function. Further investigations are needed to clarify the involvement of cannabinoid in HCC progression after HBV infection.

Ileal neuroendocrine tumors show elevated activation of mammalian target of rapamycin complex.

BACKGROUND Neuroendocrine tumors (NETs) of the ileum are sporadic tumors derived from submucosal gastrointestinal stem cells. They often show clinical symptoms only after hepatic metastasation when curative therapy is limited or impossible. In this study, we analyzed the expression of the candidate genes mammalian target of rapamycin (mTOR), alpha thalassemia/mental retardation syndrome X-linked (ATRX), and death domain-associated protein (DAXX) to investigate the specific oncogenetics and potential therapeutic options for ileal NETs. METHODS In a prospective database, all patients who underwent surgical removal of a NET of the ileum between 2001 and 2011 were specified. Expression analysis was performed for mTOR, ATRX, and DAXX by immunohistochemistry of paraffin-embedded tumor samples. To evaluate the results the immunoreactive score was applied. Normal tissue and tumor tissue were analyzed for the comparison of gene expression levels using quantitative-real-time polymerase chain reaction for ATRX and mTOR genes. Results were correlated under pathologic and clinical aspects. RESULTS A total of 69 patients were admitted to the study. Positive cytosolic expression of the potential oncogene mTOR was immunohistochemically detected in 76.2% of the human probes. A loss of nuclear ATRX expression was detected in 13.0% of the samples. A nonexpression of the DAXX-protein in cell nuclei was not found (0%). Gene transcript levels did not show a significant alteration in ileal NETs in comparison with normal tissue. CONCLUSIONS mTOR is overexpressed in ileal NETs. Additionally, the loss of ATRX expression was registered, thus underlying a tumorigenic role in a subgroup of these tumors. To enable potential therapeutic application of mTOR inhibitors, further trials with larger study groups are needed.

Epithelial cell types and their proposed roles in maintaining the mucosal barrier in human chagasic–megacolonic mucosa

Patients suffering from chagasic megacolon must have an intact mucosal barrier as they survive this chronic disease for decades. A key structure of the mucosal barrier are epithelial cells. Vasoactive-intestinal-peptide (VIP)-positive nerve fibres are involved in influencing, e.g., epithelial cell proliferation, mucus secretion (e.g., mucin 2 and trefoil factor 3 of goblet cells) and inflammation or autoimmunity, all putative and/or known factors altered in chagasic megacolon. We analyzed qualitatively and quantitatively goblet cells, their specific markers, such as mucin 2 (MUC2) and trefoil factor 3 (TFF3) and enterocytes, the relation of VIP-immunoreactive nerve fibres to the epithelia, the distribution of gelsolin, a protein involved in chronic inflammation processes in the epithelia, and the proliferation rate of epithelial cells by combined 4′,6-diamidino-2-phenylindole (DAPI) and phosphohistone-H3 (PHH3) staining. Goblet cells were the dominating epithelial cell type. They accounted for 38.4% of all epithelial cells in controls and changed to 58.9% in the megacolonic parts. In contrast to the overall expression in goblet cells of control epithelia, TFF3 was confined to goblet cells at the base of the crypts whereas MUC2 was found only in luminal goblet cells. Gelsolin-positive goblet cells were predominantly recognized within the controls. Finally, the mean value of mitosis increased from 1.5% within the controls up to 2.6% in the anal parts of the chagasic sepcimens. Taken together, increased cell proliferation, preponderance of goblet cells, differential MUC 2, and TFF 3 expression might all be factors maintaining an intact mucosal barrier within chagasic megacolon.