Preservation of HUGL-1 expression as a favourable prognostic factor in pancreatic carcinoma.

AIM: The expression of the human homologue of Drosophila tumour suppressor gene lgl (HUGL-1) in pancreatic cancer was retrospectively assessed in 97 patients with surgically treated pancreatic cancer in order to correlate the HUGL-1 profile with patients' survival. MATERIALS AND METHODS: Immunohistochemistry was performed on 4-µm-thick paraffin sections from representative tumour blocks using a standard protocol. The expression of HUGL-1 was evaluated semiquantitatively as negative (0), weak (1), medium (2) or strong (3). The results were correlated with clinicopathological parameters and with patients' survival, considering an observation period of 17 (mean) ± 16 (SD) months. RESULTS: In normal and inflammatory tissue, a uniform and relatively strong staining was observed in ductal epithelium, ganglion cells and some acinar epithelia. The endocrine islets exhibited a weak positivity. Human pancreatic cancer revealed variable intensities of HUGL-1 expression. A total of 69 tumour specimens were classified as negative and 28 as positive. The HUGL-1 expression was not correlated with clinical variables (age, gender), staging or tumour grading. HUGL-1 positivity proved to be prognostically favourable (p=0.0241) conferring a higher survival rate, especially for patients who had survived more than 12 months. The presence of distant metastases (M1) at diagnosis had a weak significant influence on survival (p=0.0474). The other staging parameters (T, N, UICC stage), tumour grading and clinical variables (age, gender) gave no significant prognostic information. In a multivariate Cox model, only HUGL-1 expression passed the entry limits. CONCLUSION: Preservation of HUGL-1 expression in pancreatic adenocarcinoma is a good prognostic factor that contributes to a better overall survival.

IFN-alpha-induced apoptosis in hepatocellular carcinoma involves promyelocytic leukemia protein and TRAIL independently of p53.

IFNs are pleiotropic cytokines that have been shown to be important regulators of cell growth. IFN-alpha has recently been recognized to harbor therapeutic potential in prevention and treatment of hepatocellular carcinoma (HCC). However, HCC cells respond differentially to IFN treatment, the mechanism of which is largely unknown. To address this issue, we analyzed the effect of IFN-alpha on different liver tumor cell lines. We found that growth inhibiting effects of IFN-alpha in hepatoma cells require PML-NB induction and, moreover, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression on the mRNA and protein level. RNAi silencing of PML down-regulates TRAIL expression in hepatoma cells and correspondingly blocks IFN-alpha-induced apoptosis. In addition, PML-deficient primary hepatocytes fail to up-regulate TRAIL upon IFN-alpha-treatment in contrast to their wild-type counterparts. These data identify TRAIL as a novel downstream transcriptional target of PML-mediated apoptosis in hepatomas and suggest that PML and TRAIL play important roles in IFN-regulated apoptosis in HCC. Furthermore, the mechanism is independent of the p53 status of the tumor cells. In summary, our results identify central molecules mediating IFN-alpha induced apoptosis in liver tumors, shed light on the differential response of hepatoma cells to IFN exposure and, thus, may contribute to an efficient application of this substance in the treatment of liver cancer.

Bcl-x(L) and Myeloid cell leukaemia-1 contribute to apoptosis resistance of colorectal cancer cells.

AIM: To explore the role of Bcl-x(L) and Myeloid cell leukaemia (Mcl)-1 for the apoptosis resistance of colorectal carcinoma (CRC) cells towards current treatment modalities. METHODS: Bcl-x(L) and Mcl-1 mRNA and protein expression were analyzed in CRC cell lines as well as human CRC tissue by Western blot, quantitative PCR and immunohistochemistry. Bcl-x(L) and Mcl-1 protein expression was knocked down or increased in CRC cell lines by applying specific siRNAs or expression plasmids, respectively. After modulation of protein expression, CRC cells were treated with chemotherapeutic agents, an antagonistic epidermal growth factor receptor (EGFR1) antibody, an EGFR1 tyrosine kinase inhibitor, or with the death receptor ligand TRAIL. Apoptosis induction and cell viability were analyzed. RESULTS: Here we show that in human CRC tissue and various CRC cell lines both Bcl-x(L) and Mcl-1 are expressed. Bcl-x(L) expression was higher in CRC tissue than in surrounding non-malignant tissue, both on protein and mRNA level. Mcl-1 mRNA expression was significantly lower in malignant tissues. However, protein expression was slightly higher. Viability rates of CRC cells were significantly decreased after knock down of Bcl-x(L) expression, and, to a lower extent, after knock down of Mcl-1 expression. Furthermore, cells with reduced Bcl-x(L) or Mcl-1 expression was more sensitive towards oxaliplatin- and irinotecan-induced apoptosis, and in the case of Bcl-x(L) also towards 5-FU-induced apoptosis. On the other hand, upregulation of Bcl-x(L) by transfection of an expression plasmid decreased chemotherapeutic drug-induced apoptosis. EGF treatment clearly induced Bcl-x(L) and Mcl-1 expression in CRC cells. Apoptosis induction upon EGFR1 blockage by cetuximab or PD168393 was increased by inhibiting Mcl-1 and Bcl-x(L) expression. More strikingly, CD95- and TRAIL-induced apoptosis was increased by Bcl-x(L) knock down. CONCLUSION: Our data suggest that Bcl-x(L) and, to a lower extent, Mcl-1, are important anti-apoptotic factors in CRC. Specific downregulation of Bcl-x(L) is a promising approach to sensitize CRC cells towards chemotherapy and targeted therapy.

The human protein Hugl-1 substitutes for Drosophila lethal giant larvae tumour suppressor function in vivo.

Drosophila lethal giant larvae: (lgl), discs large (dlg) and scribble (scrib) are tumour suppressor genes acting in a common pathway, whose loss of function leads to disruption of cell polarity and tissue architecture, uncontrolled proliferation and growth of neoplastic lesions. Mammalian homologues of these genes are highly conserved and evidence is emerging concerning their role in cell proliferation control and tumorigenesis in humans. Here we investigate the functional conservation between Drosophila lethal giant larvae and its human homologue Hugl-1(Llgl1). We first show that Hugl-1 is lost in human solid malignancies, supporting its role as a tumour suppressor in humans. Hugl-1 expression in homozygous lgl Drosophila mutants is able to rescue larval lethality; imaginal tissues do not show any neoplastic features, with Dlg and Scrib exhibiting the correct localization; animals undergo a complete metamorphosis and hatch as viable adults. These data demonstrate that Hugl-1 can act as a tumour suppressor in Drosophila and thus is the functional homologue of lgl. Furthermore, our data suggest that the genetic pathway including the tumour suppressors lgl, dlg and scrib may be conserved in mammals, since human scrib and mammalian dlg can also rescue their respective Drosophila mutations. Our results highlight the usefulness of fruit fly as a model system for investigating in vivo the mechanisms linking loss of cell polarity and cell proliferation control in human cancers.

Prognostic value of disseminated colorectal tumor cells in the liver: results of follow-up examinations.

BACKGROUND AND AIMS: Dissemination of tumor cells is an initial step in metastatic disease. Detection of disseminated tumor cells (DTC) in blood, bone marrow and lymph nodes has been associated with reduced disease-free survival, but to date there are no data for hepatic DTC. We investigated the prognostic relevance of hepatic DTC that are present in patients with colorectal cancer (CRC) at the time of surgery. PATIENTS AND METHODS: In 121 patients with CRC clinically diagnosed for liver metastasis by ultrasound, CT, and exploration during surgery DNA from liver biopsy specimens obtained during surgery was examined by a PCR-RFLP assay for K-ras mutations as a marker for DTC. At the time of surgery 54 of the 121 were mutated in K-ras codons 12 or 13. After a median follow-up of 405 days all survivors were reevaluated by ultrasound/CT. RESULTS: Patients with a K-ras mutation in their primary tumor had a significantly lower probability of survival and higher risk of harboring a synchronous second colorectal carcinoma than patients with a K-ras wild-type tumor. Based on specimens taken intraoperatively DTC were found in the liver of 14 of 54 patients (26%). At follow-up only 10 of 40 patients (25%) with DTC-free liver had died of their disease but 9 of the 14 patients with hepatic DTC. Among the 14 patients with hepatic DTC 10 (71%) had developed new liver metastasis, compared to 12 of 40 (30%) in those without hepatic DTC. CONCLUSION: Hepatic DTC in colorectal cancer patients is associated with reduced overall survival and increased risk of hepatic metastasis development. Further studies are necessary to corroborate our results since the number of patients studied is still limited.

Hepatic disseminated tumor cells in colorectal cancer UICC stage 4 patients: prognostic implications.

Approximately 30-50% of all colorectal cancer patients with a resectable primary tumor will subsequently develop metastatic disease due to tumor cell dissemination. In the case of limited solid hepatic metastasis, resection of the primary tumor and the respective hepatic metastasis can be curative. Our aim was to evaluate the incidence of hepatic DTC in patients with solid liver metastasis and to describe their prognostic impact. Therefore, we applied a sensitive PCR-RFLP assay detecting one K-ras mutant among one million wild-type cells. Tumor tissue and liver biopsies from 32 colorectal cancer patients staged UICC 4 undergoing palliative surgery could be obtained intra-operatively and were thereupon screened for the presence of hepatic DTC. The primary tumor of 16 patients (50%) harbored a K-ras mutation and survival of K-ras positive patients was reduced as compared to K-ras negatives (P=0.039). In 8 of the patients (50%) with a K-ras mutated primary, no hepatic DTC were detected, whereas the liver of the remaining 8 patients (50%), showed a coincidence of solid liver metastasis and hepatic DTC. Median survival of patients with solid liver metastasis and hepatic DTC was decreased as compared with patients without any hepatic DTC burden (median survival 165 vs. 240 days; log-rank P=0.951). A subgroup analysis revealed that survival was significantly decreased in the case of bilobal DTC affection as compared with monolobal hepatic DTC affection (median survival 68 vs. 355 days; log-rank P=0.002). We conclude that the detection of hepatic DTC is a powerful prognostic factor. Furthermore, bilobal hepatic DTC involvement might be a contraindication for the resection of solid liver metastasis.