let-7 MicroRNA transfer in pancreatic cancer-derived cells inhibits in vitro cell proliferation but fails to alter tumor progression.

Pancreatic ductal adenocarcinoma (PDAC) is still the fourth leading cause of cancer-related deaths in Western countries, with increasing incidence. Neither effective prognostic markers nor therapies exist for this cancer. MicroRNAs are potent inhibitors of protein translation, and aberrantly expressed in many cancers. Because let-7 microRNA targets the K-ras oncogene, we aimed to characterize let-7 expression and function in PDAC in vitro and in vivo. Let-7 expression was quantified by real-time RT-PCR from resected tumors and matching adjacent tissue, and in endoscopic ultrasound-guided fine needle aspiration material from patients with PDAC. Let-7 is detected by reverse transcription in situ PCR in a PDAC tissue microarray. PDAC-derived cells were transfected with plasmid-based synthetic microRNAs or by lentiviral transduction, in vitro and in vivo. Let-7 microRNA expression is strongly reduced in PDAC samples, as compared with adjacent tissue. Let-7 is present in normal acinar pancreatic cells, and lost in poorly differentiated cancer samples. In addition, let-7 expression was repressed in patients with PDAC not eligible for surgery. Restoring let-7 levels in cancer-derived cell lines strongly inhibits cell proliferation, K-ras expression, and mitogen-activated protein kinase activation, but fails to impede tumor growth progression after intratumoral gene transfer or after implantation of Capan-1 cells stably overexpressing let-7 microRNA. We describe here for the first time the extensive loss of expression of let-7 in PDAC. In addition, this study provides the initial steps for a microRNA replacement therapy for this cancer.

Expression of the transcription factor Klf6 in cirrhosis, macronodules, and hepatocellular carcinoma.

BACKGROUND AND AIMS: Macronodules (MN) occurring in cirrhosis are considered to be precursor lesions for hepatocellular carcinoma (HCC). However, early molecular events in hepatocellular carcinogenesis are poorly understood. The aim of this study was to compare gene expression profiling between cirrhotic tissues, MN, and HCC, to identify genes early involved in liver carcinogenesis. METHODS: Tissues were obtained from explanted livers: nine cirrhosis, 10 MN, and seven HCC. Total RNAs were extracted by RNeasy and reverse transcribed with labelled [(33)P]-alpha ATP. Hybridations were performed on Atlas Human Cancer 1.2 membranes (1176 genes). RESULTS: A two-way hierarchical clustering algorithm successfully isolated specific gene expression profiles when comparing MN, cirrhosis, and HCC. A total of 16 and 14 genes were up- and down-expressed, respectively, in HCC as compared to cirrhotic tissues. The molecular signature of MN was characterized by the down-expression of 23 and 42 genes as compared to cirrhosis and HCC, respectively. Among them, Klf6 was down-expressed in all MN samples whereas it was over-expressed in cirrhosis and HCC. This result was confirmed at RNA level by quantitative real time-polymerase chain reaction and at protein level by Western blotting. However, no mutation in the exon 2 of Klf6 was detected. CONCLUSION: We identified a molecular signature of MN characterized by a down-expression of several genes. One of them, Klf6 was found to be down-expressed in all MN without evidence of somatic mutations in the exon 2. This gene could be involved at an early stage of hepatocarcinogenesis.

Identification of biomarkers of human pancreatic adenocarcinomas by expression profiling and validation with gene expression analysis in endoscopic ultrasound-guided fine needle aspiration samples.

AIM: To compare gene expression profiles of pancreatic adenocarcinoma tissue specimens, human pancreatic and colon adenocarcinoma and leukemia cell lines and normal pancreas samples in order to distinguish differentially expressed genes and to validate the differential expression of a subset of genes by quantitative real-time RT-PCR (RT-QPCR) in endoscopic ultrasound-guided fine needle aspiration (EUS-guided FNA) specimens. METHODS: Commercially dedicated cancer cDNA macroarrays (Atlas Human Cancer 1.2) containing 1176 genes were used. Different statistical approaches (hierarchical clustering, principal component analysis (PCA) and SAM) were used to analyze the expression data. RT-QPCR and immunohistochemical studies were used for validation of results. RESULTS: RT-QPCR validated the increased expression of LCN2 (lipocalin 2) and for the first time PLAT (tissue-type plasminogen activator or tPA) in malignant pancreas as compared with normal pancreas. Immunohistochemical analysis confirmed the increased expression of LCN2 protein localized in epithelial cells of ducts invaded by carcinoma. The analysis of PLAT and LCN2 transcripts in 12 samples obtained through EUS-guided FNA from patients with pancreatic adenocarcinoma showed significantly increased expression levels in comparison with those found in normal tissues, indicating that a sufficient amount of high quality RNA can be obtained with this technique. CONCLUSION: Expression profiling is a useful method to identify biomarkers and potential target genes. Molecular analysis of EUS-guided FNA samples in pancreatic cancer appears as a valuable strategy for the diagnosis of pancreatic adenocarcinomas.

Clinical utility of telomerase for the diagnosis of malignant well-differentiated endocrine tumours.

OBJECTIVE: The distinction between benign and malignant well-differentiated endocrine tumours is hard to achieve. The aim of the present study was to determine whether detection of telomerase or quantification of human telomerase reverse transcriptase protein subunit (hTERT) differ between benign and malignant endocrine tumours. PATIENTS AND METHODS: This retrospective study investigated 31 well-differentiated primary endocrine tumours. Based on clinical and histopathological criteria, tumours were categorized with the most recent WHO classification as 'benign' (n = 14), 'uncertain' (n = 5) or 'malignant' (n = 12) with (n = 7) or without (n = 5) metastasis after a mean follow-up of 40.4 +/- 25.8 months (4-122 months). All these tumours were assayed for telomerase activity and hTERT mRNA expression [real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)]. RESULTS: Telomerase activity was detected in 7 malignant and metastatic tumours, in 1 malignant tumour without metastases, in 1 uncertain tumour and in 1 benign tumour. hTERT mRNA levels were significantly higher in malignant endocrine tumours with or without metastases (P = 0.001) when compared to benign tumours. The negative predictive value of hTERT mRNA quantification for the diagnosis of malignancy was 88.9%, whereas the positive predictive value was 68.7%. CONCLUSION: The presence of telomerase activity within the primary endocrine tumour might indicate a malignant tumour and might suggest the need for an attentive search for concomitant metastases. Quantification of hTERT mRNA could be used in clinical practice to exclude malignancy in most endocrine tumours.

Frequent deletions of tumor suppressor genes in pure pancreatic juice from patients with tumoral or nontumoral pancreatic diseases.

BACKGROUND/AIMS: K-ras codon 12 mutation is the most frequent genetic alteration in pancreatic cancer. Sensitivity and specificity of K-ras are not high enough to detect all pancreatic cancers, especially at early stage. This study investigated whether detection of p16 and/or DPC4 deletions along with K-ras mutation in DNA samples could improve the definition of patients at risk of pancreatic cancer. METHODS: K-ras mutations were investigated by sequencing. p16 and DPC4 homozygous deletions were studied using comparative multiplex polymerase chain reaction of DNA in pancreatic juice sampled during endoscopic retrograde pancreatography in 57 patients with either pancreatic cancer (group I, 18 patients), chronic pancreatitis (group II, 20 patients), or nontumoral pancreatobiliary disease (group III, 19 patients). RESULTS: The frequencies of Ki-ras mutations were 61% in group I, 10% in group II, and 10.5% in group III. The frequencies of p16 exon 2 and DPC4 deletions were, respectively, 28 and 36% in group I, 50 and 58% in group II, and 24 and 36% in group III. CONCLUSIONS: The combination of p16 and DPC4 deletions with K-ras mutation does not improve the diagnosis of pancreatic cancer based on K-ras mutation alone. These data suggest that tumor suppressor gene inactivation can occur with a high frequency during nonmalignant pancreatic diseases.

Expression of CCK2 receptors in the murine pancreas: proliferation, transdifferentiation of acinar cells, and neoplasia.

BACKGROUND & AIMS: To explore the pancreatic function of CCK2/gastrin receptor, we created ElasCCK2 transgenic mice expressing the human receptor in pancreatic exocrine cells. In previous studies, the transgenic CCK2/gastrin receptor was demonstrated to mediate enzyme release and protein synthesis. We now report results of phenotypic and long-term studies. METHODS: Pancreas was characterized using morphometry and immunohistochemistry. ElasCCK2 mice were crossed with INS-GAS mice expressing gastrin in pancreatic beta cells to achieve continuous stimulation of the CCK2/gastrin receptor. RESULTS: The pancreatic weight of ElasCCK2 mice was increased by 40% and correlated with an increase in the area of exocrine tissue. Alterations in pancreatic histology were apparent from postnatal day 50. Crossing the ElasCCK2 mice with INS-GAS mice resulted in development of morphologic changes in younger animals. Malignant transformation occurred in 3 of 20 homozygous ElasCCK2 mice. Although tumors had different phenotypes, they all developed through an acinar-ductal carcinoma sequence. CONCLUSIONS: Our data show that transgenic expression of a G protein-coupled receptor can lead to cancer. This study also supports a key role of the CCK2/gastrin receptor in the development of pre- and neoplastic lesions of the pancreas. ElasCCK2 mice provide a model for carcinogenesis by transformation and dedifferentiation of acinar cells.