Identification of methylation-associated gene expression in neuroendocrine pancreatic tumor cells.

BACKGROUND: CpG islands methylation is the main epigenetic modification found in human tumors leading to transcriptional silencing of certain tumor suppressor genes. Reacquisition of p16/CDKN2A tumor suppressor gene expression by 5-aza-2'-deoxycytidine results in concurrent growth inhibition of neuroendocrine pancreatic tumor cells. However, the growth suppressive effects of 5-aza-2'-deoxycytidine is unlikely to be solely attributable to the restored p16/CDKN2A function, but rather a consequence of re-expression of additional genes silenced by de novo methylation. In an effort to validate DNA methylation as an important mechanism in neuroendocrine tumorigenesis and metastatic spread, we attempted to isolate methylation-specific transcripts in neuroendocrine pancreatic tumor cells. METHODS: Differentially expressed methylation-associated genes were identified by cDNA-representational difference analysis (cDNA-RDA). Differential expression was confirmed by semiquantitative RT-PCR using insert specific primers. RESULTS: We identified 48 differently expressed gene fragments and methylation-associated expression was confirmed by semi-quantitative RT-PCR. 52,3% (25 of 48) showed elevated expression levels after 5-aza-2'-deoxycytidine treatment, whereas 47.7% revealed lower expression levels. 7 fragments showed homology to genes with unknown function. Interestingly, 5-aza-2'-deoxycytidine treatment led to re-expression of cofillin whereas matriptase expression levels were significantly lower. Both genes have been associated with metastatic spread and tissue invasion. The other differentially expressed genes play an unknown role in the course of neuroendocrine tumorigenesis. CONCLUSION: DNA methylation appears to be an important molecular mechanism in the process of neuroendocrine pancreatic tumorigenesis and metastatic spread. The definition of DNA methylation patterns associated with neuroendocrine pancreatic tumors might open up the potential for a new sensitive diagnostic tool and might serve as a new antitumor target.

Implication of the INK4a/ARF locus in gastroenteropancreatic neuroendocrine tumorigenesis.

The INK4a/ARF locus on chromosome 9p21 is one of the important defenses against tumor development and engages both the Rb and the p53 tumor suppressor pathways through its capacity to encode two distinct proteins, p16(INK4a) and p14(ARF). Despite controversial reports, the body of present data suggests that tumor suppressors p16(INK4a) and p14(ARF) are targets of in-activation in GEP-NETs. Moreover, tumor type-specific aberrant p16(INK4a) silencing appears to be associated with advanced tumor stage and may function as a predictor of patients' outcome after surgical resection. Since conventional histological and biochemical assessment are limited with respect to predicting GEP-NET behavior or outcome, methylation profiles including INK4a/ARF might offer a tool to refine future diagnosis and therapeutic management of GEP-NET patients.

Radioiodide treatment after sodium iodide symporter gene transfer is a highly effective therapy in neuroendocrine tumor cells.

This study evaluates the possibility of treating Bon1 and QGP pancreatic neuroendocrine tumor cells with radioactive iodide ((131)I) after stable transfection with the thyroid sodium iodide symporter (NIS). NIS expression was driven either by the strong viral cytomegalovirus promoter or by the tissue-specific chromogranin A promoter. Using either approach, NIS expression was confirmed by reverse transcription-PCR and Western blotting. Uptake of radioactive iodide was increased approximately 20-fold by chromogranin A promoter-driven NIS expression and approximately 50-fold by cytomegalovirus promoter-driven NIS expression. Maximal uptake was reached within 15 min in QGP cells and 30 min in Bon1 cells. Effective half-life was 5 min in QGP and 30 min in Bon1 cells. No evidence of organification was detected by high-performance liquid chromatography and gel filtration chromatography. (131)I was a highly effective treatment in NIS-expressing QGP and Bon1 cells, reducing clone formation by 99.83 and 98.75%, respectively, in the in vitro clonogenic assay. In contrast, clone formation was not reduced in QGP and Bon1 cells without NIS expression after incubation with the same activity concentration of (131)I as compared with mock treated cells. Absorbed doses to QGP and Bon1 cells are up to 150 and 30 Gy, respectively. In addition, a direct cytotoxic effect of radioiodide was demonstrated in NIS-expressing Bon1 cells after (131)I incubation. In conclusion, radioiodide treatment after NIS gene transfer appears to be a promising novel approach in the therapy of neuroendocrine tumors if its highly encouraging in vitro effectiveness can be transferred to the in vivo situation.

Frequent methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene in pancreatic endocrine tumors.

Molecular mechanisms contributing to the tumorigenesis of pancreatic endocrine tumors (PETs) are still not well understood. Allelic deletions at chromosome 22q12.3 were detected in about 30-60% of PETs, suggesting that inactivation of one or more tumor suppressor genes on this chromosomal arm is important for their pathogenesis. Because the putative tumor suppressor gene tissue inhibitor of metalloproteinase-3 (TIMP-3) has been located at 22q12.3, we undertook a genetic analysis of TIMP-3 to determine its role in the tumorigenesis of PETs. Single-strand conformational polymorphism analysis, methylation-specific PCR, RNA expression analysis, and immunohistochemistry of TIMP-3 were performed in 21 sporadic PETs. Thirteen of 21 PETs (62%) revealed TIMP-3 alterations, including promoter hypermethylation and homozygous deletion. The predominant TIMP-3 alteration was promoter hypermethylation, identified in 8 of 18 (44%) PETs. It was tumor-specific and corresponded to loss or strong reduction of TIMP-3 protein expression. Notably, 11 of 14 (79%) PETs with metastases had TIMP-3 alterations, compared with only 1 of 7 (14%) PETs without metastases (P < 0.02). These data suggest a possibly important role of TIMP-3 in the tumorigenesis of human PETs, especially in the development of metastases, which has to be further evaluated in large-scale studies.

Expression spectrum and methylation-dependent regulation of melanoma antigen-encoding gene family members in pancreatic cancer cells.

Human MAGE and GAGE genes encode tumor-specific antigens presented by HLA I molecules recognized on tumor cells by cytolytic T lymphocytes. To determine if pancreatic cancer patients would be suitable for MAGE- or GAGE-based immunotherapy, the expression frequency of MAGE-A1, -A2, -A3, -A4, -A6 and GAGE1-8 genes was assessed in 15 pancreatic tumor cell lines and 23 pancreatic tumor specimens using reverse transcription-polymerase chain reaction (RT-PCR). In 67% of the cell lines at least one of the MAGE-A genes was detected, 53% revealed concomitant expression of two or more genes. GAGE1-8 expression was detected in 47% of the cell lines. In the primary pancreatic tumors, MAGE-A analysis revealed exclusive MAGE-A1 and MAGE-A2 gene expression in 26 and 30% of the specimens, respectively, independent from clinicopathologic factors. Treatment of MAGE-A expression-negative pancreatic tumor cells with the demethylating agent 5-aza-2'-deoxycytidine could activate MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4 and GAGE transcription suggesting silencing due to promoter methylation. Interestingly, a metastatic lesion to the liver revealed concomittant expression of MAGE-A1, -A2, -A3 and -A6 consistent with a more pronounced genome-wide hypomethylation in metastases. Therefore, a subset of pancreatic cancer patients could be eligible for active, specific immunotherapy directed against MAGE-A antigens and demethylating agents could increase the number of candidate patients.