Gene expression profiles in primary pancreatic tumors and metastatic lesions of Ela-c-myc transgenic mice

Abstract

Background: Pancreatic carcinoma usually is a fatal disease with no cure, mainly due to its invasion and metastasis prior to diagnosis. We analyzed the gene expression profiles of paired primary pancreatic tumors and metastatic lesions from Ela-c-myc transgenic mice in order to identify genes that may be involved in the pancreatic cancer progression. Differentially expressed selected genes were verified by semi-quantitative and quantitative RT-PCR. To further evaluate the relevance of some of the selected differentially expressed genes, we investigated their expression pattern in human pancreatic cancer cell lines with high and low metastatic potentials.

Results: Data indicate that genes involved in posttranscriptional regulation were a major functional category of upregulated genes in both primary pancreatic tumors (PT) and liver metastatic lesions (LM) compared to normal pancreas (NP). In particular, differential expression for splicing factors, RNA binding/pre-mRNA processing factors and spliceosome related genes were observed, indicating that RNA processing and editing related events may play critical roles in pancreatic tumor development and progression. High expression of insulin growth factor binding protein-1 (Igfbp1) and Serine proteinase inhibitor A1 (Serpina1), and low levels or absence of Wt1 gene expression were exclusive to liver metastatic lesion samples.

Conclusion: We identified Igfbp1, Serpina1 and Wt1 genes that are likely to be clinically useful biomarkers for prognostic or therapeutic purposes in metastatic pancreatic cancer, particularly in pancreatic cancer where c-Myc is overexpressed.

Figure 1

Gene expression profiles. A) Histogram showing a similar (left) and differential (right) gene expression profiles of primary pancreatic tumors and liver metastatic lesions from Ela-c-Myc transgenic mice compared to normal pancreas from wild type littermates. B) Hierarchical clustering of differentially expressed genes. Clustering tree illustrate the expression pattern and similarity in primary pancreatic tumors (labeled as PT) and liver metastatic lesions (labeled as LM) compared to normal pancreas (labeled as NP) indicated by color bars. C) Shows only the differentially expressed gene profile with at least a four-fold change (≤4 or ≥4) indicated by color bars. (blue-down regulated and red up-regulated).

Figure 2

Selected genes showing up- or down regulation of mRNA expression by semi quantitative RT-PCR. A) All selected genes showed expression pattern similar to microarray data upon confirmation by sqRT-PCR. A representative data from four Ela-c-myc pancreatic tumors, liver metastatic lesions and normal pancreas is presented. B) RT-PCR showing representative differentially expressed genes in liver metastatic lesions compared to primary pancreatic tumors and normal pancreas. C) Two genes, Igfbp1 and Serpina1a, were verified in human pancreatic cancer cell lines with high (High-met) and low metastatic (Low-met) potentials. Expression patterns of both genes were consistent with the murine microarray and RT-PCR data. D) RT-PCR was performed on RNA from primary pancreatic tumors (PT), liver metastatic lesions (LM) and normal pancreas (NP) with three overlapping primer sets spanning the region from exon 1 to 10. Primary pancreatic tumors showed presence of both wild type Wt1 and Wt1 variant without exon 5, while metastatic lesions either lacked expression or had low levels of Wt1 gene expression (showed a smaller size non-specific PCR product only).

Figure 3

Expression of IGF family genes and proteins. A) Microarray data show that expression of Igf2 is about 10 fold higher in pancreatic tumors compared to liver metastatic lesions and normal pancreas from Ela-myc transgenic mice. While other IGF family proteins only showed modest change. B) Western blot analysis of Insulin like growth factors and their receptor proteins. Western blot was performed in cell lysates prepared from primary pancreatic tumors (PT), liver metastatic lesions (LM) from Ela-c-myc transgenic mice and normal pancreas (NP) from wild type littermates. Consistent with microarray data, PT samples showed noticeably higher protein levels compared to NP samples. A representative data from four PT and four NP samples are presented.