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

Pancreatic cancer (PC) is the fourth leading cause of cancer death in the United States and has no cure, partly because the tumor is at advanced stage or has already metastasized at the time of diagnosis [1]. Like many other types of cancer, pancreatic cancer also shows high frequencies of overexpression and/or amplification of the c-myc oncogene. In one study, 43.5% of primary tumors and 31.6% of metastases showed c-Myc overexpression, in association with 32.5% and 29.4% of gene amplification in the primary and metastatic lesions, respectively [2]. c-Myc and cyclin D1 gene amplification was report 54% and 28% in 31 pancreatic cancer cell lines, respectively, indicating a high frequency of concomitant amplification of both genes [3]. Moreover, simultaneous amplification of activated k-ras and c-myc has been found in both primary tumor and lymph node metastasis, suggesting that c-Myc may collaborate with other oncogenes to promote development and progression of pancreatic cancer [4]. More direct evidence for a critical role for c-Myc in pancreatic carcinogenesis comes from Ela-c-myc transgenic mice that develop PC between 2–7 months of age with 100% incidence rate [5]. One-half of the pancreatic tumors that form in this mouse model are acinar cell adenocarcinomas, while the remaining half of the tumors are mixed ductal and acinar cell carcinomas embedded in dense stroma. We have recently described detailed morphological traits of the pancreatic tumors developed in this transgenic model [6, 7] and, for the first time, observed spontaneous metastasis to the liver in this model. These transgenic mice are among the few animal models of liver metastasis of spontaneous PC. The whole carcinogenic process, from initiation to metastasis, is short (in only a few months time) and is initiated by only one gene.

The most devastating aspect of all types of cancer, particularly pancreatic cancer, is the emergence of metastases in organs distant from the primary tumor, and this remains the primary cause for the poor survival of patients with pancreatic cancer [8]. Therefore, a search for molecular markers that can predict poor prognosis and also serve as novel targets for the development of therapies against this most aggressive disease is warranted. Transgenic animals have been widely used to dissect the role of genes and molecular pathways in cancer [9]. Our transgenic model will help in understanding the molecular mechanisms by which metastases are generated, which is crucial for the prevention and treatment of metastatic disease. In this study we attempted to identify genes that may be responsible for the liver metastasis of pancreatic tumors in Ela-myc transgenic mice.

In this study, we report the genome-wide expression profiles of primary pancreatic tumors and liver metastatic lesions from Ela-c-myc transgenic mice, or normal pancreas from wild-type mice. cDNA microarray analysis showed several gene clusters under various functional categories in primary or metastatic pancreatic tumors of Ela-c-myc transgenic mice that differ from normal pancreas of non-transgenic littermates. Notably, increased expression was observed for a large number of genes related to ribosomal biogenesis, maturation and ribosome assembly in primary or metastatic pancreatic tumors. Previous studies by others have also shown enhanced expression of genes related to ribosomal proteins, rRNA maturation and ribosome assembly, in addition to enhanced expression of many translation initiation and elongation factors in c-Myc overexpressing cells [33‐35]. Thus, our model recapitulates the experimental observations and key features of c-Myc overexpressing tumors.

Genes involved in posttranscriptional regulation was a major functional category of upregulated genes in both PT and LM compared to NP samples, we observed changes in expression for splicing factors, RNA binding/pre-mRNA processing factors and spliceosome related genes, indicating that events related to RNA processing may play critical roles in pancreatic tumor development and progression induced by c-Myc. More than 50% of human genes undergo alternative splicing, and this type of RNA process has recently become an emerging topic in molecular and clinical oncology [36‐38]. Our data showed upregulation of several splicing factors from the SR family such as Sfrs1, Sfrs2, Sfrs3, Sf3b in both primary and metastatic tumors compared to normal pancreas. SR proteins represent a family of essential splicing factors, which are characterized by extensively phosphorylated serine-arginine rich domains [39]. SR proteins recognize splice sites and, depending on their relative levels, these proteins can influence alternative RNA processing [40].

Other groups of genes that were upregulated are involved in DNA replication, cell proliferation and cell cycle regulation; chromosome organization and biogenesis; and signal transduction. Many genes are related to the maintenance of chromosomal structure and integrity such as minichromosome maintenance (Mcm)2, Mcm5, Mcm10, structural maintenance of chromosome (Smc)2l1, Smc4l1, Smc5l1, Rad51, Brca1 and Centromere component (Cenp-I). The entire Mcm protein family (Mcm2-7) is essential in regulating the replication of DNA. Amplification of genes in the Mcm family has been detected in various cancer cells [41]. Their upregulation may deregulate the complete and accurate DNA replication and thus result in failure to maintain the genetic integrity of affected cells. Smc family proteins are integral components of the machinery that modulates chromosome structure for mitosis [42]. Similarly, Rad51, brca1 and Cenp-I play a role in maintenance of genetic integrity [43, 44]. We also noticed increased expression of some X-linked genes related to signal transduction such as Rsk4, Dusp9 and S100g, which have not been reported previously in pancreatic tumors.

Intriguingly, we observed highly upregulated expression of Igfbp1 and Serpina1 in liver metastatic tissues compared to primary pancreatic tumors and normal pancreas. Verification of Igfbp1 and Serpina1 by RT-PCR and quantitative PCR showed strong expression in liver metastatic lesions but there was a lack of expression of these genes in primary pancreatic tumors or normal pancreas. Similarly, both these genes also showed higher expression in highly metastatic human pancreatic cell lines (PANC28, CoLo357fg, L3.6pl) and lower expression levels in less-metastatic cell lines (PANC1 and BxPC3). Several studies have described the inhibitory and potentiating activities of both Serpina1 and Igfbp1 in a variety of cells [45‐47]. Igfbp1 interacts with α₅β₁ integrin, influencing cell adhesion and migration. Jones et al. [48] first reported the increased migration of Chinese hamster ovary cells transfected to express human Igfbp1. Increased expression of several Igfbps has also been reported in human pancreatic cancer [28‐31]. Serpins are endogenous inhibitors of serine protease activity in vivo [49, 50] and a large number of studies support the notion that proteases play an important role in the progression of malignant tumors. Therefore, the expression of proteinase inhibitors is considered to be an anti-malignant event. Serpina1, a major inhibitor of human serine proteases in serum, is produced mainly by the liver, but also by extra-hepatic cells, including neutrophils and certain cancer cells [51, 52]. However, clinical studies have shown that high circulating levels of Serpina1 directly correlate with tumor progression [53, 54]. Immunohistochemical studies revealed that patients with Serpina1-positive lung adenocarcinomas had a worse prognosis than Serpina1-negative ones [55]. More interestingly, both Serpina1 and Igfbp1 have been demonstrated to play a role in human invasive and metastatic pancreatic cancer. Together these studies and our findings suggest that Igfbp1 and Serpina1 may play critical roles in tumor progression in vivo, and are potential candidates for therapeutic interventions.

We also compared our gene expression profiles with published data on human pancreatic cancer tissues or cell lines. Gene expression pattern of many genes such as Serpina1, Igfbp1, Wt1, CD44, MMP12, CXCR4, Muc2, Dek, Capza1, Bcra1, Birc5, S100g, Claudin-18, RAD51, Mcm2, Mcm4, Mcm7, Cyclin B2, splicing factor 3b, Nap1l1 etc. (please see Tables 1, 2, 3 and 4for references) was similarly reported in other studies and therefore provide a validation for our model.

We show differential gene expression profiles under several functional categories in normal pancreas, primary pancreatic tumors and liver metastases. We identified two genes, Igfbp1 and Serpina1, which were overexpressed only in liver metastatic lesions suggesting that these genes are likely to be involved in the establishment of metastases in Ela-myc transgenic animal model. In addition, metastatic lesions appear to have low levels or absence of Wt1 gene expression while primary tumors express at least two major variants (+ exon 5 or - exon 5) Wt1 transcripts. Igfbp1 and Serpina1 may serve as clinically interesting biomarkers are likely to be useful for prognostic or therapeutic purposes in metastatic pancreatic cancer.