Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma has a median survival of 6 months and a 5-year survival of <5%, making it one of the most lethal human cancers (Warshaw and Fernandez-del Castillo 1992). This poor prognosis relates to the uniformly advanced disease stage at the time of diagnosis and to its profound resistance to existing therapies. A number of key challenges must be addressed to permit improvements in patient outcome, including the need to understand more definitively the cellular origins of this disease, to elucidate the biological interactions of the tumor cell and stromal components, to determine the role of specific genetic lesions and their signaling surrogates in the initiation and progression of the tumor, and to uncover the basis for the intense therapeutic resistance of these cancers (Kern et al. 2001). This malignancy is thought to arise from the pancreatic ducts on the basis of its histological and immunohistochemical relationship to this cell type (Solcia et al. 1995). Consistent with a ductal origin, premalignant lesions, known as pancreatic intraepithelial neoplasms (PanINs)—which are thought to arise from the smaller pancreatic ducts—are found in close physical contiguity with advanced malignant tumors (Cubilla and Fitzgerald 1976; Hruban et al. 2001). PanINs appear to progress toward increasingly atypical histological stages and display the accumulation of clonal genetic changes, suggesting that they are precursors of ductal adenocarcinoma (Moskaluk et al. 1997; Yamano et al. 2000; Luttges et al. 2001; Klein et al. 2002). The cell-of-origin question is complicated by the developmental plasticity of the pancreas that enables transdifferentiation between cell lineages (Sharma et al. 1999; Meszoely et al. 2001; Bardeesy and DePinho 2002). Acinar cells have been shown to undergo metaplastic conversion to duct-like cells, both in culture and under a variety of stresses in vivo (Jhappan et al. 1990; Sandgren et al. 1990; Hall and Lemoine 1992; Rooman et al. 2000). The development of pancreatic tumors with ductal features following a process of acinarductal metaplasia—in transgenic mice expressing TGF-α in the acini—has suggested a progenitor role for acinar cells in this malignancy (Meszoely et al. 2001; Wagner et al. 2001). Other experimental studies have suggested that islets cells or a putative pancreatic stem cell population may also give rise to pancreatic adenocarcinomas (Yoshida and Hanahan 1994; Pour et al. 2003). Finally, it remains possible that pancreatic adenocarcinoma arises from any one of these differentiated cell types or from tissue stem cells and, rather, that specific genetic lesions dictate the phenotypic endpoint of the tumor regardless of the originating cellular compartment. This paradigm has been previously suggested in malignant glioma (Holland et al. 1998; Bachoo et al. 2002). Activating KRAS mutations, present in virtually all pancreatic adenocarcinomas, occur with increasing frequency in progressively later stage PanINs (Klimstra and Longnecker 1994; Moskaluk et al. 1997; Rozenblum et al. 1997). The early onset of KRAS mutations suggests a role in tumor initiation. In support of this notion, transgenic mice expressing an activated Kras allele under the control of the acinar-specific elastase-1 promoter develop premalignant ductal lesions (Grippo et al. 2003). Loss of function of the G1 cyclin-dependent kinase inhibitor, INK4A, also appears to be a near universal event in human pancreatic adenocarcinoma, and its pathogenetic relevance is underscored by the increased susceptibility in kindreds harboring germline INK4A mutations (Goldstein et al. 1995; Whelan et al. 1995; Rozenblum et al. 1997). Moreover, in sporadic tumors, the frequent inactivation of the INK4A locus by homozygous deletion has raised the possibility that the physically linked ARF tumor suppressor, encoded in an alternate reading frame and distinct first exon in the INK4A/ARF locus (Quelle et al. 1995), may also play a role in human pancreatic adenocarcinoma. Although ARF is a known activator of the p53 pathway (Kamijo et al. 1998; Pomerantz et al. 1998; Stott et al. 1998; Zhang et al. 1998) and its loss clearly attenuates the function of that critical tumor suppressor, more recent studies have demonstrated that ARF has p53-independent functions—including the repression of ribosomal RNA synthesis, the inhibition of NF-κB activity, and the targeting of E2F for degradation—suggesting that a number of other pathways are impaired by INK4A/ARF loss (Martelli et al. 2001; Rocha et al. 2003; Sugimoto et al. 2003). INK4A/ARF mutations occur in moderately advanced PanIN lesions (at a later stage than do KRAS mutations), an observation consistent with a role for INK4A/ARF in constraining malignant progression (Moskaluk et al. 1997; Wilentz et al. 1998). Finally, mutations in SMAD4/DPC4 and p53—encountered in >50% of tumors—are late events, implying a role in full malignant progression, including acquisition of an invasive phenotype (DiGiuseppe et al. 1994; Wilentz et al. 2000; Luttges et al. 2001). The extensive genetic and molecular data in human tumors provide a strong framework in which to model how a given genetic lesion—or combinations of mutations—governs specific tumor biological features of the disease during its evolution. In this regard, genetically engineered mouse models have proven useful in the systematic dissection of these and related issues for a number of cancer types (Van Dyke and Jacks 2002). In the case of modeling cancer of the exocrine pancreas, transgenic mouse lines targeting a series of oncogenes to the acinar cell compartment have produced acinar carcinomas, mixed acinar-ductal tumors, or cystic tumors (Ornitz et al. 1987; Quaife et al. 1987; Sandgren et al. 1991; Glasner et al. 1992; Bardeesy et al. 2002a; Grippo et al. 2003). An important advance was provided by the observation of pancreatic tumors with ductal features in a subset of Elastase-TGFα; p53+/- mice (Wagner et al. 2001). These tumors were associated with widespread acinar-ductal metaplasia, a phenomenon whose relevance to human adenocarcinoma remains an area of active investigation. A notable recent report has described the impact of activated Kras expression directed to the differentiated pancreatic ductal epithelium by the cytokeratin 19 (Ck-19) promoter (Brembeck et al. 2003). These transgenic mice did not develop overt neoplastic lesions of the ducts, suggesting that cooperating mutations are needed for tumor initiation or that the differentiated ducts are insensitive to the transforming potential of the activated Kras allele. With regard to the latter possibility, there is a notable lack of tumors in transgenic mice expressing activated Hras in the β-cells of the pancreatic islet (Efrat et al. 1990). Mice with constitutive deletion of both or either component of the Ink4a/Arf locus do not develop spontaneous pancreatic adenocarcinoma; however, the rapid onset of lymphomas and sarcomas in these mice may preclude the study of more latent effects of deletions of this locus on pancreatic neoplasia (Serrano et al. 1996; Kamijo et al. 1997; Krimpenfort et al. 2001; Sharpless et al. 2001). These previous studies have provided significant insights into disease pathogenesis and underscore the need for continued efforts directed toward the construction of multiallelic mouse models that recapitulate the genesis and progression of the human disease.