Heparin-binding epidermal growth factor-like growth factor eliminates constraints on activated Kras to promote rapid onset of pancreatic neoplasia.

Pancreatic cancer remains as one of the most deadly cancers with few treatment options at late stages and little information about how it develops through earlier stages. Activating mutation of the Kras gene has been implicated in, but is not sufficient for, tumorigenesis. In mouse models of pancreatic cancer, loss of tumor suppressor genes in conjunction with Kras mutation leads to gradual stochastic acquisition of neoplastic precursors and carcinomas, whereas many cells remain phenotypically unaltered in younger mice. Here, we demonstrate that two oncogenic events, mutation of Kras and production of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF), are sufficient for rapid and complete neoplastic transformation of the exocrine pancreas. We found that macrophages are the major source of HB-EGF production in pancreatic cancer tissue samples, and that macrophages are present in high density and in close association with human pancreatic cancer lesions. In a mouse model, high macrophage density was observed at the earliest stages of neoplastic transformation. The consequence of elevated HB-EGF signaling was investigated without the confounding effects of other macrophage-produced factors via transgenic overexpression of the active form of HB-EGF. In this model, HB-EGF was sufficient to promote Kras-initiated tumorigenesis, inducing rapid and complete neoplastic transformation of the entire exocrine pancreas shortly after birth. HB-EGF overexpression and Kras(G12D) together, but neither alone, increased proliferation with increased cyclinD1 and decreased Cdkn2a/2d (p16/p19(Ink4A/Arf)). These findings establish the importance of oncogenic synergy in cancer initiation and promotion, and establish a molecular link between inflammation and the earliest stages of tumor induction.

Developmental aspects of early pancreatic cancer.

The specific cell of origin responsible for generating pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma remains unknown. During development, epithelial stem cells within embryonic pancreatic epithelium give riseto mature acinar, ductal, and islet elements. Emerging evidence suggests that cells with precursor potential also exist within adult pancreas, resulting in significant developmental plasticity among both endocrine and exocrine cell types. In this review, the contribution of developmental plasticity in initiating pancreatic metaplasia and neoplasia is considered, and evidence supporting a role for epithelial stem cells in pancreatic cancer is discussed.

Lineage commitment and cellular differentiation in exocrine pancreas.

Exocrine pancreatic cell types comprise greater than 90% of parenchymal cell mass in the adult pancreas. However, the factors regulating differentiation of acinar and ductal epithelial cells remain incompletely characterized. Like pancreatic islet cells, acinar and ductal cells arise from pluripotent precursors within embryonic pancreatic epithelium. Recent studies have suggested that a common pool of pluripotent stem cells is responsible for generating both endocrine and exocrine cell types, and that specific signaling pathways regulate a critical balance between endocrine and exocrine lineage commitment.

p53-dependent acinar cell apoptosis triggers epithelial proliferation in duct-ligated murine pancreas.

The mechanisms linking acinar cell apoptosis and ductal epithelial proliferation remain unknown. To determine the relationship between these events, pancreatic duct ligation (PDL) was performed on p53(+/+) and p53(-/-) mice. In mice bearing a wild-type p53 allele, PDL resulted in upregulation of p53 protein in both acinar cells and proliferating duct-like epithelium. In contrast, upregulation of Bcl-2 occurred only in duct-like epithelium. Both p21(WAF1/CIP1) and Bax were also upregulated in duct-ligated lobes. After PDL in p53(+/+) mice, acinar cells underwent widespread apoptosis, while duct-like epithelium underwent proliferative expansion. In the absence of p53, upregulation of p53 target genes and acinar cell apoptosis did not occur. The absence of acinar cell apoptosis in p53(-/-) mice also eliminated the proliferative response to duct ligation. These data demonstrate that PDL-induced acinar cell apoptosis is a p53-dependent event and suggest a direct link between acinar cell apoptosis and proliferation of duct-like epithelium in duct-ligated pancreas.

Transcriptional regulation of the cellular retinoic acid binding protein I gene in F9 teratocarcinoma cells.

Retinoic acid (RA) induces the differentiation of many murine teratocarcinoma cell lines such as F9 and P19. In F9 cells, the level of the cellular retinoic acid binding protein I (CRABP I) mRNA is greatly reduced after exposure of the cultured cells to exogenous RA. In P19 cells, the level of CRABP I mRNA is greatly increased after RA exposure. We have identified a 176-bp region in the murine CRABP I promoter, between -2.9 and -2.7 kb 5' of the start site of transcription, which acts as an enhancer in undifferentiated F9 stem cells and through which RA effects inhibition of CRABP I transcription. Within this region are two footprinted sites at -2763 and -2834. This 176-bp regulatory region does not function to enhance CRABP I transcription in P19 stem cells. Several DNA sequences within these two footprinted regions bind proteins from F9 nuclear extracts but not from P19 nuclear extracts (e.g., FP1B, FP1A, and FP2B), as assessed by gel shift assays. This 176-bp CRABP I genomic region has not been sequenced previously and functionally analyzed in cultured cells because it was not present in the murine CRABP I clones used for the promoter analyses reported earlier by another laboratory. The function of this enhancer may be to reduce the expression of the CRABP I gene in specific embryonic cell types in order to regulate the amount of RA to which the cells are exposed.

The CRABP I gene contains two separable, redundant regulatory regions active in neural tissues in transgenic mouse embryos.

The CRABP I gene is expressed in a spatiotemporal pattern in neural and mesenchymal tissues at the onset of organogenesis. The neural pattern of CRABP I expression includes specific rhombomeres of the hindbrain, neural crest cells and their derivatives the optic stalk, and the central area of the neural retina. We have created transgenic mouse lines with CRABP I 5' and transcribed regions fused to the lacZ structural gene that recapitulate much of this neural pattern of expression. Sequences 5' of the transcription initiation site between -7.8 and -3.2 kb confer beta-galactosidase expression to specific rhombomeres, migrating neural crest cells, trigeminal ganglion, the optic stalk, and the neural retina. We have also defined a region located between exon 1 and exon 8 that confers a portion of this expression pattern, including the mesencephalic projections of the trigeminal ganglion, the inner layer of the neural retina, and the peripheral layer of the posterior hindbrain. CRABP I expression in mesenchyme appears to require sequences in addition to or outside of those examined here.

FGF-2, BMP-2, and BMP-4 regulate retinoid binding proteins and receptors in 3T3 cells.

An Important part of intercellular signalling is the ability of responding cells to regulate multiple signal transduction pathways. Just as retinoic acid exposure alters expression of many peptide growth factors and their receptors, we have found that peptide growth factors alter the expression of cellular retinoic acid binding proteins (CRABPs) and retinoic acid receptors (RARs). FGF-2 (basic fibroblast growth factor) treatment of BALB 3T3 fibroblasts increased the level of CRABP I RNA, whereas bone morphogenetic protein (BMP)-2 and BMP-4 reduced this level as well as the levels of CRABP II and RAR beta 1/beta 3 transcripts. Regulation of the CRABP I gene by FGF-2 occurred posttranscriptionally by increasing RNA stability. However, BMP-2 down-regulated CRABP I message without affecting message stability. Neither of these mechanisms was dominant, with intermediate levels of CRABP I RNA occurring in the presence of both FGF-2 and BMP-2 or BMP-4. These two different modes of regulation thus allow different levels of CRABP I RNA accumulation in the presence of different ratios of FGF-2 and BMP-2 or BMP-4.

The roles of retinoids in vertebrate development.

Several lines of experimentation suggest that endogenous retinoids, metabolites of vitamin A, play a role in the anterior/posterior development of the central body axis and the limbs of vertebrates. High levels of endogenous retinoids have been detected in proximity to these developing axes in a variety of vertebrate fetuses. Teratogenesis studies suggest that both retinoid excess and deficiency are capable of disrupting the development of these axes. Finally, retinoic acid receptors regulate many developmental control genes, including homeobox genes and growth factor genes.

The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter.

The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to +11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the G1/S boundary.

Sequences downstream of the transcription initiation site modulate the activity of the murine dihydrofolate reductase promoter.

The murine dihydrofolate reductase gene is regulated by a bidirectional promoter that lacks a TATA box. To identify the DNA sequences required for dihydrofolate reductase transcription, the activities of various templates were determined by in vitro transcription analysis. Our data indicate that sequences both upstream and downstream of the transcription initiation site modulate the activity of the dihydrofolate reductase promoter. We have focused on two regions downstream of the transcription initiation site that are important in determining the overall efficiency of the promoter. Region 1, which included exon 1 and part of intron 1, could stimulate transcription when placed in either orientation in the normal downstream position and when inserted upstream of the transcription start site. This region could also stimulate transcription in trans when the enhancer was physically separate from the promoter. Deletion of region 2, spanning 46 nucleotides of the 5' untranslated region, reduced transcriptional activity by fivefold. DNase I footprinting reactions identified protein-binding sites in both downstream stimulatory regions. Protein bound to two sites in region 1, both of which contain an inverted CCAAT box. The protein-binding site in the 5' untranslated region has extensive homology to binding sites in promoters that both lack (simian virus 40 late) and contain (adenovirus type 2 major late promoter and c-myc) TATA boxes.

Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site.

We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).