Acinar cells contribute to the molecular heterogeneity of pancreatic intraepithelial neoplasia - PubMed (original) (raw)

Acinar cells contribute to the molecular heterogeneity of pancreatic intraepithelial neoplasia

Liqin Zhu et al. Am J Pathol. 2007 Jul.

Abstract

A number of studies have shown that pancreatic ductal adenocarcinoma develops through precursor lesions termed pancreatic intraepithelial neoplasia (PanIN). PanINs are thought to initiate in the small ducts of the pancreas through activating mutations in the KRAS proto-oncogene. What remains unanswered is the identification of the individual cell type(s) that contributes to pancreatic ductal adenocarcinoma formation. To follow the cellular and molecular changes that occur in acinar and duct cell properties on Kras(G12D) expression, we took advantage of LSL-Kras(G12D/+)/p48(Cre/+) mice, which faithfully mimic the human disease. In young animals (4 weeks), the predominant cellular alteration in the exocrine pancreas was acinar metaplasia in which individual acini consisted of acinar cells and duct-like cells. Metaplastic acinar structures were highly proliferative, expressed Notch target genes, and exhibited mosaic expression patterns for epidermal growth factor receptor, ErbB2, and pErk. This expression pattern paralleled the expression pattern detected in mouse PanINs, suggesting that mouse PanINs and acinar-ductal metaplasia follow similar molecular pathways. Indeed, immunofluorescence studies confirmed the presence of acinar cells within mPanIN lesions, raising the possibility that Kras(G12D)-induced mPanINs develop from acinar cells that undergo acinar-ductal metaplasia. Identification of an acinar contribution to PanIN formation offers new directions for successful targeted therapeutic approaches to combat this disease.

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Figures

Figure 1

Figure 1

Acinar metaplasia is an early event in KrasG12D expression. A: PanINs in humans are often associated with lobulocentric atrophy producing three zones of cells on H&E staining—central PanIN lesions, acinar-ductal metaplasia (ac-to-d), and normal acinar (ac) cells. B: Higher resolution of human acinar-ductal metaplasia showing metaplastic units that are composed of acinar cells (arrows) and duct-like cells. C: LSL-Kras G12D/+/p48 Cre/+ pancreas section containing the same three zones of exocrine cells as observed in human samples. D: Higher resolution images of mouse acinar-ductal metaplastic units revealing the contribution of acinar (arrows) and duct-like cells to these structures. E: H&E staining of control wild-type (WT) pancreas samples reveal normal acinar and duct (d) structures. F: Normal histological section from a 4-week-old LSL-Kras G12D/+/p48 Cre/+ mouse. These areas are indistinguishable from the control sample in E. G and H: Early metaplastic acinar units from 4-week LSL-Kras G12D/+/p48 Cre/+ pancreas sections revealing the contribution of acinar (arrows) cells and duct-like cells. I: An early acinar metaplasia area from a 4-week LSL-Kras G12D/+/p48 Cre/+ sample costained for K19 (brown) and amylase (purple). These individual structures reveal biphenotypic acinar cells (arrows) expressing duct cell markers. Note that these structures are surrounded by normal acinar tissue. J: Control WT pancreas costained for K19 and amylase expression. Note the mutually exclusive expression pattern of K19 and amylase in duct and acinar cells, respectively. K: Immunoblot analysis on 4-month control LSL-Kras G12D/+ (Cre−) and LSL-Kras G12D/+/p48 Cre/+ (Cre+) pancreas samples. Pancreata that activate the Kras G12D allele exhibit a decrease in acinar cell products (amylase) and an increase in duct cell products (K19, CA-II). The Hsp90 blot serves as a loading control.

Figure 2

Figure 2

Ductal cells within acinar-ductal metaplastic units are highly proliferative. A: Control 4-week pancreas immunolabeled for Ki67. A small percentage of acinar cells (black arrows) and duct cells (white arrows) are Ki67-positive. B: Morphologically normal areas from a 4-week LSL-Kras G12D/+/p48 Cre/+ mouse reveal an increase in the percentage of Ki67-expressing acinar (black arrows) and duct (white arrows) cells. d, duct. C–F: Early metaplastic structures from 4-week LSL-Kras G12D/+/p48 Cre/+ mice reveal heterogeneity with respect to Ki67 expression. Whereas the acinar component (white arrows) within the metaplastic units remain Ki67-negative, the duct-like components (black arrows) are invariably Ki67-positive. Note that as the metaplastic units increase in size, the number of proliferating cells also increase. The dotted line in C highlights a single metaplastic acinar unit.

Figure 3

Figure 3

Hes1-negative acinar cells and Hes1-positive duct-like cells are found within acinar-ductal metaplastic structures. A: Semiquantitative reverse transcriptase-PCR showing that the Notch downstream target genes Hes1, Hey1, and Hey2 are transcriptionally active in the LSL-Kras G12D/+/p48 Cre/+ model. B and C: Adjacent sections from a WT pancreas sample immunolabeled with antibodies to Hes1 and Mist1. Centroacinar cells (arrows) express Hes1 but not Mist1 (an acinar cell-restricted transcription factor). By contrast, normal acinar cells are Mist1-positive but Hes1-negative. D–F: Early acinar metaplastic units from LSL-Kras G12D/+/p48 Cre/+ mice reveal that Hes1 protein (arrows) is found in the duct-like cells. Note the normal acinar cells that surround these localized transition events. G and H: Adjacent serial sections from LSL-Kras G12D/+/p48 Cre/+ pancreas tissue stained with anti-Mist1 and anti-Hes1. A single acinar-ductal metaplastic unit reveals cells that are both Hes1- and Mist1-positive (arrows), confirming that activation of Hes1 expression initiates in metaplastic acinar cells. The surrounding normal acinar cells remain Hes1-negative but Mist1-positive. I: A single mPanIN lesion showing that mPanIN cells express Hes1 whereas the surrounding acinar tissue is Hes1-negative. J–L: Human pancreas sections from PDA patients stained for hMist1 protein. The boxed-in area in J is shown at higher magnification in K. Note the Mist1-positive (acinar) cells within the metaplastic ductal lesions (red arrows). The black arrow in L points to an early metaplastic acinus. The extreme edge of a PanIN-2 lesion is shown in J (asterisk). M: A control WT section showing that PDX1 is primarily expressed in islet cells. d, duct. N and O: PDX1 expression (red arrows) is up-regulated during acinar-ductal metaplasia in the LSL-Kras G12D/+/p48 Cre/+ mice. Adjacent normal acinar tissue (black arrows) remains PDX1-negative. PDX1 is also up-regulated in mPanINs (inset) in this model.

Figure 4

Figure 4

Acinar-ductal metaplasia leads to heterogeneity in EGFR, pErk, and ErbB2 expression profiles. A–C: LSL-Kras G12D/+/p48 Cre/+ pancreas serial sections stained with anti-pErk, anti-EGFR, and anti-Mist1 reveal that mPanIN lesions are pErk-positive but EGFR- and Mist1-negative. Boxed areas are shown at higher resolution in the insets. D–F: Analysis of acinar metaplasia reveals a distinct gene expression pattern in which Mist1-positive cells (red arrows) coexpress EGFR, whereas Mist1-negative cells (black arrows) express pErk but are EGFR-negative. These results confirm that mPanINs and duct-like cells within acinar metaplastic units exhibit a similar EGF-signaling expression pattern. G and H: Serial sections from LSL-Kras G12D/+/p48 Cre/+ pancreas samples were labeled with antibodies to EGFR and to ErbB2. EGFR and ErbB2 exhibit opposite expression patterns in acinar metaplastic structures where nuclear ErbB2 protein is restricted to the EGFR-negative duct-like cell compartment. Note that although ErbB2 protein levels are greatly elevated in all metaplastic acinar cells, nuclear ErbB2 is found only in the more advanced EGFR-negative cells. Boxed areas are shown at higher resolution in the insets. I and J: Similar sections as in G and H showing that mPanIN lesions are nuclear ErbB2-positive but EGFR-negative, reflecting an identical expression pattern as observed in acinar-ductal metaplastic structures. K and L: Immunoblot analysis on protein extracts isolated from control LSL-Kras G12D/+ (Cre−) and LSL-Kras G12D/+/p48 Cre/+ (Cre+) pancreas samples from 2- and 4-month animals. EGFR and ErbB2 protein levels are not detected in the control samples but dramatically increase with advancing age in the LSL-Kras G12D/+/p48 Cre/+ animals. The EGFR and ErbB2 receptors are active in these samples as revealed by the presence of phosphotyrosine residues 845/992 and 1221, respectively. The Hsp90 blot serves as a loading control.

Figure 5

Figure 5

Acinar cells contribute to the molecular heterogeneity of mPanINs. A and B: Ki67 labeling of mPanIN-1 lesions from LSL-Kras G12D/+/p48 Cre/+ mice revealing the high percentage of proliferating cells in these structures. Examination of mPanIN lesions often reveal a subset of Ki67-negative cells (boxed areas), which exhibit acinar cell properties and zymogen granules (arrows in inset). C: Amylase and K19 coimmunofluorescence showing a single mPanIN-1 lesion that contains cells coexpressing both products (inset). D–F: Serial sections from LSL-Kras G12D/+/p48 Cre/+ pancreas samples were stained with antibodies to EGFR, pErk, and Mist1. EGFR and pErk exhibit opposite expression patterns in mPanIN lesions where Mist1-positive PanIN cells are EGFR-negative but pErk-positive. G: H&E staining of a large mPanIN lesion that is associated with areas of acinar-ductal metaplasia. Arrows indicate areas where acinar metaplasia is part of the mPanIN epithelial cell layer. H and I: Acinar-ductal/mPanIN hybrid structures from a 4-week LSL-Kras G12D/+/p48 Cre/+ pancreas. These structures have properties that are common to both acinar-ductal metaplasia and mPanINs and are composed of both duct-like cells and acinar cells. The arrows point to several acinar cells (zymogen-positive) that are within these early acinar-ductal/mPanIN-1 hybrid structures.

Figure 6

Figure 6

Model of acinar-ductal conversion in mPanINs. Acinar cells can be identified in mPanIN-1 structures by several molecular markers, including expression of the acinar gene products amylase and Mist1. These cells remain proliferatively quiescent. During acinar-ductal metaplasia, the cells undergo a switch from acinar cell properties to duct cell properties, activating expression of K19, Muc1, and CA-II. During this transition, the Hes1 and PDX1 transcription factors are similarly up-regulated. There is also a change in EGF signaling activity in which acinar cells are EGFR-, ErbB2-, and pERK-negative. At early stages of metaplasia, EGFR and cytoplasmic ErbB2 protein can be detected. As these cells transition to duct-like cells they repress EGFR expression and accumulate nuclear ErbB2 and pErk while entering a proliferative phase. Molecularly, the ductal component of acinar metaplastic units are identical to what is detected in PanIN lesions, suggesting that acinar cells convert to mPanIN cells during this process.

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