Role of the ductal transcription factors HNF6 and Sox9 in pancreatic acinar-to-ductal metaplasia

Abstract

Objective: Growing evidence suggests that a phenotypic switch converting pancreatic acinar cells to duct-like cells can lead to pancreatic intraepithelial neoplasia and eventually to invasive pancreatic ductal adenocarcinoma. Histologically, the onset of this switch is characterised by the co-expression of acinar and ductal markers in acini, a lesion called acinar-to-ductal metaplasia (ADM). The transcriptional regulators required to initiate ADM are unknown, but need to be identified to characterise the regulatory networks that drive ADM. In this study, the role of the ductal transcription factors hepatocyte nuclear factor 6 (HNF6, also known as Onecut1) and SRY-related HMG box factor 9 (Sox9) in ADM was investigated.

Design: Expression of HNF6 and Sox9 was measured by immunostaining in normal and diseased human pancreas. The function of the factors was tested in cultured cells and in mouse models of ADM by a combination of gain and loss of function experiments.

Results: Expression of HNF6 and Sox9 was ectopically induced in acinar cells in human ADM as well as in mouse models of ADM. HNF6 and, to a lesser extent, Sox9 were required for repression of acinar genes, for modulation of ADM-associated changes in cell polarity and for activation of ductal genes in metaplastic acinar cells.

Conclusions: HNF6 and Sox9 are new biomarkers of ADM and constitute candidate targets for preventive treatment in cases when ADM may lead to cancer. This work also shows that ectopic activation of transcription factors may underlie metaplastic processes occurring in other organs.

Figure 1

HNF6 and Sox9 are expressed in human acinar-to-ductal metaplasia. (A–D) Expression of HNF6 and Sox9 in normal (A,C) and metaplastic (B,D) human pancreas. HNF6 and Sox9 are detected in duct cells, but not in acinar cells. Metaplastic areas associated with pancreatic ductal adenocarcinoma show wide expression of HNF6 and Sox9. (E–H) Immunofluorescent labelling of Amylase, HNF6, Sox9 and CK7 in metaplastic areas. HNF6 and Sox9 are coexpressed in ductal cells (cyan), and HNF6 is detected in a large proportion of metaplastic acinar cells (E). Cells positive for both Amylase and HNF6 are in most cases negative for the ductal marker CK7 (F). Sox9 is coexpressed with Amylase in a subset of metaplastic acinar cells (arrow in G) and several of these Sox9+/Amy+ cells coexpress CK7 (arrowhead). Duct-like structures positive for Sox9 and negative for HNF6 are also found (H). Dotted lines delineate a duct lumen (H).

Figure 2

Ectopic expression of HNF6 in acinar cells is associated with induction of ductal genes and repression of acinar genes in vitro. (A) Relative expression of ductal (left) and acinar (right) genes in cultured 266-6 cells. Cells were transduced with an adenovirus expressing GFP (Ad-GFP), HNF6 (Ad-HNF6), or Sox9 (Ad-Sox9) and gene expression was analyzed by Q-RT-PCR (data are means +/− SEM; n= 4; *, p<0.05; **, p<0.01). (B, C) Immunostaining for GFP, HNF6 and Amylase and Western blot for HNF6 and Amylase in cultured 266-6 cells transduced with Ad-GFP or Ad-HNF6 shows that HNF6 represses Amylase expression. Arrows, HNF6+/Amylase- cells; Arrowhead, HNF6-/Amylase+ cells. Multiplicity of infection (MOI) was 100 for Ad-GFP, 30 and 100 for Ad-HNF6. Amy, Amylase; Ela, Elastase.

Figure 3

Ectopic expression of HNF6 in acinar cells is associated with induction of ductal genes and repression of acinar genes in vivo. (A–D) Immunolabeling and immunohistochemical staining of sections of wild-type (A–C) and ElaC-Sox9^f/f (D) pancreata 3 days after infection by Ad-GFP (A), Ad-Sox9 (B), or Ad-HNF6 (C,D). Metaplasia is detected in wild-type pancreas infected with Ad-HNF6 (C). Ad-HNF6 induces Sox9, and induces the ductal marker CK19 in the cytoplasm of acinar cells, while repressing Amylase. In acinar cells, Ad-HNF6 relocates Ezrin from the apical pole to the cytoplasm. After Ad-Sox9 infection, HNF6, Amylase and Ezrin are unaffected, but marginal induction of CK19 is detectable in acinar cells (B). No induction of Sox9 is observed in ElaC-Sox9^f/f after infection with Ad-HNF6 (D). Ad-HNF6 repressed Amylase in ElaC-Sox9^f/f pancreata, and relocated Ezrin and CK19. Mislocalisation of Ezrin and CK19 was less severe than in the presence of Sox9 (see suppl. figure 3). (E) Percentage of GFP-positive cells that coexpressed HNF6 or Sox9, two (green) or three (yellow) days after Ad-HNF6 infection (left panel). Quantification of the percentage of Sox9-positive cells that coexpressed HNF6 at the same time points. These quantifications show that HNF6 expression is less stable than GFP and Sox9 (right panel). (F) GFP/HNF6 immunolabeling of pancreas two or three days after transduction with Ad-HNF6, which codes for both HNF6 and GFP. HNF6 is less stably expressed than GFP. (G) Cleaved Caspase3/GFP and Cleaved Caspase3/HNF6 immunolabelling of pancreas infected by Ad-GFP and Ad-HNF6, respectively. Many apoptotic cells are detected in pancreas infected with Ad-HNF6. Cl. Caspase3, Cleaved Caspase3.

Figure 4

In the absence of HNF6 or Sox9, acinar cells are resistant to metaplasia induced by pancreatic duct ligation. (A–E) Haematoxylin-eosin-stained sections of pancreata, in normal condition or five days after PDL, show that acini are replaced by tubular complexes in wild-type pancreas (C), whereas they are better preserved in the absence of HNF6 (D) or less affected in the absence of Sox9 (E). (F–J) In wild-type pancreas, expression of HNF6 is only found in ducts (F), but strongly induced in metaplastic acini after PDL (H). The lack of Sox9 prevents induction of HNF6 after PDL (J), except for a few cells (arrowheads in J and O). (K–O) Sox9 expression is strongly induced in wild-type metaplastic acinar cells after PDL (M), whereas only weak induction of Sox9 is detected in the absence of HNF6 (N). Sox9 is efficiently deleted in a subset of ElaC-Sox9^f/f acinar cells, and this is associated with partial resistance to acinar destruction (O). (P–T) Expression of CK19 is induced in metaplastic cells five days after PDL in wild-type (R) but not in hnf6^{− −} (S) and to a lesser extent in ElaC-Sox9^f/f (T) pancreas. Panels H and M and panels J and O show the same sections, with Sox9 labelling reset from blue to green. In panels H and M, metaplastic acini are surrounded by dotted lines. Arrows point to ducts.

Figure 5

In the absence of HNF6 or Sox9 acinar cells are resistant to pancreatic duct ligation-induced loss of polarity and apoptosis. (A–E) Expression of Ezrin is located at the apical pole of acinar cells in untreated wild-type pancreas (A), but extended into the cytoplasm of metaplastic cells after PDL (C). In hnf6^−/− pancreas (B), as well as in duct-ligated hnf6^−/− (D) and ElaC-Sox9^f/f pancreas (E), Ezrin location remains apical. Insets show magnified views of acini. (F–J) Mucin1a expression is observed at the apical pole of acinar cells in wild-type and hnf6^−/− pancreas (F, G), but extended into the cytoplasm of metaplastic cells after PDL in wild-type (H) or ElaC-Sox9^f/f (J) pancreas. In duct-ligated hnf6^−/− pancreas (I), Mucin1a location remains apical. (K–O) Cleaved Caspase3 was induced 5 days after PDL in several acinar cells in wild-type pancreas (M), but not in hnf6^−/− pancreas (N). In ElaC-Sox9^f/f pancreas, the number of Cleaved Caspase3-positive cells was lower than in wild-type pancreas after PDL (O). Arrowheads point to metaplastic acini, arrows to ducts.

Figure 6

HNF6 modulates acinar-to-ductal metaplasia induced by DMBA. (A–H) Hematoxilin- and eosin-stained sections of control (A–D) and hnf6^−/− (E–H) pancreas treated during 20 days with DMBA. (I–L) HNF6 expression was stimulated in ducts and induced in metaplastic acinar cells after 20 days of DMBA treatment (I). This was associated with induction of Sox9 (J) and CK19 (K) in metaplastic cells (arrowheads) and with dispersal of Mucin1a (arrowheads) at a distance from the apical pole of cells (L). In I, cells in yellow are non-specifically labelled blood cells. (M–O) A number of acinar cells persisted in inflammatory regions of DMBA-treated hnf6^−/− mice. The absence of HNF6 did not influence the induction of Sox9 (M) but prevented the induction of CK19 (N; arrow points to duct)) and the mislocalisation of Mucin1a (O). In wild-type sections, lobules adjacent to the inflammatory regions (area 1 in A, magnified in B) were immunostained, whereas in hnf6^−/− sections, acini surrounded by a large inflammatory region were immunostained (H).