AGR2 is a SMAD4-suppressible gene that modulates MUC1 levels and promotes the initiation and progression of pancreatic intraepithelial neoplasia

Abstract

The mechanisms controlling expression of the putative oncogene Anterior gradient 2 (AGR2) in pancreatic ductal adenocarcinoma (PDAC) are not well understood. We now show that AGR2 is a transforming growth factor-β (TGF-β)-responsive gene in human pancreatic cancer cells, whose downregulation is SMAD4 dependent. We also provide evidence supporting a role for AGR2 as an ER-chaperone for the cancer-associated mucin, MUC1. AGR2 is both sufficient and required for MUC1 expression in pancreatic cancer cells. Furthermore, AGR2 is coexpressed with MUC1 in mouse pancreatic intraepithelial neoplasia (mPanIN)-like lesions and in the cancer cells of four distinct genetically engineered mouse models of PDAC. We also show that Pdx1-Cre/LSL-Kras(G12D)/Smad4(lox/lox) mice heterozygous for Agr2 exhibit a delay in mPanIN initiation and progression to PDAC. It is proposed that loss of Smad4 may convert TGF-β from a tumor suppressor to a tumor promoter by causing the upregulation of AGR2, which then leads to increased MUC1 expression, at which point both AGR2 and MUC1 facilitate mPanIN initiation and progression to PDAC.

Figure 1. AGR2 is a downstream target of TGF-β signaling

(A) Quantitative RT-PCR of AGR2 mRNA, 24 hrs after addition of 500 pM TGF-β1, in ASPC-1, BxPC3, COLO-357, and PANC-1 cells. Data are the means ± SEM from at least three experiments. *p < 0.01, compared with respective controls. (B) The levels of AGR2 RNA were determined by quantitative RT-PCR following addition of 500 pM TGF-β1 for 0, 1, 3, 8, 12, 16, and 24 hrs in COLO-357 (white) and PANC-1 (black). The points plotted are the average of two experiments at each time point. (C) Western blot of AGR2, SMAD4, and ERK2 (loading control) in ASPC-1, BxPC3, COLO-357, and PANC-1 cells after 48 hrs of incubation with 500 pM TGF-β1. T3M4 cells had no detectable levels of AGR2 protein. (D) Densitometry of AGR2 immunoreactivity following 48 hrs of TGF-β in ASPC-1, BxPC3, COLO-357, and PANC-1. The mean pixel density of AGR2 was quantitated and normalized to its corresponding ERK2 (loading control). Data are the means ± SEM from at least three experiments.*p < 0.01, compared to untreated control. (E) Western blot of AGR2 and ERK2 (loading control) in PANC-1 cells after 16, 24, and 48 hrs incubation with TGF-β1. (F) Western blot of AGR2 and ERK2 (loading control) in COLO-357 after 16, 24, and 48 hrs incubation with 500 pM TGF-β1.

Figure 2. AGR2 is a transcriptional target of SMAD4

(A) Quantitative RT-PCR of AGR2 RNA levels in ASPC-1, BxPC3, COLO-357, and PANC-1 cells with CMV-HA sham alone, CMV-SMAD4, CMV-HA sham/TGF-β, or CMV-SMAD4/TGF-β. Data are the means ± SEM from at least three experiments. *p < 0.05, and **p < 0.01 compared to respective controls. (B) A western blot showing SMAD4, AGR2, and ERK2 (loading control) protein levels in COLO-357 cells stably expressing either pGIPZ-SMAD4 (left) or pGIPZ-scrambled (right) shRNA silencing vectors, with or without treatment with TGF-β. (C) A luciferase assay using the AGR2-luc reporter with either wild-type, mutated SBE1, mutated SBE2, or with both SBEs mutated and with co-transfection of either CMV-HA sham or CMV-SMAD4 in PANC-1. Percent luciferase units relative to wild-type and untreated AGR2-luc control are shown, after normalization to a Renilla internal control for transfection and cell lysis. Data are the means ± SEM from three experiments. *p < 0.01 compared with respective control.

Figure 3. AGR2 localizes to the endoplasmic reticulum of pancreatic cancer cells

(A) Immunofluorescence of AGR2 and an endoplasmic reticulum (ER)-associated protein, GRP78, in PANC-1 cells. AGR2 is shown in red, GRP78 in green, DAPI (nuclear stain) in blue, and the merged image of all three on the far right. Yellow color indicates areas of co-localization of AGR2 and GRP78. Magnification: 400×; Scale bar: 10 μm. (B) Live cell confocal imaging of a PANC-1 cell expressing an AGR2-RFP fusion protein (left panel) and stained with an ER-targeted dye (ER Tracker©. The merged AGR-RFP/ER Tracker signal is shown on the far right. The brightfield picture, with merged colors, is also pictured in the middle right for reference.

Figure 4. AGR2 interacts with and is required for MUC1 protein expression

(A) Immunofluorescence of AGR2 (red), MUC1 (green), and DAPI (blue) in PANC-1 cells. The merged image is in the far right panel. Yellow color indicates where AGR2 and MUC1 co-localize. Magnification: 800×; Scale bar: five μm. (B) Immunoprecipitation using anti-AGR2, mouse IgG, or no antibody (A/G beads only) and blotting for MUC1, MUC4, MUC5AC, or AGR2. Input is 1/10 the total protein used in the precipitation. Lysates shown were prepared from PANC-1. (C) Quantitative RT-PCR of AGR2 mRNA in stable COLO-357 cells expressing pTRIPZ-AGR2 (doxycycline-inducible silencing vector) and treated with 0, 0.1, 0.5, 1.0, or 2.0 ug/mL doxycycline for 72 hrs. Data are the means ± SEM of three experiments. (D) Western blot for MUC1, AGR2, or ERK2 (loading control) in stable COLO-357-pTRIPZ-AGR2 cells treated with 0, 0.1, 0.5, 1.0, or 2.0 ug/mL doxycycline for 72 hrs. (E) Quantitative RT-PCR of human (hAGR2, top) and mouse (mAGR2, bottom) AGR2 in stable COLO-357-pTRIPZ-AGR2 cells transiently transfected either with empty pcDNA vector (sham) or a pcDNA-AGR2 expression vector (magr2) and untreated or treated with 1 ug/mL DOX for 72 hrs. Data are the average ± SD from two experiments. (F) Western blot for MUC1, MUC4, AGR2, or ERK2 (loading control) in untreated, DOX-treated stable COLO-357-pTRIPZ-scramble (pT-scrm), or COLO-357-pTRIPZ-AGR2 cells transiently transfected with either empty pcDNA vector (pT-AGR2) or pcDNA-mAGR2 (pT-AGR2 + AGR2). (G) Western blot for MUC1, AGR2, and ERK2 (loading control) in stable PANC-1 clones transfected either with empty pcDNA vector (sham; first lane) or pcDNA-AGR2 (Cl.1 and Cl.2; second and third lanes). MUC1 migrates as a doublet in PANC-1 cells. (H) Western blot of MUC1 and ERK2 (loading control) in ASPC-1, BxPC-3, COLO-357, and PANC-1 cells incubated in the absence or presence of 500 pM TGF-β1. *MUC1 was not detectable in ASPC-1 cells and migrated as a doublet in PANC-1 cells.

Figure 5. AGR2 co-localizes with MUC1 in vivo

(A) Co-immunofluorescence in a Pdx1-Cre/Kras^G12D/p53^L/L model. Shown are mouse low-grade PanIN lesions stained for AGR2 (red), MUC1 (green), and DAPI (blue). The merged image from AGR2 and MUC1 is shown in the far right panel. Yellow color indicates areas of co-localization of AGR2 and MUC1. Magnification: 100×; Scale bar: 40 μm. (B) Alcian blue staining combined with immunohistochemical detection of AGR2 in mPanIN-3 and early invasive adenocarcinoma from a Pdx1-Cre/Kras^G12D/Smad4^L/L model. Magnification: 200×; Scale bar: 20 μm. (C) Immunohistochemistry of AGR2 (left) and MUC1 (right) in serial sections of human pancreas, with the same area of the mPanIN structure magnified in an enlarged view. An mPanIN-2 lesion is seen on the left, within a larger area of ADM. Early adenocarcinoma shown on the right, and is enlarged in the box. Invasive clusters surround the larger lesion and stain highly for AGR2. Magnification: 100×; Scale bar: 40 μm.

Figure 6. AGR2 is required for MUC1 expression in vivo

This figure shows serial sections of the same lesion, stained with either hematoxylin/eosin (column 1), co-IF for CK19 and amylase (column 2), Alcian blue (column 3), IF for AGR2 (column 4), or IF for MUC1 (column 5). (A) An mPanIN-1 lesion from Pdx1-Cre/Kras^G12D/Smad4^L/L (AGR2 positive). (B) An mPanIN-1 lesion from Pdx1-Cre/Kras^G12D/Smad4^L/L/Agr2^−/− (AGR2 negative). (C) Low-grade adenocarcinoma from Pdx1-Cre/Kras^G12D/Smad4^L/L/Agr2^+/− (AGR2 heterogeneous). (D) mPanIN-1/PanIN-2 lesion from Pdx1-Cre/Kras^G12D/Smad4^L/L/Agr2^+/− (AGR2 heterogenous). Magnification: 100×; Scale bar: 40 μm.

Figure 7. Model of AGR2 regulation by TGF-β and its involvement with MUC1 in pancreatic cancer cells

We describe a model in which, through one of potentially many mechanisms, AGR2 is negatively regulated by TGF-β signaling. In the presence of TGF-β, SMAD4 translocates to the nucleus, binds to SMAD-binding elements, and interacts with nuclear effectors of transcription. In the case of AGR2, SMAD4 facilitates a repression of AGR2 transcription and prevents its downstream stabilization of MUC1 in the endoplasmic reticulum.