Acinar-to-Ductal Metaplasia Induced by Transforming Growth Factor Beta Facilitates KRASG12D-driven Pancreatic Tumorigenesis

Abstract

Background & aims: Transforming growth factor beta (TGFβ) acts either as a tumor suppressor or as an oncogene, depending on the cellular context and time of activation. TGFβ activates the canonical SMAD pathway through its interaction with the serine/threonine kinase type I and II heterotetrameric receptors. Previous studies investigating TGFβ-mediated signaling in the pancreas relied either on loss-of-function approaches or on ligand overexpression, and its effects on acinar cells have so far remained elusive.

Methods: We developed a transgenic mouse model allowing tamoxifen-inducible and Cre-mediated conditional activation of a constitutively active type I TGFβ receptor (TβRI^CA) in the pancreatic acinar compartment.

Results: We observed that TβRI^CA expression induced acinar-to-ductal metaplasia (ADM) reprogramming, eventually facilitating the onset of KRAS^G12D-induced pre-cancerous pancreatic intraepithelial neoplasia. This phenotype was characterized by the cellular activation of apoptosis and dedifferentiation, two hallmarks of ADM, whereas at the molecular level, we evidenced a modulation in the expression of transcription factors such as Hnf1β, Sox9, and Hes1.

Conclusions: We demonstrate that TGFβ pathway activation plays a crucial role in pancreatic tumor initiation through its capacity to induce ADM, providing a favorable environment for KRAS^G12D-dependent carcinogenesis. Such findings are highly relevant for the development of early detection markers and of potentially novel treatments for pancreatic cancer patients.

Keywords: ADM, acinar-to-ductal metaplasia; AFI, acinar fatty infiltration; Acinar-to-Ductal Metaplasia; Cancer; EMT, epithelial-to-mesenchymal transition; KRASG12D; PBS, phosphate-buffered saline; PDA, pancreatic ductal adenocarcinoma; PanIN, pancreatic intraepithelial neoplasia; Pancreas; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TGFβ; TGFβ, transforming growth factor beta; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Graphical abstract

Figure 1

KRAS^G12Dpotentiates TGFβ-mediated cell growth inhibition of AR42J rat pancreatic acinar cells. (A) Immunoblot of RAS-GTP and total RAS on AR42J-WT and AR42J-KRAS^G12D protein extracts. (B) Phase microscopy of AR42J-WT and AR42J-KRAS^G12D cells 48 hours after TGFβ treatment. Insets represent higher magnifications fields. Scale bar, 50 μm/L. NT, untreated. *Apoptotic cells. (C) Cell count of AR42J-WT and AR42J-KRAS^G12D cells at indicated times after TGFβ treatment. Representative experiment performed in duplicate (means ± standard deviation) is shown. (D) Immunoblot of SMAD proteins after 2-hour TGFβ treatment.

Figure 2

KRAS^G12Dand TGFβ cooperate to activate apoptosis of AR42J rat acinar cells. (A) Caspase-3 assay after 12-hour TGFβ treatment. (B) TUNEL assay after 24-hour TGFβ treatment. Upper panel, fluorescence microscopy. Lower panel, graphs showing percentage of TUNEL-positive cells under fluorescence microscopy and counted in each condition (>1000 cells). (C) Annexin-V/propidium iodide (PI) assay after 48-hour TGFβ treatment. Upper panel, raw FACS data. Upper right box drawn on each plot corresponds to apoptotic cell population (cells positive for both annexin-V and PI). Lower panel, quantification of apoptotic cells in upper right boxes drawn on plots above. For (A–C), representative experiment performed in triplicate (means ± standard deviation) is shown. (D) RT-qPCR of proapoptotic Bmf (BCL2-modifying factor) marker. Cells were treated (or not) with TGFβ ± TβRI inhibitor (SB431542) for 48 hours. For each condition, mRNA level is represented as mean ± standard deviation of 1 representative experiment performed in triplicate.

Figure 3

KRAS^G12Dand TGFβ cooperate to activate dedifferentiation of AR42J rat acinar cells. (A) High-magnification microscopy of rat acinar cells (AR42J). Phase-contrast microscopy of AR42J-WT and AR42J-KRAS^G12D cells treated or not with TGFβ for 48 hours. NT, untreated. Scale bars, 100 μm. (B) RT-qPCR of acinar (Ela-1, Cpa1, Mist1), progenitor (Hnf1β, Hes1), and ductal (Sox9) markers after 48-hour TGFβ treatment. For each condition, expression is represented as mean ± standard deviation of at least 3 independent experiments. Statistical analyses were performed by using Mann-Whitney test: *P < .05, **P < .01, ***P < .001. Non-significant (ns) if P > .05. (C) Signaling pathways involved in TGFβ-induced Hnf1β mRNA activation. AR42J-WT cells were cultured in presence of TGFβ along with different kinase inhibitors (inh) of canonical and non-canonical TGFβ pathways: TβRI inh, SB431542; MEK inh, U0126 monoethanolate; JNK inh, JNK-IN-8; P38 inh, SB203580; PI3K inh, LY294002. Hnf1β mRNA was detected by RT-qPCR after 48-hour treatment. For each condition, folds are represented as mean ± standard deviation of at least 3 independent experiments. Statistical analyses were performed by using Mann-Whitney U test: **P < .01. Nonsignificant (ns) if P > .05.

Figure 4

Early activation of TβRI^CAduring mouse development is embryonically lethal. [E2A-Cre^+/+] or [E2A-Cre^+/-] mice were crossed with [LSL-TβRI^CA] (R) mice. Total number of litters, pups, and offspring genotype distribution are presented. Fisher’s exact test was performed to statistically confirm absence of [E2A-Cre^+/-; LSL-TβRI^CA] mice. Nonsignificant (ns), P > .05; *P < .05.

Figure 5

Expression of TβRI^CAin mouse embryo compromises development of acinar compartment. (A) Breeding strategy to target TβRI^CA expression in whole adult body by using tamoxifen-inducible Rosa26-Cre^ERT2 allele. (B) Overall survival (Kaplan-Meier analysis) of wild-type (WT) and [Rosa26-Cre^ERT2; LSL-TβRI^CA] mice after tamoxifen injection. Log-rank (Mantel-Cox) test. **P = .0047. (C) Histology of pancreata prepared from WT and [Rosa26-Cre^ERT2; LSL-TβRI^CA] mice 5 days after tamoxifen injection. White arrows, apoptotic cells. Scale bars, 200 μm. (D) Breeding strategy to target TβRI^CA expression in all epithelial pancreatic lineages from embryonic day 8.5 (E8.5) by using Pdx1-Cre allele. (E) Histology of pancreata prepared from WT and [Pdx1-Cre; LSL-TβRI^CA] (CR) E19.5 embryos. Scale bars, 200 μm. mag, magnification; TAM, tamoxifen.

Figure 6

Inducible TβRI^CAexpression in pancreatic acinar cells after birth results in SMAD pathway activation in vivo. (A) Breeding strategy to express TβRI^CA after birth in pancreatic acinar cells by using tamoxifen-inducible Pft1a-Cre^ERT2 allele. Black arrows represent genotyping primers. Sizes of expected fragments are shown. (B) PCR on genomic DNA to assess excision of STOP signal in C^ERR pancreas after tamoxifen injection. (C) RT-qPCR of TβRI^CA and Serpine-1 on total RNA from pancreata prepared from mice treated with tamoxifen. Expression level in R mice was arbitrarily set at 1 (mean ± standard deviation; 2 independent experiments). (D) Immunoprecipitation of SMAD2/3 and Western blot analysis of P-SMAD2 (phospho-SMAD2) and total SMAD2. Total lysate was assessed for β-tubulin and SMAD2. (E) RNAscope detection of TβRI^CA and Smad7 mRNA on pancreatic sections from WT and C^ERR mice. Scale bars, 100 μm. (A–E): WT, wild-type; R, [LSL-TβRI^CA]; C^ERR, [Pft1a-Cre^ERT2; LSL-TβRI^CA]. mag, magnification; TAM, tamoxifen.

Figure 7

TβRI^CAexpression in acinar cells induces apoptosis and ductal-like differentiation 3 days after induction. (A) Diagram of experimental design for 5-week-old mice injected with tamoxifen and euthanized 3 days later. (B) H&E staining, TUNEL assay, and immunofluorescence of amylase, CK19, and SOX9. Black arrowheads, apoptotic cells; white arrowheads, CK19/amylase and SOX9/amylase double-positive cells; WT, wild-type; [Pft1a-Cre^ERT-; LSL-TβRI^CA], C^ERR; [Pft1a-Cre^ERT2; LSL-Kras^G12D], C^ERK; [Pft1a-Cre^ERT2; LSL-TβRI^CA; LSL-Kras^G12D], C^ERKR. TAM, tamoxifen. Scale bars, 50 μm.

Figure 8

TβRI^CAexpression in acinar cells leads to regenerative ADM 3 weeks after induction. (A) Diagram of experimental design for 5-week-old mice injected with tamoxifen and euthanized 3 weeks later. (B) H&E staining and immunofluorescence of amylase and CK19. WT, wild-type; [Pft1a-Cre^ERT-; LSL-TβRI^CA], C^ERR; [Pft1a-Cre^ERT2; LSL-Kras^G12D], C^ERK; [Pft1a-Cre^ERT2; LSL-TβRI^CA; LSL-Kras^G12D], C^ERKR. TAM, tamoxifen. Scale bars, 100 μm.

Figure 9

TβRI^CAexpression in acinar cells accelerates KRAS^G12D-induced tumorigenesis several months after induction. (A) Diagram of experimental design representing 5-week-old mice injected with tamoxifen and euthanized 2 or 6 months later. (B) H&E staining. WT, wild-type; [Pft1a-Cre^ERT-; LSL-TβRI^CA], C^ERR; [Pft1a-Cre^ERT2; LSL-Kras^G12D], C^ERK; [Pft1a-Cre^ERT2; LSL-TβRI^CA; LSL-Kras^G12D], C^ERKR. Scale bars, 100 μm. (C) Quantification of pancreatic epithelial lesions at different grades and observed in C^ERK and C^ERKR pancreata 2 months and 6 months after TAM injection. TAM, tamoxifen.

Figure 10

KRAS^G12Dand TGFβ activation cooperate to accelerate pancreatic tumorigenesis without affecting endocrine compartment. Immunohistochemical detection of CK19 and INSULIN in mice of indicated genotypes 2 and 6 months after tamoxifen induction. [Pft1a-Cre^ERT2; LSL-TβRI^CA] (C^ERR); [Pft1a-Cre^ERT2; LSL-Kras^G12D] (C^ERK); [Pft1a-Cre^ERT2; LSL-TβRI^CA; LSL-Kras^G12D] (C^ERKR). Scale bars, 200 μm.

Figure 11

Schematic diagram showing effect of TGFβ signaling activation (alone or in combination with KRAS^G12D) on pancreatic acinar compartment. A few days after tamoxifen induction, TβRI^CA induces acinar cell apoptosis and subsequent regenerative metaplasia (ADM). In aging mice, ADM is overcome and replaced by AFI. KRAS^G12D activation alone induces ADM and PanINs several months after induction. When TβRI^CA and KRAS^G12D are simultaneously induced, PanINs develop much earlier. In the context of TβRI^CA-induced ADM, KRAS^G12D induces regenerative cells to undergo the ADM>ADR>PanIN sequence (ADR, acinar-to-ductal reprogramming). Hence, TGFβ signaling activation enhances the properties of KRAS^G12D through its capacity to prime a suitable surrounding for transformation. TAM, tamoxifen. [Pft1a-Cre^ERT-; LSL-TβRI^CA], C^ERR; [Pft1a-Cre^ERT2; LSL-Kras^G12D], C^ERK; [Pft1a-Cre^ERT2; LSL-TβRI^CA; LSL-Kras^G12D], C^ERKR. TAM, tamoxifen. WT, wild-type.