Ectopic overexpression of Sonic Hedgehog (Shh) induces stromal expansion and metaplasia in the adult murine pancreas

Abstract

Ligand-dependent activation of the Hedgehog (Hh) signaling pathway has been implicated in both tumor initiation and metastasis of pancreatic ductal adenocarcinoma (PDAC). Prior studies in genetically engineered mouse models (GEMMs) have assessed the role of Hh signaling by cell autonomous expression of a constitutively active Gli2 within epithelial cells. On the contrary, aberrant pathway reactivation in the human exocrine pancreas occurs principally as a consequence of Sonic Hh ligand (Shh) overexpression from epithelial cells. To recapitulate the cognate pathophysiology of Hh signaling observed in the human pancreas, we examined GEMM where Hh ligand is conditionally overexpressed within the mature exocrine pancreas using a tamoxifen-inducible Elastase-Cre promoter (Ela-CreERT2;LSL-mShh). We also facilitated potential cell autonomous epithelial responsiveness to secreted Hh ligand by generating compound transgenic mice with concomitant expression of the Hh receptor Smoothened (Ela-CreERT2;LSL-mShh;LSL-mSmo). Of interest, none of these mice developed intraductal precursor lesions or PDAC during the follow-up period of up to 12 months after tamoxifen induction. Instead, all animals demonstrated marked expansion of stromal cells, consistent with the previously described epithelial-to-stromal paracrine Hh signaling. Hh responsiveness was mirrored by the expression of primary cilia within the expanded mesenchymal compartment and the absence within mature acinar cells. In the absence of cooperating mutations, Hh ligand overexpression in the mature exocrine pancreas is insufficient to induce neoplasia, even when epithelial cells coexpress the Smo receptor. This autochthonous model serves as a platform for studying epithelial stromal interactions in pancreatic carcinogenesis.

Figure 1

Targeting endogenous Shh expression to the adult murine pancreas. (A) The presence of the transgenic construct in somatic cells can be verified by enhanced green fluorescent protein expression. Cre-mediated recombination of the LSL cassette leads to excision of the enhanced green fluorescent protein cassette and expression of Shh (described previously in Hingorani et al. [17]). (B) Positive X-Gal staining revealed robust Cre-mediated recombination in the exocrine pancreas of Ela-CreERT2;LSL-mShh;Rosa26R reporter mice after induction with tamoxifen. (C) Uninduced mouse. (D) Cerebellum of heterozygous Ptch-lacZ mice served as a positive control for X-Gal staining.

Figure 2

Hh misexpression in the adult murine pancreas leads to mesenchymal expansion in the absence of intraductal lesions. (A) No morphologic abnormalities were observed in the pancreata of Ela-CreERT2;LSL-mShh mice in the absence of tamoxifen induction. (B–D) After tamoxifen induction, morphologic changes in the Ela-CreERT2;LSL-mShh pancreata were observed as early as 2 months and persisted at 1 year of follow-up, at which time the study was terminated. The most conspicuous alteration was a marked expansion of stromal cells between existing acinar lobules, displacing the acinar cells over time. Photomicrographs were obtained at 2 (B), 6 (C), and 12 months (D) after tamoxifen induction, respectively. In contrast, no epithelial alterations such as intraductal precursor lesions or exocrine cancers were found. (E and F) Comparable histologic alterations were observed in tamoxifen-induced Ela-CreERT2;LSL-mShh; LSL-mSmo mice, beginning as early as 2 months after induction (E) and persisting up to 1 year (F). No intraductal precursor lesions or exocrine cancers were seen in these cohorts either.

Figure 3

Confirmation of stromal Hh pathway activity on Cre-mediated recombination in the exocrine pancreas. (A) Immunohistochemistry confirmed the expression of Shh ligand in most acinar cells in tamoxifen-induced Ela-CreERT2;LSL-mShh mice. (B) In contrast, Smo expression was essentially restricted to the expanded periacinar stromal compartment in tamoxifen-induced Ela-CreERT2; LSL-mShh mice. (C) The expanded stromal compartment had evidence of Hh activation, as confirmed by expression of beta-galactosidase (blue) in tamoxifen-induced Ela-CreERT2;LSL-mShh;Ptch-lacZ reporter mice. This pattern overlapped with that of Smo receptor expression in B. (D) The stromal population was negative for E-cadherin and expressed nestin, a feature of mesenchymal cells in the adult pancreas.

Figure 4

The expanded stromal compartment expresses markers of primary cilia. Acetylated tubulin expression was observed in (A) islets marked by insulin and in (B) ducts marked by pan-cytokeratin (Pan CK) from Ela-CreERT2; LSL-mShh murine pancreata. In contrast, acetylated tubulin could not be detected by staining in amylase expressing acinar cells (C). (D) Stromal cells (white arrows) intermixed within exocrine structures were identified using Nomarski optics. Expression of acetylated tubulin in these cells was consistent with the presence of primary cilia within the stroma. Staining for insulin, pan CK, acetylated tubulin, and amylase as indicated. Scale bar, 10 µm.