Sonic hedgehog acts at multiple stages during pancreatic tumorigenesis

Abstract

Activation of sonic hedgehog (Shh) signaling occurs in the majority of pancreatic ductal adenocarcinomas. Here we investigate the mechanisms by which Shh contributes to pancreatic tumorigenesis. We find that Shh expression enhances proliferation of pancreatic duct epithelial cells, potentially through the transcriptional regulation of the cell cycle regulators cyclin D1 and p21. We further show that Shh protects pancreatic duct epithelial cells from apoptosis through the activation of phosphatidylinositol 3-kinase signaling and the stabilization of Bcl-2 and Bcl-X(L). Significantly, Shh also cooperates with activated K-Ras to promote pancreatic tumor development. Finally, Shh signaling enhances K-Ras-induced pancreatic tumorigenesis by reducing the dependence of tumor cells on the sustained activation of the MAPK and phosphatidylinositol 3-kinase/Akt/mTOR signaling pathways. Thus, our data suggest that Shh signaling contributes to tumor initiation in the pancreas through at least two mechanisms and additionally enhances tumor cell resistance to therapeutic intervention. Collectively, our findings demonstrate crucial roles for Shh signaling in multiple stages of pancreatic carcinogenesis.

Fig. 1.

Shh expression and signaling in PDECs. (A) RCAS-GFP-infected PDECs in culture. (Left) Phase contrast image. (Right) Fluorescent image. (B) Formation of duct-like structures by GFP-expressing Trp53, Ink4a/Arf null PDECs embedded in matrigel. (C) Detection of Shh expression in RCAS-Shh- and RCAS-GFP-infected PDECs by immunoblotting with an anti-myc monoclonal antibody. Tumor suppressor genotypes are indicated. β-actin serves as a control. (D) RT-PCR analysis of Shh pathway components in RCAS-Shh- and RCAS-GFP-infected PDECs. β-actin serves as a control. w.t., wild type.

Fig. 2.

Shh enhances the proliferation of PDECs. (A) Cell numbers for Shh- or GFP-expressing PDECs of the indicated tumor suppressor genotypes at the indicated times after plating. Dotted lines, Shh-expressing PDECs; solid lines, GFP-expressing PDECs. Results are representative of at least two experiments. (B) RT-PCR analysis of cell cycle-related transcripts in RCAS-Shh- and RCAS-GFP-infected PDECs. β-actin serves as a control. (C) Immunoblot analysis of cell cycle- and Ras-regulated signaling molecules in RCAS-Shh- and RCAS-GFP-infected PDECs. β-actin serves as a control. w.t., wild type.

Fig. 3.

Shh promotes the survival of PDECs. (A) Viability of PDECs of the indicated tumor suppressor genotypes after treatment with a 100 μM concentration of CHX or vehicle as measured by trypan blue exclusion. Shaded bars, Shh-expressing PDECs; open bars, GFP-expressing PDECs. Results are representative of at least two experiments. (B) Immunoblot analysis of cleaved and uncleaved caspase 3 in protein lysates from RCAS-GFP- and RCAS-Shh-infected Ink4a/Arf, Trp53 null PDECs before and after a 24-hour treatment with 100 μM CHX. β-actin serves as a control. (C) Immunoblot analysis of Bcl-2 family members in protein lysates from RCAS-GFP- and RCAS-Shh-infected Ink4a/Arf, Trp53 PDECs treated with a 100 μM concentration of CHX or vehicle. β-actin serves as a control. (D) RT-PCR demonstrating Bcl-X_L mRNA levels in untreated and CHX-treated Ink4a/Arf, Trp53 null PDECs. β-actin serves as a control. (E) Viability of Ink4a/Arf, Trp53 null PDECs after treatment with CHX and Bcl-2, MAPK, and PI3-kinase inhibitors. Shaded bars, Shh-expressing PDECs; open bars, GFP-expressing PDECs. Results are representative of two experiments. ∗, P < 0.01 for CHX + inhibitors compared with CHX alone.

Fig. 4.

Cooperative interaction between Shh and activated K-Ras. (A) Proliferation curves for Ink4a/Arf, Trp53 null PDECs expressing GFP, Shh, K-Ras, or both Shh and K-Ras. (B) Cystic ductal lesion induced after orthotopic transplantation of K-Ras- and Shh-expressing Trp53 null PDECs. Note abundant reactive stroma. (C) H&E-stained tissue section illustrating the histology of tumors induced after orthotopic transplantation of Ink4a/Arf, Trp53 null PDECs expressing both Shh and activated K-Ras. Tumors are mostly undifferentiated (D), with regions of ductal differentiation (box in C, and E). (F) RT-PCR analysis of Shh pathway components in tumors induced after orthotopic transplantation of RCAS-k-ras-infected PDECs (R1–R6) or RCAS-Shh- and RCAS-k-ras-infected PDECs (RS1–RS7). Tumors R1–R3 and RS1–RS3 lack Ink4a/Arf. Tumors R4–R6 and RS4–RS7 lack Trp53 and Ink4a/Arf. β-actin serves as a control.

Fig. 5.

Shh and K-Ras in therapeutic intervention. Proliferation curves of untreated cell lines (dashed lines) or cells treated with specific pathway inhibitors (solid lines) after treatment with the MAPK inhibitor PD98059 (PD) (A), the PI3-kinase inhibitor LY294002 (LY) (B), the smoothened antagonist cyclopamine (CYC) (C), or LY294002 and cyclopamine (D). Immunoblotting of lysates from treated cells for phospho-ERK and phospho-Akt, and RT-PCR for Gli1, confirms comparable pathway inhibition in all cell lines.