Inactivation of Patched1 in mice leads to development of gastrointestinal stromal-like tumors that express Pdgfrα but not kit

Abstract

Background & aims: A fraction of gastrointestinal stromal tumor (GIST) cells overexpress the platelet-derived growth factor receptor (PDGFR)A, although most overexpress KIT. It is not known if this is because these receptor tyrosine kinases have complementary oncogenic potential, or because of heterogeneity in the cellular origin of GIST. Little also is known about why Hedgehog (HH) signaling is activated in some GIST. HH binds to and inactivates the receptor protein patched homolog (PTCH).

Methods: Ptch was conditionally inactivated in mice (to achieve constitutive HH signaling) using a Cre recombinase regulated by the lysozyme M promoter. Cre-expressing cells were traced using R26R-LacZ reporter mice. Tumors were characterized by in situ hybridization, immunohistochemistry, immunoblot, and quantitative reverse-transcriptase polymerase chain reaction analyses. Cell transformation was assessed by soft agar assay.

Results: Loss of Ptch from lysozyme M-expressing cells resulted in the development of tumors of GIST-like localization and histology; these were reduced when mice were given imatinib, a drug that targets KIT and PDGFRA. The Hh signaling pathway was activated in the tumor cells, and Pdgfrα, but not Kit, was overexpressed and activated. Lineage tracing revealed that Cre-expressing intestinal cells were Kit-negative. These cells sometimes expressed Pdgfrα and were located near Kit-positive interstitial cells of Cajal. In contrast to KIT, activation of PDGFRA increased anchorage-independent proliferation and was required for tumor formation in mice by cells with activated HH signaling.

Conclusions: Inactivation of Ptch in mice leads to formation of GIST-like tumors that express Pdgfrα, but not Kit. Activation of Pdgfrα signaling appears to facilitate tumorigenesis.

Figure 1. Ptch mutant mice develop gastrointestinal tumors

(A) Gross appearance of tumors of Ptch^flox/floxLysMcre^+/− mice. (B) Tumors either have a solid (arrow) or cystic appearance (asterisks). The cystic appearance is associated with intratumoral bleeding. (C) Shows a precursor lesion (arrow). mus, muscularis (*: longitudinal muscle layer; **: circular muscle layer); muc, mucosa; lu, lumen of the intestine. (D) Arrows point to the intact myenteric plexus. (E) When precursor lesions become larger (arrows) they adopt a (F) GIST-like histology. Scale bars in μm: (B,C,E) 500; (D) 100; (F) 50.

Figure 2. Activation of Hh signaling in tumors of Ptch mutant mice

(A) In situ hybridization of the Hh target genes Gli1 and (B) Ptch in tumors of Ptch^flox/floxLysMcre^+/− mice. The 477 bp Ptch probe identifies both wild type and mutant Ptch transcripts, in which exon 7 is spliced to exon 10 due to Cre-mediated deletion of exon 8 and 9. The 250 bp Ptch probe exclusively binds to wild type Ptch transcripts. The Figure demonstrates that the tumors overexpress exclusively mutant Ptch transcripts, since wild type Ptch transcripts were not detected. Scale bar in μm: 200.

Figure 3. Lineage tracing identifies LysM-expressing cells in tumors of Ptch mutant mice and in the normal intestine, where they may co-express Pdgfra

(A) The figure shows LacZ expression (blue color) in tumors of Ptch^flox/floxLysMcre^+/−R26R-LacZ^+/− mice. Since LacZ expression in Ptch^flox/floxLysMcre^+/−R26R-LacZ^+/− mice mark LysM-expressing cells, tumors are likely derived from LysM-positive cells. Dotted line marks the tumor (Tu)/normal muscle border. Left: H&E staining, Right: LacZ staining. (B) and (C) show LacZ-staining in normal intestine of LysMcre^+/−R26R-LacZ^+/− mice. Positive cells are detected between the longitudinal muscle layer and the serosa (B, left panel), the circular and longitudinal muscle layer (B, right panel) and within the muscle layer (C, left and right panels). Sections in (C) were co-stained with antibodies against Pdgfrα (left and middle panel) or Kit (right panel). Inset in the left panel shows a LacZ-positive, but Pdgfrα-negative cell. Inset in the middle panel shows a LacZ/Pdgfrα double-positive cell. Inset in the right panel shows a LacZ-positive cell juxtaposed to a Kit-positive cell (a similar cell is marked by an arrowhead). Please also note the LacZ-positive muscle fiber (asterisk, right panel) not juxtaposed to Kit-positive cells. Scale bars in μm: (A) 500; (B) 25; (C, left and middle panel) 50; (C, right panel) 25.

Figure 4. Tumors of Ptch mutant mice express Pdgfrα but not Kit

(A) Immunofluorescence analysis of tumors of Ptch^flox/floxLysMcre^+/− mice using an anti-Pdgfrα (green) and anti-Kit antibody (red). No Pdgfrα/Kit double-positive cells were detected in the tumors. Scale bar 50 μm. (B) Western Blot analysis of tumors (Tu) and normal small intestine (In) of Ptch^flox/floxLysMcre^+/− mice using anti-Pdgfrα and anti-pPdgfrα (phosphorylated Pdgfrα) antibodies. GIST-T1 and NIH/3T3 cells were used as control samples. (C) qRT-PCR analysis of Pdgf-a, b and c in normal intestine and in tumors. Expression levels in tumors are shown in relation to normal intestine, which was set=1. (black bars: intestine; grey bars: tumors).

Figure 5. Differentiation of the tumors from LMS and responsiveness to imatinib

(A) qRT-PCR analysis of Hh-target genes in 20 human GISTs compared to 7 human LMS samples (upper panel), and in tumors of Ptch^flox/floxLysMcre^+/− mice (Tu) compared to murine LMS derived from mice harboring a Pten/p53 mutation (lower panel). Expression levels in human and murine samples were normalized to the expression of β-actin and 18S rRNA, respectively. Data shows Box-Whisker-Plots of the relative gene expression. (B) Shown is the mean tumor number per animal for Ptch^flox/floxLysMcre^+/− mice after treatment with imatinib or with vehicle control (the asterisk indicates p<0.05 by unpaired t-test). Due to frequent clustering of tumor nodules (see Fig. 1A and 1E), each cluster was counted as one separate tumor. (C) shows the intestine of a vehicle-treated and (D) of an imatinib-treated animal.

Figure 6. HH signaling cooperates with PDGFRA signaling in cellular transformation

(A) Quantification of anchorage-independent clonogenic growth of human non-tumorigenic HaCaT keratinocytes after activation of either HH signaling (by overexpression of GLI1) or KIT signaling (overexpression of the constitutively active Kit D816V mutant) with or without stimulation of PDGFR signaling by PDGFB. Either signal alone has little or no activity towards cellular transformation, whereas GLI1 in combination with PDGFB treatment, but not in combination with KIT activation, leads to formation of clonogenic colonies (spheres) in 3D cultures (asterisks indicate p<0.03 by unpaired t-test). (B) PDGFRA signaling in HaCaT cells after incubation with PDGFB. Western blot analysis shows activation of PDGFRA/ERK/JUN signaling in response to PDGFB treatment.