Time-point and dosage of gene inactivation determine the tumor spectrum in conditional Ptch knockouts

Abstract

Mutations in Patched (PTCH) have been associated with tumors characteristic both for children [medulloblastoma (MB) and rhabdomyosarcoma (RMS)] and for elderly [basal cell carcinoma (BCC)]. The determinants of the variability in tumor onset and histology are unknown. We investigated the effects of the time-point and dosage of Ptch inactivation on tumor spectrum using conditional Ptch-knockout mice. Ptch heterozygosity induced prenatally resulted in the formation of RMS, which was accompanied by the silencing of the remaining wild-type Ptch allele. In contrast, RMS was observed neither after mono- nor biallelic postnatal deletion of Ptch. Postnatal biallelic deletion of Ptch led to BCC precancerous lesions of the gastrointestinal epithelium and mesenteric tumors. Hamartomatous gastrointestinal cystic tumors were induced by monoallelic, but not biallelic Ptch mutations, independently of the time-point of mutation induction. These data suggest that the expressivity of Ptch deficiency is largely determined by the time-point, the gene dose and mode of Ptch inactivation. Furthermore, they point to key differences in the tumorigenic mechanisms underlying adult and childhood tumors. The latter ones are unique among all tumors since their occurrence decreases rather than increases with age. A better understanding of mechanisms underlying this ontological restriction is of potential therapeutic value.

Uncommitted precursor cells might contribute to increased incidence of embryonal rhabdomyosarcoma in heterozygous Patched1-mutant mice

“…Unexpectedly, we found that only a minority of ERMS originated from committed muscle cells, which express the myogenic factor Myf5, suggesting that uncommitted precursor cells represent the main source of ERMS. Previous studies from our laboratory revealed that postnatal monoallelic inactivation of Ptch does not suffice to initiate ERMS (Zibat et al, 2009) indicating that cell populations that arise postnatally, such as side population cells, circulating myeloid cells or mesenchymal stem cells of the bone marrow, and satellite cells of the adult skeletal muscle do not contribute significantly to ERMS. Satellite cells have also been excluded by another study in which Cre/Lox inactivation of Ptch using a Pax7-Cre knock-in did not cause ERMS (Taniguchi et al, 2009).…”

“…Injection of tamoxifen at any embryonal stage resulted in expression of Ptch del transcripts in all examined organs in the adult (Figure 1d). Expression intensity of mutant Ptch del transcripts in these organs was indistinguishable from Ptch del/ þ mice, in which exons 8 and 9 of Ptch had been deleted in the germline (data for Ptch del/ þ mice have been published before (Zibat et al, 2009)). …”

“…Genotyping of the Ptch flox , Ptch del and ERT2 alleles was performed as described recently (Uhmann et al, 2007;Zibat et al, 2009). For genotyping of Myf5Cre, the primer combination 5 0 -CCAGGCTAAGTGCCTTCTCTACA-3 0 /5 0 -AATG CTTCTGTCCGTTTGCCGGT-3 0 was used.…”

“…The standard curve method for relative quantification was used for data analysis. The deletion efficiency was calculated as the ratio of the values for the deleted allele to the total value from the floxed allele plus the deleted allele and is denoted in percent (see also Zibat et al, 2009). Each sample was measured in triplicates.…”

“…We recently showed that ERMSs of Ptch-mutant mice are not derived from cells constituting postnatal muscle but are most likely initiated before birth (Zibat et al, 2009). Here, we investigated the cell type and the time point of ERMS initiation during embryogenesis using a conditional Ptch mutation and different Cre-recombinase driver strains.…”