Loss of Blm enhances basal cell carcinoma and rhabdomyosarcoma tumorigenesis in Ptch1+/- mice

Abstract

Basal cell carcinomas (BCCs) have relative genomic stability and relatively benign clinical behavior but whether these two are related causally is unknown. To investigate the effects of introducing genomic instability into murine BCCs, we have compared ionizing radiation-induced tumorigenesis in Ptch1(+/-) mice versus that in Ptch1(+/-) mice carrying mutant Blm alleles. We found that BCCs in Ptch1(+/-) Blm(tm3Brd/tm3Brd) mice had a trend toward greater genomic instability as measured by array comprehensive genomic hybridization and that these mice developed significantly more microscopic BCCs than did Ptch1(+/-) Blm(+/tm3Brd) or Ptch1(+/-) Blm(+/+) mice. The mutant Blm alleles also markedly enhanced the formation of rhabdomyosarcomas (RMSs), another cancer to which Ptch1(+/)(-) mice and PTCH1(+/)(-) (basal cell nevus syndrome) patients are susceptible. Highly recurrent but different copy number changes were associated with the two tumor types and included losses of chromosomes 4 and 10 in all BCCs and gain of chromosome 10 in 80% of RMSs. Loss of chromosome 11 and 13, including the Trp53 and Ptch1 loci, respectively, occurred frequently in BCCs, suggesting tissue-specific selection for genes or pathways that collaborate with Ptch deficiency in tumorigenesis. Despite the quantitative differences, there was no dramatic qualititative difference in the BCC or RMS tumors associated with the mutant Blm genotype.

Fig. 1.

Influence of Blm genotype on survival and tumor latency. (a) Overall survival for Ptch1^+/− mice that were also Blm^+/+, Blm^+/tm3Brd and Blm^{tm3Brd/tm3Brd}. Overall survival for Blm^+/+, Blm^+/tm3Brd and Blm^{tm3Brd/tm3Brd} were 598 ± 39, 450 ± 32 and 409 ± 34 days, respectively. Significant differences were observed between Blm^+/+ versus Blm^+/tm3Brd (P value: 0.04) and Blm^+/+ and Blm^{tm3Brd/tm3Brd} (P value: 0.002) but not between Blm^+/tm3Brd and Blm^{tm3Brd/tm3Brd} (P value: 0.2). (b) Visible BCC free duration for Blm^+/+, Blm^+/tm3Brd and Blm^{tm3Brd/tm3Brd} mice. Mean visible BCC free duration was 607 ± 42 days for Blm^+/+, 533 ± 26 days for Blm^+/tm3Brd and 536 ± 34 days for Blm^{tm3Brd/tm3Brd}. No statistically significant differences in the age of onset of visible BCCs were observed between Blm^+/+ and Blm^+/tm3Brd mice (P value: 0.7) or Blm^+/+ or Blm^{tm3Brd/tm3Brd} mice (P value: 0.6). (c) Rhabdomyosarcoma free duration for Blm^+/+, Blm^+/tm3Brd and Blm^{tm3Brd/tm3Brd} mice. The average RMS free duration for Blm^+/+, Blm^+/tm3Brd and Blm^{tm3Brd/tm3Brd} were 739 ± 22, 481 ± 31 and 440 ± 35 days, respectively. Tumors appeared significantly earlier in Ptch1^+/− Blm^+/tm3Brd (P value: 0.01) and in Blm^{tm3Brd/tm3Brd} (P value: 0.001) than in Blm^+/+mice.

Fig. 2.

Histology of microscopic BCC-like and RMS tumors. (a) Morphology of microscopic BCC-like tumors varies from almost no follicular differentiation to trichoblastoma-like with advanced follicular differentiation. (b). RMS tumor is composed of sheets of eosinophilic spindle cells with abundant cytoplasm growing in intersecting fascicles confirming rhabdomyoblastic differentiation. Higher magnification demonstrates dense eosinophilic cytoplasm, and cells varying from strap-like cytomorphology to larger, bizarre epithelioid cells often with multinucleation characteristic of atypical rhabdomyoblasts. Mitotic figures are present. (Hematoxylin and eosin stain).

Fig. 3.

Characteristics of microscopic BCC-like tumors in Ptch1^+/− mice of differing Blm genotypes. (a) Tumor size. Mean size of BCC-like tumors was higher in Blm-deficient mice compared with wild-type mice. The difference was significant between Blm^{tm3Brd/tm3Brd} and Blm^+/+ mice (*P value: 0.01). (b) BCC-like tumor number. Blm-deficient mice developed significantly more BCC-like tumors compared with Blm^+/+ mice (*P value: 0.006 and 0.0003, respectively, for Blm^+/tm3Brd and Blm^{tm3Brd/tm3Brd}). (c) BCC-like tumor area. A larger BCC-like tumor involved area was observed in Blm-deficient mice. The difference was considerable between Blm^+/+ and Blm^{tm3Brd/tm3Brd} mice (*P value: 0.0004) as well as Blm^+/tm3Brd and Blm^{tm3Brd/tm3Brd} mice (*P value: 0.01). Data are shown as mean ± SD.

Fig. 4.

Hierarchical clustering of BCC and RMS samples. Columns represent individual tumor samples. Rows represent individual genome probes (bacterial artificial chromosome and P1 clones) that are ordered from chromosome 1 to 19 and then chromosome X from proximal to distal. Genome copy number losses are indicated in red and copy number gains are indicated in green. Yellow dots represent high-level genome amplifications. The positions of some hedgehog pathway genes are indicated on the right of the heatmap. The Blm genotypes of the samples are indicated by +/+ = wild-type, +/− = Blm^+/tm3Brd and −/− = Blm^tm3Brd/tm3B.