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Basal cell carcinoma (BCC) is the most common skin tumor in humans. Although BCCs rarely metastasize, they can cause significant morbidity due to local aggressiveness. Approximately 20% of BCCs show signs of spontaneous regression. The understanding of molecular events mediating spontaneous regression has the potential to reduce morbidity of BCC and, potentially, other tumors, if translated into tumor therapies. We show that BCCs induced in conditional Ptchflox/floxERT2+/− knockout mice regress with time and show a more differentiated phenotype. Differentiation is accompanied by Wnt5a expression in the tumor stroma, which is first detectable at the fully developed tumor stage. Coculture experiments revealed that Wnt5a is upregulated in tumor-adjacent macrophages by soluble signals derived from BCC cells. In turn, Wnt5a induces the expression of the differentiation marker K10 in tumor cells, which is mediated by Wnt/Ca2+ signaling in a CaMKII-dependent manner. These data support a role of stromal Wnt5a in BCC differentiation and regression, which may have important implications for development of new treatment strategies for this tumor. Taken together, our results establish BCC as an easily accessible model of tumor regression. The regression of BCC despite sustained Hedgehog signaling activity seems to be mediated by tumor-stromal interactions via Wnt5a signaling. Cancer Res; 70(7); 2739–48

Patched (Ptch) heterozygous mice develop medulloblastoma (MB) and rhabdomyosarcoma (RMS) resembling the corresponding human tumors. We have previously shown that epigenetic silencing of the intact Ptch allele contributes to tumor formation in this model. Here, we investigated whether targeting of epigenetic silencing mechanisms could be useful in the treatment of Ptch-associated cancers. A reduction of endogenous DNA methyltransferase1 (Dnmt1) activity significantly reduced tumor incidence in heterozygous Ptch knockout mice. A combined treatment with the Dnmt inhibitor 5-aza-2′deoxycytidine (5-aza-dC) and the histone deacetlyase (HDAC) inhibitor valproic acid (VPA) efficiently prevented MB and RMS formation, whereas monotherapies with either drug were less effective. Wild-type Ptch expression was efficiently reactivated in tumors by 5-aza-dC/VPA combination therapy. This was associated with reduced methylation of the Ptch promoter and induction of histone hyperacetylation suggesting inhibition of HDACs in vivo. However, the treatment was not effective in clinically overt, advanced stage tumors. This is a first in vivo demonstration that targeting of Dnmt and HDAC activities is highly effective in preventing formation of Ptch-associated tumors. The results suggest a novel clinical strategy for consolidation therapy of corresponding tumors in humans after completion of conventional treatment. Our data also suggest that epigenetic therapy may be less effective in treating advanced stages of tumors, at least in this tumor model. [Cancer Res 2009;69(3):887–95]

Loss-of-function mutations in Patched (Ptch1) are implicated in constitutive activation of the Sonic hedgehog pathway in human basal cell carcinomas (BCCs), and inherited Ptch1 mutations underlie basal cell nevus syndrome in which a typical feature is multiple BCC occurring with greater incidence in portals of radiotherapy. Mice in which one copy of Ptch1 is inactivated show increased susceptibility to spontaneous tumor development and hypersensitivity to radiation-induced tumorigenesis, providing an ideal in vivo model to study the typical pathologies associated with basal cell nevus syndrome. We therefore examined BCC development in control and irradiated Ptch1neo67/+ mice. We show that unirradiated mice develop putative BCC precursor lesions, i.e., basaloid hyperproliferation areas arising from both follicular and interfollicular epithelium, and that these lesions progress to nodular and infiltrative BCCs only in irradiated mice. Data of BCC incidence, multiplicity, and latency support the notion of epidermal hyperproliferations, nodular and infiltrative BCC-like tumors representing different stages of tumor development. This is additionally supported by the pattern of p53 protein expression observed in BCC subtypes and by the finding of retention of the normal remaining Ptch1 allele in all nodular, circumscribed BCCs analyzed compared with its constant loss in infiltrative BCCs. Our data suggest chronological tumor progression from basaloid hyperproliferations to nodular and then infiltrative BCC occurring in a stepwise fashion through the accumulation of sequential genetic alterations.

We described previously a basal cell carcinoma (BCC) and medulloblastoma (MB) phenotype for CD1Ptch1neo67/+ mice exposed to ionizing radiation. Ptch1 heterozygous mice mimic the predisposition to BCC and MB development of patients affected by nevoid BCC syndrome that inherit a mutant Patched (Ptch1) allele. To examine the impact of genetic background on development of BCCs and other tumors we used two outbred mouse lines characterized by extremely high, carcinogenesis-susceptible (Car-S), and low, carcinogenesis-resistant (Car-R), susceptibility to skin carcinogenesis. Crosses between Ptch1neo67/+ mice and Car-S (F1S) or Car-R mice (F1R) were exposed to ionizing radiation. F1SPtch1neo67/+ mice were highly susceptible to radiation-induced BCCs, whereas F1RPtch1neo67/+ mice were completely resistant, indicating that tumor penetrance can be modulated by genetic background. Development of microscopic and macroscopic BCC lesions was influenced by Car-S and Car-R genotypes, suggesting a genetic-background effect on both initiation and progression of BCC. Susceptibility was additionally increased in N2 backcross mice (Car-S x F1SPtch1neo67/+), showing a contribution from recessive-acting Car-S modifiers. The modifying effects of Car-S-derived susceptibility alleles were tissue specific. In fact, despite higher susceptibility to BCC induction, Car-S-derived lines had lower MB incidence compared with CD1Ptch1neo67/+ mice. BCC-associated somatic events were not influenced by genetic background, as shown by similar rate of wild-type Ptch1 loss in BCCs from F1SPtch1neo67/+ (93%) and CD1Ptch1neo67/+ mice (100%). Finally, microsatellite analysis of BCCs showed Ptch1 loss through interstitial deletion. These results are relevant to humans, in which BCC is the commonest malignancy, because this model system may be used to study genes modifying BCC development.

Loss-of-function mutations in Patched (Ptch1) are implicated in constitutive activation of the Sonic hedgehog pathway in human basal cell carcinomas (BCCs), and inherited Ptch1 mutations underlie basal cell nevus syndrome in which a typical feature is multiple BCC occurring with greater incidence in portals of radiotherapy. Mice in which one copy of Ptch1 is inactivated show increased susceptibility to spontaneous tumor development and hypersensitivity to radiation-induced tumorigenesis, providing an ideal in vivo model to study the typical pathologies associated with basal cell nevus syndrome. We therefore examined BCC development in control and irradiated Ptch1(neo67/+) mice. We show that unirradiated mice develop putative BCC precursor lesions, i.e., basaloid hyperproliferation areas arising from both follicular and interfollicular epithelium, and that these lesions progress to nodular and infiltrative BCCs only in irradiated mice. Data of BCC incidence, multiplicity, and latency support the notion of epidermal hyperproliferations, nodular and infiltrative BCC-like tumors representing different stages of tumor development. This is additionally supported by the pattern of p53 protein expression observed in BCC subtypes and by the finding of retention of the normal remaining Ptch1 allele in all nodular, circumscribed BCCs analyzed compared with its constant loss in infiltrative BCCs. Our data suggest chronological tumor progression from basaloid hyperproliferations to nodular and then infiltrative BCC occurring in a stepwise fashion through the accumulation of sequential genetic alterations.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and comprises two major histological subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). The hedgehog (Hh) pathway has recently been implied in tumor formation and progression of various cancers. In the current study we investigated whether Hh pathway activation presents a general feature also of RMS and whether it bears prognostic impact. Here, we report that marker genes of active Hh signaling, i.e. Patched1 (Ptch1), Gli1, Gli3 and Myf5, are elevated in two distinct cohorts of RMS patients with a total number of 235 primary samples. Interestingly, all these marker genes of active Hh signaling were expressed at significantly higher levels in specific subtypes of RMS, i.e. ERMS and fusion gene negative ARMS, compared to fusion gene positive ARMS. Consistently, Gli1 expression correlates with Ptch1 expression in ERMS and fusion gene negative ARMS, but not in fusion gene positive ARMS. In addition, expression levels of MyoD1 are significantly lower in ERMS and fusion gene negative ARMS, pointing to an inverse association of Hh activation and early muscle differentiation in primary RMS samples. In support of this notion, the addition of Shh inhibits muscle differentiation in parallel experiments in muscle satellite cells and myoblasts. Moreover, we identify Myf5 is as a novel excellent class predictor for RMS by receiver operating characteristic (ROC) analysis. Most importantly, high expression of Ptch1 or low MyoD1 expression significantly correlate with reduced cumulative survival in fusion gene negative RMS (P< 0.004) underscoring the clinical relevance of these findings. By identifying aberrant Hh activation in specific subgroups of RMS, our study has important implications for the development of molecular targeted therapies, such as small molecule Hh inhibitors, in RMS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5343. doi:10.1158/1538-7445.AM2011-5343