Requirement of TGFβ Signaling for SMO-mediated Carcinogenesis

Abstract

Hedgehog (Hh) signaling, via the key signal transducer Smoothened (SMO) and Gli transcription factors, is essential for embryonic development and carcinogenesis. At present, the molecular mechanism of Hh signaling-mediated carcinogenesis is not completely understood. Using a mouse model (K14cre/ R26SmoM2) of SMO-mediated basal cell carcinoma development, we identified TGF␤2 as a major Hh-regulated gene. TGF␤2 expression was high in the keratinocytes, with activated TGF␤ signaling (indicated by elevated expression of phosphorylated SMAD2/3) detected in both tumor and stroma. The significance of TGF␤ signaling for SMO function was demonstrated in two assays. Down-regulation of TGF␤2 expression prevented Hh signaling-dependent osteoblast differentiation and motor neuron differentiation. Furthermore, inhibition of TGF␤ signaling by TGF␤ receptor I inhibitor SD208 significantly reduced tumor area in K14cre/R26SmoM2 mice. Tumor shrinkage in mice was associated with an increased number of lymphocytes, suggesting an immune suppression role of TGF␤ signaling. The relevance of our results to human cancer is reflected by the fact that human basal cell carcinomas, which almost always harbor activated Hh signaling, have activated TGF␤ signaling, as indicated by high levels of phosphorylated SMAD2 and SMAD3 in tumor and stroma. Together, our data indicate that TGF␤ signaling is critical for Hh signaling-mediated carcinogenesis.The Hedgehog pathway plays an important role in cell differentiation, tissue polarity, cell proliferation, and carcinogenesis (1-4). The seven-transmembrane domain-containing protein Smoothened (SMO) 4 serves as the key player for signal transduction of this pathway, whose function is inhibited by another transmembrane protein, Patched (PTC), in the absence of Hh ligands. Binding of Hh to its receptor PTC releases this inhibition, allowing SMO to signal downstream, leading to formation of active forms of Gli transcription factors. As transcription factors, Gli molecules can regulate target gene expression by direct association with a specific consensus sequence located at the promoter region of the target genes (5). In addition to the canonical pathways (ligand overexpression, altered expression of Hh signaling molecules, or gene mutations), recent studies indicate that Hh signaling can also be activated by other signaling pathways, such as K-Ras. Both canonical and non-canonical Hh signaling activation are found in many types of human cancer, including brain tumors, gastrointestinal, prostate, lung, and breast cancers (6 -8).Mounting evidence indicates that Hh signaling activation occurs frequently in a number of human cancers (9), but the underlying molecular basis remains largely elusive. To understand the molecular basis by which Hh signaling regulates carcinogenesis, we analyzed gene expression of a mouse model of basal cell carcinoma in which an activated form of SMO (SmoM2) replaces the wild type SMO allele and is expressed under the control of the keratin 14 promoter. Our results indicated that ...

A Role for Transcription Factor STAT3 Signaling in Oncogene Smoothened-driven Carcinogenesis

“…We showed previously that addition of Smo agonist purmorphamine in these two cell lines induces Hh target gene expression (33). As shown in Fig.…”

“…To stimulate Hh signaling activation, the Smo agonist purmorphamine (at 5 M) was added to the culture medium. Protocols for motor neuron differentiation and induction of alkaline phosphatase have been described previously (33).…”

“…Cell Culture and ShRNA Expression-C3H10T1/2 and ES14 cells were cultured as described previously (33). ShRNAs to IL-11R␣ were purchased from Sigma and were introduced into cells via lentivirus-mediated gene expression.…”

“…Skin-specific expression of mutant Smo, SmoM2, via mating of R26-SmoM2 YFP mice (28) with K14-cre mice (25) exhibits phenotypes of BCCs (33) and is regarded as an important model for studying Hh-mediated carcinogenesis. To further understand molecular bases of Hh-mediated carcinogenesis, we performed gene expression profiling of SmoM2 YFP -expressing keratinocytes using Affymetrix arrays and revealed changes in several cytokine molecules that are known to associate with STAT3 signaling (33). To confirm the (Fig.…”

Smoothened is a master regulator of adult liver repair

“…Activated SMO promotes stabilization and nuclear localization of glioma (Gli) family transcription factors that regulate the expression of Hh target genes controlling cell viability, growth, and differentiation (9). In addition to Hh ligand-PTC interactions, SMO activity is modulated via multiple pathways, including G protein-coupled receptors and TGF-β/ TGF-β-receptor interactions (15)(16)(17). Gli factors are also controlled by SMO-independent (i.e., noncanonical) mechanisms that are initiated by other injury-related factors, including the key profibrogenic cytokine, TGF-β (16)(17)(18).…”