The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

The Hedgehog (Hh) signaling pathway has been implicated in acquired chemoresistance. However, it remains unclear whether and how the Hh pathway may maintain the chemoresistant phenotype by controlling the tumor-initiating cell-like properties of acquired chemoresistant cancer cells. In this study, using well-established acquired chemoresistant cancer cells and chemosensitive KB cancer cells with artificially elevated Hh pathway activity, we found that Hh pathway activity may transcriptionally control the expression of twist1 and snail, thereby maintaining the tumor-initiating cell-like properties and consequently the chemoresistant phenotype. Meanwhile, we obtained direct evidence that twist1, which may amplify Hh signaling activity and plays an essential role in limb development, is a direct transcriptional target of Gli, similar to snail. We further observed that the expression of ATP-binding cassette (ABC) transporters was dispensable for the chemoresistance mediated by twist1 and snail. Collectively, these findings demonstrate that twist1, together with snail, links the Hh pathway to the tumor-initiating cell-like properties of chemoresistant cells. This consequently promotes chemoresistance independently of ABC transporters, thereby contributing to future development of strategies for combating chemoresistance through Hh pathway interference. Furthermore, our finding that twist1 is a direct target of the transcription factor Gli improves the interpretation of the association between twist1 and the Hh pathway and the nature of the signaling transduction of the Hh pathway. Stem Cells 2015;33:1063–1074

Multiple drug resistance (MDR) occurring during chemotherapy is a major obstacle for treatment of cancers using chemotherapeutic drugs; thus, the mechanisms underlying MDR have attracted intensive attention. Many studies have shown that tumor-initiating cells exhibit a chemotherapeutic tolerance characteristic. However, whether the MDR cells possess tumor-initiating cells properties and its underlying mechanisms remain to be fully elucidated. In this study, we utilized a well-established MDR cell line K562/A02 enriched by doxorubicin from K562 cells to determine if the K562/A02 cells possess tumor-initiating properties and investigated its potential molecular mechanisms. We observed that the expressions of Oct4, Sox2, and Nanog, all of which are well-characterized stem cell markers, in K562/A02 cells were elevated in comparison to parental K562 cells; in addition, we found that K562/A02 cells exhibited more potent in vitro and in vivo tumor-initiating properties, as revealed by sphere assay, self-renewal assay, soft agar assay, and animal studies. Furthermore, our data suggest that snail and twist1, two well known transcriptional factors for the epithelial-mesenchymal transition (EMT) program, may be potentially involved in the acquisition of tumor-initiating properties of K562/A02 cells. Thus, our study demonstrates that MDR K562/A02 cells possess tumor-initiating properties, most likely due to the elevated expressions of snail and twist1.

Berberine (BBR), a natural alkaloid compound, is used as a non-prescription drug in China for treating diarrhea and gastroenteritis. Many studies have revealed that BBR possesses anticancer effect. However, the molecular mechanisms underlying its anticancer action is far from being fully elucidated. This study is aimed to determine the effect of BBR on the hedgehog (Hh) activity and the growth of cancers addiction to Hh activity. The Hh activity was determined by dual luciferase assays and quantitative RT-PCR analyses. The growth inhibition of BBR on medulloblastoma which was obtained from ptch+/−;p53−/− mice was analyzed by 5-bromo-2-deoxyuridine (Brdu) assays and by allografting the medulloblastoma into nude mice. The data were statistically analyzed by one-way analysis of variance (ANOVA), and multiple comparison between the groups was performed using Dunnett’s method. In this study, we found that BBR significantly inhibited the Hh pathway activity. Meanwhile, we observed that BBR failed to affect the transcriptional factors activities provoked by tumor necrosis factor-α (TNF-α) and Prostaglandin E2 (PGE2), thus suggesting its unique property against Hh pathway activity. Further studies revealed that BBR inhibited the Hh pathway activity by potentially targeting the critical component Smoothened (Smo) and most likely shared the same binding site on Smo with cyclopamine, a classical Smo inhibitor. Finally, we demonstrated that BBR obviously suppressed the Hh-dependent medulloblastoma growth in vitro and in vivo. Collectively, our study uncovered a novel molecular mechanism responsible for the anticancer action of BBR, thus opening the way for the usage of BBR for therapeutics of cancers addiction to aberrant Hh pathway activity.