A Novel Mechanism for Activation of GLI1 by Nuclear SMO That Escapes Anti-SMO Inhibitors

Abstract

Small-molecule inhibitors of the Hedgehog (HH) pathway receptor Smoothened (SMO) have been effective in treating some patients with basal cell carcinoma (BCC), where the HH pathway is often activated, but many patients respond poorly. In this study, we report the results of investigations on PTCH1 signaling in the HH pathway that suggest why most patients with BCC respond poorly to SMO inhibitors. In immortalized human keratinocytes, PTCH1 silencing led to the generation of a compact, holoclone-like morphology with increased expression of SMO and the downstream HH pathway transcription factor GLI1. Notably, although siRNA silencing of SMO in PTCH1-silenced cells was sufficient to suppress GLI1 activity, this effect was not phenocopied by pharmacologic inhibition of SMO, suggesting the presence of a second undefined pathway through which SMO can induce GLI1. Consistent with this possibility, we observed increased nuclear localization of SMO in PTCH1-silenced cells as mediated by a putative SMO nuclear/nucleolar localization signal [N(o)LS]. Mutational inactivation of the N(o)LS ablated this increase and suppressed GLI1 induction. Immunohistologic analysis of human and mouse BCC confirmed evidence of nuclear SMO, although the pattern was heterogeneous between tumors. In PTCH1-silenced cells, >80% of the genes found to be differentially expressed were unaffected by SMO inhibitors, including the putative BCC driver gene CXCL11. Our results demonstrate how PTCH1 loss results in aberrant regulation of SMO-independent mechanisms important for BCC biology and highlights a novel nuclear mechanism of SMO-GLI1 signaling that is unresponsive to SMO inhibitors. This study describes novel noncanonical Hedgehog signaling, where SMO enters the nucleus to activate GLI1, a mode that is unaffected by SMO inhibitors, thus prompting re-evaluation of current BCC treatment as well as new potential therapies targeting nuclear SMO. .

SMO-M2 mutation does not support cell-autonomous Hedgehog activity in cerebellar granule cell precursors

“…Although not investigated in this paper, the occurrence of Gli-independent non-canonical Hh signaling ignited by Ptch1 inhibition (or loss), but independent from canonical Smo signaling 47–49 , should be taken into account when considering these data. Also, a nuclear localization of SMO, which drives GLI1 activation and is unresponsive to SMO inhibitors, has been reported to occur in Ptch1-silenced Basal Cell Carcinoma cells, again suggesting the activity of alternative non-canonical Hh-signaling 50 . Further work will be required to shed more light on this issue.…”

Polysaccharides extracted from balanophora polyandra Griff (BPP) ameliorate renal Fibrosis and EMT via inhibiting the Hedgehog pathway

“…In the absence of Shh, Ptch1 generally inhibits Smo activity. On the contrary, the inhibition of Smo can be alleviated when Shh is combined with Ptch1 33 . The accumulation of Smo receptors opened a complex network, activated Gli1 and induced the expression of α‐SMA, desmin, Snail1, fibronectin and collagen I, which promoted the proliferation of renal fibroblasts and stimulated the induction of a variety of proliferation related genes 12,34 …”

“…On the contrary, the inhibition of Smo can be alleviated when Shh is combined with Ptch1. 33 The accumulation of Smo receptors opened a complex network, activated Gli1 and induced the expression of α-SMA, desmin, Snail1, fibronectin and collagen I, which promoted the proliferation of renal fibroblasts and stimulated the induction of a variety of proliferation related genes. 12,34 The current study provided further the experimental evidence showing that Shh signalling pathway is activated during the pathogenesis of the renal fibrosis.…”

An Endogenous Anti-aging Factor, Sonic Hedgehog, Suppresses Endometrial Stem Cell Aging through SERPINB2

“…13 Subsequently, activated SMO releases glioma (GLI)-associated transcription factors from cytoplasmic sequestration, which in turn results in the nuclear localization of GLI to regulate various gene expressions. 14 Interestingly, in addition to this canonical function, particular attention has been devoted to the noncanonical functions of SHH signaling in various aging-related diseases, such as neurodegenerative diseases 15 and atherosclerosis, 16 as its signaling integrity declines during the development of these age-associated chronic diseases. These findings suggest that SHH might act as an age-associated factor in addition to its canonical function.…”