Dual degradation signals control Gli protein stability and tumor formation

Abstract

Regulated protein destruction controls many key cellular processes with aberrant regulation increasingly found during carcinogenesis. Gli proteins mediate the transcriptional effects of the Sonic hedgehog pathway, which is implicated in up to 25% of human tumors. Here we show that Gli is rapidly destroyed by the proteasome and that mouse basal cell carcinoma induction correlates with Gli protein accumulation. We identify two independent destruction signals in Gli1, D N and D C , and show that removal of these signals stabilizes Gli1 protein and rapidly accelerates tumor formation in transgenic animals. These data argue that control of Gli protein accumulation underlies tumorigenesis and suggest a new avenue for antitumor therapy. Factors controlling protein destruction are critical for the timing of key processes such as the cell cycle, apoptosis, and cell fate decisions, with aberrant regulation increasingly found during carcinogenesis (Pickart 2004;Yamasaki and Pagano 2004). Inappropriate Sonic hedgehog (Shh) signaling results in a panoply of birth defects and is implicated in up to 25% of human tumors (Callahan and Oro 2001;Lum and Beachy 2004). While the Gli family of proteins mediates the transcriptional effects of Shh (Methot and Basler 2001;Ruiz i Altaba et al. 2002), the mechanism by which Gli proteins are regulated to achieve changes in pathway output remains poorly understood. Studies in mice and humans show that Shh target gene induction is sufficient to induce a variety of tumors including basal cell carcinomas (BCCs) (Oro et al. 1997;Nilsson et al. 2000;Hutchin et al. 2005). However, there is a wide variability in the onset and severity of phenotypes among patients with mutations in the Shh pathway (Wicking et al. 1997), and a noticeably wide variability of tumor onset in animal models (Oro and Higgins 2003;Hutchin et al. 2005). This suggests the possibility that additional, previously uncharacterized, cellular processes regulate pathway output. Here we show that Gli protein accumulation correlates with tumor formation and stabilizing mutations in Gli protein dramatically accelerate tumor induction. Results and DiscussionWhile expression of either Gli1 or Gli2 in the epidermis of transgenic mice induces BCCs (Fig. 1a), we have observed a considerable delay in the appearance of Gli-dependent tumors. Analysis of transgenic mice expressing Gli2 revealed an average latency of 7 mo before tumor appearance (Fig. 1b). We ruled out changes in transcription of the transgene with age as a cause of the tumors, as similar levels of RNA are seen in both age groups as measured by quantitative PCR (Fig. 1c). This suggested the existence in keratinocytes of additional processes, whose loss or dysregulation is required to permit Gli activity and direct tumor formation. Our previous studies indicated that differential accumulation of Gli protein plays an important role in restricting Shh target gene induction in interfollicular epithelium (Oro and Higgins 2003). Indeed, we detected no transgenic Gli protein in normal...

Hedgehog signaling is restricted to the stromal compartment during pancreatic carcinogenesis

“…CLEG2-expressing mice may develop pancreatic tumors because this dominant active Gli2 allele is downstream or resistant to key posttranslational regulators of canonical Hh signaling. Posttranslational regulators downstream of Smo have been shown previously to be key determinants in the temporal and spatial control of Hh target-gene expression in tumors (32). In another study, mutations in the GLI transcription factors were identified in human PDA tumor epithelium (33).…”

Activation of the hedgehog-signaling pathway in human cancer and the clinical implications

“…For example, protein kinase A is shown to retain Gli1 proteins in the cytoplasm (through a PKA site in the nuclear localization signal peptide) whereas active Ras signaling promotes Gli nuclear localization (Stecca et al, 2007). Second, ubiquitination and protein degradation of Gli molecules is also regulated by several distinct mechanisms, including b-TRCP-, cul3/BTBand numb/Itch-mediated Gli ubiquitination (Di Marcotullio et al, 2006;Huntzicker et al, 2006;Jiang, 2006;Pan et al, 2006;Wang and Li, 2006). In addition to protein degradation, Gli3 and (Gli2 to a less extent) can be processed into transcriptional repressors, which may be mediated by the b-TRCP E3 ligase (Wang and Li, 2006).…”

The EWS/FLI1 oncogenic transcription factor deregulates GLI1

“…These findings serve as a precedent for deregulation of GLI1 in the absence of aberrancy of HH or PATCHED. More recently, GLI1 protein stability has been shown to be another HHindependent pathway deregulation (Di Marcotullio et al, 2006;Huntzicker et al, 2006). Furthermore, MEK kinase deregulation has been shown to affect GLI1 protein stability and deregulation of this pathway (Lauth et al, 2007).…”

“…It is also conceivable that EWS/FLI1 might target GLI1 directly for transcriptional activation. However, transcriptional activation alone might not be sufficient to activate this pathway in NIH3T3 due to a very rapid clearance of GLI1 that has been reported (Huntzicker et al, 2006). Experiments to address possible indirect effects such as alterations in protein stability are currently underway.…”