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[A] Various exon 24 clones generated from heterogeneous populations of PTCH1 suppressed NEB1 cells show reduced PTCH1 and increased GLI1 mRNA expression. [B] Both human and mouse SMO protein sequences contain predicted nuclear and nucleolar localisation signals located within the C-terminal region, non-matching amino acids are highlighted in red. Site directed mutagenesis was employed to alter the N(o)LS by changing each amino acid to alanine for the mNLS construct, mNLS2 was used for Figures 2B and 2C. [C] NEB1-shCON cells and to a greater extent NEB1-shPTCH1 cells transfected with EGFP-SMO and EGFP-SMO-M2 show upregulation of GLI1 mRNA expression that is then reduced to below basal levels upon mutation of N(o)LS sequences, particularly mNLS2. qPCR analysis for GLI1 expression in [D] NEB1 and [E] N/Tert exon 3 and exon 24 shPTCH1 clones treated with both KAAD-Cyc and SANT-1 show all shPTCH1 clones are unresponsive to SMO pharmacological inhibitors. [F] qPCR on NEB1-shPTCH1 exon 3 clone and N/Tert-shPTCH1 cells to validate the results for cancer related genes obtained from the microarray analysis. [G] Immunofluorescence staining of NEB1 cells with three different SMO antibodies. [Table 1] List of differentially expressed zinc-finger protein genes. *P{less than or equal to}0.05 and **P{less than or equal to}0.01 (as calculated by Student's t test), error bars represent standard deviation, n=6.

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.Significance: 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. Cancer Res; 78(10); 2577–88. ©2018 AACR.

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Basal cell carcinoma (BCC) is the most prevalent cancer in the Western world and its incidence is increasing. The pathogenesis of BCC involves deregulated Sonic hedgehog signaling, leading to activation of the Gli transcription factors. Most BCCs have a nodular growth pattern, and are indolent, slow-growing, and considered “low-risk” lesions. In contrast, the “high-risk” morphoeic variant, which causes significant morbidity, has an infiltrative growth pattern, and is so-called because of its densely fibrous stroma. As αvβ6 is capable of promoting both carcinoma invasion and fibrosis, we examined the expression of this integrin in BCCs and found that the morphoeic type showed significantly higher αvβ6 expression than the nodular type (P = 0.0009). In order to examine the function of αvβ6, we transfected the transcription factors Gli1 or Gli2 into NTERT, human keratinocytes to generate a BCC model. These cells expressed αvβ6 and were invasive, although inhibition of αvβ6 had no direct effect on cell invasion. However, the cells showed αvβ6-dependent activation of transforming growth factor-β1, which induced transdifferentiation of human fibroblasts into myofibroblasts. Paracrine secretion of hepatocyte growth factor/scatter factor by these myofibroblasts promoted c-Met–dependent tumor invasion in both Transwell and three-dimensional organotypic assays. These experimental in vitro findings were confirmed using human clinical samples in which we showed that the stroma of morphoeic BCC is myofibroblast-rich compared with nodular BCC (P = 0.0036), that myofibroblasts express hepatocyte growth factor/scatter factor, and that morphoeic BCCs are strongly c-Met–positive. These data suggest that αvβ6-dependent transforming growth factor-β1 activation induces both the infiltrative growth pattern and fibrotic stroma so characteristic of morphoeic BCC. [Cancer Res 2008;68(9):3295–303]

Aberrant activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of basal cell carcinoma (BCC). The zinc finger transcription factors GLI1 and GLI2 are considered mediators of the HH signal in epidermal cells, although their tumorigenic nature and their relative contribution to tumorigenesis are only poorly understood. To shed light on the respective role of these transcription factors in epidermal neoplasia, we screened for genes preferentially regulated either by GLI1 or GLI2 in human epidermal cells. We show here that expression of the key antiapoptotic factor BCL2 is predominantly activated by GLI2 compared with GLI1. Detailed promoter analysis and gel shift assays identified three GLI binding sites in the human BCL2 cis-regulatory region. We found that one of these binding sites is critical for conferring GLI2-specific activation of the human BCL2 promoter and that the selective induction of BCL2 expression depends on the zinc finger DNA binding domain of GLI2. In vivo, GLI2 and BCL2 were coexpressed in the outer root sheath of hair follicles and BCC and in plasma cells that infiltrated BCC tumor islands. On the basis of the latter observation, we analyzed plasma cell-derived tumors and found strong expression of GLI2 and BCL2 in neoplastic cells of plasmacytoma patients, implicating HH/GLI signaling in the development of plasma cell-derived malignancies. The results reveal a central role for GLI2 in activating the prosurvival factor BCL2, which may represent an important mechanism in the development or maintenance of cancers associated with inappropriate HH signaling.

Supplementary Figure 2 from Activation of the BCL2 Promoter in Response to Hedgehog/GLI Signal Transduction Is Predominantly Mediated by GLI2

Supplementary Figure Legends 1-2 from αvβ6 Integrin Promotes the Invasion of Morphoeic Basal Cell Carcinoma through Stromal Modulation

Supplementary Figure 2 from αvβ6 Integrin Promotes the Invasion of Morphoeic Basal Cell Carcinoma through Stromal Modulation

Supplementary Figure 1 from αvβ6 Integrin Promotes the Invasion of Morphoeic Basal Cell Carcinoma through Stromal Modulation

Supplementary Figure 1 from Activation of the BCL2 Promoter in Response to Hedgehog/GLI Signal Transduction Is Predominantly Mediated by GLI2

Basal cell carcinoma (BCC) is associated with aberrant Hedgehog (HH) signalling through mutational inactivation of PTCH1; however, there is conflicting data regarding MEK/ERK signalling in BCC and the signalling pathway interactions in these carcinomas. To address this, expression of active phospho (p) MEK and ERK was examined in a panel of 15 non-aggressive and 14 aggressive BCCs. Although not uniformly expressed, both phospho-proteins were detected in the nuclei and/or cytoplasm of normal and tumour-associated epidermal cells however, whereas phospho-MEK (pMEK) was present in all non-aggressive BCCs (14/14), phospho-ERK (pERK) was rarely expressed (2/14). In contrast pERK expression was more prevalent in aggressive tumours (11/14). Interestingly, pMEK was only localized to the tumour mass whereas pERK was expressed in tumours and stroma of aggressive BCCs. Similarly, pERK (but not pMEK) was absent in mouse BCC-like tumours derived from X-ray irradiated Ptch1+/− mice with stromal pERK observed in myofibroblasts of the aggressive variant as well as in the tumour mass. RNA sequencing analysis of tumour epithelium and stroma of aggressive and non-aggressive BCC revealed the upregulation of epidermal growth factor receptor- and ERK-related pathways. Angiogenesis and immune response pathways were also upregulated in the stroma compared with the tumour. PTCH1 suppressed NEB1 immortalized keratinocytes (shPTCH1) display upregulated pERK that can be independent of MEK expression. Furthermore, epidermal growth factor pathway inhibitors affect the HH pathway by suppressing GLI1. These studies reveal differential expression of pERK between human BCC subtypes that maybe active by a pathway independent of MEK.

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. Significance: 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. Cancer Res; 78(10); 2577–88. ©2018 AACR.

Aberrant Hedgehog (Hh) signalling has been reported in a number of malignancies, particularly basal cell carcinoma (BCC) of the skin. Clinical trials of Hh inhibitors are underway in many cancers, and these have produced significant clinical benefit in BCC patients, although regrowth of new, or clinically aggressive, variants, as well as development of secondary malignancies, has been reported. ?v?6 integrin is expressed in many cancers, where it has been shown to correlate with an aggressive tumour phenotype and poor prognosis. We have previously reported ?v?6 up-regulation in aggressive, morphoeic BCC variants, where it modulates the stromal response and induces invasion. To examine a possible link between Hh and ?v?6 function, we generated BCC models, overexpressing Gli1 in immortalized keratinocytes (NTert1, HaCaT). Unexpectedly, we found that suppressing Gli1 significantly increased ?v?6 expression. This promoted tumour cell motility and also stromal myofibroblast differentiation through integrin-dependent TGF-?1 activation. Gli1 inhibited ?v?6 expression by suppressing TGF-?1-induced Smad2/3 activation, blocking a positive feedback loop maintaining high ?v?6 levels. A similar mechanism was observed in AsPC1 pancreatic cancer cells expressing endogenous Gli1, suggesting a common mechanism across tumour types. In vitro findings were supported using human clinical samples, where we showed an inverse correlation between ?v?6 and Gli1 expression in different BCC subtypes and pancreatic cancers. In summary, we show that expression of Gli1 and ?v?6 inversely correlates in tumours in vivo, and Hh targeting up-regulates TGF-?1/Smad2/3-dependent ?v?6 expression, promoting pro-tumourigenic cell functions in vitro. These results have potential clinical significance, given the reported recurrence of BCC variants and secondary malignancies in patients treated by Hh targeting.

Ultraviolet B (UVB) light is the principal aetiological factor associated with non-melanoma skin cancer, the most prevalent group of malignancies in the Caucasian population. Exposure to environmental chemicals has also been shown to promote skin carcinogenesis and, as for UVB, this is associated with the acquisition of genomic DNA damage. Cells respond to DNA damage by inducing cell cycle arrest to facilitate DNA repair, although apoptosis will occur if the damage is excessive. Oncogenes may drive carcinogenesis by disrupting the balanced control of cell cycle progression, DNA repair and apoptosis, allowing for the propagation of cells with damaged DNA. The transcription factors GLI1 and GLI2 have been implicated in both the initiation and progression of several cancers, including basal cell carcinoma. Here we show that GLI1 and an active mutant of GLI2 (ΔNGLI2) promote apoptotic resistance in N/TERT human keratinocytes upon exposure to UVB and the DNA-alkylating chemicals such as methyl methanesulphonate (MMS) and N-ethyl-N-nitrosurea. Compared with control and untreated N/TERT-GLI1 and -GLI2 cells, those that survived genotoxic insult formed significantly more colonies in soft agar and were significantly more invasive when grown in three-dimensional organotypic collagen gel cultures. Indeed, surviving N/TERT-GLI1 and -GLI2 cells expressed higher levels of the epithelial-to-mesenchymal transition markers Snail and vimentin, and a subpopulation of MMS-treated cells displayed an elongated fibroblast-like morphology with decreased levels of E-cadherin. Finally, whereas Bcl2 was strongly increased in N/TERT-GLI2 cells, the level of induction was weak in N/TERT-GLI1 cells, indicating that GLI1 may activate anti-apoptotic mechanisms(s) independently of Bcl2. In summary, our results show that GLI1 and GLI2 facilitate the propagation of cells with damaged DNA, and thus their expression may be naturally higher in cells that form the earliest precursor tumour lesions.

Although deregulated Hedgehog signalling and elevated Gli transcription factor expression are known to promote the development of basal cell carcinoma (BCC), little is known about molecular mechanisms driving the development of specific growth pattern subtypes. Using gene array analysis, we have previously observed that over-expression of GLI1 in human keratinocytes promotes increased expression of the neuronal differentiation markers ARC and ULK1. We asked whether neuronal differentiation is a characteristic of BCC and whether there is any correlation with BCC subtype. Using RT-PCR and immunohistochemistry, we confirmed that the neuronal markers ARC, β-tubulin III, GAP-43 and Neurofilament are expressed in human BCC but not in normal epidermis. Moreover, we found that expression of these neuronal differentiation markers showed strong correlation to BCC subtype, with more aggressive infiltrative and morphoeic BCC showing low levels or lack of expression compared to nodular, superficial and micronodular subtypes. Primary human keratinocytes retrovirally expressing GLI1− and GLI2− showed elevated levels of β-tubulin III and ARC but not Neurofilament or GAP-43, suggesting that β-tubulin III and Arc may be early targets of aberrant Gli expression in BCC, whereas expression of Neurofilament and GAP-43 are either later, downstream targets or under control of alternative pathways. We propose that neuronal differentiation is a feature of BCC and that expression of these markers is in part due to aberrant Hedgehog signalling. Moreover, we suggest that correlation between loss of expression of neuronal markers in infiltrative and morphoeic BCC subtypes reflects dedifferentiation of more aggressive BCC subtypes. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Basal cell carcinoma (BCC) of the skin is a highly compact, non-metastatic epithelial tumour type that may arise from the aberrant propagation of epidermal or progenitor stem cell (SC) populations. Increased expression of GLI1 is a common feature of BCC and is linked to the induction of epidermal SC markers in immortalized N/Tert-1 keratinocytes. Here, we demonstrate that GLI1 over-expression is linked to additional SC characteristics in N/Tert-1 cells including reduced epidermal growth factor receptor (EGFR) expression and compact colony formation that is associated with repressed extracellular signal-regulated kinase (ERK) activity. Colony formation and repressed ERK activity remain evident when EGFR is increased exogenously to the basal levels in GLI1 cells revealing that ERK is additionally inhibited downstream of the receptor. Exposure to epidermal growth factor (EGF) to increase ERK activity and promote migration negates GLI1 colony formation with cells displaying an elongated, fibroblast-like morphology. However, as determined by Snail messenger RNA and E-cadherin protein expression this is not associated with epithelial-mesenchymal transition (EMT), and GLI1 actually represses induction of the EMT marker vimentin in EGF-stimulated cells. Instead, live cell imaging revealed that the elongated morphology of EGF/GLI1 keratinocytes stems from their being 'stretched' due to migrating cells displaying inefficient cell-cell detachment and impaired tail retraction. Taken together, these data suggest that GLI1 opposes EGFR signalling to maintain the epithelial phenotype. Finally, ERK activity was predominantly negative in 13/14 BCCs (superficial/nodular), indicating that GLI1 does not routinely co-operate with ERK to induce the formation of this common skin tumour.

Aberrant activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of basal cell carcinoma (BCC). The zinc finger transcription factors GLI1 and GLI2 are considered mediators of the HH signal in epidermal cells, although their tumorigenic nature and their relative contribution to tumorigenesis are only poorly understood. To shed light on the respective role of these transcription factors in epidermal neoplasia, we screened for genes preferentially regulated either by GLI1 or GLI2 in human epidermal cells. We show here that expression of the key antiapoptotic factor BCL2 is predominantly activated by GLI2 compared with GLI1. Detailed promoter analysis and gel shift assays identified three GLI binding sites in the human BCL2 cis-regulatory region. We found that one of these binding sites is critical for conferring GLI2-specific activation of the human BCL2 promoter and that the selective induction of BCL2 expression depends on the zinc finger DNA binding domain of GLI2. In vivo, GLI2 and BCL2 were coexpressed in the outer root sheath of hair follicles and BCC and in plasma cells that infiltrated BCC tumor islands. On the basis of the latter observation, we analyzed plasma cell-derived tumors and found strong expression of GLI2 and BCL2 in neoplastic cells of plasmacytoma patients, implicating HH/GLI signaling in the development of plasma cell-derived malignancies. The results reveal a central role for GLI2 in activating the prosurvival factor BCL2, which may represent an important mechanism in the development or maintenance of cancers associated with inappropriate HH signaling.

Sonic hedgehog (Shh) binds to its receptor patched (PTCH), leading to the activation and repression of target genes via the GLI family of zinc-finger transcription factors. Deregulation of the Shh pathway is associated with basal cell carcinoma (BCC) due to upregulation of GLI1 and GLI2. We recently demonstrated a positive feedback loop between GLI1 and GLI2, which revealed that GLI1 may be a direct target of GLI2. Using band shift and luciferase reporter assays, we now show that GLI2 binds the GLI-binding consensus sequence in the GLI1 promoter. These data suggest that GLI2 directly activates GLI1 and that retrovirally expressed GLI2 induces expression of endogenous GLI1 in human primary keratinocytes. Finally, using in situ hybridization, we show that GLI2 is expressed in the interfollicular epidermis and the outer root sheath of hair follicles in normal skin as well as in BCC tumor islands. These results suggest an important role for GLI2 in regulating epidermal proliferation and skin tumorigenesis.

In stratified epidermis, activation of the Hh/Gli signal transduction pathway has been implicated in the control of cell proliferation and tumorigenesis. The zinc-finger transcription factor Gli2 has been identified as critical mediator of the Hh signal at the distal end of the pathway, but the molecular mechanisms by which Gli2 regulates cell proliferation or induces epidermal malignancies such as basal cell carcinoma are still unclear. Here, we provide evidence for a role of human GLI2 in antagonizing contact inhibition and epidermal differentiation. We show by gene expression profiling that activation of the GLI2 oncogene in human keratinocytes activates the transcription of a number of genes involved in cell cycle progression such as E2F1, CCND1, CDC2 and CDC45L, while it represses genes associated with epidermal differentiation. Analysis of the proliferative effect of GLI2 revealed that GLI2 is able to induce G1-S phase progression in contact-inhibited keratinocytes. Detailed time-course experiments identified E2F1 as early transcriptional target of GLI2. Further, we show that GLI2 expression in human keratinocytes results in a marked downregulation of epidermal differentiation markers. The data suggest a role for GLI2 in Hh-induced epidermal neoplasia by opposing epithelial cell cycle arrest signals and epidermal differentiation.

The mechanism underlying the tumour-suppressor activity of the neurofibromatosis type 2 (NF2) gene product, merlin, is largely undefined but there is evidence that the biological function of the protein might be mediated partly through interactions with the cytoskeleton. Merlin is expressed predominantly as two isoforms that differ at their C-termini owing to alternative splicing of exon 16. By expressing merlin isoform I as bait in a yeast two-hybrid screen, we isolated a clone encoding a region of the cytoskeletal protein β-fodrin. Confirmation of the merlin–fodrin interaction was provided by using the mammalian two-hybrid system and binding assays in vitro. In addition, these assays and co-immunoprecipitation from mammalian cells revealed that the binding site for fodrin is located in the C-terminal half of merlin at a site that is masked in the native protein. Co-expression of the N-terminus of merlin decreased the interaction of its C-terminus with fodrin, implicating homophilic interactions of merlin isoform I in masking the fodrin-binding site. The effect of three disease-associated mutations on the merlin–fodrin interaction and merlin dimerization was also investigated. The mutation L535P, but not L360P or K413E, significantly decreased the merlin–fodrin interaction but not dimerization, indicating that the tumour suppressor ability of merlin might reside partly in its ability to interact with the cytoskeleton via fodrin.

Aberrant Hedgehog (Hh) signalling has been reported in a number of malignancies, particularly basal cell carcinoma (BCC) of the skin. Clinical trials of Hh inhibitors are underway in many cancers, and these have produced significant clinical benefit in BCC patients, although regrowth of new, or clinically aggressive, variants, as well as development of secondary malignancies, has been reported. αvβ6 integrin is expressed in many cancers, where it has been shown to correlate with an aggressive tumour phenotype and poor prognosis. We have previously reported αvβ6 up-regulation in aggressive, morphoeic BCC variants, where it modulates the stromal response and induces invasion. To examine a possible link between Hh and αvβ6 function, we generated BCC models, overexpressing Gli1 in immortalized keratinocytes (NTert1, HaCaT). Unexpectedly, we found that suppressing Gli1 significantly increased αvβ6 expression. This promoted tumour cell motility and also stromal myofibroblast differentiation through integrin-dependent TGF-β1 activation. Gli1 inhibited αvβ6 expression by suppressing TGF-β1-induced Smad2/3 activation, blocking a positive feedback loop maintaining high αvβ6 levels. A similar mechanism was observed in AsPC1 pancreatic cancer cells expressing endogenous Gli1, suggesting a common mechanism across tumour types. In vitro findings were supported using human clinical samples, where we showed an inverse correlation between αvβ6 and Gli1 expression in different BCC subtypes and pancreatic cancers. In summary, we show that expression of Gli1 and αvβ6 inversely correlates in tumours in vivo, and Hh targeting up-regulates TGF-β1/Smad2/3-dependent αvβ6 expression, promoting pro-tumourigenic cell functions in vitro. These results have potential clinical significance, given the reported recurrence of BCC variants and secondary malignancies in patients treated by Hh targeting.

The GLI (GLI1/GLI2) transcription factors have been implicated in the development and progression of prostate cancer although our understanding of how they actually contribute to the biology of these common tumours is limited. We observed that GLI reporter activity was higher in normal (PNT-2) and tumourigenic (DU145 and PC-3) androgen-independent cells compared to androgen-dependent LNCaP prostate cancer cells and, accordingly, GLI mRNA levels were also elevated. Ectopic expression of GLI1 or the constitutively active NGLI2 mutant induced a distinct cobblestone-like morphology in LNCaP cells that, regarding the former, correlated with increased GLI2 as well as expression of the basal/stem-like markers CD44, beta1-integrin, Np63 and BMI1, and decreased expression of the luminal marker AR (androgen receptor). LNCaP-GLI1 cells were viable in the presence of the AR inhibitor bicalutamide and gene expression profiling revealed that the transcriptome of LNCaP-GLI1 cells was significantly closer to DU145 and PC-3 cells than to control LNCaP-pBP (empty vector) cells, as well as identifying LCN2/NGAL as a highly induced transcript which is associated with hormone independence in breast and prostate cancer. Functionally, LNCaP-GLI1 cells displayed greater clonal growth and were more invasive than control cells but they did not form colonies in soft agar or prostaspheres in suspension suggesting that they do not possess inherent stem cell properties. Moreover, targeted suppression of GLI1 or GLI2 with siRNA did not reverse the transformed phenotype of LNCaP-GLI1 cells nor did double GLI1/GLI2 knockdowns activate AR expression in DU145 or PC-3 cells. As such, early targeting of the GLI oncoproteins may hinder progression to a hormone independent state but a more detailed understanding of the mechanisms that maintain this phenotype is required to determine if their inhibition will enhance the efficacy of anti-hormonal therapy through the induction of a luminal phenotype and increased dependency upon AR function.

Although deregulated Hedgehog signalling and elevated Gli transcription factor expression are known to promote the development of basal cell carcinoma (BCC), little is known about molecular mechanisms driving the development of specific growth pattern subtypes. Using gene array analysis, we have previously observed that over-expression of GLI1 in human keratinocytes promotes increased expression of the neuronal differentiation markers ARC and ULK1. We asked whether neuronal differentiation is a characteristic of BCC and whether there is any correlation with BCC subtype. Using RT-PCR and immunohistochemistry, we confirmed that the neuronal markers ARC, beta-tubulin III, GAP-43 and Neurofilament are expressed in human BCC but not in normal epidermis. Moreover, we found that expression of these neuronal differentiation markers showed strong correlation to BCC subtype, with more aggressive infiltrative and morphoeic BCC showing low levels or lack of expression compared to nodular, superficial and micronodular subtypes. Primary human keratinocytes retrovirally expressing GLI1(-) and GLI2(-) showed elevated levels of beta-tubulin III and ARC but not Neurofilament or GAP-43, suggesting that beta-tubulin III and Arc may be early targets of aberrant Gli expression in BCC, whereas expression of Neurofilament and GAP-43 are either later, downstream targets or under control of alternative pathways. We propose that neuronal differentiation is a feature of BCC and that expression of these markers is in part due to aberrant Hedgehog signalling. Moreover, we suggest that correlation between loss of expression of neuronal markers in infiltrative and morphoeic BCC subtypes reflects dedifferentiation of more aggressive BCC subtypes.

Basal cell carcinoma (BCC) is the most prevalent cancer in the Western world and its incidence is increasing. The pathogenesis of BCC involves deregulated Sonic hedgehog signaling, leading to activation of the Gli transcription factors. Most BCCs have a nodular growth pattern, and are indolent, slow-growing, and considered “low-risk” lesions. In contrast, the “high-risk” morphoeic variant, which causes significant morbidity, has an infiltrative growth pattern, and is so-called because of its densely fibrous stroma. As αvβ6 is capable of promoting both carcinoma invasion and fibrosis, we examined the expression of this integrin in BCCs and found that the morphoeic type showed significantly higher αvβ6 expression than the nodular type (P = 0.0009). In order to examine the function of αvβ6, we transfected the transcription factors Gli1 or Gli2 into NTERT, human keratinocytes to generate a BCC model. These cells expressed αvβ6 and were invasive, although inhibition of αvβ6 had no direct effect on cell invasion. However, the cells showed αvβ6-dependent activation of transforming growth factor-β1, which induced transdifferentiation of human fibroblasts into myofibroblasts. Paracrine secretion of hepatocyte growth factor/scatter factor by these myofibroblasts promoted c-Met–dependent tumor invasion in both Transwell and three-dimensional organotypic assays. These experimental in vitro findings were confirmed using human clinical samples in which we showed that the stroma of morphoeic BCC is myofibroblast-rich compared with nodular BCC (P = 0.0036), that myofibroblasts express hepatocyte growth factor/scatter factor, and that morphoeic BCCs are strongly c-Met–positive. These data suggest that αvβ6-dependent transforming growth factor-β1 activation induces both the infiltrative growth pattern and fibrotic stroma so characteristic of morphoeic BCC. [Cancer Res 2008;68(9):3295–303]

Hedgehog (HH)/GLI signaling plays a critical role in epidermal development and basal cell carcinoma. Here, we provide evidence that epidermal growth factor receptor (EGFR) signaling modulates the target gene expression profile of GLI transcription factors in epidermal cells. Using expression profiling and quantitative reverse transcriptase PCR, we identified a set of 19 genes whose transcription is synergistically induced by GLI1 and parallel EGF treatment. Promoter studies of a subset of GLI/EGF-regulated genes, including the genes encoding interleukin-1 antagonist IL1R2, Jagged 2, cyclin D1, S100A7, and S100A9, suggest convergence of EGFR and HH/GLI signaling at the level of promoters of selected direct GLI target genes. Inhibition of EGFR and MEK/ERK but not of phosphatidylinositol 3-kinase/AKT abrogated synergistic activation of GLI/EGF target genes, showing that EGFR can signal via RAF/MEK/ERK to cooperate with GLI proteins in selective target gene regulation. Coexpression of the GLI/EGF target IL1R2, EGFR, and activated ERK1/2 in human anagen hair follicles argues for a cooperative role of EGFR and HH/GLI signaling in specifying the fate of outer root sheath (ORS) cells. We also show that EGF treatment neutralizes GLI-mediated induction of epidermal stem cell marker expression and provide evidence that EGFR signaling is essential for GLI-induced cell cycle progression in epidermal cells. The results suggest that EGFR signaling modulates GLI target gene profiles which may play an important regulatory role in ORS specification, hair growth, and possibly cancer.

Activation of the Sonic hedgehog signaling pathway, primarily through mutational inactivation of the PTCH1 gene, is associated with the development of basal cell carcinoma (BCC). Gli1, a member of the Gli family of transcription factors, is expressed in BCC and in transgenic mice targeted expression of Gli1 in basal keratinocytes leads to BCC development. In addition to BCC, previous studies have shown that Gli1 is expressed in the outer root sheath (ORS) of the hair follicle but is absent in interfollicular epidermis. In this study, we have characterized the expression pattern of two protein kinase C (PKC) isoforms expressed in BCC and hair follicles. We have then used reporter assays to investigate the effects of these isoforms on Gli1 transcriptional activity. We report that in BCC sections, PKCalpha but not PKCdelta was weakly expressed in the epidermis, whereas in the hair follicle, PKCalpha was expressed in the ORS and PKCdelta in the inner root sheath. In contrast, neither PKCalpha nor PKCdelta was expressed in BCC tumor islands, although both isoforms were often expressed in the surrounding stroma. In mammalian 293T cells, coexpression of constitutively active PKCalpha reduced the activity of Gli1 in a dose-dependent manner, whereas constitutively active PKCdelta increased the activity of Gli1, although this required higher expression levels. Regulation of mutant Gli1 protein localized exclusively to the nucleus was similar to that of the wild-type protein, indicating that nuclear-cytoplasmic shuttling is not a determinant of Gli1 control by either PKC isoform. Furthermore, PKC regulation of Gli1 did not involve activation of mitogen-activated protein kinase signaling. Finally, we show that exogenous Gli1 does not alter the expression of PKCalpha in human primary keratinocytes, suggesting that loss of this isoform in BCC is not via Hedgehog signaling. As BCCs have been proposed to originate from the ORS, loss of PKCalpha expression may be relevant to tumor formation; this may, in part, be because of the predicted increase in Gli1 transcriptional activity.

Forkhead box (FOX) proteins have been shown to play important roles in regulating the expression of genes involved in cell growth, proliferation, differentiation, longevity, and transformation. The functional importance of this gene family in normal human skin physiology and disease processes is not well understood. Activation of Sonic Hedgehog (Shh) signaling plays a key role in the development of basal cell carcinomas (BCCs) of the skin in humans. Recent studies have established that some FOX genes are downstream targets of Shh signaling. We have investigated the role of FOX proteins in transducing Shh effects in human skin by using degenerate PCR to identify FOX genes differentially expressed in BCCs. All three known FOXM1 isoforms (a, b, and c) were detected in human skin and cultured keratinocytes, and the transcriptionally active FOXM1b isoform was found to be up-regulated in BCCs. Real-time quantitative RT-PCR showed that the increase in FOXM1 mRNA levels was specific for BCCs and not a reflection of increased cell proliferation in that no up-regulation was seen in squamous cell carcinomas or proliferating primary human keratinocyte cultures. Immunostaining studies showed intense nuclear and cytoplasmic staining throughout BCC tumor islands and not confined to the periphery regions of the tumor where proliferating Ki-67-immunopositive cells are predominantly localized. Expression of the Shh target glioma transcription factor-1 (Gli1) in primary keratinocytes and other cell lines caused a significant elevation of FOXM1 mRNA level and transcriptional activity, indicating that FOXM1 is a downstream target of Gli1. Our data provide the first evidence that activation of Shh signaling via Gli1 is an important determinant of FOXM1 expression in mammalian cells. Given the role of FOXM1 in cell proliferation, the up-regulation of FOXM1 in BCCs may be one of the mechanisms whereby Shh signaling exerts its mitogenic effect on basal keratinocytes, leading to the development of this common human cancer.

Transgenic mouse models have provided evidence that activation of the zinc-finger transcription factor GLI1 by Hedgehog (Hh)-signalling is a key step in the initiation of the tumorigenic programme leading to Basal Cell Carcinoma (BCC). However, the downstream events underlying Hh/GLI-induced BCC development are still obscure. Using in vitro model systems to analyse the effect of Hh/GLI-signalling in human keratinocytes, we identified a positive feedback mechanism involving the zinc finger transcription factors GLI1 and GLI2. Expression of GLI1 in human keratinocytes induced the transcriptional activator isoforms GLI2alpha and GLI2beta. Both isoforms were also shown to be expressed at elevated levels in 21 BCCs compared to normal skin. Detailed time course experiments monitoring the transcriptional response of keratinocytes either to GLI1 or to GLI2 suggest that GLI1 is a direct target of GLI2, while activation of GLI2 by GLI1 is likely to be indirect. Furthermore, expression of either GLI2 or GLI1 led to an increase in DNA-synthesis in confluent human keratinocytes. Taken together, these results suggest an important role of the positive GLI1-GLI2 feedback loop in Hh-mediated epidermal cell proliferation.

Introduction: Basal cell carcinoma (BCC) is the most common skin cancer in humans. Its development is associated with dysregulated hedgehog signalling and increased expression of Gli transcription factors. Of three vertebrate Gli proteins, Gli1 and Gli2 appear to be important in carcinogenesis. Currently little is known about their action but it has been shown in an animal model that the magnitude of hedgehog signalling influences tumour invasion. We have observed that cultured keratinocytes, which over-express Gli1 undergo morphological change to a fibroblastoid phenotype. We propose that genes that are over-expressed in these cells may serve as markers for BCC and that these cells may act as a model for BCC biology. Methods

The implementation of microarray (cDNA and oligonucleotide) technology is enabling investigators to assess gene expression at a tissue/cellular level by quantification of messenger RNA (mRNA) transcripts. Once assimilated, these data should provide gene-expression profiles that are unique to specific cells, their responses to specific stimuli, and also help unravel complex signalling pathways. With regards to a more holistic approach, comparisons between diseased and normal tissues could identify genes that are directly implicated in the disease pathology and hence identify targets for therapeutic intervention. However, as with all systems, there remains the possibility of generating false positives and, therefore, differential gene expression should be verified using other techniques such as real-time PCR with reverse-transcription or Northern blot analysis. Furthermore, certain genes can be upregulated without an increase in actual protein levels and, therefore, the use of tissue microarray technology allows high-throughput immunostaining to correlate protein and mRNA data.These technologies have recently been applied to dissect the molecular basis of the most common malignancy in men (with the exception of non-melanoma skin cancer) – prostate cancer. Dhanasekaran et al. 1xDelineation of prognostic biomarkers in prostate cancer. Dhanasekaran, S.M. et al. Nature. 2001; 412: 822–826Crossref | PubMed | Scopus (1213)See all References1 have used both cDNA and tissue microarrays to great effect in comparing and characterising gene-expression profiles in normal and neoplastic prostate specimens. Having generated a vast amount of data, the authors focused on the hepsin and PIM1 genes that encode a transmembrane protease and serine/threonine kinase respectively. Both proteins are overexpressed in prostate tumours and interestingly PIM1 appears to be co-regulated with the oncogene MYC, supporting the possibility of a synergistic oncogenic effect. Interestingly, immunostaining revealed that metastatic prostate samples expressed hepsin at levels in between malignant and benign prostate tumours. Men with elevated prostate-specific antigen (PSA) levels – a clinical marker for prostate cancer – are prone to die from metastases following radical prostatectomy. Extensive statistical analyses subsequently revealed that men conforming to this group have low or absent hepsin protein levels. The same trend was also demonstrated for PIM1 indicating that these two proteins can now be considered as additional clinical biomarkers for prostate cancer.In the past few months, there has been an array of similar studies reporting on the application of the expression array technology to study prostate cancer. Two such studies also identified hepsin as a useful biomarker 2xHuman prostate cancer and benign prostatic hyperplasia: molecular dissection by gene expression profiling. Luo, J. et al. Cancer Res. 2001; 61: 4683–4688PubMedSee all References, 3xExpression profiling reveals hepsin overexpression in prostate cancer. Magee, J.A. et al. Cancer Res. 2001; 61: 5692–5696PubMedSee all References. Further mining of all these microarray data sets will lead to the generation of a large panel of useful biomarkers to aid in the detection and prognosis of prostate cancer. In addition, they could also reveal new ‘molecular targets’ for potential therapeutic intervention towards improved treatment and management of this malignancy.

Sonic hedgehog (Hh) signaling plays a key role in epidermal development and skin cancer. Mutational inactivation of the tumor suppressor gene patched (PTCH) leads to constitutive activation of the Hh signaling pathway, resulting in activation of target gene transcription by the zinc finger transcription factors GLI1 and GLI2. Recent experiments in mice point to GLI2 as the key mediator of Hh signaling in skin. We have concentrated on the identification of candidate mediators of GLI2 function in the human epidermis. We show here that the forkhead/winged-helix domain transcription factor FOXE1 is likely to be a direct GLI2 target gene. The kinetics of FOXE1 induction are similar to the known direct target PTCH, and a 2.5 kb upstream fragment containing five GLI-binding sites activates transcription in a reporter assay. We show by in situ hybridization that FOXE1 is expressed in the outer root sheath of the hair follicle, where murine Gli2 is also expressed. FOXE1 expression is also found in basal keratinocytes of the human epidermis and basal cell carcinoma (BCC). These data point to a putative role of FOXE1 in mediating Hh signaling in the human epidermis downstream of GLI2.