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GLI1 confers profound phenotypic changes upon LNCaP prostate cancer cells that include the acquisition of a hormone independent state.

Nadendla SK, Hazan A, Ward M, Harper LJ, Moutasim K, Bianchi LS, Naase M, Ghali L, Thomas GJ, Prowse DM, Philpott MP, Neill GW - PLoS ONE (2011)

Bottom Line: Ectopic expression of GLI1 or the constitutively active ΔNGLI2 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, β1-integrin, ΔNp63 and BMI1, and decreased expression of the luminal marker AR (androgen receptor).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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cutaneous Research, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.

ABSTRACT
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 ΔNGLI2 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, β1-integrin, ΔNp63 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.

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GLI suppression does not induce a luminal-like phenotype in                            androgen-independent cells.(A) The transformed morphology of LNCaP-GLI1 cells does not reverse upon                            transfection with GLI1 or GLI2 siRNA. (B) qPCR analysis of GLI1 and GLI2                            mRNA in LNCaP-GLI1 cells transfected with GLI1 or GLI2 siRNA. (C) RT-PCR                            analysis of ΔNp63 and AR mRNA in LNCaP-GLI1 cells transfected with                            GLI1 or GLI2 siRNA. (D) qPCR analysis of GLI1 and GLI2 mRNA in DU145 and                            PC-3 cells transfected with GLI1 and GLI2 siRNA. (E) GLI reporter                            activity is suppressed in DU145 and PC-3 cells transfected with GLI1 and                            GLI2 siRNA (n.b. reporter activity may be influenced by GLI3 expression                            in PC-3 cells [17]). (F) RT-PCR analysis of ΔNp63 and AR                            mRNA in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA.
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pone-0020271-g005: GLI suppression does not induce a luminal-like phenotype in androgen-independent cells.(A) The transformed morphology of LNCaP-GLI1 cells does not reverse upon transfection with GLI1 or GLI2 siRNA. (B) qPCR analysis of GLI1 and GLI2 mRNA in LNCaP-GLI1 cells transfected with GLI1 or GLI2 siRNA. (C) RT-PCR analysis of ΔNp63 and AR mRNA in LNCaP-GLI1 cells transfected with GLI1 or GLI2 siRNA. (D) qPCR analysis of GLI1 and GLI2 mRNA in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA. (E) GLI reporter activity is suppressed in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA (n.b. reporter activity may be influenced by GLI3 expression in PC-3 cells [17]). (F) RT-PCR analysis of ΔNp63 and AR mRNA in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA.

Mentions: To investigate a putative role for GLI in prostate cancer, we first determined the level of GLI reporter activity in various prostate cell lines. GLI reporter activity was higher in the AI DU145 and PC-3 prostate cancer cell lines compared to the AD LNCaP prostate cancer cell line and reporter activity was also higher in the AI PNT-2 normal epithelial prostate cell line (Fig. 1A). Accordingly, GLI1 and GLI2 mRNA expression was higher in all AI cell lines compared to LNCaP cells (Fig. 1B). As such, we analysed the effect of over-expressing GLI1 and the active ΔNGLI2 mutant upon LNCaP cell biology. The most striking effect of ectopic GLI1 (eGLI1) and ΔNGLI2 related to cell morphology: in contrast to the characteristic spindle-like morphology of parental or control LNCaP-pBP (empty vector) cells, within a few days post-transduction cells/colonies with a cobblestone-like morphology were evident in LNCaP cells over-expressing eGLI1 or ΔNGLI2 (Fig. 1C). After drug selection, both LNCaP-GLI1 and LNCaP-ΔNGLI2 cells had completely transformed adopting a morphology reminiscent of PNT-2 or DU145 cells (refer Fig. 5A to view the fully transformed morphology). Ectopic GLI1 and ΔNGLI2 protein activity was confirmed by induction of PTCH1 mRNA (Fig. 1D). In addition, endogenous GLI2 mRNA was induced in LNCaP-GLI1 cells whereas, unexpectedly, endogenous GLI1 mRNA was suppressed in LNCaP-ΔNGLI2 cells revealing that the morphological change may be mediated by GLI2 (Fig. 1E). As DU145 and PC-3 cells express high levels of both GLI1 and GLI2 compared to LNCaP cells (Fig. 1B), we chose to further investigate the biology of LNCaP-GLI1 cells.


GLI1 confers profound phenotypic changes upon LNCaP prostate cancer cells that include the acquisition of a hormone independent state.

Nadendla SK, Hazan A, Ward M, Harper LJ, Moutasim K, Bianchi LS, Naase M, Ghali L, Thomas GJ, Prowse DM, Philpott MP, Neill GW - PLoS ONE (2011)

GLI suppression does not induce a luminal-like phenotype in                            androgen-independent cells.(A) The transformed morphology of LNCaP-GLI1 cells does not reverse upon                            transfection with GLI1 or GLI2 siRNA. (B) qPCR analysis of GLI1 and GLI2                            mRNA in LNCaP-GLI1 cells transfected with GLI1 or GLI2 siRNA. (C) RT-PCR                            analysis of ΔNp63 and AR mRNA in LNCaP-GLI1 cells transfected with                            GLI1 or GLI2 siRNA. (D) qPCR analysis of GLI1 and GLI2 mRNA in DU145 and                            PC-3 cells transfected with GLI1 and GLI2 siRNA. (E) GLI reporter                            activity is suppressed in DU145 and PC-3 cells transfected with GLI1 and                            GLI2 siRNA (n.b. reporter activity may be influenced by GLI3 expression                            in PC-3 cells [17]). (F) RT-PCR analysis of ΔNp63 and AR                            mRNA in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102098&req=5

pone-0020271-g005: GLI suppression does not induce a luminal-like phenotype in androgen-independent cells.(A) The transformed morphology of LNCaP-GLI1 cells does not reverse upon transfection with GLI1 or GLI2 siRNA. (B) qPCR analysis of GLI1 and GLI2 mRNA in LNCaP-GLI1 cells transfected with GLI1 or GLI2 siRNA. (C) RT-PCR analysis of ΔNp63 and AR mRNA in LNCaP-GLI1 cells transfected with GLI1 or GLI2 siRNA. (D) qPCR analysis of GLI1 and GLI2 mRNA in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA. (E) GLI reporter activity is suppressed in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA (n.b. reporter activity may be influenced by GLI3 expression in PC-3 cells [17]). (F) RT-PCR analysis of ΔNp63 and AR mRNA in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA.
Mentions: To investigate a putative role for GLI in prostate cancer, we first determined the level of GLI reporter activity in various prostate cell lines. GLI reporter activity was higher in the AI DU145 and PC-3 prostate cancer cell lines compared to the AD LNCaP prostate cancer cell line and reporter activity was also higher in the AI PNT-2 normal epithelial prostate cell line (Fig. 1A). Accordingly, GLI1 and GLI2 mRNA expression was higher in all AI cell lines compared to LNCaP cells (Fig. 1B). As such, we analysed the effect of over-expressing GLI1 and the active ΔNGLI2 mutant upon LNCaP cell biology. The most striking effect of ectopic GLI1 (eGLI1) and ΔNGLI2 related to cell morphology: in contrast to the characteristic spindle-like morphology of parental or control LNCaP-pBP (empty vector) cells, within a few days post-transduction cells/colonies with a cobblestone-like morphology were evident in LNCaP cells over-expressing eGLI1 or ΔNGLI2 (Fig. 1C). After drug selection, both LNCaP-GLI1 and LNCaP-ΔNGLI2 cells had completely transformed adopting a morphology reminiscent of PNT-2 or DU145 cells (refer Fig. 5A to view the fully transformed morphology). Ectopic GLI1 and ΔNGLI2 protein activity was confirmed by induction of PTCH1 mRNA (Fig. 1D). In addition, endogenous GLI2 mRNA was induced in LNCaP-GLI1 cells whereas, unexpectedly, endogenous GLI1 mRNA was suppressed in LNCaP-ΔNGLI2 cells revealing that the morphological change may be mediated by GLI2 (Fig. 1E). As DU145 and PC-3 cells express high levels of both GLI1 and GLI2 compared to LNCaP cells (Fig. 1B), we chose to further investigate the biology of LNCaP-GLI1 cells.

Bottom Line: Ectopic expression of GLI1 or the constitutively active ΔNGLI2 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, β1-integrin, ΔNp63 and BMI1, and decreased expression of the luminal marker AR (androgen receptor).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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cutaneous Research, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.

ABSTRACT
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 ΔNGLI2 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, β1-integrin, ΔNp63 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.

Show MeSH
Related in: MedlinePlus