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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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Ectopic GLI1 induces global changes in the gene expression profile of                            LNCaP cells.(A) A statistical comparison of global gene expression profiles to                            determine the percentage of transcripts that are expressed at                            significantly different levels in LNCaP-pBP, DU145 and PC-3 cells                            compared to LNCaP-GLI1 (Pearson correlation co-efficient ≥0.7,                            p<0.05) (B) Heat map denoting transcripts in LNCaP-GLI1 cells where                            the change in expression is both >10-fold and highly significantly                            different when compared to LNCaP-pBP cells (student's                                t-test, p<0.01): left panel lists increased                            genes, right panel lists decreased genes and DU145 and PC-3 cells are                            shown for comparison (* denotes transcript variants of the same                            gene). (C) Western blot analysis comparing the expression of certain                            signalling proteins between LNCaP-pBP and LNCaP-GLI1 cells with DU145                            and PC-3 lysates included for comparison. (D) Phosphorylation of the                            cytoskeletal protein MLC2 is mediated by ROCK in LNCaP-GLI1 cells (n.b.                            the antibody for total MLC did not work in our hands).
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pone-0020271-g003: Ectopic GLI1 induces global changes in the gene expression profile of LNCaP cells.(A) A statistical comparison of global gene expression profiles to determine the percentage of transcripts that are expressed at significantly different levels in LNCaP-pBP, DU145 and PC-3 cells compared to LNCaP-GLI1 (Pearson correlation co-efficient ≥0.7, p<0.05) (B) Heat map denoting transcripts in LNCaP-GLI1 cells where the change in expression is both >10-fold and highly significantly different when compared to LNCaP-pBP cells (student's t-test, p<0.01): left panel lists increased genes, right panel lists decreased genes and DU145 and PC-3 cells are shown for comparison (* denotes transcript variants of the same gene). (C) Western blot analysis comparing the expression of certain signalling proteins between LNCaP-pBP and LNCaP-GLI1 cells with DU145 and PC-3 lysates included for comparison. (D) Phosphorylation of the cytoskeletal protein MLC2 is mediated by ROCK in LNCaP-GLI1 cells (n.b. the antibody for total MLC did not work in our hands).

Mentions: To investigate this further, LNCaP-pBP, LNCaP-GLI1, DU145 and PC-3 cells were analysed by DNA microarrays: global array profiling revealed that the transcriptome of LNCaP-GLI1 cells was more similar to DU145 and PC-3 cells than to LNCaP-pBP cells thus revealing the extent to which LNCaP-GLI1 cells have changed phenotype (Fig. 3A). In direct comparison to LNCaP-pBP cells, the expression of 260 transcripts differed more than 10-fold (144 up and 116 down) in LNCaP-GLI1 cells (Fig. 3B and Figures S1 and S2). Functional classification of these transcripts produced 15 ontological groups including those associated with tumour biology such as cell-cell adhesion, cell motility, EMT (epithelial-mesenchymal transition) and hormone independence (Figure S3); the latter group including LCN2 (lipocalin 2) and CAV2 (caveolin 2) which were previously identified as part of a common signature for hormone independence in breast and prostate cancer [29]. The majority of the 144 increased transcripts were expressed at similar levels in LNCaP-GLI1 cells when compared to DU145 and/or PC-3 cells (<3-fold difference), whereas the expression of 12 transcripts (including LCN2) was >3-fold higher in LNCaP-GLI cells when compared to both cell types (Figure S1 and Table 1). Reciprocally, of the 116 decreased transcripts only one, MRPL23, was expressed >3-fold lower in LNCaP-GLI1 cells compared to both DU145 and PC-3 cells (Figure S2).


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)

Ectopic GLI1 induces global changes in the gene expression profile of                            LNCaP cells.(A) A statistical comparison of global gene expression profiles to                            determine the percentage of transcripts that are expressed at                            significantly different levels in LNCaP-pBP, DU145 and PC-3 cells                            compared to LNCaP-GLI1 (Pearson correlation co-efficient ≥0.7,                            p<0.05) (B) Heat map denoting transcripts in LNCaP-GLI1 cells where                            the change in expression is both >10-fold and highly significantly                            different when compared to LNCaP-pBP cells (student's                                t-test, p<0.01): left panel lists increased                            genes, right panel lists decreased genes and DU145 and PC-3 cells are                            shown for comparison (* denotes transcript variants of the same                            gene). (C) Western blot analysis comparing the expression of certain                            signalling proteins between LNCaP-pBP and LNCaP-GLI1 cells with DU145                            and PC-3 lysates included for comparison. (D) Phosphorylation of the                            cytoskeletal protein MLC2 is mediated by ROCK in LNCaP-GLI1 cells (n.b.                            the antibody for total MLC did not work in our hands).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102098&req=5

pone-0020271-g003: Ectopic GLI1 induces global changes in the gene expression profile of LNCaP cells.(A) A statistical comparison of global gene expression profiles to determine the percentage of transcripts that are expressed at significantly different levels in LNCaP-pBP, DU145 and PC-3 cells compared to LNCaP-GLI1 (Pearson correlation co-efficient ≥0.7, p<0.05) (B) Heat map denoting transcripts in LNCaP-GLI1 cells where the change in expression is both >10-fold and highly significantly different when compared to LNCaP-pBP cells (student's t-test, p<0.01): left panel lists increased genes, right panel lists decreased genes and DU145 and PC-3 cells are shown for comparison (* denotes transcript variants of the same gene). (C) Western blot analysis comparing the expression of certain signalling proteins between LNCaP-pBP and LNCaP-GLI1 cells with DU145 and PC-3 lysates included for comparison. (D) Phosphorylation of the cytoskeletal protein MLC2 is mediated by ROCK in LNCaP-GLI1 cells (n.b. the antibody for total MLC did not work in our hands).
Mentions: To investigate this further, LNCaP-pBP, LNCaP-GLI1, DU145 and PC-3 cells were analysed by DNA microarrays: global array profiling revealed that the transcriptome of LNCaP-GLI1 cells was more similar to DU145 and PC-3 cells than to LNCaP-pBP cells thus revealing the extent to which LNCaP-GLI1 cells have changed phenotype (Fig. 3A). In direct comparison to LNCaP-pBP cells, the expression of 260 transcripts differed more than 10-fold (144 up and 116 down) in LNCaP-GLI1 cells (Fig. 3B and Figures S1 and S2). Functional classification of these transcripts produced 15 ontological groups including those associated with tumour biology such as cell-cell adhesion, cell motility, EMT (epithelial-mesenchymal transition) and hormone independence (Figure S3); the latter group including LCN2 (lipocalin 2) and CAV2 (caveolin 2) which were previously identified as part of a common signature for hormone independence in breast and prostate cancer [29]. The majority of the 144 increased transcripts were expressed at similar levels in LNCaP-GLI1 cells when compared to DU145 and/or PC-3 cells (<3-fold difference), whereas the expression of 12 transcripts (including LCN2) was >3-fold higher in LNCaP-GLI cells when compared to both cell types (Figure S1 and Table 1). Reciprocally, of the 116 decreased transcripts only one, MRPL23, was expressed >3-fold lower in LNCaP-GLI1 cells compared to both DU145 and PC-3 cells (Figure S2).

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