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Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM.

Biswas NK, Chandra V, Sarkar-Roy N, Das T, Bhattacharya RN, Tripathy LN, Basu SK, Kumar S, Das S, Chatterjee A, Mukherjee A, Basu P, Maitra A, Chattopadhyay A, Basu A, Dhara S - Sci Rep (2015)

Bottom Line: Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect.Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells.Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.

ABSTRACT
Neoplastic cells of Glioblastoma multiforme (GBM) may or may not show sustained response to temozolomide (TMZ) chemotherapy. We hypothesize that TMZ chemotherapy response in GBM is predetermined in its neoplastic clones via a specific set of mutations that alter relevant pathways. We describe exome-wide enrichment of variant allele frequencies (VAFs) in neurospheres displaying contrasting phenotypes of sustained versus reversible TMZ-responses in vitro. Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect. Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells. Next, we validated the likely involvement of the Hh-pathway in TMZ-response on additional GBM neurospheres as well as on GBM patients, by extracting RNA-sequencing-based gene expression data from the TCGA-GBM database. Finally, we demonstrated TMZ-sensitization of a TMZ non-responder neurosphere in vitro by treating them with the FDA-approved pharmacological Hh-pathway inhibitor vismodegib. Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.

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(a), mRNA expressions of GLI1, SNAI1and MGMT in 6 neurospheres. (b), Correlation matrix of 11 Hh-pathway component genes and MGMT (/r/ = 0.96 FDR 0.1) on 5 cells except M45481. (c), Regression model fitting with the expressions of GLI1 with MGMT and (d), SNAI1 with MGMT. Red dots representing non-responder cells and black dots responder cells.
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f5: (a), mRNA expressions of GLI1, SNAI1and MGMT in 6 neurospheres. (b), Correlation matrix of 11 Hh-pathway component genes and MGMT (/r/ = 0.96 FDR 0.1) on 5 cells except M45481. (c), Regression model fitting with the expressions of GLI1 with MGMT and (d), SNAI1 with MGMT. Red dots representing non-responder cells and black dots responder cells.

Mentions: Next, we validated the likely involvement of Hh-pathway in TMZ-response on additional neurospheres from our repository. We estimated the expression correlations of MGMT – as a covariate for TMZ response – with the set of 11 Hh-pathway component genes on A49910, M45481, B0027, B0043, B0048 and B0051 neurospheres. As shown in Figure 5, MGMT expression correlated well with the expressions of GLI1 and SNAI1, in all the neurospheres only except in M45481. Unlike the other 5 neurospheres, expressions of GLI1/SNAI1 vs. MGMT were clearly discordant in this neurosphere. As shown in Figure 5 a, GLI1/SNAI1 expression in M45481 was as high as B0048 but MGMT expression was 61.3 fold lower than B0048. Therefore, we estimated expression correlation of the 11 Hh-pathway component genes with MGMT on the 5 neurospheres excluding M45481. For the rest of 5 neurospheres, A49910 and B0027 were responders whereas B0043, B0050 and B0051 were non-responders as revealed by annexin V and CFSE staining experiments (data not shown). We found statistically significant correlations of expression of MGMT with that of the key component genes of the Hh-pathway GLI1 (r = 0.9939), SNAI1 (r = 0.9917) and SUFU (r = 0.964); (/r/ = 0.96 is the 10% False Discovery Rate cutoff for significance). Linear regression of GLI1 on MGMT (Adjusted R2 = 0.9839, p-value 0.00056) and that of SNAI1 on MGMT (Adjusted R2 = 0.9752, p-value 0.008286), as in Figures 5 c and d respectively, suggest the Hh-pathway to be playing a significant role in TMZ-response of the GBM neurospheres.


Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM.

Biswas NK, Chandra V, Sarkar-Roy N, Das T, Bhattacharya RN, Tripathy LN, Basu SK, Kumar S, Das S, Chatterjee A, Mukherjee A, Basu P, Maitra A, Chattopadhyay A, Basu A, Dhara S - Sci Rep (2015)

(a), mRNA expressions of GLI1, SNAI1and MGMT in 6 neurospheres. (b), Correlation matrix of 11 Hh-pathway component genes and MGMT (/r/ = 0.96 FDR 0.1) on 5 cells except M45481. (c), Regression model fitting with the expressions of GLI1 with MGMT and (d), SNAI1 with MGMT. Red dots representing non-responder cells and black dots responder cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300501&req=5

f5: (a), mRNA expressions of GLI1, SNAI1and MGMT in 6 neurospheres. (b), Correlation matrix of 11 Hh-pathway component genes and MGMT (/r/ = 0.96 FDR 0.1) on 5 cells except M45481. (c), Regression model fitting with the expressions of GLI1 with MGMT and (d), SNAI1 with MGMT. Red dots representing non-responder cells and black dots responder cells.
Mentions: Next, we validated the likely involvement of Hh-pathway in TMZ-response on additional neurospheres from our repository. We estimated the expression correlations of MGMT – as a covariate for TMZ response – with the set of 11 Hh-pathway component genes on A49910, M45481, B0027, B0043, B0048 and B0051 neurospheres. As shown in Figure 5, MGMT expression correlated well with the expressions of GLI1 and SNAI1, in all the neurospheres only except in M45481. Unlike the other 5 neurospheres, expressions of GLI1/SNAI1 vs. MGMT were clearly discordant in this neurosphere. As shown in Figure 5 a, GLI1/SNAI1 expression in M45481 was as high as B0048 but MGMT expression was 61.3 fold lower than B0048. Therefore, we estimated expression correlation of the 11 Hh-pathway component genes with MGMT on the 5 neurospheres excluding M45481. For the rest of 5 neurospheres, A49910 and B0027 were responders whereas B0043, B0050 and B0051 were non-responders as revealed by annexin V and CFSE staining experiments (data not shown). We found statistically significant correlations of expression of MGMT with that of the key component genes of the Hh-pathway GLI1 (r = 0.9939), SNAI1 (r = 0.9917) and SUFU (r = 0.964); (/r/ = 0.96 is the 10% False Discovery Rate cutoff for significance). Linear regression of GLI1 on MGMT (Adjusted R2 = 0.9839, p-value 0.00056) and that of SNAI1 on MGMT (Adjusted R2 = 0.9752, p-value 0.008286), as in Figures 5 c and d respectively, suggest the Hh-pathway to be playing a significant role in TMZ-response of the GBM neurospheres.

Bottom Line: Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect.Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells.Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.

ABSTRACT
Neoplastic cells of Glioblastoma multiforme (GBM) may or may not show sustained response to temozolomide (TMZ) chemotherapy. We hypothesize that TMZ chemotherapy response in GBM is predetermined in its neoplastic clones via a specific set of mutations that alter relevant pathways. We describe exome-wide enrichment of variant allele frequencies (VAFs) in neurospheres displaying contrasting phenotypes of sustained versus reversible TMZ-responses in vitro. Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect. Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells. Next, we validated the likely involvement of the Hh-pathway in TMZ-response on additional GBM neurospheres as well as on GBM patients, by extracting RNA-sequencing-based gene expression data from the TCGA-GBM database. Finally, we demonstrated TMZ-sensitization of a TMZ non-responder neurosphere in vitro by treating them with the FDA-approved pharmacological Hh-pathway inhibitor vismodegib. Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.

Show MeSH
Related in: MedlinePlus