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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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Flow cytometry analysis of annexin-V and propidium iodide (PI) staining of apoptotic cells following vismodegib (50 μM) and TMZ (50 μM) treatment to B0048 neurosphere.a), DMSO-treated control, b), TMZ treatment alone, c) vismodegib treatment alone, d), TMZ treatment along with vismodegib treatment and e), showing % of apoptotic cells (annexin-V positive + PI positive + annexin-V and PI double positive cells). (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001).
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f6: Flow cytometry analysis of annexin-V and propidium iodide (PI) staining of apoptotic cells following vismodegib (50 μM) and TMZ (50 μM) treatment to B0048 neurosphere.a), DMSO-treated control, b), TMZ treatment alone, c) vismodegib treatment alone, d), TMZ treatment along with vismodegib treatment and e), showing % of apoptotic cells (annexin-V positive + PI positive + annexin-V and PI double positive cells). (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001).

Mentions: However, in order to further understand the link between TMZ-response and the Hh-pathway in GBM we performed a proof of principle experiment in vitro. We performed this experiment on the most TMZ-resistant neurosphere (B0048) from our repository. As shown in Figure 6 a, b and the first two columns of e, there was no significant apoptosis induced by TMZ-treatment alone to this neurosphere in vitro (p-value 0.179). But a 3.2 fold increase (p-value 0.0004) in the total number of apoptotic cells in the same neurosphere was observed when the TMZ-treatment was done along with the FDA-approved Hh-pathway inhibitor drug vismodegib treatment in vitro (Fig. 6 e). Vismodegib treatment alone induced 2.2 fold (p-value 0.0011) apoptosis compared to the DMSO-treated control (Fig. 6 e). These results suggest the Hh-pathway could be a potential therapeutic target to enhance TMZ-response in this malignancy.


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)

Flow cytometry analysis of annexin-V and propidium iodide (PI) staining of apoptotic cells following vismodegib (50 μM) and TMZ (50 μM) treatment to B0048 neurosphere.a), DMSO-treated control, b), TMZ treatment alone, c) vismodegib treatment alone, d), TMZ treatment along with vismodegib treatment and e), showing % of apoptotic cells (annexin-V positive + PI positive + annexin-V and PI double positive cells). (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Flow cytometry analysis of annexin-V and propidium iodide (PI) staining of apoptotic cells following vismodegib (50 μM) and TMZ (50 μM) treatment to B0048 neurosphere.a), DMSO-treated control, b), TMZ treatment alone, c) vismodegib treatment alone, d), TMZ treatment along with vismodegib treatment and e), showing % of apoptotic cells (annexin-V positive + PI positive + annexin-V and PI double positive cells). (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001).
Mentions: However, in order to further understand the link between TMZ-response and the Hh-pathway in GBM we performed a proof of principle experiment in vitro. We performed this experiment on the most TMZ-resistant neurosphere (B0048) from our repository. As shown in Figure 6 a, b and the first two columns of e, there was no significant apoptosis induced by TMZ-treatment alone to this neurosphere in vitro (p-value 0.179). But a 3.2 fold increase (p-value 0.0004) in the total number of apoptotic cells in the same neurosphere was observed when the TMZ-treatment was done along with the FDA-approved Hh-pathway inhibitor drug vismodegib treatment in vitro (Fig. 6 e). Vismodegib treatment alone induced 2.2 fold (p-value 0.0011) apoptosis compared to the DMSO-treated control (Fig. 6 e). These results suggest the Hh-pathway could be a potential therapeutic target to enhance TMZ-response in this malignancy.

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