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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) Light microscopic images (200 μm bar) and viable cell count of the neurospheres following TMZ treatment and post-treatment recovery. (a), DMSO treated control and (b), 5 days TMZ-treated neurospheres in A49910. (c), % viable cells in A49910 with DMSO- and 5 days of TMZ- treatment. (d), DMSO-treated control and (e), 5 days TMZ-treated neurospheres in M45481. (f), % viable cells in M45481 with DMSO- and 5 days of TMZ- treatment. (g), DMSO-treated control and (h), 28 days of post-TMZ-treated neurospheres in A49910. (i), % viable cells of DMSO-treated controls and 28 days of post-TMZ-treated cells in A49910. (j), DMSO treated control and (k), 28 days post-TMZ-treated neurospheres in M45481. (l), % viable cells of DMSO-treated controls and 28 days of post-TMZ-treated cells in M45481. (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001). (B) Growth curves (MTS assay) following 5 days of TMZ treatment and 28 days of post-treatment recovery. (a), A49910 DMSO-treated control and 5 days of TMZ-treated cells; (b), M45481 DMSO-treated control and 5 days of TMZ-treated cells; (c), A49910 DMSO-treated control and 28 days of post-TMZ-treated cells; (d), M45481 DMSO-treated control and 28 days of post-TMZ-treated cells. (C), flow cytometry analysis of apoptosis using annexin V and propidium iodide staining. (a), DMSO-treated A49910 cells, (b), 5 days of TMZ-treated A49910 cells, (c), DMSO treated M45481 cells and (d), 5 days of TMZ-treated M45481 cells. (D) Tracking cell division by CFSE staining. (a), growth arrest of TMZ-treated A49910 cells at day 5 of treatment (yellow line) compared to corresponding DMSO-treated control (blue line). The TMZ-treated A49910 cells remained arrested till day 28 post-treatment recovery (pink line) while the DMSO-treated control cells proliferated (green line). (b), Growth arrest in TMZ-treated M45481 cells at day 5 of treatment (yellow line) compared to corresponding DMSO-treated controls (blue line). At day 28 post-treatment recovery one subpopulation of TMZ-treated M45481 cells showed growth arrest while other subpopulation proliferated (pink line) showing a staining intensity almost similar to their corresponding DMSO-treated control cells (green line).
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f3: (A) Light microscopic images (200 μm bar) and viable cell count of the neurospheres following TMZ treatment and post-treatment recovery. (a), DMSO treated control and (b), 5 days TMZ-treated neurospheres in A49910. (c), % viable cells in A49910 with DMSO- and 5 days of TMZ- treatment. (d), DMSO-treated control and (e), 5 days TMZ-treated neurospheres in M45481. (f), % viable cells in M45481 with DMSO- and 5 days of TMZ- treatment. (g), DMSO-treated control and (h), 28 days of post-TMZ-treated neurospheres in A49910. (i), % viable cells of DMSO-treated controls and 28 days of post-TMZ-treated cells in A49910. (j), DMSO treated control and (k), 28 days post-TMZ-treated neurospheres in M45481. (l), % viable cells of DMSO-treated controls and 28 days of post-TMZ-treated cells in M45481. (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001). (B) Growth curves (MTS assay) following 5 days of TMZ treatment and 28 days of post-treatment recovery. (a), A49910 DMSO-treated control and 5 days of TMZ-treated cells; (b), M45481 DMSO-treated control and 5 days of TMZ-treated cells; (c), A49910 DMSO-treated control and 28 days of post-TMZ-treated cells; (d), M45481 DMSO-treated control and 28 days of post-TMZ-treated cells. (C), flow cytometry analysis of apoptosis using annexin V and propidium iodide staining. (a), DMSO-treated A49910 cells, (b), 5 days of TMZ-treated A49910 cells, (c), DMSO treated M45481 cells and (d), 5 days of TMZ-treated M45481 cells. (D) Tracking cell division by CFSE staining. (a), growth arrest of TMZ-treated A49910 cells at day 5 of treatment (yellow line) compared to corresponding DMSO-treated control (blue line). The TMZ-treated A49910 cells remained arrested till day 28 post-treatment recovery (pink line) while the DMSO-treated control cells proliferated (green line). (b), Growth arrest in TMZ-treated M45481 cells at day 5 of treatment (yellow line) compared to corresponding DMSO-treated controls (blue line). At day 28 post-treatment recovery one subpopulation of TMZ-treated M45481 cells showed growth arrest while other subpopulation proliferated (pink line) showing a staining intensity almost similar to their corresponding DMSO-treated control cells (green line).

Mentions: All the in vitro TMZ-treatment experiments were done on the neurospheres A49910 and M45481 between passages 4–16. Results were consistent in the neurospheres irrespective of their cellular passages in vitro. To simulate the 28-day cycle of TMZ chemotherapy in patients we treated all of the isolated neurospheres with the maximum clinically achievable dose of TMZ (50 μM) in vitro for 5 consecutive days and grew them for another 23 days without any drug intervention (post-treatment recovery). After 5 days, in A49910 viable cell count was 54.6 ± 22.9% (p-value 1.067e-05) and in M45481 it was 32.3 ± 6.6% (p-value 9.883e-07) compared to their corresponding DMSO-treated controls (as 100%) as shown in Figure 3A c and f. But after 28th day the viable cell count was 3.5 ± 3.2% in A49910 (p-value1.231e-10) and 52.1 ± 14.6% in M45481 (p-value 0.001117) suggesting sustained effect of the drug only in A49910, but not in M45481 (Fig 3A i and l). TMZ-treatment experiments in vitro were repeated 6 times on A49910 and 3 times on M45481 with technical replicates. Error bars were generated and statistical significances (p-values) were calculated including all the independently repeated experiments as biological replicates.


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) Light microscopic images (200 μm bar) and viable cell count of the neurospheres following TMZ treatment and post-treatment recovery. (a), DMSO treated control and (b), 5 days TMZ-treated neurospheres in A49910. (c), % viable cells in A49910 with DMSO- and 5 days of TMZ- treatment. (d), DMSO-treated control and (e), 5 days TMZ-treated neurospheres in M45481. (f), % viable cells in M45481 with DMSO- and 5 days of TMZ- treatment. (g), DMSO-treated control and (h), 28 days of post-TMZ-treated neurospheres in A49910. (i), % viable cells of DMSO-treated controls and 28 days of post-TMZ-treated cells in A49910. (j), DMSO treated control and (k), 28 days post-TMZ-treated neurospheres in M45481. (l), % viable cells of DMSO-treated controls and 28 days of post-TMZ-treated cells in M45481. (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001). (B) Growth curves (MTS assay) following 5 days of TMZ treatment and 28 days of post-treatment recovery. (a), A49910 DMSO-treated control and 5 days of TMZ-treated cells; (b), M45481 DMSO-treated control and 5 days of TMZ-treated cells; (c), A49910 DMSO-treated control and 28 days of post-TMZ-treated cells; (d), M45481 DMSO-treated control and 28 days of post-TMZ-treated cells. (C), flow cytometry analysis of apoptosis using annexin V and propidium iodide staining. (a), DMSO-treated A49910 cells, (b), 5 days of TMZ-treated A49910 cells, (c), DMSO treated M45481 cells and (d), 5 days of TMZ-treated M45481 cells. (D) Tracking cell division by CFSE staining. (a), growth arrest of TMZ-treated A49910 cells at day 5 of treatment (yellow line) compared to corresponding DMSO-treated control (blue line). The TMZ-treated A49910 cells remained arrested till day 28 post-treatment recovery (pink line) while the DMSO-treated control cells proliferated (green line). (b), Growth arrest in TMZ-treated M45481 cells at day 5 of treatment (yellow line) compared to corresponding DMSO-treated controls (blue line). At day 28 post-treatment recovery one subpopulation of TMZ-treated M45481 cells showed growth arrest while other subpopulation proliferated (pink line) showing a staining intensity almost similar to their corresponding DMSO-treated control cells (green line).
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f3: (A) Light microscopic images (200 μm bar) and viable cell count of the neurospheres following TMZ treatment and post-treatment recovery. (a), DMSO treated control and (b), 5 days TMZ-treated neurospheres in A49910. (c), % viable cells in A49910 with DMSO- and 5 days of TMZ- treatment. (d), DMSO-treated control and (e), 5 days TMZ-treated neurospheres in M45481. (f), % viable cells in M45481 with DMSO- and 5 days of TMZ- treatment. (g), DMSO-treated control and (h), 28 days of post-TMZ-treated neurospheres in A49910. (i), % viable cells of DMSO-treated controls and 28 days of post-TMZ-treated cells in A49910. (j), DMSO treated control and (k), 28 days post-TMZ-treated neurospheres in M45481. (l), % viable cells of DMSO-treated controls and 28 days of post-TMZ-treated cells in M45481. (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001). (B) Growth curves (MTS assay) following 5 days of TMZ treatment and 28 days of post-treatment recovery. (a), A49910 DMSO-treated control and 5 days of TMZ-treated cells; (b), M45481 DMSO-treated control and 5 days of TMZ-treated cells; (c), A49910 DMSO-treated control and 28 days of post-TMZ-treated cells; (d), M45481 DMSO-treated control and 28 days of post-TMZ-treated cells. (C), flow cytometry analysis of apoptosis using annexin V and propidium iodide staining. (a), DMSO-treated A49910 cells, (b), 5 days of TMZ-treated A49910 cells, (c), DMSO treated M45481 cells and (d), 5 days of TMZ-treated M45481 cells. (D) Tracking cell division by CFSE staining. (a), growth arrest of TMZ-treated A49910 cells at day 5 of treatment (yellow line) compared to corresponding DMSO-treated control (blue line). The TMZ-treated A49910 cells remained arrested till day 28 post-treatment recovery (pink line) while the DMSO-treated control cells proliferated (green line). (b), Growth arrest in TMZ-treated M45481 cells at day 5 of treatment (yellow line) compared to corresponding DMSO-treated controls (blue line). At day 28 post-treatment recovery one subpopulation of TMZ-treated M45481 cells showed growth arrest while other subpopulation proliferated (pink line) showing a staining intensity almost similar to their corresponding DMSO-treated control cells (green line).
Mentions: All the in vitro TMZ-treatment experiments were done on the neurospheres A49910 and M45481 between passages 4–16. Results were consistent in the neurospheres irrespective of their cellular passages in vitro. To simulate the 28-day cycle of TMZ chemotherapy in patients we treated all of the isolated neurospheres with the maximum clinically achievable dose of TMZ (50 μM) in vitro for 5 consecutive days and grew them for another 23 days without any drug intervention (post-treatment recovery). After 5 days, in A49910 viable cell count was 54.6 ± 22.9% (p-value 1.067e-05) and in M45481 it was 32.3 ± 6.6% (p-value 9.883e-07) compared to their corresponding DMSO-treated controls (as 100%) as shown in Figure 3A c and f. But after 28th day the viable cell count was 3.5 ± 3.2% in A49910 (p-value1.231e-10) and 52.1 ± 14.6% in M45481 (p-value 0.001117) suggesting sustained effect of the drug only in A49910, but not in M45481 (Fig 3A i and l). TMZ-treatment experiments in vitro were repeated 6 times on A49910 and 3 times on M45481 with technical replicates. Error bars were generated and statistical significances (p-values) were calculated including all the independently repeated experiments as biological replicates.

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