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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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Related in: MedlinePlus

(A) Light microscopic images (50 μm bar) at single cellular level. (a), day 5 DMSO-treated control cells of A49910, (b), day 28 DMSO-treated control cells of A49910. (c), day 5 DMSO-treated control cells of M45481, (d), day 28 DMSO-treated control cells of M4548, (e), day 5 TMZ-treated cells of A49910, (f), day 28 post-TMZ-treated cells of A49910, (g), day 5 TMZ-treated cells of M45481, and (h), day 28 post-TMZ-treated cells of M45481. (B) Light microscopic images (50 μm bar) of the cells showing SA-β-Gal staining following TMZ treatment and post-treatment recovery. (a), day 5 DMSO-treated control cells of A49910, (b), day 28 DMSO-treated control cells of A49910. (c), day 5 DMSO-treated control cells of M45481, (d), day 28 DMSO-treated control cells of M4548, (e), day 5 TMZ-treated cells of A49910, (f), day 28 post-TMZ-treated cells of A49910, (g), day 5 TMZ-treated cells of M45481, and (h), day 28 post-TMZ-treated cells of M45481. (C) VAFs showing biphasic trends where the first phase is the comparison of VAFs between C5 to T5 and second phase is the comparison of VAFs between T5 to T28. Red arrows are showing enriching VAFs in upward direction, blue showing downward direction and black showing no significant change. The green and purple horizontal bars on the right side represent the number of genes (N) in each category in A49910 and in M45481 respectively. (D) Sanger sequencing chromatogram showing a G to A transition on STK36 gene (arrow mark) in M45481 (lower panel) but not in A49910 (upper panel). (E) Showing mRNA expression patterns of Hh-pathway component genes, STK36 (a), GLI1 (b), GLI2 (c), GLI3 (d), Hh-pathway target gene SNAI1 (e) and MGMT (f) following TMZ-treatment and post-treatment recovery (C5, DMSO treated control; T5, day-5 TMZ-treated; T28, day-28 post-treatment recovery, ✶p-value < 0.05, ✶✶p-value < 0.01 and ✶✶✶p-value < 0.001).
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f4: (A) Light microscopic images (50 μm bar) at single cellular level. (a), day 5 DMSO-treated control cells of A49910, (b), day 28 DMSO-treated control cells of A49910. (c), day 5 DMSO-treated control cells of M45481, (d), day 28 DMSO-treated control cells of M4548, (e), day 5 TMZ-treated cells of A49910, (f), day 28 post-TMZ-treated cells of A49910, (g), day 5 TMZ-treated cells of M45481, and (h), day 28 post-TMZ-treated cells of M45481. (B) Light microscopic images (50 μm bar) of the cells showing SA-β-Gal staining following TMZ treatment and post-treatment recovery. (a), day 5 DMSO-treated control cells of A49910, (b), day 28 DMSO-treated control cells of A49910. (c), day 5 DMSO-treated control cells of M45481, (d), day 28 DMSO-treated control cells of M4548, (e), day 5 TMZ-treated cells of A49910, (f), day 28 post-TMZ-treated cells of A49910, (g), day 5 TMZ-treated cells of M45481, and (h), day 28 post-TMZ-treated cells of M45481. (C) VAFs showing biphasic trends where the first phase is the comparison of VAFs between C5 to T5 and second phase is the comparison of VAFs between T5 to T28. Red arrows are showing enriching VAFs in upward direction, blue showing downward direction and black showing no significant change. The green and purple horizontal bars on the right side represent the number of genes (N) in each category in A49910 and in M45481 respectively. (D) Sanger sequencing chromatogram showing a G to A transition on STK36 gene (arrow mark) in M45481 (lower panel) but not in A49910 (upper panel). (E) Showing mRNA expression patterns of Hh-pathway component genes, STK36 (a), GLI1 (b), GLI2 (c), GLI3 (d), Hh-pathway target gene SNAI1 (e) and MGMT (f) following TMZ-treatment and post-treatment recovery (C5, DMSO treated control; T5, day-5 TMZ-treated; T28, day-28 post-treatment recovery, ✶p-value < 0.05, ✶✶p-value < 0.01 and ✶✶✶p-value < 0.001).

Mentions: Sustained growth arrest in A49910 is due to induction of cellular senescence as revealed by flattened cellular morphology and SA-β-Gal staining. Morphologies at the single cellular levels as repeatedly observed in all the in vitro TMZ-treatment experiments are represented in Figure 4A. At day 5, qualitatively, there were no morphological differences observed between the TMZ-treated cells of A49910 (Fig. 4A e) and their corresponding DMSO treated cells (Fig. 4A a) but at day 28, large and flattened appearance was visible only in the TMZ-treated cells (Fig. 4A f) but not in the corresponding DMSO-treated cells (Fig. 4A b). Unlike A49910, at day-5, the TMZ-treated cells of M45481 (Fig 4A g) were appearing slightly bigger than their corresponding DMSO-treated control cells (Fig 4A c) but at day 28, both the TMZ-treated (Fig. 4A h) and the DMSO-treated (Fig. 4A d) cells appeared almost similar in sizes. By a qualitative SA-β-Gal colorimetric staining, the TMZ-treated A49910 cells showed intense blue cytoplasmic staining of SA-β-Gal at day 28 post-treatment recovery (Fig. 4B f), suggesting induction of cellular senescence by TMZ-treatment. Some degree of blue cytoplasmic staining was also observed in DMSO-treated control cells of A49910 at day 28, but unlike the TMZ-treated cells, the control cells did not show any big and flat morphology. However, M45481 cells did not show any SA-β-Gal staining, neither after 5 days of TMZ-treatment (Fig. 4B c vs. g) nor after 28 days of post-treatment recovery (Fig. 4B d vs. h).


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 (50 μm bar) at single cellular level. (a), day 5 DMSO-treated control cells of A49910, (b), day 28 DMSO-treated control cells of A49910. (c), day 5 DMSO-treated control cells of M45481, (d), day 28 DMSO-treated control cells of M4548, (e), day 5 TMZ-treated cells of A49910, (f), day 28 post-TMZ-treated cells of A49910, (g), day 5 TMZ-treated cells of M45481, and (h), day 28 post-TMZ-treated cells of M45481. (B) Light microscopic images (50 μm bar) of the cells showing SA-β-Gal staining following TMZ treatment and post-treatment recovery. (a), day 5 DMSO-treated control cells of A49910, (b), day 28 DMSO-treated control cells of A49910. (c), day 5 DMSO-treated control cells of M45481, (d), day 28 DMSO-treated control cells of M4548, (e), day 5 TMZ-treated cells of A49910, (f), day 28 post-TMZ-treated cells of A49910, (g), day 5 TMZ-treated cells of M45481, and (h), day 28 post-TMZ-treated cells of M45481. (C) VAFs showing biphasic trends where the first phase is the comparison of VAFs between C5 to T5 and second phase is the comparison of VAFs between T5 to T28. Red arrows are showing enriching VAFs in upward direction, blue showing downward direction and black showing no significant change. The green and purple horizontal bars on the right side represent the number of genes (N) in each category in A49910 and in M45481 respectively. (D) Sanger sequencing chromatogram showing a G to A transition on STK36 gene (arrow mark) in M45481 (lower panel) but not in A49910 (upper panel). (E) Showing mRNA expression patterns of Hh-pathway component genes, STK36 (a), GLI1 (b), GLI2 (c), GLI3 (d), Hh-pathway target gene SNAI1 (e) and MGMT (f) following TMZ-treatment and post-treatment recovery (C5, DMSO treated control; T5, day-5 TMZ-treated; T28, day-28 post-treatment recovery, ✶p-value < 0.05, ✶✶p-value < 0.01 and ✶✶✶p-value < 0.001).
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Related In: Results  -  Collection

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f4: (A) Light microscopic images (50 μm bar) at single cellular level. (a), day 5 DMSO-treated control cells of A49910, (b), day 28 DMSO-treated control cells of A49910. (c), day 5 DMSO-treated control cells of M45481, (d), day 28 DMSO-treated control cells of M4548, (e), day 5 TMZ-treated cells of A49910, (f), day 28 post-TMZ-treated cells of A49910, (g), day 5 TMZ-treated cells of M45481, and (h), day 28 post-TMZ-treated cells of M45481. (B) Light microscopic images (50 μm bar) of the cells showing SA-β-Gal staining following TMZ treatment and post-treatment recovery. (a), day 5 DMSO-treated control cells of A49910, (b), day 28 DMSO-treated control cells of A49910. (c), day 5 DMSO-treated control cells of M45481, (d), day 28 DMSO-treated control cells of M4548, (e), day 5 TMZ-treated cells of A49910, (f), day 28 post-TMZ-treated cells of A49910, (g), day 5 TMZ-treated cells of M45481, and (h), day 28 post-TMZ-treated cells of M45481. (C) VAFs showing biphasic trends where the first phase is the comparison of VAFs between C5 to T5 and second phase is the comparison of VAFs between T5 to T28. Red arrows are showing enriching VAFs in upward direction, blue showing downward direction and black showing no significant change. The green and purple horizontal bars on the right side represent the number of genes (N) in each category in A49910 and in M45481 respectively. (D) Sanger sequencing chromatogram showing a G to A transition on STK36 gene (arrow mark) in M45481 (lower panel) but not in A49910 (upper panel). (E) Showing mRNA expression patterns of Hh-pathway component genes, STK36 (a), GLI1 (b), GLI2 (c), GLI3 (d), Hh-pathway target gene SNAI1 (e) and MGMT (f) following TMZ-treatment and post-treatment recovery (C5, DMSO treated control; T5, day-5 TMZ-treated; T28, day-28 post-treatment recovery, ✶p-value < 0.05, ✶✶p-value < 0.01 and ✶✶✶p-value < 0.001).
Mentions: Sustained growth arrest in A49910 is due to induction of cellular senescence as revealed by flattened cellular morphology and SA-β-Gal staining. Morphologies at the single cellular levels as repeatedly observed in all the in vitro TMZ-treatment experiments are represented in Figure 4A. At day 5, qualitatively, there were no morphological differences observed between the TMZ-treated cells of A49910 (Fig. 4A e) and their corresponding DMSO treated cells (Fig. 4A a) but at day 28, large and flattened appearance was visible only in the TMZ-treated cells (Fig. 4A f) but not in the corresponding DMSO-treated cells (Fig. 4A b). Unlike A49910, at day-5, the TMZ-treated cells of M45481 (Fig 4A g) were appearing slightly bigger than their corresponding DMSO-treated control cells (Fig 4A c) but at day 28, both the TMZ-treated (Fig. 4A h) and the DMSO-treated (Fig. 4A d) cells appeared almost similar in sizes. By a qualitative SA-β-Gal colorimetric staining, the TMZ-treated A49910 cells showed intense blue cytoplasmic staining of SA-β-Gal at day 28 post-treatment recovery (Fig. 4B f), suggesting induction of cellular senescence by TMZ-treatment. Some degree of blue cytoplasmic staining was also observed in DMSO-treated control cells of A49910 at day 28, but unlike the TMZ-treated cells, the control cells did not show any big and flat morphology. However, M45481 cells did not show any SA-β-Gal staining, neither after 5 days of TMZ-treatment (Fig. 4B c vs. g) nor after 28 days of post-treatment recovery (Fig. 4B d vs. h).

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