Limits...
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.

Show MeSH

Related in: MedlinePlus

Radiological images of responder GBM patient A49910 and MGMT mRNA expressions of 6 GBM patient-derived tumor biopsy tissues and isolated neurospheres.(a), Diagnostic MRI scan of the brain showing a space-occupying lesion (SOL) in insular cortex in A49910. (b), Postoperative CT scan showing gross total resection in A49910. (c), MRI scan image after the 2nd cycle of chemotherapy showing recurrence of the tumor in A49910. (d), Follow-up scan after 6 cycles of chemotherapy showing decrease in the size of the lesion in A49910. qRT-PCR estimation of MGMT mRNA expression relative to GAPDH mRNA expression in (e), GBM tumor biopsies and (f), in corresponding neurospheres isolated from the tumors. (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4300501&req=5

f2: Radiological images of responder GBM patient A49910 and MGMT mRNA expressions of 6 GBM patient-derived tumor biopsy tissues and isolated neurospheres.(a), Diagnostic MRI scan of the brain showing a space-occupying lesion (SOL) in insular cortex in A49910. (b), Postoperative CT scan showing gross total resection in A49910. (c), MRI scan image after the 2nd cycle of chemotherapy showing recurrence of the tumor in A49910. (d), Follow-up scan after 6 cycles of chemotherapy showing decrease in the size of the lesion in A49910. qRT-PCR estimation of MGMT mRNA expression relative to GAPDH mRNA expression in (e), GBM tumor biopsies and (f), in corresponding neurospheres isolated from the tumors. (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001).

Mentions: Clinical responses of the two lead GBM patients, from whom we identified the phenotypes, are briefly described here. The first patient A49910 was a TMZ-responder. Recurrence of the tumor along the periphery of the surgically resected area was revealed in this patient after the 2nd cycle of chemotherapy (Fig. 2c). This mass appeared to decrease in size as observed in the follow-up scans done 5 months later after the completion of all the 6 cycles of TMZ chemotherapy (Fig. 2d). This is suggestive of good response to TMZ chemotherapy. The patient appeared to be doing well and free from clinical progression during follow-up visits for more than 38 months post-surgery. The second patient M45481 died in three months after surgery despite a similar treatment regimen.


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)

Radiological images of responder GBM patient A49910 and MGMT mRNA expressions of 6 GBM patient-derived tumor biopsy tissues and isolated neurospheres.(a), Diagnostic MRI scan of the brain showing a space-occupying lesion (SOL) in insular cortex in A49910. (b), Postoperative CT scan showing gross total resection in A49910. (c), MRI scan image after the 2nd cycle of chemotherapy showing recurrence of the tumor in A49910. (d), Follow-up scan after 6 cycles of chemotherapy showing decrease in the size of the lesion in A49910. qRT-PCR estimation of MGMT mRNA expression relative to GAPDH mRNA expression in (e), GBM tumor biopsies and (f), in corresponding neurospheres isolated from the tumors. (✶ 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

f2: Radiological images of responder GBM patient A49910 and MGMT mRNA expressions of 6 GBM patient-derived tumor biopsy tissues and isolated neurospheres.(a), Diagnostic MRI scan of the brain showing a space-occupying lesion (SOL) in insular cortex in A49910. (b), Postoperative CT scan showing gross total resection in A49910. (c), MRI scan image after the 2nd cycle of chemotherapy showing recurrence of the tumor in A49910. (d), Follow-up scan after 6 cycles of chemotherapy showing decrease in the size of the lesion in A49910. qRT-PCR estimation of MGMT mRNA expression relative to GAPDH mRNA expression in (e), GBM tumor biopsies and (f), in corresponding neurospheres isolated from the tumors. (✶ p-value < 0.05, ✶✶ p-value < 0.01 and ✶✶✶ p-value < 0.001).
Mentions: Clinical responses of the two lead GBM patients, from whom we identified the phenotypes, are briefly described here. The first patient A49910 was a TMZ-responder. Recurrence of the tumor along the periphery of the surgically resected area was revealed in this patient after the 2nd cycle of chemotherapy (Fig. 2c). This mass appeared to decrease in size as observed in the follow-up scans done 5 months later after the completion of all the 6 cycles of TMZ chemotherapy (Fig. 2d). This is suggestive of good response to TMZ chemotherapy. The patient appeared to be doing well and free from clinical progression during follow-up visits for more than 38 months post-surgery. The second patient M45481 died in three months after surgery despite a similar treatment regimen.

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