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Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

Munoz JL, Rodriguez-Cruz V, Ramkissoon SH, Ligon KL, Greco SJ, Rameshwar P - Oncotarget (2015)

Bottom Line: We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance.TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein.Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells.

View Article: PubMed Central - PubMed

Affiliation: New Jersey Medical School, Rutgers, Newark, NJ, USA.

ABSTRACT
Glioblastoma Multiforme (GBM), the most common and lethal adult primary tumor of the brain, showed a link between Sonic Hedgehog (SHH) pathway in the resistance to temozolomide (TMZ). PTCH1, the SHH receptor, can tonically represses signaling by endocytosis. We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance. TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein. Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells. Computational studies, real-time PCR, reporter gene studies, western blots, target protector oligos and ectopic expression identified miR-9 as the target of PTCH1 in resistant GBM cells with concomitant activation of SHH signaling. MiR-9 mediated increases in the drug efflux transporters, MDR1 and ABCG2. MiR-9 was increased in the tissues from GBM patients and in an early passage GBM cell line from a patient with recurrent GBM but not from a naïve patient. Pharmacological inhibition of SHH signaling sensitized the GBM cells to TMZ. Taken together, miR-9 targets PTCH1 in GBM cells by a SHH-independent method in GBM cells for TMZ resistance. The identified pathways could lead to new strategies to target GBM with combinations of drugs.

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Related in: MedlinePlus

PTCH1 repression in TMZ-treated GBM cells involved miRNAU87 and T98G cells were treated with 200 μM TMZ. At 72 h, real time PCR was performed for PTCH1 mRNA (A) and western blot for PTCH1 and SHH (precursor (P) and N-terminus (N)) and β-actin. The values for the untreated group were assigned 1 and are represented by an open bar. The changes in the treated cells were presented as fold change over the vehicle-treated cells. (B) U87 and T98G were knocked down for SHH with siRNA and then analyzed for SHH (precursor, P and mature, N) and β-actin by western blot. (C) The knocked down cells in ‘C’ were treated with TMZ. At 72 h, the cells were assessed for viability with Cell Titer Blue. The data are presented as the mean % viability relative to vehicle treated siRNA transfected cells ±SD, n = 4. (D) U87 and T98G were knocked down for Dicer1. Control was transfected with non-targeting oligo. Western blot was performed for Dicer. The membrane was stripped and reprobed for β-actin. (E) The cells in ‘E’ were treated with 200 μM TMZ. At 72 h, western blot was performed for PTCH1. The membrane was stripped and reprobed for β-actin (F) and assessed for viability. (G)   p < 0.05 vs. control and non-targeting oligo.
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Figure 1: PTCH1 repression in TMZ-treated GBM cells involved miRNAU87 and T98G cells were treated with 200 μM TMZ. At 72 h, real time PCR was performed for PTCH1 mRNA (A) and western blot for PTCH1 and SHH (precursor (P) and N-terminus (N)) and β-actin. The values for the untreated group were assigned 1 and are represented by an open bar. The changes in the treated cells were presented as fold change over the vehicle-treated cells. (B) U87 and T98G were knocked down for SHH with siRNA and then analyzed for SHH (precursor, P and mature, N) and β-actin by western blot. (C) The knocked down cells in ‘C’ were treated with TMZ. At 72 h, the cells were assessed for viability with Cell Titer Blue. The data are presented as the mean % viability relative to vehicle treated siRNA transfected cells ±SD, n = 4. (D) U87 and T98G were knocked down for Dicer1. Control was transfected with non-targeting oligo. Western blot was performed for Dicer. The membrane was stripped and reprobed for β-actin. (E) The cells in ‘E’ were treated with 200 μM TMZ. At 72 h, western blot was performed for PTCH1. The membrane was stripped and reprobed for β-actin (F) and assessed for viability. (G)   p < 0.05 vs. control and non-targeting oligo.

Mentions: SHH signaling has been proposed as a target of anti-neoplastic agents, such as TMZ [23, 24]. SHH signaling occurs with decreased PTCH1 [25]. This study investigated if TMZ resistant cells showed a decrease in PTCH1. GBM cells treated for 72 h resulted in ~30% viability. The surviving cells expressed high levels of the MDR1 gene and resisted TMZ [26]. We established TMZ-resistant U87 and T98G cells and then studied them for PTCH mRNA and protein by qPCR and western blot, respectively. Figure 1A show the fold change in PTCH mRNA. The vehicle-treated point for each cell line was normalized to 1. Since a similar normalization was performed for each cell line, one bar is shown to represent both cell lines. The changes in TMZ-treated cells were presented as fold change over the vehicle-treatment. PTCH mRNA was significantly (p < 0.05) increased in the TMZ resistant cells (Figure 1A) whereas its corresponding protein was decreased (Figures 1B, S1). These results suggested that TMZ could induce post-transcriptional regulation of PTCH1.


Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

Munoz JL, Rodriguez-Cruz V, Ramkissoon SH, Ligon KL, Greco SJ, Rameshwar P - Oncotarget (2015)

PTCH1 repression in TMZ-treated GBM cells involved miRNAU87 and T98G cells were treated with 200 μM TMZ. At 72 h, real time PCR was performed for PTCH1 mRNA (A) and western blot for PTCH1 and SHH (precursor (P) and N-terminus (N)) and β-actin. The values for the untreated group were assigned 1 and are represented by an open bar. The changes in the treated cells were presented as fold change over the vehicle-treated cells. (B) U87 and T98G were knocked down for SHH with siRNA and then analyzed for SHH (precursor, P and mature, N) and β-actin by western blot. (C) The knocked down cells in ‘C’ were treated with TMZ. At 72 h, the cells were assessed for viability with Cell Titer Blue. The data are presented as the mean % viability relative to vehicle treated siRNA transfected cells ±SD, n = 4. (D) U87 and T98G were knocked down for Dicer1. Control was transfected with non-targeting oligo. Western blot was performed for Dicer. The membrane was stripped and reprobed for β-actin. (E) The cells in ‘E’ were treated with 200 μM TMZ. At 72 h, western blot was performed for PTCH1. The membrane was stripped and reprobed for β-actin (F) and assessed for viability. (G)   p < 0.05 vs. control and non-targeting oligo.
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: PTCH1 repression in TMZ-treated GBM cells involved miRNAU87 and T98G cells were treated with 200 μM TMZ. At 72 h, real time PCR was performed for PTCH1 mRNA (A) and western blot for PTCH1 and SHH (precursor (P) and N-terminus (N)) and β-actin. The values for the untreated group were assigned 1 and are represented by an open bar. The changes in the treated cells were presented as fold change over the vehicle-treated cells. (B) U87 and T98G were knocked down for SHH with siRNA and then analyzed for SHH (precursor, P and mature, N) and β-actin by western blot. (C) The knocked down cells in ‘C’ were treated with TMZ. At 72 h, the cells were assessed for viability with Cell Titer Blue. The data are presented as the mean % viability relative to vehicle treated siRNA transfected cells ±SD, n = 4. (D) U87 and T98G were knocked down for Dicer1. Control was transfected with non-targeting oligo. Western blot was performed for Dicer. The membrane was stripped and reprobed for β-actin. (E) The cells in ‘E’ were treated with 200 μM TMZ. At 72 h, western blot was performed for PTCH1. The membrane was stripped and reprobed for β-actin (F) and assessed for viability. (G)   p < 0.05 vs. control and non-targeting oligo.
Mentions: SHH signaling has been proposed as a target of anti-neoplastic agents, such as TMZ [23, 24]. SHH signaling occurs with decreased PTCH1 [25]. This study investigated if TMZ resistant cells showed a decrease in PTCH1. GBM cells treated for 72 h resulted in ~30% viability. The surviving cells expressed high levels of the MDR1 gene and resisted TMZ [26]. We established TMZ-resistant U87 and T98G cells and then studied them for PTCH mRNA and protein by qPCR and western blot, respectively. Figure 1A show the fold change in PTCH mRNA. The vehicle-treated point for each cell line was normalized to 1. Since a similar normalization was performed for each cell line, one bar is shown to represent both cell lines. The changes in TMZ-treated cells were presented as fold change over the vehicle-treatment. PTCH mRNA was significantly (p < 0.05) increased in the TMZ resistant cells (Figure 1A) whereas its corresponding protein was decreased (Figures 1B, S1). These results suggested that TMZ could induce post-transcriptional regulation of PTCH1.

Bottom Line: We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance.TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein.Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells.

View Article: PubMed Central - PubMed

Affiliation: New Jersey Medical School, Rutgers, Newark, NJ, USA.

ABSTRACT
Glioblastoma Multiforme (GBM), the most common and lethal adult primary tumor of the brain, showed a link between Sonic Hedgehog (SHH) pathway in the resistance to temozolomide (TMZ). PTCH1, the SHH receptor, can tonically represses signaling by endocytosis. We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance. TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein. Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells. Computational studies, real-time PCR, reporter gene studies, western blots, target protector oligos and ectopic expression identified miR-9 as the target of PTCH1 in resistant GBM cells with concomitant activation of SHH signaling. MiR-9 mediated increases in the drug efflux transporters, MDR1 and ABCG2. MiR-9 was increased in the tissues from GBM patients and in an early passage GBM cell line from a patient with recurrent GBM but not from a naïve patient. Pharmacological inhibition of SHH signaling sensitized the GBM cells to TMZ. Taken together, miR-9 targets PTCH1 in GBM cells by a SHH-independent method in GBM cells for TMZ resistance. The identified pathways could lead to new strategies to target GBM with combinations of drugs.

Show MeSH
Related in: MedlinePlus