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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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MiR-9 targets PTCH1 and activates SHH signaling and drug transporters(A & B) CCL64, stably transfected with pPTCH-UTR-JM, was transiently transfected with pre-miR-9, non-targeting oligo and/or target protection (TP) oligo. After 48 h, luciferase activities were quantitated and then presented as normalized luciferase ±SD, n = 4; TP: Target Protector (A). Western blots were performed with whole cell extracts for PTCH1 and β-actin. The normalized band densities are shown at the bottom of the images (B). (C) U87 and T98G, stably transfected with pPTCH-UTR-JM or pPTCH-UTR™-JM, were transiently transfected with anti-miR-9 and then treated with 200 μM TMZ or vehicle. After 48 h, luciferase activities were quantitated and the results are presented as mean RLU ±SD, n = 4. (D & E) MiR-9 was ectopically expressed in U87 and T98G cells. Total RNA was isolated and then studied by real time PCR for PTCH1 mRNA (D) or Gli1 (E). (F) Diagram show increased miR-9 in TMZ-treated GBM cells leading to activated SHH signaling. (G) Western blots with whole cell extracts were performed for Gli1, PTCH1 and β-actin using miR-9-transfected or vector-transfected GBM cells. (H–K) U87 and T98G cells were ectopically expressed for miR-9 or transfected with vector alone. Real-time PCR was performed for MDR1 (H) and ABCG2 (I). The values for control vector in the real time were normalized to 1 for fold-change of the miR-9 transfectants, mean±SD, n = 4; flow cytometry for membrane MDR1 (J) and ABCG2 (K). *p < 0.05 vs. Pre-mir-9 and TP, **p < 0.05 vs. pPTCH-UTR-JM. ***p < 0.05 vs. vector alone.
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Figure 3: MiR-9 targets PTCH1 and activates SHH signaling and drug transporters(A & B) CCL64, stably transfected with pPTCH-UTR-JM, was transiently transfected with pre-miR-9, non-targeting oligo and/or target protection (TP) oligo. After 48 h, luciferase activities were quantitated and then presented as normalized luciferase ±SD, n = 4; TP: Target Protector (A). Western blots were performed with whole cell extracts for PTCH1 and β-actin. The normalized band densities are shown at the bottom of the images (B). (C) U87 and T98G, stably transfected with pPTCH-UTR-JM or pPTCH-UTR™-JM, were transiently transfected with anti-miR-9 and then treated with 200 μM TMZ or vehicle. After 48 h, luciferase activities were quantitated and the results are presented as mean RLU ±SD, n = 4. (D & E) MiR-9 was ectopically expressed in U87 and T98G cells. Total RNA was isolated and then studied by real time PCR for PTCH1 mRNA (D) or Gli1 (E). (F) Diagram show increased miR-9 in TMZ-treated GBM cells leading to activated SHH signaling. (G) Western blots with whole cell extracts were performed for Gli1, PTCH1 and β-actin using miR-9-transfected or vector-transfected GBM cells. (H–K) U87 and T98G cells were ectopically expressed for miR-9 or transfected with vector alone. Real-time PCR was performed for MDR1 (H) and ABCG2 (I). The values for control vector in the real time were normalized to 1 for fold-change of the miR-9 transfectants, mean±SD, n = 4; flow cytometry for membrane MDR1 (J) and ABCG2 (K). *p < 0.05 vs. Pre-mir-9 and TP, **p < 0.05 vs. pPTCH-UTR-JM. ***p < 0.05 vs. vector alone.

Mentions: CCL64 cells were stably transfected with a reporter gene vector in which the 3′UTR was inserted downstream of luciferase (pPTCH-UTR-JM). Parallel cultures were transfected with vector alone. The stable transfectants were transiently transfected with pre-miR-9 or a non-targeting oligo (control). Pre-miR-9 significantly (p < 0.05) decreased luciferase as compared to the control (Figure 3A). The specificity of the pre-miR-9 was studied with a target protection oligo (TP). The TP competed with pre-miR-9 for the interacting site on the 3′ UTR of PTCH1. The TP prevented miR-9 to decrease luciferase levels (Figure 3A, hatched bar). Western blots for PTCH1 with whole cell extracts from the reporter gene transfectants indicated a decrease in PTCH1 protein by transfected anti-miR-9 (Figure 3B). This was specific since the effect was reversed with TP (Figure 3B). Together there was consistency between the reporter gene studies and western blot with regards to PTCH1 being a target of miR-9.


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)

MiR-9 targets PTCH1 and activates SHH signaling and drug transporters(A & B) CCL64, stably transfected with pPTCH-UTR-JM, was transiently transfected with pre-miR-9, non-targeting oligo and/or target protection (TP) oligo. After 48 h, luciferase activities were quantitated and then presented as normalized luciferase ±SD, n = 4; TP: Target Protector (A). Western blots were performed with whole cell extracts for PTCH1 and β-actin. The normalized band densities are shown at the bottom of the images (B). (C) U87 and T98G, stably transfected with pPTCH-UTR-JM or pPTCH-UTR™-JM, were transiently transfected with anti-miR-9 and then treated with 200 μM TMZ or vehicle. After 48 h, luciferase activities were quantitated and the results are presented as mean RLU ±SD, n = 4. (D & E) MiR-9 was ectopically expressed in U87 and T98G cells. Total RNA was isolated and then studied by real time PCR for PTCH1 mRNA (D) or Gli1 (E). (F) Diagram show increased miR-9 in TMZ-treated GBM cells leading to activated SHH signaling. (G) Western blots with whole cell extracts were performed for Gli1, PTCH1 and β-actin using miR-9-transfected or vector-transfected GBM cells. (H–K) U87 and T98G cells were ectopically expressed for miR-9 or transfected with vector alone. Real-time PCR was performed for MDR1 (H) and ABCG2 (I). The values for control vector in the real time were normalized to 1 for fold-change of the miR-9 transfectants, mean±SD, n = 4; flow cytometry for membrane MDR1 (J) and ABCG2 (K). *p < 0.05 vs. Pre-mir-9 and TP, **p < 0.05 vs. pPTCH-UTR-JM. ***p < 0.05 vs. vector alone.
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Figure 3: MiR-9 targets PTCH1 and activates SHH signaling and drug transporters(A & B) CCL64, stably transfected with pPTCH-UTR-JM, was transiently transfected with pre-miR-9, non-targeting oligo and/or target protection (TP) oligo. After 48 h, luciferase activities were quantitated and then presented as normalized luciferase ±SD, n = 4; TP: Target Protector (A). Western blots were performed with whole cell extracts for PTCH1 and β-actin. The normalized band densities are shown at the bottom of the images (B). (C) U87 and T98G, stably transfected with pPTCH-UTR-JM or pPTCH-UTR™-JM, were transiently transfected with anti-miR-9 and then treated with 200 μM TMZ or vehicle. After 48 h, luciferase activities were quantitated and the results are presented as mean RLU ±SD, n = 4. (D & E) MiR-9 was ectopically expressed in U87 and T98G cells. Total RNA was isolated and then studied by real time PCR for PTCH1 mRNA (D) or Gli1 (E). (F) Diagram show increased miR-9 in TMZ-treated GBM cells leading to activated SHH signaling. (G) Western blots with whole cell extracts were performed for Gli1, PTCH1 and β-actin using miR-9-transfected or vector-transfected GBM cells. (H–K) U87 and T98G cells were ectopically expressed for miR-9 or transfected with vector alone. Real-time PCR was performed for MDR1 (H) and ABCG2 (I). The values for control vector in the real time were normalized to 1 for fold-change of the miR-9 transfectants, mean±SD, n = 4; flow cytometry for membrane MDR1 (J) and ABCG2 (K). *p < 0.05 vs. Pre-mir-9 and TP, **p < 0.05 vs. pPTCH-UTR-JM. ***p < 0.05 vs. vector alone.
Mentions: CCL64 cells were stably transfected with a reporter gene vector in which the 3′UTR was inserted downstream of luciferase (pPTCH-UTR-JM). Parallel cultures were transfected with vector alone. The stable transfectants were transiently transfected with pre-miR-9 or a non-targeting oligo (control). Pre-miR-9 significantly (p < 0.05) decreased luciferase as compared to the control (Figure 3A). The specificity of the pre-miR-9 was studied with a target protection oligo (TP). The TP competed with pre-miR-9 for the interacting site on the 3′ UTR of PTCH1. The TP prevented miR-9 to decrease luciferase levels (Figure 3A, hatched bar). Western blots for PTCH1 with whole cell extracts from the reporter gene transfectants indicated a decrease in PTCH1 protein by transfected anti-miR-9 (Figure 3B). This was specific since the effect was reversed with TP (Figure 3B). Together there was consistency between the reporter gene studies and western blot with regards to PTCH1 being a target of miR-9.

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