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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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Expressions of PTCH1, miR-9-2 and drug transporters in naïve and recurrent GBM cells from patients(A) Real-time PCR was performed for the miRNA, predicted for PTCH1 (Figure 2A). The results are presented as the mean fold change ±SD, n = 4. (B) Real time PCR for miR-9-2 was performed in four independent studies using RNA from BT145 and BT164 cells. The results of BT145 were normalized to 1 and then used to calculate the fold change for the values of BT164, mean±SD. (C) Real time PCR were performed for PTCH1 as for ‘B’ and the results are similarly presented. (D) Whole cell extracts from BT145 and BT164 were studied by western blot for PTCH1, SHH-P (precursor), SHH-N (mature) and β-actin. (E) Real time PCR was performed for MDR1 and ABCG2 mRNA with total RNA from BT145 and BT164. (F & G) Flow cytometry was performed for membrane P-glycoprotein (MDR1) (F) and ABCG2 (G). The right panels show the overlay between the two patients. *p < 0.05 vs. BT145.
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Figure 4: Expressions of PTCH1, miR-9-2 and drug transporters in naïve and recurrent GBM cells from patients(A) Real-time PCR was performed for the miRNA, predicted for PTCH1 (Figure 2A). The results are presented as the mean fold change ±SD, n = 4. (B) Real time PCR for miR-9-2 was performed in four independent studies using RNA from BT145 and BT164 cells. The results of BT145 were normalized to 1 and then used to calculate the fold change for the values of BT164, mean±SD. (C) Real time PCR were performed for PTCH1 as for ‘B’ and the results are similarly presented. (D) Whole cell extracts from BT145 and BT164 were studied by western blot for PTCH1, SHH-P (precursor), SHH-N (mature) and β-actin. (E) Real time PCR was performed for MDR1 and ABCG2 mRNA with total RNA from BT145 and BT164. (F & G) Flow cytometry was performed for membrane P-glycoprotein (MDR1) (F) and ABCG2 (G). The right panels show the overlay between the two patients. *p < 0.05 vs. BT145.

Mentions: Real-time PCR showed increases in miR-9 and miR-16 in the recurrent BT164 cells (Figure 4A). MiR-9*, which is complementary to miR-9, was also upregulated, indicating that the increase in miR-9 was due to transcription and not stability. The increase in miR-9-2 was similar to the data from the two cell lines, U87 and T98G (Figure 2C). Real-time PCR also showed an increase in miR-9-2 in the recurrent BT164 cells (Figure 4B). We did not detect miR-9-1 or miR-9-3.


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)

Expressions of PTCH1, miR-9-2 and drug transporters in naïve and recurrent GBM cells from patients(A) Real-time PCR was performed for the miRNA, predicted for PTCH1 (Figure 2A). The results are presented as the mean fold change ±SD, n = 4. (B) Real time PCR for miR-9-2 was performed in four independent studies using RNA from BT145 and BT164 cells. The results of BT145 were normalized to 1 and then used to calculate the fold change for the values of BT164, mean±SD. (C) Real time PCR were performed for PTCH1 as for ‘B’ and the results are similarly presented. (D) Whole cell extracts from BT145 and BT164 were studied by western blot for PTCH1, SHH-P (precursor), SHH-N (mature) and β-actin. (E) Real time PCR was performed for MDR1 and ABCG2 mRNA with total RNA from BT145 and BT164. (F & G) Flow cytometry was performed for membrane P-glycoprotein (MDR1) (F) and ABCG2 (G). The right panels show the overlay between the two patients. *p < 0.05 vs. BT145.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359226&req=5

Figure 4: Expressions of PTCH1, miR-9-2 and drug transporters in naïve and recurrent GBM cells from patients(A) Real-time PCR was performed for the miRNA, predicted for PTCH1 (Figure 2A). The results are presented as the mean fold change ±SD, n = 4. (B) Real time PCR for miR-9-2 was performed in four independent studies using RNA from BT145 and BT164 cells. The results of BT145 were normalized to 1 and then used to calculate the fold change for the values of BT164, mean±SD. (C) Real time PCR were performed for PTCH1 as for ‘B’ and the results are similarly presented. (D) Whole cell extracts from BT145 and BT164 were studied by western blot for PTCH1, SHH-P (precursor), SHH-N (mature) and β-actin. (E) Real time PCR was performed for MDR1 and ABCG2 mRNA with total RNA from BT145 and BT164. (F & G) Flow cytometry was performed for membrane P-glycoprotein (MDR1) (F) and ABCG2 (G). The right panels show the overlay between the two patients. *p < 0.05 vs. BT145.
Mentions: Real-time PCR showed increases in miR-9 and miR-16 in the recurrent BT164 cells (Figure 4A). MiR-9*, which is complementary to miR-9, was also upregulated, indicating that the increase in miR-9 was due to transcription and not stability. The increase in miR-9-2 was similar to the data from the two cell lines, U87 and T98G (Figure 2C). Real-time PCR also showed an increase in miR-9-2 in the recurrent BT164 cells (Figure 4B). We did not detect miR-9-1 or miR-9-3.

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