ORY-1001 inhibits glioblastoma cell growth by downregulating the Notch/HES1 pathway via suppressing lysine-specific demethylase 1 expression

doi:10.12122/j.issn.1673-4254.2024.08.22

Abstract

Abstract:

Objective To explore the inhibitory effect ORY-1001, a lysine-specific histone demethylase 1 (LSD1) inhibitor, on growth of glioblastoma (GBM) and the underlying mechanism. Methods We analyzed LSD1 expressions in GBM and normal brain tissues based on data from TCGA and HPA databases. Female BALB/c mouse models bearing xenografts derived from U87 cells or cells with lentivirus-mediated LSD1 silencing or Notch overexpression were treated with saline or 400 µg/kg ORY-1001 by gavage every 7 days, and GBM formation and survival time of the mice were recorded. The effect of ORY-1001 on GBM cell viability was assessed, and its effect on LSD1 expression was analyzed with Western blotting. The genes and pathways associated with LSD1 were analyzed using bioinformatics methods. Western blotting and qRT-PCR were used to detect Notch/HES1 pathway expression after LSD1 silencing and ORY-1001 treatment. The impact of ORY-1001 on viability of U87 cells with Notch1 silencing or overexpression was assessed, and the regulatory effects of ORY-1001 on Notch/HES1 pathway were analyzed using chromatin immunoprecipitation assay. Results A high expression of LSD1 in GBM was negatively correlated with patient survival (P<0.001). ORY-1001 and LSD1 silencing obviously reduced tumor burden and prolonged the survival time of GBM-bearing mice. ORY-1001 treatment significantly inhibited the viability and dose-dependently decreased LSD1 expression in GBM cells, and such inhibitory effect of ORY-1001 was attenuated by LSD1 silencing. The Notch pathway enriched the differential genes related to LSD1, and Notch/HES1 pathway expression was significantly down-regulated after LSD1 silencing and ORY-1001 treatment. Notch1 overexpression significantly attenuated the anti-tumor effect of ORY-1001 on GBM. Mechanistically, ORY-1001 disrupted the interaction between LSD1 and the Notch pathway target genes including Notch3, HES1 and CR2. Conclusion ORY-1001 down-regulates the Notch/HES1 pathway by inhibiting LSD1 expression to suppress the growth of GBM in mice.

Key words: Lysine-specific histone demethylase 1, bioinformatics analysis, glioblastoma, Notch/HES1 pathway, cancer treatment

Hongli YANG, Yayun XIANG, Tingting TAN, Yang LEI. ORY-1001 inhibits glioblastoma cell growth by downregulating the Notch/HES1 pathway via suppressing lysine-specific demethylase 1 expression[J]. Journal of Southern Medical University, 2024, 44(8): 1620-1630.

Figures/Tables 5

Tab.1 Sequence of primers used for qRT-PCR

Primer

Sequences 5'-3'

Notch1

Forward

Reverse

CTGAAGAACGGGGCTAACAA

CAGGTTGTACTCGTCCAGCA

HES1

Forward

Reverse

GCAGATGACGGCTGCGCTGA

AAGCGGGTCACCTCGTTCATGC

HES4

Forward

Reverse

GCTCAGCTCAAAACCCTCATC

TTCACCTCCGCCAGACACTCG

HEY1

Forward

Reverse

TGCATACGGC AGGAGGGAAAG

AGTCGAACTCGAAGCGGGTCA

HEY2

Forward

Reverse

AGGCGTCGGGATCGGATAAAT

AAGAGCGTGTGCGTCAAAGTA

GAPDH

Forward

Reverse

GGAGTCCACTGGCGTCTTCACC

GAGGAGTGGGTGTCGCTGTTG

LSD1

Forward

Reverse

AGACGACAGTTCTGGAGGGTA

TCTTGAGAAGTCATCCGGTCA

Tab.2 Primer sequences of Notch target gene promoters used for ChIP

Primer n

Sequences 5'-3'

HES1

-2200

Forward

Reverse

AGG TCA CCC AGA GTC AGG AA

CCA GCG TCT TGT TTG ATG TG

HES1

TSS

Forward

Reverse

CGT GTC TCC TCC TCC CAT T

GAG AGG TAG ACG GGG GAT TC

HES1

+500

Forward

Reverse

TCA ACA CGA CAC CGG ATA AA

TCA GCT GGC TCA GAC TTT CA

HES1

+2600

Forward

Reverse

GGC TTT TGG TGG AAT TTG AA

TCA TGG AGG ATT GGT GAA AAG

Notch3

-5000

Forward

Reverse

TAG CCC CTG GTC AGT CAT TC

GGT GCA TCG TAT CAG GAG GT

Notch3

TSS

Forward

Reverse

TGG CCT CAG TTT CCA GAG TT

CAC ACC CAA CCT CGT GAA C

Notch3

+3000

Forward

Reverse

GTC TCA GCA CAC CCC ATT CT

AAC CAC AAA GCA GGG GAA G

Notch3

+28600

Forward

Reverse

GGG GGC TAA AGA CAC AAA CA

GTT CCT TCT CTC CCC ACT CC

DTX1

-1700

Forward

Reverse

TGT GAA TGA CAT GGC AGA GG

TGA ATC TCC TGC CAG TAC CC

DTX1

+30000

Forward

Reverse

ACA TGC CAG ACA GCA GAA CA

AAC CTT CCA GAC CCT GTG TG

CR2

+5000

Forward

Reverse

GCC GGA AGG ATG TTC TTG TA

CAG GGA AGG CCA TGA AAA TA

CR2

+21000

Forward

Reverse

CCC CAC AGT GCT TAC GAT CT

AAG CCA GGA TTG CAG TCA AC

Fig.1 LSD1 inhibition prolongs the survival of GBM-bearing mice. A: LSD1 mRNA expression in glioblastoma and normal brain tissues. B: Expression of LSD1 in glioblastoma and normal brain tissues (Scale bar=200 μm). C: Kaplan-Meier survival analysis showing negative correlation between LSD1 expression and patient survival. D: ROC analysis of specificity and sensitivity of LSD1 expression for predicting GBM. E: General comparison of subcutaneous tumors in treated group and control group on day 21 after tumor cell engraftment. F: Tumor growth in the treated and control group (n=12). G: Survival curves of treated group and control group. H: Body weight curves of ORY-1001-treated group and control group. I: Cell viability of U87 cells treated with ORY-1001. J: Cell viability of U251 treated with ORY-1001. K: Cell viability of A172 treated with ORY-1001. L: Changes of LSD1 enzyme activity. *P<0.05, **P<0.01, ***P<0.001 vs the control group, vehicle.

Fig.2 Genetic targeting of LSD1 mirrors LSD1 pharmacological inhibition in GBM. A, B: Western blotting of the expression of LSD1 in ORY-1001-treated U87 cells. C, D: Western blotting of H3K4me2 expression in in ORY-1001-treated U87 cells with H3 expression as the internal reference. E: qPCR for detecting relative expression of LSD1 mRNA in sh-LSD1 and sh-NC cells. F: LSD1 expression of sh-LSD1 and sh-NC cells verified by Western blotting. G: Quantification of LSD1 expression. H: Cell viability of sh-LSD1-U87 cells compared with sh-NC-U87 cells. I, J: Cleaved caspase-3 expression upon LSD1 silencing. K: Effect of different ORY-1001 concentrations on sh-LSD1 and sh-NC cells. L: The subcutaneous tumor growth curves of LSD1-silenced group and control group. M: Survival curves of mice injected with LSD1-silenced and control GBM cells. N: Relative expression of LSD1 mRNA in LSD1-silenced group and control group at the time of death. *P<0.05, **P<0.01, ***P<0.001 vs 0 nmol, sh-NC.

Fig.5 ORY-1001 affects the binding of LSD1 to the Notch target gene promoter regions in GBM. ChIP analysis was performed on the chromatin from GBM U87 cells treated with ORY-1001 (16 nmol/L) for 24 h. A: LSD1 binds to the promoter sequence of the Notch target gene (red). B: Changes of LSD1 occupancy in Notch target gene promoter region after ORY-1001treatment. *P<0.05, **P<0.01 vs Vehicle group.

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