Effect on tumor cells of selective inhibitorof ER stress response

Sakae Saito1, Hae-Ryong Park2, Kazuo Shin-ya2,Takashi Tsuruo1, and Akihiro Tomida1
1Cancer Chemother. Ctr., Jpn. Fdn. Cancer Res.
2Inst. Mol. Cell. Biosci., Univ. Tokyo1

Introduction
In poorly vascularized solid tumors, cancer cells are exposed to microenvironmental stresses, such as glucose deprivation. Glucose deprivation
disrupt protein folding in the endoplasmic reticulum (ER), and activate theunfolded protein response (UPR) which influences cell survival duringmicroenvironmental stress. Therefore, the UPR to the unique microenvironmental conditions would be exploited for the selective killing ofsolid tumor cells.
Here, we demonstrate that VST, a novel compound, disrupts the network ofUPR transcriptional activation during glucose deprivation. Consequently,
VST exhibits a striking selective cytotoxicity towards glucose-starved tumorcells. We also show the in vivo potential of the antitumor activity of VST.
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Chemical: Versipelostatin (VST)
H3C
HOO

H3C
O
H3COCH3O
H3C

OHOOHO

CH3O
H3C

Park, et. al. J Natl Cancer Inst. (2004)
96:1300-10.

OO
CH3 O
CH3

Molecular formular: C61H94O17

FAB-MS: 1098

H3CHOHO
Molecular Weight: 1099.39

CH3

H3CO
H3COH
3

Result 1.
Glucose-dependent effect of VST on cell viability.
Under normal growth conditions, 24hours of VST treatment of HT-29
cells had only a weak effect on cellviability, with approximately 30 M
VST required to inhibit colony
formation

by50%(IC50).

By

contrast, VST was highly toxic incells exposed to glucose free or 2DGcontaining medium, resulting inapproximately 30-fold lower IC50 (1M). Under hypoglycemic conditions,
Colony formation analysis of HT-29 cells after a 24-hour VST
treatment under normal growth (control) or stress conditions, asindicated. 2DG = 2-deoxyglucose (20 mM); TM = tunicamycin (5μg/ml). Data (mean values with 95% confidence intervals in
triplicate determinations) are representative of at least two
independent experiments.

the cytotoxic activity of VST wasassociated with the inhibition ofGRP expression (Result 1, 2). By
contrast, there was no consistent
combined effect of VST with thechemical stressor TM.

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Response to unfolded protein

199 genes, named “UPR-CS (CellSurvival) signature”, which remarkably
changed their expression by glucosedeprivation. Moreover, it is showedthat about 90% of glucose deprivationHighinducible gene expression (enclosed byyellow line) was inhibited by VST.
Gene Ontology showed that VSTsensitive genes have ER stress-related
GO term, such as “ER”, “protein disulfide isomerase activity”, “unfoldedprotein binding”, or “response toLow

Unfolded protein binding

Using microarray analysis, we selectedExpression Level (Signal Log Ratio)-5-1
015UPR-CS Signature
246 probes (199 genes)

Endoplasmic reticulumStress+VST

HT1080HT1080-2HeLa
HT-29
HT1080 GF
HT1080 2DG
HeLa GF
HeLa 2DG
HeLa GF-2
HT-29 GF
HT-29 GF-2
MKN74 GF
HT1080 GF+VST
HeLa GF+VST
HT-29 GF+VST
MKN74 GF+VST

StressProtein disulfide isomerase activity

Result 2.
Stress-induced expression was widely inhibited by VST.

unfolded protein”.

Microarray analysis of 4 cell lines cultured for 18 hours under normal growth (black) or stress condition (red) as indicated. 2DG = 2deoxyglucose (HT1080, 10 mM; HeLa, 5 mM); GF = glucose free. VST (HT-29, 10 μM; HT1080, 3 μM; HeLa and MKN74, 1 μM) was treatedfor 18 hours under glucose free (blue). cRNA targets were prepared from 5 μg of total RNA, and hybridized to GeneChip Human Genome
U133 Plus 2.0 arrays (Affymetrix). The 246 probes (vertical axis) sorted by cluster analysis displayed with 16 samples (horizontal axis).

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Result 3.
ER stress signaling was repressed by VST.
ERER stressstress sensorsensor
ATF6

PERK

Pathway analysis using KeyMolNet
theERdj4

eIF2a

ATF4

GRP78

SLC7A1XBP1EDEMPDIPDI-P5

GRP94
p58IPK

BclBcl-2

UPR-CSsignatureincludedgenes which acted in ATF4/ATF6/

PDI

CHOP

software (IMMD Inc.) shows that

IRE1

GADD34

Mif1

IRE1 signaling pathways.
Microarray analysis showed that, in
ATF4/ATF6/IRE1 signaling pathways, the induction of expression by
glucose deprivation (red circle) wasextensively repressed by VST.

Armet

… Gene showed >2-fold expression increase by glucose deprivation… Gene showed <2-fold expression change by glucose deprivation… Induced expression… Decreased expression

6Conclusion■ VST shows highly selective cytotoxicity to glucose-deprived tumor cells
that is associated with inhibition of the UPR.

■ VST inhibited expression of the UPR target genes GRP78 and GRP94,
repressed the production of the UPR transcriptional activators XBP1 andATF4 during glucose deprivation, and suppressed the activation ofATF4/ATF6/ IRE1 signaling pathway stimulated by glucose deprivation.

■ VST showed in vivo antitumor activity at well-tolerated doses.

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