Biochemical and Biophysical Research Communications xxx (2018) 1e7

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Biochemical and Biophysical Research Communications

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Isoprenylcysteine carboxylmethyltransferase regulates ovarian cancer

cell response to chemotherapy and Ras activation
Qiong Liu a, 1, Jun Chen b, 1, Bingbing Fu a, Jie Dai a, Ying Zhao a, Lin Lai b, *
a
Department of Obstetrics and Gynecology, Xiangyang Central Hospital, Hubei University of Arts and Science, Xiangyang, Hubei Province, China
b
The First Department of Oncology, The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture, Enshi, Hubei Province, China

a r t i c l e i n f o a b s t r a c t

Article history: Inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step of
Received 3 May 2018 oncoproteins' prenylation, targets growth and survival of various cancers. In this work, we systematically
Accepted 6 May 2018 studied the expression, functions and molecular signaling of Icmt in ovarian cancer. We show that the
Available online xxx
upregulation of Icmt expression is a common feature in patients with epithelial ovarian cancer regardless
of age and disease stage. In line with the observations in ovarian cancer patients, a panel of epithelial
Keywords:
ovarian cancer cell lines also demonstrates the significant increase on Icmt transcript and protein levels
Ovarian cancer
than normal ovarian epithelial cells. In addition, ovarian cancer cell lines with higher Icmt levels are
Icmt
Ras
more resistant to chemotherapeutic agents. We further show that Icmt inhibition by siRNA or inhibitor
Cysmethynil cysmethynil suppresses growth and induces apoptosis in ovarian cancer cells. Importantly, Icmt inhi-
Cancer therapy bition significantly augments chemotherapeutic agent's efficacy in vitro and in vivo, demonstrating the
translational potential of Icmt inhibition in ovarian cancer. Mechanistically, we show that Ras activation
is a critical effector of Icmt in ovarian cancer cells. Using cell culturing system, mouse model and patient
samples, our work is the first to demonstrate the essential roles of Icmt in ovarian cancer via Ras
signaling, particularly on its response to chemotherapy. Our findings suggest that Icmt inhibition is a
promising therapeutic strategy to overcome chemoresistance in ovarian cancer, in particular, those pa-
tients with high Icmt expression.
© 2018 Elsevier Inc. All rights reserved.

1. Introduction The biological function of several oncoproteins largely depends

Epithelial ovarian cancer is the most lethal gynecological cancer
that affects women worldwide, and its prognosis has not been
changed significantly during several decades [1]. Surgery and
platinum-based chemotherapy are the major treatment for epithelial
ovarian cancer. However, most epithelial ovarian cancer patients are
diagnosed at an advanced stage and develop resistance to chemo-
therapy in a fast manner [2]. Ovarian cancer has high intertumor and
intratumor heterogeneity at the molecular and epigenetic levels [3].
The molecular pathogenesis of epithelial ovarian cancer is not well
elucidated and might include functional loss of tumor suppressors
and activation of oncogenic pathways (eg, RAS and BRAF) [4].
* Corresponding author. The First Department of Oncology, The Central Hospital
of Enshi Tujia and Miao Autonomous Prefecture, No. 158 Wuyang Road, Enshi,
Hubei Province, 445000, China.
E-mail address: lailin75@163.com (L. Lai).
1
These two authors equally contributed to this work and are co-first authors.
on prenylation, a post-translational modification process [5,6].
Isoprenylcysteine carboxylmethyltransferase (Icmt) has garnered
attention in recent years because it is the only enzyme known to
catalyze the carboxymethylation step of prenylation [7]. The
essential roles of Icmt have been increasingly recognized in cancer
transformation, migration, growth and survival [8e12]. Recent
studies demonstrated that Icmt also contributes to chemo-
resistance in cervical cancer [13], regulates mitochondrial respira-
tion, autophagy and cancer cell metabolism [7,14]. Inhibiting Icmt
by either a small molecule inhibitor termed as cysmethynil or
inhibitory RNA effectively targets multiple biological functions of
cancer [9,15].
In this study, we analyzed the expression, function and molec-
ular mechanism of Icmt using ovarian cancer cell models and pa-
tient tissues. We found that upregulation of Icmt expression is a
common feature in ovarian cancer cells and is associated with their
sensitivity to chemotherapy. We further found regulation of che-
moresistance via Ras activation as the predominant role of Icmt in
ovarian cancer.

https://doi.org/10.1016/j.bbrc.2018.05.038
0006-291X/© 2018 Elsevier Inc. All rights reserved.

Please cite this article in press as: Q. Liu, et al., Isoprenylcysteine carboxylmethyltransferase regulates ovarian cancer cell response to
chemotherapy and Ras activation, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.038
2 Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7

2. Materials and methods cysmethynil (Cayman Chemical, US) were reconstituted in DMSO.
Cisplatin (Sigma, US) was reconstituted in water containing 0.9%
2.1. Patient, tissue specimens and immunohistochemistry NaCl and kept in dark. Antibodies for total and phosphor-Raf, -ERK
and -Akt were from Cell Signaling Inc.
This study was approved by the ethics committee of Xiangyang
Central Hospital. Paraffin block of normal (n ¼ 6) and malignant
2.3. Measurement of proliferation and apoptosis
ovarian tissues (n ¼ 15) were obtained from the Department of
Tissue Repository. Paraffin blocks were sectioned and deparaffi-
Cells were plated on 96-well-plates (for proliferation assay) or
nised with xylene and hydrated through ethanol into the water.
12-well-plate (for apoptosis assay) for overnight. Cells were treated
After antigen retrieval using 10% citrate acid buffer, immunohisto-
with drugs for 72 h. Cell proliferation activity was evaluated by
chemistry stained were performed using Icmt (cell signaling, US)
using the CellTiter 96 AQueous One Solution Cell Proliferation assay
antibody and the corresponding secondary antibody conjugated
kit (Promega, US) according to manufacturer's instructions.
with HRP. Sections were finally counterstained with haematoxylin.
Apoptosis was evaluated using Annexin V-FITC and 7-AAD (BD
Quantification of the staining density was performed using Image J.
Pharmingen, US) followed by flow cytometry on a Beckman Coulter
FC500.
2.2. Cell cultures and materials

Human Primary Ovarian Surface Epithelial Cells (HPOVEC) and
Immortalized Human Ovarian Epithelial Cells (IHOEC) were pur-
chased from abm@ Inc. HPOVECs were cultured using Prigrow X
Series Medium (Catalog #: TM4198) and PriGrow I medium
(Catolog#: TM001) supplemented with a final concentration of 10%
fetal bovine serum and 1% Penicillin/streptomycin (Invitrogen, US).
Seven human ovarian cancer cell lines were cultured strictly ac-
cording to ATCC culture methods. Paclitaxel (Sigma, US) and
2.4. Transfection
Plasmid or siRNA were transfected into cells using Dharmafect
Transfection Reagent according to the manufacturer's instructions.
Two independent ICMT siRNAs targeting different region and
control siRNA were purchased from Santa Cruz, US. Control plasmid
(#70539) and the plasmid containing human HRAS Q61L (# 83186)
were from Addgene, US.

Fig. 1. Chemotherapy increases Icmt levels in cervical cancer cells. (A) Representative photos of immunohistochemistry staining for Icmt (brown colour) in normal and malignant
ovarian tissues. (B) Quantification of immunohistochemistry staining of Icmt in normal and malignant ovarian tissues derived from healthy donor (n ¼ 6) or patients with ovarian
epithelial carcinoma (n ¼ 15). Quantification of staining was conducted by using ImageJ software. Icmt mRNA (C) and protein (D) levels are significantly increased in ovarian cancer
than normal ovarian cells. Normal ovarian cells are HPOVEC (Human Primary Ovarian Surface Epithelial Cells) and IHOEC (Immortalized Human Ovarian Epithelial Cells). *, p < 0.05,
compared to normal ovarian tissue #1 or HPOVEC.

Please cite this article in press as: Q. Liu, et al., Isoprenylcysteine carboxylmethyltransferase regulates ovarian cancer cell response to
chemotherapy and Ras activation, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.038
 Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7 3

2.5. Real-time (RT) RCR
The total cellular RNA was isolated with TRIzol Reagent
(Ambion, US). The first-strand complementary DNA was synthe-
sized using iScript cDNA Synthesis Kit (Bio-rad, CA). ICMT transcript
levels were determined using SsoAdvanced SYBR Green Supermix
on CFX96 RT PCR detection system. ICMT primers were obtained
from BioRad. Quantitative analysis was carried out by normalizing
the amount of ICMT DNA to that of b-actin.
2.6. Ras activity assay
Cells were treated with drugs for 24 h. Ras activity was evalu-
ated using Ras activation ELISA Assay Kit (Merck Millipore, US) as
per the manufacturer's protocol.
80%). Tumor size (length x width2 x 0.5) was measured twice per
week. After three weeks of drug treatment, mice were sacrificed.
2.8. Statistical analyses
Data are expressed as the mean and standard deviation (SD).
Statistical analyses of the differences between two groups were
performed using the one-way analysis of variance (ANOVA) and
subsequently by unpaired Student's t-test. P values < 0.05 is
considered statistically significant.
3. Results
3.1. Icmt upregulation is a common feature in ovarian epithelial
carcinoma

2.7. In vivo cervical cancer xenograft
SW626 cells (1  106) were injected subcutaneously into the
flanks of sublethally irradiated (2 Gy) NOD/SCID mice (Shanghai
Laboratory Animal Center). Six days after injection, once tumor size
is at ~100 mm3, mice were treated with cysmethynil (5 mg/kg/d),
cisplatin (1 mg/kg, twice per week), combination of cysmethynil
and cisplatin by oral gavage or vehicle control (DMSO/Saline, 20%/
We firstly investigated the expression level of Icmt using
immunohistochemistry approach in normal ovarian tissues from
six healthy donors and malignant ovarian tissues from fifteen pa-
tients with ovarian epithelial carcinoma. We observed that Icmt
expression level is similar among normal ovarian tissues but
significantly varies among malignant ovarian tissues (Fig. 1A).
Quantification using Image J software demonstrated that Icmt
expression level is significantly increased in 93% (14 out of 15)

Fig. 2. Icmt expression in ovarian cells is associated with their sensitivity to chemotherapeutic agents. Chemotherapeutic agents inhibit significantly more proliferation (A) and
induce significantly more apoptosis (B) in high Icmt-expressing (PA1, Caov3 and SW626) than low Icmt-expressing (SKOV3 and TOV-112D) ovarian cancer cells. ICMT specific siRNA
knockdown augments the anti-proliferation (C) and pro-apoptotic (D) effects of paclitaxel and cisplatin in ovarian cancer cells SKOV3 and SW626. Two independent siRNA targeting
different regions of ICMT were used. Paclitaxel at 10 nM and cisplatin at 1 mM were used. Cells were treated with drugs for 72 h prior to proliferation and apoptosis analysis,
respectively. *p < 0.05, compared to paclitaxel or cisplatin alone.

Please cite this article in press as: Q. Liu, et al., Isoprenylcysteine carboxylmethyltransferase regulates ovarian cancer cell response to
chemotherapy and Ras activation, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.038
4 Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7

malignant ovarian tissues compared to normal counterparts
(Fig. 1B), demonstrating that the upregulation of Icmt is a common
phenomenon in ovarian epithelial carcinoma. We further analyzed
the Icmt expression level together with patients' age and disease
stage. We found that the upregulation of Icmt in malignant ovarian
tissues are regardless of patient age and disease stage (data not
shown). In addition, 20% malignant ovarian tissues (# 2e4) has 2e5
fold increase and 70% malignant ovarian tissues (# 5e15) has 8e12
fold increase, suggesting that the majority of ovarian epithelial
carcinoma expresses the high level of Icmt.
We further demonstrated that both Icmt mRNA and protein are
increased in a panel of ovarian epithelial carcinoma cell lines than
normal ovarian epithelial cells (Fig. 1C and D). Similar to ovarian
epithelial carcinoma tissues, Icmt expression levels also vary in
ovarian epithelial carcinoma cell lines (Fig. 1C and D), indicating
that ovarian cancer cell lines used in our work are representative of
ovarian epithelial carcinoma tissues.
3.2. Icmt expression in ovarian cancer cells is associated with their
sensitivity to chemotherapy
Icmt has been identified to play a role in tumor transformation
and development [16]. We investigated whether Icmt is involved in
ovarian cancer cell response to chemotherapy. We demonstrated
that SKOV3 and TOV-112D which have the lower Icmt expression
level are significantly more sensitive than PA1, Caov3 and SW626
which have the higher Icmt expression level to paclitaxel and
cisplatin treatment (Fig. 2A and B). These suggest that Icmt
expression in ovarian cancer cells is associated with their sensi-
tivity to chemotherapy.
We next depleted Icmt in SKOV3 (a representative cell line
expressing the low level of Icmt) and SW626 (a representative cell
line with expressing the high level of Icmt) using two independent
siRNA and exposed Icmt-depleted cells to paclitaxel and cisplatin.
We found that SKOV3 and SW626 cells responded similarly to Icmt
depletion (Fig. 2C and D). In contrast, SKOV3 cells are significantly
more sensitive than SW626 to paclitaxel and cisplatin treatment.
These results suggest regulation of chemoresistance as the pre-
dominant role of Icmt in ovarian cancer. This is further confirmed
by the results that Icmt depletion significantly augments the effi-
cacy of paclitaxel and cisplatin in SKOV3 and SW626 (Fig. 2C and D).
Notably, almost complete inhibition of proliferation and survival
was achieved by chemotherapeutic agents in Icmt-depleted ovarian
cancer cells.
3.3. Icmt inhibition augments the in vitro and in vivo efficacy of
chemotherapeutic agent in ovarian cancer
Cysmethynil is a specific Icmt inhibitor and has been used in
various studies to inhibit Icmt enzyme activity [9,14]. Using

Fig. 3. Pharmacological inhibition of Icmt sensitizes ovarian cancer cell to chemotherapy. Specific Icmt inhibitor cysmethynil (cys) at 5, 10 and 20 mM significantly inhibits
proliferation (A) and induces apoptosis (B) in SW626 ovarian cancer cells. Cys significantly sensitizes SW626 cells to paclitaxel or cisplatin in inhibiting proliferation (C) and
inducing apoptosis (D). Cys at 10 mM, paclitaxel at 10 nM and cisplatin at 1 mM were used. (E) Combination of cys and cisplatin is superior to cisplatin alone in inhibiting SW626
tumor growth in mice. *p < 0.05, compared to control, paclitaxel or cisplatin alone.

Please cite this article in press as: Q. Liu, et al., Isoprenylcysteine carboxylmethyltransferase regulates ovarian cancer cell response to
chemotherapy and Ras activation, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.038
 Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7 5

cysmethynil at concentrations that inhibit Icmt but not other
related methyltransferases [11], we found that cysmethynil signif-
icantly inhibited proliferation and induced apoptosis of SW626
ovarian cancer cells (Fig. 3A and B). In line with Icmt depletion
using siRNA, the combination of cysmethynil with paclitaxel or
cisplatin also achieved complete inhibition of growth and survival
in ovarian cancer cells (Fig. 3C and D).
We further challenged cysmethynil in ovarian cancer mouse
model using the most chemoresistant cell line SW626. In order to
observe the in vivo combinatory efficacy of cysmethynil and
cisplatin, we used the sublethal concentration of cysmethynil
(5 mg/kg/d) and cisplatin (1 mg/kg, twice per week). We did not
observe weight loss or abnormal appearance of the mice during the
drug treatment period, indicating that the mice tolerate the treat-
ment well. As expected, cysmethynil or cisplatin marginally
inhibited ovarian cancer growth (Fig. 3E). However, the combina-
tion of cysmethynil and cisplatin displayed potent inhibitory effi-
cacy on ovarian cancer growth starting from the day 6 treatment
(Fig. 3E). Our findings clearly demonstrate the synergistic effects of
Icmt inhibition and chemotherapy in ovarian cancer.
3.4. Icmt regulates Ras signaling in ovarian cancer cells
Ras protein carries the CaaX-motif and its activity depends on
post-translation modification prenylation. Since Icmt catalyzes the
last step of prenylation, various studies have demonstrated the
importance of Icmt in Ras function in cancer cells [12,17]. We
therefore analyzed Ras activity and its downstream signaling in
ovarian cancer cells that lack Icmt. We found that Icmt inhibition by
either genetic or pharmacological approach resulted in a significant
reduction in Ras activation in ovarian cancer cells (Fig. 4A and B).
We further found that the active Ras levels correlated well with the
Icmt expression in ovarian cancer cells (Fig. 4C). Importantly,
overexpression of constitutively active form of Ras (Q61L) signifi-
cantly abolished the inhibitory effects of cysmethynil in ovarian
cancer cells (Fig. 4D and E), demonstrating that Ras inhibition is
required for the action of cysmethynil. Consistently, cysmethynil
decreased the level of p-Raf, p-ERK and p-Akt in ovarian cancer
cells (Fig. 4F), which are the essential molecules involved in the
downstream of Ras signaling.
4. Discussion
The proper function of many CaaX proteins implicated in
oncogenesis and tumor progression require processing via the
prenylation pathway. With emerging evidence for the importance
of Icmt in the prenylation, Icmt has again attention as a target in
oncogenesis. Although Icmt has been shown to contribute to cancer
transformation, growth, survival and metastasis in various cancers
[12,18], the role of Icmt in cancer chemoresistance remains to be

Fig. 4. Icmt regulates Ras signaling in ovarian cancer cells. (A) ICMT siRNA knockdown significantly decreases Ras activity in SW626 cells. (B) Cysmethynil at 10 and 20 mM
significantly decreases Ras activity in SW626 cells. (C) The Ras activity level in normal (HPOVEC and IHOEC) and malignant (SKOV3, ES2, TOV-112D, OV90, PA1, Caov-3 and SW626)
ovarian cells. Overexpression of constitutively active Ras (Q61L) significantly reverses cysmethynil's effects in inhibiting proliferation (D) and inducing apoptosis (E). Cells were
processed for cellular assays at 48 h post-transfection. (F) Representative western blots showing the decreased phosphorylation of Raf, ERK and Akt levels in SW626 cells exposed to
cysmethynil. *p < 0.05, compared to HPOVEC, control or p-Vec.

Please cite this article in press as: Q. Liu, et al., Isoprenylcysteine carboxylmethyltransferase regulates ovarian cancer cell response to
chemotherapy and Ras activation, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.038
6 Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
elucidated. One study recently reported that Icmt is involved in
cancer cell response to chemotherapy [13]. They found that Icmt
expression is upregulated in cervical cancer cells after chemo-
therapeutic agents' treatment and inhibition of Icmt significantly
enhances chemosensitivity of cervical cancer cells. In agreement
with this finding, our work also demonstrated the therapeutic
value of inhibiting Icmt to overcome chemoresistance in cancer.
The expression pattern of Icmt in tumor versus normal is largely
unknown. Using a panel of ovarian cancer cell lines and patient
samples, we are the first to show that Icmt expression is upregu-
lated in epithelial ovarian cancer compared to normal counterparts
(Fig. 1). Notably, we observed the varying expression levels of Icmt
in both ovarian cancer cell lines and ovarian cancer tissues from
patients, suggesting the heterogeneity of Icmt expression in ovarian
cancer. Interestingly, the cells with high level of Icmt (eg, SW626)
responses similarly to specific Icmt inhibition compared to the cells
with low level of Icmt (eg, SKOV3) (Fig. 2). In contrast, the cells with
high level of Icmt are significantly more resistant to paclitaxel and
cisplatin compared to the cells with low level of Icmt (Fig. 2A and
B). This finding demonstrates that the predominant role of Icmt in
ovarian cancer is in chemotherapy response rather than cancer
maintenance. Icmt has been shown to play essential roles in Ras- or
Raf-induced tumor initiation and maintenance in in vitro and in vivo
tumorigenesis studies [12,19]. In addition, inactivating Icmt inhibits
tumor growth, survival, angiogenesis and malignant pleural effu-
sion [9,18e20]. In contrast, our work demonstrates the essential
role of Icmt in ovarian cancer chemoresistance. This is also sup-
ported by the recent study that inhibition of Icmt sensitizes com-
mon chemotherapies in cervical cancer [13]. Our work extends the
previous studies on the multiple roles of Icmt in cancer.
We further demonstrated that Icmt inhibition by pharmaco-
logical inhibitor cysmethynil is effective in augmenting chemo-
therapeutic agents' inhibitory effects in ovarian cancer (Fig. 3).
Since cysmethynil was identified as a specific inhibitor of Icmt, the
functional consequence of deactivating of Icmt via cysmethynil has
been extensively investigated and the anti-cancer activities of
cysmethynil has been shown in various cancers, including leuke-
mia, breast, cervical and prostate cancer [11,13e15]. Importantly,
we showed that the combination of cysmethynil and cisplatin at
sublethal dose completely inhibited ovarian cancer growth in
mouse xenograft model (Fig. 3E). Synergism between cysmethynil
with other anticancer agents has been reported in various cancers
[13,15]. Our work together with the previous work suggest that
cysmethynil is a useful addition to the treatment of cancer.
A significant finding of our work, in agreement with the previ-
ous findings, is that Icmt regulates Ras activity and signaling in
ovarian cancer. The Icmt expression pattern correlates well with the
Ras activity (Fig. 4C). In addition, Icmt inhibition decreases Ras
activity and its downstream signaling pathway Ras/Raf/MEK/ERK
(Fig. 4A, B and F). The reverse of the effects of Icmt inhibition by
overexpression of constitutively active Ras confirms Ras as an
effector of Icmt (Fig. 4D and E). The high prevalence of Ras activa-
tion and its causal role in tumorigenesis and progression has been
reported in various cancers, including ovarian cancer [21,22]. In
agreement with this, we also demonstrate the significant increase
on active Ras level in a panel of ovarian cancer cell lines than
normal ovarian epithelial cells (Fig. 4C). However, there has been
limited progress in the development of direct Ras inhibitor [23].
Our work provides preclinical evidence that targeting Ras-driven
processes by inhibiting Icmt is a strategy to circumvent the diffi-
culties in targeting RAS directly.
In conclusion, the expression pattern of Icmt in normal and
malignant ovarian tissues and the functional analysis of Icmt in
ovarian cancer cells and xenograft mouse model suggest that Icmt
might be a selective and effective target to sensitize cancer cells to
Please cite this article in press as: Q. Liu, et al., Isoprenylcysteine carboxylmethyltransferase regulates ovarian cancer cell response to
chemotherapy and Ras activation, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.038
chemotherapy, particularly in those patients with the high
expression level of Icmt. Our work also adds cancer chemo-
resistance to the growing list of Ras-driving processes.
Conflicts of interest
All authors report no conflict of interest.
Acknowledgement
This work was supported by a research grant provided by Nat-
ural Science Foundation of Hubei Province of China (2016CFB598).
Transparency document
Transparency document related to this article can be found
online at https://doi.org/10.1016/j.bbrc.2018.05.038
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Please cite this article in press as: Q. Liu, et al., Isoprenylcysteine carboxylmethyltransferase regulates ovarian cancer cell response to
chemotherapy and Ras activation, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.038