antioxidants

Review
Antioxidants and Therapeutic Targets in Ovarian Clear Cell
Carcinoma
Tsukuru Amano 1, * , Atsushi Murakami 1 , Takashi Murakami 1 and Tokuhiro Chano 2, *

1 Department of Obstetrics & Gynecology, Shiga University of Medical Science, Tsukinowa-cho, Seta, Otsu,
Shiga 520-2192, Japan; [email protected] (A.M.); [email protected] (T.M.)
2 Department of Clinical Laboratory Medicine and Medical Genetics, Shiga University of Medical Science,
Tsukinowa-cho, Seta, Otsu, Shiga 520-2192, Japan
* Correspondence: [email protected] (T.A.); [email protected] (T.C.);
Tel.: +81-77-548-2267 (T.A.); +81-77-548-2621 (T.C.)

Abstract: Ovarian clear cell carcinomas (OCCCs) are resistant to conventional anti-cancer drugs;
moreover, the prognoses of advanced or recurrent patients are extremely poor. OCCCs often arise
from endometriosis associated with strong oxidative stress. Of note, the stress involved in OCCCs can
be divided into the following two categories: (a) carcinogenesis from endometriosis to OCCC and (b)
factors related to treatment after carcinogenesis. Antioxidants can reduce the risk of OCCC formation
by quenching reactive oxygen species (ROS); however, the oxidant stress-tolerant properties assist
in the survival of OCCC cells when the malignant transformation has already occurred. Moreover,
the acquisition of oxidative stress resistance is also involved in the cancer stemness of OCCC.
This review summarizes the recent advances in the process and prevention of carcinogenesis, the
characteristic nature of tumors, and the treatment of post-refractory OCCCs, which are highly linked
to oxidative stress. Although therapeutic approaches should still be improved against OCCCs,





 multi-combinatorial treatments including nucleic acid-based drugs directed to the transcriptional
Citation: Amano, T.; Murakami, A.;
profile of each OCCC are expected to improve the outcomes of patients.
Murakami, T.; Chano, T. Antioxidants
and Therapeutic Targets in Ovarian Keywords: ovarian clear cell carcinoma; endometriosis; antioxidant; cancer stemness
Clear Cell Carcinoma. Antioxidants
2021, 10, 187. https://doi.org/
antiox10020187
1. Introduction
Academic Editor: Ovarian cancer is the eighth most common cancer affecting women worldwide, with
Jose Manuel Martinez-Martos
an estimated 295,000 new cases in 2018 [1] and has a high mortality rate. In 2018 alone,
Received: 30 December 2020
approximately 185,000, 14,000, and 4800 deaths were reported worldwide, in the United
Accepted: 24 January 2021
States and Japan due to ovarian cancer, respectively [2,3]. Ovarian cancers can be classified
Published: 28 January 2021
into the following five pathological types: high-grade serous carcinomas (HGSCs), low-
grade serous carcinomas (LGSCs), mucinous carcinomas (MCs), endometrioid carcinomas
Publisher’s Note: MDPI stays neutral
(ECs), and clear cell carcinomas (CCCs). The percentages of each pathological type among
with regard to jurisdictional claims in
all ovarian cancers in the United States and Japan are as follows: serous carcinomas (com-
published maps and institutional affil-
bining HGSCs and LGSCs) 70%/36%, MCs 11%/11%, CCCs 5%/24%, and ECs 10%/17%,
iations.
respectively. CCCs and ECs are much more common and great concerns in Japan and East
Asian nations than in Europe or the United States [4,5]. HGSCs, which account for most
of the serous carcinomas, are generally sensitive to chemotherapy. Furthermore, many
studies have been conducted, providing new information. TP53 mutations are detected in
Copyright: © 2021 by the authors.
the majority of cases, and homologous recombination deficiency (HRD), including BRCA
Licensee MDPI, Basel, Switzerland.
inactivation, have also been reported in approximately 50% of cases [6,7]. In this regard,
This article is an open access article
novel treatments, such as the application of poly ADP-ribose polymerase (PARP) inhibitors,
distributed under the terms and
are being developed for HGSCs. LGSCs and MCs are relatively rare. LGSCs account for
conditions of the Creative Commons
Attribution (CC BY) license (https://
only 5–10% of ovarian serous carcinoma, and the majority of ovarian MCs are considered
creativecommons.org/licenses/by/
metastatic tumors derived from the gastrointestinal tract, while only ≤3% of MCs are
4.0/). regarded to be truly originating from the ovary [8,9].

Antioxidants 2021, 10, 187. https://doi.org/10.3390/antiox10020187 https://www.mdpi.com/journal/antioxidants

Antioxidants 2021, 10, 187 2 of 15

Ovarian ECs and ovarian clear cell carcinomas (OCCCs) are both known as
endometriosis-associated ovarian cancers, and are similar in that they are often asso-
ciated with genetic mutations such as ARID1A and PIC3CA. Differences between these
two are also apparent. ECs display estrogen and progesterone receptors [10] and are not
significantly associated with antioxidant molecules such as hepatocyte nuclear factor 1
homeobox B (HNF1B) or mitochondrial superoxide dismutase (SOD2). The prognosis of
ECs is relatively good, because they are sensitive to chemotherapy [11]. By contrast, OCCCs
rarely express estrogen or progesterone receptors, and often overexpress HNF1B and SOD2.
As overexpression of HNF1B and SOD2 confers resistance to oxidative stress in cancer cells,
OCCCs possess strong resistance to oxidative stress caused by cancer treatments such as
chemotherapy. Therefore, conventional anti-cancer drugs are ineffective against OCCCs,
which often progress to advanced stages, and prognosis is extremely poor [12–14]. OCCC
frequency is especially high in East Asian countries including Japan, and no effective
treatment exists for OCCCs. As such, studies of molecular signatures of this type of cancer,
and establishment of novel treatment methods, are urgently needed.
Among the various pathological types of ovarian cancers, OCCCs are most strongly
linked to oxidative stress tolerance. Oxidative stress causes cancer by directly damaging
DNA. Recently, it has been elucidated that molecular abnormalities involved in oxidative
stress generation are deeply involved not only in carcinogenesis but also in cancer progres-
sion, such as cancer invasion and metastasis. The involvement of oxidative stress in OCCCs
can be divided into the following two categories: (a) carcinogenesis from endometriosis to
OCCC and (b) factors related to treatment after carcinogenesis.
This review focuses on the relationship between OCCCs and oxidative stress, the process
and prevention of carcinogenesis, the nature of tumors, and treatment after carcinogenesis.

2. Linking Oxidative Stress and Carcinogenesis from Endometriosis to OCCCs

A strong association between epithelial ovarian cancer and endometriosis, a common
gynecologic disorder affecting approximately 10% of reproductive-age women [15], has
long been suggested. Among epithelial ovarian cancers, OCCCs are considered the most
closely associated with endometriosis. A pooled analysis of case-control studies indicated
that a self-reported history of endometriosis is associated with a higher increased risk
of OCCCs (odds ratio: 3.05) than the other histologic subtypes of ovarian cancers [16].
Additionally, the coexistent rate of endometriosis in the same ovary in each histologic
subtype of ovarian cancer is reported to be 35.9% in OCCCs, 19% in ECs, 4.5% in SCs, and
1.4% in MCs [17].
Endometriosis contributes to the carcinogenesis of epithelial ovarian cancers through
chronic inflammation, local hyperestrogenism, and oxidative stress. As represented by
colitis-associated colorectal cancer, chronic inflammation has long been known to cause ma-
lignant transformation of cells and carcinogenesis [18,19]. Several inflammatory mediators,
such as TNF-α, IL-6, and TGF-β, have been shown to participate in both the initiation and
progression of endometriosis-associated ovarian cancers [20]. The abundance of estradiol
in endometriotic lesions is caused by the locally increased expression of aromatase and
steroidogenic acute regulatory protein combined with the decreased expression of 17β-
hydroxysteroid dehydrogenase 2 [21]. These hormonal pathways are thought to be more
closely associated with hormonal receptor-positive ovarian endometrioid carcinomas. On
the other hand, as gene abnormalities associated with oxidative stress response and reactive
oxygen species (ROS) metabolism are often detected [22], oxidative stress is considered
to be most involved in the carcinogenesis of endometriosis into OCCCs. Endometriosis
often results in chocolate (endometriotic) cyst formation, containing old blood with excess
iron in the ovary. Iron and its metabolites contribute to the generation of ROS through the
Fenton reaction, acting as inducers of DNA damage and sugar, lipid, and protein modifica-
tions, leading to carcinogenesis [23,24]. The carcinogenicity of iron compounds has been
clearly demonstrated in previous animal experiments. Interestingly, several studies have
shown that renal clear-cell carcinomas, which often share molecular features with OCCCs,
Antioxidants 2021, 10, 187 3 of 15

were produced by intraperitoneal iron chelate injection [25,26]. Oxidative stress induced
by excess heme production and iron accumulation could also be an important trigger in
malignant transformation of endometriosis to OCCCs [27].

3. Attempts to Prevent OCCCs Developing from Endometriosis

In OCCCs, abnormalities are often found in genes associated with oxidative stress and
ROS metabolism [22]. The elimination of persistent inflammation and ROS is important to
prevent carcinogenesis from endometriosis to OCCC. Surgery is a useful tool to prevent
endometriosis. A nested case-control study in Sweden revealed that compared to controls,
a one-sided oophorectomy or radical extirpation of all visible endometriosis reduced the
risk of later development of ovarian cancer to 19% and 30%, respectively. However, hor-
monal treatments, such as combined oral contraceptives, gestagens (including oral drugs
or levonorgestrel-containing intrauterine devices), danazol, and gonadotropin-releasing
hormone agonists, did not mitigate cancer risk [28]. Several cases of OCCCs arising from
endometrioma during hormonal treatment have also been reported [29]. On the other
hand, several studies reported that oral contraception reduces the risk of ovarian cancer
among women with and without endometriosis by suppressing ovulation [30,31]. Through
the collaborative analysis of data from 45 epidemiological studies, Basel et al. reported
that after 5 years of oral contraceptive use, the risk of OCCC was reduced by 21.3% [31].
However, a few study limitations are attributed to the retrospective and contain data from
areas with a low frequency of OCCCs among the population. Hormonal therapy using low-
dose estrogen-progestin or dienogest (progestin medication) may suppress the progression
of endometriosis to OCCC. A large-scale prospective study by the Japan Endometrioma
Malignant-transformation Study (JEMS) is currently underway in Japan, where OCCCs are
frequent in the population. Moreover, further research is needed to clarify this point.
Since oxidative stress is involved in the formation of OCCCs, antioxidant intake may
be effective in preventing its development. VitaminA (carotenoid), C, E, flavonoids, and
isothiocyanate are known antioxidant supplements [32–35]. Thus, diets rich in vegetables
and fruits, which are good sources of antioxidants, are considered healthy. Antioxidants
may prevent or delay various steps associated with carcinogenesis [36–38]. Carotenoid
astaxanthin reduces oxidative stress, and inflammation [39,40] exerts a highly protective
antioxidant effect [41]. Astaxanthin has been shown to decrease DNA damage and improve
the immune response in healthy women after 8 weeks of intake [42]. In both in vitro and
in vivo experiments, the use of astaxanthin significantly inhibited tumor formation and
growth and exhibited anticancer properties [43–46]. Flavonoids inhibit multiple enzymes
involved in cancer cell growth and arrest the cell cycle and tumor regression by activating
the mitochondrial pathway of apoptosis [47,48]. Isothiocyanate inhibits the growth of
ovarian cancer cells by inducing apoptosis in in vitro experiments [49]. Animal experiments
have demonstrated that isothiocyanate exerts inhibitory effects on the carcinogenesis of
both forestomach and lung cancers induced by the carcinogen benzopyrene [50].
As an epidemiological investigation, the effects of the daily intake of antioxidant
supplements on ovarian cancer were investigated in multiple population-based case-
control and prospective cohort studies, and several meta-analyses have been published.
These studies indicated that the intake of dietary vitamins C, D, E, and isothiocyanate
are not associated with the risk of ovarian cancers [51–53]. On the other hand, a higher
dietary intake of vitamin A, including carotenoids and flavonoids, may lower the risk of
ovarian cancer [54–56] (Table 1). In these studies, the subjects were members of the general
population and did not necessarily have increased levels of oxidative stress. Antioxidant
intake may be more effective in preventing carcinogenesis in people with endometriosis,
as endometriosis treatment reduces the risk of developing ovarian cancers later. Because
OCCC carcinogenesis is particularly affected by oxidative stress, antioxidants may be
effective for its prevention. However, it should be noted that these studies did not consider
the pathological type of ovarian cancer; thus, whether this leads to the prevention of
Antioxidants 2021, 10, 187 4 of 15

OCCCs remains unknown. Furthermore, it must also be taken into account that the intake
of vitamin A and beta-carotene has a negative effect on other carcinomas [54].

Table 1. Antioxidant supplements and foods against ovarian cancers.

Clinical Research Antioxidant Result Reference

Meta-analysis Vitamin A Reduced the risk of ovarian cancer. [52]
Meta-analysis Vitamin C No significant effect on the risk of ovarian cancer. [53]
Systematic review Vitamin D No significant effect on the risk of ovarian cancer. [55]
Meta-analysis Vitamin E No significant effect on the risk of ovarian cancer. [51]
Meta-analysis Flavonoid Reduced the risk of ovarian cancer. [56]
Cohort study Flavonoid Reduced the risk of ovarian cancer. [57]
Isothiocyanate No significant effect on the risk of ovarian cancer.
A recent systematic review, cohort study, and meta-analyses indicated that vitamin A and flavonoid intake might reduce the risk of ovarian
cancers, although vitamins C, D, E, and isothiocyanate have no significant effects on the risks. The preventive effects of flavonoids and
vitamin A on OCCC (ovarian clear cell carcinoma) remain unclarified.

Currently, surgery is suitable to prevent carcinogenesis from chocolate cysts. However,

the effects of hormonal therapies and dietary additives consisting of strong antioxidants,
flavonoids, and isothiocyanates will require further investigation.

4. Molecular Characteristics in OCCCs Related to Anti-Oxidative Pathway

As mentioned above, in OCCCs, which arise from endometriosis under massive ox-
idative stress, abnormalities are often detected in genes associated with the oxidative stress
response and ROS metabolism [22]. Several antioxidant molecules are involved in OCCC
carcinogenesis. Among them, the overexpression of hepatocyte nuclear factor 1 homeobox
B (HNF1B), a major homeobox-containing protein, also known as transcription factor-2, is
highly important. Under hypoxia and acidosis, HNF1B can modify and adapt cancer cells
to survive through a process between gluconeogenesis and glycolysis, commonly known as
the Warburg effect [58]. Tsuchiya et al. [14] first reported HNF1B overexpression in OCCCs
and showed that reduced HNF1B expression considerably increased the apoptosis rate
in two OCCC cell lines. Overexpression of HNF1B was observed in endometrial tissues
adjacent to OCCC tumors, suggesting that HNF1B overexpression is an early event in
OCCC carcinogenesis. Kato et al. [59] found that hypomethylation of the CpG island of
HNF1B induced its overexpression in OCCCs, indicating that overexpression in OCCC
was also caused by epigenetic changes rather than by mutations. Moreover, recent research
has revealed that HNF1B promotes the dedifferentiation of cancer stem-like cells (CSCs)
via activation of the Notch pathway and enhancing the invasive potential and epithelial–
mesenchymal transition in cancer cells [60]. Anti-oxidative pathways are deeply involved
in carcinogenesis and therapeutic resistance in OCCCs. As oxidative stress tolerance repre-
sents therapeutic resistance, OCCCs usually exhibit poor and fatal prognoses, even during
gradual progression. OCCC has low sensitivity to platinum- and taxane-based chemother-
apy. Therefore, the prognosis of OCCCs is extremely poor, particularly in its advanced
stages [61,62]. Previous studies have revealed the role of HNF1B in driving the expres-
sion of several characteristic genes associated with OCCCs [63], stimulating metabolic
changes to promote gluconeogenesis, glycogen accumulation, and aerobic glycolysis [64],
inducing chemotherapeutic resistance by suppressing sulfatase-1 (Sulf-1), an extracellular
sulfatase catalyzing the 6-O desulfation of heparan sulfate glycosaminoglycans [65], and
reducing the activity of immunological checkpoints against tumors. Thus, HNF1B plays an
important role in therapeutic resistance via oxidative stress tolerance in OCCCs (Figure 1).
 in driving the expression of several characteristic genes associated with OCCCs [63], stim-
ulating metabolic changes to promote gluconeogenesis, glycogen accumulation, and aer-
obic glycolysis [64], inducing chemotherapeutic resistance by suppressing sulfatase-1
(Sulf-1), an extracellular sulfatase catalyzing the 6-O desulfation of heparan sulfate gly-
Antioxidants 2021, 10, 187
cosaminoglycans [65], and reducing the activity of immunological checkpoints against tu- 5 of 15
mors. Thus, HNF1B plays an important role in therapeutic resistance via oxidative stress
tolerance in OCCCs (Figure 1).

Figure
Figure 1. Activating 1. Activating
pathways pathwaysproposals
and targeting and targeting proposals
in ovarian clearincell
ovarian clear cell
carcinomas. Incarcinomas.
ovarian clearIncell
ovarian
carcinomas,
clear cell carcinomas, downstream of receptor tyrosine kinases (RTKs), AT-rich interactive domain
downstream of receptor tyrosine kinases (RTKs), AT-rich interactive domain 1A (ARID1A)-related chromatin remodeling
1A (ARID1A)-related chromatin remodeling factors, and genomic instability, including MSI-H, are
factors, and genomic instability, including MSI-H, are activated. These are currently being targeted. However, other
activated. These are currently being targeted. However, other therapeutic strategies, such as nu-
therapeutic strategies, such as nucleic
cleic acid-based drugs, acid-based drugs, RDH10,
RDH10, RECQL1, WRN, and RECQL1,
HNF1B,WRN, shouldand
beHNF1B,
targetedshould be targeted
in the future to in the
future to reducereduce
cancercancer
stemness, induce cancer-specific synthetic lethality, and reduce gluconeogenesis,
stemness, induce cancer-specific synthetic lethality, and reduce gluconeogenesis, together with a
drug repositioning strategy against SOD2 anti-oxidative stress molecules.
together with a drug repositioning strategy against SOD2 anti-oxidative stress molecules.

MitochondrialMitochondrial
superoxidesuperoxide dismutase
dismutase (SOD2) (SOD2)
is an is an antioxidant
antioxidant enzyme that enzyme
metabo-that metabo-
lizes superoxide in mitochondria and plays an important role in maintaining
lizes superoxide in mitochondria and plays an important role in maintaining mitochon- mitochondrial
function
drial function through
through oxidative
oxidative stress stress tolerance.
tolerance. SOD2 is SOD2 is highly
highly expressed
expressed in the ectopic en-
in the ectopic
dometrium compared to normal endometrium, promoting cell proliferation and migration
in ovarian endometriosis [66]. SOD2 is also highly expressed in OCCCs, and its oxida-
tive stress tolerance appears to contribute to carcinogenesis [67,68]. SOD2 overexpression
also promotes tumor growth and metastasis in OCCCs. Hemachandra et al. [67] found
that SOD2 was more highly expressed in OCCCs than in any other epithelial ovarian
cancer subtypes, and its overexpression contributes to tumor growth and metastasis in
a chorioallantoic membrane model. The study also indicated that SOD2 expression was
associated with increased cell proliferation, migration, outgrowth on collagen, spheroid
attachment, and Akt phosphorylation in ES-2 OCCC cells. Therefore, SOD2 is regarded as
a pro-tumorigenic or metastatic factor in OCCCs. Clinical studies have also demonstrated
that high SOD2 expression was observed in 76% (33 out of 41) of OCCCs, and SOD2
overexpression was correlated with poor prognoses for OCCCs [68]. Accordingly, SOD2 is
considered to be involved in therapeutic refraction through oxidative stress resistance in
OCCCs (Figure 1).

5. Oxidative Stress and Cancer Stemness of OCCC

Recently, CSCs resistant to oxidative stress have been associated with the recurrence
and metastasis of malignant tumors [69]. In other words, CSCs can survive severe oxida-
tive stress induced by radiation therapy or chemotherapy and contribute to recurrence
and metastasis. Because OCCC is often refractory to chemotherapy or relapse even after
remission, it is suggested that CSCs are involved in the recurrence or metastasis of OCCC.
High expression levels of aldehyde dehydrogenase 1 (ALDH1), a CSC marker, and Nrf2,
Antioxidants 2021, 10, 187 6 of 15

a key transcriptional factor of the antioxidant system, were both associated with a poor
prognosis in OCCC [70]. Furthermore, our group recently found that retinol dehydroge-
nase 10 (RHD10), enzymes related to vitamin A metabolism and gluconeogenesis, can
reflect cancer stemness through precise analyses of the RAB39A (a member of the RAS
oncogene family)-RXRB (retinoid X receptor beta) axis. The RAB39A-RXRB axis drives can-
cer stemness and tumorigenesis; consequently, the downregulation of this pathway leads
to poor sphere formation and xenotransplantable function in several types of malignan-
cies, such as sarcomas, adrenal, lymphoid, and testicular tumors [71]. On the other hand,
some subpopulations of cancer cells could produce vividly growing spheres regardless
of RAB39A repression. Therefore, under continuous RAB39A repression, we compared
vividly growing cancer spheres to poor ones via RNA-seq transcriptional analysis, whose
original sequencing data have been deposited in DDBJ/EMBL/GenBank (accession no.
DRA010748). As a result, vividly growing cancer spheres were found to be significantly
related to the upregulation of 79 genes (Supplementary Table S1) and of signaling path-
ways on retinoic acid (RA), vitamin A, and carotenoid metabolism, which were listed in
statistically high orders of ranking (Table 2). In the pathway contributing to nuclear RXRB
function (WP716_83589), retinol dehydrogenase 10 (RDH10), which converts retinol to
all-trans RA and is indispensable for RA synthesis as the predominant enzyme [72,73], was
focused on as a member of the 79 genes.

Table 2. Upregulated pathways in vividly growing cancer spheres.

p-Value
Pathway
(RNAseq from Vividly Growing Cancer Spheres)
Hs_Integrated_Cancer_Pathway_WP1971_82939 1.02 × 104
Hs_Intrinsic_Pathway_for_Apoptosis_WP1841_83332 0.002799811
Hs_Signaling_by_Retinoic_Acid_WP3323_83286 0.003082763
Hs_Vitamin_A_and_Carotenoid_Metabolism_WP716_83589 0.003229062
Hs_BMAL1-CLOCK, NPAS2_activates_circadian_gene_expression_WP3355_83343 0.003687094
Hs_Integrated_Breast_Cancer_Pathway_WP1984_82941 0.004416806
Hs_Pre-NOTCH_Expression_and_Processing_WP2786_83418 0.004513508
Hs_Lipid_storage_and_perilipins_in_skeletal_muscle_WP2887_85092 0.011662396
Hs_Uptake_and_function_of_anthrax_toxins_WP3390_83389 0.013593029
Hs_miRNA_Regulation_of_DNA_Damage_Response_WP1530_84694 0.013785134
Pathways were explicitly upregulated in vividly growing cancer spheres, irrespective of continuous RAB39A repression. Note: The
pathway of retinoic acid signaling, namely vitamin A and carotenoid metabolism, is listed in the superior order of the top 10 rankings.

Interestingly, RDH10 is involved in insulin signaling and contributes to gluconeogene-

sis via conversion from retinal to all-trans RA [74]. Gluconeogenesis and carbon hydrate
storage are characteristics of OCCC phenotypes. Irrespective of RAB39A downregulation,
RDH10 overexpression can result in the continuous activation of nuclear RXRB function
and connect to CSC and carbon hydrate storage characteristics. OCCC cell lines specifically
express abundant RDH10, rather than other types of ovarian cancer cells (Figure 2). RA is
known to suppress cancer stemness and tumorigenesis because RA promotes cell differen-
tiation, cell cycle arrest, and apoptosis via the heterodimer of retinoic acid receptor (RAR)
and retinoid X receptor (RXR) [75–77]. In contrast, Schung et al. demonstrated that RA
promotes cell survival in fatty acid-binding protein 5 (FABP5) cells through peroxisome
proliferator-activated receptor beta (PPARβ/δ) [78]. Interestingly, the overexpression of
FABP5 is an unfavorable prognostic marker in renal clear-cell carcinoma, which shows
pathological similarities to OCCC [79]. Furthermore, RDH10 overexpression promotes
tumor cell proliferation and correlates with patient survival time in gliomas [80].
 noma, which shows pathological similarities to OCCC [79]. Furthermore, RDH10 overex-
pression promotes tumor cell proliferation and correlates with patient survival time in
gliomas [80].
Antioxidants 2021, 10, 187
Overall, RDH10 overexpression may imply cancer stemness and tumorigenesis in 7 of 15
OCCC, resulting in a difficult prognosis refractory to existing therapies. RDH10 may serve
as a novel diagnostic and therapeutic target for OCCC.

Figure 2. retinol
Figure 2. Abundant Abundant retinol dehydrogenase
dehydrogenase 10 (RDH10) 10 (RDH10)in
expression expression in ovarian
ovarian clear clear cell carcino-
cell carcinomas. In contrast to SK-OV-3,
mas.cell
ovarian serous In contrast
carcinomato SK-OV-3, ovarian
(A), ovarian clearserous cell carcinoma
cell carcinoma (A), ovarian
cells, OVISE clearTOV-21
(B), and cell carcinoma cells,high levels of
(C) express
OVISE (B), and TOV-21 (C) express high levels of retinol dehydrogenase 10 (RDH10). Scale bars:
retinol dehydrogenase 10 (RDH10). Scale bars: 100 µm.
100 μm.
Overall, RDH10 overexpression may imply cancer stemness and tumorigenesis in
6. Therapeutic OCCC,
Targetsresulting
for OCCCs in a in the Present
difficult prognosis and refractory
Future to existing therapies. RDH10 may serve
As mentioned above,diagnostic
as a novel conventionaland standard
therapeutic treatments
target forare less effective for OCCCs
OCCC.
because of their strong tolerance to oxidative stress. Thus, to overcome the therapeutic
6. Therapeutic
difficulties associated Targets
with ovarian for OCCCs
cancers, in the
especially forPresent
OCCCs, and Future
novel therapeutics for
recurrent or refractory cases are urgently
As mentioned needed. At present,
above, conventional standardseveral molecular
treatments are lesstargets
effective for OCCCs
because
have been proposed forofOCCCs,
their strong
whichtolerance to oxidative
are categorized stress.
into the Thus, to
following overcome
groups: path-the therapeutic
ways related difficulties
to receptorassociated
tyrosine with ovarian
kinases cancers,
(RTKs), especially
AT-rich for OCCCs,
interactive domainnovel1A therapeutics for
(ARID1A)-related chromatin remodeling factors, and molecules associated with immune targets have
recurrent or refractory cases are urgently needed. At present, several molecular
checkpoints. Somebeenclinical
proposedtrialsfor OCCCs,
have alreadywhich
beenare categorized
completed or areinto the following
currently conducting,groups: pathways
related to receptor
and we have summarized them (Tabletyrosine
3). kinases (RTKs), AT-rich interactive domain 1A (ARID1A)-
related
RTK receptors arechromatin
located onremodeling factors,
the cell surface andandplaymolecules associated
an important role inwith immune checkpoints.
regulating
Some
cell proliferation, clinical trialssurvival,
differentiation, have already been completed
metabolism, or are currently
and migration. Both theconducting,
phospho- and we have
summarized them (Table
inositide 3-kinase/AKT/mammalian 3). of rapamycin (PI3K/AKT/mTOR) and epider-
target
RTK receptors are
mal growth factor/Ras/mitogen-activated protein located on kinase
the cell(EGF/Ras/MAPK)
surface and playpathways
an importantare role in regu-
lating cell proliferation, differentiation, survival,
downstream pathways of RTKs. Mutations in phosphatidylinositol-4,5-bisphosphate 3- metabolism, and migration. Both the
phosphoinositide 3-kinase/AKT/mammalian target
kinase catalytic subunit alpha (PIK3CA), the loss of phosphatase and tensin homolog of rapamycin (PI3K/AKT/mTOR)
and epidermal
(PTEN), the amplification growthepidermal
of human factor/Ras/mitogen-activated
growth factor receptorprotein kinase
2 (HER2), (EGF/Ras/MAPK)
overex-
pathways are downstream pathways of RTKs.
pression of MET (also known as hepatocyte growth factor receptor; HGFR), and ADP- Mutations in phosphatidylinositol-4,5-
bisphosphate
ribosylation factor-like 3-kinase catalytic
4C (ARL4C) have beensubunitshown alphato (PIK3CA), the loss
activate these of phosphatase
pathways in and tensin
homolog (PTEN), the amplification of human epidermal
OCCCs [10,81–83]. In several studies, the inhibition of these molecules has shown the po- growth factor receptor 2 (HER2),
overexpression of MET (also known as hepatocyte
tential to suppress OCCCs. For example, MET inhibitors significantly decreased the pro-growth factor receptor; HGFR), and
ADP-ribosylation factor-like 4C (ARL4C) have been
liferation and increased the apoptosis of OCCC cells in vitro, and suppressed tumor shown to activate these pathways in
OCCCs [10,81–83]. In several studies, the inhibition
growth in xenograft models of OCCC in vivo [82]. Despite its effectiveness in vitro, no of these molecules has shown the
potential to suppress OCCCs. For example, MET inhibitors significantly decreased the
clinical advantages have been observed for inhibitors of downstream pathways of RTKs
proliferation and increased the apoptosis of OCCC cells in vitro, and suppressed tumor
in treating OCCCs. The MET inhibitor cabozantinib was clinically ineffective in treating
growth in xenograft models of OCCC in vivo [82]. Despite its effectiveness in vitro, no
13 patients with recurrent OCCCs [84]. The combination of temsirolimus and carboplatin
clinical advantages have been observed for inhibitors of downstream pathways of RTKs
or paclitaxel was also investigated in patients with advanced OCCCs. However, com-
in treating OCCCs. The MET inhibitor cabozantinib was clinically ineffective in treating
pared to conventional treatments, this regimen did not significantly increase the rate of
13 patients with recurrent OCCCs [84]. The combination of temsirolimus and carboplatin
progression-free survival [85]. Sunitinib, another RTK targeting inhibitor of VEGF and
or paclitaxel was also investigated in patients with advanced OCCCs. However, com-
PDGF signaling, demonstrated minimal activity in second- and third-line treatments of
pared to conventional treatments, this regimen did not significantly increase the rate of
persistent or recurrent OCCCs [86]. Thus far, no RTK inhibitors have demonstrated effi-
progression-free survival [85]. Sunitinib, another RTK targeting inhibitor of VEGF and
cacy against OCCC in clinical trials. Further studies are needed to identify more effective
PDGF signaling, demonstrated minimal activity in second- and third-line treatments of
persistent or recurrent OCCCs [86]. Thus far, no RTK inhibitors have demonstrated efficacy
against OCCC in clinical trials. Further studies are needed to identify more effective drugs
combined with PI3K/AKT/mTOR inhibitors and the mutations associated with OCCC
that can be targeted by PI3K/AKT/mTOR inhibitors.
Antioxidants 2021, 10, 187 8 of 15

Table 3. Molecular targeting drugs and the clinical trials to treat OCCCs (ovarian clear cell carcinomas).

Category Target Molecules Clinical Research Result Reference

MET already completed minimal activity [84]
PI3K/AKT/mTOR already completed clinically ineffective [85]
RTKs and related molecules
VEGFR/PDGFR already completed minimal activity [86]
VEGFR/PDGFR/FGFR Currently conducting — NCT02866370
ARID1A chromatin remodeling factor EZH/glutathione — — [87–89]
PD-L1 Currently conducting — NCT03405454
Immune checkpoint proteins PD-1/CTLA-4 Currently conducting — NCT03355976
TIM-1 Currently conducting — NCT02837991
Clinical trials have been completed for several inhibitors of RTK-related pathways, but no significant effect against OCCCs has been found.
Multiple clinical trials are currently underway to evaluate immune checkpoint inhibitors in OCCC.

ARID1A chromatin remodeling abnormalities are also useful therapeutic targets

for OCCC [13]. The ARID1A gene encodes BAF250a, a subunit of the switch/sucrose
non-fermentable (SWI/SNF) chromatin remodeling complex modifies the structure of
chromatin via histone octamer ejection, octamer sliding, or local chromatin unwrapping
to allow for the binding of other transcription factors [13]. Additionally, mutations in
ARID1A contribute to AKT phosphorylation and induce PI3K pathway activation [90]. In
ARID1A-mutated OCCCs, inhibition of the enhancer of zeste homolog 2 (EZH2) histone
methyltransferase activity could induce synthetic lethality, including the suppression of
PI3K/AKT signaling [87]. Additionally, Berns et al. [88] recently found that small-molecule
inhibitors of the bromodomain and extra-terminal domain (BET) family of proteins inhibit
the proliferation of ARID1A-mutated OCCC cells by reducing the expression of multiple
SWI/SNF members in vitro and in vivo. Furthermore, recent research has demonstrated
that ARID1A-deficient cancer cells have low levels of glutathione due to the decreased
expression of SLC7A11 and are specifically vulnerable to the inhibition of the antioxidant
glutathione and glutamate-cysteine ligase synthetase catalytic subunit, a rate-limiting en-
zyme for glutathione synthesis [89]. APR-246, a glutathione inhibitor, can act as an effective
agent to induce synthetic lethality in ARID1A-deficient cancer cells [89]. Thus, EZH2, BET,
and APR-246 are promising new drugs for the treatment of ARID1A-mutated OCCCs.
In recent years, significant progress has been made in targeting therapy to immune
checkpoint proteins in cancers. In particular, immune checkpoint inhibitors (ICIs) by anti-
programmed death receptor-1/programmed death-ligand 1 (PD-1/PD-L1) antibodies for
high microsatellite instability (MSI-H) and high tumor mutation burden (TMB-H) tumors
are effective in multiple studies [91–94]. The objective response rate and progression-free
survival rate by PD-1 blockade were reported to be 40% and 78% for mismatch repair-
deficient colorectal cancers and 0% and 11% for mismatch repair-proficient colorectal
cancers, respectively [92]. Moreover, immunotherapy with PD-1 blockade for patients
with advanced mismatch repair-deficient cancers across 12 different types achieved a 53%
objective response rate and 21% complete response rate [91]. Furthermore, recent studies
suggest that TMB can also predict the response to PD-1 inhibitors [93] and is associated
with improved survival in patients receiving ICI across various cancer types [94].
As for OCCCs, recent research demonstrated that about 7% (4 out of 57) of OCCC
cases had MSI-H cancers without any MMR mutations [95]. Feinberg reported that despite
the presence of only four (1.6%) MSI-H tumors in 254 OCCC cases, 23 (9.0%) tumors with
high TMB were found [96]. Additionally, it was also revealed that ARID1A alterations
were associated with high TMB levels across cancer types and may cooperate with ICI
treatment [97]. However, the relationship between ARID1A-mutated OCCC and efficacy
of ICI treatment remains unknown. In summary, approximately 10% of OCCCs carry
properties of MSI-H or TMB-H and may benefit from ICIs. For some OCCC patients,
immunotherapy with anti-PD-1/PD-L1 antibodies has a high potential to be used as an
effective treatment strategy. Multiple clinical trials are currently underway to evaluate PD-
Antioxidants 2021, 10, 187 9 of 15

1/PD-L1 inhibitors in OCCCs. In tumor immunotherapy, another therapeutic candidate

is T-cell immunoglobulin and mucin domain protein 1 (TIM-1), which regulates immune
responses on human T cell surfaces. TIM-1, expressed on a high percentage of OCCC cells,
exhausts T cell immunity in cancer microenvironments [98]. Clinical trials of CDX-014, an
anti-TIM-1 antibody covalently linked to the potent cytotoxin, monomethyl auristatin E
(MMAE), are currently being conducted in OCCC patients. Further studies of targeted
therapies for immune checkpoint proteins are eagerly awaited.
As mentioned under the headings “Molecular characteristics in OCCCs related to
anti-oxidative pathway” and “Oxidative stress and cancer stemness of OCCC,” molecules
conferring oxidative stress resistance, including HNF1B, SOD2, and RDH10, are deeply
implicated in therapeutic refractivity. Together with SOD2, HNF1B and RDH10 are ma-
jor potential targets for OCCC treatment. However, molecular inhibitors of the latter
two molecules have not been identified, and difficulties have arisen in development of
chemical agents that inhibit them. In cancer cell subpopulations, especially in CSCs, the
mitochondrial respiratory chain relies heavily on bioenergetic and biosynthetic processes.
Therefore, mitochondrial function can be a target for therapeutic strategies in several can-
cers [86,99–103]. Since SOD2 plays an important role in maintaining mitochondrial function
through oxidative stress tolerance, drugs suppressing mitochondrial function should be
effective in treating SOD2-abundant OCCC. Replacement therapy or drug repositioning
using biguanides, agents for treating diabetes mellitus that inhibit complex 1 of the mito-
chondrial respiratory chain may target tumor cell mitochondria and thereby improve the
therapeutic effect on OCCCs [104]. It has been confirmed that metformin, commonly used
as a first-line biguanide treatment for type 2 diabetes, can selectively inhibit mitochondrial
respiratory chain complex 1 in various cancer cell lines [105–108]. As metformin is not
metabolized, but almost all are excreted by the kidney, the half-life of plasma levels is
long. Therefore, metformin accumulates in organs, and tissue concentration is consid-
ered to be higher than that of plasma [107]. Furthermore, many studies have shown that
metformin accumulates at high concentrations in the mitochondria [107,109]. In addition
to mitochondrial inhibition, metformin also demonstrates an anti-tumor effect through
several routes, including immune-mediated, mammalian target of rapamycin (m-TOR),
and AMP-activated protein kinase (AMPK) [110]. The follow-up program post-surgical
resection of OCCC may involve drug repositioning using metformin; however, this will
need to be verified by clinical cohort studies in the future. In addition, Molina et al. recently
discovered IACS-010759, a new clinical-grade small-molecule inhibitor of complex I of the
mitochondrial electron transport chain, which inhibits tumor growth in models of brain
cancer and AML at well-tolerated doses in vivo [103]. SOD2-abundant OCCC may also be
an adequate candidate for this new inhibitor.
Targeting DNA helicases is another therapeutic strategy for MSI-H tumors, which
may be suitable for use in combination with immune checkpoint inhibitors for cancer
chemotherapy. Human DNA helicases RECQL1 and WRN proteins have been reported
as therapeutic targets for several cancers, including ovarian cancers [111,112]. As for
OCCCs, 9 out of 21 (43%) clinical cases showed high levels of RECQL1 expression by
immunohistopathology, and RECQL1-siRNA significantly inhibited the proliferation of
OCCC cell lines [112]. Importantly, recent research revealed that WRN induced double-
stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI-H
cancer models [113]. In other words, WRN was selectively essential for MSI-H cancers, and
the inhibition of WRN-induced synthetic lethality in MSI-H cancer models. These findings
indicate that at least some OCCC patients benefit from RECQL1 or WRN inhibition.
In the future, further therapeutic options will need to be developed to improve
OCCC achievements. In addition to drug repositioning against SOD2 anti-oxidative
stress molecules and targeting RECQL1 and WRN to induce cancer-specific synthetic
lethality, it is advisable to implement nucleic acid-based drugs, such as siRNA and antisense
oligonucleotides, to treat OCCCs effectively and to adapt them to the transcriptional profile
of individual tumor characteristics in the era of precision medicine. Integrative in silico
Antioxidants 2021, 10, 187 10 of 15

approaches have indicated that only 10–15% of human proteins are druggable and that
only 10–15% of human proteins are disease-modifying [114]. Hence, developing chemical
agents that inhibit RDH10, HNF1B, and ARID1A is a challenge. However, considering the
implementation of nucleic acid-based drugs, RDH10, HNF1B, ARID1A, RECQ, and WRN
helicases should be included in future therapeutic targets.

7. Conclusions
In this review, we have briefly summarized recent advances in the process and pre-
vention of carcinogenesis, the characteristic nature of tumors, and the treatment of post-
refractory OCCCs, which are highly linked to oxidative stress. The removal of oxidative
stress suppresses the development of OCCCs in endometriosis. Strong antioxidants, such
as vitamin A, carotenoids, or flavonoids, may help prevent carcinogenesis of OCCCs.
However, the stress tolerance properties of OCCCs induce therapeutic resistance, making
their treatment difficult. Antioxidants display bidirectional effects toward endometriosis
and OCCCs. Elimination of oxidative stress, including by uptake of antioxidants, is highly
effective in preventing progression from endometriosis to OCCCs, but, antioxidants are
not suitable for treatment of established OCCCs, in which oxidative stress tolerance has
accrued, providing therapeutic resistance. In OCCC therapeutics, inhibition of oxidative
stress tolerance molecules is essential. The genetic and biological characteristics of OCCCs
are being gradually evaluated, and the therapeutic effects of various anti-cancer drugs,
molecular targeting drugs, drug repositioning strategies, and immunotherapies are being
verified. Further studies will be needed to identify novel molecular targets, and studies
on precision medicine, combining multiple treatments based on the genetic and molecular
characteristics of individual tumors, will need to be conducted. Since the development
of small molecular inhibitors for some undruggable molecules remains a challenge, it
is essential that therapeutic approaches against OCCCs be improved, and that nucleic
acid-based drugs and multi-combinatorial treatments corresponding to the transcriptional
profile of each tumor be implemented.

Supplementary Materials: The following are available online at https://www.mdpi.com/2076-392

1/10/2/187/s1, Table S1: Upregulated 79 genes in vividly growing cancer spheres.
Author Contributions: T.A.: Writing—Original Draft, A.M.: Investigation, T.M.: Supervision, T.C.:
Writing—Review & Editing, Project administration. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

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