Introduction
Despite significant improvements in colorectal
cancer (CRC) treatment, especially in countries with long-standing
screening programs (1), CRC is
still the third most frequently diagnosed oncologic disease,
ranking second among causes of cancer-related mortality (2,3). A
further rise in CRC incidence is predicted. The burden of CRC is
attributed to increasing early-onset CRC, ageing of the population
and a lifestyle shift toward a Western diet combined with low
physical activity (3,4).
Interestingly, the age-standardized incidence of CRC
is consistently higher in males compared to females, including CRC
cases with early onset (5). CRC is
more often diagnosed in the proximal colon in women, whereas distal
anatomical CRC sites are detected more often in males (6–8). The
most pronounced difference is observed in the incidence of rectal
cancer, which is in 75% of cases diagnosed in males (2,3,9).
Among the possible reasons for this phenomenon are
an unfavourable diet, obesity, alcohol consumption, smoking and low
physical activity (10). These
factors influence CRC progression differently depending on the sex
of the patient and tumour localisation (11–13).
Although sex hormones have been implicated in the
generation of differences in CRC prevalence between males and
females, their precise role has not been completely elucidated
(7,8,14). In
particular, it is assumed that the most potent oestrogen,
17β-oestradiol (E2), influences CRC progression (8,15,16).
E2 signalling is mediated predominantly by two types
of nuclear receptors, ESR1 and ESR2, and the G protein-coupled
membrane receptor Gper1 (previous symbols GPER, GPR30) (15). Among the ligands of ESR1, ESR2 and
Gper1 are, in addition to oestrogens, tamoxifen, raloxifene,
bisphenols, dioxins, phthalates, quercetin, genistein, resveratrol
and others (17,18). The ligands mentioned above can
usually activate both nuclear and membrane-bound receptors,
although their affinity can differ (19). Gper1 has a lower affinity for E2
than ESR1 and ESR2 (20). Direct
binding of E2 to Gper1 was even questioned recently (21,22).
However, there is substantial in vivo and in vitro
evidence that Gper1 is involved in E2-mediated regulation (23). Recently, the binding of E2 to Gper1
was confirmed, and aldosterone was also proposed as a Gper1 agonist
(24).
Nuclear E2 receptors exert most of their effects via
transcriptional gene regulation. ESR1 and ESR2 activate thousands
of genes overlapping by 30% via a specific DNA sequence called
oestrogen response element (ERE) (25).
Compared to nuclear E2 receptors, Gper1 affects
intracellular processes much faster thanks to the prompt activation
of several intracellular signalling pathways. Signalling mediated
by Gper1 includes an increase in cAMP synthesis and consequent
activation of protein kinase A, Src-like nonreceptor tyrosine
kinase (Src) and sphingosine kinase (SphK). Src and SphK can
contribute to the activation of epidermal growth factor receptors
(EGFR). Gper1 signalling can also lead to activation of protein
kinase C and calcium mobilisation (17). Among Gper1-driven regulatory
pathways also belongs Gper1/HIFα activation of NOTCH and consequent
induction of VEGF transcription (26) and some others (18). Tissue-specific distribution and/or
changes in the tissue-specific distribution of E2 receptors in
disease can influence E2 signalling and facilitate its oncostatic
or tumour suppressor effects (17,18,25).
Epidemiological studies of patients using hormone
response therapy (HRT) implicated protective role of E2 in respect
to CRC incidence. It was shown that oestrogen/progestin HRT is
negatively associated with the risk of CRC (27). Similarly, the incidence of CRC in
postmenopausal women exposed to E2 HRT was lower compared to women
of the same age without HRT (28).
Other studies later strengthened the evidence of E2′s protective
role regarding CRC incidence (15,16,29,30).
It is of interest that patients with high levels of endogenous
circulating E2 (31) and those
exposed to E2 therapy whilst diagnosed with CRC demonstrated a
worse disease prognosis (32). On
the other hand, Mori et al (33) did not confirm the association of
high circulating E2 levels with increased incidence and/or
prognosis of CRC in postmenopausal women.
Most of the beneficial effects of E2 for patients
are attributed to ESR2 (15,34–37),
which is far more abundant in the gastrointestinal tract compared
to ESR1 (38–40). The beneficial E2 effect mediated via
ESR2 receptors was proven with the use of several in vivo
experimental models. Apcmin/+mice bearing a mutation in
the Apc gene are prone to developing multiple intestinal
tumours. Ovariectomy (OVX) significantly increased the number of
adenomas detected in the gut compared to control, and this effect
was reversed by E2 administration. E2 treatment was also
accompanied by an increase in ESR2 expression in the intestine
(41). ESR2 deficiency in
Apcmin/+ mice was associated with a higher adenoma
number compared to control (42).
The protective role of E2 executed via ESR2 was also demonstrated
in OVX mice where tumorigenesis was induced by azoxymethane (AOM)
(43), by combined treatment of AOM
and dextran sulphate sodium (44,45)
and in immunodeficient mice implanted with SW480 cells
overexpressing ESR2 (46).
Interestingly, the expression of Gper1 mRNA exceeds
that of ESR2 in healthy gut tissue (40,47).
Similarly, it is the predominant E2 receptor in several colorectal
cell lines (48). Despite that, the
role of the membrane receptor Gper1 in E2-mediated effects on
cancer progression has not been completely elucidated.
The tumour suppressor as well as the oncogenic role
of Gper1 has been demonstrated for several types of tissues.
Perhaps the most promising results were achieved in melanoma
treatment. However, in most cancer types, including CRC, the role
of Gper1 remains inconclusive (17,49,50).
It was shown that G-1, an agonist of Gper1, inhibits
the upregulation of JUN oncogene expression in HT29 cells (48). G-1 administered at a concentration
of 1 µM significantly inhibited the proliferation of HCT-116 and
SW480 cells, increased the number of cells in the G2/M phase and
stimulated apoptosis. The decrease in cell viability induced by G-1
was prevented by the administration of the ROS scavenger NAC.
Growth of tumour xenografts in nude mice was inhibited by G-1
administration (51).
On the other hand, Gper1 silencing prevented
chromosomal instability induced by diethylstilbestrol and the
occurrence of supernumerary centrosomes in HCT116 and control CCD
841 CoN cell lines. In these cell lines, Gper1 activation led to
the presence of multipolar mitotic spindles, an increased number of
cells with lagging chromosomes and aneuploidy. Interestingly,
manipulation of Gper1 levels did not influence the rate of
proliferation and cell cycle distribution under in vitro
conditions (52). The oncogenic
role of Gper1 was confirmed in HT-29 cells as Gper1 silencing
prevented an E2-induced decrease in ATM. Under normoxic conditions,
Gper1 activation caused down regulation of VEGF while the opposite
effect was observed in a hypoxic microenvironment and prevented by
Gper1 silencing. Under hypoxic conditions, Gper1 mediated an
E2-induced increase in HT-29 and DLD1 cell migration (53). E2 and its agonist G-1, via Gper1,
induced the expression of fatty acid synthase (FASN), which can
promote CRC progression. FASN upregulation is executed by the
epidermal growth factor receptor EGFR/ERK/AP1 pathway (54). Oncogenic properties of Gper1 were
demonstrated in the CRC cell lines COLO205 and SW480 as the Gper1
antagonist G15 was able to reverse the effect of the E2-related
endocrine disruptor nonylphenol, which induced proliferation, the
expression of cyclin D1, c-myc and ERK1/2. Administration of G15
also inhibited the growth of colon carcinoma xenografts in mice
(55). Disagreement in the results
of performed studies can be partly related to different
concentrations of G-1 used in experiments. In addition to
experimental designs, the biological context seems to be of special
importance in effects mediated by Gper1 (17,34,48,52,53).
In human studies, the results are also inconclusive.
Up- as well as down-regulated Gper1 expression in CRC tissue was
indicated (17), e.g. a strong
trend toward increased Gper1 levels in CRC cancer tissue compared
to adjacent tissue was reported by Gilligan et al (56) while down-regulated expression of
Gper1 was reported by Liu et al (51). High expression of Gper1 was
associated with better survival compared to low expression
(51); by contrast, low expression
of Gper1 was associated with better survival compared to high
expression (56). High expression
of Gper1 was also associated with worse survival by Bustos et
al (53) and Abancens et
al (48) but only in females in
stages 3–4 of CRC. Worse survival of CRC patients exhibiting high
expression of Gper1 is indicated by the Human Protein Atlas
(47).
As there is evidence suggesting that manipulation of
E2 signalling could be effective in clinical use, several agonists
and antagonists of nuclear and Gper1 receptors have been developed,
and some of them have been clinically tested (18,25,57).
The most promising results were obtained in the clinical study
NCT04130516 evaluating the effects of Gper1 agonist LNS8801 that
proved beneficial effects of Gper1 signalling in patients diagnosed
with cutaneous (58) and uveal
(59) melanoma. However, it was
implied that G-1 can cause effects independent of Gper1 signalling
(60–65). Interestingly, several clinically
approved antagonists of nuclear E2 receptors also act as Gper1
agonists (18).
CRC is among the most frequently diagnosed oncologic
diseases worldwide. To facilitate novel CRC treatment strategies,
we focused on Gper1 functioning in CRC tumours to define the
conditions under which Gper1 upregulation could be beneficial for
patients.
Despite ongoing clinical trials aimed at the
manipulation of Gper1 signalling in solid cancers, the involvement
of Gper1 in the generation of sex-dependent CRC incidence is
largely unknown. Similarly, the biological context that determines
the effect of Gper1 has not been described sufficiently. Therefore,
the present study is aimed to analyse the abundance of Gper1 in
healthy and tumour tissues of males and females diagnosed with CRC
according to disease stage and survival. Mutation in p53 has been
identified in more than 50% of CRC patients and at a higher
frequency in males compared to females (66). Therefore, using the CRC cell lines
LoVo and DLD1, which differ in p53 functionality (67), we tested the hypothesis that the
effects of Gper1 depend on the presence of wild-type p53.
Materials and methods
Tumour and adjacent tissues were obtained in
cooperation with the First Surgery Department, University Hospital,
Comenius University, Bratislava. The study included patients who
had to undergo surgery for CRC treatment (for details see Table SI) and agreed to sign an informed
consent. Patients were not subjected to any CRC treatment before
surgery and during hospitalization were exposed to a standard
hospital practice with lights on from 6:00 a.m. to 9:00 p.m.
Surgery was performed in the morning hours (before noon). Adjacent
tissues were collected ≥10 cm proximally and ≥2 cm distally from
the tumour. After tissue excision, samples were promptly frozen in
liquid nitrogen and stored at −70°C until analysis. The
experimental protocol was approved by the Ethics Committee of
Comenius University in Bratislava (ECH 19001).
To extract mRNA from tissues and cells, RNAzol RT
(Molecular Research Centre) was used as described previously
(40). One microgram of RNA
template from tissues and 0.02 µg from cells was used to synthesize
cDNA with the ImProm-II Reverse Transcription System and random
hexamers (Promega), according to the manufacturer's
instructions.
To analyse gene expression, the QuantiTect SYBR
Green kit (Qiagen, Germany) was used. Real-time PCR was performed
with the StepOnePlus™ Real-Time PCR System (Applied Biosystems).
The PCR conditions were activation of hot start polymerase at 95°C
for 15 min followed by 40 cycles at 94°C for 15 sec, 53°C for 30
sec and 72°C for 30 sec. As a last step of PCR, samples were
subjected to melting curve analysis. The sequences of primers for
the amplification of particular genes are provided in Table SII. The expression of nuclear
u6 mRNA was used for normalisation.
The human colorectal carcinoma cell lines DLD1 and
LoVo obtained from the American Type Culture Collection (ATCC) were
used to reveal how gper1 silencing influences cell migration
and metabolism. DLD1 cells were cultured in RPMI 1640 GlutaMax
medium (Gibco; Thermo Fisher Scientific, Inc.), and LoVo cells were
incubated in F-12K medium (Bioconcept). Both cell lines were
supplemented with 2% or 10% foetal bovine serum (FBS, Biosera)
depending on the experiment; 2% FBS supplementation was used for
the scratch assay while 10% FBS was used for the MTS test. The
culture medium also contained penicillin (50 U/ml) (Gibco),
streptomycin (50 µl/ml) (Gibco, USA) and ampicillin (50 µg/ml)
(Oasis-lab). All experiments were performed in a biological
Celculture® Incubator CCL-050B-8 (Esco Medical) with a
humidified atmosphere containing 5% CO2 at 37°C. The
cells were cultured in 96-well plates coated with 1% sterile
gelatine.
To test the effect of gper1 silencing,
DharmaFECT 1 reagent was used to transfect cells with siGENOME
Human Gper1siRNA-SMART pool or siGENOME non-targeting siRNA Pool #2
(Horizon) at a concentration of 100 nM.
The effect of siGper1 on wound healing was
determined by scratch assay performed when the cell culture reached
a confluence of 80–90% with the use of a 10-ul sterile tip. Images
were taken immediately after scratch and later as indicated in the
figure legends with the use of an inverted fluorescence microscope
NIB-100F (Nanjing Jiangnan Novel Optics Co., Ltd.) and BEL Capture
3.2 software (BEL Engineering s.r.l.). Wound closure was evaluated
with ImageJ software (68).
The rate of metabolism was measured by MTS test
according to the manufacturer's instructions (CellTiter 96 AQueous
Cell Proliferation Assay, Promega) employing a modified tetrazolium
compound to produce water-soluble formazan. The absorbance was
measured at 490 nm using a UV spectrophotometer (Epoch, Agilent
Technologies, Inc.).
Statistical analysis
To evaluate Gper1 mRNA expression in human samples,
the cohort was split into three groups according to TNM
classification. Group 1 included distant metastasis-free patients
without nodal involvement (T1-4N0M0), group 2 consisted of patients
with nodal involvement and without distant metastases (T3-4N1-2M0)
and the 3rd group was composed of patients with distant
metastases (T3-4N0-2M1). Gene expression between three groups was
compared by ANOVA followed by Tukey's post hoc test.
To compare E2 receptor expression between two
groups, a Student's paired t-test was used. ANOVA followed by
Tukey's post hoc multiple comparisons test was used to compare the
expression of three types of oestrogen receptors (ESR1, ESR2 and
Gper1) in human tissues and cells.
To analyse Gper1 mRNA expression with respect to
overall survival, a Kaplan-Meier survival curve and a log-rank test
were performed. Values were split according to the median; high
expression > median, low expression ≤ median. The starting point
for the log-rank test was the day of the surgery. The association
of Gper1 and VEGFA mRNA expression was analysed by correlation
analysis.
Data are provided as mean ± standard error of the
mean (SEM) in relative units (r.u.). The threshold for significance
for all statistical tests was set at P<0.05. Statistical
analyses were performed using GraphPad Prism 6 (GraphPad Software,
Inc.).
Results
E2 receptors show robust differences in expression
between colorectal tumours and adjacent tissues. In tumour tissue,
the expression of gper1 was higher compared to adjacent
tissue, whereas the opposite pattern was observed in levels of
nuclear E2 receptors. The expression of esr1 and esr2
was significantly lower in cancer tissue compared to adjacent
tissues (Fig. 1A). The expression
of gper1 was higher in cancer tissue compared to adjacent
distal and proximal tissues (Fig.
1B). Comparison of the absolute level of expression based on
the threshold value of the PCR revealed that gper1 is far
most abundant in cancer as well as in the adjacent tissues compared
to esr1 and esr2 mRNA levels (Fig. 1B).
The expression of gper1 was higher in cancer
tissue compared to adjacent tissue in males, but in females this
pattern was observed only as a non-significant trend (Fig. 2). The expression of esr1 was
higher in the adjacent compared to cancer tissue and this
difference, again, achieved a level of significance only in males
(Fig. 2). The most robust
difference in expression between cancer and adjacent tissue was
observed in the expression of esr2. Unlike other receptors,
this pattern was present in both sexes (Fig. 2).
In the whole cohort an increase in gper1
expression in cancer tissue compared to adjacent tissue was
observed in patients without nodal involvement and distal
metastases (Fig. 3A). However, when
the cohort was split into male and female patients, this difference
was observed only in males (Fig. 3B and
C). gper1 expression did not differ between cancer and
adjacent tissues in more advanced stages of disease (Fig. 3).
When the cohort was split according to nodal
involvement only (without considering TNM staging as a whole) the
sex-dependent difference in gper1 expression became even
more pronounced, and there was a highly significant increase in
gper1 expression in cancer tissue compared to adjacent
tissue in males without nodal involvement (Fig. 4A). Similarly, male patients without
distant metastases displayed a pronounced increase in gper1
expression in cancer tissue compared to adjacent tissue that was
not observed in females (Fig.
4B).
In accordance with the sex-dependent expression of
gper1 in CRC patients, the association of overall survival
and gper1 expression exerted a sex-dependent pattern. In the
whole cohort, better survival was correlated with low gper1
expression in patients without nodal involvement (Fig. 5A) but not in patients in higher
stages of disease (Fig. 5B). When
the cohort was split according to sex, a log-rank test revealed
that gper1 association with survival was generated by the
male subcohort (Fig. 5C). In
females the correlation between survival and gper1
expression did not reach significance (Fig. 5D).
In human samples from our cohort, the expression of
gper1 significantly correlated with the mRNA expression of
VEGFA in cancer tissue. This relationship was not observed in
proximal and distal adjacent tissues (Fig. S1).
Transfection of siRNA targeting gper1
expression successfully inhibited Gper1 mRNA expression in DLD1 as
well as LoVo cells compared to control (Fig. S2A and B, respectively).
The distribution of E2 receptors in CRC cell lines
LoVo and DLD1 resembled that of human cancer tissue. The expression
of the membrane gper1 receptor was much higher compared to
mRNA levels of nuclear E2 receptors, and the expression of ESR1
mRNA was nearly undetectable (Fig.
6).
Silencing of gper1 expression significantly
stimulated metabolism in LoVo cells in a time-dependent manner that
implicates the tumour-suppressor capacity of Gper1. In DLD1 cells
we did not observe this effect (Fig.
7).
Evaluation by the scratch test demonstrated a
time-dependent decrease in wound closure in DLD1 cells with
silenced Gper1 expression compared to control. Whereas 24h after
Gper1 silencing there was no effect on the rate of wound closure,
48 h after transfection the width of the closed area was different
between control and transfected cells with P=0.057 (Fig. 8A). Inhibition of wound healing by
Gper1 silencing implicates the oncogenic potential of Gper1 in DLD1
cells.
Unlike in DLD1 cells, the administration of siRNA
interfering with Gper1 expression did not influence the rate of
wound closure in LoVo cells (Fig.
8B).
The expression of p53 mRNA did not differ
significantly between DLD1 and LoVo cells. However, as DLD1 cells
generate a mutated form of the p53 protein, expression of
p53-inducible gene p21 was significantly lover in DLD1 cells
compared to LoVo cells (Fig.
S3).
The expression of gper1 and tp53
showed a significant correlation in males (A) but not in females
(B) undergoing surgery for CRC treatment (Fig. S4) (TCGA, Colorectal Adenocarcinoma,
Nature 2012).
Discussion
The relative ratio of E2 receptors in CRC tissue
undergoes remodelling. While the expression of nuclear E2 receptors
decreases in cancer tissue compared to adjacent tissue, the
opposite pattern is observed in Gper1 mRNA expression. Therefore,
Gper1 is by far the most abundant E2 receptor in the CRC tumour
followed by the ESR2 and very low ESR1 mRNA expression. The
increase in gper1 expression in tumours is most pronounced
in males in the lower stages of disease. A higher expression of
Gper1 mRNA was associated with worse survival in the whole cohort
and in males without metastases in nodes. This dependency was not
detected in males with nodal involvement and females. We suppose
that sex-dependent differences in E2 receptor expression can
contribute to sex-dependent features of CRC progression.
In accordance with our data, Gilligan et al
(56) showed an increase in Gper1
expression in CRC tissue compared to adjacent tissue. Several
factors can induce an increase in Gper1 mRNA levels in cancer
tissue. Firstly, Gper1 belongs to HIF-1 targeted genes that are
expressed in response to hypoxia (69). The stimulatory effect of hypoxia on
Gper1 mRNA expression was also demonstrated in the colon cancer
cell line HT-29 and the rectal cancer cell line C80 (53). The oncogenic role of Gper1 was
implicated as it was demonstrated that Gper1 cooperated with HIF-1α
in the activation of VEGF expression in hypoxia (26,70).
In line with this finding, we detected a positive correlation
between Gper1 and VEGFA mRNA expression in cancer but not in the
adjacent tissues.
We observed a decrease in ESR2 mRNA expression in
CRC tumours compared to proximal and distal parts of the gut, which
is consistent with previous studies (71–77)
that reported lower ESR2 expression in CRC tissue compared to
adjacent tissues at the protein and mRNA levels.
According to our results, the expression of ESR1
mRNA is lower in cancer tissue compared to adjacent tissues, and
this difference is more pronounced in males than in females.
Previously, a decrease was observed in ESR1 expression in
colorectal cancer tissue, which was attributed to CpG island
methylation (78). No significant
differences between cancer and adjacent tissue have also been
reported (38). According to Jiang
et al (79), the expression
of the dominant ESR1 isoform does not differ in its mRNA levels
between tumour and matched normal tissues. However, the ER-α46
isoform that shares most of its sequence with the dominant isoform
was down-regulated in cancer tissue compared to adjacent tissue. On
the other hand, an increase in esr1 mRNA in cancer compared
to adjacent healthy tissue has been reported lately (80). Although data referring to ESR1
expression in CRC seem to be inconclusive, there is a consensus on
the decrease in nuclear ESR2 and increase in Gper1 expression in
cancer compared to adjacent tissue.
The expression of all three types of receptors in
one set of samples has rarely been studied. There is a strong
evidence that the expression of ESR2 in the gut is more abundant
compared to that of ESR1 (71,76,81).
The assumption that ESR2 is the predominant form of the E2 receptor
in the gut can, to some extent, be caused by later identification
of the Gper1 receptor compared to nuclear E2 receptors (82). Previously, it has been shown that
the expression of Gper1 mRNA is more abundant compared to that of
both ESR1 and ESR2 in the rat colon (40). According to datasets available from
the Human Protein Atlas, the expression of E2 receptors is
Gper1>ESR2>>ESR1 (47),
which is in line with Harvey and Harvey (83).
The density of E2 receptors changes in CRC tumour
tissue. In CRC the abundance of E2 receptors shows the pattern
Gper1>>ESR2>ESR1. The order of E2 receptor density in the
healthy colon is similar, but there is an abrupt decrease in ESR2
and an increase in Gper1 expression in the tumour compared to the
healthy gut. Changes in the abundance of E2 receptors in CRC are
more pronounced in males than in females.
As CRC occurs to a lesser extent in females compared
to males (83), and sex-dependent
differences in nuclear E2 receptor expression have been revealed
(72,74–76,84),
female sex hormones were suggested to be involved in the regulation
of CRC progression (85).
Beneficial effects of E2 mediated via ESR2 receptors have been
convincingly demonstrated (76,86);
however, contradictory reports are available concerning the role of
Gper1 in CRC progression (34,53,56,83).
Similarly, little is known about sex-related differences in Gper1
expression, although they have been implied (70,83).
According to our results, there are more pronounced differences in
Gper1 expression in cancer tissue compared to adjacent tissue in
males than in females. This difference is noticeable mainly in
males without nodal metastases.
In our cohort poor survival correlated with high
Gper1 mRNA expression more significantly in males without nodal
involvement than in males in higher stages of disease or females.
These results are in accordance with Gilligan et al
(56), who reported an association
of worse survival in patients with high Gper1 expression.
Information about the correlation in males and females separately
was not provided. Bustos et al (53) reported sex-dependent differences in
relapse-free survival and Gper1 expression; however, as we do not
have data allowing this type of analysis, a comparison was not
possible. Our results are in accordance with the Human Protein
Atlas (47), according to which
there is a stronger association between Gper1 and survival in males
compared to females and worse survival in patients with high Gper1
expression. The association reaches significance only in males in
stage I–II and not in patients with higher stages of disease.
Interestingly, when the survival of males and females together are
correlated with Gper1 expression, the association does not reach
significance, which also implicates a sex-specific dichotomy in
regulation.
There are reports implicating Gper1 as a tumour
suppressor in pancreatic cancer, melanoma and adrenocortical cancer
and as a tumour promoter in glioblastoma and endometrial and
ovarian cancers, whereas in the case of lung, prostate, breast and
colorectal cancers, information about the regulatory impact of
Gper1 is inconclusive (17,18,34,87,88).
Gper1-mediated effects are dependent on the biological context. Our
results imply that intracellular conditions are vital for the
interpretation of Gper1 signalling, even at the level of one type
of solid cancer.
Gper1 is known to induce signalisation mediated via
the EGFR receptor with tyrosine kinase activity (17,89,90). A
deregulated EGFR pathway has been associated with the progression
of many types of cancer, including CRC (91,92).
Therefore, several tyrosine kinase inhibitors and EFGR antibodies
have been developed and introduced into clinical practice (92). Inhibition of EGFR signalling was
shown to benefit patients diagnosed with CRC, and the EFGR
monoclonal antibodies Cetuximab and Panitumumab are now routinely
used to treat this type of cancer (93). In vitro studies elucidated
the molecular mechanisms of cancer inhibition caused by EFGR
repression in several cancer cell lines, including DLD1 (94–97).
Gper1 signalling mediated via the Gs-coupled cAMP/PKA/CREB pathway
has also been shown to promote cell proliferation (98). Therefore, as Gper1 is known to
induce both EGFR- and cAMP-mediated regulation and is reciprocally
involved in VEGFA release, presumably, an oncogenic role of Gper1
might be expected in CRC.
However, signalling mediated by Gper1 is highly
complex. The same group that discovered the connection between
Gper1 and EGFR two years later revealed that Gper1 inhibits EGFR
signalling via the cAMP/PKA pathway by Raf-1 inactivation (89). To further elucidate the ambiguous
effects of Gper1 on CRC progression, we investigated the effect of
Gper1 silencing in CRC cell lines LoVo and DLD1.
According to our results, the distribution of E2
receptors in the CRC cell lines DLD1 and LoVo resembles that
observed in human tissues: the highest mRNA expression is that of
gper1, and the expression of ESR1 is nearly undetectable.
Gper1 silencing in the DLD1 cell line, which expresses mutated
tp53, inhibited the wound closure, implicating the oncogenic
potential of Gper1. By contrast, the decrease in Gper1 availability
caused an increase in the metabolic rate in LoVo cells that
preserved p53 functionality (99).
The p53 protein, encoded by the gene tp53, is
a well-known tumour suppressor that inhibits cell cycle progression
and initiates DNA repair and/or apoptosis and autophagy, in
response to DNA damage, hypoxia, nutrition deficiency, oxidative
stress and some hormones. Cell cycle inhibition is executed by
induction of p21 expression. p21 inhibits the activity of several
types of cyclin/CDK complexes that release repression of
retinoblastoma protein (RB). As cyclin/CDK complexes are
inactivated by p21 and cannot phosphorylate RB, hypophosphorylated
RB inhibits E2F-induced transcription, which is necessary for cell
proliferation (100,101).
The p53 protein is frequently mutated in many types
of cancer, including CRC. According to TCGA, more than 50% of CRC
patients carry a mutated form of tp53. The presence of a
mutated form of p53 is accompanied by worse survival than that of
wild-type tp53. Interestingly, tp53 mutations are
more frequent in males compared to females, which may also
contribute to the higher CRC incidence in males compared to females
(66).
We hypothesize that the signalling pathway
downstream of p53 influences the outcome of Gper1 signalling.
Previously, it was shown that Gper1 expression in the
triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468,
which express a mutated form of tp53, had been induced by
γ-radiation, whereas in MCF-7 cells expressing wild-type
tp53, the opposite pattern was observed. Therefore, a tumour
suppressor role dependent on p53 was attributed to Gper1 (102). Our results agree with this
statement as Gper1 was associated with oncostatic functions in LoVo
but not in DLD1 cell lines. Previously, the oncogenic potential of
Gper1 has been described in the CRC cell lines HT-29, DLD1, COLO205
and SW480 (53,55) carrying mutations in the tp53
gene (66,103–106).
Although there are reports implicating a functional
relationship between p53 and oestradiol, the exact mechanism has
either not been completely elucidated or, more likely, comprises
several ways by which E2 and p53 signalling interfere (107). These interferences can differ in a
tissue-dependent manner, e.g. E2 administration decreases p53
expression in lung cancer cells by induction of methyltransferase 1
expression, which increases methylation of the tp53 promoter
(108). On the other hand, a
protective role of E2 in non-malignant colonocytes executed via
p53-mediated regulation has been implicated (109).
The effects of E2 on p53 expression have been
previously investigated mainly with respect to the effects of ERα
on breast cancer progression. ERα interacts with tp53 and
influences its expression. Most of studies report an increase in
tp53 expression in response to ERα binding (107,110–113). On the other hand, there are also
results implicating ERα-induced suppression of p53-regulated gene
expression (114,115). However, in DLD1 cells, ESR1
expression is nearly undetectable; therefore, we do not suppose
that a substantial increase in p53 expression can be induced via
ERα. The results are inconclusive with respect to ERβ signalling
and p53 expression. Although induction of p53 activity in response
to ERβ activation has been reported (107,113), a stimulatory effect of ERβ
interacting with the tp53 promoter on tp53 expression
has not been detected (111).
In silico analysis showed a positive
correlation between Gper1 and tp53 expression in CRC tissues
of males (TCGA) (116). The E2
receptor ligands bisphenol A (117) and G-1 (102,118,119) have also been shown to induce
tp53 expression. Silencing of Gper1 caused a decrease in
tp53 expression in uveal melanoma cell lines (88). The exact mechanism of how Gper1
executes its effect on p53 transcription is unknown. A cAMP
response element (CREB) was identified in the human tp53
gene (120,121) and CREB binding protein plays a key
role in p53 activation (122).
However, whether Gper1 executes its stimulatory effect on
tp53 expression via this region remains to be
elucidated.
To conclude, the expression of E2 receptors in
healthy tissue follows the descending order
Gper1>ESR2>>ESR1 with nearly undetectable expression of
ESR1 mRNA. In CRC patients the ratio of receptors changes more in
males than in females; the expression of Gper1 mRNA increases while
the expression of ESR2 and ESR1 decreases, resulting in the
descending order Gper1>>ESR2>ESR1. Therefore, under
conditions of cancer progression, most E2 signalling is mediated
via the membrane E2 receptor Gper1. High expression of Gper1 is
associated with worse survival in males without nodal involvement
in comparison with other subcohorts (Fig. 9).
 | Figure 9.Possible mechanism of how Gper1
signalling contributes to sex-dependent differences in CRC. In
cancer tissue, the expression of ESR2, which inhibits CRC
progression, decreases in the earlier stages of disease in males
compared to females (74). E2
signalling is further modulated by an increase in Gper1 expression
in cancer tissue in males in the early stages of disease, which is
not observed in females. Therefore, in the CRC tumour
Gper1-mediated regulation strongly influences how E2 signalling
will be interpreted by the cell. Gper1 demonstrates an oncostatic
effect in the LoVo cell line carrying wild-type tp53, which
is not observed in DLD1 cells with mutated tp53. It was
concluded that Gper1 functioning is dependent on the p53
intracellular context. As the mutated form of the gene is more
frequent in males compared to females, and the two most important
gut receptors, Gper1 and ESR2, show a sex-dependent pattern of
expression, we suppose that sex-dependent interpretation of E2
signalling and the availability of a functional p53 pathway
contribute cooperatively to the differences in CRC progression
observed between sexes. ESR, oestrogen receptor; Gper1, G
protein-coupled oestrogen receptor 1; E2, 17β-oestradiol; CRC,
colorectal cancer; wt, wild-type. |
The relative expression of E2 receptors in DLD1 and
LoVo cells is similar to that observed in human CRC tumours. In
LoVo cells with wild-type tp53, a tumour suppressor effect
of Gper1 was observed. This effect was not detected in DLD1 cells
with the mutated form of tp53. We suppose that Gper1
activates the EFGR/RAS oncogenic pathway as well as the p53
pathway. In case the p53 pathway is not functional, the oncogenic
potential of Gper1 overwhelms its tumour suppressor effects. As the
frequency of tp53 mutations as well as changes in Gper1
expression are more robust in males compared to females, we suppose
that these effects can contribute to sex-dependent differences in
CRC incidence.
Supplementary Material
Supporting Data
Supporting Data
Acknowledgements
The authors would like to thank Dr. Soňa Olejarova
(Department of Animal Physiology and Ethology, Faculty of Natural
Sciences, Comenius University in Bratislava, Bratislava, Slovak
Republic) for help with laboratory analyses.
Funding
The research was supported by projects APVV-16-0209 and
APVV-20-0241 provided by The Slovak Research and Development Agency
and project VEGA 1/0455/23 provided by grant agency of the Ministry
of Education, Science, Research and Sport of the Slovak
Republic.
Availability of data and materials
The datasets are available from the corresponding
author on reasonable request.
Authors' contributions
IH and RR designed and administered the human
study. IH and RR confirm the authenticity of all the raw data. RR
obtained samples and organized their transport to the laboratory.
IH performed analysis of human samples. IH and DV performed cell
culture experiments. IH performed in silico analysis,
interpreted results, wrote the manuscript and prepared the figures.
IH, RR and DV reviewed and revised the manuscript for the
scientific content. All authors have read and approved the final
manuscript.
Ethics approval and consent to
participate
The design of the study describing the sampling of
human tissues was approved by the Ethics Committee of the Comenius
University in Bratislava (approval no. ECH 19001). All patients
included in the study agreed to sign an informed consent. The
manuscript does not contain experiments using animals or embryonic
cells.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
Authors' information
Professor Iveta Herichová, ORCID:
0000-0002-0475-0461.
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