Abstract
In normal colon tissue, oestrogen receptor alpha (ERα) is expressed at low levels, while oestrogen receptor beta (ERβ) is considered the dominant subtype. However, in colon carcinomas, the ERα/β ratio is often increased, an observation that prompted us to further investigate ERα’s role in colorectal cancer (CRC). Here, we assessed ERα nuclear expression in 351 CRC patients. Among them, 119 exhibited positive ERα nuclear expression, which was significantly higher in cancer tissues than in matched normal tissues. Importantly, patients with positive nuclear ERα expression had a poor prognosis. Furthermore, positive ERα expression correlated with increased levels of the G-protein coupled cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear β-catenin, both known tumour promoters. In mouse models, ERα expression was decreased in Cysltr1−/− CAC (colitis-associated colon cancer) mice but increased in ApcMin/+ mice with wild-type Cysltr1. In cell experiments, an ERα-specific agonist (PPT) increased cell survival via WNT/β-catenin signalling. ERα activation also promoted metastasis in a zebrafish xenograft model by affecting the tight junction proteins ZO-1 and Occludin. Pharmacological blockade or siRNA silencing of ERα limited cell survival and metastasis while restoring tight junction protein expression. In conclusion, these findings highlight the potential of ERα as a prognostic marker for CRC and its role in metastasis.
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Introduction
Oestrogen receptor alpha (ERα), one of the two primary oestrogen receptors encoded by the ESR1 gene located on chromosome 6, plays a crucial role in the tumorigenesis of various cancers, such as breast, prostate, uterine, and ovarian cancers [1,2,3,4]. In normal colon mucosa, ERα expression is generally low [5, 6]. However, studies have shown an increased ERα/β ratio in colon carcinomas [7, 8]. Despite this discrepancy, there have been limited investigations of the prognostic significance of ERα in colorectal cancer (CRC) patients [9,10,11]. Therefore, further research is needed to elucidate the prognostic implications of ERα in CRC.
CRC is currently the third most common cancer worldwide, claiming thousands of lives annually [12, 13]. The development of CRC is associated with genetic alterations and inflammation [14, 15]. Mutation of the tumour suppressor gene adenomatous polyposis coli (APC) is present in approximately 85% of sporadic CRC cases [16]. Somatic loss of APC results in aberrant activation of the Wnt/β-catenin pathway, which is known to play a pivotal role in the development and prognosis of CRC [16, 17]. Additionally, cysteinyl leukotrienes (CysLTs) have been implicated in colon tumorigenesis [18]. Previous studies have investigated the tumour-promoting effects of CysLT1R and have demonstrated its association with poor prognosis in CRC patients, as well as its tumour-promoting effects in colon cancer (CC) cell lines and several mouse models [19,20,21].
In this study, using CRC patient material, animal models, and CC cell lines, we investigated how ERα influences the prognosis of CRC patients and promotes metastasis in CRC. We report that an increase in ERα expression leads to poor outcomes in patients with CRC and that activated ERα expression stimulates the expression of tumour promoters and drives metastasis via regulation of tight junction proteins. Furthermore, we demonstrate that targeting ERα either through pharmacological inhibition or siRNA silencing could decrease the metastatic burden in preclinical models of CC.
Materials and methods
Patients
The patients involved in this study were represented from two cohorts: the Kvinno cohort, composed of a total of 333 CRC samples collected between January 1, 2008, and June 30, 2012; and the Malmö cohort, comprising 120 CRC samples collected during 1990. All the samples collected were incorporated into tissue microarrays (TMAs). Details about the study design and patient follow-up for each cohort were provided earlier [22, 23]. Both studies were approved by the Ethics Committee at Lund University. The flowchart of patient inclusion in the study is shown in Supplementary fig. S1.
Tissue microarray (TMA) and immunohistochemical (IHC) analyses
The tissues from CRC samples incorporated into the TMAs were stained to evaluate the expression of ERα using a mouse anti-ERα antibody cocktail (overnight incubation, pH 6.0) and an anti-ERα monoclonal antibody D-12 (overnight incubation, pH 6.0) as mentioned earlier [24]. Additionally, anti-CysLT1R (overnight incubation) and anti-total β-catenin antibodies were used. Immunoreactivity was assessed by two blinded independent investigators (GT and RE) using the immunoreactive score (IRS) calculated as follows: IRS = (staining intensity) × (% of stained cells) [22, 24]. Only the nuclear ERα expression was taken into consideration. All the cores with positive nuclear staining in more than 10% of the cells, regardless of the staining intensity, were considered positive for ERα expression. Tissue cores with less than 10% of positive nuclear staining were considered negative for ERα expression. For antibody dilutions and details, see Table 1.
Cell lines
The human CC cell lines HT-29 and Caco-2 were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). HT-29 cells were cultured in McCoy’s 5A medium supplemented with 10% foetal bovine serum (FBS), 1% L-glutamine and 100 μg/ml penicillin/streptomycin. Caco-2 cells were maintained in MEM supplemented with 20% foetal bovine serum (FBS), 1% L-glutamine, 1% non-essential amino acids, and 100 μg/ml penicillin/streptomycin. Both cell lines were incubated at 37 °C and 5% CO2. Cells were treated for 48 h with the ERα-selective agonist, PPT (40 nM) with or without treatment with the ERα-selective antagonist AZD9496 (0.3 nM) 30 min before PPT stimulation.
Quantitative real-time PCR
The isolation of total RNA from CC cells was performed using RNeasy Mini Kit (Qiagen, Hilden, Germany), and first-strand cDNA synthesis was accomplished using a cDNA synthesis kit (Invitrogen, Carlsbad, California, USA) [25,26,27,28]. The TaqMan probes used were specific for ESR1 (ERα, Hs01046816_m1), CYSLTR1 (CysLT1R, Hs00272624-s1), and CTNNB1 (β-catenin, Hs00991818_ml). The samples were analysed, expression levels were normalized to those of the endogenous housekeeping gene HPRT1 (Hs99999909_m1), and fold changes were quantified with the 2-ΔΔCt method using MxPro software (Agilent Technologies, Santa Clara, CA, USA).
Western blotting
Western blotting was performed based on a protocol described previously with minor modifications [25,26,27,28,29]. The following primary antibodies were used: anti-ERα, anti-phospho-β-catenin (Ser45/Thr41), anti-non-phospho (active)-β-catenin, and anti-total β-catenin. Anti-α-tubulin and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were used as loading control antibodies as indicated in the figures. Visualization of protein expression was performed with enhanced chemiluminescence (ECL) reagents Millipore with a Bio-Rad ChemiDoc™ imaging system (Hercules, CA, USA), and densitometric analysis was performed using Bio-Rad Image Lab software. For antibody dilutions and details (see Table 1).
siRNA transfection in colon cancer cells
An ESR1-specific siRNA (siESR1) was employed to study ERα specificity in functional assays using CC cells in accordance with a previously reported protocol [27].
Colonosphere model
HT-29 and Caco-2 CC cell-derived colonospheres were generated as described previously [25,26,27].
Immunofluorescence analysis
Immunofluorescence analysis was performed in HT-29 and Caco-2 cells to visualize the expression of the tight junction proteins ZO-1 and Occludin as previously described [29]. Fluorescence images were captured with a Zeiss LSM 700 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using a 63× oil objective and analysed using LSM ZEN Blue software.
Immunofluorescence staining in colonospheres was performed by adopting a protocol designed for organoids with some modifications [30]. Briefly, CC cells were used to generate colonospheres as described previously [25,26,27]. After 72 h, colonospheres were harvested and cultured in Matrigel in eight-well glass-bottom chamber slides (Ibidi, GmbH, Germany). After 24 h, the medium was removed from each chamber, and the colonosphere embedded in the Matrigel was washed with 1x PBS and fixed with 4% PFA for 1 h at room temperature (RT). After fixation, the colonospheres were washed with 1x PBS and permeabilized with 0.3% Triton X-100 for 15 min at RT. Blocking was performed with 5% BSA prepared in PBS containing 0.1% Tween 20 overnight at 4 °C. Colonospheres were incubated with the primary antibody (see Table 1 for dilutions) overnight at 4 °C prior to washing with PBS containing 0.1% Tween 20. Next, incubation with a secondary antibody combined with a nuclear stain was performed for 4 h at RT prior to washing with 1x PBS containing 0.1% Tween 20. Colonospheres were mounted using Dako mounting medium (Dako, Denmark), and images were acquired with a confocal microscope (Zeiss, Germany).
Colon cancer mouse models
We evaluated the expression of ERα in two different CC mouse models. C57BL/6 J-ApcMin/+ mice (The Jackson Laboratory, Bar Harbor, Maine, USA) and in a CAC model with Cysltr1 gene disruption on the C57BL/6 N background [31]. The experimental design for the CAC model is described in detail earlier [32]. Tissues from these mice were stained with an anti-ERα antibody (1D5 + 6F11, 1:50) to evaluate ERα expression. Five mice per genotype were included, and four different sections per mouse were evaluated.
Zebrafish xenograft model
The role of ERα expression in CC cell metastasis was validated using the zebrafish xenograft model [25,26,27, 33]. HT-29 CC cells were treated with PPT with or without AZD9496 for 48 h, labelled with Vybrant DiI and microinjected into the perivitelline space of the 2 days post-fertilization zebrafish embryos. siCTRL- or siESR1-transfected cells were treated with PPT for 48 h before DiI labelling and were injected into the embryos. After 48 h, the metastatic spread of CC cells into the tail veins of the embryos was monitored and photographed using a Nikon fluorescence microscope (Nikon Eclipse 80i, USA). The mean fluorescence intensity (MFI) of the metastatic cells was measured using ImageJ software (NIH, USA). The zebrafish housing and maintenance were as described before [34].
Statistical analysis
Continuous variables were compared using the Mann-Whitney U test or Student’s t test as indicated, and categorical variables were compared using the chi-square test. Kaplan–Meier- survival curves were compared with the log-rank test. Statistical analyses were performed using SPSS version 23.0 (SPSS, IBM, Armonk, NY, USA) and GraphPad Prism version 9 (GraphPad Software, Inc., San Diego, CA, USA). All tests were two sided, and P values < 0.05 were considered to indicate a statistically significant difference.
Public CRC dataset analysis
We also used public CRC patient data. The mRNA expression based microarray data from the GSE39582 dataset were downloaded. ERα gene expression in this public database was categorised into high ERα and low ERα. Data for a total of 566 patients were included in the study. The mRNA expression data in the dataset were normalized using the TMM method and were log2 transformed for further analysis.
Results
ERα expression and its prognostic association in colorectal cancer patients
Recently, we reported that KRAS oncogenic mutation correlates with positive ERα expression in CC patients [24]. However, the involvement of ERα in CC growth and metastasis is not yet clear. Here, using paired normal and tumour samples of CRC patients, we found consistently elevated expression of ERα in the tumour tissue compared to the normal mucosal tissue (Fig. 1A). Previously, we reported that high ERα expression in CRC patients is associated with poor overall survival (OS) [24]. Here, we combined the same cohort (Kvinno cohort, n = 270) with another cohort of patients with CRC (Malmö cohort, n = 67) and grouped the patients based on ERα expression (positive or negative) (Supplementary Fig. S1). After excluding patients from both cohorts with metastatic disease or an unknown recurrence status or date, 233 patients were available for disease free survival (DFS) analysis. The median follow-up times for the Kvinno cohort were 5.8 years for OS and 5 years for DFS. For the Malmö cohort, the median follow-up time for OS was 10 years. To investigate the prognostic role of ERα expression in CRC patients, we performed Cox regression analysis and evaluated OS and DFS. Patients with negative ERα expression had a significantly reduced risk for overall mortality of 61% (HR, 0.39; CI, 0.28–0.55) after adjustment for age and TNM stage (Fig. 1B). This result was consistent in the subgroups of patients with stage I-III CRC disease and colon cancer and, interestingly, also in the subgroup of patients with rectal cancer, in which negative ERα expression showed the greatest benefit, with a reduction in overall mortality of 80% (Fig. 1B-D). Similar results were obtained for patients who did not receive any adjuvant treatment after CRC surgery, for both patients with colon or rectal cancer (Fig. 1E-G). Together these data indicates that high ERα expression is associated with poor prognosis in CRC patients.
ERα expression positively correlates with tumour promoter expression in colon cancer
In this cohort, we previously showed that the poor prognosis of the included patients was associated with high CysLT1R and nuclear β-catenin expression. These two tumour promoters in colon cancer are known to act through a CysLT1R/Wnt-β-catenin signalling axis [25, 28, 32, 35]. Here, we tested for possible associations between ERα and CysLT1R and/or β-catenin. As expected, we noted higher expression of CysLT1R and nuclear β-catenin in patients with positive ERα expression (n = 185) compared to patients with negative ERα expression (n = 82) (Fig. 2A, B). This finding was further validated by analysis of a public CRC dataset (GSE39582, n = 566), which revealed a significant positive correlation between the mRNA level of CYSLTR1 or CTNNB1 (β-catenin) and ESR1 (ERα) (Fig. 2C, D). In addition, patients with high ESR1 expression showed higher transcript levels of both CYSLTR1 and CTNNB1 (Fig. 2E, F).
Next, we used both the colitis-associated colon cancer (CAC) mouse model and spontaneous CC mouse model (ApcMin/+) with either the functional presence or functional absence of Cysltr1 (n = five mice per genotype) (Fig. 2G, H). Cysltr1−/− mice are reported to exhibit a less aggressive tumour phenotype, with fewer polyps/tumours in their colon [32, 35]. Colon tissue sections from mice lacking functional Cysltr1 (Cysltr1−/−) showed two-fold reduced ERα expression compared to those from WT mice (Fig. 2G). On the other hand, we found that ApcMin/+ mice, which have higher amount of β-catenin accumulation in the nucleus due to the Apc mutation and are reported to develop more and larger intestinal polyps [36], had >two-fold higher nuclear ERα expression than mice with wild-type Apc (Fig. 2H).
Furthermore, we evaluated the basal mRNA levels of ERα expression in different CC cell lines, using the MCF7 breast cancer cell line, with very high expression of ERα, as a positive control (data not shown). Based on these results we used the ERα expressing cell lines HT-29 and Caco-2 cells for further studies. We pharmacologically induced or blocked ERα expression in HT-29 and Caco-2 cells by treatment with the ERα-selective agonist PPT alone or ERα-specific antagonist AZD9496 or combination of both (AZD9496 + PPT) (see Materials and Methods for experimental details). We observed a more than 30–50% increase in the mRNA expression levels of both CYSLTR1 and CTNNB1 with PPT treatment, and this increase was restricted after AZD9496 alone treatment or in PPT stimulated cells pre-treated with AZD9496 (PPT + AZD9496) in both HT-29 and Caco-2 cells (Fig. 2I, J). No statistical significance was noted between AZD9496 or AZD9496 + PPT experimental groups (Fig. 2I, J). Taken together, these results indicate a direct involvement of ERα in promoting the expression of the tumorigenic markers CysLT1R and β-catenin in CC.
ERα activation in colon cancer cells promotes survival
WNT/β-catenin signalling in CC is known to support tumour growth via nuclear β-catenin. Based on the observations in Fig. 2B and H, we next sought to investigate whether activation of ERα can promote cell survival via the WNT/β-catenin pathway. Indeed, we observed a 2-fold increase in the number of colonies formed by both HT-29 and Caco-2 CC cells treated with PPT compared to untreated control cells, and the number of colonies formed by cells pretreated with AZD9496 was substantially reduced (Fig. 3A). Furthermore, western blot analysis of HT-29 and Caco-2 cells stimulated with PPT showed increased levels of non-phosphorylated active β-catenin compared to unstimulated cells (Fig. 3B). Interestingly, the increase in the β-catenin level was inhibited in cells pretreated with AZD9496 prior to PPT stimulation, possibly due to the significant increase in phosphorylated β-catenin (three-fold for HT-29 and six-fold for Caco-2 cells), which is known to be rapidly degraded by ubiquitination.
To further validate the involvement of ERα in CC cell survival, we employed ESR1 siRNA to silence functional ERα, and the results were compared with those in siCTRL-transfected cells (Fig. 3C, D). Interestingly, we noted a significantly reduced colony number in the siESR1-transfected cell group compared to the siCTRL-transfected cell group (Fig. 3C). Treatment with PPT did not significantly affect the colony number in the siESR1-transfected cell group. Moreover, the whole-cell lysates of HT-29 and Caco-2 cells transfected with siESR1 showed a reduction in the level of active β-catenin, supporting the earlier observation of the increase in active β-catenin in PPT-treated CC cells (Fig. 3D).
ERα promotes colon cancer cell metastasis by downregulating tight junction proteins
As reported earlier, ERβ activation could reduce metastasis in CC, with a reduced number of migrated cells observed in vitro and in vivo [27]. Here, we intended to explore the metastatic potential of ERα, which is known to act in an opposite manner in breast cancer [37]. We used a public dataset of CC patients with liver metastasis (n = 18) and found a strong and significant positive correlation between CYSLTR1 and ESR1 (R = 0.77, p < 0.005) as well as between CTNNB1 and ESR1 expression (R = 0.62, p < 0.041) (Fig. 4A, B). Next, we tested the impact of ERα activation on the migration capacity of CC cells in vitro using wound-healing assay (Supplementary Fig. S2A). HT-29 cells stimulated with PPT showed more than 2-fold increase in the wound closure compared to unstimulated control. Interestingly, cells pre-treated with AZD9496 showed reduced percentage (5-fold) of wound closure compared to the PPT treated cells suggesting an involvement of ERα in the migration of CC cells. Next, to validate this observation in vivo, we used a transgenic zebrafish xenograft model established with HT-29 cells, interestingly, we observed a higher number of embryos with metastasis as well as an increased metastatic burden in the tail veins of the embryos in the group injected with PPT-treated HT-29 cells (M1 = 24; M0 = 6) compared to the group injected with untreated HT-29 cells (M1 = 10; M0 = 20) (Fig. 4C, D). The increase in metastasis was inhibited in the embryos injected with AZD9496 + PPT-treated HT-29 cells (M1 = 5; M0 = 38). PPT treatment not only resulted in a greater number of embryos with metastasis but also was able to establish a high metastatic burden, as evidenced by quantification of the mean fluorescence intensity (MFI) (mean MFI in the CTRL group = 187; PPT group = 330; AZD9496 + PPT group = 172) (Fig. 4D’).
Cancer cell dissemination due to loss of cell-cell junctions is a key and early step in metastasis. Tight junction proteins are responsible for maintaining physical connections between epithelial cells. Important among tight junction proteins is the ZO-1/Occludin complex [38]. Therefore, we next sought to determine whether ERα-mediated metastasis in CC occurs via disruption of tight junctions with a possible change of the ZO-1 complex. Indeed, we found a decrease in ZO-1 expression in HT-29 (50%) and Caco-2 (40%) cells treated with PPT (Fig. 4E). This was further supported by immunofluorescence staining of HT-29 and Caco-2 cells, where it was clearly evident that there was disruption of ZO-1 expression in cells after PPT treatment, while in both untreated and AZD9496-treated cells, ZO-1 expression remained unchanged (Fig. 4F, G). Furthermore, Occludin, a member of the ZO-1 complex, was expressed at significantly lower levels in PPT-treated HT-29 or Caco-2 cells owing to disrupted cell-cell junctions (Fig. 4F, G). Moreover, analysis of ZO-1 expression in HT-29 and Caco-2 cell-derived colonospheres validated the above observations (Fig. 4H, I). These results indicate the metastasis-promoting role of ERα induction in CC cells and the beneficial effects of antagonizing ERα expression by treatment with a selective antagonist such as AZD9496.
The functional absence of ERα inhibits colon cancer cell metastasis
Next, we tested the effect of the functional absence of ESR1 on colon cancer metastasis in vitro as well as in vivo. Wound healing assay performed in the siCTRL or siESR1 transfected cells with or without PPT treatment showed > 2-fold increase in wound closure in the siCTRL group treated with PPT (Supplementary fig. S2B), while siESR1 transfected cells failed to show significant wound closure even after PPT treatment.
While injection of PPT-treated siCTRL-transfected cells resulted in metastasis in a higher number of embryos (embryos with metastasis (M1) = 3; embryos with no metastasis (M0) = 29), siESR1 transfection failed to result in metastasis even after PPT treatment (M1 = 5, M0 = 24) (Fig. 5A, A’). Zebrafish embryos injected with HT-29 cells transfected with siESR1 showed reduced tail vein metastasis compared to embryos injected with siCTRL-transfected HT-29 cells, with a 3-fold reduction in the MFI indicating the metastatic burden in embryos with metastasis (Fig. 5A’).
We observed a reduced number of embryos with tail metastasis upon ESR1 silencing (siESR1) (M1, n = 5) (Fig. 5A), we posited that cells, with ESR1 gene silencing, might be tightly adhered to each other, indicating increased ZO-1 expression. Initially, we employed western blotting to assess ZO-1 expression under the mentioned experimental conditions. As observed in Fig. 4; in Fig. 5B, we noted reduced ZO-1 expression in whole-cell lysates of siCTRL-transfected HT-29 (50%) and Caco-2 (50%) cells after PPT treatment. Surprisingly, we found a similar expression in the siESR1-transfected control group compared to the siCTRL-transfected control group in HT-29 cells but not in Caco-2 cells (Fig. 5B). However, ZO-1 expression in siESR1-transfected HT-29 and Caco-2 CC cells remained unaffected even after PPT treatment (Fig. 5B). To validate this, we performed immunofluorescence analysis in HT-29 and Caco-2 CC cells for visual confirmation. Indeed, in the immunofluorescence images, elevated ZO-1 expression was evident at the ‘cell-cell junctions’ of siESR1 transfected HT-29 and Caco-2 cells (Fig. 5C, D). Finally, HT-29 and Caco-2 CC cell-derived colonospheres lacking functional ESR1 (derived from siESR1-transfected cells) showed intact ZO-1 expression compared to their siCTRL-transfected counterparts (Fig. 5E, E’, F, F′). Taken together, these observations indicate a role of ERα in promoting and supporting cell survival and metastasis in CC (Fig. 5G).
Discussion
In this study, we identified the cellular mechanisms by which the nuclear receptor ERα drives metastasis and confers a worse prognosis of CRC patients (Fig. 1). The expression of ERα positively correlates with that of tumour promoters in CC. Activated ERα increases the level of active β-catenin (Figs. 2 and 3) and disrupts the tight junction proteins ZO-1 and Occludin (Figs. 4 and 5), leading to increased CC metastasis (Figs. 4 and 5).
Oestrogen receptors are well studied in breast and cervical cancers [2,3,4]. In colon cancer, ERβ plays an important role in limiting tumour progression and metastasis [5, 39, 40]. We have also previously reported that activating ERβ with the selective agonist ERB-041 reduced CC cell survival, induced apoptosis, and inhibited metastatic spread [27]. However, few studies have explored ERα expression levels in CC tissues. In our earlier report [24], we showed the prognostic relevance of ERα expression in CRC patients with higher ERα expression in tumour tissue than in normal mucosa. In addition, we established the importance of considering both ERα and ERβ protein expression for better predicting the prognosis of CRC patients [24].
Here, we investigated the correlation of ERα expression with that of tumour promoters in CRC. We noted a poorer prognosis in CRC patients with high ERα expression compared to the patients with low ERα expression (Fig. 1), which could be due to the significant correlations between ERα and both CysLT1R and β-catenin, all known tumour promoters [18, 20, 25, 28, 32, 35]. Our results were supported by mRNA expression data from public databases (Fig. 2C, D), which showed positive correlations between ESR1 and both CYSLTR1 and CTNNB1 mRNA levels in CRC patients. Moreover, we have reported earlier that CysLT1R, and β-catenin expression are positively correlated in CRC patients and patients with high CysLT1R, and high nuclear β-catenin have poorer prognosis [22]. To strengthen our findings, we examined the expression of ERα in two different CC mouse models. Cysltr1−/− mice, known to have a less aggressive phenotype, exhibited decreased ERα expression, while ApcMin/+ mice showed higher ERα expression (Fig. 2G, H). The ApcMin/+ mouse model exhibits activation of Wnt/β-catenin signalling, which promotes the translocation of β-catenin into the nucleus and positively correlates with CysLT1R expression as per our earlier report [32]. In addition, using both colon and breast cancer cells, Kouzmenko et al. have shown interaction between ERα and β-catenin via immunoprecipitation [41]. Taken together this indicate the possibility of an ERα/CysLT1R/Wnt signalling axis.
To maintain normal epithelial tissue integrity and cell polarity, tight junctions sustain cell-cell adhesion and regulates intercellular signalling [42]. Loss of tight junctions in a cluster of cancer cells in the primary tumour could result in dissemination and metastasis [42]. ZO-1 is a member of the tight-junction family of proteins (including Occludin) expressed in epithelial and endothelial cells and positively associated with β-catenin expression [43, 44]. Similar to ZO-1, loss of membrane β-catenin is associated with the migratory phenotype of cancer cells. Previously, the expression levels of the tight junction proteins ZO-1 and Occludin were reported to be reduced after oestrogen treatment, resulting in ERβ activation in human gut tissue [45]. Zhou et al. used both male and female gut tissues to highlight that ZO-1 is crucial for maintaining epithelial integrity in human gut tissue. Moreover, ZO-1 loss increases gut permeability and confers vulnerability to mucosal pathogens [45]. In a more recent study, Li et al. emphasized the metastasis-promoting role of ZO-1 in KRASmut CRC patients in a sex-specific manner [46].
We noted that pharmacological induction of ERα expression with a specific agonist, PPT, reduced the protein levels of ZO-1 and Occludin, which were restored using the specific antagonist AZD9496 (Fig. 4F, G). This is in coordination with the alteration noted in the β-catenin expression in the mRNA or protein level (Figs. 2I, J; 3C). Silencing of ESR1 (by siESR1 transfection) in CC cells also protected ZO-1 and Occludin expression. This was also reflected in the number of embryos with metastasis in the zebrafish model (Fig. 4C). Compared to injection of either AZD9496-treated cells or siESR1-transfected cells, injection of CC cells with reduced expression of tight junction proteins upon PPT treatment resulted in an increased number of embryos with tail vein metastasis (Figs. 4C and 5A). This indicates that tight junction disruption is a prerequisite step in ERα-mediated CC metastasis.
Taken together, our findings show that positive expression of ERα correlates with poor prognosis and metastasis in CRC patients. These findings highlight the potential therapeutic opportunities of blocking ERα expression in CRC [47]. However, further mechanistic studies are needed to analyse the pathway in more detail. The mechanistic insights provided here could be used as a framework for the development of precision therapeutic agents targeting ERα in metastatic CRC, particularly in cases where its expression is increased, such as cases in female patients with Lynch syndrome and breast or colorectal cancer.
Change history
02 May 2024
A Correction to this paper has been published: https://doi.org/10.1186/s12964-024-01631-9
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Acknowledgements
We would like to acknowledge Kristina Ekstöm-Holka from the Tissue Micro-Array Centre at Lund University, Malmö, Sweden, for her assistance with immunohistochemistry analysis.
Funding
Open access funding provided by Lund University. This study was supported by grants to AS from the Malmö University Hospital Cancer Foundation, the Swedish Cancer Foundation Grant (21 1453), and the Governmental Funding of Clinical Research within the National Health Services and to SRS from the Royal Physiographic Society in Lund (Per Eric and Ulla Schyberg research grant) and the Crafoord Foundation. The funding agencies had no influence on the study design, data collection, data analysis, data interpretation or report writing.
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Conception and design: GT, SRS, and AS. Development of methodology: GT, SRS, and SG. Acquisition of data: GT, SRS, SG, KH, FE, and RO. Analysis and interpretation of data: GT, SRS, and SG. Writing, review, and/or revision of the manuscript: SRS, GT, and AS. Study supervision: SRS and AS. All authors have read and approved the final version of the manuscript.
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Topi, G., Satapathy, S.R., Ghatak, S. et al. High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer. Cell Commun Signal 22, 198 (2024). https://doi.org/10.1186/s12964-024-01582-1
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DOI: https://doi.org/10.1186/s12964-024-01582-1