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MRP4 sustains Wnt/β-catenin signaling for pregnancy, endometriosis and 1
endometrial cancer 2
Jun-Jiang Chen1,2,3,4*, Zhi-Jie Xiao5*, Xiaojing Meng1, Yan Wang3, Mei KuenYu2,3, Wen Qing 3
Huang3, Xiao Sun3, Hao Chen3,6, Yong-Gang Duan7, Maria Pik Wong5, Hsiao Chang Chan3, Fei 4
Zou1#, Ye Chun Ruan2# 5
1Department of Occupational Health and Occupational Medicine, Guangdong Provincial Key Laboratory 6
of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China. 7
2Deparment of Biomedical Engineering, Faculty of Engineering, the Hong Kong Polytechnic University. 8
3Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, the Chinese 9
University of Hong Kong. 10
4Department of Physiology, School of Medicine, Jinan University, Guangzhou, China 11
5Department of Pathology, The University of Hong Kong, Hong Kong, Hong Kong 12
6Department of Gynecology and 7Centre of Reproductive Medicine and Andrology, Shenzhen Second 13
People’s Hospital, 518035 Shenzhen, China. 14
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*These two authors contributed equally to this work 16
#Correspondence should be sent to [email protected] (Y.C.R.) or [email protected] (F.Z.) 17
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Abstract 21
Rationale: Abnormal Wnt/β-catenin signaling in the endometrium can lead to both embryo 22
implantation failure and severe pathogenic changes of the endometrium such as endometrial cancer 23
and endometriosis, although how Wnt/β-catenin signaling is regulated in the endometrium remains 24
elusive. We explored possible regulation of Wnt/β-catenin signaling by multi-drug resistance 25
protein 4 (MRP4), a potential target in cancer chemotherapy, and investigated the mechanism. 26
Methods: Knockdown of MRP4 was performed in human endometrial cells in vitro or in a mouse 27
embryo-implantation model in vivo. Immunoprecipitation, immunoblotting and 28
immunofluorescence were used to assess protein interaction and stability. Wnt/β-catenin signaling 29
was assessed by TOPflash reporter assay and quantitative PCR array. Normal and endometriotic 30
human endometrial tissues were examined. Data from human microarray or RNAseq databases of 31
more than 100 participants with endometriosis, endometrial cancer or IVF were analyzed. In vitro 32
and in vivo tumorigenesis was performed. 33
Results: MRP4-knockdown, but not its transporter-function-inhibition, accelerates β-catenin 34
degradation in human endometrial cells. MRP4 and β-catenin are co-localized and co-35
immunoprecipitated in mouse and human endometrium. MRP4-knockdown in mouse uterus 36
reduces β-catenin levels, downregulates a series of Wnt/β-catenin targeting genes and impairs 37
embryo implantation, which are all reversed by blocking β-catenin degradation. Analysis of human 38
endometrial biopsy samples and available databases reveals significant and positive correlations 39
of MRP4 with β-catenin and Wnt/β-catenin targeting genes in the receptive endometrium in IVF, 40
ectopic endometriotic lesions and endometrial cancers. Knockdown of MRP4 also inhibited in 41
vitro and in vivo endometrial tumorigenesis. 42
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Conclusion: A previously undefined role of MRP4 in stabilizing β-catenin to sustain Wnt/β-43
catenin signaling in endometrial cells is revealed for both embryo implantation and endometrial 44
disorders, suggesting MRP4 as a theranostic target for endometrial diseases associated with Wnt/β-45
catenin signaling abnormality. 46
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Keywords: MRP4, β-catenin, endometrium, embryo implantation, endometrial disorder 48
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Introduction 50
The endometrium is a tissue layer of epithelial and stromal cells lining the uterus with its key 51
physiological function in embedding the embryo/blastocyst for implantation [1-3]. It undergoes 52
tightly regulated changes throughout the menstruation cycle and is only receptive for implantation 53
within a limited period of time [2], although such endometrial receptivity has not been fully 54
understood. On the other hand, pathogenic changes of the endometrium can lead to severe diseases 55
such as endometrial cancer and endometriosis, a painful disorder commonly seen in gynaecology 56
clinics caused by migration and growth of endometrial tissues outside the uterine cavity [4, 5]. 57
However, mechanisms underlying the detrimental transformation of the endometrium have not 58
been elucidated either. 59
The implanting embryos/placental cells are believed to share striking similarities with 60
invasive cancer cells, and common pathways are activated in the endometrium during implantation 61
and tumorigenesis [6]. For instance, Wnt/β-catenin signaling has been observed to be activated in 62
the endometrium upon the presence of the blastocyst in the uterus in mice [7-9]. Conditionally 63
ablation or overexpression of β-catenin in the mouse uterus results in fertility defects and abolishes 64
decidualization [10], an endometrial stromal cell differentiation process required for embryo 65
implantation [11]. Aberrant activation of Wnt/β-catenin has also been reported in patients with 66
endometriosis [12] or endometrial cancer [13]. However, how Wnt/β-catenin signaling is regulated 67
in the endometrium physiology or pathophysiology remains largely unexplored. 68
Multi-drug resistance protein 4 (MRP4), a member of the ATP-binding cassette (ABC) 69
transporter subfamily C, is best known for its transporter function to exclude various drugs and 70
therefore considered as a potential target to prevent drug-resistance, particularly in cancer 71
chemotherapy [14-20]. MRP4 is also known to transport endogenous signaling molecules such as 72
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prostaglandin E2 (PGE2). We have previously demonstrated that MRP4 is expressed in the 73
endometrium and transports PGE2 [21], a key player in embryo implantation [11] [22]. 74
Interestingly, PGE2 is reported to regulate Wnt/β-catenin signaling in other cell types [23, 24]. We 75
undertook the present study to investigate possible role of MRP4 in regulation of Wnt/β-catenin 76
signaling in the endometrium and discovered unexpectedly that MRP4 acts in a manner 77
independent of its PGE2-transporter function, interacting with and stabilizing β-catenin, to sustain 78
the Wnt/β-catenin signaling for endometrial receptivity during embryo implantation, as well as in 79
pathogenic transformation of the endometrium, i.e. endometriosis and endometrial cancer. 80
81
Results 82
MRP4 sustains Wnt/β-catenin signaling independent of transporter-function 83
We first examined possible involvement of MRP4 in regulating Wnt/β-catenin signaling in a 84
human endometrial epithelial cell line, Ishikawa (ISK). Knockdown of MRP4 by siRNAs (siMPR4) 85
in ISK cells reduced levels of the active form of β-catenin (non-phosphorylated), as compared to 86
cells treated with control siRNAs (siNC), in either the presence or absence of Wnt3a (100 ng/mL), 87
a Wnt ligand reported to activate endometrial Wnt/β-catenin signaling [25] (Figure 1A). We 88
further tested whether the effect of MRP4 on β-catenin activity was mediated by its PGE2-89
transporting function. To our surprise, despite successful blockage of PGE2 release (Figure 1B), 90
the treatment with MK-571, the functional blocker of MRP4, for different time periods (10 μM, 91
up to 24 h) did not affect the level of active β-catenin in ISK cells (Figure 1C). Similarly, treatment 92
with exogenous PGE2 (10 μM, up to 24 h) did not affect the level of active β-catenin either (Figure 93
1D), excluding the involvement of PGE2 in mediating the effect of MRP4. To further confirm the 94
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effect of MRP4 on Wnt/β-catenin signaling, we transfected ISK cells with a Wnt/β-catenin 95
signaling reporter, TOPflash luciferase, in conjunction with lenti-virus-packaged shRNAs 96
targeting MRP4 (shMRP4). The results showed that compared to cells treated with control 97
shRNAs (shNC), shMRP4-treated cells exhibited significantly decreased TOPflash luciferase 98
activity (Figure 1E). However, treatment with MK-571 or exogenous PGE2 did not produce 99
significant change in the TOPflash luciferase activity in ISK cells (Figure 1F-G). These results 100
revealed an unexpected role of MRP4 in regulating Wnt/β-catenin signaling, which is independent 101
of PGE2 or MRP4 transporter function. 102
103
MRP4 interacts with and stabilizes β-catenin in human endometrial cells 104
The observed effect of MRP4-knockdown, but not its transporter-functional inhibition, on β-105
catenin activity (Figure 1A and Figure 1D) suggested that the presence of MRP4 protein per ce 106
may affect the protein level of β-catenin in ISK cells. We suspected that such an effect of MRP4 107
on β-catenin might be mediated through protein-protein interaction, a well-documented 108
mechanism to stabilize proteins, by preventing them from degradation in various cell types [26-109
30]. Indeed, immunoprecipitation with an antibody against β-catenin successfully pulled down 110
both β-catenin and MRP4 from protein extracts of ISK cells (Figure 2A). To confirm the effect of 111
MRP4-knockdown on β-catenin protein stability, we treated ISK cells with cycloheximide (10 112
μM), a protein synthesis blocker, and found that the level of active β-catenin was rapidly decreased 113
(by 63%) within 8 h in siMRP4-treated cells, significantly faster than that of siNC-treated cells (by 114
5%) (Figure 2B). On the other hand, with protein synthesis blockage for 8 h, the level of 115
phosphorylated (degrading) β-catenin was reduced in control cells (siNC), whereas the siMRP4-116
treated cells showed sustained level of degrading β-catenin (Figure 2B), suggesting enhanced β-117
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catenin degradation induced by MRP4-knockdown. Moreover, blocking protein degradation by 118
incubating the cells with MG132 (a proteasome inhibitor, 10 μM) or chloroquine (a lysosome 119
inhibitor, 10 μM) for 24 h successfully recovered the active β-catenin levels in siMRP4-treated 120
cells (Figure 2C). To further confirm the role of MRP4 in preventing β-catenin degradation, we 121
used CHIR99021 (CHIR), an inhibitor of glycogen synthase kinase 3 β (GSK3β), which blocks 122
degradation-associated phosphorylation of β-catenin [31]. After treating the cells with CHIR (10 123
μM) for 24 h, the phosphorylated β-catenin, indicating degradation, was largely abolished (Figure 124
2D), the active β-catenin level (Figure 2D) was effectively reversed and the Wnt/β-catenin 125
TOPflash activity (Figure 2E) was recovered in cells with MRP4 knockdown (Figure 2E). These 126
results in together suggested that MRP4, through protein-protein interaction, may stabilize β-127
catenin from degradation and thus sustain Wnt/β-catenin signaling in endometrial epithelial cells. 128
129
MRP4 sustains Wnt/β-catenin signaling in the endometrium for embryo implantation in 130
mice 131
We next asked whether MRP4 would interact with β-catenin in vivo and thus sustain the Wnt/β-132
catenin signaling in the endometrium required for embryo implantation. Double-labeling for β-133
catenin and MRP4 showed co-localization of these two proteins in endometrial epithelial cells in 134
uterine tissues collected from pregnant mice at 5 d.p.c. (days post coitum), the day right after 135
implantation takes place at mid-night of 4 d.p.c. (Figure 3A). Consistently, β-catenin and MRP4 136
can be pulled down together by immunoprecipitation from protein extracts of mouse uterus 137
collected at 5 d.p.c. (Figure 3B), confirming protein-protein interaction between MRP4 and β-138
catenin in mouse endometrium at the period for embryo implantation. We next performed in vivo 139
knockdown of MRP4 in the uterus at implantation period by intrauterine injection with siRNAs 140
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against MRP4 (siMRP4, 50-100 pmole per uterine horn) at 3 d.p.c., shortly before implantation. 141
Such operation led to successful knockdown of MRP4 at both mRNA (Figure 3C, 4 d.p.c.) and 142
protein (Figure 3B and Figure 3D, 5 d.p.c.) levels, and significantly reduced the number of 143
implanted embryos at 7 d.p.c., as compared to the uteri treated with control siRNAs (siNC, Fig.3E). 144
Histological analysis of uterine sections (5 d.p.c.) revealed that most cells in the siMRP4-treated 145
endometrial stoma were loosely arranged and spindle-shaped, distinctively different from the 146
massive amount of compactly arranged and round-shaped decidua cells seen in siNC-treated uteri, 147
indicating defective decidualization, a prerequisite for embryo implantation (Figure 3F, 5 d.p.c.). 148
Moreover, siMRP4-treated uteri (5 d.p.c.) showed lower expression levels of the genes essential 149
to implantation, including Igf2, Lif and Pparg, which are also known to be regulated by Wnt/β-150
catenin signaling [32-34], as compared to siNC treated ones (Figure 3G). 151
We next used a quantitative PCR array for Wnt/β-catenin signaling target genes to examine the 152
mouse uterine tissues (5 d.p.c.) with MRP4 knockdown. 74 of total 84 genes in the array were 153
successfully detected in the uterine tissues with most of them (61 genes) downregulated to different 154
extend in siMRP4-treated uteri as compared to siNC-treated ones (Figure S1). Downregulation 155
(siMRP4 versus siNC) of 28 genes (i.e. Six1, Gdf5, Gdnf, Egr1, Ccnd2, Abcb1a, Ppap2b, Ntrk2, 156
Twist1, Tcf7l1, Id2, Sox9, Wisp1, Met, Dab2, Cubn, Fzd7, Fgf7, Axin2, Jag1, Cdon, Cd44, Ahr, 157
Ctgf, Fgf9, Btrc, Fn1 and Tcf7) showed statistical p value ≤ 0.05 (Figure 4A). Consistently, 158
significantly lower levels of active β-catenin (non-phosphorylated form, Figure 4B) and total β-159
catenin (Figure 4C) were found in the mouse uteri with MRP4 knockdown at 5 d.p.c., as compared 160
to the uteri treated with siNC. These results suggested that the MRP4 knockdown-impaired Wnt/β-161
catenin signaling may underlie the observed implantation defect. To further prove this, we used 162
CHIR to persistently activate Wnt/β-catenin signaling, in conjunction with the in vivo uterine 163
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MRP4 knockdown. The results showed that co-treatment with CHIR (10 μM, intrauterine injection) 164
significantly boosted up the levels of active β-catenin (Figure 4B) and total β-catenin (Figure 4C). 165
Moreover, CHIR improved decidualization (Figure 4D), recovered expression of implantation 166
genes Lif and Pparg (Figure 4E) and, in a dose-dependent manner (10-100 μM), reversed the 167
implantation rate in siMRP4-treated uteri (2.6 ± 0.9 per horn) back to normal levels (6.2 ± 1.3 per 168
horn at 10 μM, and 7.7 ± 0.9 per horn at 100 μM, day 7, Figure 4F), comparable to that of the 169
siNC-treated group (7.0 ± 0.8 per horn). These results in together indicate a critical role of MRP4 170
in sustaining Wnt/β-catenin signaling in the endometrium for embryo implantation. 171
172
MRP4 interacts and correlates with β-catenin in human endometrium 173
We next explored possible interaction/correlation between MRP4 and β-catenin in human 174
endometrial tissues. Similar to that observed in mouse endometrium, immunostaining of MRP4 175
was found to be co-localized with that of β-catenin in normal human endometrial biopsy samples 176
(Figure 5A). We also analyzed a previously published dataset from whole genome gene expression 177
microarrays in endometrium tissues collected from women at mid-secretory phase (receptive 178
window) during IVF treatment (n = 115) (accession number GSE58144) [35], which revealed 179
significant and positive correlations of MRP4 with β-catenin (r = 0.2488, p = 0.0073) and Wnt/β-180
catenin targeting genes including Birc5 (r = 0.3713, p < 0.0001), Bmp4 (r = 0.3104, p = 0.0007), 181
Ccnd1 (r = 0.4398, p < 0.0001), Gdf5 (r = 0.2655, p = 0.0041), Id2 (r = 0.2984, p = 0.0012), Igf1 182
(r = 0.3635, p < 0.0001), Il6 (r = 0.3524, p = 0.0001), Nrp1 (r = 0.3696, p < 0.0001) and Twist1 183
(r = 0.3173, p = 0.0006) (Figure 5B). These results suggest a consistent role of MRP4 in promoting 184
Wnt/β-catenin signaling in receptive endometrium in humans as well. 185
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MRP4 correlation with Wnt/β-catenin signaling in human endometriosis and endometrium 187
cancer 188
Since many of Wnt/β-catenin target genes that were affected by MRP4-kockdown in the mouse 189
endometrium (Figure 3C) are associated with tumorigenesis (e.g. Ccnd2, Twist1, Id2, Sox9, Wisp1, 190
Met, Fzd7, Axin2, Jag1, CD44, Fgf9, and Fn1) [36-47], we next examined possible correlation of 191
MRP4 with Wnt/β-catenin signaling during detrimental transformation of the endometrium (e.g. 192
endometriosis and endometrium cancer) in humans. Western blotting for MRP4 and β-catenin in 193
ectopic endometrial lesions collected from total 19 women diagnosed of ovarian endometriosis 194
(clinical data shown in Table S1) revealed a significant correlation of protein levels between MRP4 195
and total β-catenin (r = 0.7173, p = 0.0005) or active β-catenin (r = 0.5528, p = 0.0141) (Figure 196
6A). We also analysed an available human databases (TCGA Research 197
Network: http://cancergenome.nih.gov/), and found significant correlations between MRP4 with 198
β-catenin mRNA levels in endometrial cancers, in particular at stages I and IV (Figure 6B). We 199
went further to explore possible correlation of MRP4 with β-catenin in other types of cancer and 200
found significant correlations in colorectal, prostate, bladder and breast cancers (Figure S2). These 201
results therefore suggest that in addition to the role in receptive endometrium, MRP4 may also be 202
involved in regulating Wnt/β-catenin signaling in pathogenic endometrial transformation as well. 203
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MRP4 is involved in endometrial tumorigenesis in vitro and in vivo 205
We next performed in vitro and in vivo tumorigenesis with ISK cells in conjunction of MRP4 206
knockdown by designed shRNAs. As shown in Figure 7, two out of five designs of shRNAs against 207
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MRP4 (shMRP4_2 and shMRP4_4) gave high knockdown efficiency (Figure 7A) and were 208
therefore used in the following experiments. Such knockdown of MRP4 (by shMRP4_2 or 209
shMRP4_4) resulted in significantly reduced proliferation (Figure 7B) and colony formation 210
(Figure 7C) in ISK cells, as compared to cells treated with negative control shRNAs (shNC). We 211
further subcutaneously injected ISK cells with MRP4-knockdown (by shMPR4_4) in nude mice. 212
The growth of ISK xenografts in nude mice during 26 days after transplantation was significantly 213
slower in the MRP4-knockdown group (shMRP4), as compared to controls (shNC). Both tumour 214
volume and weight were significantly reduced in MRP4-knockdown group as compared to 215
controls (shNC) at the end (day 26). These results therefore confirmed a role of MRP4 in 216
endometrial tumorigenesis. 217
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Discussion 219
Taken together, the present study has revealed a role of MRP4 in sustaining Wnt/β-catenin 220
signaling in the endometrium through a previously unidentified mechanism, which is independent 221
of its transporter function but instead via interacting with and stabilizing β-catenin (Figure 8). Such 222
a role of MRP4 in the endometrium is involved in both receptivity transition for embryo 223
implantation and pathogenic transformation to develop endometriosis or endometrial cancer 224
(Figure 8). 225
The present observation of impaired decidualization and embryo implantation rate in the 226
MRP4 knockdown mice suggests that MRP4 is required for adequate endometrial receptivity for 227
a successful pregnancy, which is consistent with previously reported smaller litter size in MRP4 228
knockout mice [48]. We have previously reported that blocking MRP4’s PGE2-transport function 229
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also inhibits embryo implantation in mice [49]. In light of the present finding, it appears that the 230
involvement of MRP4 in embryo implantation may be two-fold. First, it provides the mechanism 231
for directional transport of PGE2 to stroma, a key event necessary for inducing stromal 232
decidualization for embryo implantation [11]. Second and unexpectedly, MRP4 acts to sustain the 233
Wnt/β-catenin signaling for embryo implantation, in a PGE2-independent manner. Although PGE2 234
has been reported to regulate Wnt/β-catenin signaling in other cell types, it does not seem to exert 235
detectable effect on the Wnt/β-catenin signaling in the endometrial epithelial cells. The importance 236
of MRP4-dependent Wnt/β-catenin signaling for embryo implantation is evidenced by the fact that 237
the retrieval of active β-catenin levels by blocking β-catenin degradation can rescue the MRP4 238
knockdown-induced implantation defects. This is further supported by the significant correlation 239
between MRP4 and β-catenin, as well as MRP4 and Wnt/β-catenin downstream target genes, in 240
human endometrial biopsy samples collected at mid-secretary phase during IVF treatment. 241
The present study has demonstrated a previously unsuspected protein-protein interaction 242
between MRP4 and β-catenin, which is shown to be important for sustaining the level of active β-243
catenin in the endometrium. The level of β-catenin inside the cell is a key to the canonical Wnt/β-244
catenin signaling [50]. For a successful embryo implantation, endometrial level of β-catenin seems 245
to be crucial since manipulation β-catenin expression, since either depletion or overexpression of 246
β-catenin, in the endometrium, resulted in implantation failure in mice [10]. Of note, protein-247
protein interactions have been well documented to improve protein stability by preventing them 248
from degradation in various cell types [26-29]. The interaction between β-catenin and CFTR, 249
another ABCC family member, has previously been demonstrated to stabilize β-catenin essential 250
to various physiology or pathophysiology events such as embryonic development [27] and 251
intestinal inflammation [26]. In the present study, an enhanced stability of β-catenin by MRP4 is 252
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also observed as evidenced by 1) more rapid degradation of β-catenin (increase in degrading form 253
and decrease in active form) in MRP4-knockdown cells; and 2) reversed β-catenin protein level 254
by blocking either protein degradation in general (MG132 or chloroquine) or β-catenin degradation 255
specifically (CHIR). Therefore, the observed interaction of MRP4 with β-catenin may contribute 256
to the stability of β-catenin and thus underlie the capacity of MRP4 in sustaining Wnt/β-catenin 257
signaling. 258
Apart from its demonstrated role in endometrial receptivity, the presently discovered MRP4-259
dependent Wnt/β-catenin signaling appears to be involved in pathological transformation of the 260
endometrium, i.e. endometriosis and endometrial cancer, as well. A correlation of MRP4 with β-261
catenin is consistently found in clinical samples of endometriosis and endometrial cancer, as 262
demonstrated in the present study or from big databases. Although databases are limited to mRNA 263
level analysis, interaction and correlation between MRP4 and β-catenin proteins in cancer tissues 264
are still possible. Importantly, in vitro and in vivo results confirms a role of MRP4 in endometrial 265
tumorigenesis. While both the expression of MRP4 and the involvement of Wnt/β-catenin 266
signaling have been reported in endometriosis and endometrial cancer [51-53], a possible link 267
between the two has never been suspected. Present findings suggest correlation of MRP4 with 268
Wnt/β-catenin signaling in the endometrium both at embryo implantation and in pathogenic 269
transformation. This is consistent with the general recognition that embryo implantation and 270
tumorigenesis may share common pathways [6], although the difference between the two 271
processes in MRP4 or Wnt/β-catenin signaling awaits further investigation. Interestingly, the 272
analyses of databases for cancers from other tissues, such as colon, prostate, bladder and breast, 273
also show significant correlations between MRP4 and β-catenin, indicating the possible 274
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involvement of the MRP4-dependent Wnt/β-catenin signaling in a wide spectrum of physiological 275
or pathological processes in different tissues. 276
The non-transporter role of MRP4 discovered in the present study raises an interesting 277
possibility that ABC transporters might play an important role in signal transduction independent 278
of their transporter function. The roles of ABC transporters expressed in different tissues may have 279
to be re-examined in light of the present finding. How MRP4 or multi-drug resistance family 280
proteins should be targeted/inhibited in cancer chemotherapy needs to be re-considered, since 281
simple transport-blockage may not be effective to attenuate Wnt/β-catenin signaling, a key 282
pathway activated in tumorigenesis. Moreover, given the versatile roles of Wnt/β-catenin signaling 283
in a variety of different cellular processes and the wide distribution of MRP4, as well as other ABC 284
transporters, the presently discovered novel role of MRP4 in the regulation of Wnt/β-catenin 285
signaling may have far-reaching implications beyond embryo implantation and tumorigenesis. 286
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Methods 288
Mice and intrauterine injection 289
Institute of Cancer Research (ICR) mice were obtained from the Laboratory Animal Service Centre 290
of the Chinese University of Hong Kong. All animal experiments were conducted in accordance 291
with the university guidelines on animal experimentation, and approval by the Animal Ethics 292
Committee of the Chinese University of Hong Kong was obtained for all related procedures. The 293
day a vaginal plug was found after mating was identified as 1 d.p.c.. The intrauterine injection 294
surgery under general anesthesia was performed on 3 d.p.c. after mating as previously described 295
[11]. Dorsal midline skin incision was made and followed by two small incisions into the muscle 296
wall near each ovary to expose the uterine-oviduct connecting region. Reagents were injected at 297
the uterus-oviduct junction toward the uterine lumen. Afterwards, wounds were closed by suture 298
and the mice were placed on a 37 oC warmer till woken up from the anesthesia. The mice were 299
closely monitored for 1 to 4 consecutive days after surgery. Mice were sacrificed by CO2 300
asphyxiation on 4, 5 and 7 d.p.c. 301
302
Cell culture 303
The human endometrial epithelial cell line (adenocarcinoma line), Ishikawa (ISK), was purchased 304
from ATCC (Viginia, United States) and cultured in RPMI-1640 supplemented with 10% fetal 305
bovine serum (v/v) and 1% penicillin-streptomycin (v/v) in 5% CO2 incubators at 37 oC. The cell 306
line was recently authenticated by STR profiling at the Department of Anatomical and Cellular 307
Pathology, Faculty of Medicine, The Chinese University of Hong Kong [54]. 308
309
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RNA extraction, reverse transcription and quantitative PCR 310
Total RNA of cells or uterine tissues were extracted using TRIzol reagent (Invitrogen Life 311
Technologies) according to manufacturer’s instructions. 1 μg total RNA was used for reverse 312
transcription reaction using M-MLV reverse transcriptase (Promega) according to the 313
manufacturer’s instructions. SYBR green assay was used for mouse genes: MRP4 (Primers: 314
forward 5’-TCCCTTGTTCTGGCGAAGAC-3’ and reverse 5’-315
CGAAGACGATGACTCCCTCG-3’), HoxA10 (Primers: forward 5’-316
AATGTCATGCTCGGAGAGCC-3’ and reverse 5’-CTTCATTACGCTTGCTGCCC-3’), Igf2 317
(Primers: forward 5’-GTACAATATCTGGCCCGCCC-3’ and reverse 5’-318
GGGTATGCAAACCGAACAGC-3’), Lif (Primers: forward 5’-319
GCCCAACAACGATGGTGTCA-3’ and reverse 5’-CCCGTGTTTCCAGTGCAGA-3’), Pparg 320
(Primers: forward 5’-GTCACACTCTGACAGGAGCC-3’ and reverse 5’-321
ATCACGGAGAGGTCCACAGA-3’) and Gapdh (Primers: forward 5’-322
TCTCCTGCGACTTCAACAGC-3’ and reverse 5’-AGTTGGGATAGGGCCTCTCTT-3’). 323
Quantitative PCR with SYBR Green Master Mix (Tli RNase H Plus, Takara) was carried out in 324
triplicate on a 96-well plate using a QuantStudio 7 Flex Real-time PCR system (Applied 325
Biosystems). Gapdh was used as an internal control for normalization and data were calculated 326
using the ∆∆CT method. 327
328
Quantitative PCR array 329
1 µg total RNA extracted from each mouse uterine tissue sample was used for reserve transcription 330
by RT2 First Strand Kit (Qiagen), and followed with the RT2 Profiler PCR array for Wnt signaling 331
17
target genes (SABiosciences array PAMM-243Z), according to manufacturer’s guide. The array 332
was performed using SYBR Green PCR Master Mix (Applied Biosystems) in QuantStudio 7 Flex 333
Real-time PCR system (Applied Biosystems). Data were analyzed using the online SABioscience 334
Array data analysis software. 335
336
Protein stability/degradation assay 337
ISK cells with or without MRP4 knockdown were incubated with cycloheximide (10 μM), a 338
protein synthesis blocker, for 0, 2, 4, 6 or 8 h before proteins were extracted and analyzed by 339
immunoblotting. In some experiments, cells were treated with MG132 (a proteasome inhibitor, 10 340
μM) or chloroquine (a lysosome inhibitor, 10 μM) or CHIR99021 (CHIR, 10 μM), an inhibitor of 341
glycogen synthase kinase 3 β (GSK3β), which blocks degradation of β-catenin. 342
343
Immunoblotting 344
The cells or tissues were lysed in ice-cold RIPA lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM 345
NaCl, 1% NP-40, 0.5% DOC and 0.1% SDS) with protease and phosphatase inhibitor cocktail 346
(catalog #78443, Thermo Scientific) for 30 min on ice. Supernatant was collected after 347
centrifugation at 13,000 rpm for 30 min. Equal amounts of protein were resolved by SDS-348
polyacrylamide gel electrophoresis and electro-blotted onto equilibrated nitrocellulose membrane. 349
After blocking in Tris-buffered saline (TBS) containing 5% non-fat milk, the membranes were 350
immunoblotted with primary antibody of target proteins overnight at 4 oC. Antibodies against 351
MRP4 (1:100, Abcam, ab15602); Non-phospho (Active) β-catenin (1:1000, Cell Signaling, 8814), 352
β-catenin (1:1000, Cell Signaling, 9562), p-β-catenin (1:1000, Cell Signaling, 9561), β-tubulin 353
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(1:2000, Santa Cruz, sc-9104) and β-actin (1:5000, Sigma, A1978) were used. After three washes 354
in TBS containing 0.1% Tween 20 (TBST), membranes were further incubated with HRP-355
conjugated antibodies and visualized by the enhanced chemiluminescence assay (GE Healthcare, 356
UK) following manufacturer’s instructions. Densitometry of Western blots was performed by 357
Image J software. 358
359
Immunoprecipitation 360
Cells were lysed with 1 ml of ice-cold lysis buffer (50 mM HEPES, 420 mM KCl, 0.1% NP-40 361
and 1 mM EDTA) with protease and phosphatase inhibitor cocktail for 30 min to 1 h on ice. After 362
centrifugation at 13,000 rpm for 30 min at 4 oC, supernatants were collected as protein extracts, 363
which were re-suspended in 1 ml immunoprecipitation binding buffer (50 mM Tris, pH 7.5, 150 364
mM NaCl) with protease and phosphatase inhibitor cocktail and incubated with primary anti-β-365
catenin (1:50, Cell Signaling, 2677) or mouse IgG (1:100, Santa Cruz, sc-2025) as the control in 366
conjunction with protein A/G beads (GE Healthcare) on rotator overnight at 4 oC. The protein-367
antibody-bead complexes were washed three times with binding buffer using a magnetic separator. 368
In the end, proteins complexes were eluted from the beads by SDS sample buffer (Invitrogen) with 369
4% β-mercaptoethanol, and further analyzed by immunoblotting. 370
371
Immunofluorescence 372
Uterus tissues were harvested and fixed by immersion in 4% paraformaldehyde overnight at 4 oC. 373
After three times washed in PBS, uterine tissues were cryoprotected in 30% sucrose in PBS at 4 374
oC for 24 h, mounted in OCT embedding media (Tissue-Tek, 4583, Sakura), and cryo-sectioned 375
19
into 5 μm sections. Sections were rehydrated in PBS for 5 mins and microwaved in citrate buffer 376
(pH 6.0) for 20 mins to retrieve antigens. After cooled down to room temperature and treated with 377
1% SDS in PBS for 4 mins, sections were blocked with 1% bovine serum albumin in PBS for 15 378
mins, incubated with primary antibody (MRP4, 1:20, Abcam, ab15602) and/or β-catenin (1:100, 379
Cell Signaling, 2677) overnight at 4 oC and subsequently fluorochrome-conjugated secondary 380
antibody (invitrogen) for 1 h at room temperature. DAPI was used to stain cell nuclei. Images were 381
acquired with a confocal microscope (Zeiss, Germany). 382
383
RNA interference and gene Knockdown 384
siRNAs against MRP4 (siMRP4, Cat#1299001, Assay-ID HSS115675, Invitrogen; 5'-385
GAGAAAGAAGGAGAUUUCCAAGAUU-3') and a negative control Med GC Duplex (siNC, 386
Cat#12935300, Invitrogen) were used for knockdown of MRP4. For in vivo knockdown, 50-100 387
pmole siRNAs together with Lipofectamine 2000 (5 μl) in 10 μl Opti-MEM were injected into 388
each uterine horn. In cells, 100 nM siRNAs were transfected with Lipofectamine 2000. Lenti-virus 389
(LV3) packaged shRNAs targeting human MRP4 (shMRP4_1, 5'-390
GAGAAAGAAGGAGATTTCCAAG-3') and scrambled non-coding shRNAs (shNC, 5'-391
TTCTCCGAACGTGTCACGTTTC-3') were purchased from GenePharma (China). The viruses 392
(2×107 TU/ml) were infected into ISK cells with Polybrene (5 μg/ml). The cells were cultured for 393
three passages with puromycin (4 µg/ml) and selected stable clones for further experiments. For 394
tumorigenesis assays, additional 4 designs of shRNAs (shMRP4_2: 5'-395
CCACCAGTTAAATGCCGTCTA-3', shMRP4_3: 5'-GCTCCGGTATTATTCTTTGAT-3', 396
shMRP4_4: 5'-GCCTTCTTTAACAAGAGCAAT-3' and shMRP4_5: 5'-397
GCCTTACAAGAGGTACAACTT-3') together with the shMRP4_1 were purchased. To package 398
20
the shRNAs into lentivirus, envelope vector pMD2.G (12259, Addgene) and packaging vectors 399
psPAX2 (12260, Addgene) together with the shRNAs were transfected into 293T cells using 400
lipofectamine 2000 (Invitrogen). Packaged virus was harvested 72 h afterwards and used for next 401
transfection into ISK cells for 48 h. Virus-transfected ISK cells were grown in the presence of 402
puromycin (4 µg/ml) for 14 days to select cells with stable expression of shRNAs. 403
404
Luciferase assay 405
Cells were transiently transfected with TOPflash reporter gene (TCF reporter plasmid, 21-170, 406
Millipore) and Renilla-Luc as an internal control, respectively. Cells were harvested 48 h after 407
transfection in passive lysis buffer (Promega) and assayed by the Dual Luciferase Reporter Assay 408
System (Promega) using GloMax luminometer (Promega). Renilla luciferase activity was used 409
for normalization. 410
411
PGE2 ELISA 412
ISK cells were incubated with 1 % FBS in DMEM culture medium for 8 h to synchronize the cells 413
before FBS-free DMEM medium was used for all the treatments. Cell-free supernatant with PGE2 414
content was collected and measured using an EIA kit (Cayman Chemical, 514010). 415
416
Human tissue collection 417
Women at the Second People's Hospital of Shenzhen diagnosed of ovarian endometriosis were 418
recruited for the study. Tissues were collected at the proliferative phase from walls of the ovarian 419
21
endometriomas during gynecological laparoscopic surgery. All samples were collected with 420
informed consent from each woman and approval by the Institutional Review Board of the Second 421
People's Hospital of Shenzhen (Ethics Approval No. 201306015). Individuals receiving hormone 422
therapy or anti-inflammatory agents were excluded. Clinical data are shown in Table S1. 423
424
MTT assay 425
Cells were seeded in 96-well plates at a density of 2000 cells per well and incubated for 1-4 days 426
at 37 °C. Directly after removal of the medium, 200 μl of MTT (0.5 mg/mL, Sigma-Aldrich) was 427
added in each well for incubation at 37 °C for 4 h. The medium and MTT solution were removed 428
from each well and formazan crystals were dissolved in 100 μl of DMSO before the absorbance 429
was measured at 570 nm using a plate spectrophotometer. 430
431
Colony formation assay 432
Cells were seeded into 6-well plates at a density of 500 cells per well. The growth medium was 433
replaced with fresh ones every 2 days. After culturing for 10 days, cells were fixed with 434
methanol and stained with crystal violet. Colonies comprising > 50 cells were counted. 435
436
In vivo tumorigenicity 437
Ncr-nu/nu-nude mice were provided by Laboratory Animal Unite, the University of Hong Kong. 438
The protocols were performed after approval by the Animal Ethics Committee, the University of 439
Hong Kong according to issued guidelines. 3 × 106 of cells mixed with an equal volume of 440
22
matrigel (BD Pharmingen) were injected subcutaneously at the back of 6-week-old female Ncr-441
nu/nu-nude mice. Tumor sizes were monitored every 3 days using digital vernier calipers, and 442
tumor volumes were calculated using the formula [sagittal dimension (mm) × cross dimension 443
(mm)2]/2 and expressed in mm3. 444
445
Data availability 446
Human gene expression datasets of IVF endometrium (accession number: GSE58144), colorectal 447
cancer (GSE24551, GSE24549 and GSE75315), prostate cancer (GSE46691 and GSE21034), 448
bladder cancer (GSE57933) and breast cancer (GSE12093) were retrieved from the Gene 449
Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo). Datasets of endometrial 450
carcinoma (ID: UCEC) and colon carcinoma (ID: COAD) were retrieved from The Cancer 451
Genome Atlas (TCGA, http://cancergenome.nih.gov/). Data from normal or undetermined biopsy 452
samples were excluded for analyzing cancer datasets. R2 platform (http://r2.amc.nl) was used for 453
data retrieval and analysis. Correlation analysis was performed using Log2 transformed microarray 454
data. 455
456
Statistical analysis 457
The software GraphPad Prism 6.0 was used for graphing and statistical analyzing the data. Data 458
are shown as mean ± SEM. Student’s paired or unpaired t-test was used for two-group comparison. 459
One-way or Two-way ANOVA was used for comparing three or more groups. Pearson test was 460
used for correlation analysis. P < 0.05 was considered as statistically significant. The variance was 461
calculated to be similar between comparison groups or otherwise corrected for particular tests. 462
23
463
Acknowledgement 464
The work was supported in part by Early Career Scheme (Y.C.R. No.24104517) and General 465
Research Fund of Hong Kong (Y.C.R. No.14112814), Natural Science Foundation of China 466
(Y.C.R, No. 81471460; F.Z, No. 81671860; Y.W, No. 81571390), Health and Medical Research 467
Fund of Hong Kong (Y.C.R. 15161441), Theme-based Research Scheme of Hong Kong (Y.C.R. 468
No. T13-402/17N), and Start-up fund at the Hong Kong Polytechnic University. 469
470
Author contribution 471
Y.C.R., H.C.C. and J.J.C.: conception and design; J.J.C., Z.J.X., Y.C.R., Y.W., M.K.Y., W.Q.H. 472
and X.S., experiments and/or data analysis; H.C., and Y.G.D.: clinical materials and consultancy; 473
M.P.W., X.J.M., and F.Z.: intellectual input and supervision; J.J.C, H.C.C. and Y.C.R.: article 474
writing with contributions from other authors. 475
476
Reference 477
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610
611
27
Figure legends 612
Figure 1. MRP4 sustains Wnt/β-catenin signaling independent of transporting PGE2 in human 613
endometrial epithelial cells. 614
A) Western blots with quantification for MRP4 and active β-catenin in human endometrial epithelial cells 615
(ISK) treated with siRNAs targeting to MRP4 (siMRP4) or non-silencing controls (siNC) in the presence 616
or absence of Wnt3a (100 ng/ml). n = 6. *P < 0.05, **P < 0.01 by Student’s t test. 617
B) ELISA detection of PGE2 levels in medium incubated with or without MK-571 (10 μM, MRP4 618
transporter function blocker) cells, n = 4 biological replicates. **P < 0.01 by Student’s t test. 619
C-D) Western blots with quantification for active β-catenin in cells incubated for 0-24 h with MK-571 620
(10 µM, C) or PGE2 (10 µM, D). n = 4. ns: not significant with P > 0.05, by Student’s t test. 621
E-G) Measurement of luciferase activity in TOPflash (β-catenin/TCF reporter)-transfected (48 h) in cells 622
treated with shRNAs against MRP4 (shMRP4) or shNC (E, n = 3), MK-571 (10 µM, F, n = 4) or PGE2 623
(10 µM, G, n =4). **P < 0.01, ns: not significant with P > 0.05, by Student’s t test. 624
625
Figure 2. MRP4 stabilizes β-catenin in human endometrial epithelial cells 626
A) Representative western blots for MRP4 and active β-catenin in ISK cells before (Total lysate) and 627
after immuno-precipitated (IP) for β-catenin or IgG as the IP control. n = 3. 628
B) Western blots with quantification for MRP4, active (non-phosphorylated), degrading (phosphorylated, 629
p-β-catenin) and total (phosphorylated and non-phosphorylated) β-catenin in siNC- or siMPR4-treated 630
cells after incubation with cycloheximide 0-8 h (10 μM, a protein synthesis inhibitor). n = 3. **P < 0.01, 631
***P < 0.01 by two-way ANOVA with Bonferroni post hoc test. 632
28
C) Western blots and corresponding quantification for MRP4 and active β-catenin in cells transfected 633
with siMRP4 or siNC, treated with MG132 (10 μM, 24 h, a proteasome inhibitor) or Chloroquine (10 μM, 634
24 h, a lysosome inhibitor). n = 3. *P < 0.05, **P < 0.01 by Student’s t test. 635
D) Western blots with corresponding quantification for active β-catenin and p-β-catenin in cells 636
transfected with siNC or siMRP4 (100 pmole per uterine horn) and co-treated with CHIR-99021 (CHIR, 637
10 µM, 24 h, an inhibitor of glycogen synthase kinase-3β blocking degradation-associated 638
phosphorylation of β-catenin) or DMSO as the control. n = 5. **P < 0.01, ns > 0.05 by Student’s t test. 639
E) Measurement of luciferase activity in TOPflash (β-catenin/TCF reporter)-transfected in human 640
endometrial epithelial cells treated with shMRP4 or shNC, co-treated with or without CHIR (10 µM, 24 641
h), n = 4. **P < 0.01, ns > 0.05 by Student’s t test. 642
643
Figure 3. MRP4 interacts with β-catenin in the endometrium required for embryo implantation in 644
mice. 645
A) Confocal images of immunofluorescence labelling for MRP4 (red) and β-catenin (green) in mouse 646
endometrium at 5 d.p.c. (days post coitum). Bars = 5 μm. 647
B) Representative western blots for MRP4 and β-catenin in mouse uterine tissues (48 h after 648
intrauterinally injected with siMRP4 or siNC, 100 pmole per uterine horn) before (Total lysate) and after 649
immuno-precipitated (IP) for β-catenin or IgG as the IP control. n = 3. 650
C-D) Quantitative PCR (qPCR) analysis of (C, 4 d.p.c, ) and immunofluorescence staining (D, 5 d.p.c.) 651
for MRP4 in mouse uteri treated with siMRP4 or siNC (100 pmole per uterine horn). n = 3. ***P < 0.001 652
by Student’s t test. 653
E) Implanted embryo numbers counted at 7 d.p.c. in uteri treated with siNC or siMRP4 (50-100 pmole 654
per uterine horn). Left: Representative photograph of a mouse uterus injected with siMRP4 (100 pmole, 655
29
right horn) or siNC (100 pmole, left horn) with arrows indicating implantation sites. n = 4-7. *P < 0.05, 656
**P < 0.01 by Student’s t test. 657
F-G) Representative images of hematoxylin & eosin staining (F, 5 d.p.c, n = 4 mice) and qPCR analysis 658
of Hoxa10, Igf2, Lif, Pparg (G, 5 d.p.c, n = 3-5 mice) in mouse uteri treated with siMRP4 or siNC (100 659
pmole per uterine horn). *P < 0.05, **P < 0.01 by Student’s t test. Scale bars = 5 (A, D) and 50 (F) μm. 660
661
Figure 4. MRP4 sustains Wnt/β-catenin signaling in the endometrium for embryo implantation in 662
mice 663
A) qPCR array for Wnt/β-catenin target genes showing downregulation of Six1, Gdf5, Gdnf, Egr1, Ccnd2, 664
Abcb1a, Ppap2b, Ntrk2, Twist1, Tcf7l1, Id2, Sox9, Wisp1, Met, Dab2, Cubn, Fzd7, Fgf7, Cebpd, Axin2, 665
Igf1, Jag1, Cdon, Cd44, Ahr, Ctgf, Cdh1, Fgf9, Lrp1, Btrc, Fn1 and Tcf7 in 3 pairs of mouse uterine 666
horns (5 d.p.c.) treated with siMRP4 or siNC (100 pmole per uterine horn). n = 3 mice. P values are *(< 667
0.05), **(< 0.01), ***(< 0.001), or as indicated by paired Student’s t test. 668
B-C) Representative western blots (upper) with quantification (lower) for active β-catenin (B) and 669
immunofluorescence staining for total β-catenin (C) in mouse uteri (5 d.p.c.) injected with siNC or 670
siMRP4 (100 pmole per uterine horn) and co-treated with CHIR-99021(CHIR, 10 µM, a Wnt/β-catenin 671
activator) or DMSO as the control. n = 3. **P < 0.01, ns > 0.05 by Student’s t test. 672
D) Representative images of hematoxylin & eosin staining in mouse uteri (5 d.p.c) with MRP4 673
knockdown (siMRP4) and treatment with DMSO or CHIR (10 µM). n = 4 mice. 674
E) qPCR analysis of Igf2, Lif and Pparg in 5 d.p.c. mouse uteri injected with siNC or siMRP4 (100 pmole 675
per uterine horn) co-treated with CHIR (10 µM) or DMSO as the control. n = 3-5, *P < 0.05 by Student’s 676
t test. 677
30
F) Implanted embryo numbers counted at 7 d.p.c. in uteri treated with siNC or siMRP4 (100 pmole per 678
uterine horn) and CHIR (10-100 µM) or DMSO as the control. n = 6-7, *P < 0.05, **P < 0.01 by One-679
way ANOVA. 680
681
Figure 5. MRP4 interacts with β-catenin and correlates with Wnt/β-catenin signaling genes in 682
human endometrium 683
A) Confocal images of immunofluorescence labelling for MRP4 (red) and β-catenin (green) in normal 684
human endometrial tissues. Bars = 10 μm. 685
B) Correlation analysis of mRNA levels of MRP4 and β-catenin or Wnt/β-catenin targeting genes (i.e. 686
Birc5, Bmp4, Ccnd1, Gdf5, Id2, Igf1, Il6, Nrp1 and Twist1) in human endometrial tissues collected at 687
mid-secretory phase during IVF treatment (n = 115). Data were retrieved from a previously published 688
dataset (GSE58144, http://www.ncbi.nlm.nih.gov/geo). Values of r and P are shown for each analysis by 689
Pearson correlation test. 690
691
Figure 6. MRP4 correlates with β-catenin in human endometriosis and endometrial cancer. 692
A) Western blots for MRP4, β-catenin, active β-catenin and correlation analysis in human endometriotic 693
samples (n = 19). Values of r and P are shown for each analysis by Pearson correlation test. 694
B) Correlation analysis of MRP4 and β-catenin in human endometrial cancer (dataset ID: UCEC from 695
TCGA Research Network: http://cancergenome.nih.gov/) 696
697
Figure 7. MRP4 is involved in endometrial tumorigenesis in vitro and in vivo. 698
31
A) Western blots for MRP4 in ISK cells after stable expression of 5 designs of shRNAs against MRP4 699
(shMRP4_1, 2, 3, 4 and 5) or non-silencing controls (shNC). GAPDH was used as the loading control. 700
B-C) MTT assay (B) and colony formation (C) in ISK cells with MRP4 knockdown by shMRP4_2 or 701
shMRP4_4, or shNC controls. n = 3. *** P < 0.001 by two-way ANOVA in B and one-way AVOVA in 702
C. 703
D-F) Photographs of dissected tumors (D), measurement of tumor volume (E) and weight (F) after ISK 704
cells treated with shNC or MRP4 knockdown by shMRP4_4 were subcutaneously inoculated into the 705
flanks of nude mice (3 x 106 cells per mouse) and grown for 26 days. n = 8. *** P < 0.001 by two-way 706
ANOVA in E, * P < 0.05 by Student’s t test in F. 707
708
Figure 8. Schematic picture showing the role MRP4 in regulation of Wnt/β-catenin signaling 709
in the endometrium. In endometrial epithelial cells, MRP4, through protein-protein interaction, 710
stabilizes β-catenin (β-Cat) from degradation, which accumulates sufficient amount of β-catenin 711
to translocate into the nucleus leading to the transcription of target genes of Wnt/β-catenin 712
signaling pathway. Such a role of MRP4 in the endometrium is involved in both receptivity 713
transition for embryo implantation and pathogenic transformation to develop endometriosis or 714
endometrial cancer. 715
716
Supplementary Figures 717
Supplementary Figure 1. Identification of Wnt Signaling Targets in mouse uterine tissues with 718
MRP4 knockdown 719
32
qRT-PCR array for transcriptional profiling of Wnt signaling target genes in 3 pairs of uterine horns 720
treated with siMRP4 or siNC (100 pmole per uterine horn). The heat map (A) provides a visualization of 721
the fold changes (%) in genes expression and the volcano plot (B) displays statistical significance versus 722
fold-change on the y- and x-axes, respectively between the control siNC groups and siMRP4 treated 723
groups (n = 3 mice). 724
725
Supplementary Figure 2. MRP4 is correlated with β-catenin in other cancers. 726
Correlation analysis of MRP4 and β-catenin in human colorectal cancer (A-C, GSE24551, GSE24549 and 727
GSE75315, http://www.ncbi.nlm.nih.gov/geo), colon adenocarcinoma (D, TCGA-ID: COAD, 728
http://cancergenome.nih.gov/), prostate cancer (E-F, GSE46691 and GSE21034, 729
http://www.ncbi.nlm.nih.gov/geo), bladder cancer (G, GSE57933, http://www.ncbi.nlm.nih.gov/geo) and 730
breast cancer (H, GSE12093, http://www.ncbi.nlm.nih.gov/geo). n is indicated for each dataset. Values of 731
r and P are shown for each analysis by Pearson correlation test. 732
β‐Cat
β‐Catβ‐Cat
Nucleus
β‐Cat
β‐Cat
Inhibited degradation
β‐Cat
Target genesexpression
Interaction & stabilization
Cytoplasm
β‐Cat
MRP4
Accumulation & nuclear translocation
Degradation complex
MRP4
MRP4
GSK3β
Endometrial epithelial cells
Blastocyst
Pathogenic transformation in endometriosis & endometrial cancer
Receptivity transition forEmbryo implantation & pregnancy
β‐Cat
MRP4
Wnt/β‐Cat Signaling
Interaction & stabilization
MRP4 interacts with and stabilizes β-catenin to sustain Wnt/β-catenin signaling in the endometrium, contributing to both receptivity transition for embryo implantation and pathogenic transformation in endometriosis or endometrial cancer.
Graphical Abstract
Ctrl
MK-5
710.0
0.5
1.0
1.5
TO
Pfla
sh-
luci
fera
se a
ctiv
ity(F
old
of
cha
ng
e)
ns
Ctrl
PGE 2
0.0
0.4
0.8
1.2
TO
Pfla
shlu
cife
rase
act
ivity
(Fo
ld o
f ch
an
ge
)
nsG
E
D
FA
ctiv
e-c
aten
in(v
s.-t
ubul
in)
GAPDH
Hours0 2 4 8 12 24
100
MW(kDa)
35
Activeβ-catenin
PGE2
Ctrl
PGE 2
0.0
0.2
0.4
0.6
0.8ns
A
Figure 1
0
1
2
3
4
5 **
siNC siMRP4
MR
P4
(vs.
-tub
ulin
)
B C
Ctrl
MK-5
71
PG
E2
conc
. (pg
/mL)
GAPDH
100
MW(kDa)
35
Hours24120 6
MK-571
Activeβ-catenin
Ctrl
MK-5
710.0
0.2
0.4
0.6
0.8A
ctiv
e-
cate
nin
(vs.
GA
PD
H)
ns
MRP4
β-tubulin
MW(kDa)
siMRP4siNC
WNT3a +
-
++
+++ --
--
-
170
100
55
Activeβ-catenin
shNC
shM
RP40.0
0.4
0.8
1.2 ***
A B
Figure 2
0 2 4 6 80.0
0.5
1.0
1.5
2.0
Time (hour)
Act
ive
-cat
enin
/-a
ctin
**
siNCsiMRP4 ***
170
100
40
MW(kDa)
Active β-catenin
β-actin
siMRP4siNC-++ - + -
++ -- +-
++- - - --- -- ++ Chloroquine
MG132
MRP4
C
D
170
100
35
MRP4
β-catenin
IgG
MW(kDa)
-MG132
Chloroquine0.0
0.4
0.8
1.2
***
ns
ns
*
p-β-catenin(degrading)
0 2 4 6 8
siNC siMRP4
Activeβ-catenin
β-actin
Hours
100
40
MW(kDa)
β-tubulin
Totalβ-catenin100
70
55
170 MRP4
0 2 4 6 8
Act
ive
-cat
enin
(vs.
-tub
ulin
)
**
p--c
aten
in(v
s.-t
ubul
in)
100
70
40
MW(kDa)
Active β-catenin
p-β-catenin
β-tubulin
siMRP4siNC
CHIR+
-
++
+++ --
--
-
DMSO CHIR0
1
2
3siNCsiMRP4
**
ns
E
Impl
ante
d em
bryo
num
ber
in e
ach
horn
Ovary
siNC siNC
siMRP4 siMRP4
** ***
siNC siMRP4
siNC
siMRP4
0.0
0.4
0.8
1.2
MR
P4
mR
NA
(Fol
d of
Cha
nge)
***
A B
C
E
D F
G
Figure 3
β-catenin
MRP4
IgG/non-specific
MW(kDa)
170
100
130
100
40 β-actin
IP: β-catenin-- -+IP: IgG-+ --siNC-+ ++siMRP4+- --
Total lysateIP
0
4
8
12
****
Impl
ante
d em
bryo
num
ber
in e
ach
horn
-
10+
+
-- +
-
-
-
100+siMRP4
siNC
CHIR (μM)
Ovary
A B
E
-++ - -++ -++ -- ++ --
CHIR (10 µM)DMSO
Activeβ-catenin
β-tubulin
siMRP4siNC
MW(kDa)
100
55
siM
RP
4 +
DM
SO
siM
RP
4 +
CH
IR
siNC
siNC + CHIR siMRP4 + CHIR
siMRP4
**
MR
P4
knoc
kdow
n-in
duce
dch
ag
ne
(%
)
Figure 4
C
D
**
P value*************************0.050*0.051******0.051*0.054***
F
Figure 5
AMRP4 β-catenin Merge
BIVF (n=115)
-2 -1 0 1-2
-1
0
1
2r =p =
0.24880.0073
MRP4 (Log2 fold change)
-ca
teni
n (L
og 2
fol
d ch
ange
) IVF (n=115)
-2 -1 0 1-4
-3
-2
-1
0r =p
0.3713< 0.0001
MRP4 (Log2 fold change)
Bir
c5 (
Log
2 fo
ld c
hang
e)IVF (n=115)
-2 -1 0 1-2
-1
0
1r =p =
0.31040.0007
MRP4 (Log2 fold change)
Bm
p4 (
Log
2 fo
ld c
hang
e)
IVF (n=115)
-2 -1 0 1-4
-3
-2
-1
0r =p
0.4398< 0.0001
MRP4 (Log2 fold change)
Ccn
d1 (
Log
2 fo
ld c
hang
e)
IVF (n=115)
-2 -1 0 1-2
-1
0
1
2r =p =
0.26550.0041
MRP4 (Log2 fold change)
Gdf
5 (L
og 2
fol
d ch
ange
)
IVF (n=115)
-2 -1 0 1-1
0
1
2
3r =p =
0.29840.0012
MRP4 (Log2 fold change)
Id2
(Log
2 fol
d ch
ange
)
IVF (n=115)
-2 -1 0 1-1
0
1
2
3r =p =
0.31730.0006
MRP4 (Log2 fold change)
Twis
t1 (
Log
2 fo
ld c
hang
e)
IVF (n=115)
-2 -1 0 11
2
3
4
5r =p
0.3635< 0.0001
MRP4 (Log2 fold change)
Igf1
(Lo
g 2
fold
cha
nge)
IVF (n=115)
-2 -1 0 1-1.0
-0.5
0.0
0.5
1.0r =p =
0.35240.0001
MRP4 (Log2 fold change)
Il6 (
Log
2 fo
ld c
hang
e)
IVF (n=115)
-2 -1 0 1-2
-1
0
1
2r =p
0.3696< 0.0001
MRP4 (Log2 fold change)
Nrp
1 (L
og 2
fol
d ch
ange
)
Active β-catenin
β-catenin
β-actin
MRP4170
MW (kDa)
40
100
100
0.55280.3056
0.71730.0005
Figure 6
A
B
0.23960.0014
-cat
enin
(Lo
g2 fo
ld c
han
ge) Endometrial cancer
(stage 1, n = 97)
0 5 10 1510
11
12
13
14
150.3994
< 0.0001
r =p
MRP4 (Log2 fold change)
Endometrial cancer(stage 4, n = 10)
6 7 8 9 10 1111
12
13
14
150.64460.0442
r =p =
MRP4 (Log2 fold change)
-cat
enin
(Lo
g2 fo
ld c
han
ge)
Endometrial cancer(stage 2, n = 24)
0 5 10 1510
11
12
13
14
150.17910.4024
r =p =
MRP4 (Log2 fold change)
-cat
enin
(Lo
g2 fo
ld c
han
ge)
0 5 10 1510111213141516 -0.07800
0.6105r =p =
MRP4 (Log2 fold change)
-cat
enin
(Lo
g2 fo
ld c
han
ge) Endometrial cancer
(stage 3, n = 45)
Figure 7
0.0
0.5
1.0
1.5
2.0
MTT
24 48 72 96
shNCshMRP4_2shMRP4_4
******
shNC
shMRP4
***
Num
ber
of C
olon
ies
MRP4
GAPDH
shNC 1 2 3 4 5
shMRP4
shMRP4_2
shMRP4_4
shNC
A B
C
D
E F
β‐Cat
β‐Catβ‐Cat
Nucleus
β‐Cat
β‐Cat
Inhibited degradation
β‐Cat
Target genesexpression
Interaction & stabilization
Cytoplasm
β‐Cat
MRP4
Accumulation & nuclear translocation
Degradation complex
MRP4
MRP4
GSK3β
Endometrial epithelial cells
Blastocyst
Pathogenic transformation in endometriosis & endometrial cancer
Receptivity transition forEmbryo implantation & pregnancy
β‐Cat
MRP4
Wnt/β‐Cat Signaling
Interaction & stabilization
Figure 8
Supplementary Table 1
Subject# AgeEndometriosis
Stage
Affected ovary (Unilateral or
bilateral)
Number of gravidity (G) and parity (P)
Chronic pelvic pain (No/mild/medium/severe)
1 25 III Unilateral/Left G0P0 Severe2 27 Undetermined Unilateral/Left G0P0 Medium3 25 II Unilateral/Left G0P0 Medium4 25 IV Unilateral/Right G0P0 Severe5 42 II Unilateral/Left G3P1 Mild6 34 II Unilateral/Right G0P0 Mild7 32 III Unilateral/Right G1P0 Medium8 39 III Unilateral/Right G1P0 Mild9 31 III Bilateral G0P0 No
10 25 III Unilateral/Left G0P0 No11 27 II Unilateral/Left G1P0 No12 27 III Unilateral/Left G1P0 Medium13 29 III Unilateral/Right G0P0 Medium14 30 III Bilateral G0P0 No15 41 III Unilateral/Left G3P0 No16 34 II Bilateral G2P0 No17 29 IV Unilateral/Left G1P0 No18 35 III Unilateral/Left G1P0 No19 42 III Unilateral/Right G2P1 No
Clinical data of subjects diagnosed of ovarian endometriosis
Mouse #1
Mouse #2
Mouse #3
-2 -1 10
1
2
3
46
Log 2 (Fold change: siMRP4/siNC)
-Log
10
(p-v
alue
)
No
chan
ge
p = 0.05
0
Supplementary Figure 1
A B
0.3462< 0.0001
0.4936< 0.0001
-ca
teni
n (
Log
2 fo
ld c
hang
e)
0.6119< 0.0001
-ca
teni
n (
Log
2 fo
ld c
hang
e)
0.2380P<0.0001
-ca
teni
n (
Log
2 fo
ld c
hang
e)
0.6181< 0.0001
-cat
eni
n (L
og2
fold
cha
nge)
0.4294< 0.0001
-cat
eni
n (L
og2
fold
cha
nge)
0.3256< 0.0001
-ca
teni
n (L
og2
fold
cha
nge
)
0.17870.0374
-ca
teni
n (L
og2
fold
cha
nge
)
Supplementary Figure 2
A B C
D E F
G H