1

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 sharon.yc.ruan@polyu.edu.hk (Y.C.R.) or zfei@smu.edu.cn (F.Z.) 17

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2

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

3

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

47

Keywords: MRP4, β-catenin, endometrium, embryo implantation, endometrial disorder 48

49

4

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

5

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

6

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

7

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

13

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

14

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

18

(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