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Targeting cancer stem cell signature gene SMOC-2 Overcomes chemoresistance and inhibits cell proliferation of endometrial carcinoma

Background Endometrial cancer is one of the most common gynecological malignancies and has exhibited an increasing incidence rate in recent years. Cancer stem cells (CSCs), which are responsible for tumor growth and chemoresistance, have been confirmed in endometrial cancer. However, it is still challenging to identify endometrial cancer stem cells to then target for therapy. Methods Flow cytometry was used to identify the endometrial cancer stem cells. Sphere formation assay, western blotting, qRT-PCR assay, cell viability assay, xenograft assay and immunohistochemistry staining analysis were utilized to evaluate the effect of SPARC-related modular calcium binding 2 (SMOC-2) on the cells proliferation and drug resistance. Cell viability assay, qRT-PCR assay, immunofluorescence staining, Co-IP assay and luciferase reporter gene assay were performed to explore the possible molecular mechanism by which SMOC-2 activates WNT/β-catenin pathway. Findings We found the expression of SPARC-related modular calcium binding 2 (SMOC-2), a member of SPARC family, was higher in endometrial CSCs than that in non-CSCs. SMOC-2 was also more highly expressed in spheres than in monolayer cultures. The silencing of SMOC-2 suppressed cell sphere ability; reduced the expression of the stemness-associated genes SOX2, OCT4 and NANOG; and enhanced chemosensitivity in endometrial cancer cells. By co-culture IP assay, we demonstrated that SMOC-2 directly interacted with WNT receptors (Fzd6 and LRP6), enhanced ligand-receptor interaction with canonical WNT ligands (Wnt3a and Wnt10b), and finally, activated the WNT/β-catenin pathway in endometrial cancer. SMOC-2 expression was closely correlated with CSC markers CD133 and CD44 expression in endometrial cancer tissue. Interpretation Taken together, we conclude that SMOC-2 might be a novel endometrial cancer stem cell signature gene and therapeutic target for endometrial cancer. Fund National Natural Science Foundation of China, Scientific and Technological Innovation Act Program of Shanghai Science and Technology Commission, Scientific and Technological Innovation Act Program of Fengxian Science and Technology Commission, Natural Science Foundation of Shanghai.


Introduction
Endometrial cancer is the second most common gynecological malignancy among women worldwide with an estimated 61,380 new cases per year in the United States alone [1,2]. Recently, endometrial cancer was classified into four subtypes, POLE ultramutated, microsatellite instability hypermutated (MSI), copy number low, and copy number high, through an integrated analysis of genomic, transcriptomic, and proteomic characteristics of 373 endometrial carcinomas [3]. Among of the four subgroups, POLE proofreading mutant endometrial cancers have a favorable prognosis despite a strong association with high-grade cancer cells [4]. Patients with MSI tumors were more likely to present with early-stage disease [5,6]. Further, most endometrioid tumors have few somatic copy number alterations (SCNAs) when most serous and serous-like tumors exhibit extensive SCNAs with significantly worse progression-free survival than other groups [3]. Although most patients present with early-stage disease, 15-20% of these tumors still recur after primary surgery in metastatic disease [7,8], which require novel biomarkers or targets identified for diagnosing or treating.
The human endometrium is a highly regenerative tissue that undergoes a steroid-induced monthly cycle of proliferation, differentiation and shedding [9,10]. Evidence showed that endometrial stem cells were present in the endometrium and responsible for the cyclical regeneration of the endometrium each month [11]. The endometrium undergoes regenerative alterations under the influence of circulating ovarian steroid hormones, estrogen and progesterone [12]. CD15 appears to be a marker suitable for the enrichment of basal epithelial progenitor cells demonstrating classic adult stem cell properties [13]. Endometrial cancer was also confirmed to involve stem-like cells, self-renewing cancer stem cells (CSCs) [14]. These cells with stem cell properties are responsible for tumor growth and treatment resistance [15][16][17]. Furthermore, the vast majority of endometrial cancer is estrogen-and progestinrelated [18,19]. A variety of cell surface proteins have been successfully identified as surrogate markers for these cancer stem cells. In endometrial cancer, the surface markers, CD133 and CD44, have been used to enrich CSCs [20,21]. Recently, epithelial membrane protein-2 (EMP2) has been clearly demonstrated as an endometrial cancer stem cellassociated gene [22]. SPARC-related modular calcium binding 2 (SMOC-2), a member of the SPARC family, is highly expressed during embryogenesis and wound healing [23][24][25]. The gene product is a matricellular protein that can stimulate endothelial cell proliferation and migration, as well as angiogenic activity [24,26,27]. Furthermore, SMOC-2 has been identified as the intestinal stem cell signature gene that is required for L1-mediated colon cancer progression [28]. It has been suggested that SMOC-2 may mediate intercellular signaling and cell type-specific differentiation during gonad and reproductive tract development [23]. Thus, we wonder if SMOC-2 has similar characteristics in the CSCs of endometrial cancer.
In this study, we compared the CSCs (CD133 + /CD44 + ) with non-CSCs (CD133 − /CD44 − ) flow-sorted from endometrial cancer cells and found the expression of SMOC-2 was significantly higher in CD133 + / CD44 + cells than in CD133 − /CD44 − cells. Silencing SMOC-2 suppressed the ability of the cells to form spheres and enhanced paclitaxel and cisplatin sensitivity in endometrial cancer cells. We further demonstrated that SMOC-2 physically interacted with Fzd6 and LRP6, enhanced their interaction with canonical WNT ligands and thus activated the WNT/ β-catenin pathway in endometrial CSCs. Furthermore, SMOC-2 was high expression in endometrial cancer tissues and was closely associated with CSC markers expression in endometrial cancer tissue.

Clinical samples
Human endometrial cancer and tissues normal endometrium were obtained from the Department of Obstetrics and Gynecology, Fengxian Hospital, Southern Medical University and the Department of Gynecology, Changzhou Maternal and Child Care Hospital. None of them had received radiotherapy, chemotherapy and other related anti-tumor therapies before surgery. All human tissues were obtained with informed consent and the study was approved by the Research Ethics Committee of Fengxian Hospital, Southern Medical University.

Research in context
Evidence before this study Endometrial cancer stem cells have been prospectively enriched by use of markers such as CD133, CD44 and ALDH. However, there is no conclusive evidence showing that none of these markers is the universal marker for endometrial cancer stem cells. Previously, SPARC-related modular calcium binding 2 (SMOC-2), an extracellular matrix proteins, has been found highly expressing during embryogenesis and wound healing. SMOC-2 mediated cell type-specific differentiation during gonad and reproductive tract development. It was also suggested as a stem cell signature gene in intestine.

Added value of this study
This study identifies SMOC-2 as a cancer stem cell signature gene in endometrial cancer. The expression of SMOC-2 was higher in CD133 + /CD44 + cancer stem cells (CSCs) than that in CD133 − / CD44 − non-CSCs. SMOC-2 expression is greatly increased in spheres compared to monolayer cultures. Using the sphere assay, silencing SMOC-2 reduced the clonogenic potential of endometrial cancer cells and downregulated the expression of SOX2, OCT4 and NANOG, the stemness-associated genes. SMOC-2 enhanced the chemoresistance of endometrial cancer cells. Immunohistochemical staining revealed that the expression of SMOC-2 was positively correlated with CSCs markers expression (CD133 and CD44) in patient tissues. Furthermore, we found that SMOC-2 activated WNT/β-catenin pathway through directly interacting with WNT receptors (Fzd6 and LRP6) in stem cells and enhanced the interaction between receptors (Fzd6 and LRP6) and WNT ligands (Wnt3a and Wnt10b).

Implication of all the available evidence
Together, SMOC-2 may be a signature gene for enrich endometrial cancer stem cells. SMOC-2-Wnt/β-catenin axis may play a crucial role in cell self-renewing and chemoresistance of endometrial CSCs. And SMOC-2-Wnt/β-catenin axis might be a novel target for endometrial cancer therapy.

Sphere formation assay
The spheres of endometrial cancer cells were cultured in DMEM: F12 medium containing 2% B27, 20 ng/ml bFDF, 20 ng/ml EGF and plated in 6-well Ultra-Low Attachment Plates. The growth factors were replenished every 3 days. 14-day-old spheres as primary sphere were harvested using 40 μm cell strainers, dissociated to single cells with trypsin, and then regrown for 7 days, defined as secondary sphere. The diameter and number (N50 μm) for primary and secondary spheres were evaluated and quantified [30].

Flow cytometry
Cell were prepared as single cell suspension for FACS staining. To identify endometrial CSCs, the following antibodies were used: FITC-CD133, PE -CD44 for 30 min at 4°C and second antibody AF647 goat anti-mouse IgG for 30 min at 4°C. The stained cells were acquired for analysis or sorting on LSRFortessa or AriaII (BD). Flow cytometry data was analyzed with FlowJo software (Tree Star Inc.).

Cell viability assay (CCK8 assay)
The cells were plated in 96-well plates at a density of 4000 cells per well with 100 μl of complete culture medium and cultured for 2-5 days. Each group contains five wells. 10 μl Cell Counting Kit-8 (CCK-8, WST-8, Dojindo, Japan) solution was added to each well after 48 h and 120 h. The cell cultures were sparse after 48 h or confluent after 120 h. CCK8 was metabolized to produce a colorimetric dye that was read at 450 nm using a microplate reader.

RNA isolation and real-time qPCR
Total cellular RNA was extracted using Trizol reagent (Takara). PrimeScript RT-PCR kit (Takara) was used to perform the RT according to the protocol. SYBR Premix Ex Taq (Takara) on a 7500 real-time PCR system (Applied Biosystems) was used to determine the SMOC-2 mRNA expression at the following cycling settings: one initial cycle at 95°C for 10 s followed by 40 cycles of 5 s at 95°C and 31 s at 60°C. Data were normalized to 18 s expression and represent the average of three repeated experiments. Prime sequences used for SMOC-2, MYC, CyclinD1, SOX2, OCT4, NANOG and 18 s detection are showed in Supplementary Table 2.

In vivo tumor xenograft model
Six-week-old female athymic nude (nu/nu) mice (SLAC, Shanghai, China) were randomly divided into two groups and injected subcutaneously in the right flank with the stable single cell clones of AN3CA-sh1 and control cells at 5 × 10 6 cells in 100 μl serum-free DMEM medium for each nude mouse. After 6 weeks, mice were killed. The tumors were dissected and fixed with phosphate-buffered neutral formalin for standard histologic examination. Then paraffin embedded tumor samples were cut into 4-μm-thick sections for apoptosis detection. In situ tissue apoptosis was detected according to the protocol of TUNEL kit. Mice were manipulated and housed according to protocols approved by the East China Normal University Animal Care Commission.  activities following the manufacturer's instructions. The ratio of reporter plasmids was determined, each normalized to the luciferase activities of the Renilla-TK-luciferase vector.

Paclitaxel, cisplatin and XAV-939 treatment
The stock solution of paclitaxel and cisplatin were prepared in dimethyl sulfoxide (DMSO) at 1 mM and 50 mM. Cells were treated with 0.01 nM, 0.1 nM, 0.5 nM, 2.5 nM, 12.5 nM, 200 nM, 500 nM, 1000 nM paclitaxel and 0.1 μM, 0.5 μM, 2.5 μM, 12.5 μM, 50 μM, and 200 μM, 500 μM, 1000 μM cisplatin for 48 h. Drugs in the medium were replaced every 24 h. Control cells were incubated with the same volume of DMSO. Cells in the combined paclitaxel/cisplatin with XAV-939 treatment group were cultured in the presence of 5 μM XAV-939 and drugs for 48 h. For in vivo tumor xenograft, cisplatin (5 mg/kg) or normal saline were injected intraperitoneally once every 3 days.

Co-immunoprecipitation (CO-IP) assay and co-culture immunoprecipitation assay
For co-immunoprecipitation assay, AN3CA cell lysates transfected with Flag-tagged SMOC-2 or vector control were subjected to immunoprecipitation with Flag antibody (1:1000) or control IgG for 2 h at 4°C. All immunoprecipitations were performed with protein A/G sepharose (Santa Cruz Biotechnology) on a spinning wheel at 4°C overnight. The beads were collected by centrifugation at 3000 rpm, then washed three times with lysis buffer. The immunoprecipitates were subjected to WB.
For the co-culture IP assay, the plasmids of Fzd6-HA, LRP6-HA, Wnt3a-myc, and Wnt10b-myc were all purchased from Shanghai Generay Biotech Co., Ltd. HEK293T cells were cultured in 6-well plates and individually transfected with 0.5 μg expression plasmids for SMOC-2-Flag or WNT receptor expression plasmids: LRP6-HA or Fzd6-HA, ligand expression plasmids: Wnt3a-myc or Wnt10b-myc using Lipofectamine 3000. After 24 h of co-culture, cell lysates with Fzd6 or LRP6 receptors and cell lysates with Wnt3a or Wnt10b ligands were prepared in a ratio of 1:2:2 to detect the interaction of one receptor (Fzd6 or LRP6) and two ligands (Wnt3a and Wnt10b). The consequent steps were the same as the IP assay mentioned above. Membranes were probed using anti-HA (1:1000), anti-myc (1:1000), or anti-Flag (1:1000) monoclonal antibodies.

In situ proximith ligation assay
Duolink PLA assay was performed to detect the interactions between SMOC-2 and LRP6/Fzd6 in stem cells and non-stem cells (DUO92102, Sigma). After fixation and permeabilization, cells were incubated with DuoLink blocking buffer for 60 min at 37°C, followed by incubation with primary antibodies (SMOC-2, 1:200, Abcam, ab78069; LRP6, 1:200, Abcam, ab134146; Fzd6 1:100, Bioss, bs-13218R) for overnight at 4°C. Cells were incubated with Duolink PLA Probe (anti-rabbit PLA probe PLUS and anti-mouse PLA probe MINUS)) for 1 h at 37°C in a pre-heated humidity chamber, followed by incubation with Duolink Ligation buffer and Amplification buffer according to manufacturer's protocol. Finally, mounted the slides with Duolink In Situ Mounting Medium with DAPI, and analyzed in a fluorescence microscope.

Statistical analysis
Data are presented as means ± standard deviation (SD). Statistical analyses were done using GraphPad Prism 6 for windows. Chi-square test or student's t-test were used to compare the results from different groups. The relationship between SMOC-2 expression and CSCs markers (CD133 and CD44) was analyzed using Chi-square test (χ2 test) and Pearson's correlation test. The relationship between SMOC-2 expression and nuclear localization of β-catenin was used Spearman correlation analysis. Values of P b 0.05 were considered statistically significant.

SMOC-2 is a signature gene of endometrial cancer stem cells
To determine whether SMOC-2 is related to the CSCs of endometrial cancer, CD133 + /CD44 + cells, which have been previously described as cells possessing CSC characteristics, were isolated from AN3CA and Ishikawa cells by flow cytometry. As shown in Fig. 1a, CD133 + /CD44 + was heterogeneously expressed in tested endometrial cells, 1.57% in Ishikawa and 1.66% in AC3CA cells. Then, we measured the SMOC-2 expression level in CD133 + /CD44 + and CD133 − /CD44 − cells by quantitative RT-PCR and western blotting. Our data revealed that SMOC-2 expression was much higher in sorted CD133 + /CD44 + cells than that in CD133 − /CD44 − cells (Fig. 1b-c). It is well-known that CSCs have the ability to proliferate as spheres when cultured under nonadherent conditions [31]. Thus, we applied CSC-rich spheroids as a second model to confirm our results. Quantitative RT-PCR was conducted to analyze the SMOC-2 expression in AN3CA and Ishikawa cells grown either as monolayers or as spheres. The expression of SMOC-2 was significantly increased in spheres compared to monolayer cultures (Fig. 1d). We further investigated whether SMOC-2 have any effects on the clonogenic potential of endometrial cancer. The results showed that silencing of SMOC-2 greatly reduced the clonogenic potential of endometrial cancer by sphere assay in both primary and secondary sphere (Fig. 1e, f and g). Moreover, the expression of stemness-associated genes (SOX2, OCT4, NANOG) were downregulated in SMOC-2silenced cells; overexpressioning of SMOC-2 upregulated the expression of SOX2, OCT4 and NANOG (Fig. 1h, i and Fig. S1a, 1b). These results collectively demonstrated that SMOC-2 might be a potential cancer stem cell marker for endometrial cancer.

SMOC-2 promotes endometrial cancer cell growth in vitro and in vivo
We further investigated whether SMOC-2 has any effect on the cell proliferation of endometrial cancer. The results showed that silencing SMOC-2 had no effect on the proliferation of cells grown at low density (sparse culture). However, interestingly, we found silencing SMOC-2  significantly suppressed the proliferation of cells grown at high density (confluent culture) (Fig. 2a). Normally, cancer cells lose their contact inhibition, a phenomenon that restricts the in vitro growth of normal cells at confluence, so they still grow in confluent condition. Furthermore, we found that the overexpression of SMOC-2 promoted cell proliferation in confluent culture but had no effect on cell proliferation in sparse monolayers (Fig. 2b). These data indicated that SMOC-2 might confer endometrial cancer cells with the ability to overcome their contact inhibition.
To further confirm the effects of SMOC-2 on endometrial cancer cell growth in vivo, stable cell lines were established that were transduced by the lentivirus carrying SMOC-2-short hairpin RNA in endometrial cancer cells (Fig. S1c). Then, Lenti-shSMOC-2/AN3CA cells and Lentivector cells were inoculated subcutaneously into nude mice. The weight and size of the tumors formed by Lenti-sh-SMOC-2 cells were significantly decreased in comparison with the tumors formed by Lentivector cells. The average volume and weight of the tumors in Lenti-sh-SMOC-2 mice were 0.309 ± 0.079 cm 3 and 0.329 ± 0.008 g in contrast to 0.554 ± 0.191 cm 3 and 0.590 ± 0.176 g in control mice (P = 0.042 and 0.025, (Statistical analysis was performed by Student's t-test) (Fig. 2c, d). In short, silencing SMOC-2 in endometrial cancer cells suppressed cell growth at high density in vitro and reduced xenograft tumor growth in vivo.

SMOC-2 enhances paclitaxel-and cisplatin-resistance in endometrial cancer cell
As chemotherapeutic resistance is another feature of CSCs, we next determined whether there is an association between SMOC-2 expression and resistance to two chemotherapeutic agents, paclitaxel and cisplatin, in endometrial cancer. To this end, we treated SMOC-2-silenced cells grown at high density with a series of concentrations paclitaxel or cisplatin. After 48 h treatment, cells survival rates were measured by CCK8 assay and half maximal inhibitory concentration (IC 50 ) of paclitaxel/cisplatin against cell survivability were analyzed. The results showed that IC 50 value of siSMOC-2 groups were less than siCT groups. Silencing SMOC-2 enhanced the cytotoxic effects on AN3CA and Ishikawa cells (Fig. 3a and Table 1). Moreover, overexpressioning of SMOC-2 reduced the cytotoxic effects on HEC-1A and ECC-1 cells in sphere assay (Fig. 3b). This result indicated that SMOC-2 enhanced paclitaxel-and cisplatin-resistance.
To further confirm the results in vivo, Lenti-shSMOC-2/AN3CA cells or Lenti-vector cells were subcutaneously inoculated into nude mice. Athymic mice bearing subcutaneous tumors were treated with cisplatin (3 mg/kg per mouse) or saline once a week for four weeks (four injections in total) when the tumors first became palpable and lasted for 1 week. A significant reduction in the tumor weight and volume was recorded in the Lenti-shSMOC-2 group treated with saline compared to that in the Lenti-vector group treated with saline (P = 0.0004, P = 0.001. Statistical analysis was performed by Student's t-test). Further, tumors derived from the Lenti-shSMOC-2 group treated with cisplatin were significantly smaller than those derived from Lenti-vector group treated with cisplatin (P b 0.0001. Statistical analysis was performed by Student's t-test) (Fig. 3c-d). Apoptosis was significantly increased, and proliferation was suppressed in Lenti-shSMOC-2 tumors versus Lenti-vector tumors and cisplatin treated versus Lenti-vector tumors as detected by TUNEL assay and PCNA staining, respectively. Moreover, SMOC-2 knockdown enhanced cisplatin-induced tumor cell apoptosis and anti-proliferation (Fig. 3e -f). Taken together, these data demonstrated that silencing SMOC-2 enhanced paclitaxel and cisplatin sensitivity in endometrial cancer cells.

SMOC-2 activates the WNT/β-catenin pathway
Having shown the effects of SMOC-2 on endometrial cancer cell proliferation and chemoresistance, we next explored the mechanism by which SMOC-2 alters the function of CSCs. It has been reported that related signaling pathways, such as WNT/β-catenin, Hedgehog and Notch, played essential roles in cancer stem cells. Here, we examined which pathway is involved in the function of SMOC-2 in endometrial cancer cells using a dual-luciferase reporter gene assay. A WNT/β-catenin reporter plasmid (TCF/catenin plasmid), Hedgehog reporter plasmid (GLI plasmid), Notch reporter plasmid (RBP plasmid) and Renilla were transfected into SMOC-2 silencing cells AN3CA and HEC-1A. The results showed that WNT/β-catenin signaling was significantly inhibited in SMOC-2 silenced groups, while the other signaling pathways experienced no significant change (Fig. 4a, Figs. S1d and S2a). We further confirmed that WNT/β-catenin signaling was activated by overexpressing SMOC-2 ( Fig. 4b and Fig. S1e). Moreover, the expression of downstream target genes of WNT/β-catenin signaling (cyclin-D1 and MYC) were downregulated in SMOC-2-silenced cells compared with control cells (Fig. 4c). Immunofluorescence (IF) staining of β-catenin showed that the nuclear localization of β-catenin was more evident in SMOC-2 overexpressing cells than that in control cells (Fig. 4d). Western blotting also showed nuclear β-catenin was significantly increased in SMOC-2 overexpressing cells compared to that in control cells (Fig. 4e). Moreover, immunohistochemical staining of 151 endometrial cancer tissues also showed a positive association between SMOC-2 expression and nuclear localization of β-catenin, R = 0.4266, P b 0.001 (Spearman correlation analysis) (Fig. 4f and Fig. S2b). To further illustrate that SMOC-2 activates WNT/β-catenin signaling in endometrial cancer cells, XAV-939, a small molecule inhibitor of WNT/β-catenin signaling was used for further experiments. We treated HEC-1A and ECC-1 cells with XAV-939 and 50 nM paclitaxel or 1 μM cisplatin at the same time. The data showed that the overexpression of SMOC-2 enhanced chemoresistance in both HEC-1A and ECC-1 cells. These effects were completely abrogated by XAV-939 (Fig. 4g). These results indicated that SMOC-2 activates the WNT/β-catenin pathway by promoting the nuclear translocation of β-catenin. Obstructing the WNT/β-catenin signaling can reverse the effects of SMOC-2 on endometrial cancer cell chemoresistance.

SMOC-2 enhances ligand-receptor interaction of WNT/β-catenin pathway
Next, to investigate how the SMOC-2 activate WNT/β-catenin pathway, we examined the interaction between SMOC-2 and WNT proteins. Co-immunoprecipitation (CO-IP) results showed that SMOC-2 had no direct interaction with the canonical WNT proteins (Wnt3a and Wnt10b). However, we observed an interaction between SMOC-2 and the WNT pathway co-receptors Fzd6 and LRP6 (Fig. 5a). SMOC-2 had no direct interaction with WNT pathway co-receptors Fzd3, Fzd8, LRP1 and LRP7 (Fig. S3). We further investigated the molecular mechanism of SMOC-2 modulating the WNT ligand-receptor interaction. To this end, an interaction assay was performed, which was based on the immunoprecipitation of tagged proteins from the lysates of cocultured HEK293T cells. Co-cultured HEK293T cells were transfected with the expression plasmids of SMOC-2-Flag, Fzd6-HA, LRP6-HA, Wnt3a-myc and Wnt10b-myc (co-culture IP) (Fig. 5b-e). Cell lysates were immunoprecipitated with an HA antibody and immunoblotted with anti-Flag, anti-HA or anti-Myc antibodies. As shown in Fig. 5b, Fzd6 can co-precipitate with SMOC-2, Wnt3a and Wnt10b, and SMOC-2 overexpression can enhance the binding between Wnt3a and Fzd6 (lanes 6 and 8). A similar enhancement of the WNT-FZD interaction by SMOC-2 was also observed in combinations of Wnt10b and Fzd6 (Fig. 5b, lanes 7 and 9). Next, the effect of SMOC-2 on the interaction between Wnt3a/Wnt10b and LRP6 was examined. Similarly, SMOC-2 also enhanced the interaction between Wnt3a/Wnt10b and LRP6 (Fig. 5d-e). Moreover, we tested if the interaction of SMOC-2 with Fzd6/LRP6 occurs differently in stem versus non-stem cells. Duolink in situ proximity ligation assay showed that non-stem cells showed only a few PLA foci, whereas many PLA foci were found in stem cells (Fig. 5f). To further confirm that SMOC-2 direct interacted with Fzd6/LRP6 in activating WNT/ β-catenin pathway, we treated SMOC-2 overexpressioning cells with a series of concentrations paclitaxel or cisplatin and silenced Fzd6/ LRP6 at the same time. The results showed that overexpression of SMOC-2 reduced the cytotoxic effects on both HEC-1A and ECC-1 cells. Silencing of Fzd6/LRP6 reversed the chemoresistance effects of SMOC-2 on endometrial cancer cells (Fig. 5g,Table 2). These results suggest that SMOC-2 directly interacted with Fzd6/LRP6 and enhances the interaction between the canonical WNT ligands and receptors in endometrial cancer CSCs.

SMOC-2 and CSCs markers are co-expressed in endometrial cancer
To further evaluate whether SMOC-2 is a marker for CSCs in endometrial cancer, we investigated the expression level of SMOC-2, CD133 and CD44 in 151 endometrial cancer patients, and analyzed the relevance with clinicopathological parameters. The high expression rate of SMOC-2, CD133 and CD44 were 70.19%, 50.99% and 58.27%, respectively. The results also demonstrated that SMOC-2 expression correlated with pathological grade, depth of myometrial invasion and age (P = 0.000, 0.0469 and 0.0046, Statistical analysis was performed by Student's t-test) ( Table 3). CD133 expression correlated with pathological grade, depth of myometrial invasion and vascular invasion while CD44 expression correlated with FIGO stage, pathological grade and lymph node metastasis (Tables 3-5). Moreover, based on the IHC staining, a positive correlation was found between the SMOC-2 expression and the CD133 expression (R = 0.4122, P b 0.0001, Pearson correlation analysis). The expression of SMOC-2 also had a positively correlation with the expression of CD44 (R = 0.4358, P b 0.0001. Pearson correlation analysis) (Table 6). Obviously, SMOC-2 expression was closely associated with risk factors of poor prognosis and CSC marker expression (Fig. 6).

Discussion
Cancer stem cells (CSCs) have distinct characteristics including selfrenewal, drug resistance, and expression of specific markers that enable their isolation [32]. In endometrial cancer, it is still challenging to identify CSCs. A number of cell surface markers, including CD133, CD44, etc., can identify endometrial CSCs [20,21,33,34]. However, the markers of endometrial CSCs remain controversial. Here, we identified SMOC-2 as a novel signature gene of endometrial cancer stem cells. First, the expression of SMOC-2 was higher in CD133 + /CD44 + cells than that in CD133 − /CD44 − cells. Second, SMOC-2 expression is greatly increased in spheres compared to monolayer cultures. Third, using the sphere assay, silencing SMOC-2 reduced the clonogenic potential of endometrial cancer cells and downregulated the expression of SOX2, OCT4, and NANOG, the stemness-associated genes. Fourth, SMOC-2 enhanced the chemoresistance of endometrial cancer cells. Fifth, immunohistochemical staining revealed that the expression of SMOC-2 was positively correlated with CD133 and CD44 expression in patient tissues. A previous study has also elaborately identified SMOC-2 as a colon cancer stem cell signature gene [28].
It has been reported that SMOC-2 executed its biological function through integrin-dependent signaling [35]. In this study, we demonstrated that SMOC-2 activated WNT/β-catenin signaling in endometrial cancer, which is supported by the following evidence. 1) Using the luciferase reporter assay system, it was revealed that overexpressing SMOC-2 promoted the activation of Wnt/β-catenin signaling, while silencing SMOC-2 reduced the activation of Wnt/β-catenin signaling. 2) Silencing SMOC-2 could significantly inhibit the expression of cyclinD1 and MYC, the important target genes of the Wnt/β-catenin signaling. 3) Both western blotting and immunofluorescence staining revealed that overexpressing SMOC-2 significantly increased the nuclear translocation of β-catenin, which led to activate a downstream pathway. 4) It was discovered through CO-IP assay that SMOC-2 may bridge the Fzd6 and LRP6 proteins through trimer formation, further enhancing Wnt ligand-receptor interactions. Therefore, we concluded that SMOC-2 may participate in the progression of endometrial cancer through the Wnt/β-catenin signaling pathway.  The Wnt/β-catenin signaling pathway has a crucial function in cell proliferation, differentiation, growth, survival, development and fate determination in adult organisms [36]. Moreover, Wnt signaling supports the formation and maintenance of stem and cancer stem cells [37]. In the intestine, downstream of Wnt/β-catenin, the Tcf4-driven target gene program is essential to maintain intestinal crypt stem cells [38]. In the mammary gland, a Wnt ligand, Wnt3a, functions as a ratelimiting, self-renewal factor to clonally expand mammary stem cells [39]. Several studies in skin tumors, intestinal cancer and mammary tumors have demonstrated that Wnt signaling mediates stem cell renewal and proliferation by promoting β-catenin translocation to the nucleus [40][41][42][43][44]. In endometrial cancer, targeting Wnt signaling inhibited the proliferation, migration, invasiveness and tumorigenicity of endometrial cancer stem cells [45]. Our findings identify the extracellular matrix protein SMOC-2 as a unique signaling node that drives self-renewal and therapeutic resistance through the Wnt/β-catenin signal pathway, which provides novel evidence for targeting Wnt signaling in endometrial cancer.
Our study revealed that SMOC-2 may be a stem cell signature gene in endometrial cancer, and we discovered a novel regulator of WNT/βcatenin signaling. However, a key limitation of this study is that we only examined the biological functions of SMOC-2 in an in vitro cell model and xenograft mouse model. We are establishing a patientderived xenograft (PDX) model and genetically engineered mice for further studies.
In conclusion, SMOC-2 is a cancer stem cell-associated matricellular protein in endometrial cancer and promoted cell proliferation, enhanced resistance toward chemotherapy both in vitro and in vivo. This effect was associated with the activation of WNT/β-catenin signaling. These findings suggest that the SMOC-2-Wnt/β-catenin axis may be a novel target for endometrial cancer therapy.