Abstract
Endometrial cancer (EC) is the most common gynecological malignant tumor. The canonical Wnt/β-catenin signaling pathway plays a key role in regulating carcinogenesis, and the noncanonical Wnt5a-ROR1 pathway is an important regulator of Wnt signaling. However, the molecular mechanism by which ROR1 influences Wnt signaling in EC is not known. In this study, we found that ROR1 is expressed at higher levels in tumor tissues and blood samples from patients with stage II EC compared with patients with stage I disease. In vitro, human EC cell lines stably overexpressing ROR1 proliferated more rapidly and formed larger colonies than control cells. Consistent with this, overexpression or knockdown of ROR1 increased or decreased, respectively, the percentage of EC cells in M phase of the cell cycle. Elevated levels of ROR1 were associated with increased expression of Wnt5a and of cyclin D1 and c-Myc, two components of the Wnt signaling pathway. Finally, nude mice grew significantly larger tumors after subcutaneous injection of ROR1-overexpressing EC cells compared with control cells. These findings indicate a novel role for ROR1 in promoting EC cell proliferation by upregulating Wnt5a and stimulating the Wnt/β-catenin signaling pathway.
Keywords: ROR1, Wnt5a, endometrial cancer, proliferation, Wnt signaling pathway
Introduction
Endometrial cancer (EC) is the most common gynecologic malignancy, with an incidence of 47,130 new cases and 8010 deaths in 2014 [1]. Type 1 EC, also known as endometrioid EC, accounts for 70%-80% of all EC cases [2]. Although proliferation is a common feature of the disease and hampers cancer therapy, the underlying molecular mechanisms controlling EC cell proliferation are not clear.
The receptor-tyrosine-kinase-like orphan receptor 1 (ROR1), a transmembrane protein and member of the receptor tyrosine kinase family, is involved in skeletal and neural development [3], but is rarely expressed in adult tissues [4]. However, ROR1 is overexpressed in malignant tumors [5] where it is associated with aggressive disease and poor prognosis [6].
The Wnt signaling pathway is known to be involved in endometrial carcinogenesis [7]. Wnt5a is the key ligand activating the Wnt signaling pathway, and promotes cancer cell proliferation and cell migration during organogenesis. Wnt5a is overexpressed in ovarian cancer cells in vivo and high levels are associated with increased cell proliferation in vitro [8].
Binding of Wnt5a to its receptor ROR1 [9] mediates enhanced tumor cell growth [10] through both the canonical and noncanonical Wnt signaling pathways [11]. Hence, we considered that ROR1 might have clinicopathological significance in EC.
In this study, we analyzed the relationship between ROR1 and EC by examining the expression of ROR1 in blood samples and paraffin-embedded tumor tissues from EC patients, the effect of ROR1 overexpression or knockdown on EC cell proliferation in vitro, and the correlation between expression of ROR1 and that of Wnt5a and proteins in the Wnt signaling pathway.
Materials and methods
Patient samples and EC cell lines
The protocols used in this study were approved by the Hospital’s Protection of Human Subjects Committee. Samples were obtained from the Shanghai First People’s Hospital Affiliated to Shanghai Jiaotong University. All patients provided written informed consent.
Samples were taken from 52 patients with EC, of whom 25 had stage I and 27 had stage II disease, between Januay 2014 and December 2015. The histological grade (G2-G3) and stage were established according to the criteria of the International Federation of Gynecology and Obstetrics surgical staging system (2009) [12].
Blood samples were taken from 26 patients with EC (12 satge I, 14 stage II) for PCR analysis.
The human EC cell lines Ishikawa and HEC-1B were obtained from and maintained as recommended by the American Type Culture Collection (Manassas, VA).
Immunohistochemistry
Immunohistochemistry (IHC) was performed on sections of paraffin-embedded patient tumor samples according to the manufacturer’s recommendations. Staining was scored independently by two pathologists who were blinded to the clinical and pathological data. Protein staining was evaluated as described [13].
RNA extraction and analysis
RNA extraction from blood samples and reverse transcription were performed according to the manufacturer’s protocols. Human ROR1 was amplified by real-time PCR and ROR1 mRNAs levels were normalized to β-actin using dCt values. The primer pairs used were: human ROR1 forward: 5’-TAATCGGAGAGCAACTTCA-3’, reverse: 5’-TGTAGTAATCAGCGGAGTAA-3’. β-actin forward: 5’-TTAATCTTCGCCTTAATACTT-3’, reverse: 5’-AGCCTTCATACATCTCAA-3’.
Western blot analysis
Western blotting was performed as previously described [14]. The primary antibodies were rabbit anti-ROR1 (Abcam, Cambridge, UK; 135669), rabbit anti-GAPDH (Abcam, ab9485), rabbit anti-Wnt5a (Abcam, ab72538), rabbit anti-cyclin D1 (Abcam, ab16663), rabbit anti-c-Myc (Abcam, ab32072), and rabbit anti-Ki67 (Abcam, 16667).
Establishment of transfected EC cells lines
HEC-1B cell lines transfected with an ROR1 overexpression plasmid or an ROR1 shRNA-encoding plasmid were established as previously described [15].
Cell viability assay
Viable cells was enumerated using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described [16].
Cell cycle analysis by flow cytometry
Cells were transfected with ROR1 plasmids for 48 h and then steps were performed according to the manufacturer’s protocol. After FACS analysis, the cell profiles were analyzed using ModFit software.
Plasmids
The ROR1 cDNA plasmid was purchased from Origene (Rockville, MD, USA; RC214967). The ROR1 RNAi (shRNA) plasmid was constructed by cloning the target sequence (5’-TTACTAGGAGACGCCAATA-3’) into the pCMV6-Entry vector.
Nude mouse study
BALB/c nude mice (4-6-week-old) were purchased from the Shanghai SLAC Laboratory Animal Company. Mice (n=5/group) were injected subcutaneously with 1×107 HEC-1B cells stably transfected with the ROR1 overexpression plasmid. The mice were sacrificed on day 28 after injection, and the tumors were excised and weighed.
Statistical analysis
Data are expressed as the mean ± SD. Statistical significance between two groups was determined by the Student’s t-test. P values <0.05 were considered statistically significant. All experiments were repeated in triplicate.
Results
ROR1 expression is increased in stage II EC
The clinicopathological features of the 52 EC patients in this study are shown in Table 1. We analyzed ROR1 levels in EC tissues by IHC staining of paraffin-embedded tumor tissues from the 52 patients and found that ROR1 was expressed at higher levels in stage II tissues than in stage I tissues (Figure 1A). Low expression of ROR1 was observed in the stage II samples. To confirm the tissue finding, we examined expression of ROR1 in blood samples from the same patients and obtained similar results (Figure 1C).
Table 1.
Clinical features of the patients
| Characteristic | Stage I | Stage II N (%) | Total N (%) | P |
|---|---|---|---|---|
| Age | 0.764 | |||
| <50 | 11 (21) | 13 (25) | 24 (46) | |
| ≥50 | 14 (27) | 14 (27) | 28 (54) | |
| Grade | 0.956 | |||
| П | 10 (19) | 11 (21) | 21 (40) | |
| Ш | 15 (29) | 16 (31) | 31 (60) | |
| Stage | 0.625 | |||
| II | 9 (23) | 8 (9) | 17 (33) | |
| III | 16 (77) | 19 (91) | 35 (67) |
Figure 1.
ROR1 is significantly highly expressed in stage II endometrial cancer clinical samples. A, B. Immunohistochemistry was used to assayed ROR1 expression. ROR1 is significantly highly expressed in stage II EC samples. Error bar=50 μm. C. Real-time PCR analyzed the expression of ROR1 in blood samples from stage I (n=12) and stage II (n=14). The expression was the fold change relative to a tissue (control, expression=1). The results were expressed as Log 10 (2-ΔΔCt). (*P<0.05).
Generation of stably transfected EC cells
To dissect the function of ROR1 in EC, we constructed ROR1 overexpression and knockdown plasmids to upregulate or silence ROR1 expression. We then transfected HEC-1B cells with the plasmids and selected stably transfected clones for further study. Western blot analysis was performed to confirm the increase or decrease in ROR1 expression levels (Figure 2A).
Figure 2.
ROR1 promoted the proliferation of EC cells. A. ROR1 instantaneously transfected cells were identified. Western blot was used to detect the expression of ROR1 in plasmids. B. MTT was used to analyze the cell proliferation. The overexpression of ROR1 increased the proliferation of cells. By contrast, the down expression of ROR1 decreased the proliferation. (P<0.05). C. Colony forming assays were used on EC cells which were transfected with ROR1 overexpressed or knockdown plasmids. EC cells overexpressing ROR1 formed more numerous and larger colonies, and conversely, ROR1 knockdown resulted in smaller and fewer colonies. D. The cell cycle was analyzed by flow cytometry. The percentage of cells in M phase was higher for ROR1-over-expressing HEC-1B and Ishikawa cells. (*P<0.05).
ROR1 increases the proliferation of EC cells
MTT assays were performed to study the influence of ROR1 expression on the proliferation of EC cells. HEC-1B cells were transfected with ROR1 overexpression or ROR1 shRNA plasmids for 48 h, and cell viability was analyzed after incubation for a further 24, 48, 72 or 96 h. As shown in Figure 2B, cells overexpressing ROR1 exhibited high viability compared with the control cells, whereas ROR1 knockdown lowered the viability. This result suggests that ROR1 promotes the proliferation of EC cells.
Similar effects of ROR1 modulation were obtained in colony forming assays. Thus, EC cells overexpressing ROR1 formed more numerous and larger colonies than control cells (Figure 2C), and conversely, ROR1 knockdown resulted in smaller and fewer colonies.
The EC cell cycle was analyzed by flow cytometry. The percentage of cells in M phase was higher for ROR1-over-expressing HEC-1B and Ishikawa cells (24.1±1.9% and 26.5±1.3%, respectively) than for control cells (13.2±1.3% and 15.2±1.2%, respectively), (P<0.05) (Figure 2D). In contrast, the percentage of cells in M phase was lower in ROR1 knockdown HEC-1B and Ishikawa cells (6.0±1.4% and 7.2±1.5%, respectively) than in the control cells (P<0.05) (Figure 2D).
ROR1 activates the Wnt signaling pathway in EC cells in vitro
Our results thus far indicate that high levels of ROR1 promote the proliferation of EC cells. Therefore, we next investigated the mechanism by which this might occur. Cells were transfected with ROR1 overexpression or shRNA plasmids for 48 h, and the expression of Wnt5a was investigated. As shown in Figure 3A, ROR1 overexpression correlated with Wnt5a levels in the nucleus, and conversely, downregulation of ROR1 decreased the level of nuclear Wnt5a.
Figure 3.
ROR1 activated the noncanonical Wnt pathway in HEC-1B cells. A. Immunofluorescent analysis showed that ROR1 increased the expression of Wnt5a. B, C. Western blot experiments were performed to detect the levels of proteins in Wnt signaling pathway. ROR1 overexpression increased the expression of Wnt5a, cyclinD1 and c-Myc. (*P<0.05).
To further investigate the effect of ROR1 on Wnt5a levels and the Wnt signaling pathway, we performed western blot analysis of EC cells 72 h after transfection with ROR1 overexpression or shRNA plasmids. The results indicated that levels of Wnt5a and two proteins in the Wnt pathway (cyclinD1 and c-Myc) were increased by ROR1 overexpression and decreased by ROR1 knockdown (Figure 3B and 3C).
ROR1 activates the Wnt signaling pathway in EC cells in vivo
To examine the effects of ROR1 levels on the proliferation of EC cancer cells in vivo, we injected BALB/c nude mice (n=5/group) subcutaneously with ROR1-overexpressing or knockdown HEC-1B cells (107 cells/injection). After 28 days, the mice were euthanized, and the tumors were excised and weighed. We found that tumors formed by ROR1-overexpressing cells were significantly bigger than the tumors formed by control. The growth curves also gave similar results. In addition, IHC staining confirmed the results obtained in vitro by showing that Wnt5a, cyclinD1 and c-Myc levels were higher in tumors from the mice injected with ROR1-overexpressing cells compared with control cells (Figure 4).
Figure 4.
ROR1 increased the proliferation of EC cells in vivo. A. The tumors of HEC-1B-ROR1 cells treated group were bigger than control group. B. The tumor grow curve of treated and control group. C. Weight of subcutaneous tumors of treated and control group. (*P<0.05). D. Immunohistochemistry analysis showed that ROR1 increased the expression of Ki67 in samples.
Discussion
In this study, we investigated the effects of ROR1 on the proliferation of EC cells. We first analyzed ROR1 expression in tumor and blood samples from EC patients and then examined the effects of differential ROR1 expression on human EC cells in vitro. Finally, we investigated the mechanism by which ROR1 influences the proliferation of EC cells.
ROR1 is a transmembrane protein belongs to receptor tyrosine kinase families [17] that regulates cellular processes such as proliferation, differentiation and survival, and plays critical roles in organogenesis [9]. ROR1 is rarely expressed in normal tissues [9] but is highly expressed in some human malignancies, such as ovarian cancer [18]. In this study, we found that ROR1 was expressed at high levels in blood samples and paraffin-embedded tissues from patients with stage II EC. Based on these results, we hypothesize that ROR1 may be involved in cancer pathogenesis.
ROR1 has been shown to drive the survival and proliferation of lymphoblastic leukemia [19], lung cancer [20], and breast cancer [21] cells. ROR1 inhibits the invasion of melanoma cells but promotes the epithelial-mesenchymal transition and metastasis of breast cancer cells. This discrepancy suggests that ROR1 may play distinct roles in different types of cancer, and its function in EC is currently unclear.
To investigate the effect of differential ROR1 expression on EC cell proliferation, we constructed plasmids to overexpress or knockdown ROR1, transfected them into human EC cell lines, and examined the effects on viability, colony formation and cell cycle. The proportion of cells in the M (mitotic) phase of the cycle was increased in EC cells overexpressing ROR1, as expected, and decreased when the expression of ROR1 was suppressed.
The Wnt signaling pathways play important roles in regulating many cellular activities, including cell proliferation, calcium homeostasis, and cell polarity [22]. Wnt5a, a key component of the non-canonical Wnt pathway, is highly expressed in melanoma, and is associated with poor prognosis in both melanoma [23] and gastric cancer [24]. However, in some cancers, Wnt5a downregulation associated with poor outcomes [25,26].
In the present study, we found that ROR1 increased the expression of Wnt5a. To explore the mechanisms underlying this phenomenon, we examined the expression level of cyclinD1 and c-Myc, two proteins in the Wnt signaling pathway. The results indicate that high ROR1 expression is associated with increased levels of these proteins in vitro. We confirmed this observation in vivo by examining tumors from mice xenografted with transfected EC cells. Collectively, these data suggest that ROR1 may affect EC cell proliferation through Wnt5a-mediated activation of the Wnt signaling pathway and upregulation of cyclinD1 and c-Myc. However, the precise mechanism by which ROR1 may achieve remains to be determined.
Acknowledgements
This research was supported in part by grants from National Natural Science Foundation of China (81472427 and 81672574), The leading edge technology joint research projects of Shanghai Shen kang hospital development center (SHDC12015110), the Grant (QNRC2016104) from Jiangsu Provincial Medical Youth Talent, and the Project of Invigorating Health Care through Science, Technology and Education for Jiangsu Provincial Medical Youth Talent.
Disclosure of conflict of interest
None.
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