Introduction
During the past decades, the incidence of endometrial cancer has increased in most regions of the world (1–3). In 2013, there will be ~49,500 new cases and 8,200 deaths in the United States (4). However, our knowledge of the etiology of this disease remains poor.
Generally, endometrial cancer is grouped into type I (accounting for 80% of cases, primarily endometrioid adenocarcinoma, commonly linked to unopposed exposure to oestrogen, obesity and hormone receptor positivity) and type II (accounting for <20% of cases, which consists of serous, clear cell and some undifferentiated tumors) (5–8). To date, the main treatments for endometrial cancer are surgery and/or radiation therapy, and for patients with late stage or distant metastasis, chemotherapy is also an option (9).
Unlike ovarian and cervical cancer, most endometrial cancer cases are localized in the uterus body, but lymphatic or vascular invasion rarely occur (10,11). Molecularly, endometrial cancer is mainly characterized by uncontrolled cellular proliferation and unlimited cell cycle progression, a process in which these cell cycle-related proteins [such as cyclins and cyclin-dependent kinases (CDKs)] are strongly involved (9). This phenomenon suggested it may be a potent approach to inhibit the cell cycle in endometrial cancer, through targeting these cell cycle-related factors or their manipulators (12,13). Recently, the RPRD1B gene was confirmed to be one of them, and can accelerate cell cycle by upregulating a panel of cyclins and CDKs (14).
RPRD1B, the regulation of nuclear pre-mRNA domain containing 1B gene, is a human homolog of the Rtt103 gene (14); it can regulate the binding of RNA polymerase II to the CCND1 gene (cyclin D1) and prevent degradation of the CCND1 mRNA (15). Similarly, RPRD1B also enhances the transcription of many other cell cycle-related factors (such as CDK2, CDK4, CDK6 and cyclin E) (15). Given that elevated RPRD1B was reported in a small panel of endometrial cancer types, in the present study, we explored its detailed functions in endometrial cancer (14).
Materials and methods
Tissue collection
Seventy-six endometrial cancer tissues were collected from patients who underwent surgery in our hospital from July 2010 to October 2012. The tumor stage and grade were determined following the criteria of Federation International of Gynecology and Obstetrics (FIGO, 2009)(16). Fifteen normal endometrium tissues were obtained as the control. Details given in Table I.
Immunohistochemical (IHC) staining
The IHC staining was performed as previously described (14). The primary antibodies used were: anti-Ki 67 (1:500; Wuhan Boster Biological Technology, Ltd., Wuhan, China) and anti-cyclin D1 (1:200; Cell Signaling Technology, Danvers, MA, USA). The staining was visualized using Histostain-Plus IHC kit (Shanghai Mingrui Biotech Co., Ltd., Shanghai, China). All slides were scored by two pathologists following these criteria: 0, none (totally negative staining); 1, weak (1–25% positive); 2, moderate (26–50% positive); 3, strong (>50% positive).
Cell culture
Six endometrial cancer cell lines were used: Ishikawa, KLE, RL95-2, HEC-1B, SPEC-2 and AN3CA. All cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained at 37°C in a humidified atmosphere of 5% CO2. The medium was DMEM/F12+10% fetal bovine serum (FBS) for KLE, SPEC-2 and RL95-2 and MEM +10% FBS for Ishikawa, HEC-1B and AN3CA. The medium and the FBS were purchased from Gibco (Auckland, New Zealand).
RNA isolation and quantitative real-time PCR
The tissue and cells were homogenized and total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). The mRNA expression of RPRD1B was measured by quantitative real-time PCR using SYBR-Green reaction mixture (Takara, Dalian, China). The primers for RPRD1B were: sense, 5′-ggatgctttttctcatgttgc-3′ and antisense, 5′-cgccatacacacttcgttctt-3′. The reaction conditions were: 95°C for 30 sec, 33 cycles of 95°C for 5 sec and 60°C for 32 sec. β-actin was used as the endogenous control. The relative expression of RPRD1B was calculated by the 2−ΔΔCt method.
Western blot analysis
Total protein was extracted using the RIPA buffer (Wuhan Boster Biological Technology) and the concentration was determined by the BCA assay kit (Thermo Fisher Scientific, New York, NY, USA). Equal amount (30–50 μg) of protein was separated on 12% SDS-PAGE gel and transferred to the PVDF membrane (GE Healthcare, Buckinghamshire, UK). The membrane was then incubated with primary antibodies overnight at 4°C and with secondary antibodies for a further 1 h at room temperature. The bands were then developed using an imaging system. The primary antibodies for cyclin D1, CDK4 and CDK6 were purchased from Cell Signaling Technology and anti-RPRD1B mouse monoclonal antibody was obtained from Abgent (San Diego, CA, USA). β-actin was used as the endogenous control.
MTT assay
Ishikawa cells (3×103) or 5×103 HEC-1B cells/well were seeded into 96-well plates and incubated overnight. Then, the cells were transfected with pcDNA3.1-RPRD1B plasmid or siRNA-RPRD1B using Lipofectamine® 2000 (Invitrogen) for 48 h. Cells transfected with the scramble siRNA or the empty plasmid were set as the negative control. Then, 5 μl MTT solution (5 mg/ml; Sigma, Minneapolis, MN, USA) was added into the medium and incubated at 37°C for a further 1 h. The formazan crystal was dissolved in 100 μl DMSO (Sigma) and the absorbance was measured at 570 nm on a plate reader. These procedures were repeated in triplicate.
Colony formation assay
Then, 0.2×103 cells/well were plated into 6-well plates and transfected with siRNA-RPRD1B or pcDNA3.1-RPRD1B. The cells were routinely cultured for two weeks and the colony number was counted under an inverted microscope. This experiment was repeated in triplicate.
Cell cycle analysis
Briefly, 2×105 cells/well were seeded into 6-well plates and incubated overnight. The cells were then transfected with siRNA-RPRD1B or pcDNA3.1-RPRD1B for 48 h. The cells were harvested and resuspended in phosphate- buffered saline (PBS) and fixed in chilled 90% methanol at −20°C for 24 h. The cells were then resuspended in 1 ml PI staining solution and analyzed on a FACSCalibur (BD Biosciences, Bedford, MA, USA).
Tumor growth assay
Five to six-week-old female severe combined immunodeficient mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) were used for this assay. To establish the endometrial cancer model, 1×106 HEC-1B cells stably transfected with pcDNA3.1-RPRD1B were injected subcutaneously into the right flank of mice, and the control cells with empty plasmid were injected into the left flank. One week after injection, the tumor size was checked and recorded every other five days. The tumor volume = (length × width2)/2. Twenty-eight days after injection, all the mice were sacrificed and the tumor tissue was collected for further analysis.
Alterations of cellular sensitivity to Raloxifene
Raloxifene was dissolved in DMSO and diluted into different concentrations (μM): 0.01, 0.1, 1, 10, 20, 40 and 80. The sensitivity of HEC-1B and Ishikawa cells to Raloxifene was determined by MTT assay.
Ethics statement
The present study was approved by the Human Investigation Ethics Committee of the Affiliated Hospital of Jiangnan University. Written informed consent was obtained from each patient involved in the present study. The animal research was carried out following the Guideline for the Care and Use of Laboratory Animals of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of Jiangnan University [Permit Number: JSXK (hu) 2006-0088].
Statistical analysis
The statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). χ2 test or t-test was used for categorical and quantitative data appropriately. P<0.05 was considered to indicate a statistically significant difference.
Results
RPRD1B is frequently overexpressed in human endometrial cancer
RPRD1B mRNA was significantly overexpressed in endometrial cancer tissues, compared to the normal endometrium (Fig. 1A; P=0.0012). By further analysis, we found that RPRD1B overexpression was correlated with histology type (Fig. 1B; P=0.0146), tumor stage (Fig. 1C; P=0.0004) and depth of myometrial invasion (Fig. 1D; P=0.024), but was not associated with histology grade (Fig. 1E; P=0.3612), patient age (Fig. 1F; P=0.4503), lymphovascular space invasion (LVSI; Fig. 1G; P=0.3559) or lymph node metastasis (Fig. 1H; P=0.1845).
We then detected RPRD1B mRNA levels in these 6 endometrial cancer cell lines. As shown in Fig. 1I, RPRD1B mRNA was abundant in Ishikawa and AN3CA cells, but was only slight in HEC-1B and RL95-2.
Overexpression of RPRD1B promotes cellular proliferation and accelerates the cell cycle in HEC-1B cells
As shown in Fig. 2A, both the mRNA and protein of RPRD1B were upregulated following transfection with pcDNA3.1-RPRD1B. Compared to the control, RPRD1B overexpression promoted cellular proliferation significantly (Fig. 2B and C; P=0.032 for MTT assay and P=0.018 for colony formation assay). In addition, RPRD1B also increased the expression of cyclin D1, CDK4 and CDK6 (Fig. 2D) and accelerated the cell cycle of HEC-1B cells (Fig. 2E; P=0.018 for 24 h and P=0.007 for 48 h).
Downregulation of RPRD1B suppresses cellular proliferation and leads to cell cycle arrest in Ishikawa cells
Following treatment with siRNA-RPRD1B for 48 h, both RPRD1B mRNA and protein were reduced significantly in Ishikawa cells (Fig. 3A). Loss of RPRD1B notably suppressed cellular proliferation (Fig. 3B and C; P=0.02 for MTT assay and P=0.031 for colony formation assay). Flow cytometry analysis showed that downregulation of RPRD1B inhibited the expression of cyclin D1, CDK4 and CDK6 (Fig. 3D), and led to G1 phase arrest at both 24 and 48 h (Fig. 3E; P=0.039 for 24 h and P=0.025 for 48 h).
Overexpression of RPRD1B accelerates tumor growth in vivo
We established the endometrial cancer model by injecting HEC-1B cells (with or without overexpression of RPRD1B) into nude mice. According to our results, the xenograft with RPRD1B overexpression grew much faster than the control (Fig. 4A and B; P=0.0012). We also found the tumor weight increased significantly following RPRD1B overexpression (Fig. 4C; P=0.007). By IHC staining, much higher expression of Ki-67 and cyclin D1 was detected in the group with over-abundant RPRD1B (Fig. 4D).
Downregulation of RPRD1B sensitizes Ishikawa cells to Raloxifene
As shown in Fig. 5A, the IC50 of Raloxifene was ~25 μM for Ishikawa, but >80 μM for HEC-1B cells. Loss of RPRD1B increased the sensitivity of Ishikawa cells to Raloxifene (Fig. 5B; P=0.018); however, RPRD1B over-expression had no effects on the reactions of HEC-1B to Raloxifene (data not shown).
Discussion
Due to the high incidence rate and relatively favorable prognosis, endometrial cancer is now considered a chronic disease and, hence, preventing the tumor from unscheduled growth seems to be more appropriate. Dysregulated cell cycle is a common feature in nearly all types of human cancer (17,18). To date, the relationship between cell cycle dysregulation and human cancer have been well documented. In particular, several cell cycle regulators were confirmed to be critical for the initiation and progression of endometrial cancer (19–21).
In mammals, the cell cycle is manipulated by the cyclin-dependent kinases (CDKs), which can be activated by cyclins (such as cyclin D and E) and can be inhibited by Ink4 and Cip/Kip inhibitors (13,22,23). The activities of CDKs and their regulators are often dysregulated in human tumors owing to genetic or epigenetic changes or alterations of their upstream signal pathway (24–26). Recently, RPRD1B, a manipulator of both cyclins and CDKs, was found overexpressed in endometrial cancer (14). Therefore, we hypothesized that RPRD1B might be a key factor in endometrial neoplasia and development.
In the present study, we demonstrated RPRD1B overexpression in human endometrial cancer tissues, which was closely related with tumor stage, histology type and depth of myometrial invasion. In vitro, we explored the effects of RPRD1B on tumor cells by up- and downregulating its expression in two endometrial cancer cell lines. In HEC-1B cells, RPRD1B overexpression promoted cellular growth and accelerated the process of colony formation, while in Ishikawa cells, loss of RPRD1B inhibited its growth and colony formation.
As RPRD1B has been reported to be a regulator of cell cycle-related proteins, we then investigated the cell cycle status and expression of cyclin D1, CDK4 and CDK6 in the two cell lines. Compared to the control, RPRD1B overexpression promoted 2-fold more HEC-1B cells into the S phase, and the levels of cyclin D1, CDK4 and CDK6 were also upregulated. Moreover, RPRD1B downregulation inhibited the cell cycle and caused significant G1 phase arrest, through suppressing cyclin D1, CDK4 and CDK6. These findings were consistent with those of a previous study on gastric carcinoma cells (14).
Furthermore, we established the nude mice model to explore whether RPRD1B affects the tumor growth in vivo. As our data showed, HEC-1B cells with abundant RPRD1B grew much faster than the control group, and showed much stronger staining of Ki-67 (a proliferation index) and cyclin D1.
Considering that selective estrogen receptor modulators (SERMs) work well in a group of endometrial cancer patients but soon induced drug resistance in several cases, we then investigated whether RPRD1B could affect the sensitivity of Ishikawa and HEC-1B cells to Raloxifene (27,28). We found that knockdown of RPRD1B could sensitize Ishikawa cells to Raloxifene, but overexpression of RPRD1B had no effects on HEC-1B cells. We hypothesized it could be caused by the different ER status of Ishikawa (ER positive) and HEC-1B (ER negative).
In summary, we demonstrated that RPRD1B was frequently overexpressed in human endometrial cancer. Both in vitro and in vivo, overabundant RPRD1B promoted tumor growth and accelerated cellular cell cycle through upregulating cyclin D1, CDK4 and CDK6, while knockdown of RPRD1B suppressed tumor growth and caused cell cycle arrest by decreasing cyclin D1, CDK4 and CDK6. In addition, knockdown of RPRD1B increased cells sensitivity to Raloxifene treatment. These findings may aid in the design of drugs targeting RPRD1B and its partners, which we believe will be a new strategy for curing this disease.