Adipokine RBP4 drives ovarian cancer cell migration
Obesity has been linked to several types of cancers including ovarian cancer. Retinol binding protein 4 (RBP4) is an adipokine that drives the development of hyperinsulinemia and type II diabetes in obesity patients and animals. Previously, we have identified RBP4 as a serum marker for ovarian cancer. Here we further explored the consequence of RBP4 upregulation in ovarian cancer cells and its molecular mechanism.
Our results show that RBP4 is overexpressed in ovarian cancer cells to the same extent as in adipose tissues. The overexpression of RBP4 in ovarian cancer cells promotes cancer cell migration and proliferation. At molecular level, cancer progression factors MMP2 and MMP9 are induced in response to RBP4 overexpression. We further investigated which signaling pathways are utilized by RBP4 to activate ovarian cancer cell migration. We found RhoA/Rock1 pathway is turned on and CyclinD1 is upregulated in RBP4 overexpressed cells. Inhibition of RhoA/Rock1 pathway reduces the RBP4-induced MMP2 and MMP9 expression. The RBP4 action is depend on its associated ligand vitamin A/retinol acid (RA) and possibly involves similar pathways as for conferring insulin resistance. Moreover, we show that knockdown of RBP4 significantly reduce cancer cell migration and proliferation as well as expressions of oncogenic factors.
Our results indicated that RBP4 can drive ovarian cancer cell migration and proliferation via RhoA/Rock1 and ERK pathway. It suggests that RBP4 act as a oncogene in ovarian cancer cells. Thus, RBP4 could be a molecular bridge between obesity and cancers and a potential target for treating obese cancer patients.
KeywordsOvarian cancer Obesity RBP4 Retinol acid Migration
Vitamin A/retinol acid
Retinol binding protein 4
Stimulated by RA 6
Obesity is a well-established cancer risk factor and its occurrence is strongly associated with several types of cancers, including breast, colon, endometrial, ovarian, gastric, pancreatic and liver cancers [1, 2]. However, the molecular mechanisms that link obesity and cancers remain largely elusive. Identifying metabolites and secreted factors that connect increased fat mass to tumorigenesis is one of the central questions.
Retinol binding protein 4 (RBP4) is secreted by liver and adipose tissues [3, 4]. RBP4 acts as the major transporter for vitamin A/retinol acid (RA) in serum . Under normal physical conditions, RA bound RBP4 circulates together with transthyretin (TTR) as a holo-RBP-TTR complex . Upon arrival, RA can either enter the targeted cell by passive diffusion or active transportation by Stimulated by RA 6 (STRA6) [6, 7, 8, 9, 10].
Besides its transportation function, RBP4 has recently been recognized as an adipokine . Cumulative evidences showed that overexpression of RBP4 from adipose tissues promote hyperinsulinemia and type II diabetes [11, 12, 13, 14, 15]. Several pathways have been identified mediate RBP4 signaling . RA and its oxidative products can activate retinoic acid receptors and retinoid X receptors and promote glucose production in liver . RBP4 with RA can activate STRA6, which will then recruit and activate Janus kinase and the transcription factors STAT3 or STAT5 . RBP4, independent of RA and STRA6, could promote pro-inflammatory responses possibly through pathways involving c-Jun N-terminal protein kinase (JNK)1, JNK2, or Toll-like receptor .
Ovarian cancer is the most lethal type of gynecological cancer in the world . Ovarian cancers have a high occurrence rate in obesity peoples and obesity has been shown to promote ovarian cancer metastatic . Recently, we found that RBP4 level is highly upregulated in ovarian cancer serum samples . Overexpression of RBP4 had also been reported in liver, bone, and colon cancer cells [23, 24, 25, 26, 27]. However, the consequence of RBP4 overexpression on cancers and the mechanism of action of RBP4 in cancers are not clear.
We here investigated whether RBP4 is a tumorgenic factor that connects obesity and ovarian cancer. Our data showed that RBP4 was up-regulated in ovarian cancer cells and overexpression of RBP4 promoted cancer cell migration. The MMP-2 and MMP-9, key factors in cancer metastasis, were induced by RBP4 overexpression. We further identified RhoA/Rock1 pathway as mediators for RBP4 action. RhoA and Rock1 were overexpressed in response to RBP4 and inhibition of RhoA/Rock1 reduced MMP-2 and MMP-9 expression. The RBP4 action was dependent on its associated ligand RA. Moreover, knockdown of RBP4 greatly reduced cancer migration. Our data not only established RBP4 as a direct linkage between obesity and ovarian cancer, but also suggested RBP4 was a possible target for cancer treatment, especially in those associated with obesities.
This study was approved by the Medical Ethical Committee of Beijing Chaoyang Hospital (Beijing, China). The written informed consents were obtained from all the participants enrolled in the study. Specimens were sampled from patients undergoing surgery for ovarian carcinoma or benign ovarian tissues at the Beijing Chaoyang Hospital.
All procedures were approved by the Animal Care and Use Committee of Beijing Chaoyang Hospital (Beijing, China). All experiment methods were performed in accordance with the relevant guidelines and regulations. In brief, healthy specific-pathogen-free (SPF) male SD rats were purchased from the Vital River. All rats were preserved under standard housing laboratory conditions. After one week of adaptation to the diet and the new environment, female SD rats were divided into two diet groups: the normal control (NC) group fed ad libitum a standard rodent chow, the high-fat (HF) group fed ad libitum a high-fat chow. After six weeks to induce obesity, the ovarian tissues were obtained after euthanasia.
Antibodies against RBP4 (#ab109193), actin (#ab8226), RhoA (#ab187027), p-RhoA (#ab41435), ROCK1 (#ab45171), Erk (#ab54230), p-Erk (#ab51100), Cyclin D1 (#ab134175), MMP2 (#ab37150), MMP9 (#ab38898) were obtained from Abcam, USA. ROCK1 inhibitor Y27632 was purchased from Sigma, USA. All primers for qPCR were ordered from Invitrogen (Shanghai, China).
Human ovarian cancer cell line A2780 was obtained from Prof. Haiteng Deng laboratory [28, 29]. SKOV3 was preserved in our lab . Cells were maintained in DMEM medium in incubator with 5% CO2 at 37 °C.
To upregulate the expression of RBP4, the human RBP4 full length cDNA was amplified and inserted into the pCMV-Flag vector, and a scramble sequence was inserted into the pCMV-flag vector as the control vector. To knock down the expression of RBP4, a RBP4 siRNA was designed and obtained from Jima Inc. (Shanghai, China). For transfection, the cells were seeded into 6-well plates. When cell confluency reached 50%, RBP4 siRNA or RBP4-pCMV-Flag was transfected into the cells using lipo2000 according to the manufacturer’s instructions.
Cells were lysed in RIPA buffer with protease and phosphatase inhibitor cocktail. Equal amount of protein samples was loaded onto 12% SDS-PAGE and was then transferred to PVDF membranes. After blocking with 5% BSA for 1 h at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (from Zhongshanjinqiao, China) for 1 h at room temperature. The protein bands were visualized by ChemiDoc XRS+ (BioRad, USA). Data analysis was done using Quantity one.
Quantitative reverse transcription- polymerase chain reaction (qRT-PCR)
Total RNA was isolated from the cells using trizol method. RNA was reverse transcribed using the PrimeScript RT Master Mix (B-Belife, China) according to the manufacturer’s instructions. The PCR amplifications were performed using SYBR Premix Ex Taq II (B-Belife, China). The expression level of each sample was internally normalized against that of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative quantitative value was calculated using 2−ΔΔCt method. Each experiment was performed in triplicate. The primers used in real-time PCR were as follow: RBP4 F: AGGAGAACTTCGACAAGGCTC; RBP4 R: GAGAACTCCGCGACGATGTT; GAPDH F: GGAGCGAGATCCCTCCAAAAT; GAPDH R: GGCTGTTGTCATACTTCTCATGG; RHOA F: AGCCTGTGGAAAGACATGCTT; RHOA R: TCAAACACTGTGGGCACATAC; Rock1 F: AACATGCTGCTGGATAAATCTGG; Rock1 R: TGTATCACATCGTACCATGCCT; cyclinD1(CCND1) F: GCTGCGAAGTGGAAACCATC; cyclinD1(CCND1) R: CCTCCTTCTGCACACATTTGAA; ERK1 F: CTACACGCAGTTGCAGTACAT; ERK1 R: CAGCAGGATCTGGATCTCCC; MMP2 F: TACAGGATCATTGGCTACACACC; MMP2 R: GGTCACATCGCTCCAGACT; MMP9 F: TGTACCGCTATGGTTACACTCG; MMP9 R: GGCAGGGACAGTTGCTTCT.
The expression of RBP4 was assessed using immunohistological staining as described previously . Briefly, tissue samples were fixed and cut to 5 μm thick. RBP4 antibody was applied on the sections for 30 min and incubated overnight at 4 °C then shaking at room temperature for 30 min. Antibody binding was amplified using biotin and streptavidin HRP for 10 min each and the complex was visualized using DAB. ALL sections were assessed microscopically for positive DAB staining. The immunostained sections were examined under microscopy and the expression level of RBP4 was scored on the basis of the intensity of staining.
In vitro migration assay
A 24-well Transwell chamber (Corning, #3422, USA) was used to examine the invasive ability of the ovarian cancer cells. Cells were suspended in DMEM medium and were added into the upper Transwell chamber. The lower Transwell chamber was filled with DMEM medium supplemented with 10% FBS. After incubation of 16 h at 37 °C, the non-migrated cells were removed with a sterile cotton swab, and the migrated cells were stained with 0.1% crystal violet for 20 min at room temperature. The numbers of cells were calculated under a light microscope in five random fields.
Cell proliferation was determined by MTT assay. Cells were seeded at 1000 cells/well in a 96-well plate. After incubation for indicated time, MTT was added into the plate incubated for 4 h. The optical density (OD) was measured 490 nm at designated time.
Cell cycle analysis
Cell cycle distribution was analyzed by PI staining and flow cytometry. The 1 × 105 cells / well were seeded in 6-well plates. The cells were then harvested, fixed with 70% ice cold ethanol, and stored at 4 °C until analysis. After fixation, the cells were washed twice with cold phosphate-buffered saline (PBS) and centrifuged, following which the supernatants were removed. The pellet was resuspended and stained with PBS containing 50 μg/ml PI and 100 μg/ml RNaseA for 20 min in the dark. The cell cycle data were analyzed using Modifit software.
All the continuous variables were expressed as average ± standard deviation (SD). Student’s t-test was used for the difference analysis. A P value of more than 0.01 was considered as statistical significance. Graphpad 5.0 software was used for all the statistical analyses.
Expression of RBP4 in ovarian cancer tissues and obesity tissues
RBP4 promotes migration and proliferation of ovarian cancer cells
RBP4 induces migration-related genes expression in ovarian cancer cells
RBP4 activates RhoA/Rock1 pathways and cyclin D1
RBP4 action is partially dependent on RhoA1/rock pathway
RBP4 action is dependent on RA
Obese patients are associated with high cancer risk, poor prognosis and reduced response to anti-cancer therapies [1, 2]. Obesity is intrinsically linked with metabolic syndrome that can indirectly promote cancers as metabolic reprogramming is a hallmark of cancer. Especially for ovarian cancer, obesity has been shown to promote cancer metastasis . Besides that obesity can indirectly affect cancer metastasis through increasing lipogenesis, enhancing vascularity, and decreasing infiltration . Here, we established a direct molecular linkage between adipokine RBP4 and ovarian cancer. RBP4 is a well-established obesity factors that is overexpressed by adipose tissues . We proved that high level of RBP4 can stimulate migration and proliferation of ovarian cancer cells. The overexpression of RBP4 stimulated the expression of matrix metalloproteinase MMP-2 and MMP-9, which degraded extracellular matrix and enabled cancer cells migration. Moreover, RBP4 highly expressed in ovarian cancer cells and high level of RBP4 had been documented in ovarian patient’s serum samples . We thus proposed that high level of RBP4, either from adipose tissues or cancer tissues, can promote cancer metastasis and obesity signaling, vice versa. Although currently lack of clinical data, it would be interesting to survey if ovarian cancer patients have a high rate of obesity and insulin resistance.
RBP4 and its associated RA have been shown to trigger several downstream pathways to confer insulin resistance [16, 17, 18, 19]. The pathways could also be shared in promoting cancer metastasis. The effect of RBP4 on tumor metastasis is RA dependent. RA has broad metabolic roles including stimulating lipogenesis , which has been shown to promote ovarian cancer metastasis . It has been documented that RBP4 bound RA, but not apo RBP4 can induce signaling of STRA6 . The STRA6 expression and its signaling has been proposed drive oncogenic transformation of cancer cells . STRA6 expressed in the ovarian cancer cell lines, although its level was not affected by RBP4. STRA6 could mediate, at least part of the RBP4 effect. Circulating RBP4-RA was associated with their partner protein TTR , which inhibited the RBP4-RA triggered STRA6 signaling . To our knowledge, there is no correlation of expression level of TTR with ovarian cancer. It is quite likely that when RBP4 overexpressed, the TTR was not enough to block STRA6 signaling even STRA6 level remained unchanged. On the other hand, RBP4 itself without RA can still promote metastasis, although to a lesser degree. It has been shown RBP4, independent of RA and STRA6, can induce pro-inflammation reaction .
Adding to the existing knowledge, we showed that RhoA/Rock1 pathway was turned on in response to RBP4 overexpression. RhoA/Rock1 pathway played pivot roles in cell morphogenesis, adhesion, and motility and was often activated in malignant cancers . Previous reports had shown that inhibition of RhoA/Rock1 suppressed MMP-2 and MMP-9 action [37, 38, 39]. Consistently, we observed that inhibiting RhoA/Rock1 pathway with Rock1 inhibitor Y-27632 can reduce RBP4 induced MMP-2 and MMP-9 overexpression, indicating that the migration effect of RBP4 was mediated by RhoA/Rock1 pathway. However, how RBP4 activated RhoA/Rock1 pathway was less clear. Considering RBP4 was mainly a secretive protein, novel membrane receptors was possibly involved in promoting RBP4 signaling. Further studies were in demand to fully elucidating the RBP4 signaling pathways that related to cancers.
We observed that knockdown of RBP4 can greatly suppress ovarian cancer cell migration and proliferation. Considering RBP4 as a circulating protein, targeting RBP4 could be a relative easy option for ovarian cancer treatment, especially those associated with obesities.
In conclusion, this study described the function of RBP4 in driving ovarian cancer cell migration and proliferation. Moreover, the underlying molecular mechanism of RBP4 was activation of RhoA/Rock1 pathway and CyclinD1 expression. Therefore, RBP4 could be a molecular bridge between obesity and cancers and a potential target for treating obese cancer patients.
We thank Dr.Cui from Chinese Academy of Science for data analysis.
This work was supported by National Natural Science Foundation of China (81571455).
Availability of data and materials
Yanyan Wang performed experiments, and wrote the manuscript; Yilin Wang contributed to data analysis. ZZ designed and supervised the project. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the ethical committees of Beijing Chaoyang Hospital, Capital Medical University, China.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 1.O'Flanagan CH, Bowers LW, Hursting SD. A weighty problem: metabolic perturbations and the obesity-cancer link. Horm Mol Biol Clin Invest. 2015;23:47–57.Google Scholar
- 9.Berry DC, Jacobs H, Marwarha G, Gely-Pernot A, O'Byrne SM, DeSantis D, et al. The STRA6 receptor is essential for retinol-binding protein-induced insulin resistance but not for maintaining vitamin a homeostasis in tissues other than the eye. J Biol Chem. 2013;288:24528–39.CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Fedders R, Muenzner M, Schupp M. Retinol binding protein 4 and its membrane receptors: a metabolic perspective. Horm Mol Biol Clin Invest. 2015;22:27–37.Google Scholar
- 19.Norseen J, Hosooka T, Hammarstedt A, Yore MM, Kant S, Aryal P, et al. Retinol-binding protein 4 inhibits insulin signaling in adipocytes by inducing proinflammatory cytokines in macrophages through a c-Jun N-terminal kinase- and toll-like receptor 4-dependent and retinol-independent mechanism. Mol Cell Biol. 2012;32:2010–9.CrossRefPubMedPubMedCentralGoogle Scholar
- 20.Selvaggi SM. Tumors of the ovary, maldeveloped gonads, fallopian tube, and broad ligament. Archives of pathology & laboratory medicine. 2000;124:477.Google Scholar
- 27.Noy N, Li L, Abola MV, Berger NA. Is retinol binding protein 4 a link between adiposity and cancer? Horm Mol Biol Clin Invest. 2015;23:39–46.Google Scholar
- 39.Xue F, Takahara T, Yata Y, Xia Q, Nonome K, Shinno E, et al. Blockade of rho/rho-associated coiled coil-forming kinase signaling can prevent progression of hepatocellular carcinoma in matrix metalloproteinase-dependent manner. Hepatology research: the official journal of the Japan society of. Hepatology. 2008;38:810–7.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.