YAP promotes the malignancy of endometrial cancer cells via regulation of IL-6 and IL-11
Emerging evidence shows that Hippo signal pathways can regulate the progression of various cancer. While the roles of Yes-associated protein (YAP), the key transducer of Hippo signals, in the development of endometrial cancer (EC) are rarely investigated.
The expression of YAP in endometrial cancer cells and tissues was measured. Its roles in proliferation and expression of interleukins (ILs) were investigated by use of its specific siRNA or inhibitor (verteporfin, VP).
YAP was upregulated in endometrial cancer cells and tissues. Knockdown of YAP or VP can suppress the proliferation while increase its chemo-sensitivity of EC cells. We found that targeted inhibition of YAP can decrease the expression of interleukin-6 (IL-6) and IL-11 in EC cells. Recombinant IL-6 or IL-11 can attenuate si-YAP suppressed proliferation of EC cells. Chromatin immunoprecipitation (ChIP) assay suggested that YAP can directly bind with the promoter of IL-6 and induce its transcription. As to IL-11, inhibitor of NF-κB (BAY 11–7082) can significantly down regulate the mRNA expression of IL-11. Over expression of p65 abolished si-YAP suppressed transcription of IL-11. It suggested that NF-κB was involved in the YAP regulated expression of IL-11.
YAP can regulate the proliferation and progression of EC cells. It suggested that targeted inhibition of YAP might be a potent potential approach for EC therapy.
KeywordsYAP Endometrial Cancer IL-6 IL-11 Proliferation
endometrial stromal cell
large tumor suppressor 1/2
mammalian Ste20-like kinases 1/2
tumor necrosis factor
Endometrial cancer (EC) is the most common gynecological cancer in developed countries (Dizon, 2010), with about 50,327 deaths occurring worldwide each year (Siegel et al., 2016). Further, the incidence and mortality rate are still increasing in the developed and developing countries (Rauh-Hain and Del Carmen, 2010). The 5-year overall survival ranges from 74 to 91% in patients without metastatic disease (Colombo et al., 2013). However, for EC patients with metastasis, treatment failure is still high due to the loss of opportunity for surgery. In advance stages of EC patients, the growth and systemic metastasis lead to patient morbidity and mortality (Huang et al., 2014). Previous studies indicated that some proteins are variated in the EC tissues and cells such as PTEN, KRAS, CTNNB1, PIK3CA and FGFR2 (Tsujiura et al., 2014). Therefore, one major challenge for EC treatment is to develop efficiency approaches to block the signals essential for the progression of EC cells.
As the key downstream effector in the Hippo signaling cascade, the Yes-associated protein (YAP) is a major contributor to cancer pathophysiology (Pan, 2010; Zhao et al., 2007). The Hippo signals are composed of mammalian Ste20-like kinases 1/2 (MST1/2) and large tumor suppressor 1/2 (LATS1/2), YAP and its paralog TAZ (Liu et al., 2010). After activation of Hippo signals, MST1/2 is phosphorylated and then activates LATS1/2 (Real et al., 2018). The activation of Lats1/2 can phosphorylate YAP and subsequent promote proteasome mediated degradation (Zhao et al., 2010). Dephosphorylation of YAP can allow YAP to translocate into the nucleus, bind with its transcriptional co-activator TEAD, and increase the transcription of many oncogenes (Zhao et al., 2008). Although many studies indicated that YAP functions as an oncogene in most cancers (Zhao et al., 2010), the roles and related mechanisms of YAP on the progression of EC remain unclear. Recently studies revealed that increased nuclear YAP expression was significantly associated with higher grade, stage, lympho-vascular space invasion, postoperative recurrence/metastasis and overall survival in estrogen mediated EC patients (Tsujiura et al., 2014). It suggested that YAP may also trigger the progression of EC via unknown mechanisms.
The present study investigated the potential roles and related mechanisms of YAP in the progression of EC. Our data showed that YAP is upregulated in EC cells and tissues. Knockdown of YAP or its inhibitor verteporfin can suppress the proliferation and increase the chemo-sensitivity of endometrial cancer cells. The upregulation of interleukin-6 (IL-6) and IL-11 is essential for YAP induced malignancy of EC cells.
Materials and methods
Chemicals and reagents
The doxorubicin (Dox) and other chemicals/inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human recombinant IL-6 and IL11 were obtained from R&D Systems (Sydney, Australia). Scrambled control siRNA oligonucleotide (si-NC) or siRNA for YAP were purchased from Invitrogen (Life Technologies, Grand Island, NY, USA). Primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from the Cells Signaling Technology (Danvers, MA, USA).
Cell culture and transfection
The human EC Ishikawa, RL95–2, HEC1A, AN3CA and KLE cells and the human endometrial cell line endometrial stromal cell (ESC) were purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China. After confirmed by short tandem repeat profiling, cells were cultured in medium containing 10% (v/v) FBS (Scientifix, Cheltenham, VIC, Australia) and 1% (V/V) penicillin-streptomycin solution (Sigma, St Louis, MO, USA). All cells were routinely tested as free from mycoplasma contamination. In order to knock down the expression of YAP, siRNA specific for YAP (5’GCCAGUACUGAUGCAGGUATT3’, Shanghai GenePharma Co. Ltd., Shanghai, China) was used to transfect cells by using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. Similarly, the pcDNA (vector control) and pcDNA/p65 plasmids were also transfected by use of Lipofectamine 2000.
RNA preparation and quantitative real time RT-PCR (qRT-PCR)
Total RNAs were isolated by use of TriReagent (Sigma-Aldrich) and further purified by use of the DNA free kit (Ambion) according to the manufacturer’s instructions. The cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen) and 500 ng of total RNA. Then, qRT-PCR was performed on the Bio-Rad System (Bio-Rad Laboratories Inc., Hercules, CA, USA) using 2× Fast-Start SYBR green master mix and the following primers: YAP, forward: 5′- TAGCCCTGCGTAGCCAGTTA − 3′, reverse: 5′- TCATGCTTAGTCCACTGTCTGT -3′; IL-1β, forward: 5′- ATGATGGCTTATTACAGTGGC − 3′, reverse: 5′- GTCGGAGATTCGTAGCTGGA -3′; IL-6, forward: 5′- ACTCACCTCTTCAGAACGAATTG − 3′, reverse: 5′- CCATCTTTGGAAGGTTCAGGTTG -3′; IL-8, forward: 5′- GAG AGT GAT TGA GAG TGG ACC AC − 3′, reverse: 5′- CAC AAC CCT CTG CAC CCA GTT T -3′; IL-10, forward: 5′- TCT CCG AGA TGC CTT CAG CAG A − 3′, reverse: 5′- TCA GAC AAG GCT TGG CAA CCC A -3′; IL-11, forward: 5′- GCGCTGTTCTCCTAACCCG-3′, reverse: 5′- GAGTCCAGACTGTGATCTCCG-3′; TNF-α, forward: 5′- CTC TTC TGC CTG CTG CAC TTT G − 3′, reverse: 5′- ATG GGC TAC AGG CTT GTC ACT C − 3′; p65, forward: 5′- GTGGGGACTACGACCTGAATG − 3′, reverse: 5′- GGGGCACGATTGTCAAAGATG -3′; GAPDH, forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′, reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′. GAPDH was used as the internal control for normalization. The 2−ΔΔCT method was used to quantify gene expression.
Western blot analysis
Cells or tissues were lysed by use of lysis buffer containing the protease inhibitor (2 μl/ml; ThermoScientific, Waltham, MA, USA). Then total 20 μg proteins were separated by use of a 10% SDS-PAGE gel. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the primary antibodies overnight at 4 °C. After washed three times, membranes were incubated with the secondary antibody, exposed to an enhanced chemiluminescence (ECL) western blot by use of ECL system (GE Healthcare Life Sciences), and quantified using Bio-Rad Quantity One 1-D software.
According to the permission of Ethical Committee in our hospital, seven paired tumor tissues and adjacent normal tissues were collected during July 2015 to June 2017. Informed consent was obtained from each patient. The samples were stored at − 80 °C immediately after surgery. The expression of YAP was measured by use of western blot analysis.
Cell proliferation assay
The cell proliferation was analyzed by use of the Wst-1 assay according to the previous study (Lay et al., 2012). Briefly, cells (5, 000 per well) were seeded into 96-well plates. After treatment, cells were incubated with Wst-1 dye (1:10; Roche Applied Science) for 4 h at 37 °C. The absorbance at 450 nm was measured with a Wallac Envision 2103 plate reader (Perkin Elmer).
Enzyme-linked immunoassays (ELISAs)
The expression of IL-6 and IL-11 in medium was measured by ELSIA by use of kits according to the manufacturer’s protocol (eBioscience, USA). The absorbance at 450 nm was measured with a Wallac Envision 2103 plate reader (Perkin Elmer).
Chromatin immunoprecipitation (ChIP) PCR
ChIP assays were performed with an Agarose ChIP Kit (Thermo Scientific) according to the manufacturer’s instructions. Briefly, after treatment as indicated conditions, cells were crosslinked and lysed. The DNA was extracted and sonicated to shear DNA into fragments of 500–1000 bp in length. The DNA/protein complexes were precipitated by antibody of YAP or IgG (ab171870, Abcam), and then incubated with Protein A/ G agarose beads for 2 h. The abundance of IL-6 or IL-11 promoter was analyzed by qPCR using primers as follows: IL-6, 5′-ACCCTCACCCTCCAACAAAG-3′ and 5′ -GCAGAATGAGCCTCAGACATC-3′; IL-11, 5′- CTTTGCTTCTCTGGTGTGTC − 3′ and 5′ - CTGGTGAGGTCATTGGCGT − 3′.
All results were stated as mean ± standard deviation (SD). The data analysis was performed by use of GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) for Windows. Statistical comparison was performed using the Student’s t test for two groups. ANOVA analysis with Tukey’s multiple comparison test was also used to compare three or more groups. A p-value of < 0.05 was considered statistically significantly different between groups.
The expression of YAP is upregulated in EC cells and tissues
Targeted inhibition of YAP can suppress the proliferation and increase the chemosensitivity of EC cells
YAP can regulate the expression of IL-6 and IL-11 in EC cells
Both IL-6 and IL-11 are involved in YAP regulated malignancy of EC cells
YAP can directly regulate the transcription of IL-6, while not IL-11, in EC cells
NF-κB is involved in YAP regulated transcription of IL-11 in EC cells
Although various studies indicated that YAP functions as an oncogene in most cancers (Zhao et al., 2010), the roles of YAP in the progression of EC remain unclear. Our present study revealed that the expression of YAP was upregulated in both EC cells and tissues as compared to their corresponding controls. Targeted inhibition of YAP by its siRNA or inhibitor can suppress the proliferation and increase the chemosensitivity of EC cells. Among the measured cytokines, YAP can regulate the expression of IL-6 and IL-11. While rIL-6 and rIL-11 can reverse YAP regulated proliferation and Dox sensitivity of EC cells. Mechanistically, YAP can directly bind with the promoter of IL-6 to increase its transcription. As to IL-11, the upregulation of p65 is involved in YAP regulated its expression. Collectively, our present study revealed that YAP can trigger the malignancy of EC cells via upregulation of IL-6 and IL-11.
Our study revealed that the upregulation of YAP in EC cells can trigger its proliferation and decrease its chemosensitivity. Tsujiura et al. (Tsujiura et al., 2014) reported that higher levels of nuclear YAP were associated with poor prognostic factors of EC patients. Consistently, YAP can promote the proliferation, anchorage independent growth, invasion and migration of human EC HEC-1-B cells (Tsujiura et al., 2014). The reasons responsible for upregulation of YAP in EC cells and tissues are currently unknown. It has been reported that YAP gene can be amplified in various cancers such as breast, esophageal, hepatocellular cancer, ependymoma, malignant mesothelioma and medulloblastoma (Overholtzer et al., 2006; Zender et al., 2006). However, comprehensive analyses of somatic alterations suggested that this amplification was not observed in EC cells and tissues (Getz et al., 2013). Therefor the mechanisms responsible for the upregulation of YAP in EC cells need further study.
Our data suggested that the upregulation of IL-6 and IL-11 was involved in YAP regulated proliferation and chemosensitivity of EC cells. YAP can induce the expression of IL-6 in hepatocellular carcinoma cells and then recruit tumor-associated macrophages (Zhou et al., 2018). Further, IL-6 has been proved as the transcriptional target of YAP involved in basal-like breast cancer (Kim et al., 2015). Our data confirmed that YAP can directly bind with the promoter of IL-6 to regulate its transcription in EC cells. In addition, our data also showed that YAP can regulate the expression of IL-11 in EC cells but not directly bind with its promoter. This might be due to that YAP can upregulate p65 induced transcription of IL-11. Both IL-6 and IL-11 can promote the malignancy of EC cells via triggering cell proliferation, migration and invasion (Chu et al., 2018; Lay et al., 2012; Yap et al., 2010).
We demonstrated that the upregulation of YAP can increase the proliferation and decrease the chemosensitivity of EC cells via upregulation of IL-6 and IL-11. Although further mechanisms and in vivo evidences are needed, our data suggested that targeted inhibition of YAP might be a potential therapy approach for treatment of EC patients.
Data collecting: Jing Wang, Xianchao Kong. Writing: Jing Wang, Tiefang Song. Data analysis: Suiyang Zhou, Xianchao Kong. Design: Jing Wang, Tiefang Song, Suiyang Zhou, Xianchao Kong. All authors read and approved the final manuscript.
No funding information.
Ethics approval and consent to participate
All human related experiments have been conducted according to the approve of the Ethical Committee of our hospital according to the Chinese Ethical Regulations. Informed consent was obtained from all individual participants included in the study.
Consent for publication
All authors give the consent for the publish of this study.
The authors declare that they have no competing interests.
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