A high rate of disease relapse makes epithelial ovarian cancer (EOC) the leading cause of death among all gynecologic malignancies. These relapses are often due to tumor dormancy. Here we identify the RNA polymerase II transcriptional mediator subunit 12 (MED12) as an important molecular regulator of tumor dormancy. MED12 knockout (KO) induced dormancy of EOC cells in vitro and in vivo, and microarray analysis showed that MED12 KO decreased expression of EGFR. Restoration of EGFR expression in MED12 KO cells restored proliferation. Additionally, MED12 bound to the promoter of EGFR, and correlation studies showed that MED12 expression positively correlated with EGFR expression in EOC patient samples. Clinical data demonstrated that chemotherapy-resistant patients expressed lower levels of MED12 compared with responsive patients. Overall, our data show that MED12 plays an important role in regulating dormancy of EOC through regulation of EGFR.

Significance: MED12 is identified as a novel, important regulator of tumor dormancy in human ovarian cancer. Cancer Res; 78(13); 3532–43. ©2018 AACR.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy with 125,000 deaths each year worldwide, and the 5-year overall survival rate is only 46% (1–3). The standard treatment for EOC is optimal cytoreductive surgery followed by combination chemotherapy using taxane- and platinum-based regimens (1–3). However, the majority of patients develop recurrence with latency periods that range from years to decades due to the persistence and recurrence of dormant, drug-resistant ovarian cancer cells (3). This pause can be explained by tumor dormancy, a leading factor of treatment failure, metastasis, and tumor recurrence (4, 5). Understanding the driving force of tumor dormancy has important therapeutic implications for preventing relapse in patients with a history of EOC. Tumor dormancy can be explained by several mechanisms. These include cellular dormancy; the inability of a tumor cell population to initiate angiogenesis; and immunosurveillance (5). Cellular dormancy is mediated by different signaling pathways including EGFR signaling and high p38 over ERK activity (4, 5). It was reported that loss of EGFR that transduces growth signals from the microenvironment results in stress signaling (low FAK/Ras/ERK and high CDC42/p38 activity), which in turn may lead to dormancy (4, 5). However, precisely how cancer cells enter dormancy is currently unclear.

The RNA polymerase II mediator complex subunit 12 (MED12) is a subunit of the Mediator complex, which plays essential roles in transcriptional regulation via RNA polymerase II (6). Several studies have already proposed an important role of MED12 in human malignancies. MED12 mutations frequently occur in uterine leiomyomas, as well as in breast fibroepithelial tumors and prostate adenocarcinoma (7–9). Additionally, downregulation of MED12 has been linked to drug resistance in colon and lung cancer through regulation of TGFβ receptor signaling and induction of epithelial–mesenchymal transition (10). However, the function of MED12 in EOC has not been explored.

In this study, we aim to explore the function and mechanism of MED12 in EOC. Our results indicate that MED12 plays an important role in regulating tumor dormancy of human ovarian cancer cells through EGFR. This is the first time that MED12 has been reported as an important molecular regulator of tumor dormancy.

Cell lines

The ovarian carcinoma cell lines, HO8910 and SKOV3, were obtained from Sun Yat-sen University Cancer Center. The cell lines used in this study were authenticated by short tandem repeat profiling before the beginning of the study (2015) and periodically monitored for Mycoplasma using Hoechst staining. All cell lines were thawed from early passage stocks and passaged for less than 6 months. The cells were maintained in DMEM (Invitrogen) with 10% FBS (Invitrogen) at 37 °C and 5% CO2.

CRISPR/Cas9

The sequences of guide RNAs (gRNA) targeting the human MED12 gene are as follows: gRNA#1, AGGATTGAAGCTGACGTTCT and gRNA#2, GATTGCTGCATAGTAGGCAC. HO8910 and SKOV3 cells were cultured in six-well dishes to 70% to 80% confluence and then cotransfected with 1 μg of MED12 sgRNA plasmid plus 1 μg of pSpCas9(BB)-2A-GFP plasmid and 5 μL of Lipofectamine2000 per well. GFP was used as a fluorescent marker to sort the transfected cells. At 48 hours posttransfection, the cells were sorted into 96-well plates using fluorescence-activated cell sorting. Single cells were validated as MED12 knocked-out clone by Western blot analysis and Sanger sequencing and then expanded as the knock-out (KO) cell line.

Western blot assay

Cells were lysed in NETN lysis buffer (20 mmol/L Tris-HCl at pH 8.0, 100 mmol/L NaCl, 1 mmol/L EDTA, 0.5% Nonidet P-40) containing 50 mmol/L β-glycerophosphate (14405; Merck), 1 μg/mL pepstatin A (P5318; Sigma-Aldrich), and 10 μmol/L leupeptin (L2884; Sigma-Aldrich) on ice, and the clarified lysates were resolved by SDS-PAGE and transferred to polyvinylidenedifluoride (PVDF) membranes for Western blot analysis using ECL detection reagents (Beyotime). The antibody against MED12 was obtained from Abcam. The antibody against EGFR was from Cell Signaling Technology, and the antibody against GAPDH was from Protein-Tech.

Xenografted tumor model

All in vivo experiments were in strict accordance with the institutional guidelines and approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (L102012017008R). BALB/c-nu mice (4–5 weeks of age, female, 18–20 g) were purchased from Charles River Laboratories, housed under standard conditions at the animal care facility at Center of Experimental Animal of Sun Yat-sen University. MED12 wild-type (WT) cells and MED12 KO cells of different concentration were subcutaneously injected into bilateral flanks of mice, respectively. Tumor length and width were measured with a vernier caliper every 3 days. Tumor volume was calculated using the formula V = 0.5 × (length × width2). For in vivo bioluminescent imaging, tumor cells stably expressed luciferase were suspended in 200 μL DMEM and inoculated subcutaneously into the right flanks of 4- to 6-week-old nude mice. Tumors were monitored using the IVIS Lumina Imaging System (Xenogen) every 5 days.

Colony formation assay

HO8910 and SKOV3 cells were counted and plated in triplicate at 500 cells per well in six-well plates and cultured for approximately 10 days. The colony formation efficiency was the ratio of the number of colonies formed to the number of cells plated.

Anchorage-independent growth assay

Cells were added to 1 mL of growth medium with 0.35% agar and layered onto beds of 0.7% agar (1 mL) in six-well plates. Cells were fed with 1 mL of medium with 0.35% agar every 2 days for 2 weeks, after which colonies were stained with 0.02% crystal violet and photographed. Colonies >50 μm in diameter were counted as positive for growth. Assays were conducted in duplicate in three independent experiments.

Sphere formation assay

Cells were plated in triplicate at 500 cells per well in ultra-low attachment six-well plates (Corning), and cultured with DMEM: Ham's F-12 medium (Invitrogen) mixed with 20 ng/mL EGF (R&D Systems) and B-27 supplement (Invitrogen). After culture for 10 days, spheres containing more than 50 cells were quantitated by inverted phase contrast microscopy (Nikon).

Cell-cycle assay

Cells were harvested by trypsinization and collected by centrifugation. Cells were washed once with PBS and fixed in 70% ethanol at 4 °C overnight. Then cells were washed once with PBS and incubated with 1 mL of PBS containing 30 μg/mL propidium iodide and 0.25 mg/mL RNase A for 1 hour at room temperature. Cells were analyzed for DNA content by flow cytometry using a FACS Cytomics FC 500 (Beckman). The data were analyzed using Multicycle AV for Windows (Beckman). All cell-cycle assays were performed three times and representative results are presented.

MTT cytotoxicity assay

Different concentrations of chemotherapeutic drugs were added into cells of designated wells in 96-well plates. After 72 hours of incubation, MTT solution (4 mg/mL) was added to each well, and the plate was further incubated for 4 hours, allowing viable cells to change the yellow-colored MTT into dark-blue formazan crystals. Subsequently the medium was discarded, and 100 μL of DMSO was added into each well to dissolve the formazan crystals. The absorbance was determined at 570 nm by an OPSYS MR Microplate Reader.

mRNA microarray assay

Total RNA was extracted by TRIzol (Invitrogen) and purified by RNeasy Mini Kit (QIAGEN). MED12 WT, KO and re-expressed SKOV3 cells and MED12 WT and KO HO8910 cells were used. RNA was processed and hybridized to Human Genome U133 Plus 2.0 Bead arrays (Affymetrix), and the microarray data were normalized and analyzed by Capitalbio Technology Corporation (Beijing, China). A fold-change cut-off threshold of 2 was applied to generate the gene signature lists. GSEA analysis was performed using GSEA 2.2.4 following the online protocol (http://www.broadinstitute.org/gsea/; refs. 11, 12). Microarray data are available publicly at http://www.ncbi.nlm.nih.gov/geo (GEO accession numbers: GSE112887 and GSE112888).

Chromatin immunoprecipitation assays

Briefly, 2 × 106 cells were plated per 100-mm diameter dish and treated with formaldehyde to cross-link chromatin-associated proteins to DNA. The cells were trypsinized and resuspended in lysis buffer, and nuclei were isolated and sonicated to shear the DNA to 200 to 500bp fragments (verified by agarose gel electrophoresis). Equal aliquots of chromatin supernatants were subjected to overnight IP with MED12 antibody or anti-lgG as a negative control. DNA was extracted and the EGFR promoter was amplified by PCR. All chromatin immunoprecipitation (ChIP) assays were performed three to four times and representative results are presented.

Luciferase reporter assay

The 2.5kb EGFR promoter sequence was amplified with PCR and cloned into the pGL3 vector (Promega). pGL3-EGFR promoter-luciferase construct and the control vector pRL-TK (Promega) coding for Renilla luciferase were cotransfected with MED12 or negative control into 293T cells using PEI. The luciferase activity was measured 48 hours later using the Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase values were normalized to Renilla, and the ratios of firefly/Renilla values were presented. The experiments were performed independently in triplicate.

Patient enrollment and IHC assay

A cohort of 138 patients (median age 52 years, range 23–83 years) diagnosed with EOC in Sun Yat-sen University Cancer Centre between 2004 and 2014 were selected in this study. All patients underwent primary debulking surgery and were then treated with platinum-combined chemotherapy regimens as first-line treatment after surgery. Patients with missing clinical information or insufficient paraffin-embedded material were excluded. Original H&E slides were reviewed by one pathologist to confirm the diagnosis and to select the most suitable paraffin-embedded tissue for immunohistochemical (IHC) study. This study was reviewed and approved by the Institutional Review Board of Sun Yat-sen University Cancer Center (GZR2017-053), and the study was performed in accordance with Declaration of Helsinki. All participants provided written informed consent before the study began. The distribution of disease stage was stage I, 14.5% (20 patients); stage II, 23.9% (33 patients); stage III, 53.6% (74 patients); and stage IV, 8.0% (11 patients). Formalin-fixed, paraffin-embedded tissues of transplanted tumors were sectioned at 4-μm thickness, blocked, and incubated with a primary antibody MED12 in 4°C overnight, followed by incubation with secondary antibodies. Visualization was achieved using the EnVision peroxidase system (Dako). Of each generated tumor, five fields were randomly selected according to semiquantitative scales. A semiquantitative scoring criterion was used for the IHC results, whereby both the staining intensity and positive areas were recorded. A staining index (values 0–12), obtained as the product of intensity of MED12-positive staining (negative, 0; weak, 1; moderate, 2; or strong, 3 scores) and the proportion of immunopositive cells of interest (<25%, 1; 25%–50%, 2; 50%–75%, 3; ≥75%, 4 scores), was calculated. All scores were subdivided into two categories according to a cutoff value of the ROC curve in the study cohort: low expression (≤4) and high expression (>4).

Statistical analysis

All in vitro experiments were performed in triplicate and repeated at least three times. Statistical analyses except for microarray data were performed using the SPSS 18.0 (IBM). Data represent mean ± SEM. A two-tailed, unpaired Student t test or the Mann–Whitney U test was used to compare the values between subgroups for quantitative data, and the χ2 was used for categorical data. Bivariate correlations between study variables were calculated by Pearson correlation coefficients. A P value less than 0.05 was considered statistically significant. The authenticity of this article has been validated by uploading the key raw data onto the Research Data Deposit public platform (www.researchdata.org.cn), with the approval RDD number as RDDB2018000301.

MED12 KO induces dormancy of EOC cells in vivo

To investigate the impact of MED12 on EOC, we knocked out endogenous MED12 in ovarian cancer cells, HO8910 and SKOV3, using specific gRNAs by CRISPR/Cas9 system. As demonstrated by DNA sequencing and immunoblot, both gRNAs specifically knocked out endogenous MED12 in both cell lines (Fig. 1A and B). To determine the effects of MED12 KO on tumorigenesis ability in vivo, we established subcutaneous xenograft tumors using HO8910, SKOV3 WT cells, and their MED12 KO cells in nude mice at a limiting dilution (Fig. 1C). MED12 KO resulted in a significant inhibition of tumor growth compared with wide-type cells (Fig. 1D). Surprisingly, MED12 KO SKOV3 cells could not form any subcutaneous xenograft tumors, and MED12 KO HO8910 cells could only form subcutaneous xenograft tumors by 2.5 × 107 cells (Fig. 1E and F; Supplementary Table S1). Moreover, the tumor growth curve showed that MED12 WT cells grew very fast, but MED12 KO cells nearly not grew, suggesting that MED12 KO may induce dormancy (Fig. 1G). IHC staining assay verified that MED12 was successfully knocked out (Fig. 1H). To further verify whether MED12 KO could induce tumor dormancy in vivo, we used in vivo bioluminescent imaging to visualize tumor growth. The number of tumor cells transplanted and the size of tumor correlated with the light emitted by luciferase activity, allowing us to quantitatively detect as few as 1,000 tumor cells and to noninvasively examine tumor cell growth and regression in real time. At 4.5 months after, inoculation luciferase activity was still detectable in MED12 KO cells even when the tumor was not grossly observable. Moreover, compared with cell numbers at 10 days, the cell numbers was nearly not changed at 1.5, 3, and 4.5 months in MED12 KO cells (Fig. 1I and J). In MED12 WT cells, the xenograft became much bigger at 1.5 month than at 10 days (Fig. 1I and J). Collectively, these results indicate that MED12 KO induces tumor dormancy of EOC cells in vivo.

Figure 1.

MED12 KO induced EOC dormancy in vivo. A, Genotyping results of MED12 KO cells in HO8910 and SKOV3. Cas9-mediated indels lead to frameshift of MED12. B, Western blot assay of MED12 expression in HO8910 and SKOV3 single clones after using specific gRNAs by CRISPR. C, Schematic of in vivo xenograft experiment. D, Representative mouse at week 6 injected with MED12 WT and KO cells. E and F, Limiting dilutions assay of in vivo xenograft experiment in WT and MED12 KO cells of HO8910 and SKOV3. The number of mice in each group was 6. G, Tumor growth curve of 4 × 104 MED12 WT cells and 2.5 × 107 MED12 KO cells of HO8910 in nude mice. The number of mice in each group was 6. H, IHC staining assay of MED12 expression in xenograft tumors of MED12 WT and KO HO8910 cells. Scale bars, 100 μm. I and J,In vivo bioluminescent imaging of 106 MED12 WT cells and 2.5 × 107 MED12 KO cells of HO8910 in nude mice. Luciferase activity is measured in photons per cm2 per s per steradian (p cm−2 s−1 sr−1). The number of mice in each group was three.

Figure 1.

MED12 KO induced EOC dormancy in vivo. A, Genotyping results of MED12 KO cells in HO8910 and SKOV3. Cas9-mediated indels lead to frameshift of MED12. B, Western blot assay of MED12 expression in HO8910 and SKOV3 single clones after using specific gRNAs by CRISPR. C, Schematic of in vivo xenograft experiment. D, Representative mouse at week 6 injected with MED12 WT and KO cells. E and F, Limiting dilutions assay of in vivo xenograft experiment in WT and MED12 KO cells of HO8910 and SKOV3. The number of mice in each group was 6. G, Tumor growth curve of 4 × 104 MED12 WT cells and 2.5 × 107 MED12 KO cells of HO8910 in nude mice. The number of mice in each group was 6. H, IHC staining assay of MED12 expression in xenograft tumors of MED12 WT and KO HO8910 cells. Scale bars, 100 μm. I and J,In vivo bioluminescent imaging of 106 MED12 WT cells and 2.5 × 107 MED12 KO cells of HO8910 in nude mice. Luciferase activity is measured in photons per cm2 per s per steradian (p cm−2 s−1 sr−1). The number of mice in each group was three.

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MED12 KO induces G0–G1 arrest and chemotherapy resistance in EOC cells in vitro

Tumor dormancy can lead to G0–G1 arrest and growth inhibition of cancer cells (4, 5). To test whether MED12 KO had an effect on cell growth, we performed MTT and plate colony formation assays and found that MED12 KO significantly inhibited proliferation of ovarian cancer cells (Fig. 2A and B). Soft agar colony assays and sphere formation assays also showed that MED12 KO significantly decreased the efficiency of soft agar colony formation and sphere formation (Fig. 2C and D). Cell-cycle analysis showed that MED12 KO in ovarian cancer cells induced G0–G1 arrest (Fig. 2E). These results indicate that MED12 KO induces growth inhibition via G0–G1 arrest in vitro.

Figure 2.

MED12 KO induced G0–G1 arrest and chemotherapy resistance in EOC cells. A, MTT assays in MED12 WT and KO cells of HO8910 and SKOV3. Bars, SD (n = 6). B, Colony formation assays in MED12 WT and KO cells of HO8910 and SKOV3. C, Soft agar colony formation assay in MED12 WT and KO cells of HO8910 and SKOV3. Scale bars, 100 μm. D, Sphere formation assay in MED12 WT and KO cells of HO8910 and SKOV3. Scale bars, 100 μm. E, Cell-cycle analysis in MED12 WT and KO cells of HO8910 and SKOV3 by flow cytometry. F, MTT assay of HO8910 and SKOV3 cells treated with paclitaxel, gemcitabine, topotecan, and 5-FU. Bars, SD (n = 6).

Figure 2.

MED12 KO induced G0–G1 arrest and chemotherapy resistance in EOC cells. A, MTT assays in MED12 WT and KO cells of HO8910 and SKOV3. Bars, SD (n = 6). B, Colony formation assays in MED12 WT and KO cells of HO8910 and SKOV3. C, Soft agar colony formation assay in MED12 WT and KO cells of HO8910 and SKOV3. Scale bars, 100 μm. D, Sphere formation assay in MED12 WT and KO cells of HO8910 and SKOV3. Scale bars, 100 μm. E, Cell-cycle analysis in MED12 WT and KO cells of HO8910 and SKOV3 by flow cytometry. F, MTT assay of HO8910 and SKOV3 cells treated with paclitaxel, gemcitabine, topotecan, and 5-FU. Bars, SD (n = 6).

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Because dormant cells are thought to be resistant to chemotherapy, we next explore the effect of MED12 KO on chemosensitivity of EOC cells. We found that MED12 KO could render ovarian cancer cells to get resistance to paclitaxel, gemcitabine, topotecan, and 5-FU, but had no effect on cell-cycle nonspecific agents, cisplatin and carboplatin (Fig. 2F; Supplementary Fig. S1). Taken together, these results indicate that MED12 KO induces G0–G1 arrest, which leads to tumor dormancy and chemoresistance.

Re-expression of MED12 in MED12 KO cells enables them escape from dormancy

To verify that the growth inhibition and chemotherapy resistance in EOC cells results from MED12 KO, we reconstituted MED12 expression in MED12 KO cells (Fig. 3A). MTT assay and colony formation assay showed that transfection of MED12 in MED12 KO cells resulted in enhancement of cell proliferation (Fig. 3B and C). Soft agar colony assay and sphere formation assay also showed that reconstitution of MED12 restored tumorigenesis ability in ovarian cancer cells (Fig. 3D and E). Furthermore, the reconstitution of MED12 also restored their cell cycle (Fig. 3F) and the chemosensitivity to paclitaxel, gemcitabine, topotecan, and 5-FU (Fig. 3G). Importantly, the reconstitution of MED12 restored tumorigenesis ability in nude mice (Fig. 3H). Taken together, these results indicate that reconstitution of MED12 in MED12 KO cells restore cell proliferation, tumorigenesis ability, and chemosensitivity.

Figure 3.

Re-expression of MED12 enabled MED12 KO cells escape from dormancy. A, Western blot assay of MED12 expression in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. B, MTT assays in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. Bars, SD (n = 6). C, Colony formation assays in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. D, Soft agar colony formation assay in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. Scale bars, 100 μm. E, Sphere formation assay in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. Scale bars, 100 μm. F, Cell-cycle analysis in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells by flow cytometry. G, MTT assay in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells treated with paclitaxel, gemcitabine, topotecan, and 5-FU. Bars, SD (n = 6). H,In vivo xenograft experiment of WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. The number of mice in each group was 6.

Figure 3.

Re-expression of MED12 enabled MED12 KO cells escape from dormancy. A, Western blot assay of MED12 expression in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. B, MTT assays in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. Bars, SD (n = 6). C, Colony formation assays in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. D, Soft agar colony formation assay in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. Scale bars, 100 μm. E, Sphere formation assay in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. Scale bars, 100 μm. F, Cell-cycle analysis in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells by flow cytometry. G, MTT assay in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells treated with paclitaxel, gemcitabine, topotecan, and 5-FU. Bars, SD (n = 6). H,In vivo xenograft experiment of WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. The number of mice in each group was 6.

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Dormancy induced by MED12 KO is involved in down-regulating EGFR

To investigate the molecular mechanism that tumor dormancy induced by MED12 KO, we performed microarray assay with MED12 WT cells and MED12 KO cells (Supplementary Fig. S2A). We found that the expression of EGFR was down-regulated in MED12 KO cells (Fig. 4A). Furthermore, we found that EGFR downstream target genes, CCND1, CCND2, and MYC were down-regulated and CDKN2C was up-regulated in MED12 KO cells (Fig. 4A). We also performed microarray assay in MED12 reconstitution cells of SKOV3. MED12 reconstitution restored the expression of EGFR and its downstream target genes to the levels of MED12 WT cells (Fig. 4B). Analyzing MED12 expression and EGFR-regulated gene signatures via gene set enrichment analysis (GSEA) in our microarray results, we found that MED12 levels were inversely correlated with the EGFR down-regulation gene signatures (Fig. 4C). Moreover, we found that MED12 levels were inversely correlated with the EGFR down-regulation gene signatures in ovarian cancer, breast cancer, and glioblastoma via GSEA in published patient expression profiles (Supplementary Fig. S2B-D). Real-time PCR and Western blot assays verified that EGFR was down-regulated in MED12 KO cells and restored after reconstitution of MED12 (Fig. 4D–G). It was reported that loss of EGFR resulted in stress signaling (low FAK/Ras/ERK, and high CDC42/p38 activity), which may lead to tumor dormancy (5). We found that MED12 KO decreased the phosphorylation of ERK, but had no effect on phosphorylation of p38 (Supplementary Fig. S2E).

Figure 4.

MED12 KO induced dormancy by decreasing EGFR expression. A, Hierarchical cluster assays of the differentially expressed genes between MED12 WT and KO single clones of SKOV3 cells. B, Microarray assay was performed on MED12 WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. C, GSEA plot showing that MED12 expression inversely correlated with EGFR-suppressed gene signatures (REACTOME_EGFR_DOWNREGULTION) in the microarray results. D and E, Real-time PCR and Western blot assays of EGFR in WT and MED12 KO single clones of HO8910 and SKOV3 cells. **, P < 0.01; ***, P < 0.001. F and G, Real-time PCR and Western blot assay of EGFR in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. ***, P < 0.001. H, Western blot assays of EGFR in WT, MED12 KO, and EGFR reconstitution single clones of SKOV3 cells. I, MTT assay in WT, MED12 KO, and EGFR reconstitution single clones of SKOV3 cells. Bars, SD (n = 6). J, Colony formation assays in WT, MED12 KO, and EGFR reconstitution single clones of SKOV3 cells. K, Cell-cycle analysis in WT, MED12 KO, and EGFR reconstitution single clones of SKOV3 cells by flow cytometry. L, MTT assay of SKOV3 cells treated with paclitaxel, gemcitabine, topotecan, and 5-FU. Bars, SD (n = 6).

Figure 4.

MED12 KO induced dormancy by decreasing EGFR expression. A, Hierarchical cluster assays of the differentially expressed genes between MED12 WT and KO single clones of SKOV3 cells. B, Microarray assay was performed on MED12 WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. C, GSEA plot showing that MED12 expression inversely correlated with EGFR-suppressed gene signatures (REACTOME_EGFR_DOWNREGULTION) in the microarray results. D and E, Real-time PCR and Western blot assays of EGFR in WT and MED12 KO single clones of HO8910 and SKOV3 cells. **, P < 0.01; ***, P < 0.001. F and G, Real-time PCR and Western blot assay of EGFR in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. ***, P < 0.001. H, Western blot assays of EGFR in WT, MED12 KO, and EGFR reconstitution single clones of SKOV3 cells. I, MTT assay in WT, MED12 KO, and EGFR reconstitution single clones of SKOV3 cells. Bars, SD (n = 6). J, Colony formation assays in WT, MED12 KO, and EGFR reconstitution single clones of SKOV3 cells. K, Cell-cycle analysis in WT, MED12 KO, and EGFR reconstitution single clones of SKOV3 cells by flow cytometry. L, MTT assay of SKOV3 cells treated with paclitaxel, gemcitabine, topotecan, and 5-FU. Bars, SD (n = 6).

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Previous studies suggested that EGFR signaling pathway was important for tumor dormancy (13–16). We also found that EGFR knockdown decreased the proliferation and sphere formation of SKOV3 cells (Supplementary Fig. S3A–S3D). To explore whether tumor cell dormancy induced by MED12 KO is related to EGFR, we reconstituted EGFR expression in MED12 KO cells (Fig. 4H). We found that reconstitution of EGFR in MED12 KO cells restored cell proliferation (Fig. 4I and J), cell cycle (Fig. 4K), and chemotherapy sensitivity (Fig. 4L) in ovarian cancer cells. These results suggest that EGFR is responsible for cell dormancy induced by MED12 KO.

MED12 regulates EGFR expression by binding to the EGFR promoter locus

As MED12 is a subunit of the Mediator complex, which plays essential roles in transcriptional regulation via RNA polymerase II, we speculated that MED12 stimulated EGFR expression through binding to the 2.2kb promoter of EGFR. To investigate whether MED12 could transactivate pGL3-EGFR promoter-luciferase construct, the 2.5kb EGFR promoter-luciferase construct was cotransfected with MED12 into 293T cells. EGFR luciferase activities were increased more than 1.5-fold by MED12 (Fig. 5A). Furthermore, we transfected EGFR promoter-luciferase construct into MED12 WT and KO cells. We found that luciferase activities were much lower in MED12 KO cells than MED12 WT cells (Fig. 5B). ChIP assays were performed to investigate whether MED12 associated with the EGFR promoter locus (Fig. 5C). As shown in Fig. 5D, MED12 bound to the region of the EGFR promoter from -22bp to -773bp.

Figure 5.

MED12 regulated EGFR expression by binding to the EGFR promoter locus. A, Effect of MED12 overexpression on the luciferase activity of the EGFR promoter in 293T cells. Bars, SD (n = 3). ***, P < 0.001. B, Effect of MED12 KO and reconstitution on the luciferase activity of the EGFR promoter in SKOV3 cells. Bars, SD (n = 3). *, P < 0.05. C, Schematic representation of the EGFR promoter regions with or without binding affinity for MED12. Precipitated DNA was amplified by PCR using primers specific for regions 1–11. Arrow, transcriptional start site. D, ChIP was performed by using anti-MED12 antibody or anti-lgG antibody to identify MED12 binding sites on the EGFR promoter in MED12 WT and KO cells of SKOV3. E, Luciferase activity of deletion/truncation constructs of the EGFR promoter, with and without transfected MED12 plasmid, to map the minimal region necessary for activation by MED12. ***, P < 0.001.

Figure 5.

MED12 regulated EGFR expression by binding to the EGFR promoter locus. A, Effect of MED12 overexpression on the luciferase activity of the EGFR promoter in 293T cells. Bars, SD (n = 3). ***, P < 0.001. B, Effect of MED12 KO and reconstitution on the luciferase activity of the EGFR promoter in SKOV3 cells. Bars, SD (n = 3). *, P < 0.05. C, Schematic representation of the EGFR promoter regions with or without binding affinity for MED12. Precipitated DNA was amplified by PCR using primers specific for regions 1–11. Arrow, transcriptional start site. D, ChIP was performed by using anti-MED12 antibody or anti-lgG antibody to identify MED12 binding sites on the EGFR promoter in MED12 WT and KO cells of SKOV3. E, Luciferase activity of deletion/truncation constructs of the EGFR promoter, with and without transfected MED12 plasmid, to map the minimal region necessary for activation by MED12. ***, P < 0.001.

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To investigate binding of MED12 to EGFR promoter in depth, luciferase reporter constructs containing serial deletions of the EGFR promoter were cotransfected into 293T cells along with the MED12 expression plasmids. As shown in Fig. 5E, pGL3-luc-containing nucleotides -2300 to 200bp, -1900 to 200bp, -1500 to 200bp, and -1100 to 200bp were activated about two-fold by MED12, whereas pGL3-luc-containing nucleotides -700 to 200bp, -300 to 200bp were activated at much lower levels. pGL3-luc-containing nucleotides 100 to 200bp were not activated by MED12 (Fig. 5E). These results were consistent with the ChIP assay. Together, these findings indicate that MED12 regulates EGFR expression by binding to the EGFR promoter locus.

Low expression of MED12 is correlated with chemotherapy-resistance and low EGFR expression in patients with EOC

To investigate the clinical significance of MED12 in EOC, we first evaluated MED12 expression in EOC patient samples using IHC. We found that MED12 was highly expressed in patient-derived EOC tissues, but it could be barely detected in normal ovary tissues (Fig. 6A and B). Moreover, chemotherapy-resistant patients (PFS ≤ 6) showed lower MED12 expression than chemotherapy-sensitive patients (Fig. 6C). To assess the clinical relevance of MED12 and EGFR in EOC, we evaluated the endogenous expression pattern of MED12 and EGFR in patients with EOC. We freshly collected 10 EOC specimens and found that MED12 expression positively correlated with the expression of EGFR (P < 0.001, r = 0.702; Fig. 6D and E). Taken together, these results suggested that MED12 downregulation was correlated with chemotherapy resistance and low EGFR expression in patients with EOC.

Figure 6.

Low expression of MED12 is correlated with chemotherapy resistance and low EGFR expression in patients with EOC. A, IHC analysis of MED12 expression in 12 normal ovary tissues and 83 EOC tissues. Top scale bars, 100 μm; bottom scale bars, 50 μm. B, Scatterplots representing the IHC scores of A. ***, P < 0.001. C, Differences in MED12 expression between chemotherapy-sensitive (PFS ≤ 6) and chemotherapy-resistant (PFS > 6) patients in ovarian cancer specimens are shown. ***, P < 0.001. D and E, Western blot analysis (D) and correlation analyses (E) of MED12 expression with the levels of EGFR in 10 freshly collected human EOC samples.

Figure 6.

Low expression of MED12 is correlated with chemotherapy resistance and low EGFR expression in patients with EOC. A, IHC analysis of MED12 expression in 12 normal ovary tissues and 83 EOC tissues. Top scale bars, 100 μm; bottom scale bars, 50 μm. B, Scatterplots representing the IHC scores of A. ***, P < 0.001. C, Differences in MED12 expression between chemotherapy-sensitive (PFS ≤ 6) and chemotherapy-resistant (PFS > 6) patients in ovarian cancer specimens are shown. ***, P < 0.001. D and E, Western blot analysis (D) and correlation analyses (E) of MED12 expression with the levels of EGFR in 10 freshly collected human EOC samples.

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In clinical situations, treatment failure due to chemoresistance and high rate of disease relapse is considered the major cause of mortality in EOC (3). Disease relapse in patients with cancer after clinical remission are often referred to as cancer dormancy (4, 5). However, the mechanisms underlying tumor dormancy have been elusive and not well characterized until now. In this study, we identified for the first time that MED12 was an important molecular regulator of tumor dormancy in human ovarian cancer.

Our findings suggest that depletion of MED12 was sufficient to induce tumor dormancy of ovarian cancer cells in vitro and in vivo. It has been reported that knockdown of MED12 expression arrested cell cycle in G0–G1 phase in castration-resistant prostate cancer (17). Consistently, we found that MED12 KO induced G0–G1 phase arrest in ovarian cancer cells. Ectopic overexpression of MED12 in MED12 KO cells mediated escape from tumor dormancy in concert with a cell-cycle switch. These data support that MED12 KO can induce G0–G1 phase arrest and tumor dormancy.

Despite significant advancements in cancer therapeutics over the past several decades, relapse following long periods of remission after treatment remains a persistent problem in EOC (18, 19). Fatal recurrences for patients with EOC can arise years and even decades later, often in the form of metastatic disease, the major cause of cancer-related deaths (20, 21). We found that low MED12 expression was associated with chemotherapy resistance in patients with EOC and MED12 KO in EOC cells would induce tumor cell dormancy. These results suggest that MED12 may play an important role in EOC recurrence and death because of tumor dormancy.

Experimental and clinical data suggest that dormant tumor cells exist in a nonproliferative state having exited the cell cycle (5, 22, 23). As many conventional anticancer drugs, such as 5-Fu and Taxol, target fast growing cancer cells, dormant cancer cells are thought to be resistant to multiple drugs that ultimately can lead to disease recurrence (24, 25). Recent studies have shown that downregulation of MED12 was associated with drug resistance in colon and lung cancer (10). Consistent with previous studies, our work revealed that MED12 deletion could render ovarian cancer cells to get resistance to paclitaxel, gemcitabine, topotecan, and 5-FU, which were cell-cycle–specific agents. Understanding the mechanism that chemoresistance and dormancy induced by MED12 KO is very important for EOC therapy.

Current experimental models of cancer dormancy can be subdivided into two general categories reflecting distinct growth kinetics. The first category, referred to as cellular dormancy, involves the ability for individual cancer cells to enter a state of temporary cell-cycle arrest (26–29). The second category, known as tumor mass dormancy, involves stagnation of overall tumor growth due to the equilibrium of proliferation and cell death. The models that comprise this category include angiogenic dormancy and immunologic dormancy (30, 31). Our findings suggest that MED12 involves in cellular dormancy in EOC. Whether MED12 have an effect on tumor mass dormancy remains to be explored.

Loss of surface receptors, such as uPAR, α5β1 integrin, or EGFR, that transduces growth signals from the microenvironment results in stress signaling (low FAK–Ras–ERK, and high CDC42–p38 activity), which in turn might lead to dormancy (15, 16, 32). This is one example to illustrate the theme of crosstalk between the microenvironment and receptor signaling in cellular dormancy. Through microarray assay, we found that MED12 knockout would decrease the expression of EGFR and downstream targets of this pathway significantly. We also found that phosphorylation of ERK was inhibited after MED12 knocked-out. Taken together, these results indicate that loss of MED12 could induce EOC dormancy by down-regulating EGFR expression.

MED12 has been linked to general functions of the Mediator complex and specific interactions with transcription factors (33, 34). MED12 is a subunit of the Cdk8 kinase module and has been shown to function as a transducer of Wnt/β-catenin signaling (35, 36). This module interacts transiently with the other components of the Mediator and functions as a context-dependent positive or negative regulator (37, 38). It has been previously shown that β-catenin physically and functionally targets MED12 subunit to activate transcription, and that MED12 is essential for the trans-activation of Wnt/β-catenin signaling (36, 39). However, we did not find obvious change of Wnt/β-catenin signaling after MED12 knocked-out (Supplementary Fig. S4A–S4C). In this study, we found that MED12 could bind to the promotor of EGFR and stimulate the transcription of EGFR. Furthermore, ChIP assay indicated that MED12 bound to the region of the EGFR promoter from -22bp to -773bp. Importantly, we found that MED12 KO could decrease the expression of MED13, Cyclin C, and CDK8 (Supplementary Fig. S5), convincing that MED12 is important for the function of Mediator. Previous study demonstrated that MED12 protein is essential for activating CDK8 kinase. And MED12 deletion would lead to dissociation of Cyclin C and CDK8 with Meditator (40). Thus, we assume that MED12 might bind to the EGFR promoter through Mediator–polymerase II complex and stimulate EGFR transcription (Fig. 7). Further studies are required to investigate whether there are some co-activators involved in EGFR transcription activation by MED12.

Figure 7.

Working model of dormancy induced by MED12 KO.

Figure 7.

Working model of dormancy induced by MED12 KO.

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EOC is the most lethal gynecologic malignancy. Despite several advances in treatment, including chemotherapy and cytoreductive surgery, the 5-year survival rate remains only 46% (1, 2, 3). Therefore, it is important to develop new treatment strategies against EOC. Some studies suggested that we might prevent tumor relapse by inhibiting conversion of tumor dormancy into proliferation (41, 42). Our present data indicate that loss of MED12 could induce dormancy in vitro and in vivo in ovarian cancer cells. These results indicated that it might be effective to maintain EOC cells in dormant status and prevent the relapse of EOC by treating patients with MED12 inhibitors. However, there are no inhibitors of MED12 available nowadays, and developing MED12 inhibitors will have great significance. A better understanding of mechanisms by which dormancy can be regulated by MED12 may suggest new therapeutic approaches to eliminate dormant EOC cells or maintain the status of dormancy.

In conclusion, we demonstrate that MED12 regulates tumor dormancy of human ovarian cancer cells through regulation of EGFR expression. Elucidating the mechanisms by which MED12 KO induces tumor dormancy in depth will provide valuable insight towards understanding EOC chemoresistance and recurrence and discovering novel antitumor strategies.

No potential conflicts of interest were disclosed.

Conception and design: X.-L. Luo, C.-C. Deng, J.-H. Liu, L.-W. Fu

Development of methodology: X.-L. Luo, C.-C. Deng, X.-D. Su, Z. Chen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X.-L. Luo, C.-C. Deng, S.-B. Liang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X.-L. Luo, C.-C. Deng, X.-D. Su

Writing, review, and/or revision of the manuscript: X.-L. Luo, C.-C. Deng, X.-D. Su

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): F. Wang, Z. Chen, X.-P. Wu

Study supervision: J.-H. Liu, L.-W. Fu

This work was supported in part by grants from National Natural Science Foundation of China (No. 81473233, to L.W. Fu; No. 81772782, to J.H. Liu), Guangzhou Technology Program Foundation (No. 201504010038, 201604020079, to L.W. Fu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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