Loss of MED12 Induces Tumor Dormancy in Human Epithelial

Published OnlineFirst May 7, 2018; DOI: 10.1158/0008-5472.CAN-18-0134

Cancer Molecular Cell Biology Research

Loss of MED12 Induces Tumor Dormancy in Human Epithelial Ovarian Cancer via Downregulation of EGFR Xiao-Lin Luo1,2, Cheng-Cheng Deng1, Xiao-Dong Su1, Fang Wang1, Zhen Chen1, Xing-Ping Wu1, Shao-Bo Liang1, Ji-Hong Liu2, and Li-Wu Fu1

Abstract

A high rate of disease relapse makes epithelial ovarian promoter of EGFR, and correlation studies showed that cancer (EOC) the leading cause of death among all gyneco- MED12 expression positively correlated with EGFR expression logic malignancies. These relapses are often due to tumor in EOC patient samples. Clinical data demonstrated that chemo- dormancy. Here we identify the RNA polymerase II transcrip- therapy-resistant patients expressed lower levels of MED12 com- tional mediator subunit 12 (MED12) as an important molec- pared with responsive patients. Overall, our data show that ular regulator of tumor dormancy. MED12 knockout (KO) MED12 plays an important role in regulating dormancy of EOC induced dormancy of EOC cells in vitro and in vivo,and through regulation of EGFR. microarray analysis showed that MED12 KO decreased expres- Signiﬁcance: MED12 is identiﬁed as a novel, important sion of EGFR. Restoration of EGFR expression in MED12 KO regulator of tumor dormancy in human ovarian cancer. cells restored proliferation. Additionally, MED12 bound to the Cancer Res; 78(13); 3532–43. 2018 AACR.

Introduction of EGFR that transduces growth signals from the microenvironment results in stress signaling (low FAK/Ras/ERK and Epithelial ovarian cancer (EOC) is the most lethal gynecolog- high CDC42/p38 activity), which in turn may lead to dormancy ical malignancy with 125,000 deaths each year worldwide, and (4, 5). However, precisely how cancer cells enter dormancy is the 5-year overall survival rate is only 46% (1–3). The standard currently unclear. treatment for EOC is optimal cytoreductive surgery followed by The RNA polymerase II mediator complex subunit 12 combination chemotherapy using taxane- and platinum-based (MED12) is a subunit of the Mediator complex, which plays regimens (1–3). However, the majority of patients develop recur- essential roles in transcriptional regulation via RNA polymerase rence with latency periods that range from years to decades due to II (6). Several studies have already proposed an important the persistence and recurrence of dormant, drug-resistant ovarian role of MED12 in human malignancies. MED12 mutations cancer cells (3). This pause can be explained by tumor dormancy, frequently occur in uterine leiomyomas, as well as in breast a leading factor of treatment failure, metastasis, and tumor fibroepithelial tumors and prostate adenocarcinoma (7–9). recurrence (4, 5). Understanding the driving force of tumor Additionally, downregulation of MED12 has been linked to dormancy has important therapeutic implications for preventing drug resistance in colon and lung cancer through regulation of relapse in patients with a history of EOC. Tumor dormancy can be TGFb receptor signaling and induction of epithelial–mesen- explained by several mechanisms. These include cellular dorman- chymal transition (10). However, the function of MED12 in cy; the inability of a tumor cell population to initiate angiogen- EOC has not been explored. esis; and immunosurveillance (5). Cellular dormancy is mediated In this study, we aim to explore the function and mechanism of by different signaling pathways including EGFR signaling MED12 in EOC. Our results indicate that MED12 plays an and high p38 over ERK activity (4, 5). It was reported that loss important role in regulating tumor dormancy of human ovarian cancer cells through EGFR. This is the first time that MED12 has 1Department of Experimental Research, Sun Yat-sen University Cancer Center, been reported as an important molecular regulator of tumor State Key Laboratory of Oncology in South China, Collaborative Innovation dormancy. Center for Cancer Medicine, Guangzhou, China. 2Department of Gynecologic Oncology, Sun Yat-sen University Cancer Center, Guangzhou, China. Note: Supplementary data for this article are available at Cancer Research Materials and Methods Online (http://cancerres.aacrjournals.org/). Cell lines Corresponding Author: Li-Wu Fu, Sun Yat-sen University Cancer Center, 651 The ovarian carcinoma cell lines, HO8910 and SKOV3, were Dongfeng Road East, Guangzhou, Guangdong 510060, China. Phone: 86-20- obtained from Sun Yat-sen University Cancer Center. The cell 87243163; Fax: 86-20-87343170; E-mail: [email protected]; and Ji-Hong lines used in this study were authenticated by short tandem repeat Liu, Sun Yat-sen University Cancer Center, 651 Yongfeng Road East, Guangzhou, profiling before the beginning of the study (2015) and period- Guangdong 510060, China. Phone: 86-20-87243102; Fax: 86-20-87343014; ically monitored for Mycoplasma using Hoechst staining. All cell E-mail: [email protected] lines were thawed from early passage stocks and passaged for less doi: 10.1158/0008-5472.CAN-18-0134 than 6 months. The cells were maintained in DMEM (Invitrogen) 2018 American Association for Cancer Research. with 10% FBS (Invitrogen) at 37 C and 5% CO2.

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Loss of MED12 Induces Tumor Dormancy in Ovarian Cancer

CRISPR/Cas9 Sphere formation assay The sequences of guide RNAs (gRNA) targeting the human Cells were plated in triplicate at 500 cells per well in ultra-low MED12 gene are as follows: gRNA#1, AGGATTGAAGCTGACG- attachment six-well plates (Corning), and cultured with DMEM: TTCT and gRNA#2, GATTGCTGCATAGTAGGCAC. HO8910 and Ham's F-12 medium (Invitrogen) mixed with 20 ng/mL EGF SKOV3 cells were cultured in six-well dishes to 70% to 80% (R&D Systems) and B-27 supplement (Invitrogen). After culture confluence and then cotransfected with 1 mg of MED12 sgRNA for 10 days, spheres containing more than 50 cells were quanti- plasmid plus 1 mg of pSpCas9(BB)-2A-GFP plasmid and 5 mLof tated by inverted phase contrast microscopy (Nikon). Lipofectamine2000 per well. GFP was used as a fluorescent marker to sort the transfected cells. At 48 hours posttransfection, Cell-cycle assay the cells were sorted into 96-well plates using fluorescence- Cells were harvested by trypsinization and collected by centri- activated cell sorting. Single cells were validated as MED12 fugation. Cells were washed once with PBS and fixed in 70% knocked-out clone by Western blot analysis and Sanger sequenc- ethanol at 4 C overnight. Then cells were washed once with PBS ing and then expanded as the knock-out (KO) cell line. and incubated with 1 mL of PBS containing 30 mg/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 Western blot assay FACS Cytomics FC 500 (Beckman). The data were analyzed using Cells were lysed in NETN lysis buffer (20 mmol/L Tris-HCl at Multicycle AV for Windows (Beckman). All cell-cycle assays were pH 8.0, 100 mmol/L NaCl, 1 mmol/L EDTA, 0.5% Nonidet P-40) performed three times and representative results are presented. containing 50 mmol/L b-glycerophosphate (14405; Merck), m m 1 g/mL pepstatin A (P5318; Sigma-Aldrich), and 10 mol/L MTT cytotoxicity assay fi leupeptin (L2884; Sigma-Aldrich) on ice, and the clari ed lysates Different concentrations of chemotherapeutic drugs were were resolved by SDS-PAGE and transferred to polyvinylidenedi- added into cells of designated wells in 96-well plates. After 72 fl uoride (PVDF) membranes for Western blot analysis using ECL hours of incubation, MTT solution (4 mg/mL) was added to each detection reagents (Beyotime). The antibody against MED12 was well, and the plate was further incubated for 4 hours, allowing obtained from Abcam. The antibody against EGFR was from Cell viable cells to change the yellow-colored MTT into dark-blue Signaling Technology, and the antibody against GAPDH was from formazan crystals. Subsequently the medium was discarded, and Protein-Tech. 100 mL of DMSO was added into each well to dissolve the formazan crystals. The absorbance was determined at 570 nm Xenografted tumor model by an OPSYS MR Microplate Reader. All in vivo experiments were in strict accordance with the institutional guidelines and approved by the Institutional mRNA microarray assay Animal Care and Use Committee of Sun Yat-sen University Total RNA was extracted by TRIzol (Invitrogen) and purified by (L102012017008R). BALB/c-nu mice (4–5 weeks of age, female, RNeasy Mini Kit (QIAGEN). MED12 WT, KO and re-expressed 18–20 g) were purchased from Charles River Laboratories, housed SKOV3 cells and MED12 WT and KO HO8910 cells were used. under standard conditions at the animal care facility at Center of RNA was processed and hybridized to Human Genome U133 Plus Experimental Animal of Sun Yat-sen University. MED12 wild-type 2.0 Bead arrays (Affymetrix), and the microarray data were nor- (WT) cells and MED12 KO cells of different concentration were malized and analyzed by Capitalbio Technology Corporation subcutaneously injected into bilateral flanks of mice, respectively. (Beijing, China). A fold-change cut-off threshold of 2 was applied Tumor length and width were measured with a vernier caliper to generate the gene signature lists. GSEA analysis was performed every 3 days. Tumor volume was calculated using the formula using GSEA 2.2.4 following the online protocol (http://www. V ¼ 0.5 (length width2). For in vivo bioluminescent imaging, broadinstitute.org/gsea/; refs. 11, 12). Microarray data are avail- tumor cells stably expressed luciferase were suspended in 200 mL able publicly at http://www.ncbi.nlm.nih.gov/geo (GEO acces- DMEM and inoculated subcutaneously into the right flanks of sion numbers: GSE112887 and GSE112888). 4- to 6-week-old nude mice. Tumors were monitored using the IVIS Lumina Imaging System (Xenogen) every 5 days. Chromatin immunoprecipitation assays Briefly, 2 106 cells were plated per 100-mm diameter dish and treated with formaldehyde to cross-link chromatin-associated Colony formation assay proteins to DNA. The cells were trypsinized and resuspended in HO8910 and SKOV3 cells were counted and plated in lysis buffer, and nuclei were isolated and sonicated to shear triplicate at 500 cells per well in six-well plates and cultured the DNA to 200 to 500bp fragments (verified by agarose gel for approximately 10 days. The colony formation efficiency was electrophoresis). Equal aliquots of chromatin supernatants were the ratio of the number of colonies formed to the number of subjected to overnight IP with MED12 antibody or anti-lgG as a cells plated. negative control. DNA was extracted and the EGFR promoter was amplified by PCR. All chromatin immunoprecipitation (ChIP) Anchorage-independent growth assay assays were performed three to four times and representative Cells were added to 1 mL of growth medium with 0.35% agar results are presented. 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 Luciferase reporter assay weeks, after which colonies were stained with 0.02% crystal violet The 2.5kb EGFR promoter sequence was amplified with PCR and photographed. Colonies >50 mm in diameter were counted as and cloned into the pGL3 vector (Promega). pGL3-EGFR positive for growth. Assays were conducted in duplicate in three promoter-luciferase construct and the control vector pRL-TK independent experiments. (Promega) coding for Renilla luciferase were cotransfected with

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MED12 or negative control into 293T cells using PEI. The lucif- knocked out endogenous MED12 in both cell lines (Fig. 1A and erase activity was measured 48 hours later using the Dual- B). To determine the effects of MED12 KO on tumorigenesis Luciferase Reporter Assay System (Promega). The firefly luciferase ability in vivo, we established subcutaneous xenograft tumors values were normalized to Renilla, and the ratios of firefly/Renilla using HO8910, SKOV3 WT cells, and their MED12 KO cells in values were presented. The experiments were performed indepen- nude mice at a limiting dilution (Fig. 1C). MED12 KO resulted in a dently in triplicate. significant inhibition of tumor growth compared with wide-type cells (Fig. 1D). Surprisingly, MED12 KO SKOV3 cells could not Patient enrollment and IHC assay form any subcutaneous xenograft tumors, and MED12 KO – A cohort of 138 patients (median age 52 years, range 23 HO8910 cells could only form subcutaneous xenograft tumors 83 years) diagnosed with EOC in Sun Yat-sen University Cancer by 2.5 107 cells (Fig. 1E and F; Supplementary Table S1). Centre between 2004 and 2014 were selected in this study. All Moreover, the tumor growth curve showed that MED12 WT cells patients underwent primary debulking surgery and were then grew very fast, but MED12 KO cells nearly not grew, suggesting treated with platinum-combined chemotherapy regimens as that MED12 KO may induce dormancy (Fig. 1G). IHC staining fi rst-line treatment after surgery. Patients with missing clinical assay verified that MED12 was successfully knocked out (Fig. 1H). fi fi information or insuf cient paraf n-embedded material were To further verify whether MED12 KO could induce tumor excluded. Original H&E slides were reviewed by one pathologist dormancy in vivo, we used in vivo bioluminescent imaging to fi fi to con rm the diagnosis and to select the most suitable paraf n- visualize tumor growth. The number of tumor cells transplanted embedded tissue for immunohistochemical (IHC) study. This and the size of tumor correlated with the light emitted by lucif- study was reviewed and approved by the Institutional Review erase activity, allowing us to quantitatively detect as few as 1,000 Board of Sun Yat-sen University Cancer Center (GZR2017-053), tumor cells and to noninvasively examine tumor cell growth and and the study was performed in accordance with Declaration of regression in real time. At 4.5 months after, inoculation luciferase Helsinki. All participants provided written informed consent activity was still detectable in MED12 KO cells even when the before the study began. The distribution of disease stage was stage tumor was not grossly observable. Moreover, compared with cell I, 14.5% (20 patients); stage II, 23.9% (33 patients); stage III, numbers at 10 days, the cell numbers was nearly not changed at 53.6% (74 patients); and stage IV, 8.0% (11 patients). Formalin- 1.5, 3, and 4.5 months in MED12 KO cells (Fig. 1I and J). In fi fi xed, paraf n-embedded tissues of transplanted tumors were MED12 WT cells, the xenograft became much bigger at 1.5 month sectioned at 4-mm thickness, blocked, and incubated with a than at 10 days (Fig. 1I and J). Collectively, these results indicate primary antibody MED12 in 4 C overnight, followed by incuba- that MED12 KO induces tumor dormancy of EOC cells in vivo. tion with secondary antibodies. Visualization was achieved using the EnVision peroxidase system (Dako). Of each generated tumor, MED12 KO induces G –G arrest and chemotherapy resistance fi fi 0 1 ve elds were randomly selected according to semiquantitative in EOC cells in vitro scales. A semiquantitative scoring criterion was used for the IHC Tumor dormancy can lead to G0–G1 arrest and growth inhi- results, whereby both the staining intensity and positive areas were bition of cancer cells (4, 5). To test whether MED12 KO had – recorded. A staining index (values 0 12), obtained as the product an effect on cell growth, we performed MTT and plate colony of intensity of MED12-positive staining (negative, 0; weak, 1; formation assays and found that MED12 KO significantly inhib- moderate, 2; or strong, 3 scores) and the proportion of immuno- ited proliferation of ovarian cancer cells (Fig. 2A and B). Soft agar < – – positive cells of interest ( 25%, 1; 25% 50%, 2; 50% 75%, 3; colony assays and sphere formation assays also showed that 75%, 4 scores), was calculated. All scores were subdivided into MED12 KO significantly decreased the efficiency of soft agar two categories according to a cutoff value of the ROC curve in the colony formation and sphere formation (Fig. 2C and D). Cell- > study cohort: low expression ( 4) and high expression ( 4). cycle analysis showed that MED12 KO in ovarian cancer cells induced G –G arrest (Fig. 2E). These results indicate that MED12 Statistical analysis 0 1 KO induces growth inhibition via G –G arrest in vitro. All in vitro experiments were performed in triplicate and repeat- 0 1 Because dormant cells are thought to be resistant to chemo- ed at least three times. Statistical analyses except for microarray therapy, we next explore the effect of MED12 KO on chemosensi- data were performed using the SPSS 18.0 (IBM). Data represent tivity of EOC cells. We found that MED12 KO could render ovarian mean SEM. A two-tailed, unpaired Student t test or the Mann– cancer cells to get resistance to paclitaxel, gemcitabine, topotecan, Whitney U test was used to compare the values between and 5-FU, but had no effect on cell-cycle nonspecific agents, subgroups for quantitative data, and the x2 was used for categor- cisplatin and carboplatin (Fig. 2F; Supplementary Fig. S1). Taken ical data. Bivariate correlations between study variables were together, these results indicate that MED12 KO induces G –G calculated by Pearson correlation coefficients. A P value less than 0 1 arrest, which leads to tumor dormancy and chemoresistance. 0.05 was considered statistically significant. The authenticity of this article has been validated by uploading the key raw data onto Re-expression of MED12 in MED12 KO cells enables them the Research Data Deposit public platform (www.researchdata. escape from dormancy org.cn), with the approval RDD number as RDDB2018000301. To verify that the growth inhibition and chemotherapy resistance in EOC cells results from MED12 KO, we reconstituted Results MED12 expression in MED12 KO cells (Fig. 3A). MTT assay and MED12 KO induces dormancy of EOC cells in vivo colony formation assay showed that transfection of MED12 in To investigate the impact of MED12 on EOC, we knocked out MED12 KO cells resulted in enhancement of cell proliferation endogenous MED12 in ovarian cancer cells, HO8910 and SKOV3, (Fig. 3B and C). Soft agar colony assay and sphere formation assay using specific gRNAs by CRISPR/Cas9 system. As demonstrated by also showed that reconstitution of MED12 restored tumorigenesis DNA sequencing and immunoblot, both gRNAs specifically ability in ovarian cancer cells (Fig. 3D and E). Furthermore, the

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Loss of MED12 Induces Tumor Dormancy in Ovarian Cancer

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 mm. 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 cm2 s1 sr1). The number of mice in each group was three.

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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 ¼ 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 mm. D, Sphere formation assay in MED12 WT and KO cells of HO8910 and SKOV3. Scale bars, 100 mm. 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 ¼ 6).

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Loss of MED12 Induces Tumor Dormancy in Ovarian Cancer

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 ¼ 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 mm. E, Sphere formation assay in WT, MED12 KO, and MED12 reconstitution single clones of SKOV3 cells. Scale bars, 100 mm. 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 ¼ 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.

reconstitution of MED12 also restored their cell cycle (Fig. 3F) and Dormancy induced by MED12 KO is involved in down- the chemosensitivity to paclitaxel, gemcitabine, topotecan, and regulating EGFR 5-FU (Fig. 3G). Importantly, the reconstitution of MED12 To investigate the molecular mechanism that tumor dormancy restored tumorigenesis ability in nude mice (Fig. 3H). Taken induced by MED12 KO, we performed microarray assay with together, these results indicate that reconstitution of MED12 in MED12 WT cells and MED12 KO cells (Supplementary Fig. S2A). MED12 KO cells restore cell proliferation, tumorigenesis ability, We found that the expression of EGFR was down-regulated in and chemosensitivity. MED12 KO cells (Fig. 4A). Furthermore, we found that EGFR

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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 ¼ 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 ¼ 6).

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Loss of MED12 Induces Tumor Dormancy in Ovarian Cancer

downstream target genes, CCND1, CCND2, and MYC were down- indicate that MED12 regulates EGFR expression by binding to regulated and CDKN2C was up-regulated in MED12 KO cells the EGFR promoter locus. (Fig. 4A). We also performed microarray assay in MED12 reconstitution cells of SKOV3. MED12 reconstitution restored the Low expression of MED12 is correlated with chemotherapy- expression of EGFR and its downstream target genes to the levels resistance and low EGFR expression in patients with EOC of MED12 WT cells (Fig. 4B). Analyzing MED12 expression and To investigate the clinical significance of MED12 in EOC, we EGFR-regulated gene signatures via gene set enrichment analysis first evaluated MED12 expression in EOC patient samples using (GSEA) in our microarray results, we found that MED12 levels IHC. We found that MED12 was highly expressed in patient- were inversely correlated with the EGFR down-regulation gene derived EOC tissues, but it could be barely detected in normal signatures (Fig. 4C). Moreover, we found that MED12 levels ovary tissues (Fig. 6A and B). Moreover, chemotherapy-resistant were inversely correlated with the EGFR down-regulation gene patients (PFS 6) showed lower MED12 expression than che- signatures in ovarian cancer, breast cancer, and glioblastoma via motherapy-sensitive patients (Fig. 6C). To assess the clinical GSEA in published patient expression profiles (Supplementary relevance of MED12 and EGFR in EOC, we evaluated the endog- Fig. S2B-D). Real-time PCR and Western blot assays verified that enous expression pattern of MED12 and EGFR in patients with EGFR was down-regulated in MED12 KO cells and restored after EOC. We freshly collected 10 EOC specimens and found that reconstitution of MED12 (Fig. 4D–G). It was reported that loss of MED12 expression positively correlated with the expression of EGFR resulted in stress signaling (low FAK/Ras/ERK, and high EGFR (P < 0.001, r ¼ 0.702; Fig. 6D and E). Taken together, these CDC42/p38 activity), which may lead to tumor dormancy (5). We results suggested that MED12 downregulation was correlated found that MED12 KO decreased the phosphorylation of ERK, with chemotherapy resistance and low EGFR expression in but had no effect on phosphorylation of p38 (Supplementary patients with EOC. Fig. S2E). Previous studies suggested that EGFR signaling pathway was Discussion important for tumor dormancy (13–16). We also found that EGFR knockdown decreased the proliferation and sphere forma- In clinical situations, treatment failure due to chemoresistance tion of SKOV3 cells (Supplementary Fig. S3A–S3D). To explore and high rate of disease relapse is considered the major cause of whether tumor cell dormancy induced by MED12 KO is related to mortality in EOC (3). Disease relapse in patients with cancer after EGFR, we reconstituted EGFR expression in MED12 KO cells clinical remission are often referred to as cancer dormancy (4, 5). (Fig. 4H). We found that reconstitution of EGFR in MED12 KO However, the mechanisms underlying tumor dormancy have cells restored cell proliferation (Fig. 4I and J), cell cycle (Fig. 4K), been elusive and not well characterized until now. In this study, and chemotherapy sensitivity (Fig. 4L) in ovarian cancer cells. we identified for the first time that MED12 was an important These results suggest that EGFR is responsible for cell dormancy molecular regulator of tumor dormancy in human ovarian cancer. induced by MED12 KO. Our findings suggest that depletion of MED12 was sufficient to induce tumor dormancy of ovarian cancer cells in vitro and in MED12 regulates EGFR expression by binding to the EGFR vivo. It has been reported that knockdown of MED12 expression – promoter locus arrested cell cycle in G0 G1 phase in castration-resistant pros- As MED12 is a subunit of the Mediator complex, which plays tate cancer (17). Consistently, we found that MED12 KO – essential roles in transcriptional regulation via RNA polymerase II, induced G0 G1 phase arrest in ovarian cancer cells. Ectopic we speculated that MED12 stimulated EGFR expression through overexpression of MED12 in MED12 KO cells mediated escape binding to the 2.2kb promoter of EGFR. To investigate whether from tumor dormancy in concert with a cell-cycle switch. These – MED12 could transactivate pGL3-EGFR promoter-luciferase con- data support that MED12 KO can induce G0 G1 phase arrest struct, the 2.5kb EGFR promoter-luciferase construct was cotrans- and tumor dormancy. fected with MED12 into 293T cells. EGFR luciferase activities were Despite significant advancements in cancer therapeutics over increased more than 1.5-fold by MED12 (Fig. 5A). Furthermore, the past several decades, relapse following long periods of remis- we transfected EGFR promoter-luciferase construct into MED12 sion after treatment remains a persistent problem in EOC (18, WT and KO cells. We found that luciferase activities were 19). Fatal recurrences for patients with EOC can arise years and much lower in MED12 KO cells than MED12 WT cells even decades later, often in the form of metastatic disease, the (Fig. 5B). ChIP assays were performed to investigate whether major cause of cancer-related deaths (20, 21). We found that low MED12 associated with the EGFR promoter locus (Fig. 5C). As MED12 expression was associated with chemotherapy resistance shown in Fig. 5D, MED12 bound to the region of the EGFR in patients with EOC and MED12 KO in EOC cells would induce promoter from -22bp to -773bp. tumor cell dormancy. These results suggest that MED12 may play To investigate binding of MED12 to EGFR promoter in an important role in EOC recurrence and death because of tumor depth, luciferase reporter constructs containing serial deletions dormancy. of the EGFR promoter were cotransfected into 293T cells along Experimental and clinical data suggest that dormant tumor with the MED12 expression plasmids. As shown in Fig. 5E, cells exist in a nonproliferative state having exited the cell cycle pGL3-luc-containing nucleotides -2300 to 200bp, -1900 to (5, 22, 23). As many conventional anticancer drugs, such as 5-Fu 200bp, -1500 to 200bp, and -1100 to 200bp were activated and Taxol, target fast growing cancer cells, dormant cancer cells are about two-fold by MED12, whereas pGL3-luc-containing thought to be resistant to multiple drugs that ultimately can lead nucleotides -700 to 200bp, -300 to 200bp were activated at to disease recurrence (24, 25). Recent studies have shown that much lower levels. pGL3-luc-containing nucleotides 100 to downregulation of MED12 was associated with drug resistance in 200bp were not activated by MED12 (Fig. 5E). These results colon and lung cancer (10). Consistent with previous studies, our were consistent with the ChIP assay. Together, these findings work revealed that MED12 deletion could render ovarian cancer

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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 ¼ 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 ¼ 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.

cells to get resistance to paclitaxel, gemcitabine, topotecan, and Current experimental models of cancer dormancy can be sub- 5-FU, which were cell-cycle–specific agents. Understanding the divided into two general categories reflecting distinct growth mechanism that chemoresistance and dormancy induced by kinetics. The first category, referred to as cellular dormancy, MED12 KO is very important for EOC therapy. involves the ability for individual cancer cells to enter a state of

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Loss of MED12 Induces Tumor Dormancy in Ovarian Cancer

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 mm; bottom scale bars, 50 mm. 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.

temporary cell-cycle arrest (26–29). The second category, known (33, 34). MED12 is a subunit of the Cdk8 kinase module and as tumor mass dormancy, involves stagnation of overall tumor has been shown to function as a transducer of Wnt/b-catenin growth due to the equilibrium of proliferation and cell death. The signaling (35, 36). This module interacts transiently with the models that comprise this category include angiogenic dormancy other components of the Mediator and functions as a context- and immunologic dormancy (30, 31). Our findings suggest that dependent positive or negative regulator (37, 38). It has been MED12 involves in cellular dormancy in EOC. Whether MED12 previously shown that b-catenin physically and functionally have an effect on tumor mass dormancy remains to be explored. targets MED12 subunit to activate transcription, and that MED12 Loss of surface receptors, such as uPAR, a5b1 integrin, or EGFR, is essential for the trans-activation of Wnt/b-catenin signaling that transduces growth signals from the microenvironment results (36, 39). However, we did not find obvious change of Wnt/ in stress signaling (low FAK–Ras–ERK, and high CDC42–p38 b-catenin signaling after MED12 knocked-out (Supplementary activity), which in turn might lead to dormancy (15, 16, 32). This Fig. S4A–S4C). In this study, we found that MED12 could bind to is one example to illustrate the theme of crosstalk between the the promotor of EGFR and stimulate the transcription of EGFR. microenvironment and receptor signaling in cellular dormancy. Furthermore, ChIP assay indicated that MED12 bound to the Through microarray assay, we found that MED12 knockout region of the EGFR promoter from -22bp to -773bp. Importantly, would decrease the expression of EGFR and downstream targets we found that MED12 KO could decrease the expression of of this pathway significantly. We also found that phosphorylation MED13, Cyclin C, and CDK8 (Supplementary Fig. S5), convinc- of ERK was inhibited after MED12 knocked-out. Taken together, ing that MED12 is important for the function of Mediator. these results indicate that loss of MED12 could induce EOC Previous study demonstrated that MED12 protein is essential for dormancy by down-regulating EGFR expression. activating CDK8 kinase. And MED12 deletion would lead to MED12 has been linked to general functions of the Mediator dissociation of Cyclin C and CDK8 with Meditator (40). Thus, complex and specific interactions with transcription factors we assume that MED12 might bind to the EGFR promoter

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Luo et al.

Figure 7. Working model of dormancy induced by MED12 KO.

through Mediator–polymerase II complex and stimulate EGFR Disclosure of Potential Conflicts of Interest transcription (Fig. 7). Further studies are required to investigate No potential conflicts of interest were disclosed. whether there are some co-activators involved in EGFR transcription activation by MED12. Authors' Contributions EOC is the most lethal gynecologic malignancy. Despite several Conception and design: X.-L. Luo, C.-C. Deng, J.-H. Liu, L.-W. Fu advances in treatment, including chemotherapy and cytoreduc- Development of methodology: X.-L. Luo, C.-C. Deng, X.-D. Su, Z. Chen tive surgery, the 5-year survival rate remains only 46% (1, 2, 3). Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X.-L. Luo, C.-C. Deng, S.-B. Liang Therefore, it is important to develop new treatment strategies Analysis and interpretation of data (e.g., statistical analysis, biostatistics, against EOC. Some studies suggested that we might prevent tumor computational analysis): X.-L. Luo, C.-C. Deng, X.-D. Su relapse by inhibiting conversion of tumor dormancy into prolif- Writing, review, and/or revision of the manuscript: X.-L. Luo, C.-C. Deng, eration (41, 42). Our present data indicate that loss of MED12 X.-D. Su could induce dormancy in vitro and in vivo in ovarian cancer cells. Administrative, technical, or material support (i.e., reporting or organizing These results indicated that it might be effective to maintain EOC data, constructing databases): F. Wang, Z. Chen, X.-P. Wu Study supervision: J.-H. Liu, L.-W. Fu cells in dormant status and prevent the relapse of EOC by treating patients with MED12 inhibitors. However, there are no inhibitors Acknowledgments of MED12 available nowadays, and developing MED12 inhibi- This work was supported in part by grants from National Natural Science tors will have great significance. A better understanding of Foundation of China (No. 81473233, to L.W. Fu; No. 81772782, to J.H. Liu), mechanisms by which dormancy can be regulated by MED12 Guangzhou Technology Program Foundation (No. 201504010038, may suggest new therapeutic approaches to eliminate dormant 201604020079, to L.W. Fu). EOC cells or maintain the status of dormancy. In conclusion, we demonstrate that MED12 regulates tumor The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked dormancy of human ovarian cancer cells through regulation of advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate EGFR expression. Elucidating the mechanisms by which MED12 this fact. KO induces tumor dormancy in depth will provide valuable insight towards understanding EOC chemoresistance and recur- Received January 13, 2018; revised March 24, 2018; accepted April 26, 2018; rence and discovering novel antitumor strategies. published first May 7, 2018.

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Loss of MED12 Induces Tumor Dormancy in Human Epithelial Ovarian Cancer via Downregulation of EGFR

Xiao-Lin Luo, Cheng-Cheng Deng, Xiao-Dong Su, et al.

Cancer Res 2018;78:3532-3543. Published OnlineFirst May 7, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-0134

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