Loss of PERIOD2 (PER2) induces HO-1-mediated epithelial-mesenchymal transition and cisplatin resistance in Epi-A ovarian cancer

Loss of PERIOD2 (PER2) induces HO-1-mediated epithelial-mesenchymal transition and cisplatin resistance in Epi-A ovarian cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Loss of PERIOD2 (PER2) induces HO-1-mediated epithelial-mesenchymal transition and cisplatin resistance in Epi-A ovarian cancer Wendy Hwang-Verslues, Grace Tan, Priyanka Vinothkumar, Li-Tzu Cheng, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5242949/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Rapid metastasis and chemoresistance make ovarian cancer (OC) the deadliest gynecological cancer. These malignant phenotypes are strongly associated with epithelial-mesenchymal transition (EMT). OC is highly heterogenous with five distinct gene-expression based molecular subtypes (GEMS) correlated with different EMT levels and patient survival. However, the regulatory mechanisms underlying GEMS-specific EMT and malignancy remain unclear. We found that PER2 gene expression was significantly downregulated in OC compared to normal ovarian tissues. Among the five GEMS, PER2 expression was relatively high in the Epithelial-A (Epi-A) subtype and Epi-A OC patients with higher PER2 level had better overall survival. Notably, PER2 expression was required to maintain the epithelial phenotype. Conversely, PER2 depletion drove EMT and consequently enhanced migration and invasion ability of Epi-A OC cells via upregulation of Heme oxygenase-1 (HO-1). PER2 depletion also increased cisplatin resistance. Inhibition of HO-1 in PER2-depleted Epi-A OC cells impeded invasion and migration, and re-sensitized the cells to cisplatin. Our results revealed new insights into how an epithelial-like, less malignant OC subtype acquires EMT to become more invasive and chemoresistant. These findings also indicate that prevention of PER2 downregulation and targeting HO-1 are promising strategies for treating Epi-A OC progression. Biological sciences/Cancer/Gynaecological cancer/Ovarian cancer Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ovarian cancer (OC) is the most lethal gynecological cancer. The poor prognosis of OC is due to rapid metastasis and chemoresistance, which are strongly associated with epithelial-mesenchymal transition (EMT) [ 1 ]. Acquisition of EMT is a critical process for tumor cells to gain stemness features, enhance metastasis and gain chemoresistance [ 2 ]. EMT can be induced in several ways. For example, transforming growth factor β (TGFβ)/bone morphogenetic proteins (BMPs) Wnt, Notch and receptor tyrosine kinases are known to drive EMT [ 3 – 5 ]. Also, hypoxia [ 6 ] and inflammation [ 7 ], which are common in the tumor microenvironment, as well as oxidative stress, which is prevalent in cancer cells due to intrinsic redox regulation or metabolic reprograming [ 8 ], are all potent EMT inducers. Transcription factors including TWIST, SNAIL, SLUG and ZEB are EMT executors which repress expression of epithelial markers, such as E-cadherin (ECAD), and activate expression of mesenchymal markers, such as Vimentin (VIM), Fibronectin or N‐cadherin (NCAD) [ 2 ]. Overall, EMT is regulated in a context‐dependent manner, and cancers at different EMT status have unique EMT signatures [ 9 ]. OC is highly heterogeneous and can be categorized into serous, clear cell, endometrioid and mucinous subtypes based on histological characteristics [ 10 ]. Within each histological subtype, gene expression signatures among individual patients remain highly diverse and their clinical outcomes are different in response to the same treatment. To cope with this diverse molecular heterogeneity [ 11 – 14 ], functional genomic analysis utilizing different risk factors, epidemiological patterns, molecular characteristics and clinical manifestations were used to define five distinct gene-expression based molecular subtypes (GEMS) [ 13 , 15 ]: Epithelial-A (Epi-A), Epithelial-B (Epi-B), Stem-like A (StemA), Stem-like B (StemB) and Mesenchymal (Mes). These GEMS have been correlated with EMT and patient survival [ 11 , 13 ]. The Epi-A GEMS is associated with low EMT and better survival, whereas the Mes GEMS is associated with high EMT and worse survival. However, the regulatory mechanisms underlying GEMS specific EMT remain unclear. Chronic circadian disruption has been linked to increased cancer risks [ 16 ]. Compelling evidence has suggested that core clock genes which form the transcription-translation feedback loop (TTFL) in circadian regulation also play roles in tumor progression and chemo-response [ 17 ]. Among the core clock genes, Period2 (PER2) is a key co-repressor essential for reactivation of the TTFL in circadian regulation [ 18 ] and has been shown to have tumor suppressive functions in many cancers [ 19 – 27 ]. In OC, PER2 expression has been reported to be negatively correlated with tumor growth and tumor stage [ 28 ]. However, the mechanism by which PER2 affects OC progression remain unclear. We found that PER2 exhibits an Epi-A OC subtype specific anti-malignancy role. Epi-A OC patients with tumors expressing relatively high levels of PER2 had significantly better disease-free prognosis and overall survival compared to those with PER2 -low tumors. PER2 downregulation promoted migration and invasion via upregulation of Heme oxygenase-1 ( HMOX1 /HO-1) to drive EMT. Furthermore, PER2 depletion increased cisplatin resistance in Epi-A OC cells. Conversely, HO-1 inhibition prevented the PER2-depletion mediated malignant phenotypes and sensitized PER2-depleted Epi-A OC cells to cisplatin. Together, our results revealed a new role of PER2 in regulating EMT via control of HMOX1 /HO-1 to maintain an epithelial-like phenotype and cisplatin sensitivity of Epi-A OC cells. These findings also provided a basis for treating Epi-A OC progression by preventing PER2 downregulation and targeting HO-1. Materials and Methods Cell lines Human OC cell lines PEO1 and OVCA429 were gifts from Dr. Ruby YJ Huang (National Taiwan University, Taiwan) and Dr. Noriomi Matsumura (Kindai University, Japan) respectively. PEO1 was cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 2 mM Sodium pyruvate, 10% fetal bovine serum (FBS) and 1 × antibiotics/antimycotics (anti/anti). OVCA429 was cultured in DMEM supplemented with 10% FBS and 1 × anti/anti. Cells were cultured at 37°C in a 5% CO 2 humidified incubator. Both cancer cell lines were authenticated using short tandem repeat profiling (Table S1 ) and Mycoplasma screened using a PCR-based approach (BIOTOOLS, New Taipei City, Taiwan). Plasmids and reagents The lentiviral pLKO-puro-shRNA expression vectors shCtrl (ASN0000000003), shPER2 [TRCN0000330731 (#1) and TRCN0000330809 (#2)] were purchased from the National RNAi Core Facility (Taipei, Taiwan). HO-1 inhibitors, Tin Protoporphyrin IX dichloride (SnPPIX) and Heme Oxygenase-1-IN-1 hydrochloride (HO-1i), were purchased from MedChemExpress (Monmouth Junction, NJ, USA). N-acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Immunoblot assay Immunoblot assay Whole cell lysate was prepared using RIPA buffer (Millipore, Burlington, MA, USA) with SIGMAFAST™ Protease Inhibitors (Sigma-Aldrich) and PhosSTOP EASYpack phosphatase inhibitor cocktail (Roche, Basel, Switzerland). 15–30 µg of total protein was separated by 7.5% or 10% tris–glycine polyacrylamide gel, with overnight incubation with 1:1,000 primary antibodies against PER2 (GTX129688, GeneTex, Irvine, CA, USA), E-Cadherin (G10, Santa Cruz Biotechnology, Dallas, TX, USA), N-Cadherin (13A9, Santa Cruz), Vimentin (D21H3, Cell Signaling Technology, Danvers, MA, USA), HO-1 (10701-1-AP, Proteintech Group, Rosemont, IL, USA)] or 1:5,000 GAPDH (1E6D9, Proteintech), and followed by a 1:10,000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibodies. HRP signals were detected using Immobilon Western Chemiluminescent HRP substrate (Millipore) and images captured by a UVP ChemStudio Plus BioImaging system. The densitometry of blot bands was quantified using NIH ImageJ. RNA extraction and quantitative real-time PCR (qRT-PCR) RNA was extracted using TRI Reagent (Sigma-Aldrich) according to the manufacturer’s instructions. 3 µg total RNA from each sample was reverse-transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). 10 ng 1st-strand cDNA was used for real-time PCR with appropriate primer sets (listed in Table S2 ) and SYBR Green 2X Master Mix (KAPA Biosystems, Wilmington, MA, USA) using ABI Quantstudio5 PCR system. The mRNA relative quantities were determined using relative ΔC t method with 18s rRNA as an internal control. Immunofluorescence (IF) staining Cells were seeded and left to adhere to reach 70–80% confluence on coverslips and then fixed with ice-cold 100% methanol (for PEO1 cells) or acetone (for OVCA429 cells) for 10 min at − 20°C followed by rehydration with phosphate-buffered saline (PBS). The cells were then blocked with 1% bovine serum albumin (BSA)/PBS for 1 hour at room temperature and incubated with primary antibodies against E-Cadherin (1:100, 610182, BD Biosciences, Franklin Lakes, NJ, USA), N-Cadherin (1:50, M142, Takara, Shiga, Japan) or Vimentin (1:100, M0725, Agilent-Dako, Santa Clara, CA, USA) in 1% BSA/PBS for overnight at 4°C. After washing with PBS, the cells were incubated with goat anti-mouse IgG H&L Alexa Fluor® 488 (ab150117) or goat anti-rabbit IgG H&L Alexa Fluor® 488 (ab150077, Abcam, Cambridge, UK) secondary antibody (1:300) in 1% BSA/PBS for 1 hour in the dark. DAPI was used to stain nucleus. Coverslips with stained cells were mounted onto glass slides with fluorescence mounting medium (Agilent-Dako). Images were acquired using an Andor Dragonfly 202 high speed confocal microscope system and the level of fluorescence signal was measured by fluorescence emission at 505–550 nm with excitation at 488 nm. The fluorescence intensity was quantified using the Imaris 10.1.0 software. For ROS detection, cells were stained for 30 min at 37°C with 0.5µM (for PEO1) or 1µM (for OVCA429) CM-H2DCFDA (Thermo Fisher Scientific) in PBS. Hoechst 33432 (Invitrogen, Waltham, MA, USA) was used to stain nucleus. Oxidation of the probe was detected was measured by fluorescence emission at 517–527 nm with excitation at 492–495 nm. Chromatin immunoprecipitation (ChIP) assay ChIP assay was conducted using Zymo-Spin ChIP kit (Zymo Research, Irvine, USA). Immunoprecipitation was performed using antibodies against H3K27me3 (Lys27) (C36B11, Cell Signaling Technology), H3K4me3 (Lys4) (C42D8, Cell Signaling Technology) or corresponding control IgG (ab172730, Abcam). qRT-PCR was performed to detect H3K27me3 or H3K4me3 associated promoter regions using primers listed in Table S2 . Invasion and migration assays For invasion assay [ 26 ], 1×10 4 cells were seeded in a 24-well Falcon HTS Fluoro Block (8 µm) transwell insert coated with Matrigel (Corning, NY, USA) in growth medium without serum. Complete medium was used as a chemoattractant in the lower chamber of 24-well plate. After 24 h incubation, the invaded cells were fixed with methanol and stained with DAPI. Cells were counted with fluorescence microscopy. For migration assay, two strategies were used. For transwell migration assay, 1×10 4 cells were seeded in a 24-well Falcon Transparent membrane (0.4 µm) PET inserts (Corning) in serum free media. Complete medium was used as a chemoattractant in the lower chamber. After 24 h incubation, the migrated cells were fixed with methanol, stained with 0.2% crystal violet and imaged and counted using an inverted microscope. For scratch assay, 1.75×10 5 cells were seeded in each well of a 2-well silicone insert (ibidi USA Inc., Fitchburg, WI, USA) in a 24-well plate. The cell density reached 100% confluence 24 h after seeding and the insert was removed to generate a wound field. Fresh medium was then added and the cells were cultured for additional 48 hrs. Image of the cell monolayer was taken at 0, 12, 24, 36 and 48 hours after insert removal using a phase-contrast microscope, and distance between one side of wound field and the other was measured. Intraperitoneal ( i.p. ) tumor xenograft model Animal care and experiments were approved by the Institutional Animal Care and Utilization Committee of Academia Sinica (IACUC# 20-06-1485). Female BALB/cAnN.Cg- Foxn1 nu /CrlNarl (NUDE) mice were purchased form the National Laboratory Animal Center (Taipei, Taiwan) at 6 weeks of age. 500µL of 1 × 10 7 cells in PBS were prepared and injected into the peritoneal cavity. After injection, mice were weighed and palpated weekly. Ten weeks after injection, mice were sacrificed and the intestines were collected for further assessment of tumor burden. Immunohistochemistry (IHC) Tissues were fixed in 10% formalin overnight at 4°C and embedded in paraffin. Sections were cut into 4 µm slices, dewaxed with xylene and rehydrated with descending ethanol series to water. Antigen retrieval was performed using Target Retrieval Solution (Agilent-Dako) for 20 min under high pressure condition. Slices were stained with anti-CK7 antibody (bs-1610R, Bioss, Woburn, MA, USA) overnight at 4°C, followed by incubation with DAKO REALTM EnVisionTM HRP Labeled Polymer Anti-Rabbit (Agilent-Dako) at room-temperature for 30 min. The 3.3’-diamiobenzidine (DAB) substrate was used to detect the peroxidase activity, and the slices were counterstained with hematoxylin. Slides were photographed under 40X magnification by the ZEISS Axioscan 7 and Aperio scanner machine (Leica, Wetzlar, Germany). RNA sequencing analysis Total RNA was extracted as described above and quantified using NanoDrop (Thermo Fisher Scientific). All samples with OD 260/280 ratio > 1.8 and OD 230/260 ratio > 2 were sent to BIOTOOLS for sequencing. RNA quality was measured by Qsep100 Bio-Fragment Analyzer. Samples with RQN > 6.8 were used for cDNA library construction. Paired-end sequencing (read length 150 bp) was performed on a NovaSeq 6000 platform and quality filtering and adaptor trimming of raw reads performed using Cutadapt (v1.12; [ 29 ]). Trimmed reads were aligned to Human genome (hg38) using STAR (v2.5.3a; [ 30 ]). After mapping to the reference genome, ∼20 million paired aligned reads were recovered from each RNA sequencing (RNA-seq) library. Expected read counts of each gene were estimated with RSEM (v1.2.31; [ 31 ]), followed by adjusting the CPM with the effective library size via the calcNormFactors function implemented in the R package edgeR (v.3.26.8; [ 32 ]). For each cell line, the expression profile of each sh-PER2 clone was compared with the sh-Ctrl and gene expression fold-change calculated (Table S3). To identify enriched functional pathways, we performed KEGG Analysis [ 33 ] on the ranked gene list, determined by the Log Fold-Change. Soft agar colony formation assay 2500 cells were mixed with a layer of 0.35% agar/complete growth medium over a layer of 0.5% agar/complete growth medium in a well of a 12-well plate. After culture at 37°C for 3 weeks, colonies were counted using phase contrast microscopy. Statistical analysis All data are presented as mean of three or more replicates ± SD. Student’s t-test was used to compare control and treatment groups. All statistical analyses were performed using Prism 10 software. Results Low expression of PER2 is associated with poor prognosis in Epi-A OC The clinical significance of PER2 expression in OC was evaluated with the TNMplot web tool [ 34 ]. PER2 level was significantly downregulated in ovarian tumor samples compared to the normal ovarian tissues (Fig. 1 A, p = 4.41e − 05 ). Also, the CSIOVDB database [ 14 ] showed that PER2 was differentially expressed among the five GEMS, with a significantly higher level found in the Epi-A subtype compared to other GEMS (Fig. 1 B). Moreover, within the Epi-A subtype, high PER2 expression was associated with better overall survival (Fig. 1 C, p < 0.0001). This clinical correlation was not observed in other GEMS (Fig. S1 ), suggesting an Epi-A GEMS-specific tumor suppressive role of PER2. Loss of PER2 enhances tumorigenic ability of Epi-A OC cells Two Epi-A OC cell lines, PEO1 and OVCA429 [ 13 ], were used to evaluate the function of PER2. Depletion of PER2 using a lentiviral-shRNA system (Fig. S2 A) resulted in an approximately 3-fold increase in anchorage-independent growth in PEO1 cells (Fig. 1 D) and a 2-fold increase in OVCA429 cells (Fig. 1 E), suggesting that loss of PER2 facilitates Epi-A ovarian tumor growth. PER2 depletion also had a profound impact on invasion and migration ability of Epi-A OC cells. Even for PEO1, which has low invasion ability [ 35 ], PER2 depletion significantly promoted invasion through an extracellular matrix coated porous membrane (Fig. 1 F) and migration through a transwell (Fig. 1 G). In OVCA429 cells, which exhibit an intermediate epithelial phenotype with a substantial epithelial-mesenchymal plasticity [ 36 ], PER2 depletion elevated the invasion ability by 3-fold (Fig. 1 H) and migration ability by 2-fold (Fig. 1 I). Consistent with these results, scratch assays showed that the PER2-depleted cells moved into a gap much faster than the control in both Epi-A OC cell lines (Fig. S2 B, S2C). To validate these cell-based results in vivo , several xenograft models were considered. It has been reported that PEO1 cells do not form tumors either via intraperitoneal ( i.p. ) or subcutaneous ( s.c. ) injection in NUDE mice [ 37 ]. Although OVCA429 can form tumors via s.c. [ 38 ] and intra bursa injection [ 39 ], the tumors are often small and the latency for tumorigenesis is long. However, when injected via i.p. , OVCA429 has been observed to form solid tumors in the pelvic region, intestine, and omentum [ 38 ]. Since our cell-based experiments showed that PER2 depletion increased invasion ability of Epi-A OC cells, and because i.p. xenograft model has been used for peritoneal dissemination in intra-abdominal cancers, we injected OVCA429 cells without (shCtrl) or with PER2 knockdown (shPER2) intraperitoneally using NUDE mice (Fig. 1 J- 1 O). Interestingly, mice injected with shPER2 OVCA429 cells generated many scattered (rather than clustered) tumor nodules in the intestine (Fig. 1 N, 1 O), albeit that the number of confined intestinal tumor nodules was not significantly different between the shPER2 and shCtrl groups (Fig. 1 J- 1 M). These observations suggested that loss of PER2 may drive EMT as EMT has generally been linked to the dispersion of individual primary tumor cells. These results along with the clinical correlations suggest that PER2 expression is critical to maintain a less aggressive, epithelial phenotype in Epi-A GEMS and thereby contribute to the better survival in PER2-high Epi-A OC patients. Epi-A OC cells undergo EMT upon PER2 depletion PER2 downregulation in both PEO1 and OVCA429 cells led to a morphological change from a polygonal shape with regular dimensions to an elongated, spindle-like shape (Fig. 2 A, 2 B). This further suggested that cells underwent EMT after PER2 depletion. The epithelial to mesenchymal switch was confirmed by assay of EMT markers using immunofluorescence (IF) staining (Fig. 2 C, 2 D) and immunoblotting (IB) (Fig. 2 E, 2 F). After PER2 depletion, the number of ECAD expressing cells decreased, while NCAD and VIM expressing cells increased (Fig. 2 C, 2 D). Consistent with these results, the protein level of ECAD was reduced, and NCAD and VIM were elevated after PER2 depletion (Fig. 2 E, 2 F). These molecular changes were in line with the epithelial to mesenchymal morphological changes as well as the elevated invasion (Fig. 1 F, 1 H) and migration (Fig. 1 G, 1 I, Fig. S2 B, S2C) ability in the shPER2 cells. Together these data indicated that PER2 expression was critical to maintain an epithelial phenotype in Epi-A OC cells. HO-1 upregulation is the key to PER2 depletion-mediated EMT in Epi-A OC We previously found that PER2 inhibits EMT in breast cancer cells by recruiting the Polycomb Repressive Complex 2 (PRC2) to suppress transcription of the EMT executors, TWIST1, SNAIL and SLUG [ 26 ]. Intriguingly, upregulation of these EMT executors was not as prominent in Epi-A OC cells (Fig. S3, [ 26 ]) and EMT was not observed until a much later time point (Fig. 2 A, 2 B, 2 E, 2 F). These results suggested that, unlike in breast cancer cells where PER2 directly inhibited transcription of EMT executors, PER2 loss may result in other EMT-inducing alterations in Epi-A OC cells. To understand how PER2 regulates EMT in Epi-A OC cells, RNA-seq analysis of OVCA429 without or with PER2 depletion was performed. KEGG enrichment analysis found that genes in chemical carcinogenesis - reactive oxygen species (ROS)-related processes, including HMOX1 , CYP1A1 , EPHX1 , AKR1C1 and AKR1C3 , were significantly upregulated in PER2 depleted cells (Fig. 3 A, 3 B, Table S3). Since ROS can facilitate EMT [ 40 ], these genes were further examined in shCtrl and shPER2 OVCA429. HMOX1 was found to be significantly upregulated in both PER2 knock-down cell lines and its expression level was negatively correlated with that of PER2 (Fig. 3 C). CSIOVDB database analysis of OC patient data found significantly lower HMOX1 expression in Epi-A compared to Epi-B and Mes GEMS (Fig. 3 D). This contrasted with the relatively high expression of PER2 in Epi-A GEMS (Fig. 1 B). Epi-A patients with high HMOX1 expression had poor overall survival (Fig. 3 E, p = 0.039), consistent with observations that Heme Oxygenase 1 (HO-1), which is encoded by the HMOX1 gene, inhibited apoptosis and enhanced EMT and metastasis in the Mes GEMS cell line SKOV3 and Stem-A cell line A2780 [ 41 , 42 ]. These observations suggested that PER2 depletion and consequent upregulation of HMOX1 /HO-1 may induce EMT to facilitate invasion and metastasis in Epi-A OC. Consistent with this hypothesis, HO-1 inhibition using Tin protoporphyrin IX dichloride (SnPPIX) and Heme Oxygenase-1-IN-1 hydrochloride (HO-1i) abolished the elevated migration and invasion phenotypes (Fig. 3 F- 3 I), as well as the EMT morphology and marker expression (Fig. S4) in PER2 depleted PEO-1 and OVCA429 cells. These results indicated that HMOX1 /HO-1 upregulation is the key mechanism to induce EMT, invasion and migration in PER2-depleted Epi-A OC cells. Depletion of PER2 activates HO-1 via ROS elevation and transcriptional de-repression HO-1 has multifaceted roles in tumor progression [ 43 ]. HMOX1 /HO-1 is transcriptionally induced in response to several stimuli including oxidative stress to regulate cellular ROS level [ 44 ] and the effects of ROS on tumorigenesis and cancer progression have been intensively studied [ 45 ]. In cancer cells, ROS levels are restricted to a dynamic range which is high enough to produce genetic damage necessary for tumorigenesis and induce various pathways that drive tumor cells to undergo EMT and thereby promote invasion and metastasis [ 46 ], but not so high as to cause cytotoxic effects and activate cell death pathways [ 47 ]. In PER2-depleted Epi-A OC cells, ROS level was elevated (Fig. 4 A) possibly due to downregulation of several genes with antioxidant functions, such as superoxide dismutase 2 (SOD2) [ 48 ] and peroxidasin (PXDN) [ 49 ] (Fig. S5A, Table S3). ROS level was significantly elevated within 48 hours after shPER2 transduction (Fig. 4 A, Fig. S5B), while HO-1 protein level was not significantly increased until 96 hours after PER2 knock-down (Fig. 4 B, 4 C, Fig. S5C-S5E). The observation that elevated ROS preceded HMOX1 /HO-1 induction suggested that elevated ROS may be involved in HO-1 induction in PER2 depleted cells. HO-1 is known to have a short half-life estimated as 3 hours for mRNA and 15–21 hours for protein [ 50 ]. Intriguingly, when ROS was removed using the antioxidant N-acetyl-L-cysteine (NAC), HO-1 protein level remained higher in the PER2-depleted cells than the PER2-expressing cells (Fig. 4 D- 4 F, Fig. S5F-S5H). Moreover, 72 hours after NAC treatment, HMOX1 mRNA level in the PER2-depleted cells also remained upregulated (Fig. S6A, S6B) and EMT morphology remained unchanged (Fig. S6C, S6D). These results indicated that mechanisms in addition to (or instead of) increased ROS were involved in HMOX1 /HO-1 upregulation in PER2-depleted cells. Since PER2 acts as a transcriptional co-repressor [ 26 ], it is possible that PER2 may directly suppress the promoter activity of HMOX1 to repress HO-1 level in Epi-A OC cells. In this case, PER2 depletion would allow the HMOX1 promoter to be accessible to oxidative stress-induced transcription factors such as nuclear factor erythroid 2-related factor 2 (NRF2), activator protein-1 (AP-1), nuclear factor-κB (NF-κB) and peroxisome proliferator-activated receptors (PPARs) [ 51 , 52 ]. To test this possibility, chromatin-immunoprecipitation (ChIP) assays were performed using antibodies recognizing the repressive histone mark H3K27tri-methylation (H3K27me3) or the active histone mark H3K4tri-methylation (H3K4me3) with primer sets amplifying 100- to 150-bp fragments spanning the known HMOX1 activator binding motifs within the − 1 kb proximal HMOX1 promoter (Fig. 4 G). Upon PER2 knockdown, a decrease in H3K27me3 (Fig. 4 H, Fig. S5I) and an increase in H3K4me3 were observed on the HMOX promoter (Fig. 4 I, Fig. S5J), indicating a chromatin switch from a condensed state to an open state. Together, these results suggested that PER2 depletion-mediated HMOX1 /HO-1 upregulation was due to transcriptional de-repression; although we cannot rule out ROS accumulation as a contributing factor. PER2 depletion-mediated HO-1 upregulation increases cisplatin resistance in Epi-A OC EMT can contribute to drug resistance [ 53 ] and HO-1 upregulation has been associated with cisplatin resistance in OC cells [ 54 ]. Since PER2 downregulation promoted EMT and increased HO-1 expression, loss of PER2 in Epi-A OC cells may promote cisplatin resistance. Consistent with this hypothesis, less than 10% of the PER2 expressing cells (shCtrl) survived when treated with 5 µM cisplatin while 30% of PER2 knockdown cells survived (Fig. 5 A, 5 B). When a higher dose (10 µM) of cisplatin was used, more than 95% of the PER2-expressing cells died (Fig. 5 C, 5 D), while PER2 depletion increased survival to approximately 30% for PEO1 and 15–20% for OVCA429 cells (Fig. 5 C, 5 D). In addition to cell viability, we also evaluated the effect of PER2 depletion on cisplatin-suppression of tumorigenic ability using soft-agar colony formation assay. In PER2-expressing PEO-1 and OVCA429 cells, cisplatin treatment significantly reduced colony numbers (Fig. 5 E, 5 F). In contrast, colony forming ability of PER2-depleted cells was not affected by cisplatin (Fig. 5 E, 5 F). These results indicated that PER2 loss significantly increased cisplatin resistance of Epi-A OC cells in terms of both cell survival and tumorigenic ability. We also found that inhibition of HO-1 sensitized the PER2 knocked-down cells to cisplatin (Fig. 5 G- 5 I). Treatment with either SnPPIX or HO-1i decreased the viability of cisplatin-treated shPER2 PEO1 cells from approximately 35–15% (Fig. 5 G), and that of shPER2 OVCA429 cells from 25–40% to 15–20% (Fig. 5 H). Furthermore, HO-1 inhibitor treatment resulted in a statistically significant reduction in soft-agar colony forming ability in the cisplatin-treated shPER2 cells (Fig. 5 I). These results indicated that upregulation of HO-1 was a key cause of cisplatin resistance in PER2-depleted Epi-A OC cells. Discussion PER2 is a crucial suppressor controlling the circadian oscillation that influences many physiological and biological processes. In peripheral tissues, PER2 not only has important roles in tissue-specific responses to the circadian environment, but also exhibits tumor suppressive functions. For example, mice with homozygous Per2 mutation showed higher sensitivity to radiation and had increased radiation-induced tumorigenesis compared to the wild-type mice [ 25 ]. In humans, PER2 expression is often found to be downregulated in tumors compared to normal tissues [ 55 ]. Loss of PER2 is associated with dysregulation of genes related to cell cycle, proliferation, apoptosis, DNA repair, angiogenesis, metabolism and inflammation [ 56 ]. A recent study using the SKOV3 (Mes-like GEMS) cell line, one of the very few studies to examine PER2 in ovarian cancer, reported that PER2 expression was dramatically reduced when the cells acquired cisplatin resistance [ 57 ]. Although it was not clear whether PER2 downregulation was the cause of cisplatin resistance in SKOV3 cells or a side effect of such resistance, the authors did show that PER2 overexpression suppressed PI3K/AKT pathways and enhanced cisplatin-induced apoptosis [ 57 ]. Our results demonstrate that loss of PER2 was the cause of both enhanced EMT and cisplatin resistance in Epi-A OC cells. We also identified the key factor, HO-1, whose upregulation is responsible for these malignant phenotypes in the PER2-depleted cells (Fig. 6 ). These observations further demonstrate the tumor suppressive activity of PER2 and emphasize the critical role of PER2 in Epi-A OC progression. Several animal models deficient in core clock genes Bmal1 , Clock or Npas2 exhibit chronic oxidative stress and defects in antioxidant defense [ 58 – 60 ]. However, the role of PER2 in oxidative stress response and related pathology is less clear. The increase in cellular ROS in PER2-depleted Epi-A OC cells indicated a disrupted redox balance (Fig. 4 , Fig. S5). The major site of cellular ROS production is mitochondria and PER2-depleted Epi-A OC cells had reduced expression of several genes encoding components of the mitochondrial antioxidant system, including SOD2 (Fig. S5A, Table S3). This suggested that elevated ROS in the Epi-A OC cells was due to ROS imbalance in the mitochondria. In mitochondria, SOD2 is the first line of defense against superoxide radicals generated in the process of electron transfer during oxidative phosphorylation [ 61 ]. How PER2 expression affects the antioxidant system in Epi-A OC cells, particularly SOD2 expression, is worthy of further investigation. Transcriptional up-regulation of HO-1 is mainly mediated by oxidative stress-responsive transcription factors such as NRF2, AP1, NF-κB and PPAR [ 51 , 52 ]. Intriguingly, the interplay between HO-1 and oxidative stress is complex and HO-1 can be a friend or foe to tumorigenesis [ 62 ]. HO-1 is a key enzyme to metabolize heme to generate biliverdin, carbon monoxide (CO) and ferrous iron (Fe2 + ). Biliverdin can be subsequently converted to bilirubin to act as anti-oxidant to counteract ROS whereas Fe2 + increases ROS generation via the Fenton reaction. As for CO, it participates in signal transduction including pro-angiogenesis, anti-inflammation and anti-apoptosis pathways which can contribute to tumor progression. Despite intensive research, the complex role and regulation of HO-1 in normal and tumor cells remain elusive [ 62 ]. In this study, we found that HO-1 was transcriptionally suppressed in PER2-expressing Epi-A OC cells. Our findings also imply that rhythmic expression of PER2 in normal ovarian tissues may play a critical role in modulating HO-1 expression and ROS homeostasis in response to circadian cues consistent with a recent study which found that circadian dependent HO-1 expression is involved in regulating neuroinflammation in microglial [ 63 ]. How PER2, along with the core clock machinery, regulate HO-1 and ROS to maintain normal tissue homeostasis and stress response is also worthy of further investigation. Our study demonstrated that PER2 expression is required for Epi-A OC cells to maintain an epithelial phenotype, ROS homeostasis and sensitivity to cisplatin. Loss of PER2 increased malignancy by elevating anchorage-independent growth, promoting EMT, and facilitating invasion and migration in Epi-A OC cells. These results demonstrate the underlying mechanism that explains why PER2-low Epi-A OC patients have poorer prognosis compared to the PER2-high patients. We also identified HO-1 as a key factor critical for Epi-A OC cells to gain malignancy upon PER2 loss. Thus, targeting HO-1 may provide a therapeutic strategy to prevent Epi-A OC progression due to PER2 loss. Declarations Competing interests The authors declare no competing financial interests. Author contributions WWHV and RYJH conceived the study. GYTT, PV, LTC, PYL, CLWC and YCC performed experiments. CHAY assisted data analysis. WWHV wrote the paper. Acknowledgements This work was supported by National Taiwan University-Academia Sinica Joint Program [NTU-AS-110-09] and [NTU-AS-112-11] to WWHV and RYJH, and National Science and Technology Council [NSTC-113-2813-C-001-011-B] to WWHV. The authors would like to thank Dr. Pang-Hung Hsu (National Taiwan Ocean University) and Ms. Divya Malathy Ravinath for their help during the early stage of this study. The authors would also like to thank the following core facilities at Academia Sinica: the Bioinformatics Core at Institute of Molecular Biology for providing the RNA-seq analysis services, the Advanced Optics Microscope Core Facility, funded by Academia Sinica Core Facility and Innovative Instrument Project (AS-CFII-111-208), for microscope imaging technical support, the SPF Animal Facility funded by AS-CFII-111-204 for providing animal support, and the National RNAi Core Facility for providing shRNA reagents and related services. Availability of data and materials The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Ebrahimi N, Manavi MS, Faghihkhorasani F, Fakhr SS, Baei FJ, Khorasani FF et al . Harnessing function of EMT in cancer drug resistance: a metastasis regulator determines chemotherapy response. Cancer Metastasis Rev 2024. Brabletz S, Schuhwerk H, Brabletz T, Stemmler MP. 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Circadian dependency of microglial heme oxygenase-1 expression and inflammation determine neuronal injury in hemorrhagic stroke. J Inflamm (Lond) 2023; 20: 43. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SuppData10092024.pdf TableS3.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5242949","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":366216023,"identity":"d98bacae-9775-4872-980f-b05c03af8c0d","order_by":0,"name":"Wendy Hwang-Verslues","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-0383-1710","institution":"Academia Sinica","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wendy","middleName":"","lastName":"Hwang-Verslues","suffix":""},{"id":366216024,"identity":"6696092b-d49d-498e-8249-4d999f38c768","order_by":1,"name":"Grace Tan","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Grace","middleName":"","lastName":"Tan","suffix":""},{"id":366216025,"identity":"67f33861-2538-4d52-aef2-5cc8af381c2d","order_by":2,"name":"Priyanka Vinothkumar","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Priyanka","middleName":"","lastName":"Vinothkumar","suffix":""},{"id":366216026,"identity":"0f486064-e8e9-448c-921d-6095f3176568","order_by":3,"name":"Li-Tzu Cheng","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li-Tzu","middleName":"","lastName":"Cheng","suffix":""},{"id":366216027,"identity":"eeb448d1-9bbb-45d2-8a70-528448eddcc3","order_by":4,"name":"Pei-Yi Lin","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pei-Yi","middleName":"","lastName":"Lin","suffix":""},{"id":366216028,"identity":"c519c7eb-f913-43a2-8e6e-85a68c2bf921","order_by":5,"name":"Chung-Lien William Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chung-Lien","middleName":"William","lastName":"Chen","suffix":""},{"id":366216029,"identity":"a854f21f-c204-457d-9550-ea873235955a","order_by":6,"name":"Chen-Hsin Yu","email":"","orcid":"https://orcid.org/0000-0002-5662-3026","institution":"Institute of Molecular Biology, Academia Sinica","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chen-Hsin","middleName":"","lastName":"Yu","suffix":""},{"id":366216030,"identity":"199b7e41-1959-4435-a94c-597e1fa455e4","order_by":7,"name":"Yi-Chia Chiu","email":"","orcid":"","institution":"National Taiwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi-Chia","middleName":"","lastName":"Chiu","suffix":""},{"id":366216031,"identity":"adc06732-5b20-4472-988d-86af643d4bbb","order_by":8,"name":"Ruby Huang","email":"","orcid":"https://orcid.org/0000-0001-6376-3185","institution":"National Taiwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruby","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-10-11 03:00:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5242949/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5242949/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66836937,"identity":"bd27a59d-8496-4d4b-aa47-f4d855c0fce6","added_by":"auto","created_at":"2024-10-17 04:11:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2556702,"visible":true,"origin":"","legend":"\u003cp\u003ePER2 downregulation correlates with poor survival and elevated tumorigenic ability in Epi-A OC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e PER2 gene expression in normal (n = 46) and tumor (n = 744) ovarian tissues analyzed using gene chip data of the TNMplot web tool [34]. The \u003cem\u003ep\u003c/em\u003e value was determined by Mann Whitney test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e PER2 gene expression in Epi-like (Epi-A, Epi-B) and Mesenchymal-like (Mes) OC analyzed using CSIOVDB database [14]. The \u003cem\u003ep\u003c/em\u003evalue was determined by Mann Whitney test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Kaplan−Meier overall survival analysis of Epi-A OC patients grouped by PER2 expression. \u0026nbsp;The group with PER2 level ≥ the median is indicated by red line; the group with PER2 level \u0026lt; median is indicated by blue line. \u0026nbsp;Median survival (month) and hazard ratio (HR) are indicated. n = 203. \u0026nbsp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u0026nbsp;The \u003cem\u003ep\u003c/em\u003e value was determined by log-rank test [9, 13].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD, E. \u003c/strong\u003eSoft agar colony formation (SACF) assays using PER2-depleted (shPER2#1 and #2) PEO1 (D) and OVCA429 (E) cells. \u0026nbsp;Data are shown as mean ± SD. \u0026nbsp;Significant differences are based on unpaired T-test. \u0026nbsp;****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u0026nbsp;The experiments were repeated at least two times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF, G.\u003c/strong\u003eTranswell invasion (F) and migration (G) assays using PER2-depleted PEO1 cells. \u0026nbsp;Data are shown as mean ± SD. \u0026nbsp;Significant differences are determined by unpaired T-test. \u0026nbsp;****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u0026nbsp;The experiments were repeated at least two times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH, I.\u003c/strong\u003eTranswell invasion (H) and migration (I) assays using PER2-depleted OVCA429 cells. \u0026nbsp;Replication and data formatting are as described for\u003cstrong\u003e F\u003c/strong\u003e and\u003cstrong\u003e G\u003c/strong\u003e. ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ. \u003c/strong\u003eQuantification of tumor nodules in the intestine of NUDE mice injected with shCtrl or shPER2 OVCA429 cells intraperitoneally (\u003cem\u003ei.p.\u003c/em\u003e). All data are presented as mean ± SD with significant differences detected by unpaired T-test. ns, non-significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK. \u003c/strong\u003eRepresentative image of CK7 IHC staining of the intestine from the shCtrl OVCA429 \u003cem\u003ei.p.\u003c/em\u003e injected xenograft model. Scale bar indicates 60 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL-O. \u003c/strong\u003eRepresentative images of CK7 IHC staining of the intestine from the shPER2 OVCA429 \u003cem\u003ei.p.\u003c/em\u003e injected xenograft models. Dashed circles indicate the confined tumor nodules, and red arrows indicate the scattered nodules. Scale bar indicates 60 μm.\u003c/p\u003e","description":"","filename":"MainFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5242949/v1/0b943697fa9dd75a3f51ed12.jpg"},{"id":66837088,"identity":"a409e058-20cb-48ec-990a-42e6e8d0444c","added_by":"auto","created_at":"2024-10-17 04:19:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3759220,"visible":true,"origin":"","legend":"\u003cp\u003ePER2 depletion induces EMT in Epi-A OC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA, B.\u003c/strong\u003eRepresentative morphology images of shCtrl or shPER2 (#1, #2) PEO1 (A) and OVCA429 (B) cells. \u0026nbsp;Scale bar indicates 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC, D.\u003c/strong\u003eRepresentative immunofluorescence staining images of EMT markers using shCtrl or shPER2 (#1, #2) PEO1 (C) and OVCA429 (D) cells. \u0026nbsp;ECAD was used as an epithelial marker. NCAD and VIM were used as mesenchymal markers. Scale bar indicates 70 μm in \u003cstrong\u003eC\u003c/strong\u003eand 100 μm in \u003cstrong\u003eD\u003c/strong\u003e. \u0026nbsp;Fluorescence intensity is presented as mean ± SD from three images. Significant differences are based on unpaired T-test (n=3). \u0026nbsp;*\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE, F.\u003c/strong\u003eImmunoblot of PER2, ECAD, NCAD and VIM in PEO1 (E) and OVCA429 (F) cells transduced with shPER2 lentivirus (#1, #2). \u0026nbsp;GAPDH was used as a loading control. \u0026nbsp;These experiments were repeated at least three times.\u003c/p\u003e","description":"","filename":"MainFig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5242949/v1/1bb5053e4127dbdc7408573e.jpg"},{"id":66836934,"identity":"62979d92-5feb-4679-b1e4-96a24a24fcc7","added_by":"auto","created_at":"2024-10-17 04:11:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1740526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHMOX1\u003c/em\u003e/HO-1 activation is required for enhanced migration and invasion in PER2-depleted Epi-A OC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Enrichment KEGG pathway analysis of the significantly upregulated differential expressed genes (DEGs) genes in PER2 depleted OVCA429 cells. The vertical axis represents the pathway category and the horizontal axis represents the gene ratio of the pathway. The point size corresponds to the number of genes in the KEGG pathway and the colors to the adjusted \u003cem\u003ep\u003c/em\u003e-value. \u0026nbsp;Significant pathways were according to \u003cem\u003ep\u003c/em\u003e-adjusted threshold \u0026lt; 0.05. This analysis was performed by R packages, \"clusterProfiler\".\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Heatmap of chemical carcinogenesis reactive oxygen species related genes in OVCA429 cells without or with PER2 depletion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e qRT-PCR analysis of \u003cem\u003eHMOX1\u003c/em\u003e, \u003cem\u003eCYP1A1\u003c/em\u003e, \u003cem\u003eEPHX1\u003c/em\u003e, \u003cem\u003eAKR1C1\u003c/em\u003e and \u003cem\u003eAKR1C3 \u003c/em\u003eexpression in OVCA429 cells without (C) or with PER2 depletion (#1, #2). \u0026nbsp;Data are shown as mean ± SD. \u0026nbsp;Significant differences are determined by unpaired T-test (n=3, *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ns, non-significant). Blue asterisks indicate significant downregulation, and red asterisks indicate significant upregulation. \u0026nbsp;The experiments were repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e \u003cem\u003eHMOX1\u003c/em\u003e gene expression in Epi-like (Epi-A, Epi-B) and Mesenchymal-like (Mes) OC analyzed using CSIOVDB database. \u0026nbsp;The \u003cem\u003ep\u003c/em\u003e value was determined by Mann Whitney test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Kaplan−Meier overall survival analysis of Epi-A OC patients grouped by HMOX1 expression. \u0026nbsp;The group with HMOX1 level ≥ median is indicated by red line; the group with HMOX1 level \u0026lt; median is indicated by blue line. \u0026nbsp;Median survival (month) and hazard ratio (HR) are indicated. \u0026nbsp;n = 203. \u0026nbsp;\u003cem\u003ep\u003c/em\u003e = 0.039. \u0026nbsp;The \u003cem\u003ep\u003c/em\u003e value was determined by log-rank test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF, G.\u003c/strong\u003e Migration assays using PER2-depleted PEO1 (F) and OVCA429 (G) cells treated with DMSO (control), 10 μM SnPPIX or 10 μM HO-1i HO-1 inhibitor. \u0026nbsp;Data are shown as mean ± SD. \u0026nbsp;Significant differences are determined by unpaired T-test. ****\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001. \u0026nbsp;Green asterisks indicate the comparison between shCtrl and shPER2 cells treated with DMSO. The experiment was repeated at least two times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH, I.\u003c/strong\u003e Transwell invasion assays using PER2-depleted PEO1 (H) and OVCA429 (I) cells treated with DMSO (control), 10 μM SnPPIX or 10 μM HO-1i HO-1 inhibitor. \u0026nbsp;Replication and data formatting are as described for \u003cstrong\u003eF \u003c/strong\u003eand \u003cstrong\u003eG\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"MainFig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5242949/v1/f9f8068f29447e2e2d33d269.jpg"},{"id":66836933,"identity":"1cfe149f-abac-42f5-a0f2-e241a12b5b3f","added_by":"auto","created_at":"2024-10-17 04:11:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2166624,"visible":true,"origin":"","legend":"\u003cp\u003eHO-1 is upregulated by PER2 depletion-induced ROS and transcriptional de-repression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA, B.\u003c/strong\u003eRepresentative images of ROS level detected by CM-H2DCFDA in OVCA429 cells 48h (A) or 96h (B) after transduced with shCtrl or shPER2 (#1, #2) lentiviral vectors. Scale bars indicate 100 μm. Three independent experiments were performed. \u0026nbsp;Relative CM-H2DCFDA mean fluorescence intensity is presented as fold change to control. Data are means ± SD from one representative experiment. Significant differences are based on unpaired T-test (n=3). \u0026nbsp;**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Immunoblot of PER2 and HO-1 in OVCA429 cells 48h and 96h after lentiviral transduction. \u0026nbsp;GAPDH was used as a loading control. \u0026nbsp;The experiments were repeated three times. R.E., relative expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Representative images of ROS level detected by CM-H2DCFDA in OVCA429 cells 96h after transduced with shCtrl or shPER2 lentivirus. \u0026nbsp;Cells were treated with PBS (-) or with 10 μM NAC (+) 48h after lentiviral transduction. Scale bar indicates 200 μm. Three independent experiments were performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003eRelative CM-H2DCFDA mean fluorescence intensity from \u003cstrong\u003eE\u003c/strong\u003e is presented as fold change to control. Data are means ± SD from one representative experiment. \u0026nbsp;Significant differences are based on unpaired T-test (n=3). \u0026nbsp;****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns, non-significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003eImmunoblot of HO-1 in OVCA429 cells 96h after transduced with shCtrl or shPER2 lentivirus. Cells were treated with PBS (-) or with 10 μM NAC (+) 48h after lentiviral transduction. GAPDH was used as a loading control. R.E., relative expression as fold change to the shCtrl without NAC treatment. The experiments were repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. \u003c/strong\u003eDiagram shows the positions of five primer sets (P1 to P5 regions) flanking 1 kb upstream from the transcription start site (TSS) of the \u003cem\u003eHMOX1\u003c/em\u003e promoter. \u0026nbsp;These primers were used for ChIP–qPCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH, I.\u003c/strong\u003e ChIP-qPCR analysis of histone H3K27me3 (H) and H3K4me3 (I) on the \u003cem\u003eHMOX1\u003c/em\u003e proximal promoter region in OVCA429 cells 96h after transduced with shCtrl (sh-C) or shPER2 (sh-P) lentivirus. \u0026nbsp;Fold enrichment was calculated using H3K27me3 or H3K4me3 ChIP signals normalized to IgG ChIP signals. \u0026nbsp;Data are mean ± SD with significant differences base on unpaired T-test comparing H3K27me3 or H3K4me3 fold enrichment between sh-C and sh-P (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns, non-significant). \u0026nbsp;The experiments were repeated two times.\u003c/p\u003e","description":"","filename":"MainFig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5242949/v1/cc62ef55ce8c5350efd77fca.jpg"},{"id":66836940,"identity":"5e913a93-be82-4d5f-ba2c-cc845668962b","added_by":"auto","created_at":"2024-10-17 04:11:31","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1315654,"visible":true,"origin":"","legend":"\u003cp\u003ePER2 depletion increased cisplatin resistance by upregulation of HO-1 in EpiA-OC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA, B.\u003c/strong\u003e Cell viability assays using PER2-depleted PEO1 (A) and OVCA429 (B) cells treated with PBS (0 μM) or 5 μM cisplatin. \u0026nbsp;Data are shown as mean ± SD. \u0026nbsp;Significant differences are determined by unpaired T-test. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns, non-significant. \u0026nbsp;The experiment was repeated at least two times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC, D.\u003c/strong\u003e Cell viability assays using PER2-depleted PEO1 (C) and OVCA429 (D) cells treated with PBS (0 μM) or 10 μM cisplatin. \u0026nbsp;Replication and data formatting are as described for\u003cstrong\u003eA\u003c/strong\u003e and\u003cstrong\u003e B\u003c/strong\u003e. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns, non-significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE, F.\u003c/strong\u003e Soft agar colony formation (SACF) assays using PER2-depleted (#1 and #2) PEO1 (E) and OVCA429 (F) cells treated with PBS (0 μM) or 5 μM cisplatin. \u0026nbsp;Replication and data formatting are as described for\u003cstrong\u003e A\u003c/strong\u003eand\u003cstrong\u003e B\u003c/strong\u003e. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG, H.\u003c/strong\u003e Cell viability assays using PER2-depleted PEO1 (G) and OVCA429 (H) cells treated with 5 μM cisplatin without or with 10 μM SnPPIX or 10 μM HO-1i HO-1 inhibitor. \u0026nbsp;Data are shown as mean ± SD. \u0026nbsp;Significant differences between shPER2 cells without and with SnPPIX or HO-1i treatment are determined by unpaired T-test. ****\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001. \u0026nbsp;The experiment was repeated at least two times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI.\u003c/strong\u003e SACF assays using PER2-depleted (shPER2) PEO1 (left panel) and OVCA429 (right panel) cells treated with 5 μM cisplatin without or with 10 μM SnPPIX or 10 μM HO-1i HO-1 inhibitor. \u0026nbsp;Replication and data formatting are as described for\u003cstrong\u003eG\u003c/strong\u003e and\u003cstrong\u003e H\u003c/strong\u003e. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"MainFig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5242949/v1/87ab659194357d18d95550c6.jpg"},{"id":66836939,"identity":"ad211559-3bf7-4097-9fb7-41d04fbd7014","added_by":"auto","created_at":"2024-10-17 04:11:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":227074,"visible":true,"origin":"","legend":"\u003cp\u003eProposed model. \u0026nbsp;Schematic illustrates a negative correlation between PER2 and HMOX1/HO-1 expression in EMT process (A). PER2 is required for Epi-A OC cells to maintain epithelial phenotype, ROS homeostasis and sensitivity to cisplatin. PER2 loss increased the malignancy by facilitating EMT, invasion, migration and cisplatin resistance in Epi-A OC cells. PER2 depletion activates HMOX1/HO-1 via ROS elevation and transcriptional de-repression (B). HO-1 is a key factor critical for Epi-A OC cells to gain the malignancy upon PER2 loss. \u0026nbsp;Targeting HO-1 may provide a therapeutic strategy to prevent progression and sensitize PER2-low Epi-A OC cells to cisplatin.\u003c/p\u003e","description":"","filename":"MainFig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5242949/v1/ad571d9ba920a151da15f41f.jpg"},{"id":66837817,"identity":"baf95bab-0902-4bfb-972e-76f36b037752","added_by":"auto","created_at":"2024-10-17 04:27:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12499106,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5242949/v1/70f7da92-7166-4e25-9fbf-9c3b711ab235.pdf"},{"id":66836936,"identity":"d0ec155b-c9e5-4af6-bf80-8d750e671249","added_by":"auto","created_at":"2024-10-17 04:11:30","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":7936976,"visible":true,"origin":"","legend":"","description":"","filename":"SuppData10092024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5242949/v1/676d743e974215e9cde51424.pdf"},{"id":66837087,"identity":"a681876f-d469-4ad6-a1d4-1449de8fea5b","added_by":"auto","created_at":"2024-10-17 04:19:30","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":17979,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5242949/v1/378a9858c2f8e5fa25def31e.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Loss of PERIOD2 (PER2) induces HO-1-mediated epithelial-mesenchymal transition and cisplatin resistance in Epi-A ovarian cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer (OC) is the most lethal gynecological cancer. The poor prognosis of OC is due to rapid metastasis and chemoresistance, which are strongly associated with epithelial-mesenchymal transition (EMT) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Acquisition of EMT is a critical process for tumor cells to gain stemness features, enhance metastasis and gain chemoresistance [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. EMT can be induced in several ways. For example, transforming growth factor β (TGFβ)/bone morphogenetic proteins (BMPs) Wnt, Notch and receptor tyrosine kinases are known to drive EMT [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Also, hypoxia [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and inflammation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], which are common in the tumor microenvironment, as well as oxidative stress, which is prevalent in cancer cells due to intrinsic redox regulation or metabolic reprograming [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], are all potent EMT inducers. Transcription factors including TWIST, SNAIL, SLUG and ZEB are EMT executors which repress expression of epithelial markers, such as E-cadherin (ECAD), and activate expression of mesenchymal markers, such as Vimentin (VIM), Fibronectin or N‐cadherin (NCAD) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Overall, EMT is regulated in a context‐dependent manner, and cancers at different EMT status have unique EMT signatures [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOC is highly heterogeneous and can be categorized into serous, clear cell, endometrioid and mucinous subtypes based on histological characteristics [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Within each histological subtype, gene expression signatures among individual patients remain highly diverse and their clinical outcomes are different in response to the same treatment. To cope with this diverse molecular heterogeneity [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], functional genomic analysis utilizing different risk factors, epidemiological patterns, molecular characteristics and clinical manifestations were used to define five distinct gene-expression based molecular subtypes (GEMS) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]: Epithelial-A (Epi-A), Epithelial-B (Epi-B), Stem-like A (StemA), Stem-like B (StemB) and Mesenchymal (Mes). These GEMS have been correlated with EMT and patient survival [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The Epi-A GEMS is associated with low EMT and better survival, whereas the Mes GEMS is associated with high EMT and worse survival. However, the regulatory mechanisms underlying GEMS specific EMT remain unclear.\u003c/p\u003e \u003cp\u003eChronic circadian disruption has been linked to increased cancer risks [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Compelling evidence has suggested that core clock genes which form the transcription-translation feedback loop (TTFL) in circadian regulation also play roles in tumor progression and chemo-response [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Among the core clock genes, Period2 (PER2) is a key co-repressor essential for reactivation of the TTFL in circadian regulation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and has been shown to have tumor suppressive functions in many cancers [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In OC, PER2 expression has been reported to be negatively correlated with tumor growth and tumor stage [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the mechanism by which PER2 affects OC progression remain unclear.\u003c/p\u003e \u003cp\u003eWe found that PER2 exhibits an Epi-A OC subtype specific anti-malignancy role. Epi-A OC patients with tumors expressing relatively high levels of \u003cem\u003ePER2\u003c/em\u003e had significantly better disease-free prognosis and overall survival compared to those with \u003cem\u003ePER2\u003c/em\u003e-low tumors. PER2 downregulation promoted migration and invasion via upregulation of Heme oxygenase-1 (\u003cem\u003eHMOX1\u003c/em\u003e/HO-1) to drive EMT. Furthermore, PER2 depletion increased cisplatin resistance in Epi-A OC cells. Conversely, HO-1 inhibition prevented the PER2-depletion mediated malignant phenotypes and sensitized PER2-depleted Epi-A OC cells to cisplatin. Together, our results revealed a new role of PER2 in regulating EMT via control of \u003cem\u003eHMOX1\u003c/em\u003e/HO-1 to maintain an epithelial-like phenotype and cisplatin sensitivity of Epi-A OC cells. These findings also provided a basis for treating Epi-A OC progression by preventing PER2 downregulation and targeting HO-1.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eHuman OC cell lines PEO1 and OVCA429 were gifts from Dr. Ruby YJ Huang (National Taiwan University, Taiwan) and Dr. Noriomi Matsumura (Kindai University, Japan) respectively. PEO1 was cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 2 mM Sodium pyruvate, 10% fetal bovine serum (FBS) and 1 \u0026times; antibiotics/antimycotics (anti/anti). OVCA429 was cultured in DMEM supplemented with 10% FBS and 1 \u0026times; anti/anti. Cells were cultured at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator. Both cancer cell lines were authenticated using short tandem repeat profiling (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and Mycoplasma screened using a PCR-based approach (BIOTOOLS, New Taipei City, Taiwan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmids and reagents\u003c/h3\u003e\n\u003cp\u003eThe lentiviral pLKO-puro-shRNA expression vectors shCtrl (ASN0000000003),\u003c/p\u003e \u003cp\u003eshPER2 [TRCN0000330731 (#1) and TRCN0000330809 (#2)] were purchased from the\u003c/p\u003e \u003cp\u003eNational RNAi Core Facility (Taipei, Taiwan). HO-1 inhibitors, Tin Protoporphyrin IX dichloride (SnPPIX) and Heme Oxygenase-1-IN-1 hydrochloride (HO-1i), were purchased from MedChemExpress (Monmouth Junction, NJ, USA). N-acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich (St. Louis, MO, USA).\u003c/p\u003e\n\u003ch3\u003eImmunoblot assay\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eImmunoblot assay\u003c/div\u003e \u003cp\u003eWhole cell lysate was prepared using RIPA buffer (Millipore, Burlington, MA, USA) with SIGMAFAST\u0026trade; Protease Inhibitors (Sigma-Aldrich) and PhosSTOP EASYpack phosphatase inhibitor cocktail (Roche, Basel, Switzerland). 15\u0026ndash;30 \u0026micro;g of total protein was separated by 7.5% or 10% tris\u0026ndash;glycine polyacrylamide gel, with overnight incubation with 1:1,000 primary antibodies against PER2 (GTX129688, GeneTex, Irvine, CA, USA), E-Cadherin (G10, Santa Cruz Biotechnology, Dallas, TX, USA), N-Cadherin (13A9, Santa Cruz), Vimentin (D21H3, Cell Signaling Technology, Danvers, MA, USA), HO-1 (10701-1-AP, Proteintech Group, Rosemont, IL, USA)] or 1:5,000 GAPDH (1E6D9, Proteintech), and followed by a 1:10,000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibodies. HRP signals were detected using Immobilon Western Chemiluminescent HRP substrate (Millipore) and images captured by a UVP ChemStudio Plus BioImaging system. The densitometry of blot bands was quantified using NIH ImageJ.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and quantitative real-time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eRNA was extracted using TRI Reagent (Sigma-Aldrich) according to the manufacturer\u0026rsquo;s instructions. 3 \u0026micro;g total RNA from each sample was reverse-transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). 10 ng 1st-strand cDNA was used for real-time PCR with appropriate primer sets (listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) and SYBR Green 2X Master Mix (KAPA Biosystems, Wilmington, MA, USA) using ABI Quantstudio5 PCR system. The mRNA relative quantities were determined using relative ΔC\u003csub\u003et\u003c/sub\u003e method with 18s rRNA as an internal control.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence (IF) staining\u003c/h3\u003e\n\u003cp\u003eCells were seeded and left to adhere to reach 70\u0026ndash;80% confluence on coverslips and then fixed with ice-cold 100% methanol (for PEO1 cells) or acetone (for OVCA429 cells) for 10 min at \u0026minus;\u0026thinsp;20\u0026deg;C followed by rehydration with phosphate-buffered saline (PBS). The cells were then blocked with 1% bovine serum albumin (BSA)/PBS for 1 hour at room temperature and incubated with primary antibodies against E-Cadherin (1:100, 610182, BD Biosciences, Franklin Lakes, NJ, USA), N-Cadherin (1:50, M142, Takara, Shiga, Japan) or Vimentin (1:100, M0725, Agilent-Dako, Santa Clara, CA, USA) in 1% BSA/PBS for overnight at 4\u0026deg;C. After washing with PBS, the cells were incubated with goat anti-mouse IgG H\u0026amp;L Alexa Fluor\u0026reg; 488 (ab150117) or goat anti-rabbit IgG H\u0026amp;L Alexa Fluor\u0026reg; 488 (ab150077, Abcam, Cambridge, UK) secondary antibody (1:300) in 1% BSA/PBS for 1 hour in the dark. DAPI was used to stain nucleus. Coverslips with stained cells were mounted onto glass slides with fluorescence mounting medium (Agilent-Dako). Images were acquired using an Andor Dragonfly 202 high speed confocal microscope system and the level of fluorescence signal was measured by fluorescence emission at 505\u0026ndash;550 nm with excitation at 488 nm. The fluorescence intensity was quantified using the Imaris 10.1.0 software.\u003c/p\u003e \u003cp\u003eFor ROS detection, cells were stained for 30 min at 37\u0026deg;C with 0.5\u0026micro;M (for PEO1) or 1\u0026micro;M (for OVCA429) CM-H2DCFDA (Thermo Fisher Scientific) in PBS. Hoechst 33432 (Invitrogen, Waltham, MA, USA) was used to stain nucleus. Oxidation of the probe was detected was measured by fluorescence emission at 517\u0026ndash;527 nm with excitation at 492\u0026ndash;495 nm.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP) assay\u003c/h2\u003e \u003cp\u003eChIP assay was conducted using Zymo-Spin ChIP kit (Zymo Research, Irvine, USA). Immunoprecipitation was performed using antibodies against H3K27me3 (Lys27) (C36B11, Cell Signaling Technology), H3K4me3 (Lys4) (C42D8, Cell Signaling Technology) or corresponding control IgG (ab172730, Abcam). qRT-PCR was performed to detect H3K27me3 or H3K4me3 associated promoter regions using primers listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInvasion and migration assays\u003c/h3\u003e\n\u003cp\u003eFor invasion assay [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were seeded in a 24-well Falcon HTS Fluoro Block (8 \u0026micro;m) transwell insert coated with Matrigel (Corning, NY, USA) in growth medium without serum. Complete medium was used as a chemoattractant in the lower chamber of 24-well plate. After 24 h incubation, the invaded cells were fixed with methanol and stained with DAPI. Cells were counted with fluorescence microscopy.\u003c/p\u003e \u003cp\u003eFor migration assay, two strategies were used. For transwell migration assay, 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were seeded in a 24-well Falcon Transparent membrane (0.4 \u0026micro;m) PET inserts (Corning) in serum free media. Complete medium was used as a chemoattractant in the lower chamber. After 24 h incubation, the migrated cells were fixed with methanol, stained with 0.2% crystal violet and imaged and counted using an inverted microscope. For scratch assay, 1.75\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were seeded in each well of a 2-well silicone insert (ibidi USA Inc., Fitchburg, WI, USA) in a 24-well plate. The cell density reached 100% confluence 24 h after seeding and the insert was removed to generate a wound field. Fresh medium was then added and the cells were cultured for additional 48 hrs. Image of the cell monolayer was taken at 0, 12, 24, 36 and 48 hours after insert removal using a phase-contrast microscope, and distance between one side of wound field and the other was measured.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIntraperitoneal (\u003c/b\u003e \u003cb\u003ei.p.\u003c/b\u003e \u003cb\u003e) tumor xenograft model\u003c/b\u003e \u003c/p\u003e \u003cp\u003e Animal care and experiments were approved by the Institutional Animal Care and Utilization Committee of Academia Sinica (IACUC# 20-06-1485). Female BALB/cAnN.Cg-\u003cem\u003eFoxn1\u003c/em\u003e\u003csup\u003e\u003cem\u003enu\u003c/em\u003e\u003c/sup\u003e/CrlNarl (NUDE) mice were purchased form the National Laboratory Animal Center (Taipei, Taiwan) at 6 weeks of age. 500\u0026micro;L of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells in PBS were prepared and injected into the peritoneal cavity. After injection, mice were weighed and palpated weekly. Ten weeks after injection, mice were sacrificed and the intestines were collected for further assessment of tumor burden.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eTissues were fixed in 10% formalin overnight at 4\u0026deg;C and embedded in paraffin. Sections were cut into 4 \u0026micro;m slices, dewaxed with xylene and rehydrated with descending ethanol series to water. Antigen retrieval was performed using Target Retrieval Solution (Agilent-Dako) for 20 min under high pressure condition. Slices were stained with anti-CK7 antibody (bs-1610R, Bioss, Woburn, MA, USA) overnight at 4\u0026deg;C, followed by incubation with DAKO REALTM EnVisionTM HRP Labeled Polymer Anti-Rabbit (Agilent-Dako) at room-temperature for 30 min. The 3.3\u0026rsquo;-diamiobenzidine (DAB) substrate was used to detect the peroxidase activity, and the slices were counterstained with hematoxylin. Slides were photographed under 40X magnification by the ZEISS Axioscan 7 and Aperio scanner machine (Leica, Wetzlar, Germany).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted as described above and quantified using NanoDrop (Thermo Fisher Scientific). All samples with OD 260/280 ratio\u0026thinsp;\u0026gt;\u0026thinsp;1.8 and OD 230/260 ratio\u0026thinsp;\u0026gt;\u0026thinsp;2 were sent to BIOTOOLS for sequencing. RNA quality was measured by Qsep100 Bio-Fragment Analyzer. Samples with RQN\u0026thinsp;\u0026gt;\u0026thinsp;6.8 were used for cDNA library construction. Paired-end sequencing (read length 150 bp) was performed on a NovaSeq 6000 platform and quality filtering and adaptor trimming of raw reads performed using Cutadapt (v1.12; [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]). Trimmed reads were aligned to Human genome (hg38) using STAR (v2.5.3a; [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]). After mapping to the reference genome, \u0026sim;20\u0026nbsp;million paired aligned reads were recovered from each RNA sequencing (RNA-seq) library. Expected read counts of each gene were estimated with RSEM (v1.2.31; [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]), followed by adjusting the CPM with the effective library size via the calcNormFactors function implemented in the R package edgeR (v.3.26.8; [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]). For each cell line, the expression profile of each sh-PER2 clone was compared with the sh-Ctrl and gene expression fold-change calculated (Table S3). To identify enriched functional pathways, we performed KEGG Analysis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] on the ranked gene list, determined by the Log Fold-Change.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSoft agar colony formation assay\u003c/h2\u003e \u003cp\u003e2500 cells were mixed with a layer of 0.35% agar/complete growth medium over a layer of 0.5% agar/complete growth medium in a well of a 12-well plate. After culture at 37\u0026deg;C for 3 weeks, colonies were counted using phase contrast microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean of three or more replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Student\u0026rsquo;s t-test was used to compare control and treatment groups. All statistical analyses were performed using Prism 10 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eLow expression of\u003c/b\u003e \u003cb\u003ePER2\u003c/b\u003e \u003cb\u003eis associated with poor prognosis in Epi-A OC\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe clinical significance of \u003cem\u003ePER2\u003c/em\u003e expression in OC was evaluated with the TNMplot web tool [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. \u003cem\u003ePER2\u003c/em\u003e level was significantly downregulated in ovarian tumor samples compared to the normal ovarian tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, p\u0026thinsp;=\u0026thinsp;4.41e\u003csup\u003e\u0026minus;\u0026thinsp;05\u003c/sup\u003e). Also, the CSIOVDB database [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] showed that \u003cem\u003ePER2\u003c/em\u003e was differentially expressed among the five GEMS, with a significantly higher level found in the Epi-A subtype compared to other GEMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, within the Epi-A subtype, high \u003cem\u003ePER2\u003c/em\u003e expression was associated with better overall survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This clinical correlation was not observed in other GEMS (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), suggesting an Epi-A GEMS-specific tumor suppressive role of PER2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLoss of PER2 enhances tumorigenic ability of Epi-A OC cells\u003c/h2\u003e \u003cp\u003eTwo Epi-A OC cell lines, PEO1 and OVCA429 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], were used to evaluate the function of PER2. Depletion of PER2 using a lentiviral-shRNA system (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA) resulted in an approximately 3-fold increase in anchorage-independent growth in PEO1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and a 2-fold increase in OVCA429 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), suggesting that loss of PER2 facilitates Epi-A ovarian tumor growth. PER2 depletion also had a profound impact on invasion and migration ability of Epi-A OC cells. Even for PEO1, which has low invasion ability [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], PER2 depletion significantly promoted invasion through an extracellular matrix coated porous membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) and migration through a transwell (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). In OVCA429 cells, which exhibit an intermediate epithelial phenotype with a substantial epithelial-mesenchymal plasticity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], PER2 depletion elevated the invasion ability by 3-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) and migration ability by 2-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Consistent with these results, scratch assays showed that the PER2-depleted cells moved into a gap much faster than the control in both Epi-A OC cell lines (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB, S2C).\u003c/p\u003e \u003cp\u003eTo validate these cell-based results \u003cem\u003ein vivo\u003c/em\u003e, several xenograft models were considered. It has been reported that PEO1 cells do not form tumors either via intraperitoneal (\u003cem\u003ei.p.\u003c/em\u003e) or subcutaneous (\u003cem\u003es.c.\u003c/em\u003e) injection in NUDE mice [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Although OVCA429 can form tumors via \u003cem\u003es.c.\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and \u003cem\u003eintra bursa\u003c/em\u003e injection [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], the tumors are often small and the latency for tumorigenesis is long. However, when injected via \u003cem\u003ei.p.\u003c/em\u003e, OVCA429 has been observed to form solid tumors in the pelvic region, intestine, and omentum [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Since our cell-based experiments showed that PER2 depletion increased invasion ability of Epi-A OC cells, and because \u003cem\u003ei.p.\u003c/em\u003e xenograft model has been used for peritoneal dissemination in intra-abdominal cancers, we injected OVCA429 cells without (shCtrl) or with PER2 knockdown (shPER2) intraperitoneally using NUDE mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO). Interestingly, mice injected with shPER2 OVCA429 cells generated many scattered (rather than clustered) tumor nodules in the intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO), albeit that the number of confined intestinal tumor nodules was not significantly different between the shPER2 and shCtrl groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). These observations suggested that loss of PER2 may drive EMT as EMT has generally been linked to the dispersion of individual primary tumor cells. These results along with the clinical correlations suggest that PER2 expression is critical to maintain a less aggressive, epithelial phenotype in Epi-A GEMS and thereby contribute to the better survival in PER2-high Epi-A OC patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEpi-A OC cells undergo EMT upon PER2 depletion\u003c/h2\u003e \u003cp\u003ePER2 downregulation in both PEO1 and OVCA429 cells led to a morphological change from a polygonal shape with regular dimensions to an elongated, spindle-like shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This further suggested that cells underwent EMT after PER2 depletion. The epithelial to mesenchymal switch was confirmed by assay of EMT markers using immunofluorescence (IF) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and immunoblotting (IB) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). After PER2 depletion, the number of ECAD expressing cells decreased, while NCAD and VIM expressing cells increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Consistent with these results, the protein level of ECAD was reduced, and NCAD and VIM were elevated after PER2 depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These molecular changes were in line with the epithelial to mesenchymal morphological changes as well as the elevated invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) and migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB, S2C) ability in the shPER2 cells. Together these data indicated that PER2 expression was critical to maintain an epithelial phenotype in Epi-A OC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHO-1 upregulation is the key to PER2 depletion-mediated EMT in Epi-A OC\u003c/h2\u003e \u003cp\u003eWe previously found that PER2 inhibits EMT in breast cancer cells by recruiting the Polycomb Repressive Complex 2 (PRC2) to suppress transcription of the EMT executors, TWIST1, SNAIL and SLUG [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Intriguingly, upregulation of these EMT executors was not as prominent in Epi-A OC cells (Fig. S3, [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]) and EMT was not observed until a much later time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results suggested that, unlike in breast cancer cells where PER2 directly inhibited transcription of EMT executors, PER2 loss may result in other EMT-inducing alterations in Epi-A OC cells.\u003c/p\u003e \u003cp\u003eTo understand how PER2 regulates EMT in Epi-A OC cells, RNA-seq analysis of OVCA429 without or with PER2 depletion was performed. KEGG enrichment analysis found that genes in chemical carcinogenesis - reactive oxygen species (ROS)-related processes, including \u003cem\u003eHMOX1\u003c/em\u003e, \u003cem\u003eCYP1A1\u003c/em\u003e, \u003cem\u003eEPHX1\u003c/em\u003e, \u003cem\u003eAKR1C1\u003c/em\u003e and \u003cem\u003eAKR1C3\u003c/em\u003e, were significantly upregulated in PER2 depleted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Table S3). Since ROS can facilitate EMT [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], these genes were further examined in shCtrl and shPER2 OVCA429. \u003cem\u003eHMOX1\u003c/em\u003e was found to be significantly upregulated in both PER2 knock-down cell lines and its expression level was negatively correlated with that of PER2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCSIOVDB database analysis of OC patient data found significantly lower \u003cem\u003eHMOX1\u003c/em\u003e expression in Epi-A compared to Epi-B and Mes GEMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This contrasted with the relatively high expression of PER2 in Epi-A GEMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Epi-A patients with high \u003cem\u003eHMOX1\u003c/em\u003e expression had poor overall survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, p\u0026thinsp;=\u0026thinsp;0.039), consistent with observations that Heme Oxygenase 1 (HO-1), which is encoded by the \u003cem\u003eHMOX1\u003c/em\u003e gene, inhibited apoptosis and enhanced EMT and metastasis in the Mes GEMS cell line SKOV3 and Stem-A cell line A2780 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese observations suggested that PER2 depletion and consequent upregulation of \u003cem\u003eHMOX1\u003c/em\u003e/HO-1 may induce EMT to facilitate invasion and metastasis in Epi-A OC. Consistent with this hypothesis, HO-1 inhibition using Tin protoporphyrin IX dichloride (SnPPIX) and Heme Oxygenase-1-IN-1 hydrochloride (HO-1i) abolished the elevated migration and invasion phenotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), as well as the EMT morphology and marker expression (Fig. S4) in PER2 depleted PEO-1 and OVCA429 cells. These results indicated that \u003cem\u003eHMOX1\u003c/em\u003e/HO-1 upregulation is the key mechanism to induce EMT, invasion and migration in PER2-depleted Epi-A OC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDepletion of PER2 activates HO-1 via ROS elevation and transcriptional de-repression\u003c/h2\u003e \u003cp\u003eHO-1 has multifaceted roles in tumor progression [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. \u003cem\u003eHMOX1\u003c/em\u003e/HO-1 is transcriptionally induced in response to several stimuli including oxidative stress to regulate cellular ROS level [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and the effects of ROS on tumorigenesis and cancer progression have been intensively studied [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In cancer cells, ROS levels are restricted to a dynamic range which is high enough to produce genetic damage necessary for tumorigenesis and induce various pathways that drive tumor cells to undergo EMT and thereby promote invasion and metastasis [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], but not so high as to cause cytotoxic effects and activate cell death pathways [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In PER2-depleted Epi-A OC cells, ROS level was elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) possibly due to downregulation of several genes with antioxidant functions, such as superoxide dismutase 2 (SOD2) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and peroxidasin (PXDN) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] (Fig. S5A, Table S3). ROS level was significantly elevated within 48 hours after shPER2 transduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Fig. S5B), while HO-1 protein level was not significantly increased until 96 hours after PER2 knock-down (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Fig. S5C-S5E). The observation that elevated ROS preceded \u003cem\u003eHMOX1\u003c/em\u003e/HO-1 induction suggested that elevated ROS may be involved in HO-1 induction in PER2 depleted cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHO-1 is known to have a short half-life estimated as 3 hours for mRNA and 15\u0026ndash;21 hours for protein [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Intriguingly, when ROS was removed using the antioxidant N-acetyl-L-cysteine (NAC), HO-1 protein level remained higher in the PER2-depleted cells than the PER2-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, Fig. S5F-S5H). Moreover, 72 hours after NAC treatment, \u003cem\u003eHMOX1\u003c/em\u003e mRNA level in the PER2-depleted cells also remained upregulated (Fig. S6A, S6B) and EMT morphology remained unchanged (Fig. S6C, S6D). These results indicated that mechanisms in addition to (or instead of) increased ROS were involved in \u003cem\u003eHMOX1\u003c/em\u003e/HO-1 upregulation in PER2-depleted cells.\u003c/p\u003e \u003cp\u003eSince PER2 acts as a transcriptional co-repressor [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], it is possible that PER2 may directly suppress the promoter activity of \u003cem\u003eHMOX1\u003c/em\u003e to repress HO-1 level in Epi-A OC cells. In this case, PER2 depletion would allow the \u003cem\u003eHMOX1\u003c/em\u003e promoter to be accessible to oxidative stress-induced transcription factors such as nuclear factor erythroid 2-related factor 2 (NRF2), activator protein-1 (AP-1), nuclear factor-κB (NF-κB) and peroxisome proliferator-activated receptors (PPARs) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. To test this possibility, chromatin-immunoprecipitation (ChIP) assays were performed using antibodies recognizing the repressive histone mark H3K27tri-methylation (H3K27me3) or the active histone mark H3K4tri-methylation (H3K4me3) with primer sets amplifying 100- to 150-bp fragments spanning the known \u003cem\u003eHMOX1\u003c/em\u003e activator binding motifs within the \u0026minus;\u0026thinsp;1 kb proximal \u003cem\u003eHMOX1\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Upon PER2 knockdown, a decrease in H3K27me3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, Fig. S5I) and an increase in H3K4me3 were observed on the \u003cem\u003eHMOX\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, Fig. S5J), indicating a chromatin switch from a condensed state to an open state. Together, these results suggested that PER2 depletion-mediated \u003cem\u003eHMOX1\u003c/em\u003e/HO-1 upregulation was due to transcriptional de-repression; although we cannot rule out ROS accumulation as a contributing factor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePER2 depletion-mediated HO-1 upregulation increases cisplatin resistance in Epi-A OC\u003c/h2\u003e \u003cp\u003eEMT can contribute to drug resistance [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] and HO-1 upregulation has been associated with cisplatin resistance in OC cells [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Since PER2 downregulation promoted EMT and increased HO-1 expression, loss of PER2 in Epi-A OC cells may promote cisplatin resistance. Consistent with this hypothesis, less than 10% of the PER2 expressing cells (shCtrl) survived when treated with 5 \u0026micro;M cisplatin while 30% of PER2 knockdown cells survived (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). When a higher dose (10 \u0026micro;M) of cisplatin was used, more than 95% of the PER2-expressing cells died (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), while PER2 depletion increased survival to approximately 30% for PEO1 and 15\u0026ndash;20% for OVCA429 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In addition to cell viability, we also evaluated the effect of PER2 depletion on cisplatin-suppression of tumorigenic ability using soft-agar colony formation assay. In PER2-expressing PEO-1 and OVCA429 cells, cisplatin treatment significantly reduced colony numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In contrast, colony forming ability of PER2-depleted cells was not affected by cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These results indicated that PER2 loss significantly increased cisplatin resistance of Epi-A OC cells in terms of both cell survival and tumorigenic ability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also found that inhibition of HO-1 sensitized the PER2 knocked-down cells to cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Treatment with either SnPPIX or HO-1i decreased the viability of cisplatin-treated shPER2 PEO1 cells from approximately 35\u0026ndash;15% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), and that of shPER2 OVCA429 cells from 25\u0026ndash;40% to 15\u0026ndash;20% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Furthermore, HO-1 inhibitor treatment resulted in a statistically significant reduction in soft-agar colony forming ability in the cisplatin-treated shPER2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). These results indicated that upregulation of HO-1 was a key cause of cisplatin resistance in PER2-depleted Epi-A OC cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePER2 is a crucial suppressor controlling the circadian oscillation that influences many physiological and biological processes. In peripheral tissues, PER2 not only has important roles in tissue-specific responses to the circadian environment, but also exhibits tumor suppressive functions. For example, mice with homozygous \u003cem\u003ePer2\u003c/em\u003e mutation showed higher sensitivity to radiation and had increased radiation-induced tumorigenesis compared to the wild-type mice [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In humans, PER2 expression is often found to be downregulated in tumors compared to normal tissues [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Loss of PER2 is associated with dysregulation of genes related to cell cycle, proliferation, apoptosis, DNA repair, angiogenesis, metabolism and inflammation [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA recent study using the SKOV3 (Mes-like GEMS) cell line, one of the very few studies to examine PER2 in ovarian cancer, reported that PER2 expression was dramatically reduced when the cells acquired cisplatin resistance [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Although it was not clear whether PER2 downregulation was the cause of cisplatin resistance in SKOV3 cells or a side effect of such resistance, the authors did show that PER2 overexpression suppressed PI3K/AKT pathways and enhanced cisplatin-induced apoptosis [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Our results demonstrate that loss of PER2 was the cause of both enhanced EMT and cisplatin resistance in Epi-A OC cells. We also identified the key factor, HO-1, whose upregulation is responsible for these malignant phenotypes in the PER2-depleted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These observations further demonstrate the tumor suppressive activity of PER2 and emphasize the critical role of PER2 in Epi-A OC progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeveral animal models deficient in core clock genes \u003cem\u003eBmal1\u003c/em\u003e, \u003cem\u003eClock\u003c/em\u003e or \u003cem\u003eNpas2\u003c/em\u003e exhibit chronic oxidative stress and defects in antioxidant defense [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. However, the role of PER2 in oxidative stress response and related pathology is less clear. The increase in cellular ROS in PER2-depleted Epi-A OC cells indicated a disrupted redox balance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig. S5). The major site of cellular ROS production is mitochondria and PER2-depleted Epi-A OC cells had reduced expression of several genes encoding components of the mitochondrial antioxidant system, including SOD2 (Fig. S5A, Table S3). This suggested that elevated ROS in the Epi-A OC cells was due to ROS imbalance in the mitochondria. In mitochondria, SOD2 is the first line of defense against superoxide radicals generated in the process of electron transfer during oxidative phosphorylation [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. How PER2 expression affects the antioxidant system in Epi-A OC cells, particularly SOD2 expression, is worthy of further investigation.\u003c/p\u003e \u003cp\u003eTranscriptional up-regulation of HO-1 is mainly mediated by oxidative stress-responsive transcription factors such as NRF2, AP1, NF-κB and PPAR [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Intriguingly, the interplay between HO-1 and oxidative stress is complex and HO-1 can be a friend or foe to tumorigenesis [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. HO-1 is a key enzyme to metabolize heme to generate biliverdin, carbon monoxide (CO) and ferrous iron (Fe2\u003csup\u003e+\u003c/sup\u003e). Biliverdin can be subsequently converted to bilirubin to act as anti-oxidant to counteract ROS whereas Fe2\u003csup\u003e+\u003c/sup\u003e increases ROS generation via the Fenton reaction. As for CO, it participates in signal transduction including pro-angiogenesis, anti-inflammation and anti-apoptosis pathways which can contribute to tumor progression. Despite intensive research, the complex role and regulation of HO-1 in normal and tumor cells remain elusive [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In this study, we found that HO-1 was transcriptionally suppressed in PER2-expressing Epi-A OC cells. Our findings also imply that rhythmic expression of PER2 in normal ovarian tissues may play a critical role in modulating HO-1 expression and ROS homeostasis in response to circadian cues consistent with a recent study which found that circadian dependent HO-1 expression is involved in regulating neuroinflammation in microglial [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. How PER2, along with the core clock machinery, regulate HO-1 and ROS to maintain normal tissue homeostasis and stress response is also worthy of further investigation.\u003c/p\u003e \u003cp\u003eOur study demonstrated that PER2 expression is required for Epi-A OC cells to maintain an epithelial phenotype, ROS homeostasis and sensitivity to cisplatin. Loss of PER2 increased malignancy by elevating anchorage-independent growth, promoting EMT, and facilitating invasion and migration in Epi-A OC cells. These results demonstrate the underlying mechanism that explains why PER2-low Epi-A OC patients have poorer prognosis compared to the PER2-high patients. We also identified HO-1 as a key factor critical for Epi-A OC cells to gain malignancy upon PER2 loss. Thus, targeting HO-1 may provide a therapeutic strategy to prevent Epi-A OC progression due to PER2 loss.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eWWHV and RYJH conceived the study. GYTT, PV, LTC, PYL, CLWC and YCC performed experiments. CHAY assisted data analysis. WWHV wrote the paper.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by National Taiwan University-Academia Sinica Joint Program [NTU-AS-110-09] and [NTU-AS-112-11] to WWHV and RYJH, and National Science and Technology Council [NSTC-113-2813-C-001-011-B] to WWHV. The authors would like to thank Dr. Pang-Hung Hsu (National Taiwan Ocean University) and Ms. Divya Malathy Ravinath for their help during the early stage of this study. The authors would also like to thank the following core facilities at Academia Sinica: the Bioinformatics Core at Institute of Molecular Biology for providing the RNA-seq analysis services, the Advanced Optics Microscope Core Facility, funded by Academia Sinica Core Facility and Innovative Instrument Project (AS-CFII-111-208), for microscope imaging technical support, the SPF Animal Facility funded by AS-CFII-111-204 for providing animal support, and the National RNAi Core Facility for providing shRNA reagents and related services.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEbrahimi N, Manavi MS, Faghihkhorasani F, Fakhr SS, Baei FJ, Khorasani FF \u003cem\u003eet al\u003c/em\u003e. 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J Inflamm (Lond) 2023; 20: 43.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5242949/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5242949/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRapid metastasis and chemoresistance make ovarian cancer (OC) the deadliest gynecological cancer. These malignant phenotypes are strongly associated with epithelial-mesenchymal transition (EMT). OC is highly heterogenous with five distinct gene-expression based molecular subtypes (GEMS) correlated with different EMT levels and patient survival. However, the regulatory mechanisms underlying GEMS-specific EMT and malignancy remain unclear. We found that \u003cem\u003ePER2\u003c/em\u003e gene expression was significantly downregulated in OC compared to normal ovarian tissues. Among the five GEMS, \u003cem\u003ePER2\u003c/em\u003e expression was relatively high in the Epithelial-A (Epi-A) subtype and Epi-A OC patients with higher \u003cem\u003ePER2\u003c/em\u003e level had better overall survival. Notably, PER2 expression was required to maintain the epithelial phenotype. Conversely, PER2 depletion drove EMT and consequently enhanced migration and invasion ability of Epi-A OC cells via upregulation of Heme oxygenase-1 (HO-1). PER2 depletion also increased cisplatin resistance. Inhibition of HO-1 in PER2-depleted Epi-A OC cells impeded invasion and migration, and re-sensitized the cells to cisplatin. Our results revealed new insights into how an epithelial-like, less malignant OC subtype acquires EMT to become more invasive and chemoresistant. These findings also indicate that prevention of PER2 downregulation and targeting HO-1 are promising strategies for treating Epi-A OC progression.\u003c/p\u003e","manuscriptTitle":"Loss of PERIOD2 (PER2) induces HO-1-mediated epithelial-mesenchymal transition and cisplatin resistance in Epi-A ovarian cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-17 04:11:24","doi":"10.21203/rs.3.rs-5242949/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"32342683-c8a1-4a8a-bb08-5e790a6d7f6b","owner":[],"postedDate":"October 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38960421,"name":"Biological sciences/Cancer/Gynaecological cancer/Ovarian cancer"},{"id":38960422,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2024-10-17T04:11:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-17 04:11:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5242949","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5242949","identity":"rs-5242949","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}