SMYD3 promotes endometrial cancer through epigenetic regulation of LIG4/XRCC4/XLF complex in non-homologous end joining repair

Endometrial cancer (EC) is one of the most common malignant tumors of female genital tract with rapid increased incidence worldwide. Histone methyltransferase SMYD3, which is linked to the development and progression of various cancers, however, its oncogenic function in endometrial cancer has not been investigated thus far. In the present study, we report that the expression level of SMYD3 is significantly upregulated in EC samples and associated with EC progression. In vivo and in vitro functional experiments demonstrate that SMYD3 depletion inhibits cell proliferation, migration, and invasion abilities, increasing the sensitivity of EC cells to radiation. Moreover, with pathway enrichment analysis, we find that the DNA damage repair pathway is mainly involved in regulating EC progression. Mechanically, in response to DNA damage, SMYD3 is recruited to DNA damage sites in an PARP1-dependent manner and specifically methylates LIG4. Methylated LIG4 sequentially assembles LIG4/XRCC4/XLF complex and participates in non-homologous end joining repair (NHEJ) pathway. More importantly, pharmacological targeting of SMYD3 with its specific inhibitor BCI-121, significantly represses the tumorigenicity of EC cells and greatly enhanced radiotherapy efficiency. Overall, our data suggest SMYD3 as a pivotal factor in NHEJ repair and is associated with in EC progression. Using in vitro and in vivo system, we also prove that SMYD3 is a promising pharmacological target for endometrial cancer therapy.


Introduction
Endometrial cancer is the most common gynecological cancer in women, its incidence and new diagnoses is rapidly rising worldwide, but five-year survival has not significantly improved during recent years (1)(2)(3), indicating the limitations of existing clinical treatments.Deep understanding of the development mechanism in endometrial cancer is conductive to find new targets for EC treatment.Previous large-scale sequencing studies on primary endometrial cancer samples indicated that DNA repair is involved in endometrial tumorigenesis and progression (4,5), and was prioritized for further investigation of endometrial cancer clinical trials approved by The US National Cancer Institute (6).DNA repair is an effective way for cells to maintain genome integrity under a variety of assaults from endogenous (7) and exogenous stress (8).Improper or inefficient repair of DNA damage compromises the integrity of the genomes and leads to DNA damage accumulation.Persistent DNA damage decreases the stability of genomes and promotes cancer (9).However, targeting DNA damage response related genes is also a good strategy in cancer treatment, especially combine with chemotherapy or irradiation.Because these therapies are designed to kill cancer cells mostly by inducing toxic DNA damage.So, targeting proteins related with DNA damage repair has been explored as an attractive therapeutic target (9).
According to the previous research, post-translational modifications including phosphorylation, ubiquitylation, acetylation, SUMOylating, PARylation and methylation play essential roles in modulating DNA damage repair process by affecting the expression or recruitment of DNA damage response (DDR) factors even change the dynamic status of chromatin (10)(11)(12)(13)(14). Methylation, dynamically regulated by methyltransferases and demethylases, also plays vital roles in DNA damage and repair.
Regarding to non-histone methylation, studies have shown that methylation of MRE11 and 53BP1 promotes their focal accumulation at DSB sites by enhancing their DNA binding abilities (20)(21)(22).Moreover, methylated DNA ligase 1 by G9a recruits DNMT1 to methylate the newly synthesizes DNA at the replication forks (12).Although, more and more studies have provided insights into the function of methylation in DNA damage response, the protein methylation modification in regulating NHEJ repair pathway remains largely unknown.
SET and MYND Domain containing 3 (SMYD3) is a member of the SMYD lysine methyltransferase family, participating in methylation of various histone and nonhistone substrates (23).The SET domain of SMYD proteins catalyzes lysine methylation, while the MYND domain, a cysteine-rich zinc finger motif, is involved in protein-protein interactions (24).SMYD3 plays important roles in tumorigenesis (25).

Elevated expression of SMYD3 has been found during tumorigenesis in various types
of tumors, such as colorectal cancer, hepatocellular cancer, breast cancer and ovarian cancer (26)(27)(28), suggesting its important role in tumor initiation and progression.SMYD3 was also reported to methylate the histone H2A.Z.1 at lysine 101 at the promoter of cyclin A1, promoting cyclin A1's transcription in breast cancer (29).SMYD3-mediated methylation of MAP3K2 promotes the formation of RAS-driven carcinomas by activating the RAS-ERK signal (30).Although several studies have been reported the role of SMYD3 in different kinds of tumors, its involvement in endometrial cancer has not yet been identified.
Based on the previous studies, DNA double strand break (DSB) is the most deleterious DNA damage and includes sophisticated cellular procedures (9).There are two major pathways of DSB repair in mammalian cells, homologous recombination (HR) repair and non-homologous end joining (NHEJ) repair.HR is an error-free pathway, which utilizes an intact sister chromatid as template in S/G2 phases (31).NHEJ, on the contrary, is a rapid, high-capacity pathway that joins two DNA ends with no presence of an intact template through the whole cell cycle (32).NHEJ is initiated by the binding of the KU70-KU80 heterodimer to DSB ends, then DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is recruited to chromatin and finally establishes a long-range synapse with KU70-KU80 complex (32,33).The two DNA ends become closely aligned under the processes, requiring the DNA ligase IV (LIG4)/XRCC4/XRCC4-like factor (XLF) complex involved (32,33).LIG4 is considered as a primary ligation factor, while XRCC4 is required for the stability and activity of LIG4 (34).XLF functions to increase the ligation activity of LIG4/XRCC4 (35).It has been reported that SMYD3 is involved in HR repair by promoting the translocation of RAD51 to DNA damage sites.
Co-targeting SMYD3/PARP leads to synthetic lethality in HR-proficient cancer cells (36).However, whether SMYD3 is involved in NHEJ repair and participate in endometrial cancer development remains unknown.
In this study, we report for the first time that SMYD3 has a significant role in endometrial cancer progression, and we identified a novel mechanism of SMYD3 mediated NHEJ repair in endometrial cancer.In detail, we provide evidence that SMYD3 is upregulated in endometrial cancer and associated with EC progression.Knockdown of SMYD3 inhibits the cell proliferation, migration and invasion abilities of endometrial cancer cells in vivo and in vitro.Through analyzing the TCGA database with pathway analysis, we find the DNA repair pathway is involved in endometrial cancer progression.In response to DNA damage, SMYD3 is quickly recruited to DNA damage sites in a PARP1-dependent manner and methylates LIG4 which affects the recruitment of LIG4/XRCC4/XLF complex during NHEJ repair.More importantly, inhibition of SMYD3 with its specific inhibitor, BCI-121, significantly sensitize endometrial cancer cell to radiotherapy.Our results suggest that SMYD3 might be a promising therapeutic target for endometrial cancer.
All cell lines were incubated in a 5% CO2 humidified incubator (Thermo Fisher Heracell 240i, Thermo Fisher Scientific) at 37℃.Mycoplasma testing was conducted regularly.

Plasmids, shRNA and siRNA
The pLVX-EF1α-IRES-Puro-SMYD3-3×FLAG was constructed by General Biosystems (Anhui, China) and used as the template of SMYD3.Site-directed mutagenesis was performed using KOD Kit (NEB) according to the manufacturer's instructions.The GFP-SMYD3 was subcloned into pEGFP-C2 backbone.The ORF of human XLF was amplified from cDNA of HCA2-hTERT cells and subsequently cloned into pEGFP-C2 backbone.The GFP-LIG4, GFP-XRCC4 were kindly given by professor Zhiyong Mao from Tongji University School of Medicine.The shRNAs targeting SMYD3 were subcloned into the pLKO.1 plasmid (Addgene, USA).The viruses were collected from the medium after 72h transfection.For knockdown experiments, cells were infected with viruses 24h in the presence of polybrene (3mg/ml, HY-112735, MedChemExpress).The siRNAs targeting SMYD3 were synthetized by GenePharma (Shanghai, China).Scrambled siRNA was used as negative control.

Laser micro-irradiation-coupled live-cell imaging
Cells were presensitized with 10μM BrdU (5911439, BioGems) for 16h at 37℃.Then, laser microirradition was performed using a confocal microscope (TCS SP8, Leica) coupled with the Micropoint system (Andor Technology).Cells were locally irradiated with a 365nm UV laser from the Micropoint system.The fluorescence intensity of GFP signals at laser micro-irradiated sites was measured using ImageJ software.

SDS-PAGE and Western blotting
Total cell lysates were collected in RIPA lysis buffer.Western blotting analysis was performed as described [1].Briefly, equal numbers of cells were washed with PBS twice.The cell pellet was re-suspended in same amount of loading buffer and boiled for 10 min.Equal amounts of protein were loaded on a 7.5-12.5% SDS-polyacrylamide electrophoresis gel.

Flow cytometry
EJ5-U2OS cells were transfected with 5μg of pLVX-SMYD3-3 × FLAG, or 30ul siRNAs, and 5μg of I-SceI.Then cells were incubated in the cultured medium for 72h before FACS analysis on a flow cytometry (Accuri C6, BD Biosciences).For each sample, 1 × 10 5 cells were counted.Transfection was performed on a Lonza 4D machine (DT-130 program).Each experiment was repeated at least 3 times in triplicate.

Cell Counting Kit-8 (CCK-8) assay
HEC1B and ISK cells were seeded in 96-well plates at a density of 100 cells per well.

Colony formation assay
1 × 10 3 cells were plated in each well of 6-well plates.24h later, cells were exposed toγ-ray or without treatment.After incubated for 14 days at 37℃, colonies were stained with 0.5% crystal violet and counted using ImageJ software.

Migration and invasion assay
Cell migration was determined by using the Trans-well (351157, Corning) migration assay.HEC1B and ISK cells were seeded in serum-free medium on the upper chambers at a density of 5 × 10 4 cells per well.The lower chambers were filled with cultured medium.After incubation for 16h, each chamber was stained with AM (C3100MP, Thermo Fisher Scientific).The migrated cells were imaged by fluorescence analysis (Nikon, Japan).Cell invasion was determined by using the Trans-well (3422, Corning) invasion assay.HEC1B and ISK cells were seeded into the upper chambers covered with Matrigel (356234, Corning) for 16h.The invasive cells were stained with 0.5% crystal violet and counted using ImageJ software.

Tumor growth in vivo
The animal study was approved by the Ethics Review Committee for Animal Experimentation of Tongji University.Nude mice (female, 4 weeks) were housed in our institutional pathogen free mouse facilities.HEC1B (1 × 10 6 ) cells were suspended in 100μl of PBS buffer and injected into the flanks of the nude mice.At the end of 3weeks, the mice were sacrificed, and the tumor xenografts were collected and weighted.
For the spleen injection model, HEC1B (1 × 10 5 ) cells were suspended in 40μl of PBS buffer and injected into the spleen of the nude mice.At the end of 8 weeks, the mice were sacrificed, and the livers were collected and fixed in paraformaldehyde for HE and IHC.

Statistical analysis
Data are shown as the mean ± SD for the experiments repeated at least 3 replicates.For parametric data comparisons, t-test and one-way ANOVA were conducted.All statistical analyses and graphs were generated using GraphPad Prism (v9.2.0, La Jolla, USA).

SMYD3 is upregulated in endometrial cancer and associates with tumor progression
To access the role of SMYD3 in endometrial cancer, we first collected fresh paired samples from patients with EC.Both the mRNA and protein levels of SMYD3 were increased in EC tissues compared to adjacent tissues (Fig. 1A-B).By immunohistochemistry (IHC) staining with human endometrial cancer tissue with different pathologic category, we found that, SMYD3 was significantly upregulated in different subtypes of endometrial cancer (Fig. 1C).Then, we also analyzed the expression level of SMYD3 in The Cancer Genome Atlas (TCGA) database via several websites for public analysis.The analysis revealed that the expression level of SMYD3 was significantly increased in EC tissues compared with normal controls, as well as endometrioid, serous carcinoma or mixed serous and endometrioid.(Fig. 1D-E).
Moreover, SMYD3 was amplified in 6% of EC patients (Fig. 1F).To further verified the clinical significance of high SMYD3 expression, with endometrial cancer tissue microarrays, we found that the expression level of SMYD3 was clearly correlated with EC clinical grade (Fig. 1G).Consistently, we also used a conditional deletion of Pten in mouse endometrium, therefore, we crossed Pten (Pten loxP/loxP ) floxed mice with Pr cre/+ mice and obtained the uterus for immunohistochemistry.The expression of Smyd3 in mouse endometrial carcinoma was significantly upregulated compared with normal uterus (Figure .1H).Altogether, these findings indicate that SMYD3 contributes to endometrial cancer progression.

SMYD3 enhances endometrial cancer cell proliferation, migration and invasion abilities
The observations above prompted us to explore the potential biological function of SMYD3 in endometrial cancer progression.First, we investigated the effects of SMYD3 on cancer cell proliferation by its loss-of function assay, the EC cell lines HEC1B and Ishikawa (ISK) were stably transfected with short hairpin RNA (shRNA) against SMYD3 and its corresponding negative control (shRFP), the shRNA inhibitory efficiency was examined after puromycin selection (Fig. 2A).Cell Counting Kit-8 (CCK-8) assay revealed that depletion of SMYD3 inhibited cell proliferation ability in HEC1B and ISK cells (Fig. 2B).Additionally, the effects of SMYD3 on cell migration and invasion were observed using Trans-well and Matrigel invasion assays.The results showed that downregulation of SMYD3 significantly inhibited the cell migration and invasion abilities in both the HEC1B and ISK cell lines (Fig. 2C-D).To further explore the role of SMYD3 in cell growth, we carried out colony formation assay.The colonies formed fewer and smaller compared to the control group, indicating that downregulation of SMYD3 suppresses EC cell proliferation (Fig. 2E).Supporting the role of SMYD3 in EC cells, ectopic expression of SMYD3 in HEC1B cells increased the proliferation, migration, invasion and colony formation abilities, the enzymaticdead SMYD3-F183A failed to do so (Fig. 2F-J).Collectively, these results illustrate that SMYD3 exhibits oncogenic property through promoting the abilities of proliferation, migration and invasion in EC cells and depends on its methyltransferase activity.

SMYD3 drives tumorigenicity and metastasis of Endometrial cancer in vivo
To further investigate whether SMYD3 plays the same oncogenic role in vivo, we injected HEC1B cells (shRFP, shSMYD3-1, shSMYD3-2) into the flanks of female nude mice.As we shown, the weight and volume of tumors from shSMYD3 group were much lower than those from shRFP-HEC1B injection group (Fig. 3A-D).In agreement with the reduced tumor size, the tissues of SMYD3 deficient tumors showed decreased proliferation (Ki67) and increased cell death (cleaved-Caspase3) by using immunohistochemical staining (Fig. 3E).We next evaluated the in vivo effects of SMYD3 on liver metastasis by using spleen injection.The shRFP and shSMYD3-HEC1B stable cell lines were implanted into the spleen of nude mice, and we collected the tissue at about 8 weeks.As expected, most mice from the shRFP group developed liver metastases in contrast to SMYD3 deficient group.The clone numbers of metastatic tumor burden in the liver from the SMYD3 knockdown group were much less compared with the control group, and the size of metastasis clone size shown similar trend (Fig. 3F-G).In summary, these findings are consistent with the in vitro results, indicating that SMYD3 promotes endometrial cancer growth and metastasis in vivo.

SMYD3 is recruited to DNA damage sites in an PARP1-dependent manner
To further elucidate the molecular mechanisms underlying the effects of SMYD3 in endometrial cancer, we used pathway analysis (http://metascape.org/) with the patient samples of TCGA database to find SMYD3 correlated genes and pathways.As shown in Fig. 4A, we found that SMYD3 was closely associated with DNA repair pathway.In order to explore whether SMYD3 is involved in DNA damage response, we firstly sought to determine whether DNA damage influences the total protein level or cellular localization of SMYD3.Interestingly, we found that the total protein level of SMYD3 was significantly increased upon IR or chemotherapeutics compound VP16 treatment (Fig. 4B), and the increased SMYD3 was mainly localized in chromatin-bound fraction by using detergent and chromatin fraction methods (Fig. 4C).To examine the dynamic enrichment of SMYD3 at sites of DNA damage, time-lapse imaging showed that the laser micro irradiation with a 365 nm UV laser led to a rapid accumulation (~30s) of GFP-SMYD3 at sites of DNA damage (Fig. 4D).In addition, the enzymatic dead GFP-SMYD3-F183A abolished this dramatic recruitment, suggesting that the methylation activity of SMYD3 is essential for its recruitment (Fig. 4D).To determine which key DDR proteins are involved in regulating the recruitment of SMYD3 to DNA damage sites, we used four different inhibitors, which can sequentially block different steps of DNA damage repair.We found that the recruitment of SMYD3 to DNA damage sites was impaired when the cells were treated with PARP inhibitor Olaparib.However, the ATM inhibitor KU-55933, the ATR inhibitor VE-821, and the DNA-PKcs inhibitor NU7441 have no effect on blocking this recruitment (Fig. 4E).Moreover, consistent with the effect of PARP inhibitors, knockdown of PARP1 also abolished the recruitment of SMYD3 to DNA damage sites (Fig. 4F-G).These data demonstrates that SMYD3 is recruited to chromatin in response to DNA damage through PARP1.

SMYD3 promotes NHEJ repair via interaction with NHEJ proteins and modulation of LIG4/XRCC4/XLF dynamics at DNA damage sites
DNA double strand break is the most serious form of DNA damage, DSBs are mainly repaired by HR and NHEJ pathways.Previous studies have indicated that SMYD3 can direct interact with key members which are involved in HR pathway, however, whether SMYD3 functions in NHEJ pathway remains unknown.So, we introduced SMYD3 into EJ5-U2OS cells (37), a well-established cell line for detecting NHEJ repair efficiency, we found that the wild-type SMYD3 plasmid significantly increased the NHEJ repair efficiency, but not the enzymatic-dead F183A-SMYD3 (Fig. 5A-B).
Consistently, when we depleted SMYD3 with two independent siRNAs, the decreased repair efficiency of NHEJ was observed (Fig. 5C).These results indicated that SMYD3 and its methylation activity are responsible for NHEJ repair.To further explore the potential partner which were associated with SMYD3 mediated NHEJ repair, we first detected the key DNA repair proteins (DNA-PKcs, KU70, KU80, LIG4, XRCC4, XLF) in NHEJ pathway.However, the expression levels of all the above proteins remained unchanged upon the knockdown of SMYD3 (Fig. 5D).By using coimmunoprecipitation assay, we found that only the interaction between SMYD3 and LIG4/XRCC4/XLF complex was increased upon IR/VP16 treatment, but not the KU70/KU80/DNA-PKcs complex (Fig. 5E-F).Moreover, through laser micro irradiation assay, we observed an impaired recruitment of LIG4/XRCC4/XLF complex in SMYD3 depleted cells (Fig. 5G).We also screened which protein SMYD3 directly methylated and mediated the recruitment of LIG4/XRCC4/XLF complex.In the presence of SMYD3, the methylation level of LIG4 was dramatically increased, but no methylation signals were detected from XRCC4 or XLF proteins (Fig. 5H).These results demonstrate that SMYD3 directly methylates LIG4 and effects the interaction as well as recruitment of LIG4/XRCC4/XLF complex, which eventually leads to the efficient NHEJ repair.

Pharmacological intervention of SMYD3 enhances the response of EC cells to radiotherapy
BCI-121 is regarded as a specific and effective inhibitor of SMYD3 (38), to address whether targeting SMYD3 is a translational strategy to inhibit EC progression, we used BCI-121 for in vitro and in vivo assays.Firstly, we used EJ5-U2OS reporter cells for detecting the NHEJ repair efficiency upon BCI-121 treatment.The repair efficiency of NHEJ was significantly inhibited after BCI-121 in dose-dependent manner (Fig. 6A).
Western blotting assay showed that H3K4me2 expression level decreased upon increasing dose of BCI-121 in HEC1B cells as a positive control for detecting the effect of the inhibitor (Fig. 6B).As expected, the proliferation ability and viability of HEC1B cells were significantly inhibited upon BCI-121 treatment as examined by CCK-8 assays and colony formation (Fig. 6C-D).Trans-well assay also demonstrated that EC cells treated with BCI-121 had a much lower migration and invasion ability compared to control cells (Fig. 6E-F).As SMYD3 is closely associated with DNA repair, we next investigate the SMYD3's role in radiosensitivity.As shown in Fig. 6G, combining SMYD3 inhibitor BCI-121 with radiation induced a significant synergistic effect of inhibiting the survival of EC cells.Using our xenograft in vivo model, intra-tumoral injection of BCI-121 alone or radiation alone repressed the similar tumor growth of HEC1B cells compared to control group.However, the tumor size was significantly decreased in the BCI-121 and radiation combination therapy group (Fig. 6H-J).In agreement of the reduced tumor size, we also observed decreased Ki67 and enhanced cleaved-Caspase3 IHC staining in combination treatment mice tissue (Fig. 6K).These data are consistent with our cell-based findings and support that pharmacological intervention of SMYD3 as a radio-sensitizer that would be expected to result in an improved therapeutic outcome.Taking the above results altogether, our findings suggesting that SMYD3 may be a potential therapeutic target in endometrial cancer.

Discussion
In this study, we reveal an important oncogenic role of SMYD3 in the endometrial cancer progression.In addition, SMYD3 is a potential target for EC treatment through NHEJ repair pathway.Our data support that SMYD3 is significantly upregulated in EC patient samples and associated with cancer progression.Through pathway analysis, we present for the first time, a novel mechanism in which SMYD3 is involved in regulating NHEJ repair through LIG4 methylation and affects the recruitment of LIG4/XRCC4/XLF complex recruitment.More importantly, our results indicate that targeting SMYD3 with its specific inhibitor BCI-121 combine with radiation may be a potential therapeutic strategy for endometrial cancer treatment.
As a SET domain containing methyltransferase, SMYD3 controls various cellular functions through methylating different kinds of substrates (30,(39)(40)(41) and is upregulated in various cancers (42)(43)(44)(45)(46).For example, SMYD3 mediates the methylation of MAP3K2 kinase and activates the RAS/RAF/MEK/ERK signaling, which promotes the development of RAS-driven carcinomas (30).SMYD3 can also act as a major regulator of small cell lung cancer, enhancing alkylation-based chemotherapy sensitivity in SCLC through methylation of RNF113A (41).Moreover, SMYD3 upregulates sphingosine-1-phosphate receptor 1 (S1PR1) by methylating H3K4me3 associating with the progression of hepatocellular cancer (44).Von Hippel-Lindau/hypoxia-inducible factor α (VHL/HIF-α) axis promotes SMYD3 transcription and expression, and then SMYD3 cooperates with SP1 to promote EGFR activation as an oncogenic driver for renal cell carcinoma (47).Based on the previous studies, SMYD3 is a well-established oncogenic driver, and promotes tumorigenesis and development in several kinds of cancers.In our study, we first comprehensively investigate the role of SMYD3 in endometrial cancer, knockdown of SMYD3 attenuates cell proliferation, migration and invasion abilities in EC cells.Consistently with in vitro data, in vivo mouse models also demonstrate that SMYD3 knockdown reduces both tumor growth and metastasis properties.Moreover, in accordance with previous findings, we also provide evidence that the oncogenic properties of SMYD3 in endometrial cancer likely rely on its methyltransferase activity by using enzymatic inactive SMYD3-F183A plasmid.
Through pathway analysis, we find that SMYD3 closely associates with DNA repair pathway in endometrial cancer.In response to DNA damage, SMYD3 was previously reported to be phosphorylated by ATM kinase, promoting the formation of homologous recombination complexes and the recruitment of RAD51 at DSB sites.Pharmacological inhibition of SMYD3 sensitizes HR-proficient cancer cells to PARP inhibitors (36).
Besides this, the detailed role of SMYD3 in DNA damage repair remains unknown.In our study, we provide evidence that in response to DNA damage, SMYD3 is recruited to DNA damage sites in an PARP1-dependent manner.The interaction between SMYD3 and LIG4/XRCC4/XLF complex is greatly enhanced upon damage drug treatment, moreover, the recruitment of SMYD3 to damage sites leads to the specific methylation of LIG4, but not XRCC4 or XLF.The increased methylation of LIG4 eventually promotes the recruitment of LIG4/XRCC4/XLF complex and leads to efficient NHEJ repair.Our study fulfills the missing part of SMYD3 involving in NHEJ repair pathway, which is one of the most important pathways in DNA double strand breaks.It will be interesting to further explore the detailed relationship between SMYD3 and LIG4, including the methylation sites of LIG4 by SMYD3, and the following LIG4/XRCC4/XLF complex associations, which will expand our understanding of the regulatory network of SMYD3 in NHEJ repair.Moreover, elevated level of LIG4 confers radiation therapy resistance via increased NHEJ pathway (48), indicating that LIG4 inhibitors serve as radiosensitizers for radioresistant cancer treatment.Our study further explores the SMYD3 inhibitor BCI-121 as a radiosensitizer, which achieves better effect in endometrial cancer treatment.In the future, further combination in vivo experiments should be done to enhance the potential for clinical use.
In summary, we present a previous unidentified mechanism and role of SMYD3 in endometrial cancer, which provides a better understanding of SMYD3 in cancers.Our study also provides new insights into the dynamic regulation of LIG4/XRCC4/XLF complex in NHEJ pathway, which opens a new window to target SMYD3, as a key factor for NHEJ pathway.In addition, SMYD3 represents as a promising pharmacological target combined with radio/chemotherapy for anti-cancer therapy.Immunohistochemistry of SMYD3 expression in para-tumor and tumor of EC tissues at different pathologic category; scale bar, 60μm.D. The relative mRNA expression of SMYD3 in normal endometrial tissues and endometrial cancer tissues from the TCGA cohort using GEPIA 2 (http://gepia2.cancerpku.cn/).E. SMYD3 expression levels in different histological subtypes of EC with data from the TCGA database using UALCAN (http://ualcan.path.uab.edu/).F. SMYD3 alterations in patients with EC tissues in the TCGA database using cBioPortal (https://www.cbioportal.org/).G. Immunohistochemistry tissue microarrays showed SMYD3 expression level in paratumor tissue and EC G1-G3 tissues.n=18 for EC G1, n=25 for EC G2, n=14 for EC G3.Scale bar, 100μm for 10X; 30μm for 40X.H. Immunohistochemistry of SMYD3 expression in the uteri of 30-week-old Pten knock-out and Pten wild-type mice.Scale bar, 100μm for 10X; 30μm for 40X.

Figure legends
numbers: 82172975, 81972438, 32170602 and 81971338), Natural Science Foundation of Shanghai (grant number: 22ZR1449400) and Shanghai Science and Technology Development Funds Shanghai Rising-Star Program (grant number: 22QA1407400), Clinical Science and Technology Innovation Project of Shanghai Shenkang Hospital Development Center (grant number: SHDC12020107, SHDC2020CR5003), Special Health Industry Project of Pudong New Area Health Commission (grant number: PW2021D-06), Shanghai First Maternity and Infant Hospital Talent Launch Program (grant number: 2022RC05).

Fig. 1 SMYD3
Fig.1 SMYD3 is upregulated in endometrial cancer and associated with tumor

Fig. 2 SMYD3
Fig.2 SMYD3 promotes the abilities of cell proliferation, migration, invasion and

Fig. 3
Fig.3 Knockdown of SMYD3 impairs tumorigenicity and metastasis of EC in vivo.

Fig. 4
Fig.4 SMYD3 is recruited to DNA damage sites in an PARP1-dependent manner.

Fig. 6
Fig.6 Pharmacological intervention of SMYD3 enhances the response of EC cells