雌激素通过PI3K-AKT通路上调子宫内膜癌中DNA2的表达

Estrogen upregulates DNA2 expression through the PI3K-AKT pathway in endometrial carcinoma 雌激素通过PI3K-AKT通路上调子宫内膜癌中DNA2的表达 1 equal first-author 2 equal 1 2 3 3 3 4 5 4 5 corresp equal Ruolang PAN, panrl@zju.edu.cn Ruolang PAN, panrl@zju.edu.cn panrl@zju.edu.cn https://orcid.org/0000-0002-0810-3392 3 corresp equal Yan YU, m05yuyan1@zju.edu.cn Yan YU, m05yuyan1@zju.edu.cn m05yuyan1@zju.edu.cn https://orcid.org/0000-0003-0723-5876 浙江省中医院， 浙江中医药大学第一附属医院妇科‍， 中国杭州‍市， 310006 浙江省中医院， 浙江中医药大学第一附属医院妇科‍， 中国杭州‍市， 310006 Department of Gynecology, Zhejiang Provincial Hospital of Traditional Chinese Medicine, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, China Department of Gynecology, Zhejiang Provincial Hospital of Traditional Chinese Medicine, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, China 浙江中医药大学附属杭州市中医院， 杭州市中医院妇产科‍， 中国杭州市‍， 310005 浙江中医药大学附属杭州市中医院， 杭州市中医院妇产科‍， 中国杭州市‍， 310005 Department of Gynecology and Obstetrics, Hangzhou Hospital of Traditional Chinese Medicine, Affiliated Hangzhou TCM Hospital of Zhejiang Chinese Medical University, Hangzhou 310005, China Department of Gynecology and Obstetrics, Hangzhou Hospital of Traditional Chinese Medicine, Affiliated Hangzhou TCM Hospital of Zhejiang Chinese Medical University, Hangzhou 310005, China 浙江省人民医院，杭州医学院附属人民医院妇科‍， 中国杭州‍市， 310014 浙江省人民医院，杭州医学院附属人民医院妇科‍， 中国杭州‍市， 310014 Department of Gynecology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital of Hangzhou Medical College, Hangzhou 310014, China Department of Gynecology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital of Hangzhou Medical College, Hangzhou 310014, China 浙江省细胞药物与应用技术研究重点实验室‍， 中国杭州‍市， 311122 浙江省细胞药物与应用技术研究重点实验室‍， 中国杭州‍市， 311122 Key Laboratory of Cell-Based Drug and Applied Technology Development in Zhejiang Province, Hangzhou 311122, China Key Laboratory of Cell-Based Drug and Applied Technology Development in Zhejiang Province, Hangzhou 311122, China 浙江省易文赛细胞药物和制品研究院， 中国杭州市， 311122 浙江省易文赛细胞药物和制品研究院， 中国杭州市， 311122 Institute for Cell-Based Drug Development of Zhejiang Province, S-Evans Biosciences, Hangzhou 311122, China Institute for Cell-Based Drug Development of Zhejiang Province, S-Evans Biosciences, Hangzhou 311122, China 子宫内膜癌是最常见的子宫癌类型，占子宫癌病例的90%。本文通过临床研究，检测子宫内膜癌患者标本中雌激素受体（ER）和DNA复制ATP依赖性解旋酶/核酸酶（DNA2）的表达，并分析两者之间的关系。通过细胞培养实验研究ER调控DNA2表达的机制，证实其与PI3K-AKT通路有关。研究还发现，使用短发夹RNA（shRNA）特异性靶向降低Ishikawa中DNA2的表达，会导致细胞增殖和克隆形成能力降低。总的来说，本研究证实了DNA2作为治疗靶点的可行性，并证明抑制DNA2可使Ishikawa对喜树碱（CPT）化疗增敏。我们的发现为DNA2的潜在机制提供了新的见解，这将有助于开发与子宫内膜癌诊断和治疗相关的新方法。 子宫内膜癌 雌激素受体 DNA2 PI3K-AKT通路 Endometrial cancer is the most common gynecological malignancy, affecting up to 3% of women at some point during their lifetime ( Morice et al., 2016 Morice et al., 2016 ; Li and Wang, 2021 Li and Wang, 2021 ). Based on the pathogenesis and biological behavioral characteristics, endometrial cancer can be divided into estrogen-dependent (I) and non-estrogen-dependent (II) types ( Ulrich, 2011 Ulrich, 2011 ). Type I accounts for approximately 80% of cases, of which the majority are endometrioid carcinomas, and the remaining are mucinous adenocarcinomas ( Setiawan et al., 2013 Setiawan et al., 2013 ). It is generally recognized that long-term stimulation by high estrogen levels with the lack of progesterone antagonism is the most important risk factor; meanwhile, there is no definite conclusion on the specific pathogenesis. The incidence of endometrial cancer has been on the rise during the past two decades ( Constantine et al., 2019 Constantine et al., 2019 ; Gao et al., 2022 Gao et al., 2022 ; Luo et al., 2022 Luo et al., 2022 ). Moreover, the development of assisted reproductive technology and antiprogestin therapy following breast cancer surgery has elevated the risk of developing type I endometrial cancer to a certain extent ( Vassard et al., 2019 Vassard et al., 2019 ). Therefore, investigating the influence of estrogen in type I endometrial cancer may provide novel concepts for risk assessment and adjuvant therapy, and at the same time, provide a basis for research on new drugs to treat endometrial cancer. DNA replication adenosine triphosphate (ATP)-dependent helicase/nuclease (DNA2) is an evolutionarily conserved, essential enzyme that plays critical roles in multiple DNA metabolic pathways, including Okazaki fragment maturation, DNA replication, end resection, and homologous recombination repair ( Thakar et al., 2020 Thakar et al., 2020 ). Moreover, it participates in mitochondrial genome maintenance. Thus, the uncontrolled regulation of DNA2 may lead to a cascading effect on genomic instability and telomere imbalance, resulting in DNA damage in particular cell phases. Indeed, researchers have proved that the abnormal activity of DNA2 nuclease is correlated with the growth of various cancer cells, such as in breast and ovarian cancers ( Jia et al., 2017 Jia et al., 2017 ; Han et al., 2021 Han et al., 2021 ). DNA2 is highly expressed in estrogen receptor (ER)-rich cells like those in breast cancer and ovarian cancer. Under an increased DNA2 expression level, the malignancy of tumors and incidence of metastasis will be elevated, indicating that DNA2 may be regulated by the ER pathway. However, the relevant mechanism remains unclear. Furthermore, DNA2 has recently been described as a potential marker and an attractive target in cancer therapy. Therefore, our study investigated the expression of ER and DNA2 in samples from patients with endometrial cancer and analyzed the relationship between them. A total of 32 patients with a mean age of (44.3±4.1) years were selected from the Zhejiang People’s Hospital, Hangzhou, China. They were diagnosed based on pathological examination and received surgical resection, including 16 ER-positive cases, whereas the others were ER-negative. Another 16 normal tissue samples were collected from control subjects with a mean age of (42.1±4.4) years who underwent laparoscopic surgery after informed consent. All patients, including control subjects, were free from hormonal therapy for three months and presented no serious liver or kidney disease. This study has been carried out in accordance with the Code of Ethics of the World Medical Association. The human endometrial adenocarcinoma cell lines JEC, Ishikawa, and HEC1 were obtained from the American Type Culture Collection. As mentioned above, a total of 32 patients and 16 control subjects were enrolled in this study. We observed that the DNA2 expression was significantly higher in the patient group ((1.671±0.661)‍-fold increase, P < 0.05; Fig. 1 Fig. 1 ). To further explore the correlation between ER and DNA2, we divided the patients into two groups based on ER expression: ER-negative and ER-positive. The expression of ER in the endometrial cancer tissue samples was detected by immunohistochemical analysis. A nuclear staining of > 10% was considered to indicate positive ER status. Interestingly, we observed that DNA2 expression in the ER-negative patient group was significantly lower than that in the ER-positive patient group. In addition, there was no significant difference in DNA2 expression between the ER-negative patient group and the control group. In contrast, the ER-positive patient group exhibited a (2.084±0.575)‍-fold increase in DNA2 messenger RNA (mRNA) expression compared to the control group. These results indicated that ER positivity correlates with DNA2 expression in patients with endometrial cancer. Next, we investigated the expression of DNA2 between the two types of endometrial cancer cell lines. As depicted in Figs. 2 2 a and 2 2 b, the Ishikawa cell line positively responded to β‍-estradiol (E2) treatment. After E2 administration, both the mRNA and the protein levels of DNA2 expression were significantly upregulated. The JEC cell line, which is characterized by negative ER expression, was used as control. Remarkably, the DNA2 expression levels were relatively stable in the JEC cells before and after E2 treatment, with no significant differences. Subsequently, we investigated whether treatment with raloxifene, an ER modulator, influences the DNA2 expression in endometrial cancer cells. As shown in Fig. 2 Fig. 2 c, raloxifene treatment led to a significantly reduced expression of DNA2 mRNA levels in Ishikawa cells, which exhibited high levels of ER in a concentration-dependent manner. A similar trend in the effect of raloxifene on DNA2 mRNA levels was observed in an ER medium cell line, HEC1. However, the reduction rates were less than those in Ishikawa cells. Notably, DNA2 mRNA levels in JEC cells that were negative for ER expression were not affected. Then, total proteins were extracted from cells treated with 1×10 -7 mol/L raloxifene for 48 h. The results of western blotting assays were consistent with the changes in mRNA levels (Fig. S1). Cell counting kit-8 (CCK8) assays revealed that raloxifene resulted in approximately 24% and 29% reduction in the proliferation of Ishikawa cells within 48 and 72 h, respectively, compared to those in control cells. A similar trend was found in HEC1 cells, with approximately 13% and 19% reduction within 48 and 72 h, respectively. No significant changes were found in JEC cells ( Fig. 2 Fig. 2 d). We also confirmed that treatment with tamoxifen, another ER modulator, could also downregulate DNA2 expression in Ishikawa cells, and resulted in approximately 47% reduction in the proliferation of Ishikawa cells within 72 h compared to that in control cells (Fig. S2). The phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway has been implicated in endocrine resistance. Studies have proved that the upregulation of PI3K/AKT pathway plays a critical role in endocrine treatment resistance through extranuclear ER signaling ( Hasson et al., 2013 Hasson et al., 2013 ; Bean et al., 2015 Bean et al., 2015 ; Duan et al., 2021 Duan et al., 2021 ). In the present study, the cultured Ishikawa cells were divided into four groups and treated with or without E2, GDC-0810 (an ER inhibitor), and Ly294002 (a PI3K-AKT inhibitor), separately. As shown in Fig. 3 Fig. 3 , E2 treatment significantly activated the PI3K-AKT signaling pathway. The expression levels of phosphor-AKT (pAKT/Ser473) and PI3K were upregulated by approximately 3.0- and 2.6-fold, respectively. GDC-0810, one of the most commonly used selective ER modulators, can directly antagonize ER in cells. Consequently, PI3K-AKT signaling was not activated after E2 treatment in these Ishikawa cells pretreated with GDC-0810. Moreover, we observed that the DNA2 expression levels were slightly inhibited. We next examined whether the PI3K pathway inhibitor could abolish the effect of E2 treatment on DNA2 expression in Ishikawa cells. Ly294002 was able to inhibit both PI3K/AKT activity and AKT phosphorylation in these cells, and a reduced level of DNA2 expression was noticed. Altogether, these findings suggested that estrogen regulates DNA2 expression through the PI3K-AKT pathway in endometrial cancer, which can be blocked using either ER inhibitors or PI3K-AKT inhibitors. In order to further explore the role of DNA2 in the proliferation of endometrial cells, we established a DNA2-knockdown Ishikawa cell line with specific DNA2 short hairpin RNA (shRNA) ( Fig. 4 Fig. 4 ). The knocked down level of the DNA2 protein was approximately 70%, as determined by western blotting assay (Figs. 4 4 a and 4 4 b). Consequently, a significant induction of DNA damage, indicated by increase in the DNA damage marker γ histone family member X (γH2AX), was observed in DNA2 shRNA cells (Figs. 4 4 c and 4 4 d). Subsequently, cell growth was evaluated using the CCK8 and colony-forming efficiency (CFE) assays. As anticipated, the proliferation and colony-forming ability declined when cells were transfected with DNA2 shRNA (Figs. 4 4 e and 4 4 f). Next, we assessed whether decreased DNA2 expression had any functional consequences for cellular chemoresistance. As depicted in Fig. 5 Fig. 5 a, the knockdown of DNA2 increased the sensitivity to camptothecin (CPT) in Ishikawa cells (3 nmol/L, (35.8±8.1)% vs. (9.6±2.1)% Annexin V + ; P < 0.01). Similarly, a very strong synergy was detected when we combined the treatment with CPT and C5, which is a specific inhibitor of DNA2 activity ( Fig. 5 Fig. 5 b). DNA replication failure and DNA damage response are the two major factors leading to genome instability. DNA2 is a conserved protein involved in the maintenance of mitochondrial and nuclear DNA stability, and it possesses both endonuclease/exonuclease and helicase activity ( Zheng et al., 2008 Zheng et al., 2008 ). It can play a vital role in DNA replication and repair at the same time, thereby maintaining genome stability. It has been confirmed that DNA2 is expressed at high levels in breast cancer, ovarian cancer, and other cells that are rich in ERs, and the increase in DNA2 expression correlates with the malignancy and metastasis ability of tumors ( Strauss et al., 2014 Strauss et al., 2014 ). In the present study, we selected the ER-negative JEC endometrial cancer cell line and the ER-positive Ishikawa endometrial cancer cell line, stimulated them with E2, and detected the expression of DNA2. We observed that the expression of DNA2 in ER-positive cells was significantly increased, indicating that E2 binds to ER in endometrial cancer cells and can regulate DNA2 transcription. Reducing the expression of DNA2 in endometrial cancer can slow down cell proliferation and reduce its CFE activity, which was further confirmed by the in vivo tumor growth assay (Fig. S3). Meanwhile, we observed a positive correlation between DNA2 expression levels and chemoresistance in vivo (Fig. S4). Therefore, we proposed that DNA2 plays a vital role in the estrogen-stimulation-induced DNA damage of endometrial cells, which is linked to the development of cancer. Overall, the present study confirmed the feasibility of DNA2 as a therapeutic target, and demonstrated that inhibiting DNA2 sensitizes Ishikawa cells to chemotherapy using CPT. Our findings may provide novel insights into the potential mechanisms of DNA2, which assists in developing novel approaches pertaining to the diagnosis and treatment of endometrial cancer. All data generated or analyzed during this study are included in this published article and its supplementary information files. References Bean JR , Hosford SR , Symonds LK , et al. , 2015 . The PI3K/mTOR dual inhibitor P7170 demonstrates potent activity against endocrine-sensitive and endocrine-resistant ER+ breast cancer . Breast Cancer Res Treat , 149 ( 1 ): 69 - 79 . https://doi.org/10.1007/s10549-014-3201-6 https://doi.org/10.1007/s10549-014-3201-6 The PI3K/mTOR dual inhibitor P7170 demonstrates potent activity against endocrine-sensitive and endocrine-resistant ER+ breast cancer Constantine GD , Kessler G , Graham S , et al. , 2019 . Increased incidence of endometrial cancer following the women’s health initiative: an assessment of risk factors . J Womens Health (Larchmt) , 28 ( 2 ): 237 - 243 . https://doi.org/10.1089/jwh.2018.6956 https://doi.org/10.1089/jwh.2018.6956 Increased incidence of endometrial cancer following the women’s health initiative: an assessment of risk factors Duan L , Calhoun S , Shim D , et al. , 2021 . Fatty acid oxidation and autophagy promote endoxifen resistance and counter the effect of AKT inhibition in ER-positive breast cancer cells . J Mol Cell Biol , 13 ( 6 ): 433 - 444 . https://doi.org/10.1093/jmcb/mjab018 https://doi.org/10.1093/jmcb/mjab018 Fatty acid oxidation and autophagy promote endoxifen resistance and counter the effect of AKT inhibition in ER-positive breast cancer cells Gao CX , Shi JM , Zhang JX , et al. , 2022 . Chemerin promotes proliferation and migration of ovarian cancer cells by upregulating expression of PD-L1 . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 23 ( 2 ): 164 - 170 . https://doi.org/10.1631/jzus.B2100392 https://doi.org/10.1631/jzus.B2100392 Chemerin promotes proliferation and migration of ovarian cancer cells by upregulating expression of PD-L1 Han YY , Zhang ZY , Wang Z , et al. , 2021 . Increased expression of DNA2 was linked to poor prognosis in breast cancer . Dis Markers , 2021 : 8860728 . https://doi.org/10.1155/2021/8860728 https://doi.org/10.1155/2021/8860728 Increased expression of DNA2 was linked to poor prognosis in breast cancer Hasson SP , Rubinek T , Ryvo L , et al. , 2013 . Endocrine resist Endocrine resist ance in breast cancer: focus on the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway . Breast Care (Basel) , 8 ( 4 ): 248 - 255 . https://doi.org/10.1159/000354757 https://doi.org/10.1159/000354757 ance in breast cancer: focus on the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway Jia PP , Junaid M , Ma YB , et al. , 2017 . Role of human DNA2 (hDNA2) as a potential target for cancer and other diseases: a systematic review . DNA Repair (Amst) , 59 : 9 - 19 . https://doi.org/10.1016/j.dnarep.2017.09.00 https://doi.org/10.1016/j.dnarep.2017.09.00 Role of human DNA2 (hDNA2) as a potential target for cancer and other diseases: a systematic review Li CH , Wang CH , 2021 . LG-ESSs and HG-ESSs: underlying molecular alterations and potential therapeutic strategies . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 8 ):‍ 633 - 646 . https://doi.org/10.1631/jzus.B2000797 https://doi.org/10.1631/jzus.B2000797 LG-ESSs and HG-ESSs: underlying molecular alterations and potential therapeutic strategies Luo J , Song ZM , Zhang T , et al. , 2022 . Upregulation of h-TERT and Ki-67 in ectopic endometrium is associated with recurrence of endometriosis . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 23 ( 2 ): 158 - 163 . https://doi.org/10.1631/jzus.B2100502 https://doi.org/10.1631/jzus.B2100502 Upregulation of h-TERT and Ki-67 in ectopic endometrium is associated with recurrence of endometriosis Morice P , Leary A , Creutzberg C , et al. , 2016 . Endometrial cancer . Lancet , 387 ( 10023 ): 1094 - 1108 . https://doi.org/10.1016/s0140-6736(15)00130-0 https://doi.org/10.1016/s0140-6736(15)00130-0 Endometrial cancer Setiawan VW , Yang HP , Pike MC , et al. , 2013 . Type I and II endometrial cancers: have they different risk factors? J Clin Oncol , 31 ( 20 ): 2607 - 2618 . https://doi.org/10.1200/JCO.2012.48.2596 https://doi.org/10.1200/JCO.2012.48.2596 Type I and II endometrial cancers: have they different risk factors? Strauss C , Kornowski M , Benvenisty A , et al. , 2014 . The DNA2 nuclease/helicase is an estrogen-dependent gene mutated in breast and ovarian cancers . Oncotarget , 5 ( 19 ): 9396 - 9409 . https://doi.org/10.18632/oncotarget.2414 https://doi.org/10.18632/oncotarget.2414 The DNA2 nuclease/helicase is an estrogen-dependent gene mutated in breast and ovarian cancers Thakar T , Leung W , Nicolae CM , et al. , 2020 . Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly . Nat Commun , 11 : 2147 . https://doi.org/10.1038/s41467-020-16096-w https://doi.org/10.1038/s41467-020-16096-w Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly Ulrich LSG , 2011 . Endometrial cancer, types, prognosis, female hormones and antihormones . Climacteric , 14 ( 4 ): 418 - 425 . https://doi.org/10.3109/13697137.2010.550974 https://doi.org/10.3109/13697137.2010.550974 Endometrial cancer, types, prognosis, female hormones and antihormones Vassard D , Schmidt L , Glazer CH , et al. , 2019 . Assisted reproductive technology treatment and risk of ovarian cancer—a nationwide population-based cohort study . Hum Reprod , 34 ( 11 ): 2290 - 2296 . https://doi.org/10.1093/humrep/dez165 https://doi.org/10.1093/humrep/dez165 Assisted reproductive technology treatment and risk of ovarian cancer—a nationwide population-based cohort study Zheng L , Zhou M , Guo ZG , et al. , 2008 . Human DNA2 is a mitochondrial nuclease/helicase for efficient processing of DNA replication and repair intermediates . Mol Cell , 32 ( 3 ): 325 - 336 . https://doi.org/10.1016/j.molcel.2008.09.024 https://doi.org/10.1016/j.molcel.2008.09.024 Human DNA2 is a mitochondrial nuclease/helicase for efficient processing of DNA replication and repair intermediates All authors contributed to the study conception and design. Xinyan WANG and Xiuling XU designed the experiment. Ting ZHANG and Yang JIN performed the study. Sheng XU and Lifeng CHEN drafted the manuscript. Yucheng LAI and Ling ZHANG analyzed data. Ruolang PAN and Yan YU designed the experiment, revised the manuscript, and monitored the project progression. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the General Projects of Medical and Health Science and Technology Plan in Zhejiang Province (No. 2020KY433), China. 10.1631/jzus.B2200436 1673-1581（2023）03-0262-07 王鑫炎,徐秀玲,张婷等.雌激素通过PI3K-AKT通路上调子宫内膜癌中DNA2的表达[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(03):262-268. 王鑫炎,徐秀玲,张婷等.雌激素通过PI3K-AKT通路上调子宫内膜癌中DNA2的表达[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(03):262-268. This work was supported by the General Projects of Medical and Health Science and Technology Plan in Zhejiang Province (No. 2020KY433), China. 2022-08-29 2022-12-21 2023-03-15 2023-03-14 2023-02-10 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 Xinyan WANG, Xiuling XU, Ting ZHANG, Yang JIN, Sheng XU, Lifeng CHEN, Yucheng LAI, Ling ZHANG, Ruolang PAN, and Yan YU declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for whom identifying information is included in this article. This study was granted by the Ethics Committee of Zhejiang Provincial People's Hospital (November 9, 2019; No. 2019KY204). Compliance with ethics guidelines statement Data availability Supplementary information 1 Figs. S1‍–‍S4 浙江大学学报（英文版）（B辑：生物医学和生物技术） 3 24