Liver-specific DICER1 syndrome model mice develop cystic liver tumors with defective primary cilia.

DICER1 syndrome is a tumor predisposition syndrome caused by familial genetic mutations in DICER1. Pathogenic variants of DICER1 have been discovered in many rare cancers, including cystic liver tumors. However, the molecular mechanisms underlying liver lesions induced by these variants remain unclear. In the present study, we sought to gain a better understanding of the pathogenesis of these variants by generating a mouse model of liver-specific DICER1 syndrome. The mouse model developed bile duct hyperplasia with fibrosis, similar to congenital hepatic fibrosis, as well as cystic liver tumors resembling those in Caroli's syndrome, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma. Interestingly, the mouse model of DICER1 syndrome showed abnormal formation of primary cilia in the bile duct epithelium, which is a known cause of bile duct hyperplasia and cyst formation. These results indicated that DICER1 mutations contribute to cystic liver tumors by inducing defective primary cilia. The mouse model generated in this study will be useful for elucidating the potential mechanisms of tumorigenesis induced by DICER1 variants and for obtaining a comprehensive understanding of DICER1 syndrome. © 2024 The Pathological Society of Great Britain and Ireland.

Comprehensive microRNA analyses using vitreous humor of ocular sarcoidosis.

PURPOSE: MicroRNAs (miRNAs) are non-coding RNAs which have attracted attention as biomarkers in a variety of diseases. However, extensive unbiased analysis of miRNA in vitreous humor of sarcoidosis patients has not been reported. In the present study, we comprehensively analyzed the dysregulated miRNAs in ocular sarcoidosis to search for potential biomarkers. MATERIALS AND METHODS: This study included seven patients diagnosed with ocular sarcoidosis (five definite and two presumed). Five patients with unclassified uveitis and 24 with non-inflammatory diseases served as controls. MicroRNA expression levels in vitreous humor samples were measured by microarray, and differentially expressed miRNAs between sarcoidosis and other diseases were explored. Next, pathway enrichment analysis was performed to evaluate the functions of the dysregulated miRNAs, and machine learning was used to search for candidate biomarkers. RESULTS: A total of 614 upregulated miRNAs and 8 downregulated miRNAs were detected in vitreous humor of patients with ocular sarcoidosis compared with patients with unclassified uveitis and non-inflammatory diseases. Some dysregulated miRNAs were involved in the TGF-ß signaling pathway. Furthermore, we identified miR-764 as the best predictor for ocular sarcoidosis using Boruta selection. CONCLUSIONS: In this study, comprehensive miRNA analysis of vitreous humor samples identified dysregulated miRNAs in ocular sarcoidosis. This study suggests new insights into molecular pathogenetic mechanisms of sarcoidosis and may provide useful information toward developing novel diagnostic biomarkers and therapeutic targets for sarcoidosis.

miR-27a ameliorates chemoresistance of breast cancer cells by disruption of reactive oxygen species homeostasis and impairment of autophagy.

In patients with breast cancer, primary chemotherapy often fails due to survival of chemoresistant breast cancer stem cells (BCSCs) which results in recurrence and metastasis of the tumor. However, the factors determining the chemoresistance of BCSCs have remained to be investigated. Here, we profiled a series of differentially expressed microRNAs (miRNAs) between parental adherent breast cancer cells and BCSC-mimicking mammosphere-derived cancer cells, and identified hsa-miR-27a as a negative regulator for survival and chemoresistance of BCSCs. In the mammosphere, we found that the expression of hsa-miR-27a was downregulated, and ectopic overexpression of hsa-miR-27a reduced both number and size of mammospheres. In addition, overexpression of hsa-miR-27a sensitized breast cancer cells to anticancer drugs by downregulation of genes essential for detoxification of reactive oxygen species (ROS) and impairment of autophagy. Therefore, enhancing the hsa-miR-27a signaling pathway can be a potential therapeutic modality for breast cancer.

Fascin as a Useful Marker for Identifying Neural Components in Immature Teratomas of Human Ovary and Those Derived From Murine Embryonic Stem Cells.

Immature teratoma of the human ovary is a rare disease, and its diagnosis and grading are currently based on histologic evaluation of the presence and amount of immature neural components in the tumor. Despite the importance of tumor grading, immature neural components especially without rosette formation are difficult to identify, partly because useful biomarkers for them are not yet available. Toward this goal, we investigated 16 immature teratomas from human ovaries as well as 10 of those derived from murine embryonic stem cells transplanted into immunodeficient mice. Immunohistochemistry was performed for cytokeratin, glial fibrillary acidic protein, S100, and fascin. It was demonstrated that glial fibrillary acidic protein and S100 expression was not observed in the immature neural components of immature teratomas derived from both human ovary and embryonic stem cells, although their expression was detected in mature neural tissues. In contrast, fascin immunopositivity was clearly found in both mature and immature neural components regardless of rosette formation in immature teratomas derived from both human ovary and embryonic stem cells. Assessment of immature neural components by fascin immunostaining yielded the same or slightly increased quantity than quantification based on hematoxylin and eosin staining. These results suggest that fascin immunostaining is useful as a biomarker in correctly diagnosing and grading human immature teratomas. Further, fascin immunostaining may contribute to the development of regenerative medicine through accurate assessment of the maturation status of pluripotent stem cell-derived tumors transplanted into immunodeficient mice.

Toll-like receptor 2 activation implicated in oral squamous cell carcinoma development.

Recent studies have revealed that Toll-like receptors (TLRs) are highly expressed and activated in many types of cancer. Physiologically, TLR2 recognizes bacteria and other microorganisms in the oral cavity; however, the role of TLR2 in oral squamous cell carcinoma (OSCC) is unclear. In this study, we demonstrated that TLR2 is highly expressed in OSCC in comparison with adjacent non-malignant tissue. TLR2 was also expressed in OSCC-derived cell lines, and its expression was activated by ligands derived from bacteria and mycoplasma. Furthermore, to elucidate the mechanism of OSCC progression via TLR2 signal transduction, we focused on microRNAs (miRNAs) that are induced by TLR2 activation. Interestingly, ligand activation of TLR2 induced the expression of miR-146a and we found that downregulation of caspase recruitment domain-containing protein 10 (CARD10) mRNA in OSCC-derived cell lines. Moreover, knockdown of CARD10 induced resistance to cisplatin-induced apoptosis in OSCC cells. These findings suggest that the activation of TLR2 by bacterial components can enhance the progression of OSCC and may be implicated in acquired resistance to cisplatin-induced apoptosis through regulation of the miR-146a pathway.

PRG4 expression in myxoid liposarcoma maintains tumor cell growth through suppression of an antitumor cytokine IL-24.

PRG4 is one of the downstream molecules of the myxoid liposarcoma (MLS)-specific fusion oncoproteins TLS-CHOP and EWS-CHOP. Exogenous PRG4 expression increases the tumorigenicity of cells injected in nude mice. The molecular functions of PRG4 in tumorigenesis and/or tumor progression of MLS cells, however, still remain unclear. In this report, we demonstrated that siRNA-mediated knockdown of PRG4 suppressed the growth of the MLS-derived cell lines 1955/91 and 2645/94. In addition, PRG4 knockdown promoted adipocytic differentiation in 1955/91 cells. Thus, PRG4 may play essential roles in MLS cell growth and have potential as a therapeutic target. On the other hand, our previous study has revealed that TLS-CHOP suppresses expression of an anti-tumor cytokine IL-24, contributing to tumor cell survival. In this study, we found that double knockdown of PRG4 and IL-24 did not inhibit MLS cell growth, and single knockdown of PRG4 remarkably increased IL-24 expression. These results suggest that the growth inhibitory effect of PRG4 knockdown is caused by induction of IL-24 expression, and PRG4 may contribute to maintain MLS cell growth through repression of IL-24 expression.

Development of Novel Small Hairpin RNAs That do not Require Processing by Dicer or AGO2.

The innate cytokine response to nucleic acid is the most challenging problem confronting the practical use of nucleic acid medicine. The degree of stimulation of the innate cytokine response strongly depends on the length of the nucleic acid. In this study, we developed a 30-nucleotide single-strand RNA, termed "guide hairpin RNA (ghRNA, ghR)", that has a physiological function similar to that of miRNA and siRNA. The ghR caused no innate cytokine response either in vitro or in vivo. In addition, its structure does not contain a passenger strand seed sequence, reducing the unwanted gene repression relative to existing short RNA reagents. Systemic and local injection of ghR-form miR-34a (ghR-34a) suppressed tumor growth in a mouse model of RAS-induced lung cancer. Furthermore, Dicer and AGO2 are not required for ghR-34a function. This novel RNA interference (RNAi) technology may provide a novel, safe, and effective nucleic acid drug platform that will increase the clinical usefulness of nucleic acid therapy.

Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells.

Despite the therapeutic potential of nucleic acid drugs, their clinical application has been limited in part by a lack of appropriate delivery systems. Exosomes or microvesicles are small endosomally derived vesicles that are secreted by a variety of cell types and tissues. Here, we show that exosomes can efficiently deliver microRNA (miRNA) to epidermal growth factor receptor (EGFR)-expressing breast cancer cells. Targeting was achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide. Intravenously injected exosomes delivered let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2(-/-) mice. Our results suggest that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.

Efficacy of interstitial photodynamic therapy using talaporfin sodium and a semiconductor laser for a mouse allograft glioma model.

To investigate the therapeutic potential of photodynamic therapy (PDT) for malignant gliomas arising in unresectable sites, we investigated the effect of tumor tissue damage by interstitial PDT (i-PDT) using talaporfin sodium (TPS) in a mouse glioma model in which C6 glioma cells were implanted subcutaneously. A kinetic study of TPS demonstrated that a dose of 10 mg/kg and 90 min after administration was appropriate dose and timing for i-PDT. Performing i-PDT using a small-diameter plastic optical fiber demonstrated that an irradiation energy density of 100 J/cm2 or higher was required to achieve therapeutic effects over the entire tumor tissue. The tissue damage induced apoptosis in the area close to the light source, whereas vascular effects, such as fibrin thrombus formation occurred in the area slightly distant from the light source. Furthermore, when irradiating at the same energy density, irradiation at a lower power density for a longer period of time was more effective than irradiation at a higher power density for a shorter time. When performing i-PDT, it is important to consider the rate of delivery of the irradiation light into the tumor tissue and to set irradiation conditions that achieve an optimal balance between cytotoxic and vascular effects.

Smooth muscle differentiation of coronary intima in autopsy tissues after sirolimus-eluting stent implantation.

BACKGROUND: In coronary atherosclerotic disease, the proliferation of intimal smooth muscle cells (SMCs) is regarded as beneficial with respect to stable and unstable plaques, but is thought detrimental in discussions on coronary stent restenosis. To resolve this discrepancy, we focused on the quality, not quantity, of intimal SMCs in coronary atherosclerotic disease. METHODS: Autopsied coronary artery specimens from seven patients implanted with bare metal stents (BMS), three with paclitaxel-eluting stents (PES), and 10 with sirolimus (rapamycin)-eluting stents (SES) were immunostained for SMC markers. Cultured human coronary artery SMCs were also treated with sirolimus and paclitaxel. RESULTS: Intimal SMC differentiation, estimated by the ratio of h-caldesmon+ cells to α-smooth muscle actin+ (α-SMA+) cells, was significantly increased whereas dedifferentiation, estimated from the ratio of fibroblast activation protein alpha (FAPα)+ cells to α-SMA+ cells, was significantly decreased, in tissues of SES compared with BMS cases. No difference in the degree of differentiation was found between PES and BMS cases or between the three groups in nonstented arteries used as controls. Correlation analyses for each field of view revealed a significant positive correlation between h-caldesmon and calponin staining but significant negative correlations with FAPα staining in α-SMA+ cells. Cultured SMCs were shorter (dedifferentiated) and showed an increased FAPα/α-SMA protein when treated with paclitaxel, whereas they became elongated (differentiated) and showed increased calponin/α-SMA proteins with sirolimus. CONCLUSIONS: The SMCs of the coronary intima may differentiate after SES implantation. SMC differentiation may explain both the plaque stabilization and reduced risk of reintervention associated with SES.

Identification of a novel role of Septin 10 in paclitaxel-resistance in cancers through a functional genomics screen.

Paclitaxel (also known as taxol) is a member of the taxane class of anticancer agents, which has a well-known mechanism that blocks cell mitosis and kills tumor cells, that is often used in clinics to treat cancer. However, some carcinomas such as breast, ovarian and non-small-cell lung cancers are often resistant to paclitaxel treatment. In this study, we used a lentiviral siRNA library against the entire human genomes to identify genes associated with sensitivity to paclitaxel. We isolated two paclitaxel-resistant clones carrying the siRNA specific to septin 10 (SEPT10) and to budding uninhibited by benzimidazoles 3. The relation of budding uninhibited by benzimidazoles 3 to paclitaxel sensitivity has already been established, but that of SEPT10 remains unknown. Interestingly, overexpression of SEPT10 increased cells' sensitivity to paclitaxel; we also found that SEPT10 is an important regulator for microtubule stability. Furthermore, we found that paclitaxel-resistant tumors had decreased expression of SEPT10. Thus, SEPT10 may be a novel candidate molecule that acts as a good indicator of paclitaxel-resistant carcinomas.

Isolation of miRNAs that target EGFR mRNA in human lung cancer.

Lung cancer, predominantly non-small cell lung cancer (NSCLC), remains the leading cause of cancer-related deaths worldwide. Although epidermal growth factor receptor (EGFR) signaling is important and well studied with respect to NSCLC progression, little is known about how miRNAs mediate EGFR signaling to modulate tumorigenesis. To identify miRNAs that target EGFR, we performed a bioinformatics analysis and found that miR-542-5p down-regulates EGFR mRNA and protein expression in human lung cancer cells (H3255, A549, Hcc827). We observed increases in EGFR association with Ago2 in miR-542-5p-transfected cells. Interestingly, we observed an inverse correlation of miR-542-5p expression and EGFR protein levels in human lung cancer tissue samples, suggesting that miR-542-5p directly targets EGFR mRNA. Furthermore, we found that miR-542-5p inhibited the growth of human lung cancer cells. Our findings suggest that miR-542-5p may act as an important modulator of EGFR-mediated oncogenesis, with potential applications as a novel therapeutic target in lung cancer.

TLS-CHOP represses miR-486 expression, inducing upregulation of a metastasis regulator PAI-1 in human myxoid liposarcoma.

Myxoid liposarcomas (MLSs) are characterized by t(12;16)(q13;p11) translocation and expression of TLS-CHOP chimeric oncoprotein. However, the molecular functions of TLS-CHOP have not been fully understood. On the other hand, microRNAs (miRNAs) comprise an abundant class of endogenous small non-coding RNAs that negatively regulate the expression of their target genes, and are involved in many biological processes. It is now evident that dysregulation of miRNAs is an important step in the development of many cancers. To our knowledge, however, there have been no reports of the miRNAs involved in MLS tumorigenesis and development. In this study, we have found that miR-486 expression was repressed in TLS-CHOP-expressed NIH3T3 fibroblasts and MLS tissues, and exogenous overexpression of miR-486 repressed growth of MLS cells. Thus, downregulation of miR-486 may be an important process for MLS. In addition, we have identified plasminogen activator inhibitor-1 (PAI-1) as a novel target gene of miR-486. PAI-1 is a unique type of serine protease inhibitor and is known to be one of the key regulators of tumor invasion and metastasis. Furthermore, knockdown of PAI-1 by a specific small interfering RNA (siRNA) inhibited growth of MLS cells, suggesting that increased expression of PAI-1 by miR-486 repression is critical for survival of MLS cells. Collectively, these results suggest a novel essential molecular mechanism that TLS-CHOP activates PAI-1 expression by repression of miR-486 expression in MLS tumorigenesis and development.

Nuclear microRNAs release paused Pol II via the DDX21-CDK9 complex.

RNA activation (RNAa) is an uncharacterized mechanism of transcriptional activation mediated by small RNAs, such as microRNAs (miRNAs). A critical issue in RNAa research is that it is difficult to distinguish between changes in gene expression caused indirectly by post-transcriptional regulation and direct induction of gene expression by RNAa. Therefore, in this study, we seek to identify a key factor involved in RNAa, using the induction of ZMYND10 by miR-34a as a system to evaluate RNAa. We identify the positive transcription elongation factors CDK9 and DDX21, which form a complex with nuclear AGO and TNRC6A, as important transcriptional activators of RNAa. In addition, we find that inhibition of DDX21 suppresses RNAa by miR-34a and other miRNAs without inhibiting post-transcriptional regulation. Our findings reveal a strong connection between RNAa and release of paused Pol II, facilitating RNAa research by making it possible to separately analyze post-transcriptional regulation and RNAa.

Age-dependent sex difference of non-alcoholic fatty liver disease in TSOD and db/db mice.

According to previous clinical studies, the prevalence of non-alcoholic fatty liver disease (NAFLD) is higher in men than women only during the reproductive age. Animal models of NAFLD that reflect sex differences in humans have not been established. In this study, we examined sex differences in the hepatic lesions of Tsumura Suzuki obese diabetes (TSOD) and db/db mice, which are representative genetic models of NAFLD. Male and female TSOD and db/db mice were fed with a normal diet and tap water ad libitum. Six male and female mice of each strain were sacrificed at the ages of 3 and 9 months, respectively, and serum biochemical, pathological, and molecular analyses were performed. Serum aspartate aminotransferase (AST) levels were significantly higher in male than female mice of both strains at the age of 3 months; however, at 9 months, significant sex differences were not observed. Similarly, alanine aminotransferase (ALT) levels were significantly higher in male mice than in female TSOD mice at the age of 3 months; however, at 9 months, significant sex differences were not observed. Image analysis of histological slides revealed that the frequency of the steatotic area was significantly higher in male than female db/db mice at the age of 3 months; however, significant sex differences were not observed at 9 months. The frequency of Sirius red-positive fibrotic area was significantly higher in male than female mice in both strains at the age of 3 months; however, significant sex differences were not observed at 9 months. Serum AST and ALT levels and hepatic steatosis and fibrosis in TSOD and db/db mice showed age-dependent sex differences consistent with those observed in human NAFLD. These mice may be suitable for studying sex differences of the disease.

miR-92 is a key oncogenic component of the miR-17-92 cluster in colon cancer.

MicroRNAs (miRNAs) belong to a class of endogenously expressed non-coding small RNAs that function primarily as gene regulators. Growing evidence suggests that miRNAs play a significant role in tumor development, making them potential biomarkers for cancer diagnosis and prognosis. The miR-17-92 cluster has emerged as an important locus, being highly overexpressed in several cancers in association with cancer development and progression. The miR-17-92 miRNA cluster generates a single polycistronic primary transcript that yields six mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a. In colon cancer development, the pathophysiologic roles of these transcripts and their targets are largely unknown. In the present study, we performed copy number analyses of the six miRNAs transcribed from the miR-17-92 cluster in colon tumor tissues. We determined that miR-92a was transcribed at higher levels than the other five miRNAs in both adenomas and carcinoma. In addition, miR-92a directly targeted the anti-apoptotic molecule BCL-2-interacting mediator of cell death (BIM) in colon cancer tissues. An anti-miR-92a antagomir induced apoptosis of colon cancer-derived cell lines. These data indicate that miR-92a plays a pivotal role in the development of colorectal carcinoma.

Cisplatin induces differentiation in teratomas derived from pluripotent stem cells.

INTRODUCTION: Currently, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be induced to differentiate at the cellular level but not to form mature tissues or organs suitable for transplantation. ESCs/iPSCs form immature teratomas after injection into immunodeficient mice. In humans, immature teratomas often transform into fully differentiated mature teratomas after administration of anticancer agents. METHODS: We first investigated the ability of cisplatin to induce changes in mouse ESCs/iPSCs in vitro. Next, we designed experiments to analyze ESC/iPSC-derived immature teratoma tissue in vivo after treatment of cisplatin. Groups of six mice carrying ESC- or iPSC-derived teratomas were given either low or high dose intraperitoneal injection of cisplatin, while the control group received saline for 4 weeks. RESULTS: Treatment of ESC/iPSC cultures with cisplatin for 3 days caused a dose-related decrease in cell numbers without inducing any morphological changes to the cells. ESC/iPSC-derived teratomas showed lower growth rates with a significantly higher mature components ratio in a concentration dependent manner after cisplatin treatment (P < 0.05); however, immunohistochemical analyses demonstrated a significantly reduced PCNA labelling index and an increase in an apoptosis marker on immature neural components (P < 0.05) along with emergence of h-Caldesmon+ mature smooth muscle cells in treated mice. Moreover, newly differentiated components not found in the control group, such as mature adipose tissue, cartilage, and pancreas, as well as striated muscle, salivary glands, gastric mucosa with fundic glands, and hair follicles emerged. The identities of these components were confirmed by immunostaining for specific markers. CONCLUSIONS: Cisplatin has the ability to reduce immature components in ESC/iPSC-derived teratomas, presumably through apoptosis, and also to induce them to differentiate.

Acerola exosome-like nanovesicles to systemically deliver nucleic acid medicine via oral administration.

Extracellular vesicles derived from mammalian cells could be useful carriers for drug delivery systems (DDSs); however, with regard to clinical application, there are several issues to be overcome. Acerola (Malpighia emarginata DC.) is a popular health food. In this study, the feasibility of orally administered nucleic acid drug delivery by acerola exosome-like nanoparticles (AELNs) was examined. AELNs were recovered from acerola juice using an affinity column instead of ultracentrifugation. MicroRNA (miRNA) was sufficiently encapsulated in AELNs by 30-min incubation on ice and was protected against RNase, strong acid, and base treatments. The administration of an AELN/miRNA mixture in cells achieved downregulation of the miRNA's target gene, and this mixture showed cytoplasmic localization. AELNs orally delivered small RNA to the digestive system in vivo. The target gene-suppressing effect in the small intestine and liver peaked 1 day after administration, indicating potential for use as an oral DDS for nucleic acid in the digestive system.

Expression of L-type amino acid transporter 1 is a poor prognostic factor for Non-Hodgkin's lymphoma.

L-type neutral amino acid transporter 1 (LAT1) is a heterodimeric membrane transport protein involved in neutral amino acid transport. LAT1 is highly expressed in various malignant solid tumors and plays an essential role in cell proliferation. However, its role in malignant lymphoma remains unknown. Here, we evaluated LAT1 expression level in tissues from 138 patients with Non-Hodgkin lymphoma (NHL). Overexpression of LAT1 was confirmed in all types of NHL and we found that there is a significant correlation between the level of LAT1 expression and lymphoma grade. The LAT1 expression was higher in aggressive types of lymphomas when compared with static types of lymphomas, suggesting that active tumor proliferation requires nutrient uptake via LAT1. The expression level of LAT1 was inversely correlated with patients' survival span. Furthermore, pharmacological inhibition of LAT1 by a specific inhibitor JPH203 inhibits lymphoma cell growth. In conclusion, our study demonstrated that LAT1 expression can be used as a prognostic marker for patients with NHL and targeting LAT1 by JPH203 can be a novel therapeutic modality for NHL.

WAPL induces cervical intraepithelial neoplasia modulated with estrogen signaling without HPV E6/E7.

Since cervical cancer still afflicts women around the world, it is necessary to understand the underlying mechanism of cervical cancer development. Infection with HPV is essential for the development of cervical intraepithelial neoplasia (CIN). In addition, estrogen receptor signaling is implicated in the development of cervical cancer. Previously, we have isolated human wings apart-like (WAPL), which is expected to cause chromosomal instability in the process of HPV-infected precancerous lesions to cervical cancer. However, the role of WAPL in the development of CIN is still unknown. In this study, in order to elucidate the role of WAPL in the early lesion, we established WAPL overexpressing mice (WAPL Tg mice) and HPV E6/E7 knock-in (KI) mice. WAPL Tg mice developed CIN lesion without HPV E6/E7. Interestingly, in WAPL Tg mice estrogen receptor 1 (ESR1) showed reduction as compared with the wild type, but cell growth factors MYC and Cyclin D1 controlled by ESR1 expressed at high levels. These results suggested that WAPL facilitates sensitivity of ESR1 mediated by some kind of molecule, and as a result, affects the expression of MYC and Cyclin D1 in cervical cancer cells. To detect such molecules, we performed microarray analysis of the uterine cervix in WAPL Tg mice, and focused MACROD1, a co-activator of ESR1. MACROD1 expression was increased in WAPL Tg mice compared with the wild type. In addition, knockdown of WAPL induced the downregulation of MACROD1, MYC, and Cyclin D1 but not ESR1 expression. Furthermore, ESR1 sensitivity assay showed lower activity in WAPL or MACROD1 downregulated cells than control cells. These data suggested that WAPL increases ESR1 sensitivity by activating MACROD1, and induces the expression of MYC and Cyclin D1. Therefore, we concluded that WAPL not only induces chromosomal instability in cervical cancer tumorigenesis, but also plays a key role in activating estrogen receptor signaling in early tumorigenesis.