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The cellular origin of clear cell ovarian carcinoma (CCOC), a major histological subtype of ovarian carcinoma remains elusive. Here, we explored the candidate cellular origin and identify molecular subtypes using integrated genomic/epigenomic analysis. We performed whole exome-sequencing, microarray, and DNA methylation array in 78 CCOC samples according to the original diagnosis. The findings revealed that ARID1A and/or PIK3CA mutations were mutually exclusive with DNA repair related genes, including TP53, BRCA1, and ATM. Clustering of CCOC and other ovarian carcinomas (n = 270) with normal tissues from the fallopian tube, ovarian surface epithelium, endometrial epithelium, and pelvic peritoneum mesothelium (PPM) in a methylation array showed that major CCOC subtypes (with ARID1A and/or PIK3CA mutations) were associated with the PPM-lile cluster (n = 64). This cluster was sub-divided into three clusters: (1) mismatch repair (MMR) deficient with tumor mutational burden-high (n = 2), (2) alteration of ARID1A (n = 51), and (3) ARID1A wild-type (n = 11). The remaining samples (n = 14) were subdivided into (4) ovarian surface epithelium-like (n = 11) and (5) fallopian tube-like (considered as high-grade serous histotype; n = 3). Among these, subtypes (1-3) and others (4 and 5) were found to be associated with immunoreactive signatures and epithelial-mesenchymal transition, respectively. These results contribute to the stratification of CCOC into biological subtypes.
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Adenocarcinoma de Células Claras , Metilación de ADN , Proteínas de Unión al ADN , Mutación , Neoplasias Ováricas , Factores de Transcripción , Humanos , Femenino , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patología , Genómica/métodos , Fosfatidilinositol 3-Quinasa Clase I/genética , Epigenómica/métodos , Secuenciación del Exoma , Persona de Mediana EdadRESUMEN
Histone modification, a major epigenetic mechanism regulating gene expression through chromatin remodeling, introduces dynamic changes in chromatin architecture. Protein arginine methyltransferase 6 (PRMT6) is overexpressed in various types of cancer, including prostate, lung and endometrial cancer (EC). Epigenome regulates the expression of endogenous retrovirus (ERV), which activates interferon signaling related to cancer. The antitumor effects of PRMT6 inhibition and the role of PRMT6 in EC were investigated, using epigenome multiomics analysis, including an assay for chromatin immunoprecipitation sequencing (ChIPseq) and RNA sequencing (RNAseq). The expression of PRMT6 in EC was analyzed using reverse transcriptionquantitative polymerase chain reaction (RTqPCR) and immunohistochemistry (IHC). The prognostic impact of PRMT6 expression was evaluated using IHC. The effects of PRMT6knockdown (KD) were investigated using cell viability and apoptosis assays, as well as its effects on the epigenome, using ChIPseq of H3K27ac antibodies and RNAseq. Finally, the downstream targets identified by multiomics analysis were evaluated. PRMT6 was overexpressed in EC and associated with a poor prognosis. PRMT6KD induced histone hypomethylation, while suppressing cell growth and apoptosis. ChIPseq revealed that PRMT6 regulated genomic regions related to interferons and apoptosis through histone modifications. The RNAseq data demonstrated altered interferonrelated pathways and increased expression of tumor suppressor genes, including NK6 homeobox 1 and phosphoinositide3kinase regulatory subunit 1, following PRMT6KD. RTqPCR revealed that eight ERV genes which activated interferon signaling were upregulated by PRMT6KD. The data of the present study suggested that PRMT6 inhibition induced apoptosis through interferon signaling activated by ERV. PRMT6 regulated tumor suppressor genes and may be a novel therapeutic target, to the best of our knowledge, in EC.
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Neoplasias Endometriales , Histonas , Masculino , Femenino , Humanos , Histonas/metabolismo , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Código de Histonas , Neoplasias Endometriales/genética , Apoptosis , InterferonesRESUMEN
Cancer of unknown primary (CUP) is a heterogeneous syndrome of metastatic cancer in which the primary site cannot be determined even after a standard and comprehensive search. The present report describes a case in which the spatial distribution of the lymph node metastases contributed to the identification of the primary site. While the standard workup did not identify the primary tumor, genomic profiling analysis was useful in therapeutic management. A 68-year-old woman presented with a cancerous pleural effusion (adenocarcinoma). The primary site could not be identified, and the pleural effusion resolved spontaneously. After 11 months, the patient had elevated Krebs von den Lungen-6 and cancer antigen 125 levels, and multiple enlarged lymph nodes. Pathological diagnosis based on a biopsy sample of the para-aortic lymph nodes indicated that the tumor was a high-grade serous carcinoma of possible gynecological organ origin. The patient underwent surgery, including hysterectomy, bisalpingo-oophorectomy and lymph node dissection. Although there were no primary sites in the gynecological organs, marked lymphovascular invasion was found around the left ovary, suggesting a left ovary-derived tumor. Genetic testing revealed a high loss of heterozygosity score and high tumor mutational burden (TMB). The patient received paclitaxel and carboplatin therapy followed by a poly ADP-ribose polymerase inhibitor as regimens for ovarian cancer and achieved complete remission. The unique course of the disappearance of the effusion and the absence of tumor in the adnexa might be associated with the high immunogenicity of the tumor characterized by the high TMB. This case may provide insights into the pathogenesis of CUP.
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BACKGROUND: Cervical intraepithelial neoplasia (CIN) diagnosis is based on colposcopy-aided histological examination. However, its accuracy in CIN diagnosis is limited. Some invasive cervical cancers (ICCs) are diagnosed after cervical conization. Therefore, risk stratification of undetected ICC is particularly important for the management of patients with CIN. This study aimed to identify the risk factors for undetected ICC. We especially focused on the association of human papillomavirus (HPV) genotypes. METHODS: We retrospectively reviewed the clinicopathological characteristics (including age, parity, and preoperative diagnosis) and HPV genotypes of 348 patients diagnosed with CIN or adenocarcinoma in situ (AIS) who underwent cervical conization at our hospital between 2008 and 2016. The relationship between preoperative factors, including HPV genotypes and post-conization ICC, was evaluated. RESULTS: Among the 348 patients, 322, 7, and 19 had preoperative CIN3, CIN2, and AIS, respectively; 181 were nulliparous. The median patient age was 41 (23-83) years. HPV genotyping was performed on 237 patients. Overall, post-conization ICC was detected in 16 patients (4.6%). Multivariate analysis showed that nulliparity and HPV16 positivity were the independent risk factors for post-conization ICC (OR: 6.01, P = 0.0302; OR: 5.26, P = 0.0347, respectively). The combination of HPV16 status and parity improved diagnostic accuracy. Seven of 53 HPV16-positive cases (13%) without childbirth history were diagnosed with post-conization ICC. In contrast, none of the HPV16-negative cases with childbirth history was diagnosed with post-conization ICC. CONCLUSION: HPV16 positivity and nulliparity were identified as risk factors for undetected ICC. Careful treatment selection and preoperative scrupulous examination are especially important in these cases.
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Infecciones por Papillomavirus , Displasia del Cuello del Útero , Neoplasias del Cuello Uterino , Embarazo , Femenino , Humanos , Adulto , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/cirugía , Neoplasias del Cuello Uterino/patología , Conización , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/patología , Estudios Retrospectivos , Displasia del Cuello del Útero/patología , Genotipo , Medición de Riesgo , Papillomaviridae/genéticaRESUMEN
BACKGROUND: Single-agent chemotherapy with or without bevacizumab (Bev) is a standard therapy for platinum-resistant ovarian cancer (PR-OC). However, there is a lack of literature on chemotherapy agent selection in heterogenous PR-OC. Therefore, we aimed to clarify the heterogeneous treatment effects of each chemotherapy agent. METHODS: Patients who underwent single-drug chemotherapy agents or Bev combination therapy for PR-OC between January 2009 and June 2022 were included in this study. We assessed the impact of each chemotherapy agent on the time to treatment failure (TTF) according to histological type, platinum-free interval (PFI), and Bev usage. RESULTS: A total of 158 patients received 343 different chemotherapy regimens. In patients with clear cell carcinoma/mucinous carcinoma (CC/MC), gemcitabine (GEM) had the strongest effect with a median TTF of 5.3 months, whilst nedaplatin (NDP) had the lowest effect with a median TTF of 1.4 months. In contrast, in the non-CC/MC group, irinotecan (CPT-11) and NDP had a better TTF than GEM and pegylated liposomal doxorubicin (PLD). There were notable differences in the treatment efficacy of NDP according to PFI. Specifically, NDP prolonged the TTF in patients with a PFI ≥ 3 months. Compared with GEM alone, GEM + Bev tended to prolong the TTF more effectively; however, an additive effect was not observed with PLD + Bev. CONCLUSIONS: This study demonstrated that the effect of chemotherapy agents differed according to the tumor and background characteristics of the patient. Our findings will improve selection of effective therapies for patients with PR-OC by considering their background characteristics.
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Antineoplásicos , Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/patología , Antineoplásicos/uso terapéutico , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Bevacizumab/uso terapéutico , Doxorrubicina/uso terapéutico , Gemcitabina , Irinotecán/uso terapéutico , Polietilenglicoles , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
PURPOSE: Although many patients with endometrial cancer (EC) or atypical endometrial hyperplasia (AEH) achieve complete remission (CR) after high-dose medroxyprogesterone acetate (MPA) treatment, no consensus has been reached on management after CR. Currently, patients receive estrogen-progestin maintenance therapy, but no recommendations exist regarding the duration of maintenance therapy or whether hysterectomy should be considered. This study aimed to provide insights into the management of EC/AEH after achieving CR. METHODS: We retrospectively investigated the prognosis of 50 patients with EC or AEH who achieved CR after MPA therapy. We assessed the association between disease recurrence and clinicopathological features and the pre- and post-operative histological diagnoses of patients who underwent hysterectomy. RESULTS: The median follow-up duration was 34 months (range: 1-179 months). Recurrence was observed in 17 patients. Among the clinical characteristics investigated, only the primary disease was significantly associated with disease recurrence; patients with EC had a higher risk of recurrence than those with AEH (p = 0.037). During the observation period, 27 patients attempted pregnancy, and 14 pregnancies resulted in delivery. Patients who gave birth had significantly longer relapse-free survivals than those who did not (p = 0.031). Further, 16 patients underwent hysterectomies, and AEH was detected postoperatively in 4 of 11 patients (36.4%) with no preoperative abnormalities. CONCLUSIONS: We identified several clinical features of patients with EC and AEH after CR. Given the high probability of endometrial abnormalities detected postoperatively, hysterectomy may be considered for patients who no longer want children.
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Hiperplasia Endometrial , Neoplasias Endometriales , Preservación de la Fertilidad , Embarazo , Femenino , Niño , Humanos , Hiperplasia Endometrial/tratamiento farmacológico , Hiperplasia Endometrial/cirugía , Hiperplasia Endometrial/patología , Estudios Retrospectivos , Preservación de la Fertilidad/métodos , Resultado del Tratamiento , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/cirugía , Neoplasias Endometriales/patología , Acetato de Medroxiprogesterona/uso terapéutico , PronósticoRESUMEN
The effects of post-operative adjuvant radiotherapy (RT) on intermediate-risk patients with cervical cancer have not been fully elucidated. Therefore, the present study aimed to investigate the impact of RT on intermediate-risk cervical cancer. The data of 112 patients with stage IB and IIA cervical cancer treated with radical hysterectomy between January 2009 and December 2018 were retrospectively reviewed. Overall survival (OS), progression-free survival (PFS), and the frequency of adverse events were compared between patients with and without adjuvant RT (RT+ and RT-, respectively). Subgroup analyses of PFS based on tumor size, cervical stromal invasion, lymphovascular space invasion and histology [squamous cell carcinoma (SCC) vs. non-SCC] were performed. Among the 112 patients, 41 received adjuvant RT. Although there were no significant differences in OS or PFS between the RT+ and RT- groups, the frequency of adverse events was much higher in the RT+ group. Patients in the RT+ group also had more recurrent risk factors than those in the RT- group. Based on the subgroup analyses, although no significant differences were observed between any of the groups, RT demonstrated a different impact on PFS between SCC and non-SCC: No difference was observed in the SCC group, whereas patients in the RT+ group tended to have poorer prognoses compared to those in the RT- group of the non-SCC group. These results suggest that the impact of post-operative RT on stage IB and IIA cervical cancer is limited and is accompanied by increased adverse events. The eligibility of patients for post-operative RT should be carefully determined based on the therapeutic effect of RT in each subgroup.
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Cancer of unknown primary (CUP) is a heterogeneous group of metastatic tumors with a usually unfavorable prognosis. A 33-year-old female was diagnosed with pelvic squamous cell carcinoma of unknown primary. The tumor was p16-positive, suggesting that it was human papillomavirus (HPV)-related. The tumor progressed for 4 months after concurrent chemoradiotherapy (initial treatment) and was refractory to paclitaxel plus carboplatin (second-line therapy). Liquid-based cancer genomic profiling identified five pathogenic variants, including Neurofibromin1 (NF1) (p.T1690Mfs*5); however, due to the lack of domestic clinical trials, the patient could not receive genome-based molecular-target therapies. Simultaneously, nivolumab was administered to the patient post its approval in Japan for CUP. The tumor responded to nivolumab, accompanied by decreased levels of tumor markers. NF1 mutations and HPV-related carcinogenesis may be associated with a favorable response to nivolumab treatment. It may therefore serve as a potential treatment against cancers of unknown primaries.
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Carcinoma de Células Escamosas , Neoplasias Primarias Desconocidas , Infecciones por Papillomavirus , Femenino , Humanos , Adulto , Nivolumab/uso terapéutico , Neoplasias Primarias Desconocidas/tratamiento farmacológico , Neoplasias Primarias Desconocidas/complicaciones , Infecciones por Papillomavirus/complicaciones , Carcinoma de Células Escamosas/patología , Pronóstico , Carboplatino , Paclitaxel/uso terapéuticoRESUMEN
This study aimed to comprehensively clarify the genomic landscape and its association with tumor mutational burden-high (TMB-H, ≥10 mut/Mb) and microsatellite instability-high (MSI-H) in endometrial, cervical, and ovarian cancers. We obtained genomic datasets of a comprehensive genomic profiling test, FoundationOne® CDx, with clinical information using the "Center for Cancer Genomics and Advanced Therapeutics" (C-CAT) database in Japan. Patients can undergo the tests only after standardized treatments under universal health insurance coverage. Endometrial cancers were characterized by a high frequency of TMB-H and MSI-H, especially in endometrioid carcinomas. The lower ratio of POLE exonuclease mutations and the higher ratio of TP53 mutations compared to previous reports suggested the prognostic effects of the molecular subtypes. Among the 839 cervical cancer samples, frequent mutations of KRAS, TP53, PIK3CA, STK11, CDKN2A, and ERBB2 were observed in adenocarcinomas, whereas the ratio of TMB-H was significantly higher in squamous cell carcinomas. Among the 1606 ovarian cancer samples, genomic profiling of serous, clear cell, endometrioid, and mucinous carcinomas was characterized. Pathogenic mutations in the POLE exonuclease domain were associated with high TMB, and the mutation ratio was low in both cervical and ovarian cancers. The C-CAT database is useful for determining the mutational landscape of each cancer type and histological subtype. As the dataset is exclusively collected from patients after the standardized treatments, the information on "druggable" alterations highlights the unmet needs for drug development in major gynecological cancers.
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Positive peritoneal cytology is a poor prognostic factor in patients with advanced endometrial cancer. Suspicious positive peritoneal cytology (class III) is commonly encountered in clinical practice. However, no standard treatment protocol exists for its management. Here, we investigated a possible relationship between suspicious positive peritoneal cytology, disease stage, risk factors, and endometrial cancer prognosis. We included patients diagnosed with endometrial cancer who underwent total hysterectomy and peritoneal cytology at the University of Tokyo Hospital between 2008 and 2022. Overall, 670 patients were included in the analyses; both demographic and clinical data of the patients were collected. The proportion of patients with lymph node metastasis was significantly different between peritoneal cytology groups, showing lymph node metastasis to be more extensive in patients with positive or suspicious positive peritoneal cytology than in patients with negative peritoneal cytology (p < 0.05). Thirty-nine patients had suspicious positive peritoneal cytology. Omental resection and biopsy were performed in 16 cases. No case of omental metastasis was found. Among patients with suspected ascites cytology, no patient experienced symptom recurrence or death. Therefore, monitoring lymph node metastasis in suspicious positive cases is essential. Moreover, a change of treatment method based on the finding of suspected positive peritoneal cytology is not necessary.
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Histone modification is the key epigenetic mechanism that regulates gene expression. Coactivator-associated arginine methyltransferase 1 (CARM1) is an arginine methyltransferase that catalyzes dimethylation of histone H3 (H3R17) at arginine 17. Lately, it has been suggested that CARM1 is associated with human carcinogenesis, and the CARM1-selective inhibitor, TP-064, has been shown to be a potential therapeutic agent for multiple myeloma. However, the physiological significance of CARM1 in endometrial cancer remains unclear. Therefore, we aimed to explore the role of CARM1 and the effect of TP-064 in endometrial cancer. To this end, we analyzed CARM1 expression in endometrial cancer using quantitative real-time polymerase chain reaction and examined the antitumor mechanism with CARM1 knockdown endometrial cancer cells. Moreover, we evaluated the therapeutic capability of TP-064 in endometrial cancer cells. CARM1 was remarkably overexpressed in 52 endometrial cancer tissues compared to normal endometrial tissues. The growth of CARM1 knockdown endometrial cancer cells was suppressed and CARM1 knockdown induced apoptosis. TP-064 also inhibited endometrial cancer cell growth and declined the number of endometrial cancer cell colonies. These data suggest that CARM1 may be a powerful therapeutic target for endometrial cancer.
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Neoplasias Endometriales , Histonas , Apoptosis , Arginina/metabolismo , Proteínas Adaptadoras de Señalización CARD , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/genética , Femenino , Guanilato Ciclasa , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Metilación , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
BACKGROUND: There is increasing evidence for the benefit of poly ADP ribose polymerase (PARP) inhibitors in a subset of high-grade serous ovarian carcinoma (HGSC) patients, especially those with homologous recombination (HR)-deficient tumors. However, new treatment strategies, such as immune checkpoint inhibition, are required for patients with HR-proficient tumors. METHODS: A total of 80 cases of HGSC were analyzed in this study. Whole exome and RNA sequencing was performed for these tumors. Methylation arrays were also carried out to examine BRCA1 and RAD51C promoter methylation status. Mutations, neoantigen load, antigen presentation machinery, and local immune profile were investigated, and the relationships of these factors with clinical outcome were also analyzed. RESULTS: As expected, the numbers of predicted neoAgs were lower in HR-proficient (n=46) than HR-deficient tumors (n=34). However, 40% of the patients with HR-proficient tumors still had higher than median numbers of neoAgs and better survival than patients with lower numbers of neoAgs. Incorporation of human leukocyte antigen (HLA)-class I expression status into the survival analysis revealed that patients with both high neoAg numbers and high HLA-class I expression (neoAghiHLAhi) had the best progression-free survival (PFS) in HR-proficient HGSC (p=0.0087). Gene set enrichment analysis demonstrated that the genes for effector memory CD8 T cells, TH1 T cells, the interferon-γ response, and other immune-related genes, were enriched in these patients. Interestingly, this subset of patients also had better PFS (p=0.0015) and a more T-cell-inflamed tumor phenotype than patients with the same phenotype (neoAghiHLAhi) in HR-deficient HGSC. CONCLUSIONS: Our results suggest that immune checkpoint inhibitors might be an alternative to explore in HR-proficient cases which currently do not benefit from PARP inhibition.
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Antígenos de Neoplasias/inmunología , Cistadenocarcinoma Seroso/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Recombinación Homóloga , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Ováricas/inmunología , Linfocitos T/inmunología , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/genética , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Persona de Mediana Edad , Mutación , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Fenotipo , Pronóstico , Tasa de SupervivenciaRESUMEN
INTRODUCTION: PI3K pathway signaling has received attention as a molecular target in clear cell ovarian carcinoma (CCOC). MDM2 is one of the AKT effectors in the PI3K pathway, which binds to and degrades p53. In this study, we aimed to clarify the prognostic significance of PIK3CA and MDM2 expression, and potential therapeutic effect of a dual inhibition of the PI3K pathway and MDM2. MATERIALS AND METHODS: cDNA expression was evaluated by using microarray data using 75 samples of CCOC. DS-7423 (dual inhibitor of pan-PI3K and mTOR) and RG7112 (MDM2 inhibitor) were used on CCOC cell lines to evaluate cell proliferation, expression level of MDM2 related proteins, and apoptosis by MTT assay, western blotting, and flow cytometry. DS-7423 (3â¯mg/kg) and/or RG7112 (50â¯mg/kg) were orally administrated every day for three weeks, and the anti-tumor effect was evaluated using tumor xenografts, along with immunohistochemistry. RESULTS: Tumors with high expression of both PIK3CA and MDM2 showed significantly worse prognosis in expression array of 71 CCOCs (Pâ¯=â¯0.013). Dual inhibition of the PI3K pathway by DS-7423 and MDM2 by RG7112 showed synergistic anti-proliferative effect in 4 CCOC cell lines without TP53 mutations. The combination therapy more robustly induced pro-apoptotic proteins (PUMA and cleaved PARP) with increase of sub G1 population and apoptotic cells, compared with either single agent alone. The combination therapy significantly reduced tumor volume in mice (Pâ¯<â¯0.001 in OVISE, and Pâ¯=â¯0.038 in RMG-I) without severe body weight loss. Immunohistochemistry from the xenograft tumors showed that the combination treatment significantly reduced vascularity and cell proliferation, with an increase of apoptotic cell death. CONCLUSION: A combination therapy targeting the PI3K pathway and MDM2 might be a promising therapeutic strategy in CCOC.
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Neoplasias Ováricas/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Adenina/análogos & derivados , Adenina/farmacología , Adenocarcinoma de Células Claras , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , ADN Complementario/metabolismo , Femenino , Xenoinjertos , Imidazolinas/farmacología , Ratones Desnudos , Trasplante de Neoplasias/fisiología , Neoplasias Ováricas/metabolismo , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Distribución AleatoriaRESUMEN
BACKGROUND: Peritoneal dissemination is a critical prognostic factor in ovarian cancer. Although stabilized spheroid formation promotes cancer cell peritoneal dissemination in ovarian cancer, the associated oncogenes are unknown. In this study, we assessed the role of the KRAS oncogene in ovarian cancer cell dissemination, focusing on the stability of cells in spheroid condition, as well as the modulation of intracellular signaling following spheroid transformation. METHODS: We used ID8, a murine ovarian cancer cell line, and ID8-KRAS, an oncogenic KRAS (G12 V)-transduced ID8 cell line in this study. Spheroid-forming (3D) culture and cell proliferation assays were performed to evaluate the growth characteristics of these cells. cDNA microarray analysis was performed to identify genes involved in KRAS-associated signal transduction in floating condition. A MEK inhibitor was used to evaluate the effect on cancer peritoneal dissemination. RESULTS: Cell viability and proliferation in monolayer (2D) cultures did not differ between ID8 and ID8-KRAS cells. However, the proportions of viable and proliferating ID8-KRAS cells in 3D culture were approximately 2-fold and 5-fold higher than that of ID8, respectively. Spheroid-formation was increased in ID8-KRAS cells. Analysis of peritoneal floating cells obtained from mice intra-peritoneally injected with cancer cells revealed that the proportion of proliferating cancer cells was approximately 2-fold higher with ID8-KRAS than with ID8 cells. Comprehensive cDNA microarray analysis revealed that pathways related to cell proliferation, and cell cycle checkpoint and regulation were upregulated specifically in ID8-KRAS cells in 3D culture, and that some genes partially regulated by the MEK-ERK pathway were upregulated only in ID8-KRAS cells in 3D culture. Furthermore, a MEK inhibitor, trametinib, suppressed spheroid formation in 3D culture of ID8-KRAS cells, although trametinib did not affect 2D-culture cell proliferation. Finally, we demonstrated that trametinib dramatically improved the prognosis for mice with ID8-KRAS tumors in an in vivo mouse model. CONCLUSIONS: Our data indicated that KRAS promoted ovarian cancer dissemination by stabilizing spheroid formation and that the MEK pathway is important for stabilized spheroid formation. Disruption of spheroid formation by a MEK inhibitor could be a therapeutic target for cancer peritoneal dissemination.
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Proliferación Celular/fisiología , Genes ras/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Neoplasias Ováricas/metabolismo , Esferoides Celulares/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/patologíaRESUMEN
Neoantigens derived from tumor-specific somatic mutations are excellent targets for anti-tumor immune responses. In ovarian clear cell carcinoma (OCCC), checkpoint blockade yields durable responses in a subset of patients. To approach the question of why only some patients respond, we first investigated neoantigen loads and immune signatures using exome sequencing and expression array data for 74 OCCC patients treated conventionally. Neither the number of missense mutations nor total predicted neoantigens assessed in the tumor correlated with clinical outcomes. However, the number of neoantigens per missense mutation ("neoAg frequency") did correlate with clinical outcomes. Cox multivariate regression analysis demonstrated that low neoAg frequencies correlated with increased progression-free survival (PFS) and was an independent predictive factor for PFS in OCCC (p = 0.032), especially at stage I-II (p = 0.0045). Immunity-associated genes including those related to effector memory CD8 T cells were dominantly expressed in tumors with low neoAg frequencies in stage I-II patients, suggesting CD8 T cell-mediated elimination of immunogenic sub-clones expressing neoantigens (immunoediting) had occurred. In contrast, we observed decreased HLA-A, -B, and -C expression (p = 0.036, p = 0.026, and p = 0.030, respectively) as well as increased ratios of CTLA-4, PD-1, Tim-3, and LAG3 to CD8A expression (p = 0.0064, p = 0.017, p = 0.033 and p = 0.0136, respectively) in stage I-II tumors with high neoAg frequencies. Constrained anti-tumor immunity may thus result in limited immunoediting, and poor prognosis. Our results show that neoAg frequency in OCCC is an independent prognostic factor for clinical outcome and may become a potential candidate biomarker for immunomodulatory agent-based treatments.
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MDM2, a ubiquitin ligase, suppresses wild type TP53 via proteasome-mediated degradation. We evaluated the prognostic and therapeutic value of MDM2 in ovarian clear cell carcinoma. MDM2 expression in ovarian cancer tissues was analyzed by microarray and real-time PCR, and its relationship with prognosis was evaluated by Kaplan-Meier method and log-rank test. The anti-tumor activities of MDM2 siRNA and the MDM2 inhibitor RG7112 were assessed by cell viability assay, western blotting, and flow cytometry. The anti-tumor effects of RG7112 in vivo were examined in a mouse xenograft model. MDM2 expression was significantly higher in clear cell carcinoma than in ovarian high-grade serous carcinoma (P = 0.0092) and normal tissues (P = 0.035). High MDM2 expression determined by microarray was significantly associated with poor progression-free survival and poor overall survival (P = 0.0002, and P = 0.0008, respectively). Notably, RG7112 significantly suppressed cell viability in clear cell carcinoma cell lines with wild type TP53. RG7112 also strongly induced apoptosis, increased TP53 phosphorylation, and stimulated expression of the proapoptotic protein PUMA. Similarly, siRNA knockdown of MDM2 induced apoptosis. Finally, RG7112 significantly reduced the tumor volume of xenografted RMG-I clear cell carcinoma cells (P = 0.033), and the density of microvessels (P = 0.011). Our results highlight the prognostic value of MDM2 expression in clear cell carcinoma. Thus, MDM2 inhibitors such as RG7112 may constitute a class of potential therapeutics.
Asunto(s)
Adenocarcinoma de Células Claras/genética , Antineoplásicos/farmacología , Neoplasias Ováricas/genética , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma de Células Claras/tratamiento farmacológico , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/mortalidad , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Hipoxia/tratamiento farmacológico , Hipoxia/genética , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Imidazolinas/farmacología , Ratones , Terapia Molecular Dirigida , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/mortalidad , Pronóstico , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Acyl glucuronides, which are biosynthesized by the action of glucuronosyltransferases to material for detoxification, are water-soluble and chemically active; they produce irreversible protein adducts via both the transacylation mechanism and the imine mechanism. The acyl group at the C-1 position migrates from the anomeric carbon to the C-2 position of the glucuronic acid moiety, producing the aldehyde group at the C-1 position, where the protein easily condenses through a Schiff's base, in the open-chain aldose form. The elimination of the hydroxyl group at the C-2 position therefore may prevent a protein-bound adduct via the imine mechanism. In this paper, we describe the synthesis and characterization of an acyl 2-deoxyglucuronide of deoxycholic acid as a model compound to investigate its possible utility as a water-soluble affinity labeling reagent for lipophilic carboxylic acids. The solubility of deoxycholyl 2-deoxyglucuronide in an aqueous solution was sufficient under physiological conditions, and the desired material reacted with model peptides to produce covalently bound adducts only via the transacylation mechanism.