RESUMO
Aim: Type II diabetes (T2D) stems from insulin resistance, with ß-cell dysfunction as a hallmark in its progression. Studies reveal that ß cells undergo apoptosis or dedifferentiation during T2D development. The transcription factor PAX4 is vital for ß differentiation and survival, thus may be a potential enhancer of ß-cell function in T2D islets. Materials & methods: Human PAX4 cDNA was delivered into T2D human islets with an adenoviral vector, and its effects on ß cells were examined. Results: PAX4 gene delivery significantly improved ß-cell survival, and increased ß-cell composition in the T2D human islets. Basal insulin and glucose-stimulated insulin secretion in PAX4-expressing islets were substantially higher than untreated or control-treated T2D human islets. Conclusion: Introduced PAX4 expression in T2D human islets improves ß-cell function, thus could provide therapeutic benefits for T2D treatment.
Type II diabetes (T2D) results from insulin resistance, with ß-cell dysfunction playing a pivotal role in its progression. Deficits in ß-cell mass and function have been attributed primarily to ß-cell death through apoptosis; however, recent studies suggest ß-cell failure can also arise from ß-cell dedifferentiation that is, ß cells undergo a loss of mature identity, adopting either progenitor-like or glucagon-producing α cell states during T2D development. Therefore, a strategy preventing ß-cell dedifferentiation while promoting its survival is beneficial for T2D treatment. In this study, we explored whether PAX4, a critical transcription factor for ß differentiation and survival, could alleviate ß-cell dysfunction in human islets derived from T2D patients. To accomplish that, human PAX4 cDNA was delivered into human islets isolated from T2D donors by an adenoviral vector-based vector, Ad5.Pax4 and its effects on ß-cell function were evaluated. The results showed PAX4 expression significantly improved ß-cell survival and increased ß-cell composition in the T2D islets. Notably, PAX4-treated T2D islets exhibited significantly higher basal insulin secretion and glucose-stimulated insulin secretion than control-treated islets. The data demonstrate that PAX4 gene delivery into T2D human islets enhances ß-cell mass and function, and thus may offer therapeutic benefits in the treatment of T2D.
Assuntos
Diabetes Mellitus Tipo 2 , Proteínas de Homeodomínio , Células Secretoras de Insulina , Insulina , Fatores de Transcrição Box Pareados , Humanos , Diabetes Mellitus Tipo 2/terapia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/genética , Células Secretoras de Insulina/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Secreção de Insulina , Técnicas de Transferência de Genes , Sobrevivência Celular , Ilhotas Pancreáticas/metabolismo , Terapia Genética/métodosRESUMO
Objective To evaluate the value of SOX1 and PAX1 gene methylation detection in the secondary triage of high-grade cervical lesions.Methods Exfoliated cervical cells were collected from 122 patients tested positive for human papilloma virus (HPV) and subjected to thin-prep cytologic test (TCT) and SOX1/PAX1 gene methylation tests.Results The HPV test combined with TCT showed the sensitivity of 95.24% and the specificity of 23.75% for detecting cervical intraepithelial neoplasia (CIN) grade 2 and above (CIN2+).After the addition of the SOX1/PAX1 gene methylation detection in secondary triage,the sensitivity for detecting CIN2+ was 83.33%,which had no statistically significant difference from the sensitivity of TCT combined with HPV test (P=0.078).However,the specificity reached 77.50%,which was significantly higher than that of HPV test combined with TCT (P<0.001).The SOX1/PAX1 gene methylation level in the CIN2+ group was higher than those in the normal cervical tissue and the CIN1 group(P<0.001).The cut-off values of SOX1 and PAX1 gene methylation for CIN2+ detection were -11.81 and -11.98,respectively.Conclusion Adding the detection of SOX1/PAX1 gene methylation in secondary triage significantly improves the efficiency and accuracy of CIN2+ detection.
Assuntos
Metilação de DNA , Fatores de Transcrição Box Pareados , Fatores de Transcrição SOXB1 , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Humanos , Feminino , Fatores de Transcrição Box Pareados/genética , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Fatores de Transcrição SOXB1/genética , Adulto , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: In China, the national cervical cancer screening protocol involves initial testing for high-risk human papillomavirus (hrHPV), followed by cytology for hrHPV-positive cases. This study evaluates the effectiveness of PAX1 methylation (PAX1m) analysis in identifying precancerous or cancerous lesions in cervical samples from Chinese women positive for non-16/18 hrHPV strains. METHODS: Between February 2022 and March 2023, 281 cervical samples from non-16/18 hrHPV-positive women underwent cytological examination and PAX1m analysis. The study assessed the statistical relationship between PAX1m levels and the presence of cervical lesions, comparing the diagnostic performance of PAX1m to conventional cytology. RESULTS: A significant association was found between PAX1 methylation levels and the risk of CIN2 + and CIN3 + lesions, with 47 instances of CIN2 + detected. Odds ratios (ORs) for moderate and high PAX1m levels were 8.86 (95% CI: 2.24-42.17) and 166.32 (95% CI: 47.09-784.97), respectively. The area under the ROC curve for PAX1m in identifying CIN2 + lesions was 0.948 (95% CI: 0.895-0.99). PAX1m demonstrated similar sensitivity and negative predictive value (NPV) to cytology but reduced the colposcopy referral rate from 47.7% with cytology alone to 25.6% with PAX1m, showing superior specificity and positive predictive value across age groups. CONCLUSIONS: PAX1 methylation is a strong indicator of CIN2 + and CIN3 + risk, offering diagnostic performance comparable to cytology with the added benefit of reduced unnecessary colposcopy referrals. These findings support the use of PAX1m analysis as a reliable tool for triaging non-16/18 hrHPV-positive women in outpatient settings.
Assuntos
Metilação de DNA , Detecção Precoce de Câncer , Fatores de Transcrição Box Pareados , Infecções por Papillomavirus , Triagem , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Humanos , Feminino , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Neoplasias do Colo do Útero/genética , Pessoa de Meia-Idade , Detecção Precoce de Câncer/métodos , Adulto , Fatores de Transcrição Box Pareados/genética , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Triagem/métodos , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/virologia , Displasia do Colo do Útero/genética , China/epidemiologia , Idoso , Curva ROC , Biomarcadores Tumorais/genética , Esfregaço VaginalRESUMO
BACKGROUND: Rhabdomyosarcoma (RMS) is one of the most common soft tissue sarcomas in children and adolescents, in which PAX3-FOXO1 fusion gene positive patients have very poor prognosis. PAX3-FOXO1 has been identified as an independent prognostic predictor in RMS, with no currently available targeted therapeutic intervention. The novel tyrosine kinase inhibitor anlotinib exhibits a wide range of anticancer effects in multiple types of cancers; however, there have been no relevant studies regarding its application in RMS. MATERIALS AND METHODS: We investigated the effects of PAX3-FOXO1 on the therapeutic efficacy of anlotinib using the CCK-8 assay, flow cytometry, invasion assay, wound healing assay, western blotting, quantitative polymerase chain reaction(qPCR), and xenograft experiments. RNA-seq and co-immunoprecipitation assays were conducted to determine the specific mechanism by which anlotinib regulates PAX3-FOXO1 expression. RESULTS: Anlotinib effectively inhibited RMS cell proliferation and promoted apoptosis and G2/M phase arrest while impeding tumor growth in vivo. Downregulation of PAX3-FOXO1 enhances the antitumor effects of anlotinib. Anlotinib upregulates protein kinase NEK2 and increases the degradation of PAX3-FOXO1 via the ubiquitin-proteasome pathway, leading to a reduction in PAX3-FOXO1 protein levels. CONCLUSION: Anlotinib effectively inhibited the malignant progression of RMS and promoted degradation of the fusion protein PAX3-FOXO1. Anlotinib could be a targeted therapeutic approach to treat PAX3-FOXO1 fusion-positive RMS.
Assuntos
Apoptose , Proliferação de Células , Indóis , Quinases Relacionadas a NIMA , Proteínas de Fusão Oncogênica , Quinolinas , Rabdomiossarcoma , Regulação para Cima , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Animais , Linhagem Celular Tumoral , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/patologia , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Regulação para Cima/efeitos dos fármacos , Quinolinas/farmacologia , Quinases Relacionadas a NIMA/metabolismo , Quinases Relacionadas a NIMA/genética , Apoptose/efeitos dos fármacos , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/genética , Proliferação de Células/efeitos dos fármacos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Antineoplásicos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição Box PareadosRESUMO
Renal cell carcinoma (RCC) is a significant oncological challenge due to its heterogeneous nature and limited treatment options. The PAX developmental gene family encodes nine highly conserved transcription factors that play crucial roles in embryonic development and organogenesis, which have been implicated in the occurrence and development of RCC. This review explores the molecular landscape of RCC, with a specific focus on the role of the PAX gene family in RCC tumorigenesis and disease progression. Of the various RCC subtypes, clear cell renal cell carcinoma (ccRCC) is the most prevalent, characterized by the loss of the von Hippel-Lindau (VHL) tumor suppressor gene. Here, we review the published literature on the expression patterns and functional implications of PAX genes, particularly PAX2 and PAX8, in the three most common RCC subtypes, including ccRCC, papillary RCC (PRCC), and chromophobe RCC (ChRCC). Further, we review the interactions and potential biological mechanisms involving PAX genes and VHL loss in driving the pathogenesis of RCC, including the key signaling pathways mediated by VHL in ccRCC and associated mechanisms implicating PAX. Lastly, concurrent with our update regarding PAX gene research in RCC, we review and comment on the targeting of PAX towards the development of novel RCC therapies.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Fatores de Transcrição Box Pareados , Humanos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Fator de Transcrição PAX8/genética , Fator de Transcrição PAX8/metabolismo , Transdução de Sinais/genéticaRESUMO
OBJECTIVE: The major challenge in routine endocervical curettage (ECC) among Human Papillomavirus (HPV) 16/18-positive patients is that only a small fraction benefit. Nevertheless, current reported models often overestimate the validity and necessity of ECC, making it difficult to improve benefits for patients. This research hypothesized that assessing paired boxed gene 1 methylation levels (PAX1m) and clinical characteristics could enhance the predictive accuracy of detecting additional high-grade squamous intraepithelial lesions or worse (HSIL +) through ECC that were not identified by colposcopy-directed biopsy (CDB). METHODS: Data from 134 women with HPV16/18 positivity undergoing CDB and ECC between April 2018 and April 2022 were collected and analyzed. Quantitative methylation-specific polymerase chain reaction (qMSP) was utilized to measure PAX1m, expressed as ΔCp. Univariate and multivariate regression analyses were conducted to screen variables and select predictive factors. A nomogram was constructed using multivariate logistic regression to predict additional HSIL + detected by ECC. The discrimination, calibration, and clinical utility of the nomogram were evaluated using receiver operating characteristic curves (ROC) and the calibration plot. RESULTS: Age (odds ratio [OR], 5.654; 95% confidence interval [CI], 1.131-37.700), cytology (OR, 24.978; 95% CI, 3.085-540.236), and PAX1 methylation levels by grade (PAX1m grade) (OR, 7.801; 95% CI, 1.548-44.828) were independent predictive factors for additional detection of HSIL + by ECC. In HPV16/18-positive women, the likelihood of additional detection of HSIL + through ECC increased with the severity of cytological abnormalities, peaking at 43.8% for high-grade cytological lesions. Moreover, when cytological findings indicated low-grade lesions, PAX1 methylation levels were positively correlated with the additional detection of HSIL + by ECC (P value < 0.001). A nomogram prediction model was developed (area under curve (AUC) = 0.946; 95% CI, 0.901-0.991), demonstrating high sensitivity (90.9%) and specificity (90.5%) at the optimal cutoff point of 107. Calibration analysis confirmed the model's strong agreement between predicted and observed probabilities. CONCLUSION: The clinical nomogram presented promising predictive performance for the additional detection of HSIL + through ECC among women with HPV16/18 infection. PAX1 methylation level could serve as a valuable tool in guiding individualized clinical decisions regarding ECC for patients with HPV 16/18 infection, particularly in cases of low-grade cytological findings.
Assuntos
Colposcopia , Metilação de DNA , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Nomogramas , Fatores de Transcrição Box Pareados , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Humanos , Feminino , Fatores de Transcrição Box Pareados/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Adulto , Metilação de DNA/genética , Pessoa de Meia-Idade , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/patologia , Curetagem/métodos , Curva ROC , Displasia do Colo do Útero/virologia , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/patologia , Colo do Útero/patologia , Colo do Útero/virologiaRESUMO
Objective: To explore the value of cervical cytologic DNA methylation for screening cervical cancer. Methods: This study was a prospective multicenter study conducted from May to October 2022 in Peking Union Medical College Hospital, Zhejiang Provincial People's Hospital, and the Second Affiliated Hospital of Zhejiang University School of Medicine. Women who accepted opportunistic cervical cancer screening in gynecological outpatient clinics were subjected to liquid-based thin-layer cytology testing (TCT), high-risk human papillomavirus (hrHPV) DNA testing and PAX1/JAM3 dual-genes methylation testing (PAX1m/JAM3m). Colposcopy evaluation and biopsy were offered to women according to current guidelines. The accuracies of various testing methods and their combinations were compared based on histological diagnosis. Results: A total of 1 184 samples diagnosed by histopathology were included in this study, consisting of 541 cases (45.7%) of benign cervical tissue or chronic cervicitis, 273 (23.1%) of cervical intraepithelial neoplasia (CIN) 1, 168 (14.2%) of CIN2, 140 (11.8%) of CIN3, and 62 (5.2%) of cervical cancer. The sensitivity and specificity of PAX1m/JAM3m testing for detecting CIN2 or more severe lesions (CIN2+) were 74.1% and 95.9%, respectively. The sensitivity and specificity of PAX1m/JAM3m testing for detecting CIN3+were 87.6% and 86.8%, respectively. Receiver operating characteristic curve analysis showed that, for detecting CIN3+, the area under curve of PAX1m/JAM3m testing (0.872, 95%CI: 0.847-0.897) was significantly superior to TCT testing (0.580, 95%CI: 0.551-0.610) or hrHPV testing (0.503, 95%CI: 0.479-0.515) (all P values<0.05). Conclusions: The PAX1m/JAM3m test in cervical exfoliated cells has excellent accuracy for the diagnosis of both CIN2+and CIN3+, which is superior to traditional screening protocols and screening strategies.
Assuntos
Metilação de DNA , Detecção Precoce de Câncer , Fatores de Transcrição Box Pareados , Sensibilidade e Especificidade , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Humanos , Feminino , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Estudos Prospectivos , Fatores de Transcrição Box Pareados/genética , Detecção Precoce de Câncer/métodos , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/genética , Colposcopia , Colo do Útero/patologia , Programas de Rastreamento/métodos , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , AdultoRESUMO
Methylation panels, tools for investigating epigenetic changes associated with diseases like cancer, can identify DNA methylation patterns indicative of disease, providing diagnostic or prognostic insights. However, the application of methylation panels focusing on the sex-determining region Y-box 1 (SOX1) and paired box gene 1 (PAX1) genes for diagnosing cervical lesions is under-researched. This study aims to examine the diagnostic performance of PAX1/SOX1 gene methylation as a marker for cervical precancerous lesions and its potential application in triage diagnosis. From September 2022 to April 2023, 181 patients with abnormal HPV-DNA tests or cytological exam results requiring colposcopy were studied at Hubei Maternal and Child Health Hospital, China. Data were collected from colposcopy, cytology, HPV-DNA tests, and PAX1/SOX1 methylation detection. Patients were categorized as control, cervical intraepithelial neoplasia Grade 1 (CIN1), Grade 2 (CIN2), Grade 3 (CIN3), and cervical cancer (CC) groups based on histopathology. We performed HPV testing, liquid-based cytology, and PAX1/SOX1 gene methylation testing. We evaluated the diagnostic value of methylation detection in cervical cancer using DNA methylation positivity rate, sensitivity, specificity, and area under the curve (AUC), and explored its potential for triage diagnosis. PAX1/SOX1 methylation positivity rates were: control 17.1%, CIN1 22.5%, CIN2 100.0%, CIN3 90.0%, and CC 100.0%. The AUC values for PAX1 gene methylation detection in diagnosing CIN1+, CIN2+, and CIN3+ were 0.52 (95% confidence interval [CI]: 0.43-0.62), 0.88 (95% CI: 0.80-0.97), and 0.88 (95% CI: 0.75-1.00), respectively. Corresponding AUC values for SOX1 gene methylation detection were 0.47 (95% CI: 0.40-0.58), 0.80 (95% CI: 0.68-0.93), and 0.92 (95% CI: 0.811-1.00), respectively. In HPV16/18-negative patients, methylation detection showed sensitivity of 32.4% and specificity of 83.7% for CIN1+. For CIN2+ and CIN3+, sensitivity was all 100%, with specificities of 83.0% and 81.1%. Among the patients who underwent colposcopy examination, 166 cases had cytological examination results ≤ASCUS, of which 37 cases were positive for methylation, and the colposcopy referral rate was 22.29%. PAX1/SOX1 gene methylation detection exhibits strong diagnostic efficacy for cervical precancerous lesions and holds significant value in triage diagnosis.
Assuntos
Metilação de DNA , Fatores de Transcrição Box Pareados , Infecções por Papillomavirus , Fatores de Transcrição SOXB1 , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Adulto Jovem , Biomarcadores Tumorais/genética , China , Colposcopia , Detecção Precoce de Câncer/métodos , Fatores de Transcrição Box Pareados/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/genética , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/genética , Sensibilidade e Especificidade , Fatores de Transcrição SOXB1/genética , Triagem/métodos , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genéticaRESUMO
Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 days after denervation), it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g., type-2 diabetes, cancer).
Assuntos
Fator 1 Nuclear Respiratório , Fatores de Transcrição Box Pareados , Complexo de Endopeptidases do Proteassoma , Proteólise , Animais , Masculino , Camundongos , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Camundongos Endogâmicos ICR , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismoRESUMO
During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Córtex Cerebral , Receptores ErbB , Proteínas Hedgehog , Proteínas do Tecido Nervoso , Células-Tronco Neurais , Neurogênese , Fator de Transcrição 2 de Oligodendrócitos , Fator de Transcrição PAX6 , Animais , Neurogênese/fisiologia , Córtex Cerebral/metabolismo , Córtex Cerebral/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Camundongos , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Fator de Transcrição PAX6/metabolismo , Fator de Transcrição PAX6/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Proteína Gli3 com Dedos de Zinco/metabolismo , Proteína Gli3 com Dedos de Zinco/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/genética , Neuroglia/metabolismo , Neuroglia/citologia , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Bulbo Olfatório/metabolismo , Bulbo Olfatório/citologia , Linhagem da Célula , HumanosRESUMO
Although aberrant methylation of PAX1 is closely associated with cervical cancer (CC), PAX1 methylation (PAX1m) and its role in CC remain to be elucidated. Here, we clarified the biological function of PAX1 in CC. First, PAX1m in ThinPrep cytologic test samples was measured via quantitative methylation-specific PCR. The results showed that PAX1 promoter methylation levels were significantly increased in CC patients (p < 0.001). We also found that PAX1 promoter methylation levels were positively correlated with tumor purity but negatively correlated with immune-infiltration via public databases. Then, CRISPR-based methylation perturbation tools (dCas9-Tet1) were constructed to further demonstrate that DNA methylation participates in the regulation of PAX1 expression directly. Gain- and loss-of-function experiments were used to show that PAX1 overexpression restrained proliferation, migration and improved cisplatin sensitivity by interfering with the WNT/TIMELESS axis in CC cells. Additionally, Co-immunoprecipitation assays further confirmed the interaction between PAX1 and TCF7L2. Taken together, our results suggested that a tumor suppressor role of PAX1 in CC and that CRISPR-based PAX1 demethylation editing might be a promising therapeutic strategy for CC.
Assuntos
Proliferação de Células , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias do Colo do Útero , Via de Sinalização Wnt , Feminino , Humanos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Regiões Promotoras Genéticas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
People living with HIV (PLWH) are at highest risk of anal cancer and will benefit from optimized screening for early disease detection. We compared host DNA methylation markers in high-grade squamous intraepithelial lesions (HSIL) versus samples negative for intraepithelial lesions (NILM) or low-grade intraepithelial lesions (LSIL) in PLWH. We recruited PLWH identifying as male aged ≥18 years undergoing high-resolution anoscopy (HRA) in Seattle, Washington, 2015-2016. Anal brush samples were collected for HPV detection, genotyping, and pyrosequencing methylation (host genes ASCL1, PAX1, FMN2, and ATP10A); clinical data were abstracted from medical records. We assessed associations between methylation and presence and extent of HSIL using generalized estimating equation logistic regression, adjusting for age, CD4 count and HIV viral load. Marker panels using HPV DNA and methylation were also evaluated to predict prevalent HSIL. We analyzed 125 samples from 85 participants (mean age 50.1; standard deviation 11.0 years). ASCL1 (adjusted odds ratio [aOR] per 1 unit increase mean percent methylation: 1.07, 95% CI: 1.01-1.13) and FMN2 (aOR per 1 unit increase mean percent methylation: 1.14, 95% CI: 1.08-1.20) methylation were significantly associated with HSIL versus NILM/LSIL. ASCL1 (aOR: 1.06, 95% CI: 1.01-1.11) and FMN2 (aOR: 1.13, 95% CI: 1.08-1.17) methylation were positively associated with increasing HSIL extent. A panel combining methylation (ASCL1 and FMN2) and HPV DNA (HPV16, HPV18, and HPV31) demonstrated best balance of sensitivity (78.2%) and specificity (73.9%) for HSIL detection compared with methylation or HPV alone. Increasing levels of DNA methylation of ASCL1 and FMN2 were positively associated with HSIL detection in PLWH. Host gene methylation testing shows promise for HSIL screening and triage.
Assuntos
Neoplasias do Ânus , Metilação de DNA , Detecção Precoce de Câncer , Infecções por HIV , Infecções por Papillomavirus , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias do Ânus/virologia , Neoplasias do Ânus/genética , Neoplasias do Ânus/diagnóstico , Estudos Transversais , Infecções por HIV/virologia , Infecções por HIV/complicações , Infecções por HIV/genética , Adulto , Detecção Precoce de Câncer/métodos , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/diagnóstico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores Tumorais/genética , Lesões Intraepiteliais Escamosas/virologia , Lesões Intraepiteliais Escamosas/genética , DNA Viral/genética , Idoso , Fatores de Transcrição Box PareadosRESUMO
Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central "driver" roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC). HiBiT is an 11-amino acid peptide derived from the NanoLuc luciferase that produces a luminescence signal which is ~100-fold brighter than firefly or Renilla luciferases through high-affinity binding to a complementary NanoLuc peptide fragment called LgBiT. To facilitate single-cell clonal isolation of knock-ins, the homology-directed repair template encoding HiBiT was followed by a P2A self-cleaving peptide for coexpression of an mCherry fluorescent protein as a fluorescence-activated cell sorter (FACS)-selectable marker. HiBiT tagging thus allows highly sensitive luminescence detection of endogenous PAX3-FOXO1 levels permitting quantitative high-throughput screening of large compound libraries for the discovery of PAX3-FOXO1 inhibitors and degraders.
Assuntos
Fatores de Transcrição Box Pareados , Proteína Vermelha Fluorescente , Rabdomiossarcoma , Humanos , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Sistemas CRISPR-Cas , Rabdomiossarcoma/genética , Peptídeos/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
INTRODUCTION: Methylation assays have demonstrated potential as dependable and high-precision approaches for identifying or triaging individuals with cervical cancer (CA) or cervical intraepithelial neoplasia (CIN). Our investigation aimed to assess the efficacy of the diagnosis and triage of the PAX1/SOX1 methylation panel in detecting CIN or CA. METHODS: A total of 461 patients with abnormal high-risk human papillomavirus (hrHPV) or cytology test results were recruited for this study. Each patient underwent an assortment of assessments, comprising a cytology test, hrHPV test, colposcopy examination, and PAX1 and SOX1 methylation tests. RESULTS: The extent of methylation of both genes demonstrates a positive correlation with the severity of CIN lesions and CA. To determine the correlation for patients with CIN2 or worse (CIN2+), the area under curve was 0.821 (95% CI: 0.782-0.853) for PAX1 and 0.800 (95% CI: 0.766-0.838) for SOX1, while for CIN3 or worse (CIN3+), 0.881 (95% CI: 0.839-0.908) for PAX1 and 0.867 (95% CI: 0.830-0.901) for SOX1. The PAX1/SOX1 methylation marker panel performed sensitivity and specificity of 77.16% and 91.67% for CIN2+, 84.76% and 90.50% for CIN3+, respectively. Regarding triaging hrHPV+ patients, the PAX1/SOX1 methylation test only referred 11.83% of the patients who are unnecessary for colonoscopy examination, which is comparatively lower than cytology, thereby signifying a promising triage strategy for hrHPV-positive women. Furthermore, we observed that the positive PAX1/SOX1 methylation test result for untreated CIN1 or fewer patients would result in a higher likelihood of progression upon a 24-month follow-up visit. CONCLUSION: The present investigation demonstrates that the PAX1/SOX1 methylation marker panel exhibits favorable diagnostic performance in CIN detection and holds the potential to be employed for individual CIN tests or hrHPV-positive triage.
Assuntos
Biomarcadores Tumorais , Metilação de DNA , Fatores de Transcrição Box Pareados , Fatores de Transcrição SOXB1 , Triagem , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Humanos , Feminino , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/virologia , Displasia do Colo do Útero/patologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Neoplasias do Colo do Útero/patologia , Adulto , Fatores de Transcrição Box Pareados/genética , Biomarcadores Tumorais/genética , Pessoa de Meia-Idade , Fatores de Transcrição SOXB1/genética , Triagem/métodos , Colposcopia , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/genética , Idoso , Adulto Jovem , Esfregaço Vaginal/métodos , Valor Preditivo dos TestesRESUMO
Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-ß). This overexpression of TGF-ß1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-ß also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-ß in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-ß1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Humanos , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Fator de Crescimento Transformador beta , Fator de Crescimento Transformador beta1 , Fatores de Transcrição Box Pareados/genética , Transição Epitelial-Mesenquimal , Rabdomiossarcoma/genética , Proteínas de Fusão Oncogênica/genéticaRESUMO
Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Criança , Humanos , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma Alveolar/genética , Linhagem Celular Tumoral , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases/metabolismoRESUMO
Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Animais , Criança , Humanos , Camundongos , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica , Músculo Esquelético/metabolismo , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Rabdomiossarcoma Alveolar/genéticaRESUMO
Paired box 1 (PAX1) deficiency has been reported in a small number of patients diagnosed with otofaciocervical syndrome type 2 (OFCS2). We described six new patients who demonstrated variable clinical penetrance. Reduced transcriptional activity of pathogenic variants confirmed partial or complete PAX1 deficiency. Thymic aplasia and hypoplasia were associated with impaired T cell immunity. Corrective treatment was required in 4/6 patients. Hematopoietic stem cell transplantation resulted in poor immune reconstitution with absent naïve T cells, contrasting with the superior recovery of T cell immunity after thymus transplantation. Normal ex vivo differentiation of PAX1-deficient CD34+ cells into mature T cells demonstrated the absence of a hematopoietic cell-intrinsic defect. New overlapping features with DiGeorge syndrome included primary hypoparathyroidism (n = 5) and congenital heart defects (n = 2), in line with PAX1 expression during early embryogenesis. Our results highlight new features of PAX1 deficiency, which are relevant to improving early diagnosis and identifying patients requiring corrective treatment.
Assuntos
Fatores de Transcrição Box Pareados , Imunodeficiência Combinada Severa , Humanos , Fatores de Transcrição Box Pareados/genética , Fenótipo , Linfócitos T , Timo , Imunodeficiência Combinada Severa/genéticaRESUMO
The tumor-specific chromosomal translocation product, PAX3::FOXO1, is an aberrant fusion protein that plays a key role for oncogenesis in the alveolar subtype of rhabdomyosarcoma (RMS). PAX3::FOXO1 represents a validated molecular target for alveolar RMS and successful inhibition of its oncogenic activity is likely to have significant clinical applications. Even though several PAX3::FOXO1 function-based screening studies have been successfully completed, a directly binding small-molecule inhibitor of PAX3::FOXO1 has not been reported. Therefore, we screened small-molecule libraries to identify compounds that were capable of directly binding to PAX3::FOXO1 protein using surface plasmon resonance technology. Compounds that directly bound to PAX3::FOXO1 were further evaluated in secondary transcriptional activation assays. We discovered that piperacetazine can directly bind to PAX3::FOXO1 protein and inhibit fusion protein-derived transcription in multiple alveolar RMS cell lines. Piperacetazine inhibited anchorage-independent growth of fusion-positive alveolar RMS cells but not embryonal RMS cells. On the basis of our findings, piperacetazine is a molecular scaffold upon which derivatives could be developed as specific inhibitors of PAX3::FOXO1. These novel inhibitors could potentially be evaluated in future clinical trials for recurrent or metastatic alveolar RMS as novel targeted therapy options. SIGNIFICANCE: RMS is a malignant soft-tissue tumor mainly affecting the pediatric population. A subgroup of RMS with worse prognosis harbors a unique chromosomal translocation creating an oncogenic fusion protein, PAX3::FOXO1. We identified piperacetazine as a direct inhibitor of PAX3::FOXO1, which may provide a scaffold for designing RMS-specific targeted therapy.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Humanos , Proteína Forkhead Box O1/genética , Fatores de Transcrição Box Pareados/genética , Fator de Transcrição PAX3/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma Alveolar/genética , Translocação GenéticaRESUMO
BACKGROUND: Histomorphological differentiation between pancreatic serous cystadenoma (SCA) and clear cell renal cell carcinoma (RCC) can be challenging. We aimed to study Paired box 8 protein (Pax8) expression profile in cytologic and surgical specimens with pancreatic SCA to assess its utility as a differentiating marker from clear cell RCC. METHODS: We characterized Pax8 immunohistochemistry in 33 patients with pancreatic SCA (23 surgical resections and 10 cytology specimens). Nine cytology specimens from metastatic clear cell RCC involving pancreas were used as control tissue. Electronic medical records were reviewed to retrieve clinical information. RESULTS: All 10 pancreatic SCA cytology specimens, and 16 of 23 pancreatic SCA surgical resections showed absent Pax8 immunostaining, while the remaining 7 surgical resection specimens showed 1%-2% immunoreactivities. Islet and lymphoid cells adjacent to the pancreatic SCA expressed Pax8. In contrast, the proportion of Pax8 immunoreactivity ranged from 50 to 90% (average of 76%) in nine cases of metastatic clear cell RCC involving pancreas. Using a 5% immunoreactivity cutoff, all cases of pancreatic SCA are interpreted as negative for Pax8 immunostains while all cases of metastatic clear cell RCC involving pancreas are interpreted as positive for Pax8 immunostains. CONCLUSIONS: These results suggest that Pax8 immunohistochemistry staining can be a useful adjunct marker to differentiate pancreatic SCA from clear cell RCC in clinical practice. To the best of our knowledge, this is the first large-scale study of Pax8 immunostaining on surgical and cytology specimens with pancreatic SCA.