RESUMEN
The alcohol metabolite acetaldehyde is a potent human carcinogen linked to esophageal squamous cell carcinoma (ESCC) initiation and development. Aldehyde dehydrogenase 2 (ALDH2) is the primary enzyme that detoxifies acetaldehyde in the mitochondria. Acetaldehyde accumulation causes genotoxic stress in cells expressing the dysfunctional ALDH2E487K dominant negative mutant protein linked to ALDH2*2, the single nucleotide polymorphism highly prevalent among East Asians. Heterozygous ALDH2*2 increases the risk for the development of ESCC and other alcohol-related cancers. Despite its prevalence and link to malignant transformation, how ALDH2 dysfunction influences ESCC pathobiology is incompletely understood. Herein, we characterize how ESCC and preneoplastic cells respond to alcohol exposure using cell lines, three-dimensional organoids and xenograft models. We find that alcohol exposure and ALDH2*2 cooperate to increase putative ESCC cancer stem cells with high CD44 expression (CD44H cells) linked to tumor initiation, repopulation and therapy resistance. Concurrently, ALHD2*2 augmented alcohol-induced reactive oxygen species and DNA damage to promote apoptosis in the non-CD44H cell population. Pharmacological activation of ALDH2 by Alda-1 inhibits this phenotype, suggesting that acetaldehyde is the primary driver of these changes. Additionally, we find that Aldh2 dysfunction affects the response to cisplatin, a chemotherapeutic commonly used for the treatment of ESCC. Aldh2 dysfunction facilitated enrichment of CD44H cells following cisplatin-induced oxidative stress and cell death in murine organoids, highlighting a potential mechanism driving cisplatin resistance. Together, these data provide evidence that ALDH2 dysfunction accelerates ESCC pathogenesis through enrichment of CD44H cells in response to genotoxic stressors such as environmental carcinogens and chemotherapeutic agents.
Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Ratones , Animales , Carcinoma de Células Escamosas de Esófago/genética , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Neoplasias Esofágicas/patología , Factores de Riesgo , Consumo de Bebidas Alcohólicas/genética , Cisplatino/farmacología , Aldehído Deshidrogenasa Mitocondrial/genética , Etanol/metabolismo , Acetaldehído/metabolismo , Transformación Celular Neoplásica , Células Madre Neoplásicas/patología , Alcohol Deshidrogenasa/genéticaRESUMEN
Despite advanced therapeutics, esophageal squamous cell carcinoma (ESCC) remains one of the deadliest cancers. Here, we propose a novel therapeutic strategy based on synthetic lethality combining trifluridine/tipiracil and MK1775 (WEE1 inhibitor) as a treatment for ESCC. This study demonstrates that trifluridine induces single-strand DNA damage in ESCC cells, as evidenced by phosphorylated replication protein 32. The DNA damage response includes cyclin-dependent kinase 1 (CDK1) (Tyr15) phosphorylation as CDK1 inhibition and a decrease of the proportion of phospho-histone H3 (p-hH3)-positive cells, indicating cell cycle arrest at the G2 phase before mitosis entry. The WEE1 inhibitor remarkedly suppressed CDK1 phosphorylation (Try15) and reactivated CDK1, and also increased the proportion of p-hH3-positive cells, which indicates an increase of the number of cells into mitosis. Trifluridine combined with a WEE1 inhibitor increased trifluridine-mediated DNA damage, namely DNA double-strand breaks, as shown by increased γ-H2AX expression. Moreover, the combination treatment with trifluridine/tipiracil and a WEE1 inhibitor significantly suppressed tumor growth of ESCC-derived xenograft models. Hence, our novel combination treatment with trifluridine/tipiracil and a WEE1 inhibitor is considered a candidate treatment strategy for ESCC.
Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Trifluridina/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , Fosforilación , Histonas , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proteínas Tirosina QuinasasRESUMEN
BACKGROUND: Multiple development of esophageal squamous-cell carcinoma is explained by field cancerization and is associated with alcohol consumption and smoking. We investigated the association between the development of second primary esophageal squamous-cell carcinoma after endoscopic resection for esophageal squamous-cell carcinoma and genetic polymorphisms related to alcohol and nicotine metabolism. METHODS: The study group comprised 56 patients with esophageal squamous-cell carcinoma after endoscopic resection. The main variables were the following: (i) cumulative incidence and total number of second primary esophageal squamous-cell carcinoma according to genetic polymorphisms in alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and cytochrome P450 2A6; and (ii) risk factors of second primary esophageal squamous-cell carcinoma identified using a multivariate Cox proportional-hazards model. The frequencies of alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and cytochrome P450 2A6 genetic polymorphisms in the buccal mucosa were analyzed. RESULTS: The median follow-up was 92.8 months (range: 2.7-134.2). Slow-metabolizing alcohol dehydrogenase 1B was associated with a higher 7-year cumulative incidence of second primary esophageal squamous-cell carcinoma (fast-metabolizing alcohol dehydrogenase 1B vs slow-metabolizing alcohol dehydrogenase 1B: 20.5% vs 71.4%, P = 0.006). Slow-metabolizing alcohol dehydrogenase 1B (relative risk [95% confidence interval]: 3.17 [1.49-6.73]), inactive aldehyde dehydrogenase 2 (2.17 [1.01-4.63]) and poorly-metabolizing cytochrome P450 2A6 (4.63 [1.74-12.33]) had a significantly higher total number of second primary esophageal squamous-cell carcinoma per 100 person-years. In the multivariate Cox proportional-hazards model, slow-metabolizing alcohol dehydrogenase 1B was a significant risk factor of the development of second primary esophageal squamous-cell carcinoma (hazard ratio 9.92, 95% confidence interval: 2.35-41.98, P = 0.0018). CONCLUSIONS: Slow-metabolizing alcohol dehydrogenase 1B may be a significant risk factor for the development of second primary esophageal squamous-cell carcinoma. In addition, inactive aldehyde dehydrogenase 2 and poorly-metabolizing cytochrome P450 2A6 may be important factors.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Nicotina , Alcohol Deshidrogenasa/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/cirugía , Neoplasias Esofágicas/patología , Aldehído Deshidrogenasa Mitocondrial/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Factores de Riesgo , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/complicaciones , Polimorfismo Genético , Consumo de Bebidas Alcohólicas/efectos adversos , Etanol , Sistema Enzimático del Citocromo P-450/genética , Aldehído Deshidrogenasa/genéticaRESUMEN
Tumors demonstrating deficient mismatch repair (dMMR) account for 12%-15% of colorectal cancers (CRCs), but their characteristics have not been fully elucidated. The aim of this study was to characterize dMMR CRCs in terms of clinicopathological findings and molecular alterations. Immunostaining for mismatch repair (MMR) proteins was performed to determine MMR status, and then MLH1 promoter methylation and genetic variants of 25 genes involved in colorectal carcinogenesis were analyzed by next-generation sequencing in dMMR tumors. Coexistence of precancerous lesions was histologically evaluated to characterize the type of precursors. Immunohistochemistry revealed 34 dMMR tumors in 492 CRCs. Among dMMR CRCs, there were 25 MLH1 methylation-positive, 16 BRAF V600E variant-positive, and 7 KRAS variant-positive tumors. Positive MLH1 methylation was associated with BRAF V600E, older age, and right-side tumor location. MLH1 methylated BRAF/KRAS wild-type tumors were distinct in that all 5 tumors possessed variants in ligand-independent WNT signaling genes including APC, AXIN2, and CTNNB1. Among 10 dMMR CRCs that presented with precancerous lesions, 4 BRAF variant-positive, 1 KRAS variant-positive, and 2 BRAF/KRAS wild-type MLH1 methylated tumors coexisted with serrated lesions, whereas 1 MLH1 methylated BRAF/KRAS wild-type tumor and 2 MLH1 unmethylated tumors accompanied conventional adenomas. The present study characterized distinct subgroups of dMMR CRCs based on molecular alterations including MLH1 methylation and variants in BRAF, KRAS, and ligand-independent WNT signaling genes. The existence of distinct precursor lesions including serrated lesion and conventional adenoma further illustrates the involvement of heterogeneous carcinogenetic pathways in the development of dMMR CRCs.
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Adenoma , Neoplasias Colorrectales , Lesiones Precancerosas , Humanos , Reparación de la Incompatibilidad de ADN/genética , Homólogo 1 de la Proteína MutL/genética , Proteínas Proto-Oncogénicas B-raf/genética , Ligandos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Metilación de ADN , Adenoma/genética , Adenoma/patología , Lesiones Precancerosas/patología , MutaciónRESUMEN
Clinical cancer genome sequencing detects oncogenic variants that are potential targets for cancer treatment, but it also detects variants of unknown significance. These variants may interact with each other to influence tumor pathophysiology, however, such interactions have not been fully elucidated. Additionally, the effect of target therapy for those variants also unclarified. In this study, we investigated the biological functions of a HER2 mutation (G776S mutation) of unknown pathological significance, which was detected together with APC mutation by cancer genome sequencing of samples from a colorectal cancer (CRC) patient. Transfection of the HER2 G776S mutation alone slightly increased the kinase activity and phosphorylation of HER2 protein, but did not activate HER2 downstream signaling or alter the cell phenotype. On the other hand, the HER2 G776S mutation was shown to have strong oncogenic potential when loss of APC function was accompanied. We revealed that loss of APC function increased Wnt pathway activity but also increased RAS-GTP, which increased ERK phosphorylation triggered by HER2 G776S transfection. In addition, afatinib, a pan-HER tyrosine kinase inhibitor, suppressed tumor growth in xenografts derived from HER2 G776S-transfected CRC cells. These findings suggest that this HER2 mutation in CRC may be a potential therapeutic target.
Asunto(s)
Neoplasias Colorrectales , Oncogenes , Carcinogénesis/genética , Neoplasias Colorrectales/genética , Humanos , Mutación , Fosforilación , Agitación PsicomotoraRESUMEN
Background and Aims: Multiple developments of squamous dysplasia and squamous cell carcinoma (SCC) in the upper aerodigestive tract have been explained by field cancerization phenomenon and were associated with alcohol and cigarette use. Second primary SCC development after curative treatment impairs patients' quality of life and survival; however, how these consumption and cessation affect field cancerization is still unknown. Methods: This is a multicenter cohort study including 331 patients with superficial esophageal SCC (ESCC) treated endoscopically and pooled data from 1022 healthy subjects for comparison. Physiological condition in the background esophageal mucosa was classified into 3 groups based on the number of Lugol-voiding lesions (LVLs) per endoscopic view: grade A, 0; grade B, 1-9; or grade C, ≥10 LVLs. Lifestyle surveys were conducted using a self-administered questionnaire. Patients were counseled on the need for alcohol and smoking cessation by physicians and were endoscopically surveyed every 6 months. Results: LVL grades were positively associated with alcohol drinking intensity, flushing reactions, smoking, and high-temperature food and were negatively associated with eating green and yellow vegetables and fruit. Second primary ESCC and head/neck SCC were significantly more prevalent in the grade C LVL (cumulative 5-y incidences 47.1%, 95% confidence interval [CI] = 38.0-57.2 and 13.3%, 95% CI = 8.1-21.5, respectively). Alcohol and smoking cessation significantly reduced the development of second primary ESCC (adjusted hazard ratios 0.47, 95% = CI 0.26-0.85 and 0.49, 95% CI = 0.26-0.91, respectively). Conclusion: Alcohol drinking, smoking, flushing reaction, and high-temperature food were closely associated with field cancerization, and cessation of alcohol and smoking significantly reduced the risk of development of second primary cancer. UMIN Clinical Trials Registry ID:UMIN000001676.
RESUMEN
(1) Background: CIC-DUX4 sarcoma is a rare mesenchymal small round cell tumor which belongs to rare cancers that occupy a significant percentage of cancer cases as a whole, despite each being rare. Importantly, each rare cancer type has different features, and thus there is a need to develop a model that mimics the features of each of these cancers. We evaluated the idea that the chicken chorioallantoic membrane assay (CAM), a convenient and versatile animal model, can be established for the CIC-DUX4 sarcoma. (2) Methods: Patient-derived cell lines of CIC-DUX4 were applied. These cells were transplanted onto the CAM membrane and tumor formation was examined by H&E staining, immunohistochemistry and Western blotting. The CAM tumor was transferred onto a fresh CAM and was also used to form organoids. Retention of the fusion gene was examined. (3) Results: H&E staining as well as molecular characterization demonstrated the formation of the CIC-DUX4 tumor on the CAM membrane. Expression of cyclin D2 and ETV4 was identified. The CAM tumor was transferred to a fresh CAM to form the second-generation CAM tumor. In addition, we were successful in forming tumor organoids using the CAM tumor. Retention of the fusion gene CIC-DUX4 in the CAM, second-generation CAM, and in the CAM-derived organoids was confirmed by RT-PCR. (4) Conclusions: The CAM assay provides a promising model for CIC-DUX4 sarcoma.
Asunto(s)
Membrana Corioalantoides , Proteínas de Homeodominio/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Represoras/metabolismo , Sarcoma/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Membrana Corioalantoides/metabolismo , Membrana Corioalantoides/patología , HumanosRESUMEN
Fibroblast growth factors (FGFs) and their receptors (FGFRs) are important for signaling to maintain cancer stem-like cells (CSCs) in esophageal squamous cell carcinoma (ESCC). However, which FGF receptor, 1, 2, 3, 4, and L1, is essential or whether FGFRs have distinct different roles in ESCC-CSCs is still in question. This study shows that FGFR2, particularly the IIIb isoform, is highly expressed in non-CSCs. Non-CSCs have an epithelial phenotype, and such cells are more differentiated in ESCC. Further, FGFR2 induces keratinocyte differentiation through AKT but not MAPK signaling and diminishes CSC populations. Conversely, knockdown of FGFR2 induces epithelial-mesenchymal transition (EMT) and enriches CSC populations in ESCC. Finally, data analysis using The Cancer Genome Atlas (TCGA) dataset shows that expression of FGFR2 significantly correlated with cancer cell differentiation in clinical ESCC samples. The present study shows that each FGFR has a distinct role and FGFR2-AKT signaling is a key driver of keratinocyte differentiation in ESCC. Activation of FGFR2-AKT signaling could be a future therapeutic option targeting CSC in ESCC.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Carcinoma de Células Escamosas/genética , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Transición Epitelial-Mesenquimal , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Proto-Oncogénicas c-akt , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
BACKGROUND: Visceral fat obesity can be defined quantitatively by abdominal computed tomography, however, the usefulness of measuring visceral fat area to assess the etiology of gastrointestinal reflux disease has not been fully elucidated. METHODS: A total of 433 healthy subjects aged 40-69 years (234 men, 199 women) were included in the study. The relationship between obesity-related factors (total fat area, visceral fat area, subcutaneous fat area, waist circumference, and body mass index) and the incidence of reflux erosive esophagitis was investigated. Lifestyle factors and stomach conditions relevant to the onset of erosive esophagitis were also analyzed. RESULTS: The prevalence of reflux erosive esophagitis was 27.2% (118/433; 106 men, 12 women). Visceral fat area was higher in subjects with erosive esophagitis than in those without (116.6 cm2 vs. 64.9 cm2, respectively). The incidence of erosive esophagitis was higher in subjects with visceral fat obesity (visceral fat area ≥ 100 cm2) than in those without (61.2% vs. 12.8%, respectively). Visceral fat obesity had the highest odds ratio (OR) among obesity-related factors. Multivariate analysis showed that visceral fat area was associated with the incidence of erosive esophagitis (OR = 2.18), indicating that it is an independent risk factor for erosive esophagitis. In addition, daily alcohol intake (OR = 1.54), gastric atrophy open type (OR = 0.29), and never-smoking history (OR = 0.49) were also independently associated with the development of erosive esophagitis. CONCLUSIONS: Visceral fat obesity is the key risk factor for the development of reflux erosive esophagitis in subjects aged 40-69 years.
Asunto(s)
Esofagitis Péptica , Grasa Intraabdominal , Adulto , Anciano , Estudios Transversales , Esofagitis Péptica/complicaciones , Esofagitis Péptica/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Obesidad/complicaciones , Obesidad/epidemiología , Factores de RiesgoRESUMEN
BACKGROUND: Talaporfin sodium photodynamic therapy (tPDT) is an effective salvage treatment for local failure after chemoradiotherapy for esophageal cancer. Repeated tPDT could also be indicated for local recurrence or residue after the first salvage tPDT. However, the safety and efficacy of repeated tPDT have not been elucidated. METHODS: We reviewed 52 patients with esophageal cancer who were treated with the first tPDT at Kyoto University Hospital between October 2015 and April 2020. RESULTS: Among 52 patients, repeated tPDT after the first tPDT was indicated for 13 patients (25%), of which six had residual tumor, four had local recurrence after complete response (CR) after the first tPDT at the primary site, and six had metachronous lesion. The total session of repeated tPDT was 25; 16 were for primary sites and nine were for metachronous sites. Among them, six patients (46.2%) achieved local (L)-CR and nine lesions (56.3%) achieved lesion L-CR. By session, 10 sessions (40%) achieved L-CR. There were no severe adverse events except for one patient; this patient showed grade 3 esophageal stenosis and perforation after the third tPDT on the same lesion that was previously treated with porfimer sodium photodynamic therapy four times. CONCLUSION: Repeated tPDT could be an effective and safe treatment for local failure even after salvage tPDT for esophageal cancer.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Fotoquimioterapia , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/patología , Humanos , Recurrencia Local de Neoplasia/patología , Fotoquimioterapia/efectos adversos , PorfirinasRESUMEN
OBJECTIVES: Photodynamic therapy (PDT) is an effective salvage endoscopic treatment for local failure at the primary site after chemoradiotherapy (CRT) in esophageal cancer patients. However, the contribution of local control by salvage PDT to the prognosis is unclear. We investigated whether complete response at primary site by salvage PDT could improve the prognosis. METHODS: Between January 2008 and March 2016, 34 patients received salvage PDT for local failure of esophageal cancer limited to stage T1-2 after definitive CRT or radiotherapy. Local complete response (L-CR) rate, adverse events, overall survival (OS), and progression-free survival (PFS) were assessed retrospectively. RESULTS: Local complete response rates after PDT were 68% (23/34; 95% CI, 50-83%) in all patients: 81% (17/21; 95% CI, 58-95%) for stage T1 and 46% (6/13; 95% CI, 19-75%) for stage T2 patients. Grade 3 esophageal stricture occurred in one patient. The median follow-up was 26.0 months (range, 3.7-93.6 months); 21 patients died. The median survival times were 54.3 months in patients who achieved L-CR after PDT (L-CR group) and 19.8 months in those who did not (non-CR group). The 2-year OS rates were 79% (95% CI, 54-92%) in the L-CR group and 40% (95% CI, 11-68%) in the non-CR group (P = 0.0389; log-rank test). The median PFS was 21.2 months in the L-CR group and 1.9 months in the non-CR group (P < 0.001; log-rank test). CONCLUSION: Achieving L-CR by salvage PDT for local failure after CRT in esophageal cancer was associated with good prognosis.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Fotoquimioterapia , Carcinoma de Células Escamosas/tratamiento farmacológico , Quimioradioterapia , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Humanos , Recurrencia Local de Neoplasia , Pronóstico , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: Cancer of unknown primary (CUP) is usually treated with nonselective and empirical chemotherapy; however, its prognosis is generally poor, with a median survival of less than a year. Thus, clinicians eagerly await the development of more effective treatment strategies. In recent years, advances in next-generation sequencing (NGS) have made it possible to analyze comprehensively the genome of individual cancers. NGS has identified many genomic alterations, some of which are potential molecular targets of specific agents. We report a case of CUP that was successfully treated with targeted therapy directed by the genomic data obtained from an NGS-based multiplex assay. CASE PRESENTATION: A 52-year-old Asian woman with right hip joint pain underwent fluorodeoxyglucose-positron emission tomography/computed tomography, which showed multiple metastatic tumors in her right hip joint, thyroid gland, lung, and vertebrae. Brain magnetic resonance imaging showed multiple cerebral metastases. Additional tests, including pathology examination and conventional epidermal growth factor receptor (EGFR) gene mutation analysis (single-strand conformation polymorphism assay), could not identify the primary origin of the tumors, so the patient was diagnosed with CUP. After empirical chemotherapy for CUP, an NGS-based multiplex assay performed using a resected specimen of thyroid tumor detected the EGFR mutation c.2573 T > G p.Leu858Arg (L858R). Her treatment was changed to erlotinib, an EGFR tyrosine-kinase inhibiter, which dramatically shrank the tumors and decreased her serum carcinoembryonic antigen level. She achieved long-term disease control and survived for 2 years and 9 months from the first diagnosis. CONCLUSION: This case might support the strategy that NGS-based multiplex assays could identify actionable molecular targets for individual patients with CUP.
Asunto(s)
Receptores ErbB/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Primarias Desconocidas/tratamiento farmacológico , Femenino , Humanos , Persona de Mediana Edad , MutaciónRESUMEN
During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.
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Autofagia , Esófago/citología , Queratinocitos/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Línea Celular , Células Cultivadas , Etanol/farmacología , Femenino , Queratinocitos/efectos de los fármacos , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Mitochondrial aldehyde dehydrogenase 2 (ALDH2), which is a homotetramer assembled by two equivalent dimers, is an important enzyme that metabolizes ethanol-derived acetaldehyde to acetate in a coenzyme-dependent manner. The highly reactive acetaldehyde exhibits a toxic effect, indicating that the proper functioning of ALDH2 is essential to counteract aldehyde-associated diseases. It is known that the catalytic activity of ALDH2 is drastically impaired by a frequently observed mutation, E487K, in a dominant fashion. However, the molecular basis of the inactivation mechanism is elusive because of the complex nature of the dynamic behavior. Here, we performed microsecond-timescale molecular dynamics simulations of the proteins complexed with coenzymes. The E487K mutation elevated the conformational heterogeneity of the dimer interfaces, which are relatively distal from the substituted residue. Dynamic network analyses showed that Glu487 and the dimer interface were dynamically communicated, and the dynamic community further spanned throughout all of the subunits in the wild-type; however, this network was completely rearranged by the E487K mutation. The perturbation of the dynamic properties led to alterations of the global conformational motions and destabilization of the coenzyme binding required for receiving a proton from the catalytic nucleophile. The insights into the dynamic behavior of the dominant negative mutant in this work will provide clues to restore its function.
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Etanol , Simulación de Dinámica Molecular , Aldehído Deshidrogenasa Mitocondrial/genética , Aldehído Deshidrogenasa Mitocondrial/metabolismo , MutaciónRESUMEN
Esophageal squamous cell carcinoma (ESCC) is a disease characterized by a high mutation rate of the TP53 gene, which plays pivotal roles in the DNA damage response (DDR) and is regulated by checkpoint kinase (CHK) 2. CHK1 is another key DDR-related protein, and its selective inhibition is suggested to be particularly sensitive to TP53-mutated cancers, because a loss of both pathways (CHK1 and/or CHK2-p53) is lethal due to the serious impairment of DDR. Such a therapeutic strategy is termed synthetic lethality. Here, we propose a novel therapeutic strategy based on synthetic lethality combining trifluridine/tipiracil and prexasertib (CHK1 inhibitor) as a treatment for ESCC. Trifluridine is a key component of the antitumor drug combination with trifluridine/tipiracil (an inhibitor of trifluridine degradation), also known as TAS-102. In this study, we demonstrate that trifluridine increases CHK1 phosphorylation in ESCC cells combined with a reduction of the S-phase ratio as well as the induction of ssDNA damage. Because CHK1 phosphorylation is considered to be induced as DDR for trifluridine-mediated DNA damage, we examined the effects of CHK1 inhibition on trifluridine treatment. Consequently, CHK1 inhibition by short hairpin RNA or treatment with the CHK1 inhibitor, prexasertib, markedly enhanced trifluridine-mediated DNA damage, represented by an increase of γH2AX expression. Moreover, the combination of trifluridine/tipiracil and CHK1 inhibition significantly suppressed tumor growth of ESCC-derived xenograft tumors. Furthermore, the combination of trifluridine and prexasertib enhanced radiosensitivity both in vitro and in vivo Thus, the combination of trifluridine/tipiracil and a CHK1 inhibitor exhibits effective antitumor effects, suggesting a novel therapeutic strategy for ESCC.
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Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/patología , Inhibidores de Proteínas Quinasas/farmacología , Pirrolidinas/farmacología , Mutaciones Letales Sintéticas , Timina/farmacología , Trifluridina/farmacología , Animales , Apoptosis , Proliferación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Combinación de Medicamentos , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Humanos , Masculino , Ratones , Ratones Pelados , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Radiotherapy (RT) is the primary treatment for cancer. Ionizing radiation from RT induces tumor damage at the irradiated site, and, although clinically infrequent, may cause regression of tumors distant from the irradiated site-a phenomenon known as the abscopal effect. Recently, the abscopal effect has been related to prolongation of overall survival time in cancer patients, though the factors that influence the abscopal effect are not well understood. The aim of this study is to clarify the factors influencing on abscopal effect. Here, we established a mouse model in which we induced the abscopal effect. We injected MC38 (mouse colon adenocarcinoma) cells subcutaneously into C57BL/6 mice at two sites. Only one tumor was irradiated and the sizes of both tumors were measured over time. The non-irradiated-site tumor showed regression, demonstrating the abscopal effect. This effect was enhanced by an increase in the irradiated-tumor volume and by administration of anti-PD1 antibody. When the abscopal effect was induced by a combination of RT and anti-PD1 antibody, it was also influenced by radiation dose and irradiated-tumor volume. These phenomena were also verified in other cell line, B16F10 cells (mouse melanoma cells). These findings provide further evidence of the mechanism for, and factors that influence, the abscopal effect in RT.
RESUMEN
Alcohol consumption is the key risk factor for the development of esophageal squamous cell carcinoma (ESCC), and acetaldehyde, a metabolite of alcohol, is an alcohol-derived major carcinogen that causes DNA damage. Aldehyde dehydrogenase2 (ALDH2) is an enzyme that detoxifies acetaldehyde, and its activity is reduced by ALDH2 gene polymorphism. Reduction in ALDH2 activity increases blood, salivary and breath acetaldehyde levels after alcohol intake, and it is deeply associated with the development of ESCC. Heavy alcohol consumption in individuals with ALDH2 gene polymorphism significantly elevates the risk of ESCC; however, effective prevention has not been established yet. In this study, we investigated the protective effects of Alda-1, a small molecule ALDH2 activator, on alcohol-mediated esophageal DNA damage. Here, we generated novel genetically engineered knock-in mice that express the human ALDH2*1 (wild-type allele) or ALDH2*2 gene (mutant allele). Those mice were crossed, and human ALDH2*1/*1, ALDH2*1/*2 and ALDH2*2/*2 knock-in mice were established. They were given 10% ethanol for 7 days in the presence or absence of Alda-1, and we measured the levels of esophageal DNA damage, represented by DNA adduct (N2-ethylidene-2'-deoxyguanosine). Alda-1 significantly increased hepatic ALDH2 activity both in human ALDH2*1/*2 and/or ALDH2*2/*2 knock-in mice and reduced esophageal DNA damage levels after alcohol drinking. Conversely, cyanamide, an ALDH2-inhibitor, significantly exacerbated esophageal DNA adduct level in C57BL/6N mice induced by alcohol drinking. These results indicate the protective effects of ALDH2 activation by Alda-1 on esophageal DNA damage levels in individuals with ALDH2 gene polymorphism, providing a new insight into acetaldehyde-mediated esophageal carcinogenesis and prevention.
Asunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Benzamidas/administración & dosificación , Benzodioxoles/administración & dosificación , Carcinogénesis/efectos de los fármacos , Neoplasias Esofágicas/prevención & control , Carcinoma de Células Escamosas de Esófago/prevención & control , Acetaldehído/metabolismo , Acetaldehído/toxicidad , Aldehído Deshidrogenasa Mitocondrial/antagonistas & inhibidores , Aldehído Deshidrogenasa Mitocondrial/genética , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/genética , Cianamida/administración & dosificación , Aductos de ADN/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Mucosa Esofágica/efectos de los fármacos , Mucosa Esofágica/patología , Neoplasias Esofágicas/etiología , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/etiología , Carcinoma de Células Escamosas de Esófago/patología , Etanol/metabolismo , Etanol/toxicidad , Técnicas de Sustitución del Gen , Humanos , Masculino , Ratones Transgénicos , Mutación , Neoplasias Experimentales/etiología , Neoplasias Experimentales/patología , Neoplasias Experimentales/prevención & control , Polimorfismo Genético , Factores de RiesgoRESUMEN
Tumor models are powerful tools to plan preclinical therapeutic strategies that cannot be conducted on humans. Recently, patient-derived tumor models such as patient-derived xenograft (PDX) and/or patient-derived organoid (PDO), are widely used to investigate the antitumor effects of various drugs, because those tumors resemble original tumors at the physiological and molecular level. In this chapter, we describe patient-derived tumor models of esophageal cancers and outline the latest research as well as the molecular pathology of esophageal cancers.
Asunto(s)
Neoplasias Esofágicas/patología , Animales , Modelos Animales de Enfermedad , Humanos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most intractable cancers, so the development of novel therapeutics has been required to improve patient outcomes. Curcumin, a polyphenol from Curcuma longa, exhibits various health benefits including antitumor effects, but its clinical utility is limited because of low bioavailability. Theracurmin® (THC) is a highly bioavailable curcumin dispersed with colloidal submicron particles. METHODS: We examined antitumor effects of THC on ESCC cells by cell viability assay, colony and spheroid formation assay, and xenograft models. To reveal its mechanisms, we investigated the levels of reactive oxygen species (ROS) and performed microarray gene expression analysis. According to those analyses, we focused on NQO1, which involved in the removal of ROS, and examined the effects of NQO1-knockdown or overexpression on THC treatment. Moreover, the therapeutic effect of THC and NQO1 inhibitor on ESCC patient-derived xenografts (PDX) was investigated. RESULTS: THC caused cytotoxicity in ESCC cells, and suppressed the growth of xenografted tumors more efficiently than curcumin. THC increased ROS levels and activated the NRF2-NMRAL2P-NQO1 expressions. Inhibition of NQO1 in ESCC cells by shRNA or NQO1 inhibitor resulted in an increased sensitivity of cells to THC, whereas overexpression of NQO1 antagonized it. Notably, NQO1 inhibitor significantly enhanced the antitumor effects of THC in ESCC PDX tumors. CONCLUSIONS: These findings suggest the potential usefulness of THC and its combination with NQO1 inhibitor as a therapeutic option for ESCC.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Curcumina/administración & dosificación , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Pelados , Ratones Endogámicos C57BL , Ratones SCID , NAD(P)H Deshidrogenasa (Quinona)/genética , ARN Interferente Pequeño/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Clonal expansion in aged normal tissues has been implicated in the development of cancer. However, the chronology and risk dependence of the expansion are poorly understood. Here we intensively sequence 682 micro-scale oesophageal samples and show, in physiologically normal oesophageal epithelia, the progressive age-related expansion of clones that carry mutations in driver genes (predominantly NOTCH1), which is substantially accelerated by alcohol consumption and by smoking. Driver-mutated clones emerge multifocally from early childhood and increase their number and size with ageing, and ultimately replace almost the entire oesophageal epithelium in the extremely elderly. Compared with mutations in oesophageal cancer, there is a marked overrepresentation of NOTCH1 and PPM1D mutations in physiologically normal oesophageal epithelia; these mutations can be acquired before late adolescence (as early as early infancy) and significantly increase in number with heavy smoking and drinking. The remodelling of the oesophageal epithelium by driver-mutated clones is an inevitable consequence of normal ageing, which-depending on lifestyle risks-may affect cancer development.