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The secreted protein collagen and calcium-binding EGF domain 1 (CCBE1) is critical for embryonic lymphatic development through its role in the proteolytic activation of mature vascular endothelial growth factor C (VEGFC). We previously reported that CCBE1 is overexpressed in colorectal cancer (CRC) and that its transcription is negatively regulated by the TGFß-SMAD pathway, but the transcriptional activation mechanism of CCBE1 in CRC remains unknown. Recent studies have revealed the vital role of the hippo effectors YAP/TAZ in lymphatic development; however, the role of YAP/TAZ in tumor lymphangiogenesis has not been clarified. In this study, we found that high nuclear expression of transcription factor TEAD4 is associated with lymph node metastasis and high lymphatic vessel density in patients with CRC. YAP/TAZ-TEAD4 complexes transcriptionally upregulated the expression of CCBE1 by directly binding to the enhancer region of CCBE1 in both CRC cells and cancer-associated fibroblasts, which resulted in enhanced VEGFC proteolysis and induced tube formation and migration of human lymphatic endothelial cells in vitro and lymphangiogenesis in a CRC cell-derived xenograft model in vivo. In addition, the bromodomain and extraterminal domain (BET) inhibitor JQ1 significantly inhibited the transcription of CCBE1, suppressed VEGFC proteolysis, and inhibited tumor lymphangiogenesis in vitro and in vivo. Collectively, our study reveals a new positive transcriptional regulatory mechanism of CCBE1 via YAP/TAZ-TEAD4-BRD4 complexes in CRC, which exposes the protumor lymphangiogenic role of YAP/TAZ and the potential inhibitory effect of BET inhibitors on tumor lymphangiogenesis.
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Neoplasias Colorrectales , Linfangiogénesis , Humanos , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Colágeno/metabolismo , Neoplasias Colorrectales/patología , Células Endoteliales/metabolismo , Linfangiogénesis/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Factor C de Crecimiento Endotelial Vascular/metabolismo , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/metabolismoRESUMEN
Breast cancer is one of the most common malignant tumors worldwide. SLC7A2 is abnormally expressed in multiple cancers. However, its potential in triple negative breast cancer (TNBC) is still unclear. The purpose of this study was to investigate the roles of SLC7A2 and its underlying molecular mechanisms in TNBC. mRNA expression was detected by RT-qPCR. Protein expression was detected by western blot. Co-localization of ACOX1 and TCF1 was determined using FISH assay. Histone crotonylation was performed using in vitro histone crotonylation assay. Functional analysis was performed using CCK-8 and flow cytometry assays. Xenograft assay was conducted to further verify the role of SLC7A2 in TNBC. CD8A expression was detected using immunohistochemistry. We found that SLC7A2 is downregulated in TNBC tumors. Low levels are associated with advanced stages and lymph node metastasis. SLC7A2 expression is positively correlated with CD8A. SLC7A2-mediated lysine catabolism drives the activation of CD8+ T cells. Moreover, SLC7A2 promotes histone crotonylation via upregulating ACOX1. It also promotes interaction between ACOX1 and TCF1, thus promoting antitumor T cell immunity. Additionally, overexpression of SLC7A2 activates CD8+ T cells and enhances the chemosensitivity of anti-PD-1 therapies in vivo. In conclusion, SLC7A2 may function as an antitumor gene in TNBC by activating antitumor immunity, suggesting SLC7A2/ACOX1/TCF1 signaling as a promising therapeutic strategy.
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Lisina , Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Ratones , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Lisina/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND: Lymph node (LN) metastasis confers gastric cancer (GC) progression, poor survival and cancer-related death. Aberrant activation of Wnt/ß-catenin promotes epithelial-mesenchymal transition (EMT) and LN metastasis, whereas the constitutive activation mutation of Wnt/ß-catenin is rare in GC, suggesting that the underlying mechanisms enhancing Wnt/ß-catenin activation need to be further investigated and understood. METHODS: Bioinformatics analyses and immunohistochemistry (IHC) were used to identify and detect LN metastasis-related genes in GC. Cellular functional assays and footpad inoculation mouse model illustrate the biological function of CCT5. Co-immunoprecipitation assays, western blot and qPCR elucidate the interaction between CCT5 and E-cadherin, and the regulation on ß-catenin activity. RESULTS: CCT5 is upregulated in LN metastatic GCs and correlates with poor prognosis. In vitro assays prove that CCT5 markedly promotes GC cell proliferation, anti-anoikis, invasion and lymphatic tube formation. Moreover, CCT5 enhances xenograft GC growth and popliteal lymph node metastasis in vivo. Furthermore, CCT5 binds the cytoplasmic domain of E-cadherin and abrogates the interaction between E-cadherin and ß-catenin, thereby releasing ß-catenin to the nucleus and enhancing Wnt/ß-catenin signalling activity and EMT. CONCLUSION: CCT5 promotes GC progression and LN metastasis by enhancing wnt/ß-catenin activation, suggesting a great potential of CCT5 as a biomarker for GC diagnosis and therapy.
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Chaperonina con TCP-1 , Neoplasias Gástricas , Vía de Señalización Wnt , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Chaperonina con TCP-1/genética , Chaperonina con TCP-1/metabolismo , Transición Epitelial-Mesenquimal/genética , Xenoinjertos , Humanos , Metástasis Linfática , Ratones , Metástasis de la Neoplasia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Toxin A (TcdA) and toxin B (TcdB), the two exotoxins of Clostridium difficile, are main causal agents for the colonic epithelium damage in Clostridium difficile infection (CDI). The Hippo pathway is crucial for the control of tissue homeostasis and regeneration of intestines. However, the dysregulation of Hippo pathway in CDI is unclear. Here we show that YAP and TAZ, the transcriptional coactivators downstream of the Hippo pathway, are sequestered in the cytoplasm, degraded, and inactivated by treatment with TcdA and TcdB in colonic epithelial cells. The overexpression of YAP restores the messenger RNA expressions of YAP target genes, attenuates the disruption of cytoskeleton and cell rounding, and rescues the cell proliferation of colonic epithelial cells under exposure of the two toxins. Our results demonstrate that inhibition of YAP and TAZ is involved in the pathogenesis of CDI, implicating that increasing YAP activity could be a potential therapeutic strategy for the CDI treatment.
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Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Colon/efectos de los fármacos , Enterotoxinas/toxicidad , Mucosa Intestinal/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colon/citología , HumanosRESUMEN
Siglec-15 was recently reported to be an immunosuppressive molecule that is expressed by tumor-associated macrophages and upregulated in some solid tumors. Targeting Siglec-15 is a potential strategy for normalization cancer immunotherapy. Here, we identified the important post-translational modification, N-glycosylation of Siglec-15, which is regulated by glucose uptake. Using a series of glycosidase and glycosylation inhibitors, we demonstrated that Siglec-15 was completely N-glycosylated in vitro and in vivo. The precise glycosylation site was determined. N-glycosylation stabilized Siglec-15 by decreasing its lysosome-dependent degradation. Siglec-15 subcellular distribution detected by immunofluorescence indicated that N-glycosylation promoted Siglec-15 transportation to the cell membrane. The collective observations indicate that targeting the N-glycosylation of Siglec-15 may be an effective supplement to immunotherapy.
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Inmunoglobulinas/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Celular/metabolismo , Glicosilación , Células HEK293 , Humanos , Transporte de Proteínas , ProteolisisRESUMEN
Previous literatures reported insulin-like growth factor-2 messenger RNA-binding protein 3 (IGF2BP3) is a poor prognostic marker for colorectal cancer (CRC) patients. However, basic research on the effect and biological role of IGF2BP3 in CRC was still scare. Real-time quantitative polymerase chain reaction and western blot analysis were used to examine IGF2BP3 expression level in tumors and paired normal tissues from CRC patients. Tissue microarrays with 192 CRC patients were subjected to immunohistochemical staining to analyze the prognostic value of IGF2BP3. Proliferation assays, migration assays, and xenograft tumor formation in nude mice were performed to assess the biological role of IGF2BP3 in CRC cells. IGF2BP3 expression was significantly upregulated in tumor tissues compared with the matched normal tissues both in messenger RNA and protein level and was associated with worse prognosis. IGF2BP3 knockdown made cell cycle arrest to impair the proliferation ability of CRC cells and further inhibited the xenograft tumor growth in nude mice, also inhibited the migration ability of CRC cells via inducing epithelial-mesenchymal transition. Therefore, the research demonstrated that increased IGF2BP3 expression promoted the aggressive phenotypes of CRC cells. Targeted IGF2BP3 could be a novel and effective gene therapy for CRC patients to make a better prognosis.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión al ARN/genética , Anciano , Animales , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia sin Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Células HEK293 , Humanos , Masculino , Ratones Desnudos , Persona de Mediana Edad , Análisis Multivariante , Fenotipo , Proteínas de Unión al ARN/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Yes-associated protein (YAP) is a transcription coactivator that plays a crucial role in organ size control by promoting cell proliferation and inhibiting apoptosis. The Hippo tumor suppressor pathway inhibits YAP through phosphorylation-induced cytoplasmic retention and degradation. Here we report a novel mechanism of YAP regulation by angiomotin (AMOT) family proteins via a direct interaction. Knockdown of AMOT family protein AMOTL2 in polarized Madin-Darby canine kidney (MDCK) cells leads to YAP activation, as indicated by decreased YAP tight junction localization, attenuated YAP phosphorylation, accumulation of nuclear YAP, and induction of YAP target gene expression. Transcriptional coactivator with PDZ-binding motif (TAZ), the YAP paralog, is also regulated by AMOT in a similar fashion. Furthermore, AMOTL2 knockdown results in loss of cell contact inhibition in a manner dependent on the functions of YAP and TAZ. Our results indicate a potential tumor-suppressing role of AMOT family proteins as components of the Hippo pathway, and demonstrate a novel mechanism of YAP and TAZ inhibition by AMOT-mediated tight junction localization. These observations provide a potential link between the Hippo pathway and cell contact inhibition.
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Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Angiomotinas , Animales , Proteínas de Ciclo Celular , Línea Celular , Transformación Celular Neoplásica , Perros , Epitelio/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Ratones , Proteínas de Microfilamentos , Fosforilación , Transporte de ProteínasRESUMEN
Prostate cancer is a very common malignant disease and a leading cause of death for men in the Western world. Tumorigenesis and progression of prostate cancer involves multiple signaling pathways, including the Hippo pathway. Yes-associated protein (YAP) is the downstream transcriptional co-activator of the Hippo pathway, is overexpressed in prostate cancer, and plays a vital role in the tumorigenesis and progression of prostate cancer. However, the role of the YAP paralog and another downstream effector of the Hippo pathway, transcriptional co-activator with PDZ-binding motif (TAZ), in prostate cancer has not been fully elucidated. Here, we show that TAZ is a basal cell marker for the prostate epithelium. We found that overexpression of TAZ promotes the epithelial-mesenchymal transition (EMT), cell migration, and anchorage-independent growth in the RWPE1 prostate epithelial cells. Of note, knock down of TAZ in the DU145 prostate cancer cells inhibited cell migration and metastasis. We also found that SH3 domain binding protein 1 (SH3BP1), a RhoGAP protein that drives cell motility, is a direct target gene of TAZ in the prostate cancer cells, mediating TAZ function in enhancing cell migration. Moreover, the prostate cancer-related oncogenic E26 transformation-specific (ETS) transcription factors, ETV1, ETV4, and ETV5, were required for TAZ gene transcription in PC3 prostate cancer cells. MAPK inhibitor U0126 treatment decreased TAZ expression in RWPE1 cells, and ETV4 overexpression rescued TAZ expression in RWPE1 cells with U0126 treatment. Our results show a regulatory mechanism of TAZ transcription and suggest a significant role for TAZ in the progression of prostate cancer.
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Proteínas E1A de Adenovirus/metabolismo , Movimiento Celular , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas E1A de Adenovirus/genética , Animales , Adhesión Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , Factores de Transcripción/genética , Proteínas Señalizadoras YAPRESUMEN
The Hippo pathway plays a major role in organ size control, and its dysregulation contributes to tumorigenesis. The major downstream effectors of the Hippo pathway are the YAP/TAZ transcription co-activators, which are phosphorylated and inhibited by the Hippo pathway kinase LATS1/2. Here, we report a novel mechanism of TAZ regulation by the tight junction protein PARD3. PARD3 promotes the interaction between PP1A and LATS1 to induce LATS1 dephosphorylation and inactivation, therefore leading to dephosphorylation and activation of TAZ. The cytoplasmic, but not the tight junction complex associated, PARD3 is responsible for TAZ regulation. Our study indicates a potential molecular basis for cell growth-promoting function of PARD3 by modulating the Hippo pathway signaling in response to cell contact and cell polarity signals.
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Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Adaptadoras Transductoras de Señales , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Polaridad Celular , Regulación de la Expresión Génica , Células HEK293 , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Fosforilación , Transducción de Señal , Proteínas de Uniones Estrechas/genética , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZRESUMEN
Resistance to chemotherapy is the major cause of colorectal cancer (CRC) treatment failure. The cytokine IL-22, which is produced by T cells and NK cells, is associated with tumorigenesis and tumor progression in cancers. However, the role of IL-22 in chemoresistance has not been investigated. We found that IL-22 levels in tumor tissues and peripheral blood were associated with chemoresistance and indicate poor prognosis for patients who received FOLFOX chemotherapy. In CRC cells, IL-22 was able to attenuate the cytotoxic and apoptosis-inducing effects of 5-FU and OXA by activating the STAT3 pathway and subsequently increasing the expression of anti-apoptotic genes. In addition, IL-22 conferred resistance to 5-FU and OXA by inducing IL-8 autocrine expression through STAT3 activation. Our findings identify IL-22 as a novel chemoresistance cytokine and may be a useful prognostic biomarker for CRC patients receiving FOLFOX chemotherapy.
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Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/fisiología , Interleucina-8/metabolismo , Interleucinas/metabolismo , Factor de Transcripción STAT3/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Comunicación Autocrina , Línea Celular Tumoral , Resistencia a Antineoplásicos , Fluorouracilo/uso terapéutico , Humanos , Interleucina-8/genética , Interleucinas/genética , Leucovorina/uso terapéutico , Compuestos Organoplatinos/uso terapéutico , Factor de Transcripción STAT3/genética , Insuficiencia del Tratamiento , Interleucina-22RESUMEN
A paper-based ratiometric fluorescent sensing platform has been developed for glucose detection based on a dual-emission fluorescent probe consisting of carbon quantum dots (C QDs) and CdTe QDs. When the two kinds of QDs are mixed, the fluorescence of C QDs is reversibly quenched by CdTe QDs. However, in the presence of glucose, the fluorescence of CdTe QDs is quenched by H2O2 catalyzed by glucose oxidase (GOx), which restores the fluorescence of C QDs. The proposed paper-based ratiometric fluorescent sensing platform exhibited good sensitivity and selectivity towards glucose. The working linear range was 0.1 mM to 50 mM with a limit of detection (LOD) of 0.026 mM. Additionally, the proposed paper-based sensor possesses viability for the determination of glucose in actual urine samples.
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The role of processing bodies (P-bodies) in tumorigenesis and tumor progression is not well understood. Here, we showed that the oncogenes YAP/TAZ promote P-body formation in a series of cancer cell lines. Mechanistically, both transcriptional activation of the P-body-related genes SAMD4A, AJUBA, and WTIP and transcriptional suppression of the tumor suppressor gene PNRC1 are involved in enhancing the effects of YAP/TAZ on P-body formation in colorectal cancer (CRC) cells. By reexpression of PNRC1 or knockdown of P-body core genes (DDX6, DCP1A, and LSM14A), we determined that disruption of P-bodies attenuates cell proliferation, cell migration, and tumor growth induced by overexpression of YAP5SA in CRC. Analysis of a pancancer CRISPR screen database (DepMap) revealed co-dependencies between YAP/TEAD and the P-body core genes and correlations between the mRNA levels of SAMD4A, AJUBA, WTIP, PNRC1, and YAP target genes. Our study suggests that the P-body is a new downstream effector of YAP/TAZ, which implies that reexpression of PNRC1 or disruption of P-bodies is a potential therapeutic strategy for tumors with active YAP.
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Proteínas Adaptadoras Transductoras de Señales , Carcinogénesis , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAP , Humanos , Proteínas Señalizadoras YAP/metabolismo , Proteínas Señalizadoras YAP/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Carcinogénesis/genética , Línea Celular Tumoral , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Transactivadores/metabolismo , Transactivadores/genética , Animales , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Ratones , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Regulación Neoplásica de la Expresión Génica , Movimiento Celular , Proteínas con Dominio LIMRESUMEN
BACKGROUND: Various extracellular matrix (ECM) reshaping events are involved in inflammatory bowel disease (IBD). LAMB3 is a vital subunit of laminin-332, an important ECM component. Data on the biological function of LAMB3 in intestinal inflammation are lacking. Our aim is to discuss the effect of LAMB3 in IBD. METHODS: LAMB3 expression was assessed in cultured intestinal epithelial cells, inflamed mucosal tissues of patients and mouse colitis models. RNA sequencing, quantitative real-time polymerase chain reaction and Western blotting were used to detect the LAMB3 expression distribution and potential downstream target genes. Dual-luciferase assays and chromatin immunoprecipitation-quantitative polymerase chain reaction were used to determine whether P65 could transcriptionally activate LAMB3 under tumor necrosis factor α stimulation. RESULTS: LAMB3 expression was increased in inflammatory states in intestinal epithelial cells and colonoids and was associated with adverse clinical outcomes in Crohn's disease. Knockdown of LAMB3 inhibited the expression of proinflammatory cytokines. Mechanistically, LAMB3 expression was directly transcriptionally activated by P65 and was inhibited by nuclear factor kappa B inhibitors under tumor necrosis factor α stimulation. Furthermore, RNA sequencing and replenishment experiments revealed that LAMB3 upregulated SERPINA3 to promote intestinal inflammation via the integrin α3ß1/FAK pathway. CONCLUSION: We propose that LAMB3 could serve as a potential therapeutic target of IBD and a predictor of intestinal stenosis of Crohn's disease. Our findings demonstrate the important role of ECM in the progression of IBD and offer an experimental basis for the treatment and prognosis of IBD.
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Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Serpinas , Animales , Humanos , Ratones , Enfermedad de Crohn/patología , Inflamación/patología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Serpinas/metabolismo , Serpinas/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Colorectal cancer (CRC) is the second leading cause of cancer mortality worldwide. At initial diagnosis, approximately 20% of patients are diagnosed with metastatic CRC (mCRC). Although the APCâAsef interaction is a well-established target for mCRC therapy, the discovery and development of effective and safe drugs for mCRC patients remains an urgent and challenging endeavor. In this study, we identified a novel structural scaffold based on MAI inhibitors, the first-in-class APCâAsef inhibitors we reported previously. ONIOM model-driven optimizations of the N-terminal cap and experimental evaluations of inhibitory activity were performed, and 24-fold greater potency was obtained with the best inhibitor compared to the parental compound. In addition, the cocrystal structure validated that the two-layer πâπ stacking interactions were essential for inhibitor stabilization in the bound state. Furthermore, in vitro and in vivo studies have demonstrated that novel inhibitors suppressed lung metastasis in CRC by disrupting the APCâAsef interaction. These results provide an intrinsic structural basis to further explore drug-like molecules for APCâAsef-mediated CRC therapy.
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Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast cancer. Anti-PD-1/PD-L1 treatment for advanced TNBC is still limited to PD-L1-positive patients. Ataxia telangiectasia mutated (ATM) is a switch molecule for homologous recombination and repair. In this study, we found a significant negative correlation between ATM and PD-L1 in 4 TNBC clinical specimens by single-cell RNA sequencing (scRNA-seq), which was confirmed by immunochemical staining in 86 TNBC specimens. We then established ATM knockdown TNBC stable cell lines to perform in vitro studies and animal experiments, proving the negative regulation of PD-L1 by ATM via suppression of tumor necrosis factor-alpha (TNF-α), which was confirmed by cytokine array analysis of TNBC cell line and analysis of clinical specimens. We further found that ATM inhibits TNF-α via inactivating JNK/c-Jun by scRNA-seq, Western blot and luciferase reporter assays. Finally, we identified a negative correlation between changes in phospho-ATMS1981 and PD-L1 levels in TNBC post- and pre-neoadjuvant therapy. This study reveals a novel mechanism by which ATM negatively regulates PD-L1 by downregulating JNK/c-Jun/TNF-α in TNBC, shedding light on the wide application of immune checkpoint blockade therapy for treating multi-line-resistant TNBC.
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Ataxia Telangiectasia , Neoplasias de la Mama Triple Negativas , Animales , Humanos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Antígeno B7-H1/metabolismo , Citocinas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
IκB kinase (IKK) complex is central regulators of the NF-κB pathway, and dysregulation of IKK phosphorylation leads to hyperactivation of proinflammatory response in various chronic inflammatory diseases, including inflammatory bowel disease (IBD). However, the dynamic modulation of IKK phosphorylation and dephosphorylation in intestinal inflammation remains uncharacterized. Here, we found that autophagy/beclin-1 regulator 1 (AMBRA1) was highly expressed in inflamed colons in a colitis mouse model and in clinical IBD samples. Importantly, AMBRA1 deletion significantly decreased proinflammatory cytokine expression and enhanced the therapeutic effect of infliximab on intestinal inflammation. Mechanistically, the N-term F1 domain of AMBRA1 was required for AMBRA1 to competitively interact with protein phosphatase 4 regulatory subunit 1 (PP4R1) and catalytic protein phosphatase 4 (PP4c) to suppress their interactions with IKK, promote the dissociation of the PP4R1/PP4c complex, and antagonize the dephosphorylation activity of this complex towards the IKK complex. In response to TNF-α stimulation, IKKα phosphorylates AMBRA1 at S1043 to stabilize AMBRA1 expression by impairing its binding to Cullin4A (CUL4A) to decrease its CUL4A-mediated K48-linked ubiquitination. Overall, our study identifies an autophagy-independent function of AMBRA1 as a positive modulator of IKK phosphorylation to promote intestinal inflammation, thus providing a new targeted therapeutic strategy for patients with refractory IBD.
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Proteínas Adaptadoras Transductoras de Señales , Autofagia , Quinasa I-kappa B , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/efectos de los fármacos , Colitis/metabolismo , Colitis/patología , Colitis/inducido químicamente , Células HEK293 , Quinasa I-kappa B/metabolismo , Inflamación/metabolismo , Inflamación/patología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Ratones Endogámicos C57BL , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/genética , FosforilaciónRESUMEN
Colorectal cancer (CRC) is the second most common malignant tumor world-wide. Analysis of the changes that occur during CRC progression could provide insights into the molecular mechanisms driving CRC development and identify improved treatment strategies. Here, we performed an integrated multi-omics analysis of 435 trace-tumor-samples from 148 colorectal cancer (CRC) patients, covering non-tumor (NT), intraepithelial neoplasia (IEN), infiltration (IFT), and advanced-stage CRC (A-CRC) phases. Proteogenomics analyses demonstrated that KRAS and BRAF mutations were mutually exclusive and elevated oxidation phosphorylation in the IEN phase. Chr17q loss and chr20q gain were also mutually exclusive, occurred predominantly in the IEN and IFT phases, respectively, and impacted the cell cycle. Mutation of TP53 was frequent in the A-CRC phase and associated with tumor microenvironment, including increased extracellular matrix rigidity and stromal infiltration. Analysis of the profiles of CRC based on CMS and CRIS classifications revealed the progression paths of each subtype and indicated that microsatellite instability was associated with specific subtype classifications. Additional comparison of molecular characteristics of CRC based on location showed that ANKRD22 amplification by chr10q23.31 gain enhanced glycolysis in the right-sided CRC. The AOM/DSS-induced CRC carcinogenesis mouse model in mice indicated that DDX5 deletion due to chr17q loss promoted CRC development, consistent with the findings from the patient samples. Collectively, this study provides an informative resource for understanding the driving events of different stages of CRC and identifying the potential therapeutic targets.
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Aspirin, also named acetylsalicylate, can directly acetylate the side-chain of lysine in protein, which leads to the possibility of unexplained drug effects. Here, the study used isotopic-labeling aspirin-d3 with mass spectrometry analysis to discover that aspirin directly acetylates 10 HDACs proteins, including SIRT1, the most studied NAD+-dependent deacetylase. SIRT1 is also acetylated by aspirin in vitro. It is also identified that aspirin directly acetylates lysine 408 of SIRT1, which abolishes SIRT1 deacetylation activity by impairing the substrates binding affinity. Interestingly, the lysine 408 of SIRT1 can be acetylated by CBP acetyltransferase in cells without aspirin supplement. Aspirin can inhibit SIRT1 to increase the levels of acetylated p53 and promote p53-dependent apoptosis. Moreover, the knock-in mice of the acetylation-mimic mutant of SIRT1 show the decreased production of pro-inflammatory cytokines and maintain intestinal immune homeostasis. The study indicates the importance of the acetylated internal functional site of SIRT1 in maintaining intestinal immune homeostasis.