RESUMEN
DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function.
Asunto(s)
Anhidrasa Carbónica IX , Metilación de ADN , Epigénesis Genética , Células Epiteliales , Óxido Nítrico , Humanos , Óxido Nítrico/metabolismo , Células Epiteliales/metabolismo , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Regiones Promotoras Genéticas , Células CultivadasRESUMEN
Clear cell renal cell carcinoma (ccRCC) is a lethal malignancy with high metastatic probability. Paired box 2 gene product (PAX2) carbonic anhydrase IX were biomolecules closely linked with ccRCC development and outcomes of multiple malignancies. We aim to explore the role of immunohistochemical staining of PAX2 and CAIX to predict ccRCC prognosis after nephrectomy. Surgical specimens of patients who were pathologically diagnosed as ccRCC were reviewed. Expression levels of PAX2 and CAIX were assessed via immunohistochemical staining. Recurrence-free survival (RFS) and overall survival were compared among different phenotypes. Inverse probability of treatment weighting (IPTW) was used for adjustment of confounding factors. 56 patients were included. Patients with PAX2 and CAIX high-expression (the two-high group, n=8) had significantly longer RFS and OS than those of simultaneously down-expression (the two-low group, n=31). Median RFS was 38.4 (95% CI: 32.3-NA) for the two-high group and 14.8 (95% CI: 13.4-39.0) months for the two-low group (P=0.043). IPTW confirmed PAX2 and CAIX co-expression is associated with less recurrence risk HR: 0.39, 95% CI: 0.17-0.92, P=0.031). Co-expression of PAX2 and CAIX is associated better prognosis of ccRCC. We are looking for validation by large cohort studies.
Asunto(s)
Anhidrasa Carbónica IX , Carcinoma de Células Renales , Inmunohistoquímica , Neoplasias Renales , Nefrectomía , Factor de Transcripción PAX2 , Humanos , Factor de Transcripción PAX2/metabolismo , Factor de Transcripción PAX2/genética , Carcinoma de Células Renales/cirugía , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/genética , Masculino , Femenino , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/genética , Nefrectomía/métodos , Persona de Mediana Edad , Estudios Retrospectivos , Neoplasias Renales/cirugía , Neoplasias Renales/patología , Neoplasias Renales/metabolismo , Neoplasias Renales/mortalidad , Neoplasias Renales/genética , Pronóstico , Anciano , Supervivencia sin Enfermedad , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Adulto , Antígenos de NeoplasiasRESUMEN
Microtubule-associated serine-threonine kinase-like (MASTL) has recently been identified as an oncogenic kinase given its overexpression in numerous cancers. Our group has shown that MASTL expression is upregulated in mouse models of sporadic colorectal cancer and colitis-associated cancer (CAC). CAC is one of the most severe complications of chronic inflammatory bowel disease (IBD), but a limited understanding of the mechanisms governing the switch from normal healing to neoplasia in IBD underscores the need for increased research in this area. However, MASTL levels in patients with IBD and its molecular regulation in IBD and CAC have not been studied. This study reveals that MASTL is upregulated by the cytokine interleukin (IL)-22, which promotes proliferation and has important functions in colitis recovery; however, IL-22 can also promote tumorigenesis when chronically elevated. Upon reviewing the publicly available data, we found significantly elevated MASTL and IL-22 levels in the biopsies from patients with late-stage ulcerative colitis compared with controls, and that MASTL upregulation was associated with high IL-22 expression. Our subsequent in vitro studies found that IL-22 increases MASTL expression in intestinal epithelial cell lines, which facilitates IL-22-mediated cell proliferation and downstream survival signaling. Inhibition of AKT activation abrogated IL-22-induced MASTL upregulation. We further found an increased association of carbonic anhydrase IX (CAIX) with MASTL in IL-22-treated cells, which stabilized MASTL expression. Inhibition of CAIX prevented IL-22-induced MASTL expression and cell survival. Overall, we show that IL-22/AKT signaling increases MASTL expression to promote cell survival and proliferation. Furthermore, CAIX associates with and stabilizes MASTL in response to IL-22 stimulation.NEW & NOTEWORTHY MASTL is upregulated in colorectal cancer; however, its role in colitis and colitis-associated cancer is poorly understood. This study is the first to draw a link between MASTL and IL-22, a proinflammatory/intestinal epithelial recovery-promoting cytokine that is also implicated in colon tumorigenesis. We propose that IL-22 increases MASTL protein stability by promoting its association with CAIX potentially via AKT signaling to promote cell survival and proliferation.
Asunto(s)
Interleucina-22 , Interleucinas , Mucosa Intestinal , Interleucinas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Animales , Proliferación Celular , Transducción de Señal , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Ratones , Regulación hacia Arriba , Proteínas Proto-Oncogénicas c-akt/metabolismo , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/genética , Antígenos de NeoplasiasRESUMEN
Ovarian cancer is an aggressive gynecological tumor characterized by a high relapse rate and chemoresistance. Ovarian cancer exhibits the cancer hallmark of elevated glycolysis, yet effective strategies targeting cancer cell metabolic reprogramming to overcome therapeutic resistance in ovarian cancer remain elusive. Here, we revealed that epigenetic silencing of Otubain 2 (OTUB2) is a driving force for mitochondrial metabolic reprogramming in ovarian cancer, which promotes tumorigenesis and chemoresistance. Mechanistically, OTUB2 silencing destabilizes sorting nexin 29 pseudogene 2 (SNX29P2), which subsequently prevents hypoxia-inducible factor-1 alpha (HIF-1α) from von Hippel-Lindau tumor suppressor-mediated degradation. Elevated HIF-1α activates the transcription of carbonic anhydrase 9 (CA9) and drives ovarian cancer progression and chemoresistance by promoting glycolysis. Importantly, pharmacological inhibition of CA9 substantially suppressed tumor growth and synergized with carboplatin in the treatment of OTUB2-silenced ovarian cancer. Thus, our study highlights the pivotal role of OTUB2/SNX29P2 in suppressing ovarian cancer development and proposes that targeting CA9-mediated glycolysis is an encouraging strategy for the treatment of ovarian cancer.
Asunto(s)
Anhidrasa Carbónica IX , Silenciador del Gen , Mitocondrias , Neoplasias Ováricas , Tioléster Hidrolasas , Animales , Femenino , Humanos , Ratones , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucólisis/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Reprogramación Metabólica , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/tratamiento farmacológico , Tioléster Hidrolasas/genéticaRESUMEN
One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.
Asunto(s)
Anhidrasas Carbónicas , Carcinoma de Células Renales , Neoplasias Renales , Receptores Quiméricos de Antígenos , Animales , Ratones , Humanos , Anhidrasa Carbónica IX/genética , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/patología , Receptores Quiméricos de Antígenos/genética , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/uso terapéutico , Antígenos de Neoplasias , Anticuerpos , Linfocitos T/metabolismoRESUMEN
Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia-responsive tumor-associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9-expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a "cold" tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen-specific cytotoxic T cells compared with RENCA cells. Notably, siRNA-mediated serpinB9 knockdown reclaimed this sensitivity. In vivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA-mediated knockdown of the mouse CA9 gene inhibited the hypoxia-induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia-associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as "cold."
Asunto(s)
Acidosis , Apoptosis , Anhidrasa Carbónica IX , Carcinoma de Células Renales , Neoplasias Renales , Serpinas , Animales , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/genética , Ratones , Serpinas/metabolismo , Serpinas/genética , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/inmunología , Línea Celular Tumoral , Humanos , Acidosis/metabolismo , Acidosis/patología , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
Carbonic anhydrase IX (CAIX) is a cancer-associated membrane protein frequently overexpressed in hypoxic solid tumours leading to enhanced tumour cell survival and invasion, and it has been proposed to be an attractive tumour-specific molecule for antibody-mediated targeting. This study aimed to generate a virus-like particle (VLP)-based CAIX vaccine candidate and evaluate its efficacy in a mouse model of breast cancer. The prototype murine vaccine was developed based on the ssRNA bacteriophage Qbeta VLPs with chemically coupled murine CAIX protein catalytic domains on their surfaces. The vaccine was shown to efficiently break the natural B cell tolerance against autologous murine CAIX and to induce high-titre Th1-oriented IgG responses in the BALB/c mice. This vaccine was tested in a therapeutic setting by using a triple-negative breast cancer mouse model system comprising 4T1, 4T1-Car9KI and 4T1-Car9KO cells, the latter representing positive and negative controls for murine CAIX production, respectively. The humoural immune responses induced in tumour-bearing animals were predominantly of Th1-type and higher anti-mCAIXc titres correlated with slower growth and lung metastasis development of 4T1 tumours constitutively expressing mCAIX in vivo in the syngeneic host.
Asunto(s)
Antígenos de Neoplasias , Vacunas contra el Cáncer , Anhidrasa Carbónica IX , Vacunas de Partículas Similares a Virus , Animales , Femenino , Humanos , Ratones , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/inmunología , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Ratones Endogámicos BALB C , Vacunas de Partículas Similares a Virus/inmunologíaRESUMEN
PURPOSE: Prognostic significance and gene signatures associated with carbonic anhydrase IX (CAIX) was investigated in triple negative breast cancer (TNBC) patients. METHODS: Immunohistochemistry (IHC) for CAIX was performed in tissue microarrays (TMAs) of 136 TNBC patients. In a subset of 52 patients Digital Spatial Profiler (DSP) was performed in tumour (pan-cytokeratin+) and stroma (pan-cytokeratin-). Differentially expressed genes (DEGs) with P<0.05 and and log2 fold change (FC)>(±0.25 and ±0.3, for tumour and stromal compartment, respectively) were identified. Four genes were validated at the protein level. RESULT: Cytoplasmic CAIX expression was independently associated with poor recurrence free survival in TNBC patients [hazard ratio (HR)=6.59, 95% confidence interval (CI): 1.47-29.58, P=0.014]. DEG analysis identified 4 up-regulated genes (CD68, HIF1A, pan-melanocyte, and VSIR) in the tumour region and 9 down-regulated genes in the stromal region (CD86, CD3E, MS4A1, BCL2, CCL5, NKG7, PTPRC, CD27, and FAS) when low versus high CAIX expression was explored. Employing IHC, high CD68 and HIF-1α was associated with poorer prognosis and high BCL2 and CD3 was associated with good prognosis. CONCLUSIONS: DSP technology identified DEGs in TNBC. Selected genes validated by IHC showed involvement of CD3 and BCL2 expression within stroma and HIF-1α, and CD68 expression within tumour. However, further functional analysis is warranted.
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Anhidrasas Carbónicas , Neoplasias de la Mama Triple Negativas , Humanos , Antígenos de Neoplasias/análisis , Biomarcadores de Tumor/metabolismo , Anhidrasa Carbónica IX/genética , Anhidrasas Carbónicas/análisis , Anhidrasas Carbónicas/metabolismo , Perfilación de la Expresión Génica , Queratinas , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2 , ARN , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
A strong hypoxic environment has been observed in pancreatic ductal adenocarcinoma (PDAC) cells, which contributes to drug resistance, tumor progression, and metastasis. Therefore, we performed bioinformatics analyses to investigate potential targets for the treatment of PDAC. To identify potential genes as effective PDAC treatment targets, we selected all genes whose expression level was related to worse overall survival (OS) in The Cancer Genome Atlas (TCGA) database and selected only the genes that matched with the genes upregulated due to hypoxia in pancreatic cancer cells in the dataset obtained from the Gene Expression Omnibus (GEO) database. Although the extracted 107 hypoxia-responsive genes included the genes that were slightly enriched in angiogenic factors, TCGA data analysis revealed that the expression level of endothelial cell (EC) markers did not affect OS. Finally, we selected CA9 and PRELID2 as potential targets for PDAC treatment and elucidated that a CA9 inhibitor, U-104, suppressed pancreatic cancer cell growth more effectively than 5-fluorouracil (5-FU) and PRELID2 siRNA treatment suppressed the cell growth stronger than CA9 siRNA treatment. Thus, we elucidated that specific inhibition of PRELID2 as well as CA9, extracted via exhaustive bioinformatic analyses of clinical datasets, could be a more effective strategy for PDAC treatment.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Hipoxia/metabolismo , ARN Interferente Pequeño , Biología Computacional , Neoplasias PancreáticasRESUMEN
Gastric cancer (GC) is the fifth most frequent malignancy and the fourth leading cause of worldwide cancer-related death. Despite the usage of multimodal perioperative chemotherapy (pCT), GC progressively gains chemoresistance, thereby, the identification of suitable targets to overcome drug resistance is fundamental. Amongst the potential biomarkers, carbonic anhydrase IX (CAIX) - associated with a poor prognosis of several solid cancers - has gained the most attention. In a cohort of GC patients who received perioperative FLOT (i.e., Leucovorin, 5-Fluouracil, Docetaxel, and Oxaliplatin) or FOLFOX (i.e., Leucovorin, 5-Fluouracil, and Oxaliplatin), non-responder patients showed an increased expression of tumor CAIX compared to responder group. Moreover, GC cell lines induced to be resistant to 5-Fluouracil, Paclitaxel, Cisplatin, or the combination of 5-Fluorouracil, Oxaliplatin, and Docetaxel, overexpressed CAIX compared to the control. Accordingly, CAIX-high-expressing GC cells showed increased therapy resistance compared to low-expressing cells. Notably, SLC0111 significantly improved the therapy response of both wild-type and resistant GC cells. Overall, these data suggest a correlation between CAIX and GC drug resistance highlighting the potential of SLC-0111 in re-sensitizing GC cells to pCT.
Asunto(s)
Antineoplásicos , Inhibidores de Anhidrasa Carbónica , Neoplasias Gástricas , Humanos , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Línea Celular , Docetaxel/farmacología , Fluorouracilo/farmacología , Leucovorina/farmacología , Oxaliplatino/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patología , Taxoides/farmacología , Taxoides/uso terapéutico , Línea Celular TumoralRESUMEN
PURPOSE: Ferroptosis is a type of regulatory cell death, caused by excessive lipid peroxidation This study aimed to explore whether ionizing radiation could induce ferroptosis in glioma cells and whether carbonic anhydrase 9 (CA9) knockdown could enhance the killing effect of ionizing radiation on hypoxic glioma cells through ferroptosis. MATERIALS AND METHODS: The protein levels of Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) were detected by Western blot in glioma cells irradiated by different doses of X-ray. The relative mRNA levels of ferroptosis markers and intracellular iron-associated proteins were detected by Real-time qPCR. Lipid peroxidation of glioma cells was detected by oxidation-sensitive probe C11-BODIPY581/591 staining. CCK-8 Assay was used to detect cell viability after X-ray irradiation. Cloning formation assay was used to assess the radiosensitivity of glioma cells. The exposure of cell surface calreticulin was measured by immunofluorescence staining. RESULTS: X-ray induced lipid peroxidation and ferroptosis markers expression in U251 and GL261 glioma cells. Knockdown of CA9 in hypoxic glioma cells significantly altered the expression of iron regulation-related proteins and enhanced X-ray-induced ferroptosis and radiosensitivity. The ferroptosis inhibitor significantly improved the survival of cells irradiated by X-ray, while ferroptosis inducers (FINs) enhanced the lethal effect of X-ray on cells. Enhancing ferroptosis in glioma cells promoted the exposure and release of damage-associated molecular patterns (DAMPs). CONCLUSIONS: Ionizing radiation can induce ferroptosis in glioma cells. CA9 knockdown can enhance the radiosensitivity of hypoxic glioma cells and overcome the resistance of ferroptosis under hypoxia. Enhancing ferroptosis will become a new idea to improve the efficacy of radiotherapy for glioma.
Asunto(s)
Anhidrasa Carbónica IX , Ferroptosis , Glioma , Tolerancia a Radiación , Hipoxia Tumoral , Glioma/genética , Glioma/radioterapia , Radiación Ionizante , Anhidrasa Carbónica IX/genética , Técnicas de Silenciamiento del Gen , Línea Celular Tumoral , Humanos , Animales , Ratones , Alarminas/metabolismo , Peroxidación de LípidoRESUMEN
HHLA2 has been recently demonstrated to play multifaceted roles in several types of cancers. However, its underlying mechanism in the progression of human ovarian cancer (OC) remains largely unexplored. In the present study, we aimed to determine whether downregulation of HHLA2 inhibited malignant phenotypes of human OC cells and explore its specific mechanism. Our results revealed that downregulation of HHLA2 by transfection with a lentiviral vector significantly suppressed the viability, invasion, and migration of OC cells. Interaction study showed that downregulation of HHLA2 in OC cells reduced the expression of CA9 and increased the expressions of p-IKKß and p-RelA. Conversely, the viability, invasion, and migration of HHLA2-depleted OC cells were increased when CA9 was upregulated. In vivo, we found that downregulation of HHLA2 significantly inhibited tumor growth, which was reversed by CA9 overexpression. In addition, downregulation of HHLA2 inhibited the OC progression via activating the NF-κB signaling pathway and decreasing the expression of CA9. Collectively, our data suggested a link between HHLA2 and NF-κB axis in the pathogenesis of OC, and these findings might provide valuable insights into the development of novel potential therapeutic targets for OC.
Asunto(s)
FN-kappa B , Neoplasias Ováricas , Humanos , Femenino , FN-kappa B/metabolismo , Regulación hacia Abajo , Línea Celular Tumoral , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Transducción de Señal , Movimiento Celular , Proliferación Celular , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Antígenos de Neoplasias , Inmunoglobulinas/metabolismoRESUMEN
Cutaneous melanoma is a highly aggressive skin cancer, with poor prognosis. The tumor microenvironment is characterized by areas of hypoxia. Carbonic anhydrase IX (CA-IX) is a marker of tumor hypoxia and its expression is regulated by hypoxia-inducible factor-1 (HIF-1). CA-IX has been found to be highly expressed in invasive melanomas. In this study, we investigated the effects of hypoxia on the release of small extracellular vesicles (sEVs) in two melanoma in vitro models. We demonstrated that melanoma cells release sEVs under both normoxic and hypoxic conditions, but only hypoxia-induced sEVs express CA-IX mRNA and protein. Moreover, we optimized an ELISA assay to provide evidence for CA-IX protein expression on the membranes of the sEVs. These CA-IX-positive sEVs may be exploited as potential biomarkers for liquid biopsy.
Asunto(s)
Anhidrasas Carbónicas , Melanoma , Neoplasias Cutáneas , Humanos , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Anhidrasa Carbónica IX/genética , Anhidrasas Carbónicas/metabolismo , Hipoxia , Melanoma/genética , Microambiente Tumoral , Melanoma Cutáneo MalignoRESUMEN
BACKGROUND: To clarify the research prospect and mechanism analysis of isorhamnetin as a therapeutic drug for bladder cancer. METHODS: Firstly, the effects of different concentrations of isorhamnetin on the expression of PPARγ/PTEN/Akt pathway protein, CA9, PPARγ, PTEN and AKT protein were discussed by western blot. The effects of isorhamnetin on the growth of bladder cells were also analyzed. Secondly, we verified whether the effect of isorhamnetin on CA9 was related to PPARγ/PTEN/Akt pathway by western blot, and the mechanism of isorhamnetin on the growth of bladder cells is related to this pathway by CCK8, cell cycle and ball formation experiment. Further, nude mouse model of subcutaneous tumor transplantation was constructed to analyze the effects of isorhamnetin, PPAR and PTEN on 5637 cell tumorigenesis and the effects of isorhamnetin on tumorigenesis and CA9 expression through PPARγ/PTEN/Akt pathway. RESULTS: Isorhamnetin inhibited the development of bladder cancer, and regulated the expression of PPAR, PTEN, AKT, CA9. Isorhamnetin inhibits cell proliferation and the transition of cells from G0/G1 phase to S phase, and tumor sphere formation. Carbonic anhydrase IX is a potential downstream molecule of PPARγ/PTEN/AKT pathway. Overexpression of PPARγ and PTEN inhibited expression of CA9 in bladder cancer cells and tumor tissues. Isorhamnetin reduced CA9 expression in bladder cancer via PPARγ/PTEN/AKT pathway, thereby inhibiting bladder cancer tumorigenicity. CONCLUSION: Isorhamnetin has the potential to become a therapeutic drug for bladder cancer, whose antitumor mechanism is related to PPARγ/PTEN/AKT pathway. Isorhamnetin reduced CA9 expression in bladder cancer via PPARγ/PTEN/AKT pathway, thereby inhibiting bladder cancer tumorigenicity.
Asunto(s)
PPAR gamma , Neoplasias de la Vejiga Urinaria , Ratones , Animales , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/farmacología , PPAR gamma/genética , PPAR gamma/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Carcinogénesis , Transformación Celular Neoplásica , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Línea Celular Tumoral , Proliferación CelularRESUMEN
Tumor-associated carbonic anhydrases IX (CAIX) and XII (CAXII) have long been in the spotlight as potential new targets for anti-cancer therapy. Recently, CAIX/CAXII specific inhibitor SLC-0111 has passed clinical phase I study and showed differential response among patients with colorectal cancer (CRC). CRC can be classified into four different consensus molecular subgroups (CMS) showing unique expression patterns and molecular traits. We questioned whether there is a CMS-related CAIX/CAXII expression pattern in CRC predicting response. As such, we analyzed transcriptomic data of tumor samples for CA9/CA12 expression using Cancertool. Protein expression pattern was examined in preclinical models comprising cell lines, spheroids and xenograft tumors representing the CMS groups. Impact of CAIX/CAXII knockdown and SLC-0111 treatment was investigated in 2D and 3D cell culture. The transcriptomic data revealed a characteristic CMS-related CA9/CA12 expression pattern with pronounced co-expression of both CAs as a typical feature of CMS3 tumors. Protein expression in spheroid- and xenograft tumor tissue clearly differed, ranging from close to none (CMS1) to strong CAIX/CAXII co-expression in CMS3 models (HT29, LS174T). Accordingly, response to SLC-0111 analyzed in the spheroid model ranged from no (CMS1) to clear (CMS3), with moderate in CMS2 and mixed in CMS4. Furthermore, SLC-0111 positively affected impact of single and combined chemotherapeutic treatment of CMS3 spheroids. In addition, combined CAIX/CAXII knockdown and more effective treatment with SLC-0111 reduced clonogenic survival of CMS3 modelling single cells. In conclusion, the preclinical data support the clinical approach of targeted CAIX/CAXII inhibition by showing linkage of expression with response and suggest that patients with CMS3-classified tumors would most benefit from such treatment.
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Anhidrasas Carbónicas , Neoplasias Colorrectales , Humanos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Compuestos de Fenilurea , Sulfonamidas , AnimalesRESUMEN
The overexpression of the pH regulator carbonic anhydrase IX (CAIX) due to hypoxic/metabolic stress was reported in various tumors as an adverse prognostic feature. Our retrospective study aimed to investigate the general pattern and dynamics of CAIX expression in rectal adenocarcinoma following preoperative neoadjuvant therapy (NAT) in matched initial biopsy and surgical resection samples. A total of 40/55 (72.72%) of the post-treatment samples showed partial CAIX expression, frequently in the proximity of hypoxic tumor areas. CAIX expression showed a significant increase in post-treatment tumors (mean% 21.8 ± 24.9 SD vs. 39.4 ± 29.4 SD, p < 0.0001), that was not obvious in untreated tumors (mean% 15.0 ± 21.3 SD vs. 20 ± 23.02, p = 0.073). CAIXhigh phenotype was associated with mutant KRAS status and lack of pathological regression (WHO Tumor Regression Grade 4 and 5). However, the adverse effect of CAIX on overall or progression-free survival could not be statistically confirmed. In conclusion, the dynamic upregulation of CAIX expression is a general feature of rectal adenocarcinoma following neoadjuvant chemo-radiotherapy indicating therapy-induced metabolic reprogramming and cellular adaptation. A synergism of the CAIX-associated regulatory pathways and the mutant KRAS oncogenic signaling most likely contributes to therapy resistance and survival of residual cancer.
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Adenocarcinoma , Neoplasias del Recto , Humanos , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Terapia Neoadyuvante , Estudios Retrospectivos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Biomarcadores de Tumor/metabolismo , Antígenos de Neoplasias/metabolismo , Neoplasias del Recto/tratamiento farmacológico , Neoplasias del Recto/genética , Adenocarcinoma/genética , Adenocarcinoma/terapia , QuimioradioterapiaRESUMEN
Non-invasive visualisation of the expression of hypoxia-related proteins, such as carbonic anhydrase IX (CA IX), by positron emission tomography (PET) could provide important information on the oxygenation status of tumours. Since betulinic acid derivatives bind specifically to CA IX the aim of the study was the development betulinic acid-based 68Ga-labelled PET tracers and to evaluate the hypoxia detecting properties in vitro and in vivo. The binding of betulinic acid (B-DOTA) and betulinyl-3-sulfamate (BS-DOTA) was assessed in two rat tumour cell lines (AT1 prostate and Walker-256 mammary carcinomas). AT1 cells express CA IX in a hypoxia-dependent manner whereas Walker-256 cells, expressing almost no CA IX in wildtype, were transfected with the rat Car9 gene. In vivo measurements were carried out in a small animal PET/CT in AT1 tumours in rats breathing room air, 8% or 100% O2. In AT1 cells hypoxia-induced overexpression of CA IX led to a stronger binding of BS-DOTA but not of B-DOTA. The BS-DOTA binding correlated linearly with the CA IX protein expression and could be blocked by an excess of unlabelled tracer. In the transfected Walker-256 cells no specific binding of either of the tracers was seen. In vivo the intratumoral accumulation of BS-DOTA was increased in animals kept under inspiratory hypoxia and reduced by hyperoxia. Therefore, betulinyl-3-sulfamate could be used as a PET tracer of CA IX expression in tumours and to provide information about the oxygenation status. However, accumulation data indicated that binding not only depends on hypoxia-induce CA IX expression but also on the tumour-line-specific basal expression and on the initial oxygenation status of the tumour.
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Ácido Betulínico , Tomografía Computarizada por Tomografía de Emisión de Positrones , Masculino , Animales , Ratas , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Tomografía de Emisión de Positrones/métodos , Antígenos de Neoplasias/metabolismo , Hipoxia/diagnóstico por imagenRESUMEN
BACKGROUND: Carbonic anhydrase IX (CAIX) protein has been correlated with progression and survival in patients with some tumors such as head and neck carcinoma. But renal cell carcinoma is an exception. The prognostic value of CAIX in RCC used to be associated with patients' survival according to published works. This study aimed to rectify the former conclusion. METHODS: This study was registered in PROSPERO (CRD42020160181). A literature search of the PubMed, Embase, Cochrane library and Web of Science databases was performed to retrieve original studies until April of 2022. Twenty-seven studies, including a total of 5462 patients with renal cell carcinoma, were reviewed. Standard meta-analysis methods were used to evaluate the prognostic impact of CAIX expression on patient prognosis. The hazard ratio and its 95% confidence interval were recorded for the relationship between CAIX expression and survival, and the data were analyzed using Stata 11.0. Then we verify the meta-analysis resort to bioinformatics (TCGA). RESULTS: Our initial search resulted in 908 articles in total. From PubMed, Embase, Web of Science electronic and Cochrane library databases, 493, 318 and 97 potentially relevant articles were discovered, respectively. We took the analysis between CA9 and disease-specific survival (HR = 1.18, 95% CI: 0.82-1.70, I2 = 79.3%, P<0.05), a subgroup then was performed to enhance the result (HR = 1.63, 95%CI: 1.30-2.03, I2 = 26.3%, P = 0.228); overall survival was also parallel with the former (HR = 1.13, 95%CI: 0.82-1.56, I2 = 79.8%, P<0.05), then a subgroup also be performed (HR = 0.90, 95%CI:0.75-1.07, I2 = 23.1%, P = 0.246) to verify the result; the analysis between CAIX and progression-free survival got the similar result (HR = 1.73, 95%CI:0.97-3.09, I2 = 82.4%, P<0.05), we also verify the result by subgroup analysis (HR = 1.04, 95%CI:0.79-1.36, I2 = 0.0%, P = 0.465); at last the relationship between CAIX and recurrence-free survival got the same result, too (HR = 0.99, 95%CI: 0.95-1.02, I2 = 57.8%, P = 0.050), the subgroup's result was also parallel with the former (HR = 1.01, 95%CI: 0.91-1.03, I2 = 0.00%, P = 0.704). To validate our meta-analysis, we took a bioinformatic analysis based on TCGA database, survival curve between low and high CAIX expression in four endpoints (DSS, OS, PFI, DFI) have corresponding P value (DSS:P = 0.23, OS:P = 0.77, PFI:P = 0.25, DFI:P = 0.78). CONCLUSIONS: CAIX expression in patients with RCC is an exception to predict tumor survival. Both low CAIX expression and high expression are not associated with survivals in RCC patients.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/metabolismo , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Renales/metabolismo , PronósticoRESUMEN
Carbonic anhydrase 9 (CA9) is a marker for decreased O2 concentration and acidosis, associated with poor prognosis in cancerous patients. The current study suggested that the changes in CA9 gene expression level might be used as a predictive marker to assess early prognosis at the time of detection of de novo leukemia, and then monitor tumor progress during treatment. This study highlights the level of CA9 gene expression in leukemic patients. A total of 44 cases (acute myeloid leukaemia (AML) group: 23 cases; acute lymphoid leukaemia (ALL) group: 13 cases; control group: 8 healthy volunteers) were selected for this study. The CA9 gene expression was assessed by a real-time PCR with the SYBR green assay. A high level of CA9 gene expression was noticed in AML patients compared to the control group, while the results were not significant in ALL patients. After treatment follow-up, significant differences were observed in CA9 gene expression between a complete response and no response in AML patients. As a result, the CA9 tumor gene could act as a potential early marker for acute leukemia prognosis. A low level of CA9 expression was associated with better clinical outcomes, while a high level was related to a negative prognosis in patients with AML.
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Leucemia Mieloide Aguda , Humanos , Anhidrasa Carbónica IX/genética , Pronóstico , Leucemia Mieloide Aguda/genética , Biomarcadores , Reacción en Cadena en Tiempo Real de la Polimerasa , Antígenos de Neoplasias/genéticaRESUMEN
Numerous human cancers, especially hypoxic solid tumors, express carbonic anhydrase IX (CAIX), a transmembrane protein with its catalytic domain located in the extracellular space. CAIX acidifies the tumor microenvironment, promotes metastases and invasiveness, and is therefore considered a promising anticancer target. We have designed a series of high affinity and high selectivity fluorescein-labeled compounds targeting CAIX to visualize and quantify CAIX expression in cancer cells. The competitive binding model enabled the determination of common CA inhibitors' dissociation constants for CAIX expressed in exponentially growing cancer cells. All tested sulfonamide compounds bound the proliferating cells with similar affinity as to recombinantly purified CAIX. The probes are applicable for the design of selective drug-like compounds for CAIX and the competition strategy could be applied to other drug targets.