RESUMO
N6-methyladenosine (m6A) serves as the most abundant posttranscription modification. However, the role of m6A in tumorigenesis and chemotherapeutic drugs sensitivity remains largely unclear. Present research focuses on the potential function of the m6A writer KIAA1429 in tumor development and sorafenib sensitivity in liver cancer. We found that the level of KIAA1429 was significantly elevated in liver cancer tissues and cells and was closely associated with poorer prognosis. Functionally, KIAA1429 promoted the proliferation and Warburg effect of liver cancer cells in vitro and in vivo. RNA-seq and MeRIP-seq analysis revealed the glycolysis was one of the most affected pathways by KIAA1429, and m6A-modified HK1 was the most likely targeted gene to regulate the Warburg effect. KIAA1429 depletion decreased Warburg effect and increased sorafenib sensitivity in liver cancer. Mechanistically, KIAA1429 could affect the m6A level of HK1 mRNA through directly binding with it. Moreover, KIAA1429 cooperated with the m6A reader HuR to enhance HK1 mRNA stability, thereby upregulating its expression. These findings demonstrated that KIAA1429/HK1 axis decreases the sensitivity of liver cancer cells to sorafenib by regulating the Warburg effect, which may provide a novel therapeutic target for liver cancer treatment.
Assuntos
Antineoplásicos , Hexoquinase , Neoplasias Hepáticas , Camundongos Nus , Sorafenibe , Efeito Warburg em Oncologia , Humanos , Sorafenibe/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Camundongos , Efeito Warburg em Oncologia/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Hexoquinase/metabolismo , Hexoquinase/genética , Camundongos Endogâmicos BALB C , Linhagem Celular Tumoral , MasculinoRESUMO
AIMS: The intricate molecular mechanisms underlying estrogen receptor-positive (ER+) breast carcinogenesis and resistance to endocrine therapy remain elusive. In this study, we elucidate the pivotal role of GPR81, a G protein-coupled receptor, in ER+ breast cancer (BC) by demonstrating low expression of GPR81 in tamoxifen (TAM)-resistant ER+ BC cell lines and tumor samples, along with the underlying molecular mechanisms. MAIN METHODS: Fatty acid oxidation (FAO) levels and lipid accumulation were explored using MDA and FAßO assay, BODIPY 493/503 staining, and Lipid TOX staining. Autophagy levels were assayed using CYTO-ID detection and Western blotting. The impact of GPR81 on TAM resistance in BC was investigated through CCK8 assay, colony formation assay and a xenograft mice model. RESULTS: Aberrantly low GPR81 expression in TAM-resistant BC cells disrupts the Rap1 pathway, leading to the upregulation of PPARα and CPT1. This elevation in PPARα/CPT1 enhances FAO, impedes lipid accumulation and lipid droplet (LD) formation, and subsequently inhibits cell autophagy, ultimately promoting TAM-resistant BC cell growth. Moreover, targeting GPR81 and FAO emerges as a promising therapeutic strategy, as the GPR81 agonist and the CPT1 inhibitor etomoxir effectively inhibit ER+ BC cell and tumor growth in vivo, re-sensitizing TAM-resistant ER+ cells to TAM treatment. CONCLUSION: Our data highlight the critical and functionally significant role of GPR81 in promoting ER+ breast tumorigenesis and resistance to endocrine therapy. GPR81 and FAO levels show potential as diagnostic biomarkers and therapeutic targets in clinical settings for TAM-resistant ER+ BC.
Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Ácidos Graxos , Camundongos Nus , Oxirredução , PPAR alfa , Receptores Acoplados a Proteínas G , Tamoxifeno , Tamoxifeno/farmacologia , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Feminino , Receptores Acoplados a Proteínas G/metabolismo , Animais , Ácidos Graxos/metabolismo , Camundongos , PPAR alfa/metabolismo , Antineoplásicos Hormonais/farmacologia , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Autofagia/efeitos dos fármacos , Camundongos Endogâmicos BALB CRESUMO
Pirarubicin (THP) is a new generation of cell cycle non-specific anthracycline-based anticancer drug. In the clinic, THP and THP combination therapies have been shown to be effective in hepatocellular carcinoma (HCC) patients with transcatheter arterial chemoembolization (TACE) without serious side effects. However, drug resistance limits its therapeutic efficacy. Berberine (BBR), an isoquinoline alkaloid, has been shown to possess antitumour properties against various malignancies. However, the synergistic effect of BBR and THP in the treatment of HCC is unknown. In the present study, we demonstrated for the first time that BBR sensitized HCC cells to THP, including enhancing THP-induced growth inhibition and apoptosis of HCC cells. Moreover, we found that BBR sensitized THP by reducing the expression of autophagy-related 4B (ATG4B). Mechanistically, the inhibition of HIF1α-mediated ATG4B transcription by BBR ultimately led to attenuation of THP-induced cytoprotective autophagy, accompanied by enhanced growth inhibition and apoptosis in THP-treated HCC cells. Tumor-bearing experiments in nude mice showed that the combination treatment with BBR and THP significantly suppressed the growth of HCC xenografts. These results reveal that BBR is able to strengthen the killing effect of THP on HCC cells by repressing the ATG4B-autophagy pathway, which may provide novel insights into the improvement of chemotherapeutic efficacy of THP, and may be conducive to the further clinical application of THP in HCC treatment.
Assuntos
Apoptose , Proteínas Relacionadas à Autofagia , Autofagia , Berberina , Carcinoma Hepatocelular , Doxorrubicina , Neoplasias Hepáticas , Camundongos Nus , Berberina/farmacologia , Berberina/análogos & derivados , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Autofagia/efeitos dos fármacos , Animais , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Camundongos , Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Doxorrubicina/análogos & derivados , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Camundongos Endogâmicos BALB C , Antineoplásicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Cisteína EndopeptidasesRESUMO
Persistent activation of estrogen receptor alpha (ERα)-mediated estrogen signaling plays a pivotal role in driving the progression of estrogen receptor positive (ER+) breast cancer (BC). In the current study, LINC00173, a long non-coding RNA, was found to bind both ERα and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFα) factor (LITAF), then cooperatively to inhibit ERα protein degradation by impeding the nuclear export of ERα. Concurrently, LITAF was found to attenuate TNFα transcription after binding to LINC00173, and this attenuating transcriptional effect was quite significant under lipopolysaccharide stimulation. Distinct functional disparities between estrogen subtypes emerge, with estradiol synergistically promoting ER+ BC cell growth with LINC00173, while estrone (E1) facilitated LITAF-transcriptional activation. In terms of therapeutic significance, silencing LINC00173 alongside moderate addition of E1 heightened TNFα and induced apoptosis, effectively inhibiting ER+ BC progression.
Assuntos
Neoplasias da Mama , Receptor alfa de Estrogênio , Estrona , RNA Longo não Codificante , Fatores de Transcrição , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Estrona/metabolismo , Estrona/farmacologia , Estrona/análogos & derivados , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/metabolismo , Células MCF-7 , Linhagem Celular Tumoral , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Animais , Camundongos , Inativação GênicaRESUMO
BACKGROUND: Sorafenib is the first-line therapy for patients with advanced-stage HCC, but its clinical cure rate is unsatisfactory due to adverse reactions and drug resistance. Novel alternative strategies to overcome sorafenib resistance are urgently needed. Oxyberberine (OBB), a major metabolite of berberine in vivo, exhibits potential antitumor potency in various human malignancies, including liver cancer. However, it remains unknown whether and how OBB sensitizes liver cancer cells to sorafenib. METHODS: Cell viability, trypan blue staining and flow cytometry assays were employed to determine the synergistic effect of OBB and sorafenib on killing HCC cells. PCR, western blot, co-immunoprecipitation and RNA interference assays were used to decipher the mechanism by which OBB sensitizes sorafenib. HCC xenograft models and clinical HCC samples were utilized to consolidate our findings. RESULTS: We found for the first time that OBB sensitized liver cancer cells to sorafenib, enhancing its inhibitory effect on cell growth and induction of apoptosis in vitro. Interestingly, we observed that OBB enhanced the sensitivity of HCC cells to sorafenib by reducing ubiquitin-specific peptidase 7 (USP7) expression, a well-known tumor-promoting gene. Mechanistically, OBB inhibited notch homolog 1-mediated USP7 transcription, leading to the downregulation of V-Myc avian myelocytomatosis viral oncogene homolog (c-Myc), which synergized with sorafenib to suppress liver cancer. Furthermore, animal results showed that cotreatment with OBB and sorafenib significantly inhibited the tumor growth of liver cancer xenografts in mice. CONCLUSIONS: These results indicate that OBB enhances the sensitivity of liver cancer cells to sorafenib through inhibiting notch homolog 1-USP7-c-Myc signaling pathway, which potentially provides a novel therapeutic strategy for liver cancer to improve the effectiveness of sorafenib.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animais , Camundongos , Sorafenibe/farmacologia , Peptidase 7 Específica de Ubiquitina/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/farmacologia , Transdução de Sinais , Linhagem Celular Tumoral , Receptor Notch1/uso terapêuticoRESUMO
N6-methyladenosine (m6A), a dynamically reversible modification in eukaryotic RNAs, modulates gene expression and pathological processes in various tumors. KIAA1429, the largest component of the m6A methyltransferase complex, plays an important role in m6A modification. However, the underlying mechanism of KIAA1429 in hepatocellular carcinoma (HCC) remains largely unknown. Immunohistochemical assay was performed to examine the expression of KIAA1429 in HCC tissues. Transwell, wound healing and animal experiments were used to investigate the influence of KIAA1429 on cell migration and invasion. The mRNA high-throughput sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) were performed to screen the downstream target of KIAA1429. RNA stability assays, RNA immunoprecipitation assay (RIP), MeRIP-qPCR and luciferase assay were used to evaluate the relationship between KIAA1429 and the m6A-modified genes. Results showed that the expression level of KIAA1429 was significantly higher in HCC tissues than in adjacent tissues, and the upregulation of KIAA1429 could promote HCC metastasis in vitro and in vivo. Mechanistically, we confirmed that KIAA1429 negatively regulated the tumor suppressor, Rho family GTPase 3 (RND3), by decreasing its mRNA stability in coordination with the m6A reader YTHDC1. Moreover, we demonstrated that KIAA1429 could regulate the m6A modification of RND3 mRNA via its RNA binding domain. Our data indicated that KIAA1429 exerted its oncogenic role by inhibiting RND3 expression in an m6A-dependent manner, suggesting that KIAA1429 might be a potential prognostic biomarker and therapeutic target in HCC.
Assuntos
Adenina , Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Adenina/análogos & derivados , Carcinoma Hepatocelular/genética , Regulação para Baixo , Neoplasias Hepáticas/genética , RNA , RNA Mensageiro , HumanosRESUMO
Sorafenib is currently the first-line treatment for patients with advanced liver cancer, but its therapeutic efficacy declines significantly after a few months of treatment. Therefore, it is of great importance to investigate the regulatory mechanisms of sorafenib sensitivity in liver cancer cells. In this study, we provided initial evidence demonstrating that circPHKB, a novel circRNA markedly overexpressed in sorafenib-treated liver cancer cells, attenuated the sensitivity of liver cancer cells to sorafenib. Mechanically, circPHKB sequestered miR-1234-3p, resulting in the up-regulation of cytochrome P450 family 2 subfamily W member 1 (CYP2W1), thereby reducing the killing effect of sorafenib on liver cancer cells. Moreover, knockdown of circPHKB sensitized liver cancer cells to sorafenib in vivo. The findings reveal a novel circPHKB/miR-1234-3p/CYP2W1 pathway that decreases the sensitivity of liver cancer cells to sorafenib, suggesting that circPHKB and the axis may serve as promising targets to improve the therapeutic efficacy of sorafenib against liver cancer.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Humanos , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , MicroRNAs/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Regulação para Cima , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Família 2 do Citocromo P450/genéticaRESUMO
Liver cancer is the most common and frequentlyoccurring cancer. In addition to radiotherapy, chemotherapy and surgery are recommended as part of liver cancer treatment. The efficacy of sorafenib and sorafenib-based combination treatment against tumors has been verified. Although, clinical trials have revealed that some individuals are not sensitive to sorafenib therapy, and current therapeutic approaches are ineffective. Consequently, it is urgent to explore effective drug combinations and innovative techniques for increasing the effectiveness of sorafenib in the curing of liver tumor. Herein, we show that dihydroergotamine mesylate (DHE), an anti-migraine agent, could effectively suppress liver cancer cells proliferation by inhibiting STAT3 activation. However, DHE can enhance the protein stability of Mcl-1 by activating ERK, making DHE less effective in apoptosis induction. Specifically, DHE enhances the effects of sorafenib on liver cancer cells, such as decreased viability and increased apoptosis. Furthermore, the mixture of sorafenib and DHE could enhance DHE-triggered STAT3 suppression and inhibit DHE-mediated ERK-Mcl-1 pathway activation. In vivo, the combination of sorafenib with DHE produced a substantial synergy in suppressing tumour growth and causing apoptosis, ERK inhibition and Mcl-1 degradation. These findings suggest that DHE can effectively inhibit cell proliferation and enhance sorafenib anti-cancer activity in liver cancer cells. The current study provides some new insights that DHE asa novel anti-liver cancer therapeutic agent has been shown to improve treatment outcomes of sorafenib, which might be helpful in order to advance sorafenib in liver cancer therapeutics.
Assuntos
Di-Hidroergotamina , Neoplasias Hepáticas , Humanos , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Di-Hidroergotamina/farmacologia , Di-Hidroergotamina/uso terapêutico , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias Hepáticas/metabolismo , Apoptose , Linhagem Celular Tumoral , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêuticoRESUMO
Eukaryotes initiate autophagy when facing environmental changes such as a lack of external nutrients. However, the mechanisms of autophagy initiation are still not fully elucidated. Here, we showed that deacetylation of ATG4B plays a key role in starvation-induced autophagy initiation. Specifically, we demonstrated that ATG4B is activated during starvation through deacetylation at K39 by the deacetylase SIRT2. Moreover, starvation triggers SIRT2 dephosphorylation and activation in a cyclin E/CDK2 suppression-dependent manner. Meanwhile, starvation down-regulates p300, leading to a decrease in ATG4B acetylation at K39. K39 deacetylation also enhances the interaction of ATG4B with pro-LC3, which promotes LC3-II formation. Furthermore, an in vivo experiment using Sirt2 knockout mice also confirmed that SIRT2-mediated ATG4B deacetylation at K39 promotes starvation-induced autophagy initiation. In summary, this study reveals an acetylation-dependent regulatory mechanism that controls the role of ATG4B in autophagy initiation in response to nutritional deficiency.
RESUMO
The objective of this study was to examine the effect of autophagy on stress-induced M2 macrophage polarization in the tumor microenvironment of breast cancer and to determine whether the underlying mechanism was related to the reactive oxygen species (ROS)/ERK and mTOR pathway. In vitro, we found that the basal autophagy level in mouse RAW 264.7 macrophages decreased with the incubation of tumor cell culture supernatant. Similarly, the polarization of RAW 264.7 to M2 macrophages was inhibited by the autophagy inducer rapamycin and increased by the autophagy inhibitor 3-MA or by siBeclin1. In addition, we found that not only was M2 molecule expression down-regulated but intracellular ROS generation was also blocked by autophagy induction. In vivo, we observed that mice that received an isoprenaline injection as a stress agent exhibited augmented implanted breast tumor growth, lung metastasis, intratumoral mRNA expression of M2 molecules and serum ROS generation. In contrast, the intratumoral expression of LC3-II and Beclin1 was decreased. In addition, we observed that isoprenaline induced the up-regulation of the intratumoral expression of phosphorylated mTOR, phosphorylated ERK1/2, phosphorylated Tyr705-STAT3 and HIF-1α, whereas rapamycin induced an opposite effect on the same molecules and could abolish the effects of isoprenaline. These results suggest that autophagy might suppress M2 macrophage polarization induced by isoprenaline via the ROS/ERK and mTOR signaling pathway. Our findings provide a theoretical basis for why high levels of stress hormones accelerate the progression of breast cancer, and autophagy may play a role in determining the outcomes of cancer therapy.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Sirolimo/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoproterenol/antagonistas & inibidores , Isoproterenol/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Sistema de Sinalização das MAP Quinases , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Microambiente Tumoral/efeitos dos fármacosRESUMO
In this study, we investigate the effect of miR-34a expression and biological characteristics of breast cancer stem cells (BCSCs). The mammospheres were formed from murine breast cancer cell line 4T1 and regarded as murine BCSCs. Identification of stemness molecules and cloning experiments validate the biological characteristics of BCSCs we have established. We showed that miR-34a, as a tumor suppressor, could separately reduce the stemness of BCSCs and activate the cytotoxic susceptibility of BCSCs to natural killer (NK) cells in vitro via down regulating the expression of Notch1 signaling molecules. Moreover, miR-34a could completely restrain established mice breast tumor xenografts in vivo in the NOD/SCID mice that have functional NK cells at a normal level, whereas it was less effective in NOD/SCID/ CD122/IL-2Rß mice that do not have functional NK cells. We conclude that miR-34a is a crucial, dual tumor suppressor and BCSCs-targeting immunotherapeutic agent and has shown efficacy in the treatment of murine breast cancer. The results also suggest that impaired NK cells could contribute to the resistance to therapies.
Assuntos
MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Neoplasias da Mama , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/patologia , TransfecçãoRESUMO
Fluorescence microscopy of live cells is instrumental in deciphering the molecular details of autophagy. To facilitate the routine examination of yeast Atg proteins under diverse conditions, here we provide a comprehensive tool set, including (1) plasmids for the expression of GFP chimeras at endogenous levels for most Atg proteins, (2) RFP-Atg8 constructs with improved properties as a PAS marker, and (3) plasmids for the complementation of common yeast auxotrophic markers. We hope that the availability of this tool set will further accelerate yeast autophagy research.
Assuntos
Autofagia/fisiologia , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Fluorescência Verde/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Fagossomos/metabolismo , Proteínas Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismoRESUMO
Nitrogen starvation is a universal stimulus of autophagy. At present, little is known about the relationship between carbon metabolism and autophagy under nitrogen starvation. Here, we show that yeast cells continue to consume glucose and downregulate fermentation under nitrogen starvation. Storage lipid production is increased, with concurrent proliferation of lipid droplets. Furthermore, we provide evidence that triacylglycerol synthesis is crucial for autophagosome biogenesis. It is involved in a step downstream of PAS (phagophore assembly site) scaffold assembly, and upstream of the recruitment of Atg1, Atg14, Atg5 and Atg8. Finally, we demonstrate that lipid droplets transiently interact with Atg8-containing membranes. Our study reveals a novel connection linking neutral lipid metabolism, lipid droplets and autophagy.