RESUMEN
Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by progressive loss of dopamine-producing neurons from the substantia nigra region of the brain. Mitochondrial dysfunction is one of the major causes of oxidative stress and neuronal cell death in PD. E3 ubiquitin ligases such as Parkin (PRKN) modulate mitochondrial quality control in PD; however, the role of other E3 ligases associated with mitochondria in the regulation of neuronal cell death in PD has not been explored. The current study investigated the role of TRIM32, RING E3 ligase, in sensitization to oxidative stress-induced neuronal apoptosis. The expression of TRIM32 sensitizes SH-SY5Y dopaminergic cells to rotenone and 6-OHDA-induced neuronal death, whereas the knockdown increased cell viability under PD stress conditions. The turnover of TRIM32 is enhanced under PD stress conditions and is mediated by autophagy. TRIM32 translocation to mitochondria is enhanced under PD stress conditions and localizes on the outer mitochondrial membrane. TRIM32 decreases complex-I assembly and activity as well as mitochondrial reactive oxygen species (ROS) and ATP levels under PD stress. Deletion of the RING domain of TRIM32 enhanced complex I activity and rescued ROS levels and neuronal viability under PD stress conditions. TRIM32 decreases the level of XIAP, and co-expression of XIAP with TRIM32 rescued the PD stress-induced cell death and mitochondrial ROS level. In conclusion, turnover of TRIM32 increases during stress conditions and translocation to mitochondria is enhanced, regulating mitochondrial functions and neuronal apoptosis by modulating the level of XIAP in PD.
Asunto(s)
Apoptosis , Neuronas Dopaminérgicas , Mitocondrias , Estrés Oxidativo , Enfermedad de Parkinson , Especies Reactivas de Oxígeno , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Mitocondrias/genética , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Rotenona/farmacología , Transporte de Proteínas , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Línea Celular Tumoral , Oxidopamina/farmacología , Autofagia , Adenosina Trifosfato/metabolismo , Supervivencia Celular/genéticaRESUMEN
Parkinson's Disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of midbrain dopaminergic neurons in the substantia nigra part of the brain. Pathology spread to numerous brain regions and cell types suggests that intercellular communication is essential to PD progression. Exosomes mediate intercellular communication between neurons, glia, and other cell types throughout PD-relevant brain regions. However, the mechanism remains unclear, and its implication in PD pathology, is not well understood. In the current study, we explored the role of exosomes in modulating the response to PD-relevant toxicants. In cellular models of PD, neuronal cell-derived exosomes are readily internalized by recipient neuronal cells as intact vesicles. Internalized exosomes in bystander neuronal cells localize to mitochondria and dysregulate mitochondrial functions, leading to cell death under PD stress conditions. NGS analysis of exosomes released by neuronal cells subjected to PD stress conditions showed that levels of specific miRNAs were altered in exosomes under PD stress conditions. Bioinformatic analysis of the miRNA targets revealed enriched pathways related to neuronal processes and morphogenesis, apoptosis and ageing. Levels of two miRNAs, hsa-miR-30a-5p and hsa-miR-181c-5p, were downregulated in exosomes under PD stress conditions. Expression of the identified miRNAs in neuronal cells led to their enrichment in exosomes, and exosome uptake in neuronal cells ameliorated mitochondrial dysfunction induced by PD stress conditions and rescued cell death. In conclusion, loss of enrichment of specific miRNAs, including miR-30a-5p and miR-181c-5p, under PD stress conditions causes mitochondrial dysfunction and neuronal death, and hence may lead to progression of PD.
Asunto(s)
MicroARNs , Enfermedades Mitocondriales , Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Muerte Celular , Mitocondrias/metabolismo , Neuronas Dopaminérgicas/metabolismo , Enfermedades Mitocondriales/metabolismoRESUMEN
Tumor microenvironment (TME) of solid tumors including breast cancer is complex and contains a distinct cytokine pattern including TNF-α, which determines the progression and metastasis of breast tumors. The metastatic potential of triple negative breast cancer subtypes is high as compared to other subtypes of breast cancer. NF-κB is key transcription factor regulating inflammation and mitochondrial bioenergetics including oxidative phosphorylation (OXPHOS) genes which determine its oxidative capacity and generating reducing equivalents for synthesis of key metabolites for proliferating breast cancer cells. The differential metabolic adaptation and OXPHOS function of breast cancer subtypes in inflammatory conditions and its contribution to metastasis is not well understood. Here we demonstrated that different subunits of NF-κB are differentially expressed in subtypes of breast cancer patients. RELA, one of the major subunits in regulation of the NF-κB pathway is positively correlated with high level of TNF-α in breast cancer patients. TNF-α induced NF-κB regulates the expression of LYRM7, an assembly factor for mitochondrial complex III. Downregulation of LYRM7 in MDA-MB-231 cells decreases mitochondrial super complex assembly and enhances ROS levels, which increases the invasion and migration potential of these cells. Further, in vivo studies using Infliximab, a monoclonal antibody against TNF-α showed decreased expression of LYRM7 in tumor tissue. Large scale breast cancer databases and human patient samples revealed that LYRM7 levels decreased in triple negative breast cancer patients compared to other subtypes and is determinant of survival outcome in patients. Our results indicate that TNF-α induced NF-κB is a critical regulator of LYRM7, a major factor for modulating mitochondrial functions under inflammatory conditions, which determines growth and survival of breast cancer cells.
Asunto(s)
Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Femenino , Humanos , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/genética , Microambiente Tumoral , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by dopaminergic neuronal cell death. Emerging evidence suggest exosomes as a crucial player in the progression and pathogenesis of PD via intercellular communication between different cell types in brain. Exosome release is enhanced from dysfunctional neurons/glia (source cells) under PD stress and mediates the transfer of biomolecules between different cell types (recipient) in brain leading to unique functional outcomes. Exosome release is modulated by alterations in the autophagy and lysosomal pathways; however, the molecular factors regulating these pathways remain elusive. Micro-RNAs (miRNAs) are class of non-coding RNAs that regulate gene expression post-transcriptionally by binding target mRNA and modulate its turnover and translation; however their role in modulating exosome release is not understood. Here, we analyzed the miRNAs-mRNAs network which target cellular processes regulating exosome release. hsa-miR-320a showed the maximum mRNA targets of autophagy, lysosome, mitochondria and exosome release pathways. hsa-miR-320a regulate ATG5 levels and modulate exosome release under PD stress conditions in neuronal SH-SY5Y and glial U-87 MG cells. hsa-miR-320a modulates autophagic flux, lysosomal functions, and mitochondrial ROS in neuronal SH-SY5Y and glial U-87 MG cells. Exosomes derived from hsa-miR-320a expressing source cells under PD stress conditions were actively internalized in the recipient cells and rescued cell death and mitochondrial ROS. These results suggest that hsa-miR-320a regulates autophagy and lysosomal pathways and modulates exosome release in the source cells and derived exosomes under PD stress conditions rescue cell death and mitochondrial ROS in the recipient neuronal and glial cells.
Asunto(s)
Exosomas , MicroARNs , Neuroblastoma , Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Exosomas/metabolismo , Neuroblastoma/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Neuronas/metabolismo , Muerte Celular , Neuroglía/metabolismo , ARN Mensajero/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismoRESUMEN
Triple-negative breast cancer is aggressive and metastatic breast cancer type and shows immune evasion, drug resistance, relapse and poor survival. Anti-cancer therapy like ionizing radiation and chemotherapeutic drug majorly induces DNA damage hence, alteration in DNA damage repair and downstream pathways may contribute to tumor cell survival. DNA damage during chemotherapy is sensed by cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes (STING), which determines the anti-tumor immune response by modulating the expression of programmed cell death ligand-1 (PD-L1), immune suppressor, in the tumor microenvironment. Triple-negative breast cancer cells are cGAS-STING positive and modulation of this pathway during DNA damage response for survival and immune escape mechanism is not well understood. Here we demonstrate that doxorubicin-mediated DNA damage induces STING mediated NF-κB activation in triple-negative as compared to ER/PR positive breast cancer cells. STING-mediated NF-κB induces the expression of IL-6 in triple-negative breast cancer cells and activates pSTAT3, which enhances cell survival and PD-L1 expression. Doxorubicin and STAT3 inhibitor act synergistically and inhibit cell survival and clonogenicity in triple-negative breast cancer cells. Knockdown of STING in triple-negative breast cancer cells enhances CD8 mediated immune cell death of breast cancer cells. The combinatorial treatment of triple-negative breast cells with doxorubicin and STAT3 inhibitor reduces PD-L1 expression and activates immune cell-mediated cancer cell death. Further STING and IL-6 levels show a positive correlation in breast cancer patients and poor survival outcomes. The study here strongly suggests that STING mediated activation of NF-κB enhances IL-6 mediated STAT3 in triple-negative breast cancer cells which induces cell survival and immune-suppressive mechanism.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Apoptosis , Antígeno B7-H1 , Daño del ADN/genética , Doxorrubicina/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Recurrencia Local de Neoplasia , FN-kappa B/genética , FN-kappa B/metabolismo , Nucleotidiltransferasas , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Microambiente TumoralRESUMEN
Ubiquitin E3-ligases are recruited at different steps of TNF-α-induced NF-κB activation; however, their role in temporal regulation of the pathway remains elusive. The study systematically identified TRIMs as potential feedback regulators of the TNF-α-induced NF-κB pathway. We further observed that TRIM15 is "late" response TNF-α-induced gene and inhibits the TNF-α-induced NF-κB pathway in several human cell lines. TRIM15 promotes turnover of K63-linked ubiquitin chains in a PRY/SPRY domain-dependent manner. TRIM15 interacts with TAK1 and inhibits its K63-linked ubiquitination, thus NF-κB activity. Further, TRIM15 interacts with TRIM8 and inhibits cytosolic translocation to antagonize TRIM8 modualted NF-κB. TRIM8 and TRIM15 also show functionally inverse correlation in psoriasis condition. In conclusion, TRIM15 is TNF-α-induced late response gene and inhibits TNF-α induced NF-κB pathway hence a feedback modulator to keep the proinflammatory NF-κB pathway under control.
Asunto(s)
FN-kappa B , Ubiquitina-Proteína Ligasas , Proteínas Portadoras/metabolismo , Humanos , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: Tumor necrosis factor-α (TNF-α) is an immunostimulatory cytokine that is consistently high in the breast tumor microenvironment (TME); however, its differential role in mitochondrial functions and cell survival in ER/PR +ve and ER/PR -ve breast cancer cells is not well understood. METHODS: In the current study, we investigated TNF-α modulated mitochondrial proteome using high-resolution mass spectrometry and identified the differentially expressed proteins in two different breast cancer cell lines, ER/PR positive cell line; luminal, MCF-7 and ER/PR negative cell line; basal-like, MDA-MB-231 and explored its implication in regulating the tumorigenic potential of breast cancer cells. We also compared the activity of mitochondrial complexes, ATP, and ROS levels between MCF-7 and MDA-MB-231 in the presence of TNF-α. We used Tumor Immune Estimation Resource (TIMER) webserver to analyze the correlation between TNF-α and mitochondrial proteins in basal and luminal breast cancer patients. Kaplan-Meier method was used to analyze the correlation between mitochondrial protein expression and survival of breast cancer patients. RESULTS: The proteome analysis revealed that TNF-α differentially altered the level of critical proteins of mitochondrial respiratory chain complexes both in MCF-7 and MDA-MB-231, which correlated with differential assembly and activity of mitochondrial ETC complexes. The inhibition of the glycolytic pathway in the presence of TNF-α showed that glycolysis is indispensable for the proliferation and clonogenic ability of MDA-MB-231 cells (ER/PR -ve) as compared to MCF-7 cells (ER/PR +ve). The TIMER database showed a negative correlation between the expressions of TNF-α and key regulators of mitochondrial OXPHOS complexes in basal breast vs lobular carcinoma. Conversely, patient survival analysis showed an improved relapse-free survival with increased expression of identified proteins of ETC complexes and survival of the breast cancer patients. CONCLUSION: The evidence presented in our study convincingly demonstrates that TNF-α regulates the survival and proliferation of aggressive tumor cells by modulating the levels of critical assembly factors and subunits involved in mitochondrial respiratory chain supercomplexes organization and function. This favors the rewiring of mitochondrial metabolism towards anaplerosis to support the survival and proliferation of breast cancer cells. Collectively, the results strongly suggest that TNF-α differentially regulates metabolic adaptation in ER/PR +ve (MCF-7) and ER/PR -ve (MDA-MB-231) cells by modulating the mitochondrial supercomplex assembly and activity.
RESUMEN
Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of metastasis including breast cancer. Chromosomal instability in highly metastatic cells releases fragmented chromosomal parts in the cytoplasm, hence the activation of STING via an increased level of cyclic dinucleotides (cDNs) synthesized by cGMP-AMP synthase (cGAS). Cyclic dinucleotides 2' 3'-cGAMP and it's analog can potentially activate STING mediated pathways leading to nuclear translocation of p65 and IRF-3 and transcription of inflammatory genes. The differential modulation of STING pathway via 2' 3'-cGAMP and its analog and its implication in breast tumorigenesis is still not well explored. In the current study, we demonstrated that c-di-AMP can activate type-1 IFN response in ER negative breast cancer cell lines which correlate with STING expression. c-di-AMP binds to STING and activates downstream IFN pathways in STING positive metastatic MDA-MB-231/MX-1 cells. Prolonged treatment of c-di-AMP induces cell death in STING positive metastatic MDA-MB-231/MX-1 cells mediated by IRF-3. c-di-AMP induces IRF-3 translocation to mitochondria and initiates Caspase-9 mediated cell death and inhibits clonogenicity of triple-negative breast cancer cells. This study suggests that c-di-AMP can activate and modulates STING pathway to induce mitochondrial mediated apoptosis in estrogen-receptor negative breast cancer cells.
Asunto(s)
Muerte Celular/efectos de los fármacos , Fosfatos de Dinucleósidos/farmacología , Proteínas de la Membrana/metabolismo , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Fosfatos de Dinucleósidos/metabolismo , Humanos , Inmunidad Innata/efectos de los fármacos , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Unión Proteica , Receptores de Progesterona/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder caused by an expansion of 55 to 200 CGG repeats located within 5'UTR of FMR1.These CGG repeats are transcribed into RNAs, which sequester several RNA binding proteins and alter the processing of miRNAs. CGG repeats are also translated into a toxic polyglycine-containing protein, FMRpolyG, that affects mitochondrial and nuclear functions reported in cell and animal models and patient studies. Nuclear-encoded small non-coding RNAs, including miRNAs, are transported to mitochondria; however, the role of mitochondrial miRNAs in FXTAS pathogenesis is not understood. Here, we analyzed mitochondrial miRNAs from HEK293 cells expressing expanded CGG repeats and their implication in the regulation of mitochondrial functions. The analysis of next generation sequencing (NGS) data of small RNAs from HEK293 cells expressing CGG premutation showed decreased level of cellular miRNAs and an altered pattern of association of miRNAs with mitochondria (mito-miRs). Among such mito-miRs, miR-320a was highly enriched in mitoplast and RNA immunoprecipitation of Ago2 (Argonaute-2) followed by Droplet digital PCR (ddPCR)suggested that miR-320a may form a complex with Ago2 and mitotranscripts. Finally, transfection of miR-320a mimic in cells expressing CGG permutation recovers mitochondrial functions and rescues cell death. Overall, this work reveals an altered translocation of miRNAs to mitochondria and the role of miR-320a in FXTAS pathology.
Asunto(s)
MicroARNs , Temblor , Animales , Ataxia , Muerte Celular , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil , Células HEK293 , Humanos , MicroARNs/genética , Mitocondrias/genéticaRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) pars compacta region of the brain. The main pathological hallmark involves cytoplasmic inclusions of α-synuclein and mitochondrial dysfunction, which is observed in other part of the central nervous system other than SN suggesting the spread of pathogenesis to bystander neurons. The inter-neuronal communication through exosomes may play an important role in the spread of the disease; however, the mechanisms are not well elucidated. Mitochondria and its role in inter-organellar crosstalk with multivesicular body (MVB) and lysosome and its role in modulation of exosome release in PD is not well understood. In the current study, we investigated the mitochondria-lysosome crosstalk modulating the exosome release in neuronal and glial cells. We observed that PD stress showed enhanced release of exosomes in dopaminergic neurons and glial cells. The PD stress condition in these cells showed fragmented network and mitochondrial dysfunction which further leads to functional deficit of lysosomes and hence inhibition of autophagy flux. Neuronal and glial cells treated with rapamycin showed enhanced autophagy and inhibited the exosomal release. The results here suggest that maintenance of mitochondrial function is important for the lysosomal function and hence exosomal release which is important for the pathogenesis of PD.
Asunto(s)
Exosomas/metabolismo , Lisosomas/metabolismo , Mitocondrias/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Estrés Fisiológico , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Exosomas/efectos de los fármacos , Humanos , Lisosomas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Sirolimus/farmacología , Estrés Fisiológico/efectos de los fármacosRESUMEN
PSMD10Gankyrin, a proteasome assembly chaperone, is a widely known oncoprotein which aspects many hall mark properties of cancer. However, except proteasome assembly chaperon function its role in normal cell function remains unknown. To address this issue, we induced PSMD10Gankyrin overexpression in HEK293 cells and the resultant large-scale changes in gene expression profile were analyzed. We constituted networks from microarray data of these differentially expressed genes and carried out extensive topological analyses. The overrecurring yet consistent theme that appeared throughout analysis using varied network metrics is that all genes and interactions identified as important would be involved in neurogenesis and neuronal development. Intrigued we tested the possibility that PSMD10Gankyrin may be strongly associated with cell fate decisions that commit neural stem cells to differentiate into neurons. Overexpression of PSMD10Gankyrin in human neural progenitor cells facilitated neuronal differentiation via ß-catenin Ngn1 pathway. Here for the first time we provide preliminary and yet compelling experimental evidence for the involvement of a potential oncoprotein - PSMD10Gankyrin, in neuronal differentiation.