RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of any cancer in the United States. Our previous work has shown that autophagy can promote PDAC progression. We recently established the importance of autophagy in regulating bioavailable iron to control mitochondrial metabolism in PDAC. We found that inhibition of autophagy in PDAC leads to mitochondrial dysfunction due to abrogation of succinate dehydrogenase complex iron sulfur subunit B (SDHB) expression. Additionally, we observed that cancer-associated fibroblasts (CAFs) can provide iron to autophagy-inhibited PDAC tumor cells, thereby increasing their resistance to autophagy inhibition. To impede such metabolic compensation, we used a low iron diet together with autophagy inhibition and demonstrated a significant improvement of tumor response in syngeneic PDAC models.Abbreviations: PDAC: Pancreatic ductal adenocarcinoma; CAFs: cancer-associated fibroblasts; SDHB: succinate dehydrogenase complex iron sulfur subunit B; ISCA1: iron sulfur cluster assembly protein 1; FPN: ferroportin; LIP: labile iron pool; FAC: ferric ammonium chloride; OCR: oxygen consumption rate; OXPHOS: oxidative phosphorylation, IL6: interleukin 6; Fe-S: iron sulfur; ATP: adenosine triphosphate.
RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) cells maintain a high level of autophagy, allowing them to thrive in an austere microenvironment. However, the processes through which autophagy promotes PDAC growth and survival are still not fully understood. Here, we show that autophagy inhibition in PDAC alters mitochondrial function by losing succinate dehydrogenase complex iron sulfur subunit B expression by limiting the availability of the labile iron pool. PDAC uses autophagy to maintain iron homeostasis, while other tumor types assessed require macropinocytosis, with autophagy being dispensable. We observed that cancer-associated fibroblasts can provide bioavailable iron to PDAC cells, promoting resistance to autophagy ablation. To overcome this cross-talk, we used a low-iron diet and demonstrated that this augmented the response to autophagy inhibition therapy in PDAC-bearing mice. Our work highlights a critical link between autophagy, iron metabolism, and mitochondrial function that may have implications for PDAC progression.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Ratones , Línea Celular Tumoral , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/metabolismo , Autofagia , Homeostasis , Mitocondrias/metabolismo , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Mitophagy regulates cancer stem cell (CSC) populations affecting tumorigenicity and malignancy in various cancer types. Here, we report that cisplatin treatment led to the activation of higher mitophagy through regulating CLU (clusterin) levels in oral CSCs. Moreover, both the gain-of-function and loss-of-function of CLU indicated its mitophagy-specific role in clearing damaged mitochondria. CLU also regulates mitochondrial fission by activating the Ser/Thr kinase AKT, which triggered phosphorylation of DNM1L/Drp1 at the serine 616 residue initiating mitochondrial fission. More importantly, we also demonstrated that CLU-mediated mitophagy positively regulates oral CSCs through mitophagic degradation of MSX2 (msh homeobox 2), preventing its nuclear translocation from suppressing SOX2 activity and subsequent inhibition of cancer stemness and self-renewal ability. However, CLU knockdown disturbed mitochondrial metabolism generating excessive mitochondrial superoxide, which improves the sensitivity to cisplatin in oral CSCs. Notably, our results showed that CLU-mediated cytoprotection relies on SOX2 expression. SOX2 inhibition through genetic (shSOX2) and pharmacological (KRX-0401) strategies reverses CLU-mediated cytoprotection, sensitizing oral CSCs toward cisplatin-mediated cell death.
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Neoplasias , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-akt/metabolismo , Clusterina/genética , Clusterina/metabolismo , Cisplatino/farmacología , Autofagia , Células Madre Neoplásicas/metabolismo , Dinámicas Mitocondriales/genética , Neoplasias/metabolismoRESUMEN
Autophagy, a lysosomal catabolic process, involves degradation of cellular materials, protein aggregate, and dysfunctional organelles to maintain cellular homeostasis. Strikingly, autophagy exhibits a dual-sided role in cancer; on the one hand, it promotes clearance of transformed cells and inhibits tumorigenesis, while cytoprotective autophagy has a role in sustaining cancer. The autophagy signaling in the tumor microenvironment (TME) during cancer growth and therapy is not adequately understood. The review highlights the role of autophagy signaling pathways to support cancer growth and progression in adaptation to the oxidative and hypoxic context of TME. Furthermore, autophagy contributes to regulating the metabolic switch for generating sufficient levels of high-energy metabolites, including amino acids, ketones, glutamine, and free fatty acids for cancer cell survival. Interestingly, autophagy has a critical role in modulating the tumor-associated fibroblast resulting in different cytokines and paracrine signaling mediated angiogenesis and invasion of pre-metastatic niches to secondary tumor sites. Moreover, autophagy promotes immune evasion to inhibit antitumor immunity, and autophagy inhibitors enhance response to immunotherapy with infiltration of immune cells to the TME niche. Furthermore, autophagy in TME maintains and supports the survival of cancer stem cells resulting in chemoresistance and therapy recurrence. Presently, drug repurposing has enabled the use of lysosomal inhibitor-based antimalarial drugs like chloroquine and hydroxychloroquine as clinically available autophagy inhibitors in cancer therapy. We focus on the recent developments of multiple autophagy modulators from pre-clinical trials and the challenges in developing autophagy-based cancer therapy.
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Neoplasias , Microambiente Tumoral , Humanos , Autofagia , Neoplasias/patología , Transducción de Señal , Neovascularización Patológica/metabolismoAsunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Presentación de Antígeno , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares/tratamiento farmacológicoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer. The elevated macroautophagy/autophagy in these tumors supports growth, promotes immune evasion, and increases therapeutic resistance. Therefore, targeting autophagy is a therapeutic strategy that is being pursued to treat PDAC patients. Whereas autophagy inhibition impairs mitochondrial metabolism in PDAC, the specific metabolite(s) that becomes limiting when autophagy is inhibited has not been identified. We report that loss of autophagy specifically results in intracellular cysteine depletion under nutrient-replete conditions. Mechanistically, we show that PDAC cells utilize the autophagy machinery to regulate the activity and localization of the cystine transporter SLC7A11 at the plasma membrane. Upon inhibition of autophagy, SLC7A11 is localized to lysosomes in an MTORC2-dependent manner. Our findings reveal a novel connection between autophagy and cysteine metabolism in pancreatic cancer.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Sistema de Transporte de Aminoácidos y+ , Autofagia , Línea Celular Tumoral , Cisteína , HumanosRESUMEN
The evolution of the CRISPR-Cas9 technology in cancer research has tremendous potential to shape the future of oncology. Although this gene-editing tool's pre-clinical progress is into its nascent stage, there are many unanswered questions regarding health benefits and therapy precision using CRISPR. The application of CRISPR is highly specific, economically sustainable, and is a high throughput technique, but on the other hand, its application involves measured risk of countering the toxic immune response of Cas protein, off-target effects, limitation of delivering the edited cells back into cancer patients. The current chapter highlights the possibilities and perils of the present-day CRISPR engineering in cancer that should highlight CRISPR translation to therapy.
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Sistemas CRISPR-Cas , Neoplasias , Sistemas CRISPR-Cas/genética , Edición Génica , Humanos , Neoplasias/genética , Neoplasias/terapia , TecnologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is highly refractory to current therapies. We had previously shown that PDAC can utilize its high levels of basal autophagy to support its metabolism and maintain tumor growth. Consistent with the importance of autophagy in PDAC, autophagy inhibition significantly enhances response of PDAC patients to chemotherapy in two randomized clinical trials. However, the specific metabolite(s) that autophagy provides to support PDAC growth is not yet known. In this study, we demonstrate that under nutrient-replete conditions, loss of autophagy in PDAC leads to a relatively restricted impairment of amino acid pools, with cysteine levels showing a significant drop. Additionally, we made the striking discovery that autophagy is critical for the proper membrane localization of the cystine transporter SLC7A11. Mechanistically, autophagy impairment results in the loss of SLC7A11 on the plasma membrane and increases its localization at the lysosome in an mTORC2-dependent manner. Our results demonstrate a critical link between autophagy and cysteine metabolism and provide mechanistic insights into how targeting autophagy can cause metabolic dysregulation in PDAC.
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Adenocarcinoma/genética , Sistema de Transporte de Aminoácidos y+/genética , Carcinoma Ductal Pancreático/genética , Proliferación Celular/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Autofagia/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Xenoinjertos , Homeostasis/genética , Humanos , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
AIMS: Secretory clusterin (sCLU) plays an important role in tumor development and cancer progression. However, the molecular mechanisms and physiological functions of sCLU in oral cancer is unclear. We examined the impact of sCLU-mediated autophagy in cell survival and apoptosis inhibition in oral cancer. MAIN METHODS: Immunohistochemical analysis was performed to analyze protein expression in patient samples. Autophagy and mitophagy was studied by immunofluorescence microscopy and Western blot. The gain and loss of function was studied by overexpression of plasmid and siRNA approaches respectively. Cellular protection against nutrient starvation and therapeutic stress by sCLU was studied by cell viability, caspase assay and meta-analysis. KEY FINDINGS: The data from oral cancer patients showed that the expression levels of sCLU, ATG14, ULK1, and PARKIN increased in grade-wise manners. Interestingly, sCLU overexpression promoted autophagy through AMPK/Akt/mTOR signaling pathway leading to cell survival and protection from long exposure serum starvation induced-apoptosis. Additionally, sCLU was demonstrated to interact with ULK1 and inhibition of ULK1 activity by SBI206965 was found to abolish sCLU-induced autophagy indicating critical role of ULK1 in induction of autophagy. Furthermore, sCLU was observed to promote expression of mitophagy-associated proteins in serum starvation conditions to protect cells from nutrient deprivation. The meta-analysis elucidated that high CLU expression is associated with therapy resistance in cancer and we demonstrated that sCLU-mediated mitophagy was revealed to inhibit cell death by cisplatin. SIGNIFICANCE: The present investigation has highlighted the probable implications of the clusterin-induced autophagy in cell survival and inhibition of apoptosis in oral cancer.
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Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia , Clusterina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias de la Boca/patología , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Apoptosis/genética , Autofagia/genética , Línea Celular Tumoral , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Mitofagia/genética , Neoplasias de la Boca/genética , Neoplasias de Células Escamosas/genética , Neoplasias de Células Escamosas/patologíaRESUMEN
Autophagy can either be cytoprotective or promote cell death in a context-dependent manner in response to stress. How autophagy leads to autophagy dependent cell death requires further clarification. In this study, we document a nonlinear roller coaster form of autophagy oscillation when cells are subjected to different stress conditions. Serum starvation induces an initial primary autophagic peak at 6 h, that helps to replenish cells with de novo fluxed nutrients, but protracted stress lead to a secondary autophagic peak around 48 h. Time kinetic studies indicate that the primary autophagic peak is reversible, whereas the secondary autophagic peak is irreversible and leads to cell death. Key players involved in different stages of autophagy including initiation, elongation and degradation during this oscillatory sequence were identified. A similar molecular pattern was intensified under apoptosis-deficient conditions. mTOR was the central molecule regulating this autophagic activity, and upon knockdown a steady increase of autophagy without any non-linear fluctuation was evident. An unbiased proteome screening approach was employed to identify the autophagy molecules potentially regulating these autophagic peaks. Our proteomics analysis has identified Annexin A2 as a stress-induced protein to implicate in autophagy fluctuation and its deficiency reduced autophagy. Moreover, we report that mTOR in its phosphorylated condition interacts with Annexin A2 to induce autophagy fluctuation by altering its cellular localization. The work highlights the molecular mechanism of a mTOR-dependent roller coaster fluctuation of autophagy and autophagy dependent cell death during prolong stress.
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Anexina A2/metabolismo , Autofagia , Estrés Fisiológico , Serina-Treonina Quinasas TOR/metabolismo , Células A549 , Células HeLa , Humanos , Fosforilación , Células Tumorales CultivadasRESUMEN
Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy1-3. However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB4, mutations that cause loss of MHC-I are rarely found5 despite the frequent downregulation of MHC-I expression6-8. Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8+ T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC.
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Adenocarcinoma/inmunología , Autofagia/inmunología , Carcinoma Ductal Pancreático/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias Pancreáticas/inmunología , Escape del Tumor/inmunología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Autofagia/efectos de los fármacos , Autofagia/genética , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/inmunología , Línea Celular Tumoral , Cloroquina/farmacología , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Escape del Tumor/efectos de los fármacosRESUMEN
Therapy-induced senescence in cancer cells is an irreversible antiproliferative state, which inhibits tumor growth and is therefore a potent anti-neoplastic mechanism. In this study, low doses of Abrus agglutinin (AGG)-induced senescence through autophagy in prostate carcinoma cells (PC3) and inhibited proliferation. The inhibition of autophagy with 3-methyl adenine reversed AGG-induced senescence, thus confirming that AGG-triggered senescence required autophagy. AGG treatment also led to lipophagy-mediated accumulation of free fatty acids (FFAs), with a concomitant decrease in the number of lipid droplets. Lalistat, a lysosomal acid lipase inhibitor, abrogated AGG-induced lipophagy and senescence in PC3 cells, indicating that lipophagy is essential for AGG-induced senescence. The accumulation of FFAs increased reactive oxygen species generation, a known facilitator of senescence, which was also reduced in the presence of lalistat. Furthermore, AGG upregulated silent mating type information regulator 2 homolog 1 (SIRT1), while the presence of sirtinol reduced autophagy flux and the senescent phenotype in the AGG-treated cells. Mechanistically, AGG-induced cytoplasmic SIRT1 deacetylated a Lys residue on the cytoplasmic domain of lysosome-associated membrane protein 1 (LAMP1), an autolysosomal protein, resulting in lipophagy and senescence. Taken together, our findings demonstrate a novel SIRT1/LAMP1/lipophagy axis mediating AGG-induced senescence in prostate cancer cells.
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Autofagia/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Ácidos Grasos no Esterificados/biosíntesis , Proteínas de Membrana de los Lisosomas/metabolismo , Lectinas de Plantas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Adenina/análogos & derivados , Adenina/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Autofagia/fisiología , Benzamidas/farmacología , Carbamatos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Senescencia Celular/fisiología , Humanos , Masculino , Naftoles/farmacología , Células PC-3 , Neoplasias de la Próstata/patología , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/metabolismo , Esterol Esterasa/antagonistas & inhibidores , Tiadiazoles/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Macroautophagy (autophagy) is a conserved lysosomal-based intracellular degradation pathway. Here, we present different methods used for monitoring autophagy at cellular level. The methods involve Atg8/LC3 detection and quantification by Western blot, autophagic flux measurement through Western blot, direct fluorescence microscopy or indirect immunofluorescence, and finally traffic light assay using tf-LC3-II. Monitoring autophagic flux is experimentally challenging but obviously a prerequisite for the proper investigation of the process. These methods are suitable for screening purposes and can be used for measurements in cell lysates as well as in living cells. These assays have proven useful for the identification of genes and small molecules that regulate autophagy in mammalian cells.
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Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Western Blotting , Células HeLa , Humanos , Lisosomas/metabolismo , Microscopía FluorescenteRESUMEN
OBJECTIVE: We inspected the relevance of CD44, ABCB1 and ADAM17 in OSCC stemness and deciphered the role of autophagy/mitophagy in regulating stemness and chemoresistance. MATERIAL AND METHODS: A retrospective analysis of CD44, ABCB1 and ADAM17 with respect to the various clinico-pathological factors and their correlation was analysed in sixty OSCC samples. Furthermore, the stemness and chemoresistance were studied in resistant oral cancer cells using sphere formation assay, flow cytometry and florescence microscopy. The role of autophagy/mitophagy was investigated by transient transfection of siATG14, GFP-LC3, tF-LC3, mKeima-Red-Mito7 and Western blot analysis of autophagic and mitochondrial proteins. RESULTS: In OSCC, high CD44, ABCB1 and ADAM17 expressions were correlated with higher tumour grades and poor differentiation and show significant correlation in their co-expression. In vitro and OSCC tissue double labelling confirmed that CD44+ cells co-expresses ABCB1 and ADAM17. Further, cisplatin (CDDP)-resistant FaDu cells displayed stem-like features and higher CD44, ABCB1 and ADAM17 expression. Higher autophagic flux and mitophagy were observed in resistant FaDu cells as compared to parental cells, and inhibition of autophagy led to the decrease in stemness, restoration of mitochondrial proteins and reduced expression of CD44, ABCB1 and ADAM17. CONCLUSION: The CD44+ /ABCB1+ /ADAM17+ expression in OSCC is associated with stemness and chemoresistance. Further, this study highlights the involvement of mitophagy in chemoresistance and autophagic regulation of stemness in OSCC.