RESUMO
Tumor acidosis is associated with increased invasiveness and drug resistance. Here, we take an unbiased approach to identify vulnerabilities of acid-exposed cancer cells by combining pH-dependent flow cytometry cell sorting from 3D colorectal tumor spheroids and transcriptomic profiling. Besides metabolic rewiring, we identify an increase in tetraploid cell frequency and DNA damage response as consistent hallmarks of acid-exposed cancer cells, supported by the activation of ATM and ATR signaling pathways. We find that regardless of the cell replication error status, both ATM and ATR inhibitors exert preferential growth inhibitory effects on acid-exposed cancer cells. The efficacy of a combination of these drugs with 5-FU is further documented in 3D spheroids as well as in patient-derived colorectal tumor organoids. These data position tumor acidosis as a revelator of the therapeutic potential of DNA repair blockers and as an attractive clinical biomarker to predict the response to a combination with chemotherapy.
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Neoplasias Colorretais , Tetraploidia , Humanos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Transdução de Sinais , Dano ao DNA , Reparo do DNA , Inibidores de Proteínas Quinases/farmacologiaRESUMO
BACKGROUND: Neoadjuvant chemotherapy (NAC) is the standard of care for patients with early-stage triple negative breast cancers (TNBC). However, more than half of TNBC patients do not achieve a pathological complete response (pCR) after NAC, and residual cancer burden (RCB) is associated with dismal long-term prognosis. Understanding the mechanisms underlying differential treatment outcomes is therefore critical to limit RCB and improve NAC efficiency. METHODS: Human TNBC cell lines and patient-derived organoids were used in combination with real-time metabolic assays to evaluate the effect of NAC (paclitaxel and epirubicin) on tumor cell metabolism, in particular glycolysis. Diagnostic biopsies (pre-NAC) from patients with early TNBC were analyzed by bulk RNA-sequencing to evaluate the predictive value of a glycolysis-related gene signature. RESULTS: Paclitaxel induced a consistent metabolic switch to glycolysis, correlated with a reduced mitochondrial oxidative metabolism, in TNBC cells. In pre-NAC diagnostic biopsies from TNBC patients, glycolysis was found to be upregulated in non-responders. Furthermore, glycolysis inhibition greatly improved response to NAC in TNBC organoid models. CONCLUSIONS: Our study pinpoints a metabolic adaptation to glycolysis as a mechanism driving resistance to NAC in TNBC. Our data pave the way for the use of glycolysis-related genes as predictive biomarkers for NAC response, as well as the development of inhibitors to overcome this glycolysis-driven resistance to NAC in human TNBC patients.
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Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Terapia Neoadjuvante , Prognóstico , Resultado do Tratamento , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
Pharmacological AMPK activation represents an attractive approach for the treatment of type 2 diabetes (T2D). AMPK activation increases skeletal muscle glucose uptake, but there is controversy as to whether AMPK activation also inhibits hepatic glucose production (HGP) and pharmacological AMPK activators can have off-target effects that contribute to their anti-diabetic properties. The main aim was to investigate the effects of 991 and other direct AMPK activators on HGP and determine whether the observed effects were AMPK-dependent. In incubated hepatocytes, 991 substantially decreased gluconeogenesis from lactate, pyruvate and glycerol, but not from other substrates. Hepatocytes from AMPKß1-/- mice had substantially reduced liver AMPK activity, yet the inhibition of glucose production by 991 persisted. Also, the glucose-lowering effect of 991 was still seen in AMPKß1-/- mice subjected to an intraperitoneal pyruvate tolerance test. The AMPK-independent mechanism by which 991 treatment decreased gluconeogenesis could be explained by inhibition of mitochondrial pyruvate uptake and inhibition of mitochondrial sn-glycerol-3-phosphate dehydrogenase-2. However, 991 and new-generation direct small-molecule AMPK activators antagonized glucagon-induced gluconeogenesis in an AMPK-dependent manner. Our studies support the notion that direct pharmacological activation of hepatic AMPK as well as inhibition of pyruvate uptake could be an option for the treatment of T2D-linked hyperglycemia.
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Diabetes Mellitus Tipo 2 , Glucagon , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucagon/metabolismo , Gluconeogênese , Glucose/metabolismo , Ácido Láctico/metabolismo , Fígado/metabolismo , Camundongos , Ácido Pirúvico/metabolismoRESUMO
Squamous cell carcinoma of the head and neck (SCCHN) is among the most prevalent cancer types worldwide. Despite multimodal therapeutic approaches that include surgical resection, radiation therapy or concurrent chemoradiation, targeted therapy and immunotherapy, SCCHN is still associated with a poor prognosis for patients with locally advanced or recurrent/metastatic (R/M) diseases. Although next-generation sequencing data from thousands of SCCHN patients have provided a comprehensive landscape of the somatic genomic alterations in this disease, genomic-based precision medicine is not implemented yet in routine clinical use since no satisfactory genetic biomarker has been identified for diagnosis, patient outcome prediction and selection of tailored therapeutic options. The lack of significant improvement in SCCHN patient survival over the last decades stresses the need for reliable predictive biomarkers and new therapeutic strategies for personalized clinical management of SCCHN patients. Targeting the SCCHN-associated microenvironment or the interaction of the latter with cancer cells may represent such paradigm shift in the development of new strategies to treat SCCHN patients, as exemplified by the recent implementation of immune checkpoint inhibitors to improve clinical outcomes by increasing anti-tumor immune responses in SCCHN patients. Several clinical trials are in progress in SCCHN patients to evaluate the activity of monoclonal antibodies and small-molecule inhibitors targeting the tumor microenvironment (TME) at different treatment settings, including combinations with adjuvant surgery, radiation therapy and chemotherapy. This review describes the current knowledge about the influence of the TME on intratumoral heterogeneity and clinical relapse in human SCCHN patients. More precisely, the role of hypoxia as well as the presence of non-cancer cells (e.g. cancer-associated fibroblasts and immune cells) on therapy response of SCCHN cells is highlighted. We also discuss relevant (pre)clinical models that may help integrate the microenvironment-tumor cell interplay in translational research studies for SCCHN. Finally, this review explores potential therapeutic strategies that may exploit the crosstalk between TME and SCCHN cells in order to implement fundamental changes in the tumor treatment paradigm of patients with locally advanced or R/M SCCHN.
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Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Imunoterapia , Medicina de Precisão , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Microambiente Tumoral/genéticaRESUMO
Precision oncology is the practice of matching one therapy to one specific patient, based on particular genetic tumor alterations, in order to achieve the best clinical response. Despite an expanding arsenal of targeted therapies, many patients still have a poor outcome because tumor cells show a remarkable capacity to develop drug resistance, thereby leading to tumor relapse. Besides genotype-driven resistance mechanisms, tumor microenvironment (TME) peculiarities strongly contribute to generate an intratumoral phenotypic heterogeneity that affects disease progression and treatment outcome. In this Review, we describe how TME-mediated metabolic heterogeneities actively participate to therapeutic failure. We report how a lactate-based metabolic symbiosis acts as a mechanism of adaptive resistance to targeted therapies and we describe the role of mitochondrial metabolism, in particular oxidative phosphorylation (OXPHOS), to support the growth and survival of therapy-resistant tumor cells in a variety of cancers. Finally, we detail potential metabolism-interfering therapeutic strategies aiming to eradicate OXPHOS-dependent relapse-sustaining malignant cells and we discuss relevant (pre)clinical models that may help integrate TME-driven metabolic heterogeneity in precision oncology.
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Lactatos/metabolismo , Neoplasias/genética , Oncogenes/genética , Medicina de Precisão , Antineoplásicos/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Despite an increasing arsenal of anticancer therapies, many patients continue to have poor outcomes due to the therapeutic failures and tumor relapses. Indeed, the clinical efficacy of anticancer therapies is markedly limited by intrinsic and/or acquired resistance mechanisms that can occur in any tumor type and with any treatment. Thus, there is an urgent clinical need to implement fundamental changes in the tumor treatment paradigm by the development of new experimental strategies that can help to predict the occurrence of clinical drug resistance and to identify alternative therapeutic options. Apart from mutation-driven resistance mechanisms, tumor microenvironment (TME) conditions generate an intratumoral phenotypic heterogeneity that supports disease progression and dismal outcomes. Tumor cell metabolism is a prototypical example of dynamic, heterogeneous, and adaptive phenotypic trait, resulting from the combination of intrinsic [(epi)genetic changes, tissue of origin and differentiation dependency] and extrinsic (oxygen and nutrient availability, metabolic interactions within the TME) factors, enabling cancer cells to survive, metastasize and develop resistance to anticancer therapies. In this review, we summarize the current knowledge regarding metabolism-based mechanisms conferring adaptive resistance to chemo-, radio-and immunotherapies as well as targeted therapies. Furthermore, we report the role of TME-mediated intratumoral metabolic heterogeneity in therapy resistance and how adaptations in amino acid, glucose, and lipid metabolism support the growth of therapy-resistant cancers and/or cellular subpopulations. We also report the intricate interplay between tumor signaling and metabolic pathways in cancer cells and discuss how manipulating key metabolic enzymes and/or providing dietary changes may help to eradicate relapse-sustaining cancer cells. Finally, in the current era of personalized medicine, we describe the strategies that may be applied to implement metabolic profiling for tumor imaging, biomarker identification, selection of tailored treatments and monitoring therapy response during the clinical management of cancer patients.
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Neoplasias , Microambiente Tumoral , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Medicina de PrecisãoRESUMO
Acetate is reported as a regulator of fat mass but also as lipogenic source for cancer cells. Breast cancer is surrounded by adipose tissue and has been associated with obesity. However, whether acetate contributes to cancer cell metabolism as lipogenic substrate and/or by changing fat storage and eventually obesity-induced breast cancer progression remains unknown. Therefore, we studied the contribution of acetate to breast cancer metabolism and progression. In vitro, we found that acetate is not a bioenergetic substrate under normoxia and did not result in a significant change of growth. However, by using lipidomic approaches, we discovered that acetate changes the lipid profiles of the cells under hypoxia. Moreover, while mice fed a high-fat diet (HFD) developed bigger tumours than their lean counterparts, exogenous acetate supplementation leads to a complete abolishment of fat mass gain without reverting the HFD-induced obesity-driven tumour progression. In conclusion, although acetate protects against diet-induced obesity, our data suggest that it is not affecting HFD-driven tumour progression.
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Acetatos/metabolismo , Acetatos/farmacologia , Neoplasias da Mama/metabolismo , Obesidade/metabolismo , Adipogenia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipidômica/métodos , Camundongos , Oxigênio/metabolismo , Carga Tumoral/efeitos dos fármacosRESUMO
Warburg's hypothesis that cancer cells take up a lot of glucose in the presence of ambient oxygen but convert pyruvate into lactate due to impaired mitochondrial function led to the misconception that cancer cells rely on glycolysis as their major source of energy. Most recent 13C-based metabolomic studies, including in cancer patients, indicate that cancer cells may also fully oxidize glucose. In addition to glucose-derived pyruvate, lactate, fatty acids and amino acids supply substrates to the TCA cycle to sustain mitochondrial metabolism. Here, we discuss how the metabolic flexibility afforded by these multiple mitochondrial inputs allows cancer cells to adapt according to the availability of the different fuels and the microenvironmental conditions such as hypoxia and acidosis. In particular, we focused on the role of the TCA cycle in interconnecting numerous metabolic routes in order to highlight metabolic vulnerabilities that represent attractive targets for a new generation of anticancer drugs.
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Ciclo do Ácido Cítrico/fisiologia , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Acidose/metabolismo , Acidose/patologia , Aminoácidos/metabolismo , Animais , Glicólise/fisiologia , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Mitocôndrias/fisiologia , Neoplasias/patologiaRESUMO
Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze-thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKß1-/- mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.
Assuntos
Glicemia/efeitos dos fármacos , Flavonoides/química , Flavonoides/farmacologia , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Moraceae/química , Quinases Proteína-Quinases Ativadas por AMP , Animais , Sistema Livre de Células , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Masculino , Camundongos , Proteínas Quinases/metabolismo , Ratos , Ratos WistarRESUMO
Mitochondrial metabolism is an attractive target for cancer therapy. Reprogramming metabolic pathways can potentially sensitize tumors with limited treatment options, such as triple-negative breast cancer (TNBC), to chemo- and/or radiotherapy. Dichloroacetate (DCA) is a specific inhibitor of the pyruvate dehydrogenase kinase (PDK), which leads to enhanced reactive oxygen species (ROS) production. ROS are the primary effector molecules of radiation and an increase hereof will enhance the radioresponse. In this study, we evaluated the effects of DCA and radiotherapy on two TNBC cell lines, namely EMT6 and 4T1, under aerobic and hypoxic conditions. As expected, DCA treatment decreased phosphorylated pyruvate dehydrogenase (PDH) and lowered both extracellular acidification rate (ECAR) and lactate production. Remarkably, DCA treatment led to a significant increase in ROS production (up to 15-fold) in hypoxic cancer cells but not in aerobic cells. Consistently, DCA radiosensitized hypoxic tumor cells and 3D spheroids while leaving the intrinsic radiosensitivity of the tumor cells unchanged. Our results suggest that although described as an oxidative phosphorylation (OXPHOS)-promoting drug, DCA can also increase hypoxic radioresponses. This study therefore paves the way for the targeting of mitochondrial metabolism of hypoxic cancer cells, in particular to combat radioresistance.
Assuntos
Neoplasias da Mama/metabolismo , Ácido Dicloroacético/farmacologia , Inibidores Enzimáticos/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Hipóxia Tumoral , Linhagem Celular , Feminino , Humanos , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismoRESUMO
Bacterial peritonitis remains the main cause of technique failure in peritoneal dialysis (PD). During peritonitis, the peritoneal membrane undergoes structural and functional alterations that are mediated by IL-1ß The NLRP3 inflammasome is a caspase-1-activating multiprotein complex that links sensing of microbial and stress products to activation of proinflammatory cytokines, including IL-1ß The potential roles of the NLRP3 inflammasome and IL-1ß in the peritoneal membrane during acute peritonitis have not been investigated. Here, we show that the NLRP3 inflammasome is activated during acute bacterial peritonitis in patients on PD, and this activation associates with the release of IL-1ß in the dialysate. In mice, lipopolysaccharide- or Escherichia coli-induced peritonitis led to IL-1ß release in the peritoneal membrane. The genetic deletion of Nalp3, which encodes NLRP3, abrogated defects in solute transport during acute peritonitis and restored ultrafiltration. In human umbilical vein endothelial cells, IL-1ß treatment directly enhanced endothelial cell proliferation and increased microvascular permeability. These in vitro effects require endothelial IL-1 receptors, shown by immunofluorescence to be expressed in peritoneal capillaries in mice. Furthermore, administration of the IL-1ß receptor antagonist, anakinra, efficiently decreased nitric oxide production and vascular proliferation and restored peritoneal function in mouse models of peritonitis, even in mice treated with standard-of-care antibiotherapy. These data demonstrate that NLRP3 activation and IL-1ß release have a critical role in solute transport defects and tissue remodeling during PD-related peritonitis. Blockade of the NLRP3/IL-1ß axis offers a novel method for rescuing morphologic alterations and transport defects during acute peritonitis.
Assuntos
Inflamassomos/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Diálise Peritoneal , Peritonite/etiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Interleucina-1beta/fisiologia , Masculino , Camundongos , Pessoa de Meia-IdadeRESUMO
PURPOSE OF REVIEW: Lipid metabolism in cancer cells and tumor-associated stromal cells was recently identified to contribute to disease progression particularly in response to changes in tumor microenvironment such as acidosis and hypoxia. RECENT FINDINGS: New molecular mechanisms driving lipid metabolism in various cancers were elicited through genetic silencing, pharmacological inhibition of key metabolic enzymes, including those involved in fatty acid oxidation and synthesis, and modulation of diet composition. SUMMARY: To proliferate, metastasize, or resist stress conditions imposed by the microenvironment, many cancer cells rely on fatty acid ß-oxidation to generate acetyl-CoA and fuel the TCA cycle, and on fatty acid synthesis to produce building blocks. These processes are fine-tuned through regulation of acetyl-CoA carboxylases expression and activity. Stromal cells including lymphocytes, (lymphatic) endothelial cells and adipocytes also participate through either fatty acid transfer or lipid-based signaling to cancer disease progression. Altogether, these data identify critical nodes in the orchestration of lipid metabolism in cancer that may facilitate the design of synthetic-lethal treatments.
Assuntos
Metabolismo dos Lipídeos/fisiologia , Neoplasias/patologia , Neoplasias/fisiopatologia , Acetilcoenzima A/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Ciclo do Ácido Cítrico/fisiologia , Resistencia a Medicamentos Antineoplásicos , Ácidos Graxos/metabolismo , Humanos , Metástase Neoplásica , Obesidade , Oxirredução , Células Estromais/metabolismoRESUMO
PURPOSE OF REVIEW: Glutamine and acetate were recently identified as alternatives to glucose for fueling the tricarboxylic acid (TCA) cycle in cancer cells, particularly in the context of hypoxia. RECENT FINDINGS: Molecular mechanisms orchestrating glutamine and acetate metabolism were elicited through the combination of C tracer analysis and genetic silencing, or pharmacological modulation of key metabolic enzymes including those converting glutamate into α-ketoglutarate (αKG) (and beyond) and acetate into acetyl-coenzyme A (CoA). SUMMARY: Oxidative decarboxylation and reductive carboxylation of αKG represent two options for the glutamine metabolism. The canonical forward mode of the TCA cycle fuelled by glutamine may benefit from the decarboxylation of malate into pyruvate for fueling pyruvate dehydrogenase and generating acetyl-CoA to offer a self-sustainable TCA cycle. Under hypoxia and mutations in the TCA cycle, the reductive carboxylation of glutamine-derived αKG into citrate mainly supports lipogenesis via the ATP citrate lyase that cleaves citrate into oxaloacetate and acetyl-CoA. Still, a largely unsuspected source of acetyl-CoA was shown to derive from the direct ligation of acetate to CoA by acetyl-CoA synthetases. Altogether, these findings identify critical metabolic nodes in the glutamine and acetate metabolism as new determinants of tumor metabolic plasticity that may facilitate the design of synthetic lethal treatments.
Assuntos
Acetatos/metabolismo , Glucose/metabolismo , Glutamina/metabolismo , Neoplasias/metabolismo , Acetilcoenzima A/metabolismo , Linhagem Celular Tumoral , Citratos/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Humanos , Hipóxia/sangue , Ácidos Cetoglutáricos/metabolismo , Lipogênese/efeitos dos fármacosRESUMO
Despite the implementation of personalized medicine, patients with metastatic CRC (mCRC) still have a dismal overall survival due to the frequent occurrence of acquired resistance mechanisms thereby leading to clinical relapse. Understanding molecular mechanisms that support acquired resistance to anti-EGFR targeted therapy in mCRC is therefore clinically relevant and key to improving patient outcomes. Here, we observe distinct metabolic changes between cetuximab-resistant CRC cell populations, with in particular an increased glycolytic activity in KRAS-mutant cetuximab-resistant CRC cells (LIM1215 and OXCO2) but not in KRAS-amplified resistant DiFi cells. We show that cetuximab-resistant LIM1215 and OXCO2 cells have the capacity to recycle glycolysis-derived lactate to sustain their growth capacity. This is associated with an upregulation of the lactate importer MCT1 at both transcript and protein levels. Pharmacological inhibition of MCT1, with AR-C155858, reduces the uptake and oxidation of lactate and impairs growth capacity in cetuximab-resistant LIM1215 cells both in vitro and in vivo. This study identifies MCT1-dependent lactate utilization as a clinically actionable, metabolic vulnerability to overcome KRAS-mutant-mediated acquired resistance to anti-EGFR therapy in CRC.
Assuntos
Cetuximab , Neoplasias Colorretais , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB , Ácido Láctico , Transportadores de Ácidos Monocarboxílicos , Simportadores , Humanos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Simportadores/metabolismo , Simportadores/genética , Ácido Láctico/metabolismo , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Cetuximab/farmacologia , Linhagem Celular Tumoral , Animais , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Glicólise/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de Células/efeitos dos fármacosRESUMO
The precursor of nerve growth factor (proNGF) has been described as a biologically active polypeptide able to induce apoptosis in neuronal cells, via the neurotrophin receptor p75(NTR) and the sortilin receptor. Herein, it is shown that proNGF is produced and secreted by breast cancer cells, stimulating their invasion. Using Western blotting and mass spectrometry, proNGF was detected in a panel of breast cancer cells as well as in their conditioned media. Immunohistochemical analysis indicated an overproduction of proNGF in breast tumors, when compared with benign and normal breast biopsies, and a relationship to lymph node invasion in ductal carcinomas. Interestingly, siRNA against proNGF induced a decrease of breast cancer cell invasion that was restored by the addition of non-cleavable proNGF. The activation of TrkA, Akt, and Src, but not the MAP kinases, was observed. In addition, the proNGF invasive effect was inhibited by the Trk pharmacological inhibitor K252a, a kinase-dead TrkA, and siRNA against TrkA sortilin, neurotensin, whereas siRNA against p75(NTR) and the MAP kinase inhibitor PD98059 had no impact. These data reveal the existence of an autocrine loop stimulated by proNGF and mediated by TrkA and sortilin, with the activation of Akt and Src, for the stimulation of breast cancer cell invasion.
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Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Comunicação Autócrina , Neoplasias da Mama/metabolismo , Carcinoma Ductal/metabolismo , Fator de Crescimento Neural/metabolismo , Precursores de Proteínas/metabolismo , Receptor trkA/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Biópsia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carbazóis/farmacologia , Carcinoma Ductal/genética , Carcinoma Ductal/patologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Alcaloides Indólicos/farmacologia , Metástase Linfática , Invasividade Neoplásica , Fator de Crescimento Neural/genética , Precursores de Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor trkA/genética , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
xCT overexpression in cancer cells has been linked to tumor growth, metastasis and treatment resistance. Sulfasalazine (SSZ), an FDA-approved drug for the treatment of rheumatoid sarthritis, and inflammatory bowel diseases, has anticancer properties via inhibition of xCT, leading to the disruption of redox homeostasis. Since reactive oxygen species (ROS) are pivotal for the efficacy of radiotherapy (RT), elevated levels of ROS are associated with improved RT outcomes. In this study, the influence of SSZ treatment on the radiosensitivity of human colorectal cancer (CRC) cells was investigated. Our principal finding in human HCT116 and DLD-1 cells was that SSZ enhances the radiosensitivity of hypoxic CRC cells but does not alter the intrinsic radiosensitivity. The radiosensitizing effect was attributed to the depletion of glutathione and thioredoxin reductase levels. In turn, the reduction leads to excessive levels of ROS, increased DNA damage, and ferroptosis induction. Confirmation of these findings was performed in 3D models and in DLD-1 xenografts. Taken together, this study is a stepping stone for applying SSZ as a radiosensitizer in the clinic and confirms that xCT in cancer cells is a valid radiobiological target.
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Aim: Acquired resistance to the targeted agent cetuximab poses a significant challenge in finding effective anti-cancer treatments for head and neck squamous cell carcinoma (HNSCC). To accurately study novel combination treatments, suitable preclinical mouse models for cetuximab resistance are key yet currently limited. This study aimed to optimize an acquired cetuximab-resistant mouse model, with preservation of the innate immunity, ensuring intact antibody-dependent cellular cytotoxicity (ADCC) functionality. Methods: Cetuximab-sensitive and acquired-resistant HNSCC cell lines, generated in vitro, were subcutaneously engrafted in Rag2 knock-out (KO), BALB/c Nude and CB17 Scid mice with/without Matrigel or Geltrex. Once tumor growth was established, mice were intraperitoneally injected twice a week with cetuximab for a maximum of 3 weeks. In addition, immunohistochemistry was used to evaluate the tumor and its microenvironment. Results: Despite several adjustments in cell number, cell lines and the addition of Matrigel, Rag2 KO and BALB/C Nude mice proved to be unsuitable for xenografting our HNSCC cell lines. Durable tumor growth of resistant SC263-R cells could be induced in CB17 Scid mice. However, these cells had lost their resistance phenotype in vivo. Immunohistochemistry revealed a high infiltration of macrophages in cetuximab-treated SC263-R tumors. FaDu-S and FaDu-R cells successfully engrafted into CB17 Scid mice and maintained their sensitivity/resistance to cetuximab. Conclusion: We have established in vivo HNSCC mouse models with intact ADCC functionality for cetuximab resistance and sensitivity using the FaDu-R and FaDu-S cell lines, respectively. These models serve as valuable tools for investigating cetuximab resistance mechanisms and exploring novel drug combination strategies.
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Cancer cells may stimulate glycolytic flux when O2 becomes insufficient. Increase in L-lactate release therefore appears as an escape mechanism to drugs targeting mitochondrial respiration but also represents a response that may be exploited to screen for compounds blocking either mitochondrial carriers of oxidizable substrates or the electron transport chain. Here, we developed a screening procedure based on the capacity of cancer cells to release L-lactate to gain insights on the development of mitochondrial complex I inhibitors. For this purpose, we synthesized derivatives of carboxyamidotriazole, a compound previously described as a potential OXPHOS inhibitor. Two series of derivatives were generated by cycloaddition between benzylazide and either cyanoacetamides or alkynes. A primary assay measuring L-lactate release as a compensatory mechanism upon OXPHOS inhibition led us to identify 15 hits among 28 derivatives. A secondary assay measuring O2 consumption in permeabilized cancer cells confirmed that 12 compounds among the hits exhibited reversible complex I inhibitory activity. Anticancer effects of a short list of 5 compounds identified to induce more L-lactate release than reference compound were then evaluated on cancer cells and tumor-mimicking 3D spheroids. Human and mouse cancer cell monolayers exhibiting high level of respiration in basal conditions were up to 3-fold more sensitive than less oxidative cancer cells. 3D tumor spheroids further revealed potency differences between selected compounds in terms of cytotoxicity but also radiosensitizing activity resulting from local reoxygenation. In conclusion, this study documents the feasibility to efficiently screen in 96-well plate format for mitochondrial complex I inhibitors based on the capacity of drug candidates to induce L-lactate release.
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Extracellular acidification has been shown to be an important characteristic of invasive tumors, as it promotes invasion and migration but also resistance to treatments. Targeting transporters involved in the regulation of tumor pH constitutes a promising anti-tumor approach, as it would disrupt cellular pH homeostasis and negatively impact tumor growth. In this study, we evaluated the impact of syrosingopine, an inhibitor of MCT1 and MCT4, as a modulator of tumor metabolism and extracellular acidification in human breast cancer (MDA-MB-231) and pharyngeal squamous cell carcinoma (FaDu) cell models. In both models in vitro, we observed that exposure to syrosingopine led to a decrease in the extracellular acidification rate, intracellular pH, glucose consumption, lactate secretion and tumor cell proliferation with an increase in the number of late apoptotic/necrotic cells. However, in vivo experiments using the MDA-MB-231 model treated with a daily injection of syrosingopine did not reveal any significant change in extracellular pH (pHe) (as measured using CEST-MRI) or primary tumor growth. Overall, our study suggests that targeting MCT could lead to profound changes in tumor cell metabolism and proliferation, and it warrants further research to identify candidates without off-target effects.
RESUMO
Targeting enzymes involved in tumor metabolism is a promising way to tackle cancer progression. The inhibition of carnitine palmitoyltransferase 1 (CPT1) by etomoxir (Eto) efficiently slows down the growth of various cancers. Unfortunately, the clinical use of this drug was abandoned because of hepatotoxic effects. We report the development of pH-sensitive peptide (pHLIP)-drug conjugate to deliver Eto selectively to cancer cells exposed to acidic microenvironmental conditions. A newly designed sequence for the pHLIP peptide, named pHLIPd, was compared with a previously published reference pHLIP peptide, named pHLIPr. We showed that the conjugate between pHLIPd and Eto has a better pH-dependent insertion and structuration than the pHLIPr-based conjugate inside POPC vesicles. We observed antiproliferative effects when applied on acid-adapted cancer cells, reaching a larger inhibitory activity than Eto alone. In conclusion, this study brings the first evidence that pHLIP-based conjugates with a CPT1 inhibitor has the potential to specifically target the tumor acidic compartment and exert anticancer effects while sparing healthy tissues.