RESUMEN
Metabolic rewiring is essential for tumor growth and progression to metastatic disease, yet little is known regarding how cancer cells modify their acquired metabolic programs in response to different metastatic microenvironments. We have previously shown that liver-metastatic breast cancer cells adopt an intrinsic metabolic program characterized by increased HIF-1α activity and dependence on glycolysis. Here, we confirm by in vivo stable isotope tracing analysis (SITA) that liver-metastatic breast cancer cells retain a glycolytic profile when grown as mammary tumors or liver metastases. However, hepatic metastases exhibit unique metabolic adaptations including elevated expression of genes involved in glutathione (GSH) biosynthesis and reactive oxygen species (ROS) detoxification when compared to mammary tumors. Accordingly, breast-cancer-liver-metastases exhibited enhanced de novo GSH synthesis. Confirming their increased capacity to mitigate ROS-mediated damage, liver metastases display reduced levels of 8-Oxo-2'-deoxyguanosine. Depletion of the catalytic subunit of the rate-limiting enzyme in glutathione biosynthesis, glutamate-cysteine ligase (GCLC), strongly reduced the capacity of breast cancer cells to form liver metastases, supporting the importance of these distinct metabolic adaptations. Loss of GCLC also affected the early steps of the metastatic cascade, leading to decreased numbers of circulating tumor cells (CTCs) and impaired metastasis to the liver and the lungs. Altogether, our results indicate that GSH metabolism could be targeted to prevent the dissemination of breast cancer cells.
RESUMEN
BACKGROUND: Compared to minimally invasive brain metastases (MI BrM), highly invasive (HI) lesions form abundant contacts with cells in the peritumoral brain parenchyma and are associated with poor prognosis. Reactive astrocytes (RAs) labeled by phosphorylated STAT3 (pSTAT3) have recently emerged as a promising therapeutic target for BrM. Here, we explore whether the BrM invasion pattern is influenced by pSTAT3+â RAs and may serve as a predictive biomarker for STAT3 inhibition. METHODS: We used immunohistochemistry to identify pSTAT3+ RAs in HI and MI human and patient-derived xenograft (PDX) BrM. Using PDX, syngeneic, and transgenic mouse models of HI and MI BrM, we assessed how pharmacological STAT3 inhibition or RA-specific STAT3 genetic ablation affected BrM growth in vivo. Cancer cell invasion was modeled in vitro using a brain slice-tumor co-culture assay. We performed single-cell RNA sequencing of human BrM and adjacent brain tissue. RESULTS: RAs expressing pSTAT3 are situated at the brain-tumor interface and drive BrM invasive growth. HI BrM invasion pattern was associated with delayed growth in the context of STAT3 inhibition or genetic ablation. We demonstrate that pSTAT3+ RAs secrete Chitinase 3-like-1 (CHI3L1), which is a known STAT3 transcriptional target. Furthermore, single-cell RNA sequencing identified CHI3L1-expressing RAs in human HI BrM. STAT3 activation, or recombinant CHI3L1 alone, induced cancer cell invasion into the brain parenchyma using a brain slice-tumor plug co-culture assay. CONCLUSIONS: Together, these data reveal that pSTAT3+ RA-derived CHI3L1 is associated with BrM invasion, implicating STAT3 and CHI3L1 as clinically relevant therapeutic targets for the treatment of HI BrM.
Asunto(s)
Astrocitos , Neoplasias Encefálicas , Proteína 1 Similar a Quitinasa-3 , Invasividad Neoplásica , Factor de Transcripción STAT3 , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Humanos , Proteína 1 Similar a Quitinasa-3/metabolismo , Proteína 1 Similar a Quitinasa-3/genética , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Neoplasias Encefálicas/genética , Astrocitos/metabolismo , Astrocitos/patología , Ratones , Ratones Transgénicos , Proliferación Celular , Ensayos Antitumor por Modelo de Xenoinjerto , Células Tumorales CultivadasRESUMEN
Transmembrane glycoprotein NMB (GPNMB) is a prognostic marker of poor outcome in patients with triple-negative breast cancer (TNBC). Glembatumumab Vedotin, an antibody drug conjugate targeting GPNMB, exhibits variable efficacy against GPNMB-positive metastatic TNBC as a single agent. We show that GPNMB levels increase in response to standard-of-care and experimental therapies for multiple breast cancer subtypes. While these therapeutic stressors induce GPNMB expression through differential engagement of the MiTF family of transcription factors, not all are capable of increasing GPNMB cell-surface localization required for Glembatumumab Vedotin inhibition. Using a FACS-based genetic screen, we discovered that suppression of heat shock protein 90 (HSP90) concomitantly increases GPNMB expression and cell-surface localization. Mechanistically, HSP90 inhibition resulted in lysosomal dispersion towards the cell periphery and fusion with the plasma membrane, which delivers GPNMB to the cell surface. Finally, treatment with HSP90 inhibitors sensitizes breast cancers to Glembatumumab Vedotin in vivo, suggesting that combination of HSP90 inhibitors and Glembatumumab Vedotin may be a viable treatment strategy for patients with metastatic TNBC.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Neoplasias de la Mama Triple Negativas , Anticuerpos Monoclonales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Inmunoconjugados/efectos adversos , Lisosomas/metabolismo , Glicoproteínas de Membrana/genética , Factores de Transcripción , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
Growing tumors exist in metabolically compromised environments that require activation of multiple pathways to scavenge nutrients to support accelerated rates of growth. The folliculin (FLCN) tumor suppressor complex (FLCN, FNIP1, FNIP2) is implicated in the regulation of energy homeostasis via 2 metabolic master kinases: AMPK and mTORC1. Loss-of-function mutations of the FLCN tumor suppressor complex have only been reported in renal tumors in patients with the rare Birt-Hogg-Dube syndrome. Here, we revealed that FLCN, FNIP1, and FNIP2 are downregulated in many human cancers, including poor-prognosis invasive basal-like breast carcinomas where AMPK and TFE3 targets are activated compared with the luminal, less aggressive subtypes. FLCN loss in luminal breast cancer promoted tumor growth through TFE3 activation and subsequent induction of several pathways, including autophagy, lysosomal biogenesis, aerobic glycolysis, and angiogenesis. Strikingly, induction of aerobic glycolysis and angiogenesis in FLCN-deficient cells was dictated by the activation of the PGC-1α/HIF-1α pathway, which we showed to be TFE3 dependent, directly linking TFE3 to Warburg metabolic reprogramming and angiogenesis. Conversely, FLCN overexpression in invasive basal-like breast cancer models attenuated TFE3 nuclear localization, TFE3-dependent transcriptional activity, and tumor growth. These findings support a general role of a deregulated FLCN/TFE3 tumor suppressor pathway in human cancers.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Neoplasias de la Mama/patología , Neovascularización Patológica/prevención & control , Proteínas Proto-Oncogénicas/fisiología , Proteínas Supresoras de Tumor/fisiología , Efecto Warburg en Oncología , Proteínas Quinasas Activadas por AMP/fisiología , Línea Celular Tumoral , Femenino , Humanos , Fosforilación OxidativaRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is the most frequent liver disease worldwide and can progress to non-alcoholic steatohepatitis (NASH), which is characterized by triglyceride accumulation, inflammation, and fibrosis. No pharmacological agents are currently approved to treat these conditions, but it is clear now that modulation of lipid synthesis and autophagy are key biological mechanisms that could help reduce or prevent these liver diseases. The folliculin (FLCN) protein has been recently identified as a central regulatory node governing whole body energy homeostasis, and we hypothesized that FLCN regulates highly metabolic tissues like the liver. We thus generated a liver specific Flcn knockout mouse model to study its role in liver disease progression. Using the methionine- and choline-deficient diet to mimic liver fibrosis, we demonstrate that loss of Flcn reduced triglyceride accumulation, fibrosis, and inflammation in mice. In this aggressive liver disease setting, loss of Flcn led to activation of transcription factors TFEB and TFE3 to promote autophagy, promoting the degradation of intracellular lipid stores, ultimately resulting in reduced hepatocellular damage and inflammation. Hence, the activity of FLCN could be a promising target for small molecule drugs to treat liver fibrosis by specifically activating autophagy. Collectively, these results show an unexpected role for Flcn in fatty liver disease progression and highlight new potential treatment strategies.
Asunto(s)
Autofagia/genética , Hepatitis/etiología , Hepatitis/metabolismo , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , Proteínas Proto-Oncogénicas/deficiencia , Transducción de Señal , Proteínas Supresoras de Tumor/deficiencia , Animales , Biomarcadores , Biopsia , Biología Computacional , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Hepatitis/patología , Inmunohistoquímica , Cirrosis Hepática/patología , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , TranscriptomaRESUMEN
Cell migration is central to many developmental, physiologic and pathological processes, including cancer progression. The Ral GTPases (RalA and RalB) which act down-stream the Ras oncogenes, are key players in the coordination between membrane trafficking and actin polymerization. A major direct effector of Ral, the exocyst complex, works in polarized exocytosis and is at the center of multiple protein-protein interactions that support cell migration by promoting protrusion formation, front-rear polarization, and extra-cellular matrix degradation. In this review we describe the recent advancements in deciphering the molecular mechanisms underlying this role of Ral via exocyst on cell migration. Among others, we will discuss the recently identified cross-talk between Ral and Rac1 pathways: exocyst binds to a negative regulator (the RacGAP SH3BP1) and to the major effector (the Wave Regulatory Complex, WRC) of Rac1, the master regulator of protrusions. Next challenge will be to better characterize the dynamics in space and in time of these molecular interplays, to better understand the pleiotropic functions of Ral in both normal and cancer cells.
Asunto(s)
Movimiento Celular , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Proteínas ras/metabolismo , Animales , Proteínas Activadoras de GTPasa , Humanos , Neoplasias/patologíaRESUMEN
GPNMB has emerged as an immunomodulator and an important positive mediator of tumor progression and metastasis in numerous solid cancers. Tumor intrinsic GPNMB-mediated effects on cellular signaling, coupled with the ability of GPNMB to influence the primary tumor and metastatic microenvironments in a non-cell autonomous fashion, combine to augment malignant cancer phenotypes. In addition, GPNMB is often overexpressed in a variety of cancers, making it an attractive therapeutic target. In this regard, glembatumumab vedotin, an antibody-drug conjugate (ADC) that targets GPNMB, is currently in clinical trials as a single agent in multiple cancers. In this review, we will describe the physiological functions of GPNMB in normal tissues and summarize the processes through which GPNMB augments tumor growth and metastasis. We will review the pre-clinical and clinical development of glembatumumab vedotin, evaluate on-going clinical trials, explore emerging opportunities for this agent in new disease indications and discuss exciting possibilities for this ADC in the context of combination therapies.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Inmunoconjugados/uso terapéutico , Glicoproteínas de Membrana/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Biomarcadores/metabolismo , Humanos , Inmunoconjugados/farmacología , Glicoproteínas de Membrana/químicaRESUMEN
Coordination between membrane trafficking and actin polymerization is fundamental in cell migration, but a dynamic view of the underlying molecular mechanisms is still missing. The Rac1 GTPase controls actin polymerization at protrusions by interacting with its effector, the Wave regulatory complex (WRC). The exocyst complex, which functions in polarized exocytosis, has been involved in the regulation of cell motility. Here, we show a physical and functional connection between exocyst and WRC. Purified components of exocyst and WRC directly associate in vitro, and interactions interfaces are identified. The exocyst-WRC interaction is confirmed in cells by co-immunoprecipitation and is shown to occur independently of the Arp2/3 complex. Disruption of the exocyst-WRC interaction leads to impaired migration. By using time-lapse microscopy coupled to image correlation analysis, we visualized the trafficking of the WRC towards the front of the cell in nascent protrusions. The exocyst is necessary for WRC recruitment at the leading edge and for resulting cell edge movements. This direct link between the exocyst and WRC provides a new mechanistic insight into the spatio-temporal regulation of cell migration.
Asunto(s)
Movimiento Celular , Extensiones de la Superficie Celular/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Células HEK293 , Humanos , Unión Proteica , Subunidades de Proteína/metabolismoRESUMEN
PURPOSE: To determine if BRAF and/or MEK inhibitor-induced GPNMB expression renders melanomas sensitive to CDX-011, an antibody-drug conjugate targeting GPNMB. EXPERIMENTAL DESIGN: The Cancer Genome Atlas melanoma dataset was interrogated for a panel of MITF-regulated melanosomal differentiation antigens, including GPNMB. BRAF-mutant melanoma cell lines treated with BRAF or MEK inhibitors were assessed for GPNMB expression by RT-qPCR, immunoblot, and FACS analyses. Transient siRNA-mediated knockdown approaches were used to determine if MITF is requirement for treatment-induced GPNMB upregulation. GPNMB expression was analyzed in serial biopsies and serum samples from patients with melanoma taken before, during, and after disease progression on MAPK inhibitor treatment. Subcutaneous injections were performed to test the efficacy of MAPK inhibitors alone, CDX-011 alone, or their combination in suppressing melanoma growth. RESULTS: A MITF-dependent melanosomal differentiation signature is associated with poor prognosis in patients with this disease. MITF is increased following BRAF and MEK inhibitor treatment and induces the expression of melanosomal differentiation genes, including GPNMB. GPNMB is expressed at the cell surface in MAPK inhibitor-treated melanoma cells and is also elevated in on-treatment versus pretreatment biopsies from melanoma patients receiving MAPK pathway inhibitors. Combining BRAF and/or MEK inhibitors with CDX-011, an antibody-drug conjugate targeting GPNMB, is effective in causing melanoma regression in preclinical animal models and delays the recurrent melanoma growth observed with MEK or BRAF/MEK inhibitor treatment alone. CONCLUSIONS: The combination of MAPK pathway inhibitors with an antibody-drug conjugate targeting GPNMB is an effective therapeutic option for patients with melanoma. Clin Cancer Res; 22(24); 6088-98. ©2016 AACR.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/tratamiento farmacológico , Glicoproteínas de Membrana/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Inmunoconjugados/farmacología , Melanoma/genética , Ratones , Mutación , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias Cutáneas/genética , Melanoma Cutáneo MalignoRESUMEN
RalA and RalB proteins are key mediators of oncogenic Ras signaling in human oncogenesis. Herein we investigated the mechanistic contribution of Ral proteins to invasion of lung cancer A549 cells after induction of epithelial-mesenchymal transition (EMT) with TGFß. We show that TGFß-induced EMT promotes dissemination of A549 cells in a 2/3D assay, independently of proteolysis, by activating the Rho/ROCK pathway which generates actomyosin-dependent contractility forces that actively remodel the extracellular matrix, as assessed by Traction Force microscopy. RalB, but not RalA, is required for matrix deformation and cell dissemination acting via the RhoGEF GEF-H1, which associates with the Exocyst complex, a major Ral effector. Indeed, uncoupling of the Exocyst subunit Sec5 from GEF-H1 impairs RhoA activation, generation of traction forces and cell dissemination. These results provide a novel molecular mechanism underlying the control of cell invasion by RalB via a cross-talk with the Rho pathway.
Asunto(s)
Transición Epitelial-Mesenquimal/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Proteínas de Unión al GTP ral/metabolismo , Amidas/farmacología , Línea Celular Tumoral , Humanos , Microscopía de Fuerza Atómica , Piridinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Proteínas de Unión al GTP ral/genética , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
Characterizing the migration of a population of cells remains laborious and somewhat subjective. Advances in genetics and robotics allow researchers to perform many experiments in parallel, but analyzing the large sets of data remains a bottleneck. Here we describe a rapid, fully automated correlation-based method for cell migration analysis, compatible with standard video microscopy. This method allows for the computation of quantitative migration parameters via an extensive dynamic mapping of cell displacements.
Asunto(s)
Movimiento Celular , Rastreo Celular/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Línea Celular , Humanos , Microscopía por Video/métodos , Cicatrización de HeridasRESUMEN
The coordination of the several pathways involved in cell motility is poorly understood. Here, we identify SH3BP1, belonging to the RhoGAP family, as a partner of the exocyst complex and establish a physical and functional link between two motility-driving pathways, the Ral/exocyst and Rac signaling pathways. We show that SH3BP1 localizes together with the exocyst to the leading edge of motile cells and that SH3BP1 regulates cell migration via its GAP activity upon Rac1. SH3BP1 loss of function induces abnormally high Rac1 activity at the front, as visualized by in vivo biosensors, and disorganized and instable protrusions, as revealed by cell morphodynamics analysis. Consistently, constitutively active Rac1 mimics the phenotype of SH3BP1 depletion: slow migration and aberrant cell morphodynamics. Our finding that SH3BP1 downregulates Rac1 at the motile-cell front indicates that Rac1 inactivation in this location, as well as its activation by GEF proteins, is a fundamental requirement for cell motility.