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BACKGROUND: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC). METHODS: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer. RESULTS: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer. CONCLUSIONS: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.
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Transformación Celular Neoplásica/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Dinámicas Mitocondriales/fisiología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Carboxiliasas/genética , Carboxiliasas/metabolismo , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Femenino , Humanos , Inmunosupresores/farmacología , Leflunamida/farmacología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mitocondrias/metabolismo , Mitocondrias/patología , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the present study, spermatozoon ultrastructure was documented in two species of hangingflies, Bittacus strigosus Hagen (Mecoptera: Bittacidae) and B. stigmaterus Say. Structures considered important to phylogenetic assessment that were observed in B. strigosus and B. stigmaterus included a short bilayered acrosome, elongated nucleus, tube-like glycocalyx, centriole adjunct material, accessory bodies, two mitochondrial derivatives, extra axonemal rods, globular units, and 9+2 arrangement of microtubules in the axoneme. Comparisons were made to Bittacus planus Cheng, which was previously examined by electron microscopy (Xie and Hua 2010). Similarities among the ultrastructural characteristics of the three Bittacus species support the monophyly of this genus. Displacement of a mitochondrial derivative by an accessory body was documented for the first time. This paper includes clarifications on differences between accessory bodies and extra axonemal rods, which are issues important to phylogenetic placement.
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Insectos/ultraestructura , Espermatozoides/ultraestructura , Animales , Masculino , Filogenia , Especificidad de la EspecieRESUMEN
Single cancer cells within a tumor exhibit variable levels of resistance to drugs, ultimately leading to treatment failures. While tumor heterogeneity is recognized as a major obstacle to cancer therapy, standard dose-response measurements for the potency of targeted kinase inhibitors aggregate populations of cells, obscuring intercellular variations in responses. In this work, we develop an analytical and experimental framework to quantify and model dose responses of individual cancer cells to drugs. We first explore the connection between population and single-cell dose responses using a computational model, revealing that multiple heterogeneous populations can yield nearly identical population dose responses. We demonstrate that a single-cell analysis method, which we term a threshold inhibition surface, can differentiate among these populations. To demonstrate the applicability of this method, we develop a dose-titration assay to measure dose responses in single cells. We apply this assay to breast cancer cells responding to phosphatidylinositol-3-kinase inhibition (PI3Ki), using clinically relevant PI3Kis on breast cancer cell lines expressing fluorescent biosensors for kinase activity. We demonstrate that MCF-7 breast cancer cells exhibit heterogeneous dose responses with some cells requiring over ten-fold higher concentrations than the population average to achieve inhibition. Our work reimagines dose-response relationships for cancer drugs in an emerging paradigm of single-cell tumor heterogeneity.
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Antineoplásicos , Neoplasias de la Mama , Humanos , Femenino , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Células MCF-7RESUMEN
Mitochondria produce reactive oxygen species (ROS), which function in signal transduction. Mitochondrial dynamics, encompassing morphological shifts between fission and fusion, can directly impact ROS levels in cancer cells. In this study, we identified an ROS-dependent mechanism for how enhanced mitochondrial fission inhibits triple negative breast cancer (TNBC) cell migration. We found that enforcing mitochondrial fission in TNBC resulted in an increase in intracellular ROS levels and reduced cell migration and the formation of actin-rich migratory structures. Consistent with mitochondrial fission, increasing ROS levels in cells inhibited cell migration. Conversely, reducing ROS levels with either a global or mitochondrially targeted scavenger overcame the inhibitory effects of mitochondrial fission. Mechanistically, we found that the ROS sensitive SHP-1/2 phosphatases partially regulate inhibitory effects of mitochondrial fission on TNBC migration. Overall, our work reveals the inhibitory effects of ROS in TNBC and supports mitochondrial dynamics as a potential therapeutic target for cancer.
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The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochemical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues influence transformed epithelial cells, prompting a diversity of migration modes spanning single cell and multicellular phenotypes. Purported consequences of these variations in tumor escape strategies include differential metastatic capability and therapy resistance. Therefore, understanding how cues from the peritumoral stromal microenvironment regulate migration mode has both prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel mechanics can be orthogonally tuned to investigate the contribution of these two key matrix attributes on MCF10A migration mode phenotypes, epithelial-mesenchymal transition (EMT), and invasive potential. We develop an automated computational image analysis framework to extract migratory phenotypes from fluorescent images and determine 3D migration metrics relevant to metastatic spread. Using this analysis, we find that matrix fiber density and bulk hydrogel mechanics distinctly contribute to a variety of MCF10A migration modes including amoeboid, single mesenchymal, clusters, and strands. We identify combinations of physical and soluble cues that induce a variety of migration modes originating from the same MCF10A spheroid and use these settings to examine a functional consequence of migration mode -resistance to apoptosis. We find that cells migrating as strands are more resistant to staurosporine-induced apoptosis than either disconnected clusters or individual invading cells. Improved models of the peritumoral stromal microenvironment and understanding of the relationships between matrix attributes and cell migration mode can aid ongoing efforts to identify effective cancer therapeutics that address cell plasticity-based therapy resistances. STATEMENT OF SIGNIFICANCE: Stromal extracellular matrix structure dictates both cell homeostasis and activation towards migratory phenotypes. However decoupling the effects of myriad biophysical cues has been difficult to achieve. Here, we encapsulate electrospun fiber segments within an amorphous hydrogel to create a fiber-reinforced hydrogel composite in which fiber density and hydrogel stiffness can be orthogonally tuned. Quantification of 3D cell migration reveal these two parameters uniquely contribute to a diversity of migration phenotypes spanning amoeboid, single mesenchymal, multicellular cluster, and collective strand. By tuning biophysical and biochemical cues to elicit heterogeneous migration phenotypes, we find that collective strands best resist apoptosis. This work establishes a composite approach to modulate fibrous topography and bulk hydrogel mechanics and identified biomaterial parameters to direct distinct 3D cell migration phenotypes.
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Hidrogeles , Neoplasias , Humanos , Hidrogeles/farmacología , Hidrogeles/química , Movimiento Celular , Materiales Biocompatibles/farmacología , Células Epiteliales , Matriz Extracelular , Microambiente TumoralRESUMEN
Cancer cells reprogram energy metabolism through metabolic plasticity, adapting ATP-generating pathways in response to treatment or microenvironmental changes. Such adaptations enable cancer cells to resist standard therapy. We employed a coculture model of estrogen receptor-positive (ER+) breast cancer and mesenchymal stem cells (MSC) to model interactions of cancer cells with stromal microenvironments. Using single-cell endogenous and engineered biosensors for cellular metabolism, coculture with MSCs increased oxidative phosphorylation, intracellular ATP, and resistance of cancer cells to standard therapies. Cocultured cancer cells had increased MCT4, a lactate transporter, and were sensitive to the MCT1/4 inhibitor syrosingopine. Combining syrosingopine with fulvestrant, a selective estrogen receptor degrading drug, overcame resistance of ER+ breast cancer cells in coculture with MSCs. Treatment with antiestrogenic therapy increased metabolic plasticity and maintained intracellular ATP levels, while MCT1/4 inhibition successfully limited metabolic transitions and decreased ATP levels. Furthermore, MCT1/4 inhibition decreased heterogenous metabolic treatment responses versus antiestrogenic therapy. These data establish MSCs as a mediator of cancer cell metabolic plasticity and suggest metabolic interventions as a promising strategy to treat ER+ breast cancer and overcome resistance to standard clinical therapies. IMPLICATIONS: This study reveals how MSCs reprogram metabolism of ER+ breast cancer cells and point to MCT4 as potential therapeutic target to overcome resistance to antiestrogen drugs.
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Neoplasias de la Mama , Células Madre Mesenquimatosas , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Receptores de Estrógenos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Metabolismo Energético , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Microambiente TumoralRESUMEN
Plants exhibit organ- and tissue-specific light responses. To explore the molecular basis of spatial-specific phytochrome-regulated responses, a transgenic approach for regulating the synthesis and accumulation of the phytochrome chromophore phytochromobilin (PΦB) was employed. In prior experiments, transgenic expression of the BILIVERDIN REDUCTASE (BVR) gene was used to metabolically inactivate biliverdin IXα, a key precursor in the biosynthesis of PΦB, and thereby render cells accumulating BVR phytochrome deficient. Here, we report analyses of transgenic Arabidopsis (Arabidopsis thaliana) lines with distinct patterns of BVR accumulation dependent upon constitutive or tissue-specific, promoter-driven BVR expression that have resulted in insights on a correlation between root-localized BVR accumulation and photoregulation of root elongation. Plants with BVR accumulation in roots and a PΦB-deficient elongated hypocotyl2 (hy2-1) mutant exhibit roots that are longer than those of wild-type plants under white illumination. Additional analyses of a line with root-specific BVR accumulation generated using a GAL4-dependent bipartite enhancer-trap system confirmed that PΦB or phytochromes localized in roots directly impact light-dependent root elongation under white, blue, and red illumination. Additionally, roots of plants with constitutive plastid-localized or root-specific cytosolic BVR accumulation, as well as phytochrome chromophore-deficient hy1-1 and hy2-1 mutants, exhibit reduced sensitivity to the plant hormone jasmonic acid (JA) in JA-dependent root inhibition assays, similar to the response observed for the JA-insensitive mutants jar1 and myc2. Our analyses of lines with root-localized phytochrome deficiency or root-specific phytochrome depletion have provided novel insights into the roles of root-specific PΦB, or phytochromes themselves, in the photoregulation of root development and root sensitivity to JA.
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Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación , Biliverdina/análogos & derivados , Ciclopentanos/farmacología , Luz , Oxilipinas/farmacología , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/efectos de la radiación , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Biliverdina/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes de Plantas/genética , Hipocótilo/efectos de los fármacos , Hipocótilo/crecimiento & desarrollo , Hipocótilo/efectos de la radiación , Morfogénesis/efectos de los fármacos , Morfogénesis/efectos de la radiación , Mutación/genética , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/efectos de la radiación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Fitocromo/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/enzimología , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/genética , Plantones/efectos de la radiaciónRESUMEN
Cancer cells continually sense and respond to mechanical cues from the extracellular matrix (ECM). Interaction with the ECM can alter intracellular signaling cascades, leading to changes in processes that promote cancer cell growth, migration, and survival. The present study used a recently developed composite hydrogel composed of a fibrin matrix and phase-shift emulsion, termed an acoustically responsive scaffold (ARS), to investigate effects of local mechanical properties on breast cancer cell signaling. Treatment of ARSs with focused ultrasound drives acoustic droplet vaporization (ADV) in a spatiotemporally controlled manner, inducing local compaction and stiffening of the fibrin matrix adjacent to the matrix-bubble interface. Combining ARSs and live single cell imaging of triple-negative breast cancer cells, it is discovered that both basal and growth-factor stimulated activities of protein kinase B (also known as Akt) and extracellular signal-regulated kinase (ERK), two major kinases driving cancer progression, negatively correlate with increasing distance from the ADV-induced bubble both in vitro and in a mouse model. Together, these data demonstrate that local changes in ECM compaction regulate Akt and ERK signaling in breast cancer and support further applications of the novel ARS technology to analyze spatial and temporal effects of ECM mechanics on cell signaling and cancer biology.
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Neoplasias de la Mama Triple Negativas , Animales , Línea Celular Tumoral , Fibrina , Humanos , Ratones , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , VolatilizaciónRESUMEN
Patients with estrogen receptor-positive (ER+) breast cancer, the most common subtype, remain at risk for lethal metastatic disease years after diagnosis. Recurrence arises partly because tumor cells in bone marrow become resistant to estrogen-targeted therapy. Here, we utilized a co-culture model of bone marrow mesenchymal stem cells (MSCs) and ER+ breast cancer cells to recapitulate interactions of cancer cells in bone marrow niches. ER+ breast cancer cells in direct contact with MSCs acquire cancer stem-like (CSC) phenotypes with increased resistance to standard antiestrogenic drugs. We confirmed that co-culture with MSCs increased labile iron in breast cancer cells, a phenotype associated with CSCs and disease progression. Clinically approved iron chelators and in-house lysosomal iron-targeting compounds restored sensitivity to antiestrogenic therapy. These findings establish iron modulation as a mechanism to reverse MSC-induced drug resistance and suggest iron modulation in combination with estrogen-targeted therapy as a promising, translatable strategy to treat ER+ breast cancer.
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Células Madre Mesenquimatosas , Neoplasias , Línea Celular Tumoral , Resistencia a Medicamentos , Resistencia a Antineoplásicos , Antagonistas de Estrógenos/farmacología , Estrógenos/farmacología , Hierro , Receptores de EstrógenosRESUMEN
Histopathology, the standard method to assess BM in hematologic malignancies such as myeloproliferative neoplasms (MPNs), suffers from notable limitations in both research and clinical settings. BM biopsies in patients fail to detect disease heterogeneity, may yield a nondiagnostic sample, and cannot be repeated frequently in clinical oncology. Endpoint histopathology precludes monitoring disease progression and response to therapy in the same mouse over time, missing likely variations among mice. To overcome these shortcomings, we used MRI to measure changes in cellularity, macromolecular constituents, and fat versus hematopoietic cells in BM using diffusion-weighted imaging (DWI), magnetization transfer, and chemical shift-encoded fat imaging. Combining metrics from these imaging parameters revealed dynamic alterations in BM following myeloablative radiation and transplantation. In a mouse MPLW515L BM transplant model of MPN, MRI detected effects of a JAK2 inhibitor, ruxolitinib, within 5 days of initiating treatment and identified differing kinetics of treatment responses in subregions of the tibia. Histopathology validated the MRI results for BM composition and heterogeneity. Anatomic MRI scans also showed reductions in spleen volume during treatment. These findings establish an innovative, clinically translatable MRI approach to quantify spatial and temporal changes in BM in MPN.
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Neoplasias Hematológicas , Imágenes de Resonancia Magnética Multiparamétrica , Trastornos Mieloproliferativos , Animales , Imagen por Resonancia Magnética , Ratones , Trastornos Mieloproliferativos/diagnóstico por imagenRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has had a detrimental effect on research. However, little has been done to identify and solve the unique challenges faced by early career investigators (ECIs). As a group of American Cancer Society-funded ECIs, we provide recommendations for solving these challenges in the aftermath of the pandemic.
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COVID-19 , Movilidad Laboral , Investigadores , Equilibrio entre Vida Personal y Laboral , Humanos , Tutoría , Investigadores/economía , Sociedades CientíficasRESUMEN
INTRODUCTION: CXCR4 and epidermal growth factor receptor (EGFR) represent two major families of receptors, G-protein coupled receptors and receptor tyrosine kinases, with central functions in cancer. While utilizing different upstream signaling molecules, both CXCR4 and EGFR activate kinases ERK and Akt, although single-cell activation of these kinases is markedly heterogeneous. One hypothesis regarding the origin of signaling heterogeneity proposes that intercellular variations arise from differences in pre-existing intracellular states set by extrinsic noise. While pre-existing cell states vary among cells, each pre-existing state defines deterministic signaling outputs to downstream effectors. Understanding causes of signaling heterogeneity will inform treatment of cancers with drugs targeting drivers of oncogenic signaling. METHODS: We built a single-cell computational model to predict Akt and ERK responses to CXCR4- and EGFR-mediated stimulation. We investigated signaling heterogeneity through these receptors and tested model predictions using quantitative, live-cell time-lapse imaging. RESULTS: We show that the pre-existing cell state predicts single-cell signaling through both CXCR4 and EGFR. Computational modeling reveals that the same set of pre-existing cell states explains signaling heterogeneity through both EGFR and CXCR4 at multiple doses of ligands and in two different breast cancer cell lines. The model also predicts how phosphatidylinositol-3-kinase (PI3K) targeted therapies potentiate ERK signaling in certain breast cancer cells and that low level, combined inhibition of MEK and PI3K ablates potentiated ERK signaling. CONCLUSIONS: Our data demonstrate that a conserved motif exists for EGFR and CXCR4 signaling and suggest potential clinical utility of the computational model to optimize therapy.
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The small Rho GTPases regulate important cellular processes that affect cancer metastasis, such as cell survival and proliferation, actin dynamics, adhesion, migration, invasion and transcriptional activation. The Rho GTPases function as molecular switches cycling between an active GTP-bound and inactive guanosine diphosphate (GDP)-bound conformation. It is known that Rho GTPase activities are mainly regulated by guanine nucleotide exchange factors (RhoGEFs), GTPase-activating proteins (RhoGAPs), GDP dissociation inhibitors (RhoGDIs) and guanine nucleotide exchange modifiers (GEMs). These Rho GTPase regulators are often dysregulated in cancer; however, the underlying mechanisms are not well understood. MicroRNAs (miRNAs), a large family of small non-coding RNAs that negatively regulate protein-coding gene expression, have been shown to play important roles in cancer metastasis. Recent studies showed that miRNAs are capable of directly targeting RhoGAPs, RhoGEFs, and RhoGDIs, and regulate the activities of Rho GTPases. This not only provides new evidence for the critical role of miRNA dysregulation in cancer metastasis, it also reveals novel mechanisms for Rho GTPase regulation. This review summarizes recent exciting findings showing that miRNAs play important roles in regulating Rho GTPase regulators (RhoGEFs, RhoGAPs, RhoGDIs), thus affecting Rho GTPase activities and cancer metastasis. The potential opportunities and challenges for targeting miRNAs and Rho GTPase regulators in treating cancer metastasis are also discussed. A comprehensive list of the currently validated miRNA-targeting of small Rho GTPase regulators is presented as a reference resource.
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Estrogen receptor-positive (ER+) breast cancer can recur up to 20 years after initial diagnosis. Delayed recurrences arise from disseminated tumors cells (DTCs) in sites such as bone marrow that remain quiescent during endocrine therapy and subsequently proliferate to produce clinically detectable metastases. Identifying therapies that eliminate DTCs and/or effectively target cells transitioning to proliferation promises to reduce risk of recurrence. To tackle this problem, we utilized a 3D co-culture model incorporating ER+ breast cancer cells and bone marrow mesenchymal stem cells to represent DTCs in a bone marrow niche. 3D co-cultures maintained cancer cells in a quiescent, viable state as measured by both single-cell and population-scale imaging. Single-cell imaging methods for metabolism by fluorescence lifetime (FLIM) of NADH and signaling by kinases Akt and ERK revealed that breast cancer cells utilized oxidative phosphorylation and signaling by Akt to a greater extent both in 3D co-cultures and a mouse model of ER+ breast cancer cells in bone marrow. Using our 3D co-culture model, we discovered that combination therapies targeting oxidative phosphorylation via the thioredoxin reductase (TrxR) inhibitor, D9, and the Akt inhibitor, MK-2206, preferentially eliminated breast cancer cells without altering viability of bone marrow stromal cells. Treatment of mice with disseminated ER+ human breast cancer showed that D9 plus MK-2206 blocked formation of new metastases more effectively than tamoxifen. These data establish an integrated experimental system to investigate DTCs in bone marrow and identify combination therapy against metabolic and kinase targets as a promising approach to effectively target these cells and reduce risk of recurrence in breast cancer.
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Médula Ósea/metabolismo , Neoplasias de la Mama/metabolismo , Técnicas de Cultivo de Célula/métodos , Células Neoplásicas Circulantes/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Femenino , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Células MCF-7 , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Recurrencia Local de Neoplasia , Células Neoplásicas Circulantes/efectos de los fármacos , Células Neoplásicas Circulantes/patología , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Tumor microenvironments expose cancer cells to heterogeneous, dynamic environments by shifting availability of nutrients, growth factors, and metabolites. Cells integrate various inputs to generate cellular memory that determines trajectories of subsequent phenotypes. Here we report that short-term exposure of triple-negative breast cancer cells to growth factors or targeted inhibitors regulates subsequent tumor initiation. Using breast cancer cells with different driver mutations, we conditioned cells lines with various stimuli for 4 hours before implanting these cells as tumor xenografts and quantifying tumor progression by means of bioluminescence imaging. In the orthotopic model, conditioning a low number of cancer cells with fetal bovine serum led to enhancement of tumor-initiating potential, tumor volume, and liver metastases. Epidermal growth factor and the mTORC1 inhibitor ridaforolimus produced similar but relatively reduced effects on tumorigenic potential. These data show that a short-term stimulus increases tumorigenic phenotypes based on cellular memory. Conditioning regimens failed to alter proliferation or adhesion of cancer cells in vitro or kinase signaling through Akt and ERK measured by multiphoton microscopy in vivo, suggesting that other mechanisms enhanced tumorigenesis. Given the dynamic nature of the tumor environment and time-varying concentrations of small-molecule drugs, this work highlights how variable conditions in tumor environments shape tumor formation, metastasis, and response to therapy.
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Carcinogénesis , Neoplasias de la Mama Triple Negativas/patología , Microambiente Tumoral , Animales , Adhesión Celular , Recuento de Células , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Factor de Crecimiento Epidérmico/farmacología , Quinasas MAP Reguladas por Señal Extracelular/farmacología , Femenino , Humanos , Mediciones Luminiscentes , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-akt/farmacología , Albúmina Sérica Bovina/farmacología , Sirolimus/análogos & derivados , Sirolimus/farmacologíaRESUMEN
The chemokine receptor CXCR4 regulates fundamental processes in development, normal physiology, and diseases, including cancer. Small subpopulations of CXCR4-positive cells drive the local invasion and dissemination of malignant cells during metastasis, emphasizing the need to understand the mechanisms controlling responses at the single-cell level to receptor activation by the chemokine ligand CXCL12. Using single-cell imaging, we discovered that short-term cellular memory of changes in environmental conditions tuned CXCR4 signaling to Akt and ERK, two kinases activated by this receptor. Conditioning cells with growth stimuli before CXCL12 exposure increased the number of cells that initiated CXCR4 signaling and the amplitude of Akt and ERK activation. Data-driven, single-cell computational modeling revealed that growth factor conditioning modulated CXCR4-dependent activation of Akt and ERK by decreasing extrinsic noise (preexisting cell-to-cell differences in kinase activity) in PI3K and mTORC1. Modeling established mTORC1 as critical for tuning single-cell responses to CXCL12-CXCR4 signaling. Our single-cell model predicted how combinations of extrinsic noise in PI3K, Ras, and mTORC1 superimposed on different driver mutations in the ERK and/or Akt pathways to bias CXCR4 signaling. Computational experiments correctly predicted that selected kinase inhibitors used for cancer therapy shifted subsets of cells to states that were more permissive to CXCR4 activation, suggesting that such drugs may inadvertently potentiate pro-metastatic CXCR4 signaling. Our work establishes how changing environmental inputs modulate CXCR4 signaling in single cells and provides a framework to optimize the development and use of drugs targeting this signaling pathway.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal/fisiología , Línea Celular Tumoral , Quimiocina CXCL12/farmacología , Simulación por Computador , Activación Enzimática/efectos de los fármacos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Microscopía Fluorescente/métodos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Análisis de la Célula Individual/métodos , Imagen de Lapso de Tiempo/métodosRESUMEN
Migration and invasion of cancer cells constitute fundamental processes in tumor progression and metastasis. Migratory cancer cells commonly upregulate expression of plasminogen activator inhibitor 1 (PAI1), and PAI1 correlates with poor prognosis in breast cancer. However, mechanisms by which PAI1 promotes migration of cancer cells remain incompletely defined. Here we show that increased PAI1 drives rearrangement of the actin cytoskeleton, mitochondrial fragmentation, and glycolytic metabolism in triple-negative breast cancer (TNBC) cells. In two-dimensional environments, both stable expression of PAI1 and treatment with recombinant PAI1 increased migration, which could be blocked with the specific inhibitor tiplaxtinin. PAI1 also promoted invasion into the extracellular matrix from coculture spheroids with human mammary fibroblasts in fibrin gels. Elevated cellular PAI1 enhanced cytoskeletal features associated with migration, actin-rich migratory structures, and reduced actin stress fibers. In orthotopic tumor xenografts, we discovered that TNBC cells with elevated PAI1 show collagen fibers aligned perpendicular to the tumor margin, an established marker of invasive breast tumors. Further studies revealed that PAI1 activates ERK signaling, a central regulator of motility, and promotes mitochondrial fragmentation. Consistent with known effects of mitochondrial fragmentation on metabolism, fluorescence lifetime imaging microscopy of endogenous NADH showed that PAI1 promotes glycolysis in cell-based assays, orthotopic tumor xenografts, and lung metastases. Together, these data demonstrate for the first time that PAI1 regulates cancer cell metabolism and suggest targeting metabolism to block motility and tumor progression. IMPLICATIONS: We identified a novel mechanism through which cancer cells alter their metabolism to promote tumor progression.
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Citoesqueleto de Actina/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Glucólisis , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Trasplante de Neoplasias , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Regulación hacia Arriba , Secuenciación Completa del GenomaRESUMEN
First discovered in 1993, microRNAs (miRNAs) have been one of the hottest research areas over the past two decades. Oftentimes, miRNAs levels are found to be dysregulated in cancer patients. The potential use of miRNAs in cancer therapies is an emerging and promising field, with research finding miRNAs to play a role in cancer initiation, tumor growth, and metastasis. Therefore, miRNAs could become an integral part from cancer diagnosis to treatment in future. This review aims to examine current novel research work on the potential roles of miRNAs in cancer therapies, while also discussing several current challenges and needed future research.