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Background: Neurofibromin, coded by the NF1 tumor suppressor gene, is the main negative regulator of the RAS pathway and is frequently mutated in various cancers. Women with Neurofibromatosis Type I (NF1)-a tumor predisposition syndrome caused by a germline NF1 mutation-have an increased risk of developing aggressive breast cancer with poorer prognosis. The mechanism by which NF1 mutations lead to breast cancer tumorigenesis is not well understood. Therefore, the objective of this work was to identify stromal alterations before tumor formation that result in the increased risk and poorer outcome seen among NF1 patients with breast cancer. Approach: To accurately model the germline monoallelic NF1 mutations in NF1 patients, we utilized an Nf1-deficient rat model with accelerated mammary development before presenting with highly penetrant breast cancer. Results: We identified increased collagen content in Nf1-deficient rat mammary glands before tumor formation that correlated with age of tumor onset. Additionally, gene expression analysis revealed that Nf1-deficient mature adipocytes in the rat mammary gland have increased collagen expression and shifted to a fibroblast and preadipocyte expression profile. This alteration in lineage commitment was also observed with in vitro differentiation, however, flow cytometry analysis did not show a change in mammary adipose-derived mesenchymal stem cell abundance. Conclusion: Collectively, this study uncovered the previously undescribed role of Nf1 in mammary collagen deposition and regulating adipocyte differentiation. In addition to unraveling the mechanism of tumor formation, further investigation of adipocytes and collagen modifications in preneoplastic mammary glands will create a foundation for developing early detection strategies of breast cancer among NF1 patients.
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Distinguishing key factors that drive the switch from indolent to invasive disease will make a significant impact on guiding the treatment of prostate cancer (PCa) patients. Here, we identify a novel signaling pathway linking hypoxia and PIM1 kinase to the actin cytoskeleton and cell motility. An unbiased proteomic screen identified Abl-interactor 2 (ABI2), an integral member of the wave regulatory complex (WRC), as a PIM1 substrate. Phosphorylation of ABI2 at Ser183 by PIM1 increased ABI2 protein levels and enhanced WRC formation, resulting in increased protrusive activity and cell motility. Cell protrusion induced by hypoxia and/or PIM1 was dependent on ABI2. In vivo smooth muscle invasion assays showed that overexpression of PIM1 significantly increased the depth of tumor cell invasion, and treatment with PIM inhibitors significantly reduced intramuscular PCa invasion. This research uncovers a HIF-1-independent signaling axis that is critical for hypoxia-induced invasion and establishes a novel role for PIM1 as a key regulator of the actin cytoskeleton.
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Actinas , Proteínas Adaptadoras de Transdução de Sinal , Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-pim-1 , Humanos , Masculino , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Hipóxia , Proteômica , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Transdução de Sinais , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Invasividade NeoplásicaRESUMO
We previously identified the mechanistic target of rapamycin complex 2 (mTORC2) as an effector of Ras for the control of directed cell migration in Dictyostelium. Recently, the Ras-mediated regulation of mTORC2 was found to be conserved in mammalian cells, and mTORC2 was shown to be an effector of oncogenic Ras. Interestingly, mTORC2 has been linked to cancer cell migration, and particularly in breast cancer. Here, we investigated the role of Ras in promoting the migration and invasion of breast cancer cells through mTORC2. We observed that both Ras and mTORC2 promote the migration of different breast cancer cells and breast cancer cell models. Using HER2 and oncogenic Ras-transformed breast epithelial MCF10A cells, we found that both wild-type Ras and oncogenic Ras promote mTORC2 activation and an mTORC2-dependent migration and invasion in these breast cancer models. We further observed that, whereas oncogenic Ras-transformed MCF10A cells display uncontrolled cell proliferation and invasion, disruption of mTORC2 leads to loss of invasiveness only. Together, our findings suggest that, whereas the Ras-mediated activation of mTORC2 is expected to play a minor role in breast tumor formation, the Ras-mTORC2 pathway plays an important role in promoting the migration and invasion of breast cancer cells.
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Neoplasias da Mama , Dictyostelium , Animais , Feminino , Humanos , Neoplasias da Mama/patologia , Movimento Celular/fisiologia , Dictyostelium/metabolismo , Células Epiteliais/metabolismo , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Sirolimo , Proteínas ras/metabolismoRESUMO
We describe a three-dimensional (3D) in vitro assay for quantifying cancer cell invasion into a 3D microenvironment with defined biochemical and biophysical properties. Researchers can quantify invasion dynamics (e.g., cell motility and directionality) and examine morphological changes during invasion, using live-cell and confocal imaging techniques. Together, these advantages over existing in vitro invasion assays, such as transwell-based assays, provide researchers with a valuable tool to gain insight into the mechanisms regulating cancer cell invasion. For complete details on the use and execution of this protocol, please refer to Padilla-Rodriguez et al. (2018) and Watson et al. (2021).
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Microambiente Tumoral , Biofísica , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Humanos , Invasividade NeoplásicaRESUMO
Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report ßIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific ßIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, ßIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, ßIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.
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Espectrina , Fator A de Crescimento do Endotélio Vascular , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Camundongos , Neovascularização Fisiológica , Proteômica , Transdução de Sinais , Espectrina/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The human proteome is replete with short linear motifs (SLiMs) of four to six residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of human ENAH, an actin regulator that is highly expressed in invasive cancers, we screened 36-residue proteome-derived peptides and discovered new interaction partners of ENAH and diverse mechanisms by which context influences binding. A pocket on the ENAH EVH1 domain that has diverged from other Ena/VASP paralogs recognizes extended SLiMs and favors motif-flanking proline residues. Many high-affinity ENAH binders that contain two proline-rich SLiMs use a noncanonical site on the EVH1 domain for binding and display a thermodynamic signature consistent with the two-motif chain engaging a single domain. We also found that photoreceptor cilium actin regulator (PCARE) uses an extended 23-residue region to obtain a higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of proteomic context on motif-mediated binding, revealing diverse mechanisms of control over EVH1 interactions and establishing that SLiMs can't be fully understood outside of their native context.
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Actinas/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Proteínas dos Microfilamentos/metabolismo , Prolina/metabolismo , Moléculas de Adesão Celular/metabolismo , Células HEK293 , Humanos , ProteômicaRESUMO
Metazoan proteomes contain many paralogous proteins that have evolved distinct functions. The Ena/VASP family of actin regulators consists of three members that share an EVH1 interaction domain with a 100 % conserved binding site. A proteome-wide screen revealed photoreceptor cilium actin regulator (PCARE) as a high-affinity ligand for ENAH EVH1. Here, we report the surprising observation that PCARE is ~100-fold specific for ENAH over paralogs VASP and EVL and can selectively bind ENAH and inhibit ENAH-dependent adhesion in cells. Specificity arises from a mechanism whereby PCARE stabilizes a conformation of the ENAH EVH1 domain that is inaccessible to family members VASP and EVL. Structure-based modeling rapidly identified seven residues distributed throughout EVL that are sufficient to differentiate binding by ENAH vs. EVL. By exploiting the ENAH-specific conformation, we rationally designed the tightest and most selective ENAH binder to date. Our work uncovers a conformational mechanism of interaction specificity that distinguishes highly similar paralogs and establishes tools for dissecting specific Ena/VASP functions in processes including cancer cell invasion.
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Actinas/metabolismo , Sítios de Ligação , Moléculas de Adesão Celular/metabolismo , Proteínas do Olho/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Conformação Molecular , Domínios ProteicosRESUMO
While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.
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Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Fenômenos Biomecânicos , Medula Óssea/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Mecanotransdução Celular , Invasividade Neoplásica , Microambiente TumoralRESUMO
Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P-bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P-body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P-body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P-bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin ß1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer-relevant functions and suggest that modulation of P-body activity may represent a new paradigm for cancer treatment.
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Estabilidade de RNA , Mutação , Fosforilação , RNA Mensageiro/metabolismoRESUMO
We stand against racism and discrimination in cancer research in the U.S. By sharing the stories of scientists from different ethnicities, identities, and national origins, we want to promote change through mentoring, active participation, and policy changes and to inspire the next generation of cancer researchers: we make better science together.
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Pesquisa Biomédica/estatística & dados numéricos , Diversidade Cultural , Etnicidade/estatística & dados numéricos , Tutoria/estatística & dados numéricos , Neoplasias/terapia , Pesquisadores/estatística & dados numéricos , Pesquisa Biomédica/educação , Etnicidade/psicologia , Humanos , Neoplasias/diagnóstico , Pesquisadores/psicologia , Estados UnidosRESUMO
The tumor microenvironment has been demonstrated to play a crucial role in modulating cancer progression. Amongst various cell types within the tumor microenvironment, cancer associated fibroblasts (CAFs) are in abundance, serving to modulate the biophysical properties of the stromal matrix, through excessive deposition of extracellular matrix (ECM) proteins that leads to enhanced tumor progression. There is still a critical need to develop a fundamental framework on the role of tumor-stromal cell interactions on desmoplasia and tumorigenicity. Herein, we developed a 3D microengineered organotypic tumor-stroma model incorporated with breast cancer cells surrounded by CAF-embedded collagen matrix. We further integrated our platform with atomic force microscopy (AFM) to study the dynamic changes in stromal stiffness during active tumor invasion. Our findings primarily demonstrated enhanced tumor progression in the presence of CAFs. Furthermore, we highlighted the crucial role of crosstalk between tumor cells and CAFs on stromal desmoplasia, where we identified the role of tumor-secreted PDGF-AA/-BB on elevated matrix stiffness. Inhibition of the activity of PDGFRs in CAFs led to attenuation of stromal stiffness. Overall, our work presents a well-controlled tumor microenvironment model capable of dissecting specific biophysical and biochemical signaling cues which lead to stromal desmoplasia and tumor progression.
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Fibroblastos Associados a Câncer , Neoplasias , Fibroblastos , Humanos , Transdução de Sinais , Células Estromais , Microambiente TumoralRESUMO
The mechanical properties of a cell's microenvironment influence many aspects of cellular behavior, including cell migration. Durotaxis, the migration toward increasing matrix stiffness, has been implicated in processes ranging from development to cancer. During durotaxis, mechanical stimulation by matrix rigidity leads to directed migration. Studies suggest that cells sense mechanical stimuli, or mechanosense, through the acto-myosin cytoskeleton at focal adhesions (FAs); however, FA actin cytoskeletal remodeling and its role in mechanosensing are not fully understood. Here, we show that the Ena/VASP family member, Ena/VASP-like (EVL), polymerizes actin at FAs, which promotes cell-matrix adhesion and mechanosensing. Importantly, we show that EVL regulates mechanically directed motility, and that suppression of EVL expression impedes 3D durotactic invasion. We propose a model in which EVL-mediated actin polymerization at FAs promotes mechanosensing and durotaxis by maturing, and thus reinforcing, FAs. These findings establish dynamic FA actin polymerization as a central aspect of mechanosensing and identify EVL as a crucial regulator of this process.
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Actinas/metabolismo , Actomiosina/metabolismo , Adesões Focais/metabolismo , Mecanotransdução Celular , Citoesqueleto de Actina/metabolismo , Animais , Adesão Celular , Movimento Celular , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Células NIH 3T3RESUMO
Tumor-stroma interactions significantly influence cancer cell metastasis and disease progression. These interactions are partly comprised of the cross-talk between tumor and stromal fibroblasts, but the key molecular mechanisms within the cross-talk that govern cancer invasion are still unclear. Here, we adapted our previously developed microfluidic device as a 3D in vitro organotypic model to mechanistically study tumor-stroma interactions by mimicking the spatial organization of the tumor microenvironment on a chip. We cocultured breast cancer and patient-derived fibroblast cells in 3D tumor and stroma regions, respectively, and combined functional assessments, including cancer cell migration, with transcriptome profiling to unveil the molecular influence of tumor-stroma cross-talk on invasion. This led to the observation that cancer-associated fibroblasts (CAF) enhanced invasion in 3D by inducing expression of a novel gene of interest, glycoprotein nonmetastatic B (GPNMB), in breast cancer cells, resulting in increased migration speed. Importantly, knockdown of GPNMB blunted the influence of CAF on enhanced cancer invasion. Overall, these results demonstrate the ability of our model to recapitulate patient-specific tumor microenvironments to investigate the cellular and molecular consequences of tumor-stroma interactions. SIGNIFICANCE: An organotypic model of tumor-stroma interactions on a microfluidic chip reveals that CAFs promote invasion by enhancing expression of GPNMB in breast cancer cells.
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Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Fibroblastos/patologia , Glicoproteínas de Membrana/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Organoides/patologia , Microambiente Tumoral , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Movimento Celular , Técnicas de Cocultura , Feminino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Modelos Biológicos , Invasividade Neoplásica , Organoides/metabolismoRESUMO
Estrogen promotes growth of estrogen receptor-positive (ER+) breast tumors. However, epidemiological studies examining the prognostic characteristics of breast cancer in postmenopausal women receiving hormone replacement therapy reveal a significant decrease in tumor dissemination, suggesting that estrogen has potential protective effects against cancer cell invasion. Here, we show that estrogen suppresses invasion of ER+ breast cancer cells by increasing transcription of the Ena/VASP protein, EVL, which promotes the generation of suppressive cortical actin bundles that inhibit motility dynamics, and is crucial for the ER-mediated suppression of invasion in vitro and in vivo. Interestingly, despite its benefits in suppressing tumor growth, anti-estrogenic endocrine therapy decreases EVL expression and increases local invasion in patients. Our results highlight the dichotomous effects of estrogen on tumor progression and suggest that, in contrast to its established role in promoting growth of ER+ tumors, estrogen has a significant role in suppressing invasion through actin cytoskeletal remodeling.
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Citoesqueleto de Actina/química , Actinas/química , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/química , Estrogênios/química , Invasividade Neoplásica , Animais , Células CACO-2 , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Cães , Estradiol/química , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Linfonodos/patologia , Células MCF-7 , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos NOD , Metástase Neoplásica , Transcrição GênicaRESUMO
Cancer is a major leading cause of disease-related death in the world. The severe impact of cancer can be attributed to poor understanding of the mechanisms involved in earliest steps of the metastatic cascade, specifically invasion into the surrounding stroma and intravasation into the blood capillaries. However, conducting integrated biological studies of invasion and intravasation have been challenging, within in vivo models and traditional in vitro assay, due to difficulties in establishing a precise tumor microenvironment. To that end, in this work, a novel 3D microfluidic platform comprised of concentric three-layer cell-laden hydrogels for simultaneous investigation of breast cancer cell invasion and intravasation as well as vasculature maturation influenced by tumor-vascular crosstalk is developed. It was demonstrated that the presence of spontaneously formed vasculature enhance MDA-MB-231 invasion into the 3D stroma. Following invasion, cancer cells are visualized intravasating into the outer vasculature. Additionally, invading cancer cells significantly reduce vessel diameter while increasing permeability, consistent with previous in vivo studies. Major signaling cytokines involved in tumor-vascular crosstalk that govern cancer cell invasion and intravasation are further identified. Taken together, this platform will enable unique insights of critical biological events within the metastatic cascade, with significant potential for developing efficient cancer therapeutics.
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Neoplasias da Mama , Modelos Biológicos , Neovascularização Patológica , Microambiente Tumoral , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Invasividade Neoplásica , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologiaRESUMO
INTRODUCTION: Cancer associated fibroblasts (CAFs) are known to participate in anti-cancer drug resistance by upregulating desmoplasia and pro-survival mechanisms within the tumor microenvironment. In this regard, anti-fibrotic drugs (i.e., tranilast) have been repurposed to diminish the elastic modulus of the stromal matrix and reduce tumor growth in presence of chemotherapeutics (i.e., doxorubicin). However, the quantitative assessment on impact of these stromal targeting drugs on matrix stiffness and tumor progression is still missing in the sole presence of CAFs. METHODS: We developed a high-density 3D microengineered tumor model comprised of MDA-MB-231 (highly invasive breast cancer cells) embedded microwells, surrounded by CAFs encapsulated within collagen I hydrogel. To study the influence of tranilast and doxorubicin on fibrosis, we probed the matrix using atomic force microscopy (AFM) and assessed matrix protein deposition. We further studied the combinatorial influence of the drugs on cancer cell proliferation and invasion. RESULTS: Our results demonstrated that the combinatorial action of tranilast and doxorubicin significantly diminished the stiffness of the stromal matrix compared to the control. The two drugs in synergy disrupted fibronectin assembly and reduced collagen fiber density. Furthermore, the combination of these drugs, condensed tumor growth and invasion. CONCLUSION: In this work, we utilized a 3D microengineered model to tease apart the role of tranilast and doxorubicin in the sole presence of CAFs on desmoplasia, tumor growth and invasion. Our study lay down a ground work on better understanding of the role of biomechanical properties of the matrix on anti-cancer drug efficacy in the presence of single class of stromal cells.
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In this study, to model 3D chemotactic tumor-stroma invasion in vitro, we developed an innovative microfluidic chip allowing side-by-side positioning of 3D hydrogel-based matrices. We were able to (1) create a dual matrix architecture that extended in a continuous manner, thus allowing invasion from one 3D matrix to another, and (2) establish distinct regions of tumor and stroma cell/ECM compositions, with a clearly demarcated tumor invasion front, thus allowing us to quantitatively analyze progression of cancer cells into the stroma at a tissue or single-cell level. We showed significantly enhanced cancer cell invasion in response to a transient gradient of epidermal growth factor (EGF). 3D tracking at the single-cell level displayed increased migration speed and persistence. Subsequently, we analyzed changes in expression of EGF receptors, cell aspect ratio, and protrusive activity. These findings show the unique ability of our model to quantitatively analyze 3D chemotactic invasion, both globally by tracking the progression of the invasion front, and at the single-cell level by examining changes in cellular behavior and morphology using high-resolution imaging. Taken together, we have shown a novel model recapitulating 3D tumor-stroma interactions for studies of real-time cell invasion and morphological changes within a single platform.
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Breast cancer cell invasion is a highly orchestrated process driven by a myriad of complex microenvironmental stimuli, making it difficult to isolate and assess the effects of biochemical or biophysical cues (i.e. tumor architecture, matrix stiffness) on disease progression. In this regard, physiologically relevant tumor models are becoming instrumental to perform studies of cancer cell invasion within well-controlled conditions. Herein, we explored the use of photocrosslinkable hydrogels and a novel, two-step photolithography technique to microengineer a 3D breast tumor model. The microfabrication process enabled precise localization of cell-encapsulated circular constructs adjacent to a low stiffness matrix. To validate the model, breast cancer cell lines (MDA-MB-231, MCF7) and non-tumorigenic mammary epithelial cells (MCF10A) were embedded separately within the tumor model, all of which maintained high viability throughout the experiments. MDA-MB-231 cells exhibited extensive migratory behavior and invaded the surrounding matrix, whereas MCF7 or MCF10A cells formed clusters that stayed confined within the circular tumor regions. Additionally, real-time cell tracking indicated that the speed and persistence of MDA-MB-231 cells were substantially higher within the surrounding matrix compared to the circular constructs. Z-stack imaging of F-actin/α-tubulin cytoskeletal organization revealed unique 3D protrusions in MDA-MB-231 cells and an abundance of 3D clusters formed by MCF7 and MCF10A cells. Our results indicate that gelatin methacrylate (GelMA) hydrogel, integrated with the two-step photolithography technique, has great promise in the development of 3D tumor models with well-defined architecture and tunable stiffness.
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Neoplasias da Mama/patologia , Movimento Celular , Microtecnologia/métodos , Modelos Biológicos , Actinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Forma Celular , Sobrevivência Celular , Rastreamento de Células , Citoesqueleto/metabolismo , Feminino , Fluorescência , Humanos , Indóis/metabolismo , Rodaminas/metabolismo , Imagem com Lapso de TempoRESUMO
Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.
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Citoesqueleto de Actina/metabolismo , Movimento Celular , Neoplasias/patologia , Profilinas/fisiologia , Citoesqueleto de Actina/ultraestrutura , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/ultraestrutura , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Miosinas/metabolismo , Miosinas/fisiologia , Gradação de Tumores , Invasividade Neoplásica/genética , Neoplasias/genética , Neoplasias/metabolismo , Profilinas/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Interferência de RNARESUMO
Therapies for most malignancies are generally ineffective once metastasis occurs. While tumour cells migrate through tissues using diverse strategies, the signalling networks controlling such behaviours in human tumours are poorly understood. Here we define a role for the Diaphanous-related formin-3 (DIAPH3) as a non-canonical regulator of metastasis that restrains conversion to amoeboid cell behaviour in multiple cancer types. The DIAPH3 locus is close to RB1, within a narrow consensus region of deletion on chromosome 13q in prostate, breast and hepatocellular carcinomas. DIAPH3 silencing in human carcinoma cells destabilized microtubules and induced defective endocytic trafficking, endosomal accumulation of EGFR, and hyperactivation of EGFR/MEK/ERK signalling. Silencing also evoked amoeboid properties, increased invasion and promoted metastasis in mice. In human tumours, DIAPH3 down-regulation was associated with aggressive or metastatic disease. DIAPH3-silenced cells were sensitive to MEK inhibition, but showed reduced sensitivity to EGFR inhibition. These findings have implications for understanding mechanisms of metastasis, and suggest that identifying patients with chromosomal deletions at DIAPH3 may have prognostic value.