RESUMO
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.
Assuntos
Estabilidade de RNA , Mutação , Fosforilação , RNA Mensageiro/metabolismoRESUMO
Insulin-stimulated glucose uptake is known to involve microtubules, although the function of microtubules and the microtubule-regulating proteins involved in insulin action are poorly understood. CLASP2, a plus-end tracking microtubule-associated protein (+TIP) that controls microtubule dynamics, was recently implicated as the first +TIP associated with insulin-regulated glucose uptake. Here, using protein-specific targeted quantitative phosphoproteomics within 3T3-L1 adipocytes, we discovered that insulin regulates phosphorylation of the CLASP2 network members G2L1, MARK2, CLIP2, AGAP3, and CKAP5 as well as EB1, revealing the existence of a previously unknown microtubule-associated protein system that responds to insulin. To further investigate, G2L1 interactome studies within 3T3-L1 adipocytes revealed that G2L1 coimmunoprecipitates CLASP2 and CLIP2 as well as the master integrators of +TIP assembly, the end binding (EB) proteins. Live-cell total internal reflection fluorescence microscopy in adipocytes revealed G2L1 and CLASP2 colocalize on microtubule plus-ends. We found that although insulin increases the number of CLASP2-containing plus-ends, insulin treatment simultaneously decreases CLASP2-containing plus-end velocity. In addition, we discovered that insulin stimulates redistribution of CLASP2 and G2L1 from exclusive plus-end tracking to "trailing" behind the growing tip of the microtubule. Insulin treatment increases α-tubulin Lysine 40 acetylation, a mechanism that was observed to be regulated by a counterbalance between GSK3 and mTOR, and led to microtubule stabilization. Our studies introduce insulin-stimulated microtubule stabilization and plus-end trailing of +TIPs as new modes of insulin action and reveal the likelihood that a network of microtubule-associated proteins synergize to coordinate insulin-regulated microtubule dynamics.
Assuntos
Adipócitos/metabolismo , Insulina/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Células 3T3-L1 , Acetilação/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Animais , Lisina/metabolismo , Camundongos , Microtúbulos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Transporte Proteico/efeitos dos fármacos , Tubulina (Proteína)/metabolismoRESUMO
Dynamic assembly and disassembly of actin filaments is a major driving force for cell movements. Border cells in the Drosophila ovary provide a simple and genetically tractable model to study the mechanisms regulating cell migration. To identify new genes that regulate cell movement in vivo, we screened lethal mutations on chromosome 3R for defects in border cell migration and identified two alleles of the gene psidin (psid). In vitro, purified Psid protein bound F-actin and inhibited the interaction of tropomyosin with F-actin. In vivo, psid mutations exhibited genetic interactions with the genes encoding tropomyosin and cofilin. Border cells overexpressing Psid together with GFP-actin exhibited altered protrusion/retraction dynamics. Psid knockdown in cultured S2 cells reduced, and Psid overexpression enhanced, lamellipodial dynamics. Knockdown of the human homolog of Psid reduced the speed and directionality of migration in wounded MCF10A breast epithelial monolayers, whereas overexpression of the protein increased migration speed and altered protrusion dynamics in EGF-stimulated cells. These results indicate that Psid is an actin regulatory protein that plays a conserved role in protrusion dynamics and cell migration.
Assuntos
Proteínas Sanguíneas/metabolismo , Movimento Celular , Extensões da Superfície Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Extensões da Superfície Celular/genética , Extensões da Superfície Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mutação , Ovário/citologia , Tropomiosina/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
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
Dendritic spines are the postsynaptic compartment of a neuronal synapse and are critical for synaptic connectivity and plasticity. A developmental precursor to dendritic spines, dendritic filopodia (DF), facilitate synapse formation by sampling the environment for suitable axon partners during neurodevelopment and learning. Despite the significance of the actin cytoskeleton in driving these dynamic protrusions, the actin elongation factors involved are not well characterized. We identified the Ena/VASP protein EVL as uniquely required for the morphogenesis and dynamics of DF. Using a combination of genetic and optogenetic manipulations, we demonstrated that EVL promotes protrusive motility through membrane-direct actin polymerization at DF tips. EVL forms a complex at nascent protrusions and DF tips with MIM/MTSS1, an I-BAR protein important for the initiation of DF. We proposed a model in which EVL cooperates with MIM to coalesce and elongate branched actin filaments, establishing the dynamic lamellipodia-like architecture of DF.
Assuntos
Actinas , Moléculas de Adesão Celular , Proteínas dos Microfilamentos , Pseudópodes , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Espinhas Dendríticas/metabolismo , Neurônios/metabolismo , Pseudópodes/metabolismo , Sinapses/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas dos Microfilamentos/metabolismoRESUMO
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.
Assuntos
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
Epidermal Growth Factor (EGF) is an important regulator of normal epithelial and carcinoma cell migration. The mechanism by which EGF induces cell migration is not fully understood. A recent report in Nature Cell Biology (Katz et al., 2007) demonstrates that EGF regulates migration through a switch in the expression of two tensin isoforms, weakening the association of beta1 integrin with the actin cytoskeleton in focal adhesions.
Assuntos
Movimento Celular/fisiologia , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Neoplasias da Mama/patologia , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Feminino , Adesões Focais/fisiologia , Células HeLa , Humanos , Integrina beta1/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Modelos Biológicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , TensinasRESUMO
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.
Assuntos
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
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).
Assuntos
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.
Assuntos
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
Understanding the mechanisms controlling cancer cell invasion and metastasis constitutes a fundamental step in setting new strategies for diagnosis, prognosis, and therapy of metastatic cancers. LIM kinase1 (LIMK1) is a member of a novel class of serine-threonine protein kinases. Cofilin, a LIMK1 substrate, is essential for the regulation of actin polymerization and depolymerization during cell migration. Previous studies have made opposite conclusions as to the role of LIMK1 in tumor cell motility and metastasis, claiming either an increase or decrease in cell motility and metastasis as a result of LIMK1 over expression (Zebda, N., O. Bernard, M. Bailly, S. Welti, D.S. Lawrence, and J.S. Condeelis. 2000. J. Cell Biol. 151:1119-1128; Davila, M., A.R. Frost, W.E. Grizzle, and R. Chakrabarti. 2003. J. Biol. Chem. 278:36868-36875; Yoshioka, K., V. Foletta, O. Bernard, and K. Itoh. 2003. Proc. Natl. Acad. Sci. USA. 100:7247-7252; Nishita, M., C. Tomizawa, M. Yamamoto, Y. Horita, K. Ohashi, and K. Mizuno. 2005. J. Cell Biol. 171:349-359). We resolve this paradox by showing that the effects of LIMK1 expression on migration, intravasation, and metastasis of cancer cells can be most simply explained by its regulation of the output of the cofilin pathway. LIMK1-mediated decreases or increases in the activity of the cofilin pathway are shown to cause proportional decreases or increases in motility, intravasation, and metastasis of tumor cells.
Assuntos
Cofilina 1/metabolismo , Neoplasias Mamárias Experimentais/genética , Proteínas Quinases/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Extensões da Superfície Celular/genética , Extensões da Superfície Celular/fisiologia , Quimiotaxia/genética , Quimiotaxia/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Quinases Lim , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Mutação , Invasividade Neoplásica , Metástase Neoplásica , Fosforilação , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Endogâmicos F344 , Análise de Sobrevida , TransfecçãoRESUMO
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.
Assuntos
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.
Assuntos
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
Metastatic mammary carcinoma cells, which have previously been observed to form mature, matrix degrading invadopodia on a thick ECM matrix, are able to form invadopodia with similar characteristics on glass without previously applied matrix. They form in response to epidermal growth factor (EGF), and contain the usual invadopodium core proteins N-WASP, Arp2/3, cortactin, cofilin, and F-actin. The study of invadopodia on glass allows for higher resolution analysis including the use of total internal reflection microscopy and analysis of their relationship to other cell motility events, in particular, lamellipodium extension and chemotaxis toward an EGF gradient. Invadopodium formation on glass requires N-WASP and cortactin but not microtubules. In a gradient of EGF more invadopodia form on the side of the cells facing the source of EGF. In addition, depletion of N-WASP or cortactin, which blocks invadopodium fromation, inhibits chemotaxis of cells towards EGF. This appears to be a localized defect in chemotaxis since depletion of N-WASP or cortactin via siRNA had no effect on lamellipodium protrusion or barbed end generation at the lamellipodium's leading edge. Since chemotaxis to EGF by breast tumor cells is involved in metastasis, inhibiting N-WASP activity in breast tumor cells might prevent metastasis of tumor cells while not affecting chemotaxis-dependent innate immunity which depends on WASp function in macrophages.
Assuntos
Neoplasias da Mama/patologia , Quimiotaxia , Cortactina/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Quimiotaxia/genética , Cortactina/genética , Destrina/genética , Destrina/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , RNA Interferente Pequeno/genética , Ratos , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genéticaRESUMO
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.
Assuntos
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.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias , Fibroblastos , Humanos , Transdução de Sinais , Células Estromais , Microambiente TumoralRESUMO
BACKGROUND: Previous work has led to the hypothesis that cofilin severing, as regulated by PLC, is involved in chemotactic sensing. We have tested this hypothesis by investigating whether activation of endogenous cofilin is spatially and temporally linked to sensing an EGF point source in carcinoma cells. RESULTS: We demonstrate that inhibition of endogenous cofilin activity with either siRNA or overexpression of LIMK suppresses directional sensing in carcinoma cells. LIMK siRNA knockdown, which suppresses cofilin phosphorylation, and microinjection of S3C cofilin, a cofilin mutant that is constitutively active and not phosphorylated by LIMK, also inhibits directional sensing and chemotaxis. These results indicate that phosphorylation of cofilin by LIMK, in addition to cofilin activity, is required for chemotaxis. Cofilin activity concentrates rapidly at the newly formed leading edge facing the gradient, whereas cofilin phosphorylation increases throughout the cell. Quantification of these results indicates that the amplification of asymmetric actin polymerization required for protrusion toward the EGF gradient occurs at the level of cofilin but not at the level of PLC activation by EGFR. CONCLUSIONS: These results indicate that local activation of cofilin by PLC and its global inactivation by LIMK phosphorylation combine to generate the local asymmetry of actin polymerization required for chemotaxis.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/genética , Animais , Linhagem Celular Tumoral , Fatores Quimiotáticos/metabolismo , Quimiotaxia/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Quinases Lim , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Ratos , Fosfolipases Tipo C/metabolismoRESUMO
The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.
Assuntos
Carcinoma/metabolismo , Quimiotaxia/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Proteínas dos Microfilamentos/fisiologia , Metástase Neoplásica/fisiopatologia , Fosfolipases Tipo C/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina , Actinas/biossíntese , Actinas/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Carcinoma/patologia , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Proteínas dos Microfilamentos/efeitos dos fármacos , Proteínas dos Microfilamentos/genética , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Interferência de RNA , Ratos , Fosfolipases Tipo C/efeitos dos fármacosRESUMO
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.