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1.
Proc Natl Acad Sci U S A ; 120(11): e2215553120, 2023 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-36877839

RESUMO

Mounting evidence implicates the giant, cytoskeletal protein obscurin (720 to 870 kDa), encoded by the OBSCN gene, in the predisposition and development of breast cancer. Accordingly, prior work has shown that the sole loss of OBSCN from normal breast epithelial cells increases survival and chemoresistance, induces cytoskeletal alterations, enhances cell migration and invasion, and promotes metastasis in the presence of oncogenic KRAS. Consistent with these observations, analysis of Kaplan-Meier Plotter datasets reveals that low OBSCN levels correlate with significantly reduced overall and relapse-free survival in breast cancer patients. Despite the compelling evidence implicating OBSCN loss in breast tumorigenesis and progression, its regulation remains elusive, limiting any efforts to restore its expression, a major challenge given its molecular complexity and gigantic size (~170 kb). Herein, we show that OBSCN-Antisense RNA 1 (OBSCN-AS1), a novel nuclear long-noncoding RNA (lncRNA) gene originating from the minus strand of OBSCN, and OBSCN display positively correlated expression and are downregulated in breast cancer biopsies. OBSCN-AS1 regulates OBSCN expression through chromatin remodeling involving H3 lysine 4 trimethylation enrichment, associated with open chromatin conformation, and RNA polymerase II recruitment. CRISPR-activation of OBSCN-AS1 in triple-negative breast cancer cells effectively and specifically restores OBSCN expression and markedly suppresses cell migration, invasion, and dissemination from three-dimensional spheroids in vitro and metastasis in vivo. Collectively, these results reveal the previously unknown regulation of OBSCN by an antisense lncRNA and the metastasis suppressor function of the OBSCN-AS1/OBSCN gene pair, which may be used as prognostic biomarkers and/or therapeutic targets for metastatic breast cancer.


Assuntos
RNA Longo não Codificante , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , RNA Longo não Codificante/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Recidiva Local de Neoplasia , Biópsia , Proteínas Serina-Treonina Quinases , Fatores de Troca de Nucleotídeo Guanina Rho
2.
Proc Natl Acad Sci U S A ; 117(42): 26008-26019, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33020304

RESUMO

Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.


Assuntos
Mama/patologia , Cálcio/metabolismo , Mecanotransdução Celular , NADPH Oxidase 2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPM/metabolismo , Mama/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Microtúbulos/metabolismo , NADPH Oxidase 2/genética , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Taxa de Sobrevida , Canais de Cátion TRPM/genética , Microambiente Tumoral
3.
Breast Cancer Res ; 24(1): 13, 2022 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-35164808

RESUMO

Clinical cancer imaging focuses on tumor growth rather than metastatic phenotypes. The microtubule-depolymerizing drug, Vinorelbine, reduced the metastatic phenotypes of microtentacles, reattachment and tumor cell clustering more than tumor cell viability. Treating mice with Vinorelbine for only 24 h had no significant effect on primary tumor survival, but median metastatic tumor survival was extended from 8 to 30 weeks. Microtentacle inhibition by Vinorelbine was also detectable within 1 h, using tumor cells isolated from blood samples. As few as 11 tumor cells were sufficient to yield 90% power to detect this 1 h Vinorelbine drug response, demonstrating feasibility with the small number of tumor cells available from patient biopsies. This study establishes a proof-of-concept that targeted microtubule disruption can selectively inhibit metastasis and reveals that existing FDA-approved therapies could have anti-metastatic actions that are currently overlooked when focusing exclusively on tumor growth.


Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Microtúbulos , Metástase Neoplásica , Vinorelbina/farmacologia
4.
Cancers (Basel) ; 15(3)2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36765843

RESUMO

Cytoskeletal remodeling in circulating tumor cells (CTCs) facilitates metastatic spread. Previous oncology studies examine sustained aberrant calcium (Ca2+) signaling and cytoskeletal remodeling scrutinizing long-term phenotypes such as tumorigenesis and metastasis. The significance of acute Ca2+ signaling in tumor cells that occur within seconds to minutes is overlooked. This study investigates rapid cytoplasmic Ca2+ elevation in suspended cells on actin and tubulin cytoskeletal rearrangements and the metastatic microtentacle (McTN) phenotype. The compounds Ionomycin and Thapsigargin acutely increase cytoplasmic Ca2+, suppressing McTNs in the metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-436. Functional decreases in McTN-mediated reattachment and cell clustering during the first 24 h of treatment are not attributed to cytotoxicity. Rapid cytoplasmic Ca2+ elevation was correlated to Ca2+-induced actin cortex contraction and rearrangement via myosin light chain 2 and cofilin activity, while the inhibition of actin polymerization with Latrunculin A reversed Ca2+-mediated McTN suppression. Preclinical and phase 1 and 2 clinical trial data have established Thapsigargin derivatives as cytotoxic anticancer agents. The results from this study suggest an alternative molecular mechanism by which these compounds act, and proof-of-principle Ca2+-modulating compounds can rapidly induce morphological changes in free-floating tumor cells to reduce metastatic phenotypes.

5.
Cells ; 12(9)2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37174666

RESUMO

Levels of hydrogen peroxide are highly elevated in the breast tumor microenvironment compared to normal tissue. Production of hydrogen peroxide is implicated in the mechanism of action of many anticancer therapies. Several lines of evidence suggest hydrogen peroxide mediates breast carcinogenesis and metastasis, though the molecular mechanism remains poorly understood. This study elucidates the effects of exposure to elevated hydrogen peroxide on non-tumorigenic MCF10A mammary epithelial cells, tumorigenic MCF7 cells, and metastatic MDA-MB-231 breast cancer cells. Hydrogen peroxide treatment resulted in a dose- and time-dependent induction of two α-tubulin post-translational modifications-de-tyrosination and acetylation-both of which are markers of poor patient prognosis in breast cancer. Hydrogen peroxide induced the formation of tubulin-based microtentacles in MCF10A and MDA-MB-231 cells, which were enriched in detyrosinated and acetylated α-tubulin. However, the hydrogen peroxide-induced microtentacles did not functionally promote metastatic phenotypes of cellular reattachment and homotypic cell clustering. These data establish for the first time that microtentacle formation can be separated from the functions to promote reattachment and clustering, which indicates that there are functional steps that remain to be identified. Moreover, signals in the primary tumor microenvironment may modulate α-tubulin post-translational modifications and induce microtentacles; however, the functional consequences appear to be context-dependent.


Assuntos
Neoplasias da Mama , Metástase Neoplásica , Tubulina (Proteína) , Humanos , Acetilação , Peróxido de Hidrogênio , Células MCF-7 , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo , Neoplasias da Mama/patologia
6.
bioRxiv ; 2023 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-37034765

RESUMO

The tumor microenvironment and wound healing after injury, both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it’s altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca 2+ in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca 2+ responses. The non-tumorigenic cells respond to increased Ca 2+ with actin polymerization and localization to cellular junctions, while the metastatic cells remained unaffected. The increase in intracellular Ca 2+ after ATP stimulation could be blunted using a P2Y2 antagonist, which also prevented actin mobilization in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca 2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca 2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. STATEMENT OF SIGNIFICANCE: This work shows non-tumorigenic breast epithelial cells increase intracellular Ca 2+ after ATP-P2Y2 signaling and re-localize actin, while metastatic cells lack this response, due to decreased P2Y2 expression, which correlates with poorer survival.

7.
Nat Commun ; 13(1): 6128, 2022 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253369

RESUMO

Cell migration regulates diverse (patho)physiological processes, including cancer metastasis. According to the Osmotic Engine Model, polarization of NHE1 at the leading edge of confined cells facilitates water uptake, cell protrusion and motility. The physiological relevance of the Osmotic Engine Model and the identity of molecules mediating cell rear shrinkage remain elusive. Here, we demonstrate that NHE1 and SWELL1 preferentially polarize at the cell leading and trailing edges, respectively, mediate cell volume regulation, cell dissemination from spheroids and confined migration. SWELL1 polarization confers migration direction and efficiency, as predicted mathematically and determined experimentally via optogenetic spatiotemporal regulation. Optogenetic RhoA activation at the cell front triggers SWELL1 re-distribution and migration direction reversal in SWELL1-expressing, but not SWELL1-knockdown, cells. Efficient cell reversal also requires Cdc42, which controls NHE1 repolarization. Dual NHE1/SWELL1 knockdown inhibits breast cancer cell extravasation and metastasis in vivo, thereby illustrating the physiological significance of the Osmotic Engine Model.


Assuntos
Neoplasias , Trocadores de Sódio-Hidrogênio , Movimento Celular/fisiologia , Tamanho Celular , Humanos , Água
8.
Cancers (Basel) ; 14(7)2022 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-35406479

RESUMO

Post-translational modifications (PTMs) of the microtubule network impart differential functions across normal cell types and their cancerous counterparts. The removal of the C-terminal tyrosine of α-tubulin (deTyr-Tub) as performed by the tubulin carboxypeptidase (TCP) is of particular interest in breast epithelial and breast cancer cells. The recent discovery of the genetic identity of the TCP to be a vasohibin (VASH1/2) coupled with a small vasohibin-binding protein (SVBP) allows for the functional effect of this tubulin PTM to be directly tested for the first time. Our studies revealed the immortalized breast epithelial cell line MCF10A undergoes apoptosis following transfection with TCP constructs, but the addition of oncogenic KRas or Bcl-2/Bcl-xL overexpression prevents subsequent apoptotic induction in the MCF10A background. Functionally, an increase in deTyr-Tub via TCP transfection in MDA-MB-231 and Hs578t breast cancer cells leads to enhanced focal gelatin degradation. Given the elevated deTyr-Tub at invasive tumor fronts and the correlation with poor breast cancer survival, these new discoveries help clarify how the TCP synergizes with oncogene activation, increases focal gelatin degradation, and may correspond to increased tumor cell invasion. These connections could inform more specific microtubule-directed therapies to target deTyr-tubulin.

9.
Sci Rep ; 11(1): 3214, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547369

RESUMO

Mammosphere assays are widely used in vitro to identify prospective cancer-initiating stem cells that can propagate clonally to form spheres in free-floating conditions. However, the traditional mammosphere assay inevitably introduces cell aggregation that interferes with the measurement of true mammosphere forming efficiency. We developed a method to reduce tumor cell aggregation and increase the probability that the observed mammospheres formed are clonal in origin. Tethering individual tumor cells to lipid anchors prevents cell drift while maintaining free-floating characteristics. This enables real-time monitoring of single tumor cells as they divide to form mammospheres. Monitoring tethered breast cancer cells provided detailed size information that correlates directly to previously published single cell tracking data. We observed that 71% of the Day 7 spheres in lipid-coated wells were between 50 and 150 µm compared to only 37% in traditional low attachment plates. When an equal mixture of MCF7-GFP and MCF7-mCherry cells were seeded, 65% of the mammospheres in lipid-coated wells demonstrated single color expression whereas only 32% were single-colored in low attachment wells. These results indicate that using lipid tethering for mammosphere growth assays can reduce the confounding factor of cell aggregation and increase the formation of clonal mammospheres.


Assuntos
Neoplasias da Mama/patologia , Mama/patologia , Agregação Celular , Técnicas de Cultura de Células , Feminino , Humanos , Lipídeos/química , Células MCF-7 , Esferoides Celulares/patologia , Células Tumorais Cultivadas
10.
Lab Chip ; 20(16): 2872-2888, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32744284

RESUMO

The technical challenges of imaging non-adherent tumor cells pose a critical barrier to understanding tumor cell responses to the non-adherent microenvironments of metastasis, like the bloodstream or lymphatics. In this study, we optimized a microfluidic device (TetherChip) engineered to prevent cell adhesion with an optically-clear, thermal-crosslinked polyelectrolyte multilayer nanosurface and a terminal lipid layer that simultaneously tethers the cell membrane for improved spatial immobilization. Thermal imidization of the TetherChip nanosurface on commercially-available microfluidic slides allows up to 98% of tumor cell capture by the lipid tethers. Importantly, time-lapse microscopy demonstrates that unique microtentacles on non-adherent tumor cells are rapidly destroyed during chemical fixation, but tethering microtentacles to the TetherChip surface efficiently preserves microtentacle structure post-fixation and post-blood isolation. TetherChips remain stable for more than 6 months, enabling shipment to distant sites. The broad retention capability of TetherChips allows comparison of multiple tumor cell types, revealing for the first time that carcinomas beyond breast cancer form microtentacles in suspension. Direct integration of TetherChips into the Vortex VTX-1 CTC isolation instrument shows that live CTCs from blood samples are efficiently captured on TetherChips for rapid fixation and same-day immunofluorescence analysis. Highly efficient and unbiased label-free capture of CTCs on a surface that allows rapid chemical fixation also establishes a streamlined clinical workflow to stabilize patient tumor cell samples and minimize analytical variables. While current studies focus primarily on CTC enumeration, this microfluidic device provides a novel platform for functional phenotype testing in CTCs with the ultimate goal of identifying anti-metastatic, patient-specific therapies.


Assuntos
Células Neoplásicas Circulantes , Adesão Celular , Contagem de Células , Linhagem Celular Tumoral , Membrana Celular , Separação Celular , Humanos , Polieletrólitos , Microambiente Tumoral
11.
Nat Biomed Eng ; 3(6): 452-465, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061459

RESUMO

The challenge of predicting which patients with breast cancer will develop metastases leads to the overtreatment of patients with benign disease and to the inadequate treatment of aggressive cancers. Here, we report the development and testing of a microfluidic assay that quantifies the abundance and proliferative index of migratory cells in breast cancer specimens, for the assessment of their metastatic propensity and for the rapid screening of potential antimetastatic therapeutics. On the basis of the key roles of cell motility and proliferation in cancer metastasis, the device accurately predicts the metastatic potential of breast cancer cell lines and of patient-derived xenografts. Compared with unsorted cancer cells, highly motile cells isolated by the device exhibited similar tumourigenic potential but markedly increased metastatic propensity in vivo. RNA sequencing of the highly motile cells revealed an enrichment of motility-related and survival-related genes. The approach might be developed into a companion assay for the prediction of metastasis in patients and for the selection of effective therapeutic regimens.


Assuntos
Neoplasias da Mama/patologia , Microfluídica/métodos , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Ensaios Clínicos como Assunto , Células Epiteliais/patologia , Feminino , Genótipo , Humanos , Camundongos Nus , Mutação/genética , Invasividade Neoplásica , Metástase Neoplásica , Fenótipo , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
12.
iScience ; 8: 29-39, 2018 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-30268511

RESUMO

The mammosphere assay has become widely employed to quantify stem-like cells in a population. However, the problem is there is no standard protocol employed by the field. Cell seeding densities of 1,000 to 100,000 cells/mL have been reported. These high densities lead to cellular aggregation. To address this, we have individually tracked 1,127 single MCF-7 and 696 single T47D human breast tumor cells by eye over the course of 14 days. This tracking has given us detailed information for the commonly used endpoints of 5, 7, and 14 days that is unclouded by cellular aggregation. This includes mean sphere sizes, sphere-forming efficiencies, and a well-defined minimum size for both lines. Importantly, we have correlated early cell division with eventual sphere formation. At 24 hr post seeding, we can predict the total spheres on day 14 with 98% accuracy in both lines. This approach removes cell aggregation and potentially shortens a 5- to 14-day assay to a 24 hours.

13.
Comp Med ; 55(5): 465-9, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16270904

RESUMO

There is an ongoing need to eradicate intercurrent disease from research mouse colonies. Commonly used surgical methods, however, are expensive and time-consuming. The purpose of this study was to determine the percentage of litters that could be rederived from infected mouse colonies by neonatal transfer. We immersed neonatal mice in a dilute iodine solution and transferred them to disease-free foster mothers within 48 h of birth. Donor and foster mothers were evaluated for pathogens by serology and fecal polymerase chain reaction (PCR) assay. Of 55 donor mothers, 100% were positive serologically and 59% were positive by fecal PCR for one or more tested organisms, including mouse hepatitis virus, Theiler's murine encephalomyelitis virus, mouse rotavirus, and Helicobacter hepaticus. At 4 to 6 weeks after neonatal transfer, 95% of foster mothers (which served as sentinels for the transferred pups) tested free of pathogens, the exceptions being one case of mouse parvovirus 1 and two of Helicobacter spp. We suggest that cross-fostering is a viable low-cost method for rederivation of mouse colonies contaminated with pathogens such as mouse hepatitis virus, Theiler's murine encephalomyelitis virus, mouse rotavirus, and H. hepaticus.


Assuntos
Imunocompetência , Iodo/administração & dosagem , Camundongos Endogâmicos C57BL/microbiologia , Animais , Animais Recém-Nascidos , Sequência de Bases , Primers do DNA , Transmissão de Doença Infecciosa/prevenção & controle , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL/imunologia , Camundongos Endogâmicos C57BL/virologia , Reação em Cadeia da Polimerase
14.
Oncotarget ; 6(8): 6251-66, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25749040

RESUMO

The presence of circulating tumor cells (CTCs) in blood predicts poor patient outcome and CTC frequency is correlated with higher risk of metastasis. Recently discovered, novel microtubule-based structures, microtentacles, can enhance reattachment of CTCs to the vasculature. Microtentacles are highly dynamic membrane protrusions formed in detached cells and occur when physical forces generated by the outwardly expanding microtubules overcome the contractile force of the actin cortex. Rho-associated kinase (ROCK) is a major regulator of actomyosin contractility and Rho/ROCK over-activation is implicated in tumor metastasis. ROCK inhibitors are gaining popularity as potential cancer therapeutics based on their success in reducing adherent tumor cell migration and invasion. However, the effect of ROCK inhibition on detached cells in circulation is largely unknown. In this study, we use breast tumor cells in suspension to mimic detached CTCs and show that destabilizing the actin cortex through ROCK inhibition in suspended cells promotes the formation of microtentacles and enhances reattachment of cells from suspension. Conversely, increasing actomyosin contraction by Rho over-activation reduces microtentacle frequency and reattachment. Although ROCK inhibitors may be effective in reducing adherent tumor cell behavior, our results indicate that they could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy.


Assuntos
Amidas/farmacologia , Neoplasias da Mama/patologia , Células Neoplásicas Circulantes/patologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Actomiosina/metabolismo , Neoplasias da Mama/enzimologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/patologia , Feminino , Humanos , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismo , Quinases Associadas a rho/metabolismo
15.
Oncotarget ; 6(34): 35231-46, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26497685

RESUMO

A high proportion of human tumors maintain activation of both the PI3K and Ras/MAPK pathways. In basal-like breast cancer (BBC), PTEN expression is decreased/lost in over 50% of cases, leading to aberrant activation of the PI3K pathway. Additionally, BBC cell lines and tumor models have been shown to exhibit an oncogenic Ras-like gene transcriptional signature, indicating activation of the Ras/MAPK pathway. To directly test how the PI3K and Ras/MAPK pathways contribute to tumorigenesis, we deleted PTEN and activated KRas within non-tumorigenic MCF-10A breast cells. Neither individual mutation was sufficient to promote tumorigenesis, but the combination promoted robust tumor growth in mice. However, in vivo bioluminescence reveals that each mutation has the ability to promote a persistent phenotype. Inherent in the concept of tumor cell dormancy, a stage in which residual disease is present but remains asymptomatic, viable cells with each individual mutation can persist in vivo during a period of latency. The persistent cells were excised from the mice and showed increased levels of the cell cycle arrest proteins p21 and p27 compared to the aggressively growing PTEN-/-KRAS(G12V) cells. Additionally, when these persistent cells were placed into growth-promoting conditions, they were able to re-enter the cell cycle and proliferate. These results highlight the potential for either PTEN loss or KRAS activation to promote cell survival in vivo, and the unique ability of the combined mutations to yield rapid tumor growth. This could have important implications in determining recurrence risk and disease progression in tumor subtypes where these mutations are common.


Assuntos
Neoplasias da Mama/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose/fisiologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Ativação Enzimática , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas ras/genética
16.
Cancer Res ; 74(4): 1250-60, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24371229

RESUMO

Cancer stem-like cells (CSC) and circulating tumor cells (CTC) have related properties associated with distant metastasis, but the mechanisms through which CSCs promote metastasis are unclear. In this study, we report that breast cancer cell lines with more stem-like properties display higher levels of microtentacles (McTN), a type of tubulin-based protrusion of the plasma cell membrane that forms on detached or suspended cells and aid in cell reattachment. We hypothesized that CSCs with large numbers of McTNs would more efficiently attach to distant tissues, promoting metastatic efficiency. The naturally occurring stem-like subpopulation of the human mammary epithelial (HMLE) cell line presents increased McTNs compared with its isogenic non-stem-like subpopulation. This increase was supported by elevated α-tubulin detyrosination and vimentin protein levels and organization. Increased McTNs in stem-like HMLEs promoted a faster initial reattachment of suspended cells that was inhibited by the tubulin-directed drug, colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live-cell confocal microscopy showed that McTNs persist in breast stem cell mammospheres as flexible, motile protrusions on the surface of the mammosphere. Although exposed to the environment, they also function as extensions between adjacent cells along cell-cell junctions. We found that treatment with the breast CSC-targeting compound curcumin rapidly extinguished McTN in breast CSC, preventing reattachment from suspension. Together, our results support a model in which breast CSCs with cytoskeletal alterations that promote McTNs can mediate attachment and metastasis but might be targeted by curcumin as an antimetastatic strategy.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias da Mama/patologia , Curcumina/uso terapêutico , Células-Tronco Neoplásicas/efeitos dos fármacos , Pseudópodes/patologia , Esferoides Celulares/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Feminino , Humanos , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/patologia , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/ultraestrutura , Pseudópodes/efeitos dos fármacos , Esferoides Celulares/patologia , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas
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