RESUMO
microRNAs (miRNA) are regulators of cellular pathways and alterations of normal miRNA expression levels have been shown to increase tumorigenesis. miR-24 has been demonstrated as having both tumor suppressive and oncogenic properties depending on cell context. Here, we demonstrate a possible role for pre-miR-24-2 as a tumor suppressor in the MCF-7 breast cancer cell line through the preferential processing of mature miR-24-2* over miR-24. Specifically, we show that the ectopic expression of miR-24-2* in MCF-7 breast cancer cells results in a suppression of cellular survival both in vivo and in vitro. Notably, the overexpression of miR-24-2* results in a dampening of cell survival through the targeted suppression of PKCα. In addition, a similar biological change is observed in vivo where MCF-7 cells overexpressing pre-miR-24-2 have decreased tumorigenicity and tumor incidence. Taken together our data demonstrate that when overexpressed biogenesis of the pre-miR-24-2 favors miR-24-2* in the MCF-7 breast cancer cell line and suggests a tumor suppressive role for miR-24-2* observed through the inhibition of PKCα-mediated cellular survival.
Assuntos
Neoplasias da Mama/genética , MicroRNAs/genética , Proteína Quinase C-alfa/genética , Animais , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas , Células MCF-7 , Camundongos , MicroRNAs/química , MicroRNAs/metabolismo , Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/metabolismo , Interferência de RNARESUMO
Sphingosine kinase signaling has become of increasing interest as a cancer target in recent years. Two sphingosine kinase inhibitors, sphingosine kinase inhibitor (SKI)-II and ABC294640, are promising as potential breast cancer therapies. However, evidence for their therapeutic properties in specific breast cancer subtypes is currently lacking. In this study, we characterize these drugs in luminal, endocrine-resistant (MDA-MB-361) and basal-A, triple-negative (MDA-MB-468) breast cancer cells and compare them with previously published data in other breast cancer cell models. Both SKI-II and ABC294640 demonstrated greater efficacy in basal-A compared with luminal breast cancer. ABC294640, in particular, induced apoptosis and blocked proliferation both in vitro and in vivo in this triple-negative breast cancer system. Furthermore, Sphk expression promotes survival and endocrine therapy resistance in previously sensitive breast cancer cells. Taken together, these results characterize sphingosine kinase inhibitors across breast cancer cell systems and demonstrate their therapeutic potential as anti-cancer agents.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Isoenzimas/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/efeitos dos fármacos , Adamantano/análogos & derivados , Adamantano/farmacologia , Adamantano/uso terapêutico , Aminofenóis/farmacologia , Aminofenóis/uso terapêutico , Animais , Antineoplásicos Hormonais/uso terapêutico , Sequência de Bases , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Primers do DNA , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Piridinas/farmacologia , Piridinas/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Estrogênio/metabolismo , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo. METHODS: TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology. RESULTS: Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype. CONCLUSIONS: This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/metabolismo , Proteínas Cdh1 , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Expressão Gênica , Histonas/metabolismo , Humanos , Camundongos , Camundongos SCID , Panobinostat , Distribuição Aleatória , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The development of drug resistance represents a major complication in the effective treatment of breast cancer. Epigenetic therapy, through the use of histone deacetylase inhibitors (HDACi) or demethylation agents, is an emerging area of therapeutic targeting in a number of ontological entities, particularly in the setting of aggressive therapy-resistant disease. Using the well-described HDAC inhibitor trichostatin A (TSA) we demonstrate the suppression of in vitro clonogenicity in the previously described apoptosis-resistant MCF-7TN-R breast carcinoma cell line. Additionally, recent work has demonstrated that these agents can alter the expression profile of microRNA signatures in malignant cells. Using an unbiased microRNA microarray analysis, changes in miRNA expression of MCF-7TN-R cells treated with TSA for 24 h were analyzed. We observed significant up-regulation of 22 miRNAs and down-regulation of 10 miRNAs in response to TSA treatment. Our results demonstrate that the HDACi, TSA, exerts anticancer activity in the apoptosis-resistant MCF-7TN-R breast carcinoma cell line. This activity is correlated with TSA alteration of microRNA expression profiles indicative of a less aggressive phenotype.
Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , MicroRNAs/biossíntese , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Resistance to chemotherapy remains a significant obstacle in the treatment of hormone- independent breast cancer. Recent evidence suggests that altered sphingolipid signaling through increased sphingosine kinase activity may be an important mediator of breast cancer drug resistance. Sphingosine kinase-1 (Sphk1) is a proposed key regulator of breast cancer tumorigenesis, proliferation and resistance. There is, however, conflicting data on the role of sphingosine kinase-2 (Sphk2) in cancer biology and resistance, with some suggesting that Sphk2 has an opposing role to that of Sphk1. Here, we studied the effects of the novel selective Sphk2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl) amide), on human breast cancer. ABC294640 blocked both viability and survival at low micromolar IC(50) concentrations in the endocrine therapy-resistant MDA-MB-231 and chemoresistant MCF-7TN-R cell systems. Treatment with the inhibitor significantly reduced proliferation, as seen in immunofluorescence staining of Ki-67 in vitro. Interestingly, pharmacological inhibition of Sphk2 induced apoptosis through the intrinsic programmed cell death pathway. Furthermore, ABC294640 also diminished NF-ĸB survival signaling, through decreased activation of the Ser536 phosphorylation site on the p65 subunit. Xenografts of MCF-7TN-R cells growing in immunocompromised mice were utilized to validate the therapeutic efficacy of the sphingosine kinase-2 inhibitor. Treatment with 50 mg of ABC294640/kg completely blocked tumor volume in this model. These results indicate that pharmacological inhibition of Sphk2 with the orally bioavailable selective inhibitor, ABC294640, has therapeutic potential in the treatment of chemo- and endocrine therapy- resistant breast cancer.