RESUMO
Epigenetic deregulation, especially mutagenesis or the abnormal expression of epigenetic regulatory factors (ERFs), plays an important role in malignant tumorigenesis. To screen natural inhibitors of breast cancer metastasis, we adopted small interfering RNAs (siRNAs) to transiently knock down 591 ERF-coding genes in luminal breast cancer MCF-7 cells and found that depletion of AF9 significantly promoted MCF-7 cell invasion and migration. A mouse model of metastasis further confirmed the suppressive role of AF9 in breast cancer metastasis. RNA profiling revealed enrichment of AF9 targets genes in the epithelial-mesenchymal transition (EMT). Mechanistically, tandem mass spectrometry showed that AF9 interacts with Snail, which hampers Snail transcriptional activity in basal-like breast cancer (BLBC) cells. AF9 reconstitutes an activated state on the promoter of Snail, which is a master regulator of EMT, and derepresses genes by recruiting CBP or GCN5. Additionally, microRNA-5694 (miR-5694) targeted and degraded AF9 messenger RNA (mRNA) in BLBC cells, further enhancing cell invasion and migration. Notably, AF9 and miR-5694 expression in BLBC clinical samples correlated inversely. Hence, miR-5694 mediates downregulation of AF9 and provides metastatic advantages in BLBC. Restoring expression of the metastasis suppressor AF9 is a possible therapeutic strategy against metastatic breast cancer.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Basocelular/patologia , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/secundário , MicroRNAs/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Proteínas Nucleares/genética , Prognóstico , RNA Interferente Pequeno/genética , Fatores de Transcrição da Família Snail/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer causes high mortality among females worldwide. Bufalin has recently been shown to trigger tumor cell death, although the mechanism of cytotoxicity remains unclear. The cytotoxicity of bufalin in breast cancer cells was examined using an MTT assay. The modes of death and intracellular reactive oxygen species production were measured by flow cytometry. We also observed cellular morphologic changes by Hoechst 33342 and propidium iodide staining and transmission electron microscopy. Western blotting was performed to determine the expression levels of related proteins. Our results showed that bufalin reduced cellular viability and promoted reactive oxygen species production, which could be inhibited by Nec-1 and N-acetylcysteine. Necroptosis was detected by Hoechst 33342 and propidium iodide staining and transmission electron microscopy. Western blot analysis showed that bufalin induced necroptosis by upregulating the necroptosis mediator RIP1 and the RIP1/RIP3/PGAM5 pathway. Taken together, these findings indicated that bufalin induces breast cancer cell necroptosis by targeting the RIP1/RIP3/PGAM5 pathway.
Assuntos
Neoplasias da Mama/patologia , Bufanolídeos/farmacologia , Proteínas Mitocondriais/antagonistas & inibidores , Necroptose , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Fosfoproteínas Fosfatases/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células Tumorais CultivadasRESUMO
Bufalin, a key active ingredient of the Chinese medicine Chan Su, inhibits breast cancer tumorigenesis in vitro and in vivo. Here, we found that the pan-caspase inhibitor zVAD-fmk failed to inhibit bufalin-induced cell death in MCF-7 and MDA-MB-231 human breast cancer cells, confirming that the cell death induced by bufalin is caspase-independent. Instead, bufalin increased the expression of the necroptosis mediators RIP1 and RIP3. Bufalin-induced cell death was prevented by small molecule inhibitors of RIP1 and poly (ADP-ribose) polymerase-1 (PARP-1) or genetic knockdown of RIP3 by shRNA transfection. In addition, ectopic RIP3 expression enhanced cell death by bufalin. We also found that bufalin increased intracellular reactive oxygen species levels; and cell death by bufalin was inhibited by the antioxidant NAC. In a mouse xenograft model of human breast cancer, bufalin induced PARP-1-dependent tumor cell death and inhibited tumor growth. These results demonstrated that bufalin inhibits human breast cancer tumorigenesis by inducing cell death through the reactive oxygen species-mediated RIP1/RIP3/PARP-1 pathways.