RESUMO
Ras mutations have been frequently observed in human cancer. Although there is a high degree of similarity between Ras isomers, they display preferential coupling in specific cancer types. The binding of Ras to the plasma membrane is essential for its activation and biological functions. The present study elucidated Ras isoform-specific interactions with the membrane and their role in Ras-mediated biological activities. We investigated the role of a lipid raft protein flotillin-1 (Flot-1) in the activations of Ras. We found that Flot-1 was co-localized with H-Ras, but not with N-Ras, in lipid rafts of MDA-MB-231 human breast cells. The amino-terminal hydrophobic domain (1-38) of Flot-1 interacted with the hypervariable region of H-Ras. The epidermal growth factor-stimulated activation of H-Ras required Flot-1 which was not necessary for that of N-Ras in breast cancer cells. Flot-1 interacted with son of sevenless (SOS)-1, which promotes the conversion of Ras-bound GDP to GTP. Notably, Flot-1 was crucial for the interaction between SOS1 and H-Ras/K-Ras in breast and pancreatic cancer cells. Stable knockdown of Flot-1 reduced the in vivo metastasis in a mouse xenograft model with human breast carcinoma cells. A tissue microarray composed of 61 human pancreatic cancer samples showed higher levels of Flot-1 expression in pancreatic tumor tissues compared to normal tissues, and a correlation between K-Ras and Flot-1. Taken together, our findings suggest that Flot-1 may serve as a membrane platform for the interaction of SOS1 with H-Ras/K-Ras in human cancer cells, presenting Flot-1 as a potential target for Ras-driven cancers.
Assuntos
Proteínas de Membrana , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Neoplasias Pancreáticas/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is the second most common cancer worldwide, demonstrating aggressiveness and mortality more frequently in men than in women. Despite reports regarding the inhibitory ability of estrogen receptor alpha (ERα, ESR1) in certain cancer progression, targets and the basis of underlying gender disparity in HCC worsening remain elusive. Here, we report the ability of ERα to transcriptionally inhibit G protein subunit alpha 12 (Gα12) responsible for HCC worsening. First, using human samples and public database, the expression of ERα and Gα12 in HCC was examined. Then, quantitative real-time PCR, chromatin immunoprecipitation-assay, luciferase assay and immunoblottings of liver cancer cell lines confirmed the inhibitory ability of ERα on Gα12 and HCC progression. Gα12 promoted mesenchymal characteristics and amoeboidal movement, which was antagonized by ERα overexpression. Additionally, we found microRNA-141 and microRNA-200a as downstream targets of the Gα12 signaling axis for cancer malignancy regulation under the control of ERα. As for in-depth mechanism, PTP4A1 was found to be directly inhibited by microRNA-141 and microRNA-200a. Moreover, we found the inhibitory effect of ERα on amoeboidal movement by analyzing the morphology and blebbing of liver cancer cells and the active form of MLC levels. The identified targets and ESR1 levels are inversely correlated with human specimens, as well as with sex-biased survival rates of HCC patients. Collectively, ERα-dependent repression of Gα12 and consequent changes in the Gα12 signaling may explain the gender disparity in HCC, providing pharmacological clues for the control of metastatic HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Masculino , MicroRNAs/metabolismoRESUMO
Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with tumor progression in breast cancer. Although crosstalk between breast cancer cells and CAFs has been studied, the effect of CAFs on non-neoplastic breast epithelial cells is not fully understood to date. Here, we investigated the effect of CAFs on aggressive phenotypes in non-neoplastic MCF10A breast epithelial cells. CAFs induced epithelial-to-mesenchymal transition (EMT) and invasive phenotype in MCF10A cells. S100A8, a potential prognostic marker in several cancers, was markedly increased in MCF10A cells by CAFs. S100A8 was crucial for CAFs-induced invasive phenotype of MCF10A cells. Among cytokines increased by CAFs, interleukin (IL)-8 induced S100A8 through transcription factors p65 NF-κB and C/EBPß. In a xenograft mouse model with MCF10A cells and CAFs, tumor was not developed, suggesting that coinjection with CAFs may not be sufficient for in vivo tumorigenicity of MCF10A cells. Xenograft mouse tumor models with MDA-MB-231 breast carcinoma cells provided an in vivo evidence for the effect of CAFs on breast cancer progression as well as a crucial role of IL-8 in tumor growth and S100A8 expression in vivo. Using a tissue microarray of human breast cancer, we showed that S100A8 expression was correlated with poor outcomes. S100A8 expression was more frequently detected in cancer-adjacent normal human breast tissues than in normal breast tissues. Together, this study elucidated a novel mechanism for the acquisition of invasive phenotype of non-neoplastic breast cells induced by CAFs, suggesting that targeting IL-8 and S100A8 may be an effective strategy against breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Calgranulina A/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Células Epiteliais/metabolismo , Interleucina-8/metabolismo , Comunicação Parácrina , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Calgranulina A/genética , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Movimento Celular , Técnicas de Cocultura , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Feminino , Humanos , Interleucina-8/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Fenótipo , Transdução de Sinais , Sulfonamidas/farmacologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ras proteins mediate extracellular and cytoplasmic signaling networks via receptor tyrosine kinase. The Ras pathway induces activation of signaling molecules involved in cell proliferation and growth, cell survival and apoptosis, metabolism, and motility. Although Ras mutations in breast cancer are not frequently reported, hyperactivation of Ras signaling plays an important role in breast cancer growth and progression. Oncogenic Ras activation occurs via loss of Ras GTPase-activating proteins, overexpression of growth factor receptor, and stimulation by various cytokines. Effective control of oncogenic Ras is one of the therapeutic strategies in breast cancer. The mechanisms of intracellular localization, activation, and signaling pathway of Ras in cancer have been used to develop therapeutic candidates. Recent studies have reported an effective therapy for breast cancer by inhibition of enzymes involved in the posttranslational modification of Ras, such as farnesyltransferase and geranylgeranyltransferase 1, and anti-cancer therapies targeting the epidermal growth factor receptor (EGFR). Emerging targets involved in EGF-mediated Ras activity in breast cancer have shed new insight into Ras activation in breast cancer progression. These alternative mechanisms for Ras signaling pathway may suggest novel therapeutic approaches for targeting Ras in breast cancer. In spite of the difficulties in targeting Ras protein, important discoveries highlight the direct inhibition of Ras activity. Further studies may elucidate the effects of targeting Ras protein and the clinical relevance thereof.
Assuntos
Neoplasias da Mama , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proliferação de Células , Genes ras , Humanos , Transdução de SinaisRESUMO
Activation of sterol regulatory element-binding protein 1 (SREBP-1), a master lipogenic transcription factor, is associated with cancer metabolism and metabolic disorders. Neddylation, the process of adding NEDD8 to its substrate, contributes to diverse biological processes. Here, we identified SREBP-1 as a substrate for neddylation by UBC12 and explored its impact on tumor aggressiveness. In cell-based assays, SREBP-1 neddylation prolonged SREBP-1 stability with a decrease in ubiquitination. Consequently, NEDD8 overexpression facilitated proliferation, migration, and invasion of SK-Hep1 liver tumor cells. MLN4924 (an inhibitor of the NEDD8-activating enzyme-E1) treatment or UBC12 knockdown prevented SREBP-1 neddylation and tumor cell phenotype change. This effect was corroborated in an in vivo xenograft model. In human specimens, SREBP-1, UBC12, and NEDD8 were all upregulated in hepatocellular carcinoma (HCC) compared to nontumorous regions. Moreover, SREBP-1 levels positively correlated with UBC12. In GEO database analyses, SREBP-1 levels were greater in metastatic HCC samples accompanying UBC12 upregulation. In HCC analysis, tumoral SREBP-1 and UBC12 levels discriminated overall patient survival rates. Additionally, MLN4924 treatment destabilized SREBP-1 in MDA-MB-231 breast cancer cells and in the tumor cell xenograft. SREBP-1 and UBC12 were also highly expressed in human breast cancer tissues. Moreover, most breast cancers with lymph node metastasis displayed predominant SREBP-1 and UBC12 expressions, which compromised overall patient survival rates. In summary, SREBP-1 is neddylated by UBC12, which may contribute to HCC and breast cancer aggressiveness through SREBP-1 stabilization, and these events can be intervented by MLN4924 therapy. Our findings may also provide potential reliable prognostic markers for tumor metastasis.
Assuntos
Neoplasias da Mama/mortalidade , Carcinoma Hepatocelular/mortalidade , Neoplasias Hepáticas/mortalidade , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/secundário , Linhagem Celular Tumoral , Ciclopentanos/farmacologia , Ciclopentanos/uso terapêutico , Feminino , Humanos , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Metástase Linfática/patologia , Camundongos , Proteína NEDD8/metabolismo , Prognóstico , Estabilidade Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Proteína de Ligação a Elemento Regulador de Esterol 1/análise , Taxa de Sobrevida , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/análise , Ubiquitinação/efeitos dos fármacos , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite strong evidence for the involvement of PDGF signaling in breast cancer, little is known about the PDGF ligand responsible for PDGFR activation during breast cancer progression. Here, we found PDGF-C to be highly expressed in breast carcinoma cell lines. Immunohistochemical analysis of invasive breast cancer revealed an association between increased PDGF-C expression and lymph node metastases, Ki-67 proliferation index, and poor disease-free survival. We also identified a PDGF-C splice variant encoding truncated PDGF-C (t-PDGF-C) isoform lacking the signal peptide and the N-terminal CUB domain. While t-PDGF C homodimer is retained intracellularly, it can be secreted as a heterodimer with full-length PDGF-C (FL-PDGF-C). PDGF-C downregulation reduced anchorage-independent growth and matrigel invasion of MDA-MB-231 cells. Conversely, ectopic expression of t-PDGF-C enhanced phenotypic transformation and invasion in BT-549 cells expressing endogenous FL-PDGF-C. The present study provides new insights into the functional significance of PDGF-C and its splice variant in human breast cancer.
Assuntos
Neoplasias da Mama/patologia , Metástase Linfática/genética , Linfocinas/genética , Linfocinas/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Metástase Linfática/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de SinaisRESUMO
Sex-related incidence and outcomes were reported in various cancers, including colorectal cancer. 5-Fluorouracil (5-FU) is widely used as an essential chemotherapeutic agent for colorectal cancer. However, sex-based differences in 5-FU toxicity have yet to be reported in human cancer cell lines and xenograft mouse models to date. Here, we investigated, for the first time, sex-based differences in 5-FU toxicity using human colon cancer cell lines, xenograft mouse models, and Korean patients' data. Female-derived colon cancer cell lines exhibited greater 5-FU-induced cytotoxicity than male-derived colon cancer cell lines. We established two xenograft mouse models: one with a male-derived human colon cancer cell line injected into male mice (a male-xenograft model) and another involving a female-derived human colon cancer cell line injected into female mice (a female xenograft model). Treatment with 5-FU inhibited tumor growth and led to hematological toxicity in a female xenograft model more potently than in a male xenograft model. We analyzed the data obtained from Korean patients with colorectal cancer to examine sex differences in adverse drug reactions caused by 5-FU. Korean female patients with colorectal cancer who received 5-FU chemotherapy experienced more frequent adverse drug reactions including alopecia and leukopenia than male patients. Taken together, we demonstrated that female may be associated with increased risk of toxicity to 5-FU treatment in colorectal cancer based on in vitro and in vivo investigations and clinical data analysis. Our study suggests sex as an important clinical factor, which predicts induction of toxicity related to 5-FU treatment.
Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/efeitos adversos , Idoso , Animais , Povo Asiático , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Caracteres Sexuais , Carga TumoralRESUMO
Interleukin (IL)-6 plays a crucial role in the progression, invasion, and metastasis of breast cancer. Triple-negative breast cancer (TNBC) cell line MDA-MB-231 is known for its aggressive metastasis. Epithelial to mesenchymal transition (EMT) is a critical process in cancer metastasis. The positive correlation between IL-6 and EMT in tumor microenvironment is reported. We found significantly upregulated IL-6 expression in MDA-MB-231 cells. A blockade of IL-6 expression decreased levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT3), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), phosphorylated protein kinase B (pAkt), and cell cycle-related molecules, including cyclin-dependent kinases (CDKs) and cyclins in MDA-MB-231 cells. A short-hairpin RNA (shRNA)-mediated blockade of IL-6 expression inhibited migration and N-cadherin expression and induced E-cadherin expression in MDA-MB-231 cells. Growth rate was slower for the tumors derived from IL-6 shRNA-treated MDA-MB-231 cells than for those derived from control shRNA-treated MDA-MB-231 cells. The expression of pSTAT3, phosphorylated extracellular signal-regulated kinase (pERK), PI3K, pAkt, snail, vimentin, and N-cadherin was significantly lower in tumors from IL-6 shRNA-treated MDA-MB cells. In addition, apigenin treatment significantly inhibited the growth of MDA-MB-231-derived xenograft tumors along with the protein expressions of pSTAT3, pERK, IL-6, PI3K, pAkt, and N-cadherin. Our results demonstrate that the anti-invasive effect of apigenin in MDA-MB-231-derived xenograft tumors is mediated by the inhibition of IL-6-linked downstream signaling pathway.
Assuntos
Antineoplásicos/uso terapêutico , Apigenina/uso terapêutico , Carcinoma/tratamento farmacológico , Interleucina-6/metabolismo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apigenina/farmacologia , Ciclinas/genética , Ciclinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Interleucina-6/genética , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Identifying novel biomarkers to detect nephrotoxicity is clinically important. Here, we attempted to identify new biomarkers for mercury-induced nephrotoxicity and compared their sensitivity to that of traditional biomarkers in animal models. Comparative proteomics analysis was performed in kidney tissues of Sprague-Dawley rats after oral treatment with HgCl2 (0.1, 1, or 5 mg/kg/day) for 21 days. Kidney cortex tissues were analyzed by two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionization, and differentially expressed proteins were identified. The corresponding spots were quantitated by RT-PCR. Selenium-binding protein 1 (SBP1) was found to be the most markedly upregulated protein in the kidney cortex of rats after HgCl2 administration. However, blood urea nitrogen, serum creatinine, and glucose levels increased significantly only in the 1 or 5 mg/kg HgCl2-treated groups. A number of urinary excretion proteins, including kidney injury molecule-1, clusterin, monocyte chemoattractant protein-1, and ß-microglobulin, increased dose-dependently. Histopathological examination revealed severe proximal tubular damage in high-dose (5 mg/kg) HgCl2-exposed groups. In addition, urinary excretion of SBP1 significantly increased in a dose-dependent manner. To confirm the critical role of SBP1 as a biomarker for nephrotoxicity, normal kidney proximal tubular cells were treated with HgCl2, CdCl2, or cisplatin for 24 h. SBP1 levels significantly increased in conditioned media exposed to nephrotoxicants, but decreased in cell lysates. Our investigations suggest that SBP1 may play a critical role in the pathological processes underlying chemical-induced nephrotoxicity. Thus, urinary excretion of SBP1 might be a sensitive and specific biomarker to detect early stages of kidney injury.
Assuntos
Cloreto de Cádmio/toxicidade , Nefropatias/induzido quimicamente , Cloreto de Mercúrio/toxicidade , Proteínas de Ligação a Selênio/metabolismo , Animais , Biomarcadores/metabolismo , Nitrogênio da Ureia Sanguínea , Cloreto de Cádmio/administração & dosagem , Cisplatino/administração & dosagem , Cisplatino/toxicidade , Creatinina/sangue , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional , Córtex Renal/efeitos dos fármacos , Córtex Renal/patologia , Nefropatias/patologia , Masculino , Cloreto de Mercúrio/administração & dosagem , Metais Pesados/administração & dosagem , Metais Pesados/toxicidade , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Elevated expression and aberrant activation of Ras have been implicated in breast cancer aggressiveness. H-Ras, but not N-Ras, induces breast cell invasion. A crucial link between lipid rafts and H-Ras function has been suggested. This study sought to identify the lipid raft protein(s) responsible for H-Ras-induced tumorigenicity and invasiveness of breast cancer. We conducted a comparative proteomic analysis of lipid raft proteins from invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein flotillin-1 as an important regulator of H-Ras activation and breast cell invasion. Flotillin-1 was required for epidermal growth factor-induced activation of H-Ras, but not that of N-Ras, in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Flotillin-1 knockdown inhibited the invasiveness of MDA-MB-231 and Hs578T TNBC cells in vitro and in vivo. In xenograft mouse tumor models of these TNBC cell lines, we showed that flotillin-1 played a critical role in tumor growth. Using human breast cancer samples, we provided clinical evidence for the metastatic potential of flotillin-1. Membrane staining of flotillin-1 was positively correlated with metastatic spread (p = 0.013) and inversely correlated with patient disease-free survival rates (p = 0.005). Expression of flotillin-1 was associated with H-Ras in breast cancer, especially in TNBC (p < 0.001). Our findings provide insight into the molecular basis of Ras isoform-specific interplay with flotillin-1, leading to tumorigenicity and aggressiveness of breast cancer.
Assuntos
Neoplasias da Mama/patologia , Genes ras , Proteínas de Membrana/fisiologia , Adulto , Idoso , Animais , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Movimento Celular , Receptores ErbB/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Proteômica , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de SinaisRESUMO
Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds KRS, impinged on the interaction of KRS with 67LR and suppressed metastasis in three different mouse models. The compound inhibited the KRS-67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS-67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS.
Assuntos
Lisina-tRNA Ligase/metabolismo , Metástase Neoplásica , Receptores de Laminina/metabolismo , Membrana Celular/metabolismo , Lisina-tRNA Ligase/antagonistas & inibidores , Transporte Proteico , Receptores de Laminina/antagonistas & inibidoresRESUMO
Annexin A5 belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of various pathophysiological processes. There is increasing evidence that annexin A5 is related to cytotoxicity, but the precise function of this protein has yet to be elucidated. In this study, we aimed to verify the function of annexin A5 in the apoptosis of renal epithelial cells. Real-time PCR and Western blot analysis, together with immunofluorescence analysis, showed that the expression of annexin A5 significantly increased in the presence of cisplatin in both human and rat renal epithelial cells. With regard to the mechanism of cisplatin-induced apoptosis, apoptosis-inducing factor (AIF) release into the cytosol was observed, and the underlying mechanism was identified as voltage-dependent anion channel (VDAC) oligomerization. Mitochondrial membrane potential (Δψm) was found to be greatly disrupted in cisplatin-treated cells. Moreover, cisplatin strongly induced translocation of annexin A5 into mitochondria. To understand the functional significance of annexin A5 in renal cell death, we used a siRNA-mediated approach to knock down annexin A5. Annexin A5 depletion by siRNA led to decreased annexin A5 translocation into mitochondria and significantly reduced VDAC oligomerization and AIF release. Annexin A5 siRNA also increased cell viability compared with the control. Moreover, expression of annexin A5 was induced by other nephrotoxicants such as CdCl2 and bacitracin. Taken together, our data suggest that annexin A5 may play a crucial role in cisplatin-induced toxicity by mediating the mitochondrial apoptotic pathway via the induction and oligomerization of VDAC.
Assuntos
Anexina A5/metabolismo , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Cisplatino/efeitos adversos , Células Epiteliais/metabolismo , Túbulos Renais Proximais/metabolismo , Animais , Anexina A5/genética , Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Apoptose/genética , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Bacitracina/efeitos adversos , Bacitracina/farmacologia , Cloreto de Cádmio/toxicidade , Linhagem Celular , Cisplatino/farmacologia , Citosol/metabolismo , Células Epiteliais/patologia , Técnicas de Silenciamento de Genes , Humanos , Túbulos Renais Proximais/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Ratos , Canais de Ânion Dependentes de Voltagem/genética , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
The epithelial-to-mesenchymal transition (EMT) process allows carcinoma cells to dissociate from the primary tumor thereby facilitating tumor cell invasion and metastasis. Ras-dependent hyperactive signaling is commonly associated with tumorigenesis, invasion, EMT, and metastasis. However, the downstream effectors by which Ras regulates EMT remain ill defined. In this study, we show that the H-Ras pathway leads to mesenchymal-like phenotypic changes in human breast epithelial cells by controlling the ZEB1/microRNA-200c axis. Moreover, H-Ras suppresses the expression of the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, via ZEB1, thus identifying ZEB1 as a novel transcriptional repressor of DDR1. Mutation studies on the putative promoter of the DDR1 gene revealed that bipartite Z- and E-box elements play a key role in transcriptional repression of DDR1 in Hs578T and MDA-MB-231 breast carcinoma cell lines by ZEB1. Furthermore, we found an inverse correlation between ZEB1 and DDR1 expression in various cancer cell lines and in human breast carcinoma tissues. Consistently, overexpression of DDR1 reduced the invasive phenotype of mesenchymal-like triple-negative breast cancer cells in 3D cultures and in vivo. Thus, ZEB1's role in maintenance of EMT in breast carcinoma cells is mediated in part by its ability to suppress DDR1 expression and consequently contribute to the activation of the invasive phenotype. Taken together, our results unveil a novel H-Ras/ZEB1/DDR1 network that contributes to breast cancer progression in triple-negative breast cancers.
Assuntos
Mama/patologia , Transição Epitelial-Mesenquimal , Genes ras/fisiologia , Proteínas de Homeodomínio/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores Mitogênicos/fisiologia , Fatores de Transcrição/fisiologia , Linhagem Celular Tumoral , Citoesqueleto/fisiologia , Receptores com Domínio Discoidina , Células Epiteliais/patologia , Feminino , Humanos , MicroRNAs/fisiologia , Morfogênese , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Measurable indicators of renal injury are required for the assessment of kidney function after toxicant challenge. In our previous study, pleckstrin homology-like domain, family A, member 3 (Phlda3) was a most greatly up-regulated molecule downstream from p53, culminating with kidney tubular injury. This study investigated the positive feedforward effect of Phlda3 on p53 in an effort to explain the largest increase of Phlda3 in injured tubules and the potential of its urine excretion. qRT-PCR assays confirmed a rapid and substantial increase in Phlda3 messenger RNA (mRNA) in the kidney cortex of mice treated with a single dose of cisplatin. Cisplatin overexpression of Phlda3 was verified by gene set analyses of three different microarray databases. In the immunohistochemistry, Phlda3 staining intensities were augmented in the tubules as kidney injury worsened. Moreover, the urinary content of Phlda3 was increased after cisplatin treatment, as were those of other kidney injury markers (Kim-1 and Timp-1). By contrast, cisplatin failed to increase Phlda3 mRNA in the liver despite hepatocyte necrosis and ensuing increases in serum transaminase activities. In NRK52E tubular cells, siRNA knockdown of Phlda3 enhanced the ability of cisplatin to increase p-Mdm2 presumably via Akt, enforcing the interaction between Mdm2 and p53. Consistently, a deficiency in Phlda3 abrogated p53 increase by cisplatin, indicating that Phlda3 promotes p53 accumulation. Phlda3 overexpression had the opposite effect. In addition, treatment with cyclosporine A or CdCl2, other nephrotoxicants, increased Phlda3 mRNA and protein levels in NRK52E cells, as did cisplatin treatment. Overall, Phlda3 may cause p53 accumulation through a feedforward pathway, facilitating tubular injury and its urine excretion.
Assuntos
Injúria Renal Aguda/genética , Cisplatino/toxicidade , Citotoxinas/toxicidade , Túbulos Renais/efeitos dos fármacos , Proteínas Nucleares/genética , Proteína Supressora de Tumor p53/genética , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Injúria Renal Aguda/urina , Animais , Cloreto de Cádmio/toxicidade , Linhagem Celular , Ciclosporina/toxicidade , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica , Receptor Celular 1 do Vírus da Hepatite A , Hepatócitos/efeitos dos fármacos , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Córtex Renal/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Fígado/efeitos dos fármacos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/urina , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Proteína Supressora de Tumor p53/agonistas , Proteína Supressora de Tumor p53/urinaRESUMO
Triple-negative breast cancer (TNBC) is associated with a poor prognosis and metastatic growth. TNBC cells frequently undergo macroautophagy/autophagy, contributing to tumor progression and chemotherapeutic resistance. ANXA2 (annexin A2), a potential therapeutic target for TNBC, has been reported to stimulate autophagy. In this study, we investigated the role of ANXA2 in autophagic processes in TNBC cells. TNBC patients exhibited high levels of ANXA2, which correlated with poor outcomes. ANXA2 increased LC3B-II levels following bafilomycin A1 treatment and enhanced autophagic flux in TNBC cells. Notably, ANXA2 upregulated the phosphorylation of HSF1 (heat shock transcription factor 1), resulting in the transcriptional activation of ATG7 (autophagy related 7). The mechanistic target of rapamycin kinase complex 2 (MTORC2) played an important role in ANXA2-mediated ATG7 transcription by HSF1. MTORC2 did not affect the mRNA level of ANXA2, but it was involved in the protein stability of ANXA2. HSPA (heat shock protein family A (Hsp70)) was a potential interacting protein with ANXA2, which may protect ANXA2 from lysosomal proteolysis. ANXA2 knockdown significantly increased sensitivity to doxorubicin, the first-line chemotherapeutic regimen for TNBC treatment, suggesting that the inhibition of autophagy by ANXA2 knockdown may overcome doxorubicin resistance. In a TNBC xenograft mouse model, we demonstrated that ANXA2 knockdown combined with doxorubicin administration significantly inhibited tumor growth compared to doxorubicin treatment alone, offering a promising avenue to enhance the effectiveness of chemotherapy. In summary, our study elucidated the molecular mechanism by which ANXA2 modulates autophagy, suggesting a potential therapeutic approach for TNBC treatment.Abbreviation: ATG: autophagy related; ChIP: chromatin-immunoprecipitation; HBSS: Hanks' balanced salt solution; HSF1: heat shock transcription factor 1; MTOR: mechanistic target of rapamycin kinase; TNBC: triple-negative breast cancer; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3.
Assuntos
Anexina A2 , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Autofagia/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Fatores de Transcrição de Choque Térmico/genética , Anexina A2/genética , Linhagem Celular Tumoral , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Doxorrubicina , SirolimoRESUMO
Early prediction of metastatic breast cancer is important for improvement of prognosis and survival rate. The present study aimed to identify secreted protein biomarkers for detection of invasive breast cancer. To this end, we performed a comparative proteomic analysis by a combination of 2DE and MALDI-TOF MS analysis of conditioned media from invasive H-Ras MCF10A human breast epithelial cells and noninvasive MCF10A and N-Ras MCF10A cells. We identified a list of 25 proteins that were strongly detected in media of H-Ras MCF10A and focused on annexin II, which was shown to be involved in cell motility. Invasive triple-negative human breast carcinoma cells, Hs578T, and MDA-MB-231, showed increased levels of annexin II in media, demonstrating that secretion of annexin II correlated well with the invasive phenotype of cells. We demonstrated a crucial role of annexin II in breast cell invasion/migration and actin cytoskeleton reorganization required for filopodia formation. Annexin II levels in the plasma samples and breast cancer tissues of breast cancer patients were significantly higher than those of normal groups, providing a clinical relevance to our in vitro findings. Taken together, we identified annexin II as a novel secretory biomarker candidate for invasive breast cancer, especially estrogen receptor-negative breast cancer.
Assuntos
Anexina A2/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Proteômica/métodos , Anexina A2/análise , Anexina A2/química , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/química , Mama/química , Mama/metabolismo , Neoplasias da Mama/química , Linhagem Celular Tumoral , Movimento Celular , Eletroforese em Gel Bidimensional , Feminino , Humanos , Immunoblotting , Invasividade Neoplásica , Proteínas/análise , Proteínas/química , Proteínas/classificação , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Recent evidence suggests that inflammation is involved in malignant progression of breast cancer. Sphingosine 1-phosphate (S1P), acting on the G-protein-coupled receptors, is known as a potent inflammatory mediator. In this study, the effect of the inflammatory lipid S1P on the regulation of invasive/migratory phenotypes of MCF10A human breast epithelial cells was investigated to elucidate a causal relationship between inflammation and the control of invasiveness of breast cells. We show that S1P causes induction of matrix metalloproteinase-9 (MMP-9) in vitro and in vivo, and thus enhances invasion and migration. We also show that fos plays a crucial role in the transcriptional activation of MMP-9 by S1P. In addition, activation of extracellular-signal-regulated kinases 1 and 2 (ERK1/2), p38 and alpha serine/threonine-protein kinase (Akt) are involved in the process of S1P-mediated induction of MMP-9 expression and invasion. Activation of the S1P receptor S1P3 and G(αq) are required for S1P-induced invasive/migratory responses, suggesting that the enhancement of S1P-mediated invasiveness is triggered by the specific coupling of S1P3 to the heterotrimeric G(αq) subunit. Activation of phospholipase C-ß4 and intracellular Ca²âº release are required for S1P-induced MMP-9 upregulation. Taken together, this study demonstrated that S1P regulates MMP-9 induction and invasiveness through coupling of S1P3 and G(αq) in MCF10A cells, thus providing a molecular basis for the crucial role of S1P in promoting breast cell invasion.
Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Lisofosfolipídeos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Esfingosina/análogos & derivados , Animais , Mama/citologia , Neoplasias da Mama/patologia , Linhagem Celular , Movimento Celular , Feminino , Humanos , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Proteínas Oncogênicas v-fos/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Esfingosina/metabolismoRESUMO
Mounting evidence suggests that metformin (N,N-dimethylbiguanide), a widely prescribed drug for the treatment of type II diabetes, exerts an anti-tumor effect on several cancers including breast cancer. Breast cancer has been estimated as one of the most commonly diagnosed types of cancer among women. In particular, triple-negative breast cancers are associated with poor prognosis and metastatic growth. In the present study, we synthesized a novel metformin derivative 5 (HL010183) and metformin salts, 9a, 9b, and 9c (metformin gamma-aminobutyric acid (GABA) salt, metformin pregabalin salt and metformin gabapentin salt), which exerted more potent inhibitory effects on the proliferation and invasiveness of Hs578T triple-negative breast carcinoma cells than metformin. Importantly, 5 showed approximately 100-fold more potent effects compared to metformin. In a triple-negative breast cancer xenograft model, 5 showed a comparable degree of inhibitory effect on in vivo tumor growth at the 100mg/kg dose to that of metformin at 500 mg/kg. Our results clearly demonstrate that 5 exerts a potent anti-tumor effect both in vitro and in vivo, paving the way for a strategy for treatment of triple-negative breast cancer.
Assuntos
Metformina/análogos & derivados , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Metformina/química , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Engineering approaches using antibody drug conjugates (ADCs) and bispecific antibodies (bsAbs) are designed to overcome the limitations of conventional chemotherapies and therapeutic antibodies such as drug resistance and non-specific toxicity. Cancer immunotherapies have been shown to be clinically successful with checkpoint blockade and chimeric antigen receptor T cell therapy; however, overactive immune systems still represent a major problem. Given the complexity of a tumor environment, it would be advantageous to have a strategy targeting two or more molecules. We highlight the necessity and importance of a multi-target platform strategy against cancer. Approximately 400 ADCs and over 200 bsAbs are currently being clinically developed for several indications, with promising signs of therapeutic activity. ADCs include antibodies that recognize tumor antigens, linkers that stably connect drugs, and powerful cytotoxic drugs, also known as payloads. ADCs have direct therapeutic effects by targeting cancers with a strong payload. Another type of drug that uses antibodies are bsAbs, targeting two antigens by linking to antigen recognition sites or bridging cytotoxic immune cells to tumor cells, resulting in cancer immunotherapy. Three bsAbs and one ADC have been approved for use by the FDA and the EMA in 2022. Among these, two of the bsAbs and the one ADC are used for cancers. We introduced that bsADC, a combination of ADC and bsAbs, has yet to be approved and several candidates are in the early stages of clinical development in this review. bsADCs technology helps increase the specificity of ADCs or the internalization and killing ability of bsAbs. We also briefly discuss the application of click chemistry in the efficient development of ADCs and bsAbs as a conjugation strategy. The present review summarizes the ADCs, bsAbs, and bsADCs that have been approved for anti-cancer or currently in development. These strategies selectively deliver drugs to malignant tumor cells and can be used as therapeutic approaches for various types of cancer.
Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Imunoconjugados , Neoplasias , Humanos , Imunoconjugados/uso terapêutico , Anticorpos Biespecíficos/uso terapêutico , Química Click , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêutico , Antineoplásicos/química , AntígenosRESUMO
Invasion and metastasis are important hallmarks of breast cancer and are the leading cause of patient mortality. Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by a poor prognosis and a lack of effective targeted therapies. The present study investigated the inhibitory effect of a novel FTY720 derivative on the invasive phenotype of TNBC cells. Here, we showed that a novel compound with an isoxazole ring, 4-(3-Decylisoxazol-5-yl)-1-hydroxy-2-(hydroxymethyl)butan-2-aminium chloride (CM2-II-173), significantly inhibited invasiveness of MDA-MB-231 TNBC cells. Expression of matrix metalloproteinase (MMP)-9 and invasiveness of MCF10A normal breast cells induced by sphingosine-1-phosphate (S1P) were reduced by CM2-II-173 treatment. Activations of pMEK1, pAkt, pERK, and p38 MAPK by S1P were inhibited by treatment with CM2-II-173. Proliferation and anchorage-independent growth of MDA-MB-231 TNBC cells were significantly decreased by CM2-II-173. CM2-II-173 efficiently induced apoptosis in MDA-MB-231 TNBC cells. CM2-II-173 significantly inhibited invasive phenotypes of breast, liver, prostate, and ovarian cancer cells. CM2-II-173 exhibited a more potent effect on the invasiveness of MDA-MB-231 TNBC cells compared to FTY720. Taken together, this study demonstrated that CM2-II-173 has the potential to be a lead compound that can inhibit cancer progression of not only TNBC cells, but also of liver, prostate, and ovarian cancer cells.