RESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterised by limited responses to chemoimmunotherapy attributed to highly desmoplastic tumor microenvironment. Disrupting the tumor-stromal cell crosstalk is considered as an improved PDAC treatment strategy, whereas little progress has been made due to poor understanding of its underlying mechanism. Here, we examined the cellular role of melanoma associated antigen A isoforms (MAGEA) in regulating tumor-stromal crosstalk mediated chemoresistance. METHODS: We used clinical samples to explore the correlation between MAGEA expression and patient prognosis in multiple cancers. We utilized cancer cell lines, patient derived organoids and orthotopic PDAC model to examine the function of MAGEA in chemoresistance. We performed biochemical, proteome profiler array and transcriptional analysis to uncover a mechanism that governs tumor-stromal crosstalk. We developed a multi-MAGEA antigen targeted DNA vaccine and tested its effect on PDAC tumor growth. RESULTS: We establish MAGEA as a regulator of the tumor-stromal crosstalk in PDAC. We provide strong clinical evidence indicating that high MAGEA expression, including MAGEA2, MAGEA3 and MAGEA10, correlates with worse chemotherapeutic response and poor prognosis in multiple cancers, while their expression is up-regulated in chemoresistant PDAC patient derived organoids and cancer cell lines. Mechanistically, MAGEA2 prohibits gemcitabine-induced JNK-c-Jun-p53 mediated cancer cell apoptosis, while gemcitabine stimulated pancreatic stellate cells secretes GDF15 to further enhance the gemcitabine resistance of MAGEA2 expressing cells by activating GFRAL-RET mediated Akt and ERK1/2 dependent survival pathway. Strikingly, immunization with a DNA vaccine that targeting multiple MAGEA antigens, including MAGEA2, MAGEA3 and MAGEA10, elicits robust immune responses against the growth of gemcitabine resistant tumors. CONCLUSIONS: These findings suggest that targeting MAGEA-mediated paracrine regulation of chemoresistance by immunotherapy can be an improved pancreatic cancer treatment strategy.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Vacinas de DNA , Humanos , Vacinas de DNA/metabolismo , Vacinas de DNA/farmacologia , Vacinas de DNA/uso terapêutico , Desoxicitidina/farmacologia , Linhagem Celular Tumoral , Neoplasias Pancreáticas/patologia , Gencitabina , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Imunização , Células Estromais/patologia , Resistencia a Medicamentos Antineoplásicos , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
Evidence is emerging that estrogen receptor alpha (ERalpha) is central to the rapid transduction of estrogen signaling to the downstream kinase cascades; however, the mechanisms underlying this nongenomic function are not fully understood. Here we report a paradigm of ERalpha regulation through arginine methylation by PRMT1, which transiently methylates arginine 260 within the ERalpha DNA-binding domain. This methylation event is required for mediating the extranuclear function of the receptor by triggering its interaction with the p85 subunit of PI3K and Src. Furthermore, we find that the focal adhesion kinase (FAK), a Src substrate involved in the migration process, is also recruited in this complex. Our data indicate that the methylation of ERalpha is a physiological process occurring in the cytoplasm of normal and malignant epithelial breast cells and that ERalpha is hypermethylated in a subset of breast cancers.
Assuntos
Arginina/metabolismo , Estrogênios/farmacologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Ativação Enzimática/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Genoma Humano/genética , Humanos , Metilação/efeitos dos fármacos , Camundongos , Modelos Biológicos , Células NIH 3T3 , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptor Cross-Talk/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacosRESUMO
Although the presence of nuclear estrogen receptor is widely used to guide breast cancer therapy, less attention has been paid to the receptor cytoplasmic signaling. Recently, we have shown that this pathway is operative in vivo and is activated in aggressive tumors representing a new potential target for breast cancer therapy. Here, we identified LKB1 as a partner of ERα and we explored its potential role in estrogen nongenomic signaling. The associations between LKB1 expression and the actors of this pathway, namely the methylated form of ERα (metERα), Src and PI3K, have been analyzed both in cultured cells and in 154 primary breast tumor samples. We found that LKB1 is a component of the cytoplasmic signaling complex in breast cell lines as well as in primary breast tumors. Moreover, an inverse correlation between the localization of LKB1 in nuclear and cytoplasmic compartments is observed. Importantly, high expression of cytoplasmic LKB1 is an independent marker of poor prognosis, associated with reduced overall survival (OS) and disease free survival (DFS). Conversely, the presence of nuclear LKB1 associates with increased OS and DFS. In conclusion, our results highlight that LKB1 expression in breast cancer appears to have opposite effects depending on its subcellular localization and may be used as a new prognostic biomarker.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Metilação , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Transfecção , Quinases da Família src/metabolismoRESUMO
Exon skipping mutations of the MET receptor tyrosine kinase (METex14), increasingly reported in cancers, occur in 3-4% of non-small-cell lung cancer (NSCLC). Only 50% of patients have a beneficial response to treatment with MET-tyrosine kinase inhibitors (TKIs), underlying the need to understand the mechanism of METex14 oncogenicity and sensitivity to TKIs. Whether METex14 is a driver mutation and whether it requires hepatocyte growth factor (HGF) for its oncogenicity in a range of in vitro functions and in vivo has not been fully elucidated from previous preclinical models. Using CRISPR/Cas9, we developed a METex14/WT isogenic model in nontransformed human lung cells and report that the METex14 single alteration was sufficient to drive MET-dependent in vitro anchorage-independent survival and motility and in vivo tumorigenesis, sensitising tumours to MET-TKIs. However, we also show that human HGF (hHGF) is required, as demonstrated in vivo using a humanised HGF knock-in strain of mice and further detected in tumour cells of METex14 NSCLC patient samples. Our results also suggest that METex14 oncogenicity is not a consequence of an escape from degradation in our cell model. Thus, we developed a valuable model for preclinical studies and present results that have potential clinical implication.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Éxons , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Pulmonares/patologia , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , CamundongosRESUMO
Regulation of the proteome by post-translational modifications (PTM) emerges as a major contributing factor to the functional diversity in biology regulating cellular processes. Because PTM are key to the physiologic functions of the proteins involved, it is imperative that we understand the << coding >> that these modifications impart to regulate diverse activities. As estrogen signalling mediates a plethora of PTM not only on the receptors themselves but also on their coregulators, we investigate to << crack >> the ER code. Besides the long-known phosphorylation, other covalent additions such as acetylation, ubiquitination, sumoylation and methylation have been described for estrogen receptors in recent years. These modifications affect receptor stability and activity, and provide potential mechanisms for cell- or-gene-specific regulation. A better understanding of the impact of these PTMs on estrogen receptor should help in the identification of new drugs for breast cancer treatments.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Processamento de Proteína Pós-Traducional , Neoplasias da Mama/enzimologia , Ativação Enzimática , Receptor alfa de Estrogênio/genética , Feminino , Histona Acetiltransferases/metabolismo , Humanos , Fosforilação , Proteínas Quinases/metabolismoRESUMO
Matrix vesicles (MVs) are involved in the initial step of mineralization in skeletal tissues and provide an easily model to analyze the hydroxyapatite (HA) formation. Sr stimulates bone formation and its effect was tested on MVs. Sr(2+) (15-50 microM) in the mineralization medium containing MVs, 2 mM Ca(2+) and 3.42 mM P(i), retarded HA formation. Sr(2+) (1-5 mM) in the same medium-induced other types of mineral than HA and cancelled the ATP-, ADP- or PP(i)-induced retardation in the mineral formation. Our findings suggest that the beneficial effect of Sr(2+) at a low dose (15-50 microM) is rather an inhibitor of bone resorption than an activator of mineral formation, while at high Sr(2+) concentration (1-5 mM), mineral formation, especially other types of mineral than HA, is favored.
Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Durapatita/metabolismo , Osteogênese/efeitos dos fármacos , Vesículas Secretórias/efeitos dos fármacos , Estrôncio/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Reabsorção Óssea/metabolismo , Cátions Bivalentes/farmacologia , Embrião de Galinha , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Vesículas Secretórias/metabolismo , Espectrofotometria InfravermelhoRESUMO
Oestrogen receptors can mediate rapid activation of cytoplasmic signalling cascades by recruiting Src and PI3K. However, the involvement of this pathway in breast cancer remains poorly defined. We have previously shown that methylation of ERα is required for the formation of the ERα/Src/PI3K complex and that ERα is hypermethylated in a subset of breast cancers. Here, we used Proximity Ligation Assay to demonstrate that this complex is present in the cytoplasm of breast cancer cell lines as well as formalin-fixed, paraffin-embedded tumours. Of particular interest, the analysis of 175 breast tumours showed that overexpression of this complex in a subset of breast tumours correlates to the activation of the downstream effector Akt. Survival analysis revealed that high expression of this complex is an independent marker of poor prognosis and associated with reduced disease-free survival. Our data introduces the new concept that the rapid oestrogen pathway is operative in vivo. It also provides a rationale for patient stratification defined by the activation of this pathway and the identification of target therapies.