RESUMO
Glutathione-S-transferases (GSTs) not only show cytoprotective role and their involvement in the development of anticancer drug resistance, but also transmit signals that control cell proliferation and apoptosis. However, the role of GST isoforms in chemotherapy resistance remains elusive in pancreatic cancer. Here, we demonstrated that gemcitabine treatment increased the GSTM2 expression in pancreatic cancer cell lines. Knockdown of GSTM2 by siRNA elevated apoptosis and decreased viability of pancreatic cancer cells treated with gemcitabine. Moreover, in vivo experiments further showed that shRNA induced GSTM2 downregulation enhanced drug sensitivity of gemcitabine in orthotopic pancreatic tumor mice. We also found that GSTM2 levels were lower in tumor tissues than in non-tumor tissues and higher GSTM2 expression was significantly associated with longer overall survival. In conclusion, our findings indicate that GSTM2 expression is essential for the survival of pancreatic cancer cells undergoing gemcitabine treatment and leads to chemo resistance. Downregulation of GSTM2 in pancreatic cancer may benefit gemcitabine treatment. GSTM2 expression in patients also shows significant correlation with overall survival. Thus, our study suggests that GSTM2 is a potential target for chemotherapy optimization and prognostic biomarker of pancreatic cancer.
Assuntos
Desoxicitidina/análogos & derivados , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Antineoplásicos/farmacologia , Biomarcadores Tumorais , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Marcadores Genéticos , Glutationa Transferase/genética , Humanos , Interferência de RNA , GencitabinaRESUMO
BACKGROUND/AIMS: The cell surface protein transmembrane 4 L6 family member 1 (TM4SF1) has been detected in various tumors and plays a major role in the development of cancer. We aimed to investigate the effects of TM4SF1 on the migration and invasion of pancreatic cancer in vitro and in vivo and explore its related molecular mechanisms. METHODS: qRT-PCR and immunohistochemical analyses were used to measure the expression of TM4SF1 in pancreatic cancer tissues and adjacent tissues. TM4SF1 was silenced using siRNA and shRNA to investigate the role of this protein in the proliferation and metastasis of pancreatic cancer cells. MTS and Transwell assays were used to examine the effect of TM4SF1 on pancreatic cancer cell lines. The expression and activity of MMP-2 and MMP-9 were determined by qRT-PCR, western blots and gelatin zymography. In vivo, orthotopic pancreatic tumor models were used to examine the formation of metastasis. RESULTS: qRT-PCR and immunohistochemical analyses showed that TM4SF1 was highly expressed in pancreatic cancer tissues compared with the adjacent tissues. In in vitro experiments the silencing of TM4SF1 reduced cell migration and invasion and down-regulated the expression and activity of MMP-2 and MMP-9. However, no significant difference in cell proliferation was detected after silencing TM4SF1. Additionally, knocking down TM4SF1 decreased the formation of lung and liver metastases in orthotopic pancreatic tumor models. CONCLUSION: Our results demonstrate that the expression of TM4SF1 is higher in pancreatic cancer tissues and pancreatic cancer cell lines than controls. Knockdown of TM4SF1 inhibited the migration and invasion of pancreatic cancer cells by regulating the expression and activity of MMP-2 and MMP-9, which suggests that TM4SF1 may play a significant role in metastasis in pancreatic cancer.
Assuntos
Antígenos de Superfície/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Animais , Antígenos de Superfície/metabolismo , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Interferência de RNA , Terapêutica com RNAi , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is relatively rare but extremely lethal. Standard cytotoxic therapeutics provide little benefit. To date, newer targeted therapeutics have also not been highly successful. Often novel therapeutics that have appeared to perform well in preclinical models have failed in the clinic. Many factors contribute to these failures, but the one most often attributed is the shortcomings of the preclinical models. A plethora of animal models now exist for PDAC, including cell line xenografts, patient-derived xenografts, a wide variety of genetic mouse models, and syngeneic xenografts. These models have generated a tremendous amount of information useful for the understanding of PDAC. Yet none seems to well predict clinical outcomes of new treatments. This review will discuss how genetic instability and cellular heterogeneity make this disease so difficult to model accurately. We will also discuss the strengths and weaknesses of many of the popular models. Ultimately we will argue that there is no perfect model and that the best approach to understanding clinical performance is the use of multiple preclinical models with an understanding of their salient features.
Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/fisiopatologia , Carcinoma Ductal Pancreático/terapia , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Camundongos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/fisiopatologia , Neoplasias Pancreáticas/terapia , Especificidade da EspécieRESUMO
BACKGROUND & AIMS: New-onset diabetes in patients with pancreatic cancer is likely to be a paraneoplastic phenomenon caused by tumor-secreted products. We aimed to identify the diabetogenic secretory product(s) of pancreatic cancer. METHODS: Using microarray analysis, we identified adrenomedullin as a potential mediator of diabetes in patients with pancreatic cancer. Adrenomedullin was up-regulated in pancreatic cancer cell lines, in which supernatants reduced insulin signaling in beta cell lines. We performed quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry on human pancreatic cancer and healthy pancreatic tissues (controls) to determine expression of adrenomedullin messenger RNA and protein, respectively. We studied the effects of adrenomedullin on insulin secretion by beta cell lines and whole islets from mice and on glucose tolerance in pancreatic xenografts in mice. We measured plasma levels of adrenomedullin in patients with pancreatic cancer, patients with type 2 diabetes mellitus, and individuals with normal fasting glucose levels (controls). RESULTS: Levels of adrenomedullin messenger RNA and protein were increased in human pancreatic cancer samples compared with controls. Adrenomedullin and conditioned media from pancreatic cell lines inhibited glucose-stimulated insulin secretion from beta cell lines and islets isolated from mice; the effects of conditioned media from pancreatic cancer cells were reduced by small hairpin RNA-mediated knockdown of adrenomedullin. Conversely, overexpression of adrenomedullin in mice with pancreatic cancer led to glucose intolerance. Mean plasma levels of adrenomedullin (femtomoles per liter) were higher in patients with pancreatic cancer compared with patients with diabetes or controls. Levels of adrenomedullin were higher in patients with pancreatic cancer who developed diabetes compared those who did not. CONCLUSIONS: Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in ß cells and mice.
Assuntos
Adenocarcinoma/metabolismo , Adrenomedulina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Neoplasias Pancreáticas/metabolismo , Regulação para Cima , Adenocarcinoma/patologia , Adrenomedulina/efeitos dos fármacos , Adrenomedulina/genética , Idoso , Animais , Linhagem Celular Tumoral , Células Cultivadas , Diabetes Mellitus Tipo 2/patologia , Feminino , Glucose/farmacologia , Humanos , Técnicas In Vitro , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Modelos Animais , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , RNA Interferente Pequeno/farmacologia , Ratos , Transplante HeterólogoRESUMO
BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA. Surgical resection is the only effective treatment; however, only 20% of patients are candidates for surgery. The ability to detect early PDAC would increase the availability of surgery and improve patient survival. This study assessed the feasibility of using the enzymatic activity of cathepsin E (Cath E), a protease highly and specifically expressed in PDAC, as a novel biomarker for the detection of pancreas-bearing pancreatic intraepithelial neoplasia (PanIN) lesions and PDAC. METHODS: Pancreas from normal, chronic pancreatitis and PDAC patients was assessed for Cath E expression by quantitative real-time PCR and immunohistochemistry. Human PDAC xenografts and genetically engineered mouse models (GEMM) of PDAC were injected with a Cath E activity selective fluorescent probe and imaged using an optical imaging system. RESULTS: The specificity of Cath E expression in PDAC patients and GEMM of pancreatic cancer was confirmed by quantitative real-time PCR and immunohistochemistry. The novel probe for Cath E activity specifically detected PDAC in both human xenografts and GEMM in vivo. The Cath E sensitive probe was also able to detect pancreas with PanIN lesions in GEMM before tumour formation. CONCLUSIONS: The elevated Cath E expression in PanIN and pancreatic tumours allowed in-vivo detection of human PDAC xenografts and imaging of pancreas with PanIN and PDAC tumours in GEMM. Our results support the usefulness of Cath E activity as a potential molecular target for PDAC and early detection imaging.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma in Situ/diagnóstico , Carcinoma Ductal Pancreático/diagnóstico , Catepsina E/metabolismo , Diagnóstico por Imagem/métodos , Neoplasias Pancreáticas/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Animais , Biomarcadores Tumorais/genética , Carcinoma in Situ/enzimologia , Carcinoma Ductal Pancreático/enzimologia , Catepsina E/genética , Linhagem Celular Tumoral , Primers do DNA/química , Modelos Animais de Doenças , Diagnóstico Precoce , Estudos de Viabilidade , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Sondas de Oligonucleotídeos/química , Neoplasias Pancreáticas/enzimologia , Lesões Pré-Cancerosas/enzimologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Regulação para CimaRESUMO
Effective therapeutic strategies are needed for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations that acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) mediated by epithelial-to-mesenchymal transition (EMT). We investigate cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches and identify CD70 as being highly upregulated in EMT-associated resistance. Moreover, CD70 upregulation is an early event in the evolution of resistance and occurs in drug-tolerant persister cells (DTPCs). CD70 promotes cell survival and invasiveness, and stimulation of CD70 triggers signal transduction pathways known to be re-activated with acquired TKI resistance. Anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting chimeric antigen receptor (CAR) T cell and CAR NK cells show potent activity against EGFR TKI-resistant cells and DTPCs. These results identify CD70 as a therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits clinical investigation.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Ligante CD27/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , /uso terapêuticoRESUMO
S100P expression is described in many different cancers, and its expression is associated with drug resistance, metastasis, and poor clinical outcome. S100P is member of the S100 family of small calcium-binding proteins that have been reported to have either intracellular or extracellular functions, or both. Extracellular S100P can bind with the receptor for advanced glycation end products (RAGE) and activate cellular signaling. Through RAGE, S100P has been shown to mediate tumor growth, drug resistance, and metastasis. S100P is specifically expressed in cancer cells in the adult. Therefore, S100P is a useful marker for differentiating cancer cells from normal cells, and can aid in the diagnosis of cancer by cytological examination. The expression of S100P in cancer cells has been related to hypomethylation of the gene. Multiple studies have confirmed the beneficial effects of blocking S100P/RAGE in cancer cells, and different blockers are being developed including small molecules and antagonist peptides. This review summarizes the role and significance of S100P in different cancers.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Desenho de Fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismoRESUMO
PURPOSE: Neuropilin-2 (NRP-2) is a coreceptor for vascular endothelial growth factor (VEGF) on endothelial cells. NRP-2 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) cells relative to nonmalignant ductal epithelium. This study determined the role of NRP-2 in PDAC cells. EXPERIMENTAL DESIGN: NRP-2 expression was reduced in PDAC cells with stable short-hairpin RNA (shRNA) transfection. Western blotting was done to evaluate signaling intermediates. Migration and invasion studies were carried out in Boyden chambers. Anchorage-independent growth was assessed by soft-agar colony formation. In vivo growth was evaluated using murine subcutaneous and orthotopic xenograft models. Immunohistochemical analysis evaluated in vivo proliferation and angiogenesis. RESULTS: shRNA-NRP-2 decreased NRP-2 levels without affecting neuropilin-1 levels. Akt activation was decreased in clones with reduced NRP-2 (shRNA-NRP-2). shRNA-NRP-2 cells showed decreased migration, invasion, and anchorage-independent growth compared with control cells. In vitro proliferation rates were similar in control- and shRNA-transfected cells. Subcutaneous and orthotopic xenografts from shRNA-transfected cells were significantly smaller than those resulting from control-transfected cells (P < 0.05). Furthermore, shRNA-NRP-2 tumors exhibited less cellular proliferation and decreased microvascular area relative to control tumors (P < 0.05). Constitutive expression of the angiogenic mediator Jagged-1 was reduced in shRNA-NRP-2 cells, whereas vascular endothelial growth factor levels were unchanged. CONCLUSION: Reduction of NRP-2 expression in PDAC cells decreased survival signaling, migration, invasion, and ability to grow under anchorage-independent conditions. In vivo, reduction of NRP-2 led to decreased growth of xenograft tumors and decreased vascular area, which was associated with decreased Jagged-1 levels. NRP-2 is a potential therapeutic target on PDAC cells.
Assuntos
Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Neovascularização Patológica/etiologia , Neuropilina-2/fisiologia , Neoplasias Pancreáticas/patologia , Adenocarcinoma/irrigação sanguínea , Proteínas de Ligação ao Cálcio/análise , Carcinoma Ductal Pancreático/irrigação sanguínea , Linhagem Celular Tumoral , Movimento Celular , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Proteína Jagged-1 , Proteínas de Membrana/análise , Invasividade Neoplásica , Neuropilina-2/análise , Neoplasias Pancreáticas/irrigação sanguínea , Proteínas Serrate-JaggedRESUMO
PURPOSE: Nuclear factor kappaB (NFkappaB) activity may increase survival and protect cancer cells from chemotherapy. Therefore, NFkappaB activity may be prognostic, and inhibition of NFkappaB may be useful for pancreatic cancer therapy. To test these hypotheses, we examined NFkappaB activity and the effects of inhibiting NFkappaB in several pancreatic cancer cell lines with differing sensitivities to gemcitabine. EXPERIMENTAL DESIGN: The gemcitabine sensitivity of pancreatic cancer cell lines BxPC-3, L3.6pl, CFPAC-1, MPanc-96, PANC-1, and MIA PaCa-2 were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and fluorescence-activated cell sorting assays. NFkappaB levels were determined by electrophoretic mobility shift assay and reporter assays. The effects of gemcitabine on NFkappaB activity were determined in vitro and in vivo. NFkappaB was inhibited by silencing of the p65/relA subunit using small interfering RNA in vitro and by neutral liposomal delivery of small interfering RNA in vivo, and the effects were evaluated on gemcitabine sensitivity. RESULTS: The cell lines L3.6pl, BxPC-3, and CFPAC-1 were sensitive, whereas MPanc-96, PANC-1, and MIA PaCa-2 were resistant to gemcitabine. No significant correlation was observed between basal NFkappaB activity and gemcitabine sensitivity. Gemcitabine treatment did not activate NFkappaB either in vitro or in vivo. Silencing of p65/relA induced apoptosis and increased gemcitabine killing of all gemcitabine-sensitive pancreatic cancer cells. No significant effects, however, were observed on gemcitabine-resistant pancreatic cancer cell lines either in vitro or in vivo. CONCLUSIONS: NFkappaB activity did not correlate with sensitivity to gemcitabine. Silencing of p65/relA was effective alone and in combination with gemcitabine in gemcitabine-sensitive but not gemcitabine-resistant pancreatic cancer cells. Thus, NFkappaB may be a useful therapeutic target for a subset of pancreatic cancers.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , RNA Interferente Pequeno/genética , Fator de Transcrição RelA/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Inativação Gênica , Humanos , Masculino , Camundongos , Neoplasias Pancreáticas/patologia , Fator de Transcrição RelA/genética , GencitabinaRESUMO
The current study investigated adrenomedullin as a potential autocrine regulator of pancreatic cancer cell function. Adrenomedullin was localized in the neoplastic epithelium of 90% (43 of 48) of human pancreatic adenocarcinomas analyzed by immunohistochemistry and was expressed by 100% (8 of 8) of pancreatic cancer cell lines analyzed by reverse transcription-PCR. Pancreatic cancer cell lines also secreted adrenomedullin into the culture medium as determined by ELISA (5 of 5). Exogenous adrenomedullin treatment of Panc-1, BxPC3, and MPanc96 cells in vitro stimulated cell proliferation, invasion, and nuclear factor kappaB activity, indicating the ability of the cells to respond to adrenomedullin. Treatment of the cell cultures with an adrenomedullin antagonist inhibited basal levels of proliferation and nuclear factor kappaB activity, supporting the autocrine function of this molecule. Furthermore, increasing adrenomedullin levels by gene transfer to Panc-1 cells increased, whereas adrenomedullin small hairpin RNA silencing in MPanc96 cells inhibited tumor growth and metastasis in vivo. Adrenomedullin is able to act through at least two different receptors, adrenomedullin receptor (ADMR) and calcitonin receptor-like receptor (CRLR). Reverse transcription-PCR and Western blotting indicated that pancreatic cancer cells expressed only ADMR but not CRLR. In contrast, cells found in the tumor microenvironment, primary human pancreatic stellate and endothelial (HUVEC) cells, expressed both ADMR and CRLR. Small hairpin RNA silencing of ADMR in pancreatic cancer cells blocked adrenomedullin-induced growth and invasion, indicating that this receptor is involved in the autocrine actions of adrenomedullin. These data indicate that adrenomedullin acting via ADMR increases the aggressiveness of pancreatic cancer cells and suggests that these molecules may be useful therapeutic targets.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adrenomedulina/fisiologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores de Peptídeos/metabolismo , Adenocarcinoma/genética , Adrenomedulina/antagonistas & inibidores , Adrenomedulina/biossíntese , Adrenomedulina/farmacologia , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos SCID , NF-kappa B/metabolismo , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , RNA Interferente Pequeno/genética , Receptores de Adrenomedulina , Receptores de Peptídeos/antagonistas & inibidores , Receptores de Peptídeos/genéticaRESUMO
The receptor for advanced glycation end-products (RAGE) is a multifunctional receptor with multiple ligands that is known to play a key role in several diseases, including diabetes, arthritis, and Alzheimer's disease. Recent evidence indicates that this receptor also has an important role in cancer. RAGE ligands, which include the S100/calgranulins and high-mobility group box 1 (HMGB1) ligands, are expressed and secreted by cancer cells and are associated with increased metastasis and poorer outcomes in a wide variety of tumors. These ligands can interact in an autocrine manner to directly activate cancer cells and stimulate proliferation, invasion, chemoresistance, and metastasis. RAGE ligands derived from cancer cells can also influence a variety of important cell types within the tumor microenvironment, including fibroblasts, leukocytes, and vascular cells, leading to increased fibrosis, inflammation, and angiogenesis. Several of the cells in the tumor microenvironment also produce RAGE ligands. Most of the cancer-promoting effects of RAGE ligands are the result of their interaction with RAGE. However, these ligands also often have separate intracellular roles, and some may interact with other extracellular targets, so it is not currently possible to assign all of their effects to RAGE activation. Despite these complications, the bulk of the evidence supports the premise that the ligand-RAGE axis is an important target for therapeutic intervention in cancer.
Assuntos
Neoplasias/metabolismo , Receptores Imunológicos/metabolismo , Processamento Alternativo/genética , Animais , Humanos , Ligantes , Neoplasias/terapia , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/química , Receptores Imunológicos/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and it is unclear whether its stromal infiltrate contributes to its aggressiveness. Here, we demonstrate that Dickkopf-3 (DKK3) is produced by pancreatic stellate cells and is present in most human PDAC. DKK3 stimulates PDAC growth, metastasis, and resistance to chemotherapy with both paracrine and autocrine mechanisms through NF-κB activation. Genetic ablation of DKK3 in an autochthonous model of PDAC inhibited tumor growth, induced a peritumoral infiltration of CD8+ T cells, and more than doubled survival. Treatment with a DKK3-blocking monoclonal antibody inhibited PDAC progression and chemoresistance and prolonged survival. The combination of DKK3 inhibition with immune checkpoint inhibition was more effective in reducing tumor growth than either treatment alone and resulted in a durable improvement in survival, suggesting that DKK3 neutralization may be effective as a single targeted agent or in combination with chemotherapy or immunotherapy for PDAC.
Assuntos
Carcinoma Ductal Pancreático/patologia , Progressão da Doença , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Comunicação Autócrina/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Quimiocinas , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inativação Gênica , Humanos , Imunoterapia , Camundongos Endogâmicos C57BL , Camundongos Nus , NF-kappa B/metabolismo , Testes de Neutralização , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Células Estreladas do Pâncreas/metabolismo , Células Estreladas do Pâncreas/patologia , Comunicação Parácrina/efeitos dos fármacos , Análise de Sobrevida , Gencitabina , Neoplasias PancreáticasRESUMO
Although it displays promising activity in other tumor models, the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on human pancreatic cancer cells have not been comprehensively explored. We report that a majority of human pancreatic cancer cell lines (seven of nine) underwent apoptosis when they were exposed to recombinant human TRAIL in vitro. Characterization of surface TRAIL receptors by fluorescence-activated cell sorting showed that TRAIL-resistant cells (Panc-1 and HS766T) expressed lower levels of DR4 and DR5 than did TRAIL-sensitive cells. The proteasome inhibitor bortezomib (PS-341, Velcade) further increased TRAIL responsiveness in the TRAIL-sensitive cells and synergized with TRAIL to reverse resistance in Panc-1 and HS776T cells. The effects of bortezomib were mimicked by transfection with a small interfering RNA construct specific for the p65 subunit of nuclear factor-kappaB (NF-kappaB) or exposure to a selective chemical inhibitor of IKK (PS-1145). Silencing IkappaBalpha prevented TRAIL sensitization by PS-1145, confirming that IkappaBalpha mediated the effects of PS-1145. NF-kappaB inhibition resulted in down-regulation of BCL-XL and XIAP, and silencing either restored TRAIL sensitivity in TRAIL-resistant cells. Finally, therapy with TRAIL plus PS-1145 reversed TRAIL resistance in vivo to produce synergistic growth inhibition in orthotopic Panc-1 tumors. Together, our results show that NF-kappaB inhibits TRAIL-induced apoptosis in human pancreatic cancer cells and suggest that combination therapy with TRAIL and NF-kappaB inhibitors, such as bortezomib, PS-1145, or curcumin, should be considered as a possible treatment strategy in patients with pancreatic cancer.
Assuntos
NF-kappa B/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Ácidos Borônicos/farmacologia , Bortezomib , Sinergismo Farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pirazinas/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Proteínas Recombinantes/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: In the current study, we examined the functional significance and mechanism of action of S100P in pancreatic cancer cells. EXPERIMENTAL DESIGN: S100P levels were increased in Panc-1 cells, which do not express S100P, by transfection with an S100P cDNA and S100P levels were reduced in BxPC3 cells, which express high levels of S100P, by small interfering RNA gene silencing. Effects of these manipulations on cell proliferation, resistance to apoptotic insults, cell migration, and invasion were estimated in vitro using standard assays. The influences of S100P on tumor growth in vivo were studied using xenograft mouse models. To identify the mechanisms involved in these responses, coimmunoprecipitation studies were conducted with S100P with receptor for advanced glycation end products (RAGE) and the effects of inhibiting RAGE using an antagonistic peptide were analyzed. RESULTS: S100P levels correlated with the rates of cell proliferation, survival, migration, and invasion in both cell models in vitro. In vivo, increased S100P levels increased the growth of tumors in mice with s.c.-implanted Panc-1 cells and decreased S100P levels decreased tumor growth after orthotopic implantation of BxPC-3 cells. A direct interaction between S100P and RAGE was indicated by coimmunoprecipitation of these molecules from pancreatic cancer cells. A RAGE antagonist peptide inhibited this interaction and also inhibited the biological effects of S100P on these cells in vitro. CONCLUSIONS: These data suggest that S100P plays a major role in the aggressiveness of pancreatic cancer that is likely mediated by its ability to activate RAGE. Thus, interference with S100P may provide a novel approach for treatment of pancreatic cancer.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/patologia , Receptores Imunológicos/metabolismo , Animais , Apoptose , Western Blotting , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Separação Celular , Sobrevivência Celular , Meios de Cultivo Condicionados/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Inativação Gênica , Produtos Finais de Glicação Avançada , Humanos , Imunoprecipitação , Camundongos , Camundongos Nus , Células NIH 3T3 , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Pancreáticas/metabolismo , Peptídeos/química , RNA Interferente Pequeno/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , TransfecçãoRESUMO
The molecular basis of pancreatic cancer is not understood. Previous attempts to determine the specific genes expressed in pancreatic cancer have been hampered by similarities between adenocarcinoma and chronic pancreatitis. In the current study, microarrays (Affymetrix) were used to profile gene expression in pancreatic adenocarcinoma (10), pancreatic cancer cell lines (7), chronic pancreatitis (5), and normal pancreas (5). Molecular profiling indicated a large number of genes differentially expressed between pancreatic cancer and normal pancreas but many fewer differences between pancreatic cancer and chronic pancreatitis, likely because of the shared stromal influences in the two diseases. To specifically identify genes expressed in neoplastic epithelium, we selected genes more highly expressed (>2-fold, p < 0.01) in adenocarcinoma compared with both normal pancreas and chronic pancreatitis and which were also highly expressed in pancreatic cancer cell lines. This strategy yielded 158 genes, of which 124 were not previously associated with pancreatic cancer. Quantitative-reverse transcription-PCR for two molecules, S100P and 14-3-3sigma, validated the microarray data. Support for the success of the neoplastic cell gene expression identification strategy was obtained by immunocytochemical localization of four representative genes, 14-3-3sigma, S100P, S100A6, and beta4 integrin, to neoplastic cells in pancreatic tumors. Thus, comparisons between pancreatic adenocarcinoma, pancreatic cancer cell lines, normal pancreas, and chronic pancreatitis have identified genes that are selectively expressed in the neoplastic epithelium of pancreatic adenocarcinoma. These data provide new insights into the molecular pathology of pancreatic cancer that may be useful for detection, diagnosis, and treatment.
Assuntos
Adenocarcinoma/genética , Neoplasias Pancreáticas/genética , Pancreatite/genética , Adenocarcinoma/metabolismo , Doença Crônica , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/metabolismo , Pancreatite/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Anterior gradient 2 (AGR2) promotes cancer growth, metastasis, and resistance to therapy via unknown mechanisms. We investigated the effects of extracellular AGR2 signaling through the orphan glycosylphosphatidylinositol-linked receptor C4.4A in pancreatic ductal adenocarcinoma (PDAC). Proliferation, migration, invasion, and apoptosis were measured using colorimetric, Boyden chamber, and FACS analyses. We developed blocking mAbs against AGR2 and C4.4A and tested their effects, along with siRNAs, on cancer cell functions and on orthotopic tumors in nude mice. Extracellular AGR2 stimulated proliferation, migration, invasion, and chemoresistance of PDAC cell lines. AGR2 interacted with C4.4A in cell lysates and mixtures of recombinant proteins. Knockdown of C4.4A reduced migration and resistance to gemcitabine. PDAC tissues, but not adjacent healthy pancreatic tissues, expressed high levels of AGR2 and C4.4A. AGR2 signaling through C4.4A required laminins 1 or 5 and integrin ß1. Administration of antibodies against AGR2 and C4.4A reduced growth and metastasis and caused regression of aggressive xenograft tumors, leading to increased survival of mice. These data support a model in which AGR2 binds and signals via C4.4A in an autocrine loop and promotes the growth of pancreas tumors in mice. Blocking mAbs against AGR2 and C4.4A may have therapeutic potential against PDAC.
Assuntos
Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Moléculas de Adesão Celular/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Espaço Extracelular/metabolismo , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Humanos , Cadeias beta de Integrinas/metabolismo , Laminina/metabolismo , Camundongos , Mucoproteínas , Proteínas Oncogênicas , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Ligação Proteica , Proteínas/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Calinina , Neoplasias PancreáticasRESUMO
BACKGROUND: TM4SF1 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and affects the development of this cancer. Also, multidrug resistance (MDR) is generally associated with tumor chemoresistance in pancreatic cancer. However, the correlation between TM4SF1 and MDR remains unknown. This research aims to investigate the effect of TM4SF1 on gemcitabine resistance in PDAC and explore the possible molecular mechanism between TM4SF1 and MDR. METHODS: The expression of TM4SF1 was evaluated in pancreatic cancer cell lines and human pancreatic duct epithelial (HPDE) cell lines by quantitative RT-PCR. TM4SF1 siRNA transfection was carried out using Hiperfect transfection reagent to knock down TM4SF1. The transcripts were analyzed by quantitative RT-PCR, RT-PCR and western blotting for further study. The cell proliferation and apoptosis were obtained to investigate the sensitivity to gemcitabine of pancreatic cancer cells after silencing TM4SF1 in vitro. We demonstrated that cell signaling of TM4SF1 mediated chemoresistance in cancer cells by assessing the expression of multidrug resistance (MDR) genes using quantitative RT-PCR. In vivo, we used orthotopic pancreatic tumor models to investigate the effect of proliferation after silencing TM4SF1 by a lentivirus-mediated shRNA in MIA PaCa-2 cell lines. RESULTS: The mRNA expression of TM4SF1 was higher in seven pancreatic cancer cell lines than in HPDE cell lines. In three gemcitabine-sensitive cell lines (L3.6pl, BxPC-3, SU86.86), the expression of TM4SF1 was lower than that in four gemcitabine-resistant cell lines (MIA PaCa-2, PANC-1, Hs766T, AsPC-1). We evaluated that TM4SF1 was a putative target for gemcitabine resistance in pancreatic cancer cells. Using AsPC-1, MIA PaCa-2 and PANC-1, we investigated that TM4SF1 silencing affected cell proliferation and increased the percentages of cell apoptosis mediated by treatment with gemcitabine compared with cells which were treated with negative control. This resistance was associated with the expression of multidrug resistance genes including ABCB1 and ABCC1. In vivo, silencing of TM4SF1 in MIA PaCa-2 cell lines increased the effectiveness of gemcitabine-based treatment in orthotopic pancreatic tumor models evaluated using noninvasive bioluminescent imaging. CONCLUSION: These findings suggest that TM4SF1 is a surface membrane antigen that is highly expressed in pancreatic cancer cells and increases the chemoresistance to gemcitabine. Thus, TM4SF1 may be a promising target to overcome the chemoresistance of pancreatic cancer.
Assuntos
Antígenos de Superfície/genética , Carcinoma Ductal Pancreático/genética , Desoxicitidina/análogos & derivados , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/tratamento farmacológico , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Animais , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , GencitabinaRESUMO
INTRODUCTION: Early detection of pancreatic cancer could save many thousands of lives. Non-invasive diagnostic imaging, including PET with [(18)F]FDG, has inadequate resolution for detection of small (2-3 mm) pancreatic tumours. We demonstrated the efficacy of PET imaging with an (18)F-labelled lactose derivative, [(18)F]FEDL, that targets HIP/PAP, a biomarker that is overexpressed in the peritumoural pancreas. We developed another analogue, 1-[(18)F]fluoroethyl lactose ([(18)F]FEL), which is simpler to synthesise, for the same application. We conducted a preliminary evaluation of the new probe and its efficacy in detecting orthotopic pancreatic carcinoma xenografts in mice. METHODS: Xenografts were developed in nude mice by injecting L3.6 pl/GL(+) pancreatic carcinoma cells into the pancreas of each mouse. Tumour growth was monitored by bioluminescence imaging (BLI); accuracy of BLI tumour size estimates was verified by MRI in two representative mice. When the tumour size reached approximately 2-3mm, the animals were injected with [(18)F]FEL (3.7 MBq) and underwent static PET/CT scans. Blood samples were collected at 2, 5, 10, 20 and 60 min after [(18)F]FEL injection to track blood clearance. Following imaging, animals were sacrificed and their organs and tumours/pancreatic tissue were collected and counted on a gamma counter. Pancreas, including tumour, was frozen, sliced and used for autoradiography and immunohistochemical analysis of HIP/PAP expression. RESULTS: Tumour growth was rapid, as observed by BLI and MRI. Blood clearance of [(18)F]FEL was bi-exponential, with half-lives of approximately 3.5 min and 40 min. Mean accumulation of [(18)F]FEL in the peritumoural pancreatic tissue was 1.29±0.295 %ID/g, and that in the normal pancreas of control animals was 0.090±0.101 %ID/g. [(18)F]FEL was cleared predominantly by the kidneys. Comparative analysis of autoradiographic images and immunostaining results demonstrated a correlation between [(18)F]FEL binding and HIP/PAP expression. CONCLUSION: [(18)F]FEL may be useful for non-invasive imaging of early-stage pancreatic tumours by PET. The results warrant further studies.
Assuntos
Radioisótopos de Flúor , Lactose/análogos & derivados , Neoplasias Pancreáticas/diagnóstico , Compostos Radiofarmacêuticos , Animais , Feminino , Radioisótopos de Flúor/farmacocinética , Humanos , Técnicas Imunoenzimáticas , Lactose/farmacocinética , Lactose/farmacologia , Medições Luminescentes , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Proteínas Associadas a Pancreatite , Tomografia por Emissão de Pósitrons , Proteínas/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Tomografia Computadorizada por Raios X , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sulfated non-anticoagulant heparins (S-NACHs) might be preferred for potential clinical use in cancer patients without affecting hemostasis as compared to low molecular weight heparins (LMWHs). We investigated anti-tumor effects, anti-angiogenesis effects, and mechanisms of S-NACH in a mouse model of pancreatic cancer as compared to the LMWH tinzaparin. S-NACH or tinzaparin with or without gemcitabine were administered, and tumor luminescent signal intensity, tumor weight, and histopathology were assessed at the termination of the study. S-NACH and LMWH efficiently inhibited tumor growth and metastasis, without any observed bleeding events with S-NACH as compared to tinzaparin. S-NACH distinctly increased tumor necrosis and enhanced gemcitabine response in the mouse pancreatic cancer models. These data suggest the potential implication of S-NACH as a neoadjuvant in pancreatic cancer.
Assuntos
Inibidores da Angiogênese/farmacologia , Fibrinolíticos/uso terapêutico , Heparina de Baixo Peso Molecular/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica , Neovascularização Patológica/tratamento farmacológico , Tinzaparina , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA, accounting for ~40,000 deaths annually. The dismal prognosis for PDAC is largely due to its late diagnosis. Currently, the most sensitive diagnosis of PDAC requires invasive procedures, such as endoscopic ultrasonography, which has inherent risks and accuracy that is highly operator dependent. Here we took advantage of a general characteristic of solid tumors, the acidic microenvironment that is generated as a by-product of metabolism, to develop a novel approach of using pH (Low) Insertion Peptides (pHLIPs) for imaging of PDAC. We show that fluorescently labeled pHLIPs can localize and specifically detect PDAC in human xenografts as well as PDAC and PanIN lesions in genetically engineered mouse models. This novel approach may improve detection, differential diagnosis and staging of PDAC.