RESUMO
BACKGROUND: Breast cancer is a heterogenous disease with several histological and molecular subtypes. Models that represent these subtypes are essential for translational research aimed at improving clinical strategy for targeted therapeutics. METHODS: Different combinations of genetic aberrations (Brca1 and Trp53 loss, and inhibition of proteins of the Rb family) were induced in the mammary gland by injection of adenovirus expressing Cre recombinase into the mammary ducts of adult genetically engineered mice. Mammary tumors with different genetic aberrations were classified into molecular subtypes based on expression of molecular markers and RNAseq analysis. In vitro potency assays and Western blots were used to examine their drug sensitivities. RESULTS: Induction of Brca1 and Trp53 loss in mammary ductal epithelium resulted in development of basal-like hormone receptor (HR)-negative mammary tumors. Inhibition of Rb and Trp53 loss or the combination of Rb, Trp53 and Brca1 aberrations resulted in development of luminal ductal carcinoma positive for ER, PR, and Her2 expression. HR positivity in tumors with Rb, Trp53 and Brca1 aberrations indicated that functionality of the Rb pathway rather than Brca1 status affected HR status in these models. Mammary tumor gene expression profiles recapitulated human basal-like or luminal B breast cancer signatures, but HR-positive luminal cancer models were endocrine resistant and exhibited upregulation of PI3K signaling and sensitivity to this pathway inhibition. Furthermore, both tumor subtypes were resistant to CDK4/6 inhibition. CONCLUSIONS: Examination of molecular expression profiles and drug sensitivities of tumors indicate that these breast cancer models can be utilized as a translational platform for evaluation of targeted combinations to improve chemotherapeutic response in patients that no longer respond to hormone therapy or that are resistant to CDK4/6 inhibition.
Assuntos
Neoplasias da Mama , Glândulas Mamárias Humanas , Neoplasias Mamárias Animais , Camundongos , Animais , Humanos , Feminino , Glândulas Mamárias Humanas/metabolismo , Fosfatidilinositol 3-Quinases , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias Mamárias Animais/patologia , Epitélio/metabolismo , Hormônios , Proteína BRCA1/genéticaRESUMO
Antibody-based therapies designed for human use frequently fail to cross-react with the murine isoform of their target. Because of this problem, preclinical studies of antibody-based mesothelin (Msl)-targeted therapeutics in immunocompetent systems have been limited by the lack of suitable mouse models. Here, we describe two immunocompetent humanized mesothelin transgenic mouse lines that can act as tolerant hosts for C57Bl/6-syngeneic cell lines expressing the human isoform of mesothelin. Thyroid peroxidase (TPO) mice have thyroid-restricted human mesothelin expression. Mesothelin (Msl) mice express human mesothelin in the typical serosal membrane distribution and can additionally be utilized to assess on-target, off-tumor toxicity of human mesothelin-targeted therapeutics. Both transgenic strains shed human mesothelin into the serum like human mesothelioma and patients with ovarian cancer, and serum human mesothelin can be used as a blood-based surrogate of tumor burden. Using these models, we examined the on-target toxicity and antitumor activity of human mesothelin-targeted recombinant immunotoxins. We found that immunotoxin treatment causes acute and chronic histologic changes to serosal membranes in Msl mice, while human mesothelin-expressing thyroid follicular cells in TPO mice are resistant to immunotoxin despite excellent drug delivery. Furthermore, poor delivery of immunotoxin to syngeneic orthotopic human mesothelin-expressing pancreatic adenocarcinoma limits antitumor activity both alone and in combination with immune checkpoint inhibition. In summary, we have developed two high-fidelity, immunocompetent murine models for human cancer that allow for rigorous preclinical evaluation of human mesothelin-targeted therapeutics.
Assuntos
Adenocarcinoma/terapia , Mesotelina/administração & dosagem , Mesotelioma/terapia , Neoplasias Pancreáticas/terapia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Apoptose , Proliferação de Células , Feminino , Engenharia Genética , Humanos , Masculino , Mesotelina/genética , Mesotelina/metabolismo , Mesotelioma/genética , Mesotelioma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGFß antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGFß inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGFß-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGFß inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGFß inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGFß inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGFß inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/imunologia , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Células Estromais/imunologia , Microambiente Tumoral , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proliferação de Células , Terapia Combinada , Desoxicitidina/farmacologia , Humanos , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for patients with pancreatic cancer and contributes to a high rate of recurrence. Oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. Here, we show that NRF2 contributed to chemoresistance and was associated with a poor prognosis in patients with pancreatic cancer. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. In addition, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for patients with pancreatic cancer harboring KRAS mutation. SIGNIFICANCE: These findings illuminate the mechanistic features of KRAS-mediated chemoresistance and provide a rationale for exploiting metabolic reprogramming in pancreatic cancer cells to confer therapeutic opportunities that could be translated into clinical trials. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/8/1630/F1.large.jpg.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Glutamina/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Glutaminase/antagonistas & inibidores , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Prognóstico , Distribuição Aleatória , Análise Serial de Tecidos , Regulação para Cima , GencitabinaRESUMO
PURPOSE: Metarrestin is a first-in-class small molecule clinical candidate capable of disrupting the perinucleolar compartment, a subnuclear structure unique to metastatic cancer cells. This study aims to define the pharmacokinetic (PK) profile of metarrestin and the pharmacokinetic/pharmacodynamic relationship of metarrestin-regulated markers. METHODS: PK studies included the administration of single or multiple dose of metarrestin at 3, 10, or 25 mg/kg via intravenous (IV) injection, gavage (PO) or with chow to wild-type C57BL/6 mice and KPC mice bearing autochthonous pancreatic tumors. Metarrestin concentrations were analyzed by UPLC-MS/MS. Pharmacodynamic assays included mRNA expression profiling by RNA-seq and qRT-PCR for KPC mice. RESULTS: Metarrestin had a moderate plasma clearance of 48 mL/min/kg and a large volume of distribution of 17 L/kg at 3 mg/kg IV in C57BL/6 mice. The oral bioavailability after single-dose (SD) treatment was > 80%. In KPC mice treated with SD 25 mg/kg PO, plasma AUC0-∞ of 14400 ng h/mL, Cmax of 810 ng/mL and half-life (t1/2) of 8.5 h were observed. At 24 h after SD of 25 mg/kg PO, the intratumor concentration of metarrestin was high with a mean value of 6.2 µg/g tissue (or 13 µM), well above the cell-based IC50 of 0.4 µM. At multiple dose (MD) 25 mg/kg/day PO in KPC mice, mean tissue/plasma AUC0-24h ratio for tumor, spleen and liver was 37, 30 and 31, respectively. There was a good linear relationship of dosage to AUC0-24h and C24h. AUC0-24h MD to AUC0-24h SD ratios ranged from two for liver to five for tumor indicating additional accumulation in tumors. Dose-dependent normalization of FOXA1 and FOXO6 mRNA expression was observed in KPC tumors. CONCLUSIONS: Metarrestin is an effective therapeutic candidate with a favorable PK profile achieving excellent intratumor tissue levels in a disease with known poor drug delivery.
Assuntos
Antineoplásicos/farmacocinética , Organelas/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Pirimidinas/farmacocinética , Pirróis/farmacocinética , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/sangue , Antineoplásicos/uso terapêutico , Área Sob a Curva , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fatores de Transcrição Forkhead/genética , Meia-Vida , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos , Organelas/metabolismo , Organelas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pirimidinas/administração & dosagem , Pirimidinas/sangue , Pirimidinas/uso terapêutico , Pirróis/administração & dosagem , Pirróis/sangue , Pirróis/uso terapêutico , Distribuição TecidualRESUMO
The majority of human high-grade serous epithelial ovarian cancer (SEOC) is characterized by frequent mutations in p53 and alterations in the RB and FOXM1 pathways. A subset of human SEOC harbors a combination of germline and somatic mutations as well as epigenetic dysfunction for BRCA1/2. Using Cre-conditional alleles and intrabursal induction by Cre-expressing adenovirus in genetically engineered mice, we analyzed the roles of pathway perturbations in epithelial ovarian cancer initiation and progression. Inactivation of RB-mediated tumor suppression induced surface epithelial proliferation with progression to stage I carcinoma. Additional biallelic inactivation and/or missense p53 mutation in the presence or absence of Brca1/2 caused progression to stage IV disease. As in human SEOC, mice developed peritoneal carcinomatosis, ascites, and distant metastases. Unbiased gene expression and metabolomic profiling confirmed that Rb, p53, and Brca1/2-triple mutant tumors aligned with human SEOC, and not with other intraperitoneal cancers. Together, our findings provide a novel resource for evaluating disease etiology and biomarkers, therapeutic evaluation, and improved imaging strategies in epithelial ovarian cancer.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Epitelial do Ovário , Modelos Animais de Doenças , Feminino , Deleção de Genes , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Mutação , Metástase Neoplásica , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for triacylglycerol (TAG) hydrolysis in adipocytes. The precise mechanisms whereby ATGL is regulated remain uncertain. Here, we demonstrate that a protein encoded by G(0)/G(1) switch gene 2 (G0S2) is a selective regulator of ATGL. G0S2 is highly expressed in adipose tissue and differentiated adipocytes. When overexpressed in HeLa cells, G0S2 localizes to lipid droplets and prevents their degradation mediated by ATGL. Moreover, G0S2 specifically interacts with ATGL through the hydrophobic domain of G0S2 and the patatin-like domain of ATGL. More importantly, interaction with G0S2 inhibits ATGL TAG hydrolase activity. Knockdown of endogenous G0S2 accelerates basal and stimulated lipolysis in adipocytes, whereas overexpression of G0S2 diminishes the rate of lipolysis in both adipocytes and adipose tissue explants. Thus, G0S2 functions to attenuate ATGL action both in vitro and in vivo and by this mechanism regulates TAG hydrolysis.
Assuntos
Adipócitos/enzimologia , Tecido Adiposo/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Ciclo Celular/genética , Lipase/metabolismo , Lipólise/genética , Adipócitos/ultraestrutura , Animais , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Lipídeos , Camundongos , Domínios e Motivos de Interação entre Proteínas , Vacúolos/metabolismoRESUMO
Numerous studies have implicated either the presence or absence of CD36 in the development of hypertension. In addition, hypercholesterolemia is associated with the loss of nitric oxide-induced vasodilation and the subsequent increase in blood pressure. In the current study, we tested the hypothesis that diet-induced hypercholesterolemia promotes the disruption of agonist-stimulated nitric oxide generation and vasodilation in a CD36-dependent manner. To test this, C57BL/6, apoE null, CD36 null, and apoE/CD36 null mice were maintained on chow or high fat diets. In contrast to apoE null mice fed a chow diet, apoE null mice fed a high fat diet did not respond to acetylcholine with a decrease in blood pressure. Caveolae isolated from in vivo vessels did not contain endothelial nitric-oxide synthase and were depleted of cholesterol. Age-matched apoE/CD36 null mice fed a chow or high fat diet responded to acetylcholine with a decrease in blood pressure. The mechanism underlying the vascular dysfunction was reversible because vessels isolated from apoE null high fat-fed mice regained responsiveness to acetylcholine when incubated with plasma obtained from chow-fed mice. Further analysis demonstrated that the plasma low density lipoprotein fraction was responsible for depleting caveolae of cholesterol, removing endothelial nitric-oxide synthase from caveolae, and preventing nitric oxide production. In addition, the pharmacological removal of caveola cholesterol with cyclodextrin mimicked the effects caused by the low density lipoprotein fraction. We conclude that the ablation of CD36 prevented the negative impact of hypercholesterolemia on agonist-stimulated nitric oxide-mediated vasodilation in apoE null mice. These studies provide a direct link between CD36 and the early events that underlie hypercholesterolemia-mediated hypertension and mechanistic linkages between CD36 function, nitric-oxide synthase activation, caveolae integrity, and blood pressure regulation.