RESUMO
TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.
Assuntos
Proteínas 14-3-3/genética , Proteínas Fetais/genética , Linfócitos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Tirosina Quinases/genética , Ubiquitina/genética , Proteínas 14-3-3/metabolismo , Células A549 , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas Fetais/antagonistas & inibidores , Proteínas Fetais/metabolismo , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Camundongos , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Poly(ADP-ribose) polymerases (PARPs) convert NAD to polymers of ADP-ribose that are converted to free ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG). The activation of the nuclear enzyme PARP-1 following genotoxic stress has been linked to release of apoptosis inducing factor from the mitochondria, but the mechanisms by which signals are transmitted between nuclear and mitochondrial compartments are not well understood. The study reported here has examined the relationship between PARG and mitochondria in HeLa cells. Endogenous PARG associated with the mitochondrial fraction migrated in the range of 60 kDa. Transient transfection of cells with PARG expression constructs with amino acids encoded by exon 4 at the N-terminus was targeted to the mitochondria as demonstrated by subcellular fractionation and immunofluorescence microscopy of whole cells. Deletion and missense mutants allowed identification of a canonical N-terminal mitochondrial targeting sequence consisting of the first 16 amino acids encoded by PARG exon 4. Sub-mitochondrial localization experiments indicate that this mitochondrial PARG isoform is targeted to the mitochondrial matrix. The identification of a PARG isoform as a component of the mitochondrial matrix raises several interesting possibilities concerning mechanisms of nuclear-mitochondrial cross talk involved in regulation of cell death pathways.
Assuntos
Glicosídeo Hidrolases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Substituição de Aminoácidos/fisiologia , Aminoácidos/fisiologia , Proteínas Reguladoras de Apoptose , Chaperonina 60/metabolismo , Digitonina/farmacologia , Endopeptidase K/metabolismo , Éxons/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/metabolismo , Mitocôndrias/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Transporte Proteico , Deleção de Sequência/fisiologia , TransfecçãoRESUMO
Effective treatment for AML is challenging due to the presence of clonal heterogeneity and the evolution of polyclonal drug resistance. Here, we report that TP-0903 has potent activity against protein kinases related to STAT, AKT, and ERK signaling, as well as cell cycle regulators in biochemical and cellular assays. In vitro and in vivo, TP-0903 was active in multiple models of drug-resistant FLT3 mutant AML, including those involving the F691L gatekeeper mutation and bone marrow microenvironment-mediated factors. Furthermore, TP-0903 demonstrated preclinical activity in AML models with FLT3-ITD and common co-occurring mutations in IDH2 and NRAS genes. We also showed that TP-0903 had ex vivo activity in primary AML cells with recurrent mutations including MLL-PTD, ASXL1, SRSF2, and WT1, which are associated with poor prognosis or promote clinical resistance to AML-directed therapies. Our preclinical studies demonstrate that TP-0903 is a multikinase inhibitor with potent activity against multiple drug-resistant models of AML that will have an immediate clinical impact in a heterogeneous disease like AML.
Assuntos
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Duplicação Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Camundongos , Camundongos Nus , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirimidinas/metabolismo , Sulfonamidas/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFß signaling and induced JAK1-STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1-STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFß, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFß, and JAK1-STAT3 signal activation in select tumors that may be targeted by combined AXL-JAK1 inhibition. SIGNIFICANCE: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Janus Quinase 1/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Estudos de Viabilidade , Feminino , Citometria de Fluxo/métodos , Humanos , Janus Quinase 1/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Nitrilas , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA-Seq , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única/métodos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
Purpose: The tumor microenvironment (TME) evolves to support tumor progression. One marker of more aggressive malignancy is hyaluronan (HA) accumulation. Here, we characterize biological and physical changes associated with HA-accumulating (HA-high) tumors.Experimental Design: We used immunohistochemistry, in vivo imaging of tumor pH, and microdialysis to characterize the TME of HA-high tumors, including tumor vascular structure, hypoxia, tumor perfusion by doxorubicin, pH, content of collagen. and smooth muscle actin (α-SMA). A novel method was developed to measure real-time tumor-associated soluble cytokines and growth factors. We also evaluated biopsies of murine and pancreatic cancer patients to investigate HA and collagen content, important contributors to drug resistance.Results: In immunodeficient and immunocompetent mice, increasing tumor HA content is accompanied by increasing collagen content, vascular collapse, hypoxia, and increased metastatic potential, as reflected by increased α-SMA. In vivo treatment of HA-high tumors with PEGylated recombinant human hyaluronidase (PEGPH20) dramatically reversed these changes and depleted stores of VEGF-A165, suggesting that PEGPH20 may also diminish the angiogenic potential of the TME. Finally, we observed in xenografts and in pancreatic cancer patients a coordinated increase in HA and collagen tumor content.Conclusions: The accumulation of HA in tumors is associated with high tIP, vascular collapse, hypoxia, and drug resistance. These findings may partially explain why more aggressive malignancy is observed in the HA-high phenotype. We have shown that degradation of HA by PEGPH20 partially reverses this phenotype and leads to depletion of tumor-associated VEGF-A165. These results encourage further clinical investigation of PEGPH20. Clin Cancer Res; 24(19); 4798-807. ©2018 AACR.
Assuntos
Carcinogênese/genética , Colágeno/metabolismo , Hialuronoglucosaminidase/administração & dosagem , Neoplasias/terapia , Animais , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Colágeno/genética , Humanos , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/genética , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ROCK, or Rho-associated coiled coil-containing protein kinase, is a member of the AGC kinase family and has been shown to play a role in cell migration, ECM synthesis, stress-fiber assembly, and cell contraction. Increased ROCK expression has been reported in multiple pathological conditions, including cancer. Here, we report increased expression of ROCK 1 in pancreatic tumor epithelial cells as well as in cancer associated fibroblasts (CAF). In our analysis, 62% of tumor samples exhibited ≥2+ in staining intensity by IHC analysis, versus 40% of adjacent normal tissue samples (P<0.0001). Thus, we hypothesized that ROCKs may play a significant role in pancreatic cancer progression, and may serve as a suitable target for treatment. We report a low frequency (4/34) amplification of the ROCK1 gene locus at chromosome 18q11.1 in pancreatic ductal adenocarcinoma (PDAC) patient tissue samples by aCGH analysis. Inhibition of ROCK kinase activity by a small molecule inhibitor (fasudil) resulted in moderate (IC50s of 6-71 µM) inhibition of PDAC cell proliferation, migration, and activation of co-cultured stellate cells. In the KPC mouse model for pancreatic cancer, fasudil decreased tumor collagen deposition. This translated to an enhanced overall survival of the mice and an increase in gemcitabine uptake. Though fasudil may target both the tumor epithelial cells and the CAFs, our findings are consistent with the hypothesis that inhibition of tumor stroma enhances drug penetration and efficacy in PDAC. Overall, our data suggests that ROCK1 may serve as a potential therapeutic target to enhance current treatment regimens for pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Células Estromais/patologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/genética , Hibridização Genômica Comparativa , Inativação Gênica , Humanos , Camundongos , Metástase Neoplásica , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , RNA Interferente Pequeno/genética , Quinases Associadas a rho/metabolismoRESUMO
Pancreatic cancer is the fourth leading cause of cancer death in the United States. The microenvironment of pancreatic cancer could be one of the "perfect storms" that support the growth of a cancer. Indeed, pancreatic cancer may be the poster child of a problem with the microenvironment. In this article, we review the rationale and attempts to date on modifying or targeting structural proteins in the microenvironment including hyaluronan (HA) (in primary and metastases), collagen, and SPARC (secreted protein, acidic, and rich in cysteine). Indeed, working in this area has produced a regimen that improves survival for patients with advanced pancreatic cancer (nab-paclitaxel + gemcitabine). In addition, in initial clinical trials, PEGylated hyaluronidase appears promising. We also review a new approach that is different than targeting/destroying the microenvironment and that is orchestrating, reengineering, reprogramming, or normalizing the microenvironment (including normalizing structural proteins, normalizing an immunologically tumor-friendly environment to a less friendly environment, reversing epithelial-to-mesenchymal transition, and so on). We believe this will be most effectively done by agents that have global effects on transcription. There is initial evidence that this can be done by agents such as vitamin D derivatives and other new agents. There is no doubt these opportunities can now be tried in the clinic with hopefully beneficial effects.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Microambiente Tumoral , Albuminas/administração & dosagem , Colágeno , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Transição Epitelial-Mesenquimal , Fibrose , Humanos , Ácido Hialurônico , Hialuronoglucosaminidase/administração & dosagem , Terapia de Alvo Molecular , Osteonectina , Paclitaxel/administração & dosagem , Polietilenoglicóis , Taxa de Sobrevida , GencitabinaRESUMO
PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is characterized by high levels of fibrosis, termed desmoplasia, which is thought to hamper the efficacy of therapeutics treating PDAC. Our primary focus was to evaluate differences in the extent of desmoplasia in primary tumors and metastatic lesions. As metastatic burden is a primary cause for mortality in PDAC, the extent of desmoplasia in metastases may help to determine whether desmoplasia targeting therapeutics will benefit patients with late-stage, metastatic disease. EXPERIMENTAL DESIGN: We sought to assess desmoplasia in metastatic lesions of PDAC and compare it with that of primary tumors. Fifty-three patients' primaries and 57 patients' metastases were stained using IHC staining techniques. RESULTS: We observed a significant negative correlation between patient survival and extracellular matrix deposition in primary tumors. Kaplan-Meier curves for collagen I showed median survival of 14.6 months in low collagen patients, and 6.4 months in high-level patients (log rank, P < 0.05). Low-level hyaluronan patients displayed median survival times of 24.3 months as compared with 9.3 months in high-level patients (log rank, P < 0.05). Our analysis also indicated that extracellular matrix components, such as collagen and hyaluronan, are found in high levels in both primary tumors and metastatic lesions. The difference in the level of desmoplasia between primary tumors and metastatic lesions was not statistically significant. CONCLUSIONS: Our results suggest that both primary tumors and metastases of PDAC have highly fibrotic stroma. Thus, stromal targeting agents have the potential to benefit PDAC patients, even those with metastatic disease.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Matriz Extracelular/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/patologia , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Intervalo Livre de Doença , Matriz Extracelular/patologia , Feminino , Humanos , Ácido Hialurônico/metabolismo , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Análise Serial de TecidosRESUMO
Adenosquamous carcinoma of the pancreas (ASCP) is a rare entity. Like adenocarcinoma of the pancreas, overall survival is poor. Characteristics of ASCP include central tumor necrosis, along with osteoclasts and hypercalcemia. Various theories exist as to why this histological subtype exists, as normal pancreas tissue has no benign squamous epithelium. Due to the rarity of this disease, limited molecular analysis has been performed, and those reports indicate unique molecular features of ASCP. In this paper, we characterize 23 patients diagnosed with ASCP through molecular profiling using immunohistochemistry staining, fluorescent in situ hybridization, chromogenic in situ hybridization, and gene sequencing, Additionally, we provide a comprehensive literature review of what is known to date of ASCP. Molecular characterization revealed overexpression in MRP1 (80%), MGMT (79%), TOP2A (75), RRM1 (42%), TOPO1 (42%), PTEN (45%), CMET (40%), and C-KIT (10%) among others. One hundred percent of samples tested were positive for KRAS mutations. This analysis shows heretofore unsuspected leads to be considered for treatments of this rare type of exocrine pancreas cancer. Molecular profiling may be appropriate to provide maximum information regarding the patient's tumor. Further work should be pursued to better characterize this disease.
RESUMO
Increased extracellular matrix (ECM) deposition is a characteristic observed in many solid tumors. Increased levels of one ECM component-namely, hyaluronan (HA)-leads to reduced elasticity of tumor tissue and increased interstitial fluid pressure. Multiple initial reports showed that the addition of hyaluronidase (HYAL) to chemotherapeutic regimens could greatly improve efficacy. Unfortunately, the bovine HYAL used in those studies was limited therapeutically by immunologic responses to treatment. Newly developed recombinant human HYAL has recently been introduced into clinical trials. In this article, we describe the role of HA in cancer, methods of targeting HA, and clinical studies performed to date, and we propose that targeting HA could now be an effective treatment option for patients with many different types of solid tumors.
Assuntos
Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Animais , Bovinos , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Humanos , Hialuronoglucosaminidase/farmacologia , Terapia de Alvo Molecular/métodos , Neoplasias/enzimologia , Neoplasias/patologia , Proteínas Recombinantes/farmacologiaRESUMO
Poly(ADP-ribose)glycohydrolase (PARG) is the major enzyme capable of rapidly hydrolyzing poly(ADP-ribose) (PAR) formed by the diverse members of the PARP enzyme family. This study presents an alternative splice mechanism by which two novel PARG protein isoforms of 60 kDa and 55 kDa are expressed from the human PARG gene, termed hPARG60 and hPARG55, respectively. Homologous forms were found in the mouse (mPARG63 and mPARG58) supporting the hypothesis that expression of small PARG isoforms is conserved among mammals. A PARG protein of approximately 60 kDa has been described for decades but with its genetic basis unknown, it was hypothesized to be a product of posttranslational cleavage of larger PARG isoforms. While this is not excluded entirely, isolation and expression of cDNA clones from different sources of RNA indicate that alternative splicing leads to expression of a catalytically active hPARG60 in multiple cell compartments. A second enzyme, hPARG55, that can be expressed through alternative translation initiation from hPARG60 transcripts is strictly targeted to the mitochondria. Functional studies of a mitochondrial targeting signal (MTS) in PARG exon IV suggest that hPARG60 may be capable of shuttling between nucleus and mitochondria, which would be in line with a proposed function of PAR in genotoxic stress-dependent, nuclear-mitochondrial crosstalk.