RESUMO
UNLABELLED: The hypoxia-inducible factor (HIF), HIF-1, is a central regulator of the response to low oxygen or inflammatory stress and plays an essential role in survival and function of immune cells. However, the mechanisms regulating nonhypoxic induction of HIF-1 remain unclear. Here, we assess the impact of germline heterozygosity of a novel, oxygen-independent ubiquitin ligase for HIF-1α: hypoxia-associated factor (HAF; encoded by SART1). SART1(-/-) mice were embryonic lethal, whereas male SART1(+/-) mice spontaneously recapitulated key features of nonalcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC), including steatosis, fibrosis, and inflammatory cytokine production. Male, but not female, SART1(+/-) mice showed significant up-regulation of HIF-1α in circulating and liver-infiltrating immune cells, but not in hepatocytes, before development of malignancy. Additionally, Kupffer cells derived from male, but not female, SART1(+/-) mice produced increased levels of the HIF-1-dependent chemokine, regulated on activation, normal T-cell expressed and secreted (RANTES), compared to wild type. This was associated with increased liver-neutrophilic infiltration, whereas infiltration of lymphocytes and macrophages were not significantly different. Neutralization of circulating RANTES decreased liver neutrophilic infiltration and attenuated HCC tumor initiation/growth in SART1(+/-) mice. CONCLUSION: This work establishes a new tumor-suppressor role for HAF in immune cell function by preventing inappropriate HIF-1 activation in male mice and identifies RANTES as a novel therapeutic target for NASH and NASH-driven HCC.
Assuntos
Carcinoma Hepatocelular/etiologia , Quimiocina CCL5/fisiologia , Haploinsuficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Hepáticas/etiologia , Transativadores/genética , Animais , Ácidos Graxos/metabolismo , Fígado Gorduroso/etiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Ribonucleoproteínas Nucleares PequenasRESUMO
BACKGROUND: Worldwide, gastric cancer (GC) is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. OBJECTIVE: To determine oncogenic mechanisms and novel therapeutic targets specific for GC by identifying commonly dysregulated genes from the tumours of both Asian-Pacific and Caucasian patients. METHODS: We generated transcriptomic profiles of 22 Caucasian GC tumours and their matched non-cancerous samples and performed an integrative analysis across different GC gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signalling pathways by RNAi and/or pharmacological inhibition. RESULTS: Hepatocyte nuclear factor-4α (HNF4α) upregulation was a key signalling event in gastric tumours from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumour cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signalling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest and tumour growth inhibition. HNF4α also regulated WNT signalling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumours. CONCLUSIONS: Our results indicate that HNF4α is a targetable oncoprotein in GC, is regulated by AMPK signalling through AMPKα and resides upstream of WNT signalling. HNF4α may regulate 'metabolic switch' characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signalling cascade represents a potentially targetable pathway for drug development.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias Gástricas/genética , Proteínas Wnt/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adenocarcinoma/etnologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Povo Asiático , Biomarcadores Tumorais/metabolismo , Western Blotting , Estudos de Casos e Controles , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Proteínas Proto-Oncogênicas/metabolismo , Distribuição Aleatória , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/etnologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Regulação para Cima , População Branca , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Proteína Wnt-5aRESUMO
The phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascade is an important component of the insulin signaling in normal tissues leading to glucose uptake and homeostasis and for cell survival signaling in cancer cells. Hyperglycemia is an on-target side effect of many inhibitors of PI3K/Akt signaling including the specific PI3K inhibitor PX-866. The peroxisome proliferator-activated receptor gamma agonist pioglitazone, used to treat type 2 diabetes, prevents a decrease in glucose tolerance caused by acute administration of PX-866. Our studies have shown that pioglitazone does not inhibit the antitumor activity of PX-866 in A-549 non-small cell lung cancer and HT-29 colon cancer xenografts. In vitro studies also showed that pioglitazone increases 2-[1-(14)C]deoxy-D-glucose uptake in L-6 muscle cells and prevents inhibition of 2-deoxyglucose uptake by PX-866. Neither pioglitazone nor PX-866 had an effect on 2-deoxyglucose uptake in A-549 lung cancer cells. In vivo imaging studies using [18F]2-deoxyglucose (FDG) positron emission tomography showed that pioglitazone increases FDG accumulation by normal tissue but does not significantly alter FDG uptake by A-549 xenografts. Thus, peroxisome proliferator-activated receptor gamma agonists may be useful in overcoming the increase in blood glucose caused by inhibitors of PI3K signaling by preventing the inhibition of normal tissue insulin-mediated glucose uptake without affecting antitumor activity.
Assuntos
Gonanos/farmacologia , Hiperglicemia/enzimologia , Hiperglicemia/prevenção & controle , PPAR gama/agonistas , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Progressão da Doença , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , PPAR gama/metabolismo , Pioglitazona , Tiazolidinedionas/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hypoxia inducible factor-1 (HIF-1) promotes tumor cell adaptation to microenvironmental stress. HIF-1 is up-regulated in irradiated tumors and serves as a promising target for radiosensitization. We initially confirmed that the orally bioavailable HIF-1 inhibitor PX-478 reduces HIF-1 protein levels and signaling in vitro in a dose-dependent manner and provides direct radiosensitization of hypoxic cancer cells in clonogenic survival assays using C6 glioma, HN5 and UMSCCa10 squamous cells, and Panc-1 pancreatic adenocarcinoma cell lines. However, PX-478 yields striking in vivo tumor sensitization to single-dose irradiation, which cannot be explained by incremental improvement in direct tumor cell killing. We show that PX-478 prevents postradiation HIF-1 signaling and abrogates downstream stromal adaptation in C6 and HN5 reporter xenografts as measured by serial ultrasound, vascular magnetic resonance imaging, and hypoxia response element-specific micro-positron emission tomography imaging. The primacy of indirect PX-478 in vivo effects was corroborated by our findings that (a) either concurrent or early postradiation sequencing of PX-478 provides roughly equivalent sensitization and (b) constitutive vascular endothelial growth factor expression maintains refractory tumor vessel function and progression following combined radiation and PX-478. These results confirm that disruption of postradiation adaptive HIF-1 signaling by PX-478 imparts increased therapeutic efficacy through blockade of HIF-1-dependent reconstitution of tumor stromal function. Successful translation of targeted HIF-1 radiosensitization to the clinical setting will require specific consideration of tumor microenvironmental effects and mechanisms.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Compostos de Mostarda/farmacologia , Fenilpropionatos/farmacologia , Células Estromais/efeitos da radiação , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/radioterapia , Animais , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/radioterapia , Hipóxia Celular/efeitos da radiação , Ensaio de Imunoadsorção Enzimática , Glioma/metabolismo , Glioma/patologia , Glioma/radioterapia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas Imunoenzimáticas , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/radioterapia , Tomografia por Emissão de Pósitrons , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Fator A de Crescimento do Endotélio Vascular/metabolismo , Irradiação Corporal TotalRESUMO
Cancer cells respond to hypoxia by upregulating the hypoxia-inducible factor 1α (HIF1A) transcription factor, which drives survival mechanisms that include metabolic adaptation and induction of angiogenesis by VEGF. Pancreatic tumors are poorly vascularized and severely hypoxic. To study the angiogenic role of HIF1A, and specifically probe whether tumors are able to use alternative pathways in its absence, we created a xenograft mouse tumor model of pancreatic cancer lacking HIF1A. After an initial delay of about 30 days, the HIF1A-deficient tumors grew as rapidly as the wild-type tumors and had similar vascularization. These changes were maintained in subsequent passages of tumor xenografts in vivo and in cell lines ex vivo. There were many cancer cells with a "clear-cell" phenotype in the HIF1A-deficient tumors; this was the result of accumulation of glycogen. Single-cell RNA sequencing (scRNA-seq) of the tumors identified hypoxic cancer cells with inhibited glycogen breakdown, which promoted glycogen accumulation and the secretion of inflammatory cytokines, including interleukins 1ß (IL1B) and 8 (IL8). scRNA-seq of the mouse tumor stroma showed enrichment of two subsets of myeloid dendritic cells (cDC), cDC1 and cDC2, that secreted proangiogenic cytokines. These results suggest that glycogen accumulation associated with a clear-cell phenotype in hypoxic cancer cells lacking HIF1A can initiate an alternate pathway of cytokine and DC-driven angiogenesis. Inhibiting glycogen accumulation may provide a treatment for cancers with the clear-cell phenotype. SIGNIFICANCE: These findings establish a novel mechanism by which tumors support angiogenesis in an HIF1α-independent manner.
Assuntos
Proliferação de Células/fisiologia , Glicogênio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Neovascularização Patológica/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Linhagem Celular Tumoral , Hipóxia/metabolismo , Hipóxia/patologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Patológica/patologia , Pâncreas/metabolismo , Pâncreas/patologia , Transdução de Sinais/fisiologiaRESUMO
Cnk1 (connector enhancer of kinase suppressor of Ras 1) is a pleckstrin homology (PH) domain-containing scaffold protein that increases the efficiency of Ras signaling pathways, imparting efficiency and specificity to the response of cell proliferation, survival, and migration. Mutated KRAS (mut-KRAS) is the most common proto-oncogenic event, occurring in approximately 25% of human cancers and has no effective treatment. In this study, we show that selective inhibition of Cnk1 blocks growth and Raf/Mek/Erk, Rho and RalA/B signaling in mut-KRAS lung and colon cancer cells with little effect on wild-type (wt)-KRAS cells. Cnk1 inhibition decreased anchorage-independent mut-KRas cell growth more so than growth on plastic, without the partial "addiction" to mut-KRAS seen on plastic. The PH domain of Cnk1 bound with greater affinity to PtdIns(4,5)P2 than PtdIns(3,4,5)P3, and Cnk1 localized to areas of the plasma membranes rich in PtdIns, suggesting a role for the PH domain in the biological activity of Cnk1. Through molecular modeling and structural modification, we identified a compound PHT-7.3 that bound selectively to the PH domain of Cnk1, preventing plasma membrane colocalization with mut-KRas. PHT-7.3 inhibited mut-KRas, but not wild-type KRas cancer cell and tumor growth and signaling. Thus, the PH domain of Cnk1 is a druggable target whose inhibition selectively blocks mutant KRas activation, making Cnk1 an attractive therapeutic target in patients with mut-KRAS-driven cancer. SIGNIFICANCE: These findings identify a therapeutic strategy to selectively block oncogenic KRas activity through the PH domain of Cnk1, which reduces its cell membrane binding, decreasing the efficiency of Ras signaling and tumor growth.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Domínios de Homologia à Plecstrina , Células Tumorais CultivadasRESUMO
The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here, we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional coactivator p300. Notably, of the HIF1α-activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity, and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small-molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cells, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors. Cancer Res; 76(14); 4259-69. ©2016 AACR.
Assuntos
Frutose-Bifosfato Aldolase/antagonistas & inibidores , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias/tratamento farmacológico , Transdução de Sinais/fisiologia , Proteínas Quinases Ativadas por AMP/fisiologia , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Proteína p300 Associada a E1A/fisiologia , Humanos , Camundongos , Neoplasias/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Clear-cell renal cell cancer (CRCC) is initiated typically by loss of the tumor-suppressor VHL, driving constitutive activation of hypoxia-inducible factor-1 (HIF1) and HIF2. However, whereas HIF1 has a tumor-suppressor role, HIF2 plays a distinct role in driving CRCC. In this study, we show that the HIF1α E3 ligase hypoxia-associated factor (HAF) complexes with HIF2α at DNA to promote HIF2-dependent transcription through a mechanism relying upon HAF SUMOylation. HAF SUMOylation was induced by hypoxia, whereas HAF-mediated HIF1α degradation was SUMOylation independent. HAF overexpression in mice increased CRCC growth and metastasis. Clinically, HAF overexpression was associated with poor prognosis. Taken together, our results show that HAF is a specific mediator of HIF2 activation that is critical for CRCC development and morbidity.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias Renais/metabolismo , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/genética , Proteínas de Transporte/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Renais/enzimologia , Neoplasias Renais/genética , Camundongos , Camundongos Nus , Ribonucleoproteínas Nucleares Pequenas , Sumoilação , Transativadores/genética , Ativação Transcricional , Ubiquitina-Proteína Ligases/genéticaRESUMO
PURPOSE: Vemurafenib, a selective inhibitor of BRAF(V600), has shown significant activity in BRAF(V600) melanoma but not in less than 10% of metastatic BRAF(V600) colorectal cancers (CRC), suggesting that studies of the unique hypermethylated phenotype and concurrent oncogenic activation of BRAF(mut) CRC may provide combinatorial strategies. EXPERIMENTAL DESIGN: We conducted comparative proteomic analysis of BRAF(V600E) melanoma and CRC cell lines, followed by correlation of phosphoinositide 3-kinase (PI3K) pathway activation and sensitivity to the vemurafenib analogue PLX4720. Pharmacologic inhibitors and siRNA were used in combination with PLX4720 to inhibit PI3K and methyltransferase in cell lines and murine models. RESULTS: Compared with melanoma, CRC lines show higher levels of PI3K/AKT pathway activation. CRC cell lines with mutations in PTEN or PIK3CA were less sensitive to growth inhibition by PLX4720 (P = 0.03), and knockdown of PTEN expression in sensitive CRC cells reduced growth inhibition by the drug. Combined treatment of PLX4720 with PI3K inhibitors caused synergistic growth inhibition in BRAF-mutant CRC cells with both primary and secondary resistance. In addition, methyltransferase inhibition was synergistic with PLX4720 and decreased AKT activation. In vivo, PLX4720 combined with either inhibitors of AKT or methyltransferase showed greater tumor growth inhibition than PLX4720 alone. Clones with acquired resistance to PLX4720 in vitro showed PI3K/AKT activation with EGF receptor (EGFR) or KRAS amplification. CONCLUSIONS: We show that activation of the PI3K/AKT pathway is a mechanism of both innate and acquired resistance to BRAF inhibitors in BRAF(V600E) CRC and suggest combinatorial approaches to improve outcomes in this poor prognosis subset of patients.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Indóis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Animais , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Humanos , Metilação/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , PTEN Fosfo-Hidrolase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , VemurafenibRESUMO
Compared with xenografts from previously established cell lines, patient-derived xenografts may more faithfully recapitulate the molecular diversity, cellular heterogeneity, and histology seen in patient tumors, although other limitations of murine models remain. The ability of these models to inform clinical development and answer mechanistic questions will determine their ultimate use.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Feminino , Humanos , MasculinoRESUMO
Most solid tumors and their metastases experience periods of low oxygen or hypoxia, which is of major clinical significance as it promotes both tumor progression and resistance to therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1α and HIF-2α. The HIFs are nonredundant and regulate both overlapping and unique downstream target genes. Here, we describe a novel mechanism for the switch between HIF-1α- and HIF-2α-dependent transcription during tumor hypoxia caused by the hypoxia associated factor (HAF). HAF is overexpressed in a variety of tumors and its levels are decreased during acute hypoxia, but increased following prolonged hypoxia. We have previously identified HAF as an E3 ubiquitin ligase that binds and ubiquitinates HIF-1α by an oxygen and pVHL-independent mechanism, thus targeting HIF-1α for proteasomal degradation. Here, we show that HAF also binds to HIF-2α, but at a different site than HIF-1α, and increases HIF-2α transactivation without causing its degradation. HAF, thus, switches the hypoxic response of the cancer cell from HIF-1α-dependent to HIF-2α-dependent transcription and activates genes involved in invasion such as MMP9, PAI-1, and the stem cell factor OCT-3/4. The switch to HIF-2α-dependent gene expression caused by HAF also promotes an enriched tumor stem cell population, resulting in highly aggressive tumors in vivo. Thus, HAF, by causing a switch from a HIF-1α- to HIF-2α-dependent response to hypoxia, provides a mechanism for more aggressive growth of tumors under prolonged hypoxia.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteínas de Transporte/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Neoplasias/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Nus , Análise em Microsséries , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/patologia , Ribonucleoproteínas Nucleares Pequenas , Transdução de Sinais , TransfecçãoRESUMO
PURPOSE: Hypoxia-inducible factor 1 (HIF-1) promotes cancer cell survival and tumor progression. The specific role played by HIF-1 and tumor-stromal interactions toward determining tumor resistance to radiation treatment remains undefined. We applied a multimodality preclinical imaging platform to mechanistically characterize tumor response to radiation, with a focus on HIF-1-dependent resistance pathways. METHODS: C6 glioma and HN5 human squamous carcinoma cells were stably transfected with a dual HIF-1 signaling reporter construct (dxHRE-tk/eGFP-cmvRed2XPRT). Reporter cells were serially interrogated in vitro before and after irradiation as monolayer and multicellular spheroid cultures and as subcutaneous xenografts in nu/nu mice. RESULTS: In vitro, single-dose irradiation of C6 and HN5 reporter cells modestly impacted HIF-1 signaling in normoxic monolayers and inhibited HIF-1 signaling in maturing spheroids. In contrast, irradiation of C6 or HN5 reporter xenografts with 8 Gy in vivo elicited marked upregulation of HIF-1 signaling and downstream proangiogenic signaling at 48 hours which preceded recovery of tumor growth. In situ ultrasound imaging and dynamic contrast-enhanced (DCE) MRI indicated that HIF-1 signaling followed acute disruption of stromal vascular function. High-resolution positron emission tomography and dual-contrast DCE-MRI of immobilized dorsal skin window tumors confirmed postradiotherapy HIF-1 signaling to spatiotemporally coincide with impaired stromal vascular function. Targeted disruption of HIF-1 signaling established this pathway to be a determinant of tumor radioresistance. CONCLUSIONS: Our results illustrate that tumor radioresistance is mediated by a capacity to compensate for stromal vascular disruption through HIF-1-dependent proangiogenic signaling and that clinically relevant vascular imaging techniques can spatially define mechanisms associated with tumor irradiation.
Assuntos
Fator 1 Induzível por Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/efeitos da radiação , Isquemia/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/radioterapia , Tolerância a Radiação/fisiologia , Fatores de Crescimento do Endotélio Vascular/efeitos da radiação , Adaptação Fisiológica , Animais , Hipóxia Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Humanos , Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Nus , Neoplasias/patologia , Tomografia por Emissão de Pósitrons/mortalidade , Ratos , Esferoides Celulares/efeitos da radiação , Transplante Heterólogo , Carga Tumoral/efeitos da radiação , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Growing tumors are hypoxic and respond to microenvironmental stress through increased expression of the hypoxia inducible factor-1alpha (HIF-1alpha) transcription factor, resulting in an adaptive switch to glycolytic metabolism, angiogenic signaling, survival, and metastasis. HIF-1alpha expression is associated with tumor resistance to cytotoxic therapy and inferior patient outcomes. Pancreatic cancer is the most hypoxic of all solid tumors and remains refractory to current chemoradiotherapy. We have seen nuclear HIF-1alpha in 88% of human pancreatic ductal carcinoma but in only 16% of normal pancreas. Stroma adjacent to the pancreatic ductal carcinoma also showed HIF-1alpha in 43% of cases. We investigated the novel selective HIF-1alpha inhibitor PX-478 on in vitro and in vivo radiation response of human pancreatic cancer models. Inhibition of HIF-1alpha by PX-478 increased cell killing by radiation. In mice with Panc-1, CF-PAC-1, or SU.86.86 pancreatic xenografts, concurrent administration of PX-478 potentiated the antitumor effects of fractionated radiation, with or without combined treatment with 5-fluorouracil or gemcitabine. Alternative sequencing of PX-478 with fractionated radiotherapy suggests optimal radiosensitization with concurrent or neoadjuvant administration of drug. Early tumor responses to combined PX-478/radiation treatment could be rapidly and repeatedly quantified by vascular imaging biomarkers. Dual-tracer dynamic contrast enhanced-magnetic resonance imaging and ultrasound imaging discriminated response to combined treatment prior to detection of differences in anatomic tumor size at 10 days posttreatment. Therefore, PX-478 is a mechanistically appealing and potentially clinically relevant enhancer of pancreatic cancer radiosensitivity, inhibiting tumor and stromal HIF-1 proangiogenic signaling and reducing the innate radiation resistance of hypoxic tumor cells.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Compostos de Mostarda/farmacologia , Neoplasias Pancreáticas/terapia , Fenilpropionatos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Western Blotting , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Camundongos , Camundongos SCID , Compostos de Mostarda/administração & dosagem , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fenilpropionatos/administração & dosagem , Radioterapia/métodos , Análise Serial de Tecidos , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/efeitos da radiaçãoRESUMO
Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non-small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity.
Assuntos
Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Proteína Oncogênica v-akt/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapêutico , Tiadiazóis/farmacocinética , Tiadiazóis/uso terapêutico , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Antineoplásicos/efeitos adversos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Modelos Biológicos , Proteína Oncogênica v-akt/química , Proteína Oncogênica v-akt/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A new approach is described for delivering small interfering RNA (siRNA) into cancer cells by noncovalently complexing unmodified siRNA with pristine single-walled carbon nanotubes (SWCNTs). The complexes were prepared by simple sonication of pristine SWCNTs in a solution of siRNA, which then served both as the cargo and as the suspending agent for the SWCNTs. When complexes containing siRNA targeted to hypoxia-inducible factor 1 alpha (HIF-1α) were added to cells growing in serum containing culture media, there was strong specific inhibition of cellular HIF-1α activity. The ability to obtain a biological response to SWCNT/siRNA complexes was seen in a wide variety of cancer cell types. Moreover, intratumoral administration of SWCNT-HIF-1α siRNA complexes in mice bearing MiaPaCa-2/HRE tumors significantly inhibited the activity of tumor HIF-1α. As elevated levels of HIF-1α are found in many human cancers and are associated with resistance to therapy and decreased patient survival, these results imply that SWCNT/siRNA complexes may have value as therapeutic agents.
RESUMO
The novel phosphatidylinositol-3-kinase (PI3K) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI3K (PIK3CA) and loss of PTEN activity were sufficient, but not necessary, as predictors of sensitivity to the antitumor activity of the PI3K inhibitor PX-866 in the presence of wild-type Ras, whereas mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI3K signaling measured by tumor phosphorylated Ser(473)-Akt was insufficient to predict in vivo antitumor response to PX-866. Reverse-phase protein array revealed that the Ras-dependent downstream targets c-Myc and cyclin B were elevated in cell lines resistant to PX-866 in vivo. Studies using an H-Ras construct to constitutively and preferentially activate the three best-defined downstream targets of Ras, i.e., Raf, RalGDS, and PI3K, showed that mutant Ras mediates resistance through its ability to use multiple pathways for tumorigenesis. The identification of Ras and downstream signaling pathways driving resistance to PI3K inhibition might serve as an important guide for patient selection as inhibitors enter clinical trials and for the development of rational combinations with other molecularly targeted agents.
Assuntos
Gonanos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Fosfatidilinositol 3-Quinases/genética , Proteínas ras/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Genes ras , Humanos , Camundongos , Camundongos SCID , Mutação , Neoplasias/genética , Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/metabolismo , Fator ral de Troca do Nucleotídeo Guanina/metabolismo , Proteínas ras/genéticaRESUMO
The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties.