RESUMO
There is an unmet need for better therapeutic strategies for advanced prostate cancer. Poly (ADP-ribose) polymerase-1 (PARP-1) is a chromatin-binding DNA repair enzyme overexpressed in prostate cancer. This study evaluates whether PARP-1, on account of its proximity to the cell's DNA, would be a good target for delivering high-linear energy transfer Auger radiation to induce lethal DNA damage in prostate cancer cells. We analyzed the correlation between PARP-1 expression and Gleason score in a prostate cancer tissue microarray. A radio-brominated Auger emitting inhibitor ([77Br]Br-WC-DZ) targeting PARP-1 was synthesized. The ability of [77Br]Br-WC-DZ to induce cytotoxicity and DNA damage was assessed in vitro. The antitumor efficacy of [77Br]Br-WC-DZ was investigated in prostate cancer xenograft models. PARP-1 expression was found to be positively correlated with the Gleason score, thus making it an attractive target for Auger therapy in advanced diseases. The Auger emitter, [77Br]Br-WC-DZ, induced DNA damage, G2-M cell cycle phase arrest, and cytotoxicity in PC-3 and IGR-CaP1 prostate cancer cells. A single dose of [77Br]Br-WC-DZ inhibited the growth of prostate cancer xenografts and improved the survival of tumor-bearing mice. Our studies establish the fact that PARP-1 targeting Auger emitters could have therapeutic implications in advanced prostate cancer and provides a strong rationale for future clinical investigation.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , Neoplasias da Próstata , Animais , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Dano ao DNA , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias da Próstata/metabolismo , Radioisótopos/uso terapêuticoRESUMO
The bone marrow environment is among the most hypoxic in the body, but how hypoxia affects bone formation is not known. Because low oxygen tension stabilizes hypoxia-inducible factor alpha (HIFα) proteins, we have investigated the effect of expressing a stabilized form of HIF1α in osteoblast precursors. Brief stabilization of HIF1α in SP7-positive cells in postnatal mice dramatically stimulated cancellous bone formation via marked expansion of the osteoblast population. Remarkably, concomitant deletion of vascular endothelial growth factor A (VEGFA) in the mouse did not diminish bone accrual caused by HIF1α stabilization. Thus, HIF1α-driven bone formation is independent of VEGFA up-regulation and increased angiogenesis. On the other hand, HIF1α stabilization stimulated glycolysis in bone through up-regulation of key glycolytic enzymes including pyruvate dehydrogenase kinase 1 (PDK1). Pharmacological inhibition of PDK1 completely reversed HIF1α-driven bone formation in vivo. Thus, HIF1α stimulates osteoblast formation through direct activation of glycolysis, and alterations in cellular metabolism may be a broadly applicable mechanism for regulating cell differentiation.
Assuntos
Glicólise/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Osteogênese/fisiologia , Regulação para Cima , Animais , Western Blotting , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Hipóxia Celular , Feminino , Glicólise/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
WNT signaling has been implicated in both embryonic and postnatal bone formation. However, the pertinent WNT ligands and their downstream signaling mechanisms are not well understood. To investigate the osteogenic capacity of WNT7B and WNT5A, both normally expressed in the developing bone, we engineered mouse strains to express either protein in a Cre-dependent manner. Targeted induction of WNT7B, but not WNT5A, in the osteoblast lineage dramatically enhanced bone mass due to increased osteoblast number and activity; this phenotype began in the late-stage embryo and intensified postnatally. Similarly, postnatal induction of WNT7B in Runx2-lineage cells greatly stimulated bone formation. WNT7B activated mTORC1 through PI3K-AKT signaling. Genetic disruption of mTORC1 signaling by deleting Raptor in the osteoblast lineage alleviated the WNT7B-induced high-bone-mass phenotype. Thus, WNT7B promotes bone formation in part through mTORC1 activation.
Assuntos
Complexos Multiproteicos/genética , Osteogênese/genética , Proteínas Proto-Oncogênicas/genética , Serina-Treonina Quinases TOR/genética , Proteínas Wnt/genética , Animais , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/biossíntese , Osteoblastos/citologia , Proteínas Proto-Oncogênicas/biossíntese , Serina-Treonina Quinases TOR/biossíntese , Proteínas Wnt/biossíntese , Via de Sinalização Wnt , Proteína Wnt-5aRESUMO
BACKGROUND: Gene therapy for inherited serum deficiency disorders has previously been limited by the balance between obtaining adequate expression and causing hepatic toxicity. Our group has previously described modifications of a replication deficient human adenovirus serotype 5 that increase pulmonary vasculature transgene expression. METHODS: In the present study, we use a modified pulmonary targeted adenovirus to express human alpha-1 antitrypsin (A1AT) in C57BL/6 J mice. RESULTS: Using the targeted adenovirus, we were able to achieve similar increases in serum A1AT levels with less liver viral uptake. We also increased pulmonary epithelial lining fluid A1AT levels by more than an order of magnitude compared to that of untargeted adenovirus expressing A1AT in a mouse model. These gains are achieved along with evidence of decreased systemic inflammation and no evidence for increased inflammation within the vector-targeted end organ. CONCLUSIONS: In addition to comprising a step towards clinically viable gene therapy for A1AT, maximization of protein production at the site of action represents a significant technical advancement in the field of systemically delivered pulmonary targeted gene therapy. It also provides an alternative to the previous limitations of hepatic viral transduction and associated toxicities.
Assuntos
Endotélio Vascular/metabolismo , Marcação de Genes/métodos , Terapia Genética/métodos , Pulmão/irrigação sanguínea , Deficiência de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/genética , Adenovírus Humanos , Animais , Modelos Animais de Doenças , Feminino , Marcação de Genes/efeitos adversos , Técnicas de Transferência de Genes/efeitos adversos , Vetores Genéticos/efeitos adversos , Células HEK293 , Humanos , Inflamação/etiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transgenes , alfa 1-Antitripsina/sangueRESUMO
Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.
Assuntos
Adenoviridae/genética , Endotélio Vascular/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/fisiologia , Receptores de Superfície Celular/administração & dosagem , Tropismo Viral , Adenoviridae/fisiologia , Animais , Citomegalovirus/genética , Endotélio Vascular/citologia , Endotélio Vascular/virologia , Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Células Mieloides/metabolismo , Células Mieloides/virologia , Peptídeos/administração & dosagem , Peptídeos/metabolismo , Regiões Promotoras Genéticas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/fisiologiaRESUMO
Neuropilins (NRPs) are cell surface receptors for vascular endothelial growth factor (VEGF) and SEMA3 (class 3 semaphorin) family members. The role of NRPs in neurons and endothelial cells has been investigated, but the expression and role of NRPs in epithelial cells is much less clear. Herein, the expression and localization of NRP1 was investigated in human and mouse skin and squamous cell carcinomas (SCCs). Results indicated that NRP1 mRNA and protein was expressed in the suprabasal epithelial layers of the skin sections. NRP1 staining did not overlap with that of keratin 14 (K14) or proliferating cell nuclear antigen, but did co-localize with staining for keratin 1, indicating that differentiated keratinocytes express NRP1. Similar to the expression of NRP1, VEGF-A was expressed in suprabasal epithelial cells, whereas Nrp2 and VEGFR2 were not detectable in the epidermis. The expression of NRP1 correlated with a high degree of differentiation in human SCC specimens, human SCC xenografts, and mouse K14-HPV16 transgenic SCC. UVB irradiation of mouse skin induced Nrp1 upregulation. In vitro, Nrp1 was upregulated in primary keratinocytes in response to differentiating media or epidermal growth factor-family growth factors. In conclusion, the expression of NRP1 is regulated in the skin and is selectively produced in differentiated epithelial cells. NRP1 may function as a reservoir to sequester VEGF ligand within the epithelial compartment, thereby modulating its bioactivity.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Epiderme/metabolismo , Neuropilina-1/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Células Epidérmicas , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Queratina-1/metabolismo , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Neuropilina-1/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Although our understanding of the molecular regulation of adult neovascularization has advanced tremendously, vascular-targeted therapies for tissue ischemia remain suboptimal. The master regulatory transcription factors of the hypoxia-inducible factor (HIF) family are attractive therapeutic targets because they coordinately up-regulate multiple genes controlling neovascularization. Here, we used an inducible model of epithelial HIF-1 activation, the TetON-HIF-1 mouse, to test the requirement for VEGF in HIF-1 mediated neovascularization. TetON-HIF-1, K14-Cre, and VEGF(flox/flox) alleles were combined to create TetON-HIF-1:VEGF(Δ) mice to activate HIF-1 and its target genes in adult basal keratinocytes in the absence of concomitant VEGF. HIF-1 induction failed to produce neovascularization in TetON-HIF-1:VEGF(Δ) mice despite robust up-regulation of multiple proangiogenic HIF targets, including PlGF, adrenomedullin, angiogenin, and PAI-1. In contrast, endothelial sprouting was preserved, enhanced, and more persistent, consistent with marked reduction in Dll4-Notch-1 signaling. Optical-resolution photoacoustic microscopy, which provides noninvasive, label-free, high resolution, and wide-field vascular imaging, revealed the absence of both capillary expansion and arteriovenous remodeling in serially imaged individual TetON-HIF-1:VEGF(Δ) mice. Impaired TetON-HIF-1:VEGF(Δ) neovascularization could be partially rescued by 12-O-tetradecanoylphorbol-13-acetate skin treatment. These data suggest that therapeutic angiogenesis for ischemic cardiovascular disease may require treatment with both HIF-1 and VEGF.
Assuntos
Células Endoteliais/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Deleção de Genes , Integrases/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Microvasos/efeitos dos fármacos , Microvasos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Neovascularização Patológica/genética , Tetraciclina/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Neovascularization is a crucial component of tumor growth and ischemia. Although prior work primarily used disease models, delineation of neovascularization in the absence of disease can reveal intrinsic mechanisms of microvessel regulation amenable to manipulation in illness. We created a conditional model of epithelial HIF-1 induction in adult mice (TetON-HIF-1 mice). Longitudinal photoacoustic microscopy (L-PAM) was coincidentally developed for noninvasive, label-free serial imaging of red blood cell-perfused vasculature in the same mouse for weeks to months. TetON-HIF-1 mice evidenced 3 stages of neovascularization: development, maintenance, and transgene-dependent regression. Regression occurred despite extensive and tight pericyte coverage. L-PAM mapped microvascular architecture and quantified volumetric changes in neocapillary morphogenesis, arteriovenous remodeling, and microvessel regression. Developmental stage endothelial proliferation down-regulation was associated with a DNA damage checkpoint consisting of p53, p21, and endothelial γ-H2AX induction. The neovasculature was temporally responsive to VEGFR2 immuno-blockade, with the developmental stage sensitive, and the maintenance stage resistant, to DC101 treatment. L-PAM analysis also pinpointed microvessels ablated or resistant to VEGFR2 immuno-blockade. HIF-1-recruited myeloid cells did not mediate VEGFR2 inhibitor resistance. Thus, HIF-1 neovascularization in the absence of disease is self-regulated via cell autonomous endothelial checkpoints, and resistant to angiogenesis inhibitors independent of myeloid cells.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Hemodinâmica/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Transgênicos , Microcirculação/fisiologia , Células Mieloides/fisiologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Pericitos/fisiologia , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologia , Microambiente Tumoral/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The migration of epithelial layers requires specific and coordinated organization of the cells at the leading edge of the sheet. Mice that are conditionally deleted for the c-jun protooncogene in epidermis are born at expected frequencies, but with open eyes and with defects in epidermal wound healing. Keratinocytes lacking c-Jun are unable to migrate or elongate properly in culture at the border of scratch assays. Histological analyses in vitro and in vivo demonstrate an inability to activate EGF receptor at the leading edge of wounds, and we demonstrate that this can be rescued by supplementation with conditioned medium or the EGF receptor ligand HB-EGF. Lack of c-Jun prevents EGF-induced expression of HB-EGF, indicating that c-jun controls formation of the epidermal leading edge through its control of an EGF receptor autocrine loop.
Assuntos
Epiderme/crescimento & desenvolvimento , Receptores ErbB/metabolismo , Genes jun/fisiologia , Animais , Divisão Celular , Movimento Celular , Células Cultivadas , Células Epidérmicas , Fator de Crescimento Epidérmico/metabolismo , Epiderme/lesões , Pálpebras/anormalidades , Pálpebras/embriologia , Deleção de Genes , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Peptídeos e Proteínas de Sinalização Intercelular , Queratinócitos/citologia , Queratinócitos/fisiologia , Queratinas/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Fosfoproteínas Fosfatases , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/metabolismoRESUMO
Adenoviral (Ad) vector vaccines represent one of the most promising modern vaccine platforms, and Ad vector vaccines are currently being investigated in human clinical trials for infectious disease and cancer. Our studies have shown that specific targeting of adenovirus to dendritic cells dramatically enhanced vaccine efficacy. However, this was achieved using a molecular adapter, thereby necessitating a two component vector approach. To address the mandates of clinical translation of our strategy, we here sought to accomplish the goal of DC targeting with a single-component adenovirus vector approach. To redirect the specificity of Ad vector vaccines, we replaced the Ad fiber knob with fiber-fibritin chimeras fused to DC1.8, a single-domain antibody (sdAb) specific for murine immature DC. We engineered a fiber-fibritin-sdAb chimeric molecule using the coding sequence for DC1.8, and then replaced the native Ad5 fiber knob sequence by homologous recombination. The resulting Ad5 virus, Ad5FF1.8, expresses the chimeric fiber-fibritin sdAb chimera. Infection with Ad5FF1.8 dramatically enhances transgene expression in DC2.4 dendritic cells compared with infection with native Ad5. Ad5FF1.8 infection of bone marrow-derived DC demonstrates that Ad5FF1.8 selectively infects immature DC consistent with the known specificity of DC1.8. Thus, sdAb can be used to selectively redirect the tropism of Ad5 vector vaccines, providing the opportunity to engineer Ad vector vaccines that are specifically targeted to DC, or specific DC subsets.
Assuntos
Adenoviridae , Células Dendríticas/imunologia , Vetores Genéticos , Vacinas , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Camundongos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Vacinas/genética , Vacinas/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
While modern therapies for metastatic prostate cancer (PCa) have improved survival they are associated with an increasingly prevalent entity, aggressive variant PCa (AVPCa), lacking androgen receptor (AR) expression, enriched for cancer stem cells (CSCs), and evidencing epithelial-mesenchymal plasticity with a varying extent of neuroendocrine transdifferentiation. Parallel work revealed that endothelial cells (ECs) create a perivascular CSC niche mediated by juxtacrine and membrane tethered signaling. There is increasing interest in pharmacological metastatic niche targeting, however, targeted access has been impossible. Here, we discovered that the Gleason 7 derived, androgen receptor negative, IGR-CaP1 cell line possessed some but not all of the molecular features of AVPCa. Intracardiac injection into NOD/SCID/IL2Rg -/- (NSG) mice produced a completely penetrant bone, liver, adrenal, and brain metastatic phenotype; noninvasively and histologically detectable at 2 weeks, and necessitating sacrifice 4-5 weeks post injection. Bone metastases were osteoblastic, and osteolytic. IGR-CaP1 cells expressed the neuroendocrine marker synaptophysin, near equivalent levels of vimentin and e-cadherin, all of the EMT transcription factors, and activation of NOTCH and WNT pathways. In parallel, we created a new triple-targeted adenoviral vector containing a fiber knob RGD peptide, a hexon mutation, and an EC specific ROBO4 promoter (Ad.RGD.H5/3.ROBO4). This vector was expressed in metastatic microvessels tightly juxtaposed to IGR-CaP1 cells in bone and visceral niches. Thus, the combination of IGR-CaP1 cells and NSG mice produces a completely penetrant metastatic PCa model emulating end-stage human disease. In addition, the metastatic niche access provided by our novel Ad vector could be therapeutically leveraged for future disease control or cure.
Assuntos
Adenoviridae/genética , Neoplasias Ósseas/genética , Células Endoteliais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/genética , Vísceras/metabolismo , Animais , Western Blotting , Neoplasias Ósseas/secundário , Caderinas , Linhagem Celular Tumoral , Modelos Animais de Doenças , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , Nicho de Células-Tronco , Transplante Heterólogo , Vimentina/metabolismo , Vísceras/patologiaRESUMO
BACKGROUND: Long noncoding RNAs (lncRNAs) are an emerging class of relatively underexplored oncogenic molecules with biological and clinical significance. Current inadequacies for stratifying patients with aggressive disease presents a strong rationale to systematically identify lncRNAs as clinical predictors in localized prostate cancer. OBJECTIVE: To identify RNA biomarkers associated with aggressive prostate cancer. DESIGN, SETTING, AND PARTICIPANTS: Radical prostatectomy microarray and clinical data was obtained from 910 patients in three published institutional cohorts: Mayo Clinic I (N=545, median follow-up 13.8 yr), Mayo Clinic II (N=235, median follow-up 6.7 yr), and Thomas Jefferson University (N=130, median follow-up 9.6 yr). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary clinical endpoint was distant metastasis-free survival. Secondary endpoints include prostate cancer-specific survival and overall survival. Univariate and multivariate Cox regression were used to evaluate the association of lncRNA expression and these endpoints. RESULTS AND LIMITATIONS: An integrative analysis revealed Prostate Cancer Associated Transcript-14 (PCAT-14) as the most prevalent lncRNA that is aberrantly expressed in prostate cancer patients. Down-regulation of PCAT-14 expression significantly associated with Gleason score and a greater probability of metastatic progression, overall survival, and prostate cancer-specific mortality across multiple independent datasets and ethnicities. Low PCAT-14 expression was implicated with genes involved in biological processes promoting aggressive disease. In-vitro analysis confirmed that low PCAT-14 expression increased migration while overexpressing PCAT-14 reduced cellular growth, migration, and invasion. CONCLUSIONS: We discovered that androgen-regulated PCAT-14 is overexpressed in prostate cancer, suppresses invasive phenotypes, and lower expression is significantly prognostic for multiple clinical endpoints supporting its significance for predicting metastatic disease that could be used to improve patient management. PATIENT SUMMARY: We discovered that aberrant prostate cancer associated transcript-14 expression during prostate cancer progression is prevalent across cancer patients. Prostate cancer associated transcript-14 is also prognostic for metastatic disease and survival highlighting its importance for stratifying patients that could benefit from treatment intensification.
Assuntos
Neoplasias da Próstata/genética , RNA Longo não Codificante/genética , Idoso , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Prostatectomia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/cirurgia , RNA Longo não Codificante/biossínteseRESUMO
PURPOSE: The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable. RESULTS: Hypoxia, or low oxygenation, has emerged as an important factor in tumor biology and response to cancer treatment. It has been correlated with angiogenesis, tumor aggressiveness, local recurrence, and metastasis, and it appears to be a prognostic factor for several cancers, including those of the cervix, head and neck, prostate, pancreas, and brain. The relationship between tumor oxygenation and response to radiation therapy has been well established, but hypoxia also affects and is affected by some chemotherapeutic agents. Although hypoxia is an important aspect of tumor physiology and response to treatment, the lack of simple and efficient methods to measure and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia; Eppendorf measurement of pO(2) has been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as hypoxia inducible factor 1 (HIF-1) and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques. CONCLUSIONS: This workshop yielded recommendations on using hypoxia measurement to identify patients who would respond best to radiation therapy, which would improve treatment planning. This represents a narrow focus, as hypoxia measurement might also prove useful in drug development and in increasing our understanding of tumor biology.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Diagnóstico por Imagem/métodos , Hipóxia/diagnóstico , Neoplasias/tratamento farmacológico , Oxigênio/metabolismo , Antígenos de Neoplasias/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais/análise , Anidrase Carbônica IX , Anidrases Carbônicas/metabolismo , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Isoenzimas/metabolismo , National Institutes of Health (U.S.) , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Prognóstico , Radiografia , Reprodutibilidade dos Testes , Estados UnidosRESUMO
Human cervix cancer is caused by high-risk human papillomaviruses encoding E6 and E7 oncoproteins, each of which alter function of distinct targets regulating the cell cycle, apoptosis, and differentiation. Here we determined the molecular contribution of E6 or E7 to neoplastic progression and malignant growth in a transgenic mouse model of cervical carcinogenesis. E7 increased proliferation and centrosome copy number, and produced progression to multifocal microinvasive cervical cancers. E6 elevated centrosome copy number and eliminated detectable p53 protein, but did not produce neoplasia or cancer. E6 plus E7 additionally elevated centrosome copy number and created large, extensively invasive cancers. Centrosome copy number increases and p53 loss likely contributed to malignant growth; however, dysregulated proliferation and differentiation were required for carcinogenic progression.
Assuntos
Proteínas Oncogênicas Virais/fisiologia , Proteínas Repressoras , Neoplasias do Colo do Útero/etiologia , Animais , Apoptose , Centrossomo/patologia , Modelos Animais de Doenças , Estrogênios/toxicidade , Feminino , Camundongos , Camundongos Transgênicos , Estadiamento de Neoplasias , Fases de Leitura Aberta , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Neoplasias do Colo do Útero/patologiaRESUMO
The construction of transgenic FVB/N mice targeting the PMLRARA fusion gene under the control of a human MRP8 promoter recapitulated the phenotype of acute promyelocytic leukemia but had the unexpected result of multiple squamous papillomas of the skin (Brown et al., PROC: Natl. Acad. Sci. USA, 94:2551-2556, 1997). In addition, transgenic MRP8-PMLRARA mice exhibited a skin phenotype characteristic of vitamin A deficiency. The severity of the skin phenotype and spontaneous papilloma development correlated with the level of transgene expression. Papilloma formation was preceded by follicular hyperplasia and the expression of epidermal differentiation markers in the follicular epithelium. Mutations in the Ha or Ki alleles of ras were not detected in papillomas that developed on transgenic skin, and papilloma formation was accentuated on the C57/Bl6 background, unlike the usual resistance of this strain to skin tumor induction. Analysis of liver extracts from transgenic mice indicated a deficiency in the production of retinoic acid. Furthermore, affected transgenic epidermis had reduced levels of retinoic acid receptoralpha (RARalpha) and retinoic X receptor (RXRalpha), and supplementation with exogenous retinoic acid prevented the skin phenotype. When transgenic keratinocytes were grafted to nude mice, the resulting integument was normal, and conversely, when transgenic bone marrow was grafted to normal mice, a skin phenotype did not develop. Together these results suggest that local interruption of PML and RARalpha signaling in the skin, together with a systemic retinoid deficiency, initiates a tumor induction pathway that is independent of ras activation.
Assuntos
Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Papiloma/genética , Retinoides/metabolismo , Neoplasias Cutâneas/genética , Vitamina A/análogos & derivados , Animais , Calgranulina A/genética , Diferenciação Celular/genética , Diterpenos , Genes ras/genética , Predisposição Genética para Doença , Folículo Piloso/metabolismo , Folículo Piloso/patologia , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Papiloma/metabolismo , Papiloma/patologia , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Ésteres de Retinil , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Transcrição/metabolismo , Transgenes , Tretinoína/farmacologia , Vitamina A/metabolismoRESUMO
Angiogenesis in a tumor region creates arteriovenous (AV) shunts that cause an abnormal venous blood oxygen saturation ( sO2 ) distribution. Here, we applied optical-resolution photoacoustic microscopy to study the AV shunting in vivo. First, we built a phantom to image sO2 distribution in a vessel containing converged flows from two upstream blood vessels with different sO2 values. The phantom experiment showed that the blood from the two upstream vessels maintained a clear sO2 boundary for hundreds of seconds, which is consistent with our theoretical analysis using a diffusion model. Next, we xenotransplanted O-786 tumor cells in mouse ears and observed abnormal sO2 distribution in the downstream vein from the AV shunts in vivo. Finally, we identified the tumor location by tracing the sO2 distribution. Our study suggests that abnormal sO2 distribution induced by the AV shunts in the vessel network may be used as a new functional benchmark for early tumor detection.
Assuntos
Microscopia/métodos , Neovascularização Patológica/fisiopatologia , Oximetria/métodos , Oxigênio/sangue , Técnicas Fotoacústicas/métodos , Animais , Difusão , Orelha/irrigação sanguínea , Desenho de Equipamento , Camundongos , Neoplasias , Imagens de FantasmasRESUMO
Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer.
Assuntos
Ácidos/metabolismo , Metabolismo Energético , Glucose/metabolismo , Tumores Neuroendócrinos/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Linhagem Celular Tumoral , Glicólise/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Metástase Neoplásica , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/patologia , Niclosamida/administração & dosagem , Fosforilação Oxidativa/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
The importance of neovascularization for primary and metastatic tumor growth fostered numerous clinical trials of angiogenesis inhibitors either alone or in combination with conventional antineoplastic therapies. One challenge with the use of molecularly targeted agents has been the disconnection between size reduction and tumor biologic behavior, either when the drug is efficacious or when tumor resistance emerges. Here, we report the synthesis and characterization of (64)Cu-NOTA-bevacizumab as a PET imaging agent for imaging intratumoral VEGF content in vivo. (64)Cu-NOTA-bevacizumab avidly accumulated in 786-O renal carcinoma xenografts with lower levels in host organs. RAD001 (everolimus) markedly attenuated (64)Cu-NOTA-bevacizumab accumulation within 786-O renal carcinoma xenografts. Tumor tissue and cellular molecular analysis validated PET imaging, demonstrating decreases in total and secreted VEGF content and VEGFR2 activation. Notably, (64)Cu-NOTA-bevacizumab PET imaging was concordant with the growth arrest of RAD001 tumors. These data suggest that immunoPET targeting of angiogenic factors such as VEGF could be a new class of surrogate markers complementing the RECIST criteria in patients receiving molecularly targeted therapies.
Assuntos
Anticorpos Monoclonais Humanizados , Transformação Celular Neoplásica , Radioisótopos de Cobre , Imunoconjugados , Neoplasias Renais/patologia , Tomografia por Emissão de Pósitrons , Sirolimo/farmacologia , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais Humanizados/farmacocinética , Bevacizumab , Linhagem Celular Tumoral , Everolimo , Compostos Heterocíclicos/química , Compostos Heterocíclicos com 1 Anel , Humanos , Neoplasias Renais/irrigação sanguínea , Neoplasias Renais/diagnóstico por imagem , Neoplasias Renais/terapia , Camundongos , Neovascularização Patológica , Fosforilação/efeitos dos fármacos , Sirolimo/análogos & derivados , Sirolimo/química , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
New approaches targeting metastatic neovasculature are needed. Payload capacity, cellular transduction efficiency, and first-pass cellular uptake following systemic vector administration, motivates persistent interest in tumor vascular endothelial cell (EC) adenoviral (Ad) vector targeting. While EC transductional and transcriptional targeting has been accomplished, vector administration approaches of limited clinical utility, lack of tumor-wide EC expression quantification, and failure to address avid liver sequestration, challenged prior work. Here, we intravenously injected an Ad vector containing 3 kb of the human roundabout4 (ROBO4) enhancer/promoter transcriptionally regulating an enhanced green fluorescent protein (EGFP) reporter into immunodeficient mice bearing 786-O renal cell carcinoma subcutaneous (SC) xenografts and kidney orthotopic (KO) tumors. Initial experiments performed in human coxsackie virus and adenovirus receptor (hCAR) transgenic:Rag2 knockout mice revealed multiple ECs with high-level Ad5ROBO4-EGFP expression throughout KO and SC tumors. In contrast, Ad5CMV-EGFP was sporadically expressed in a few tumor vascular ECs and stromal cells. As the hCAR transgene also facilitated Ad5ROBO4 and control Ad5CMV vector EC expression in multiple host organs, follow-on experiments engaged warfarin-mediated liver vector detargeting in hCAR non-transgenic mice. Ad5ROBO4-mediated EC expression was undetectable in most host organs, while the frequencies of vector expressing intratumoral vessels and whole tumor EGFP protein levels remained elevated. In contrast, AdCMV vector expression was only detectable in one or two stromal cells throughout the whole tumor. The Ad5ROBO4 vector, in conjunction with liver detargeting, provides tractable genetic access for in-vivo EC genetic engineering in malignancies.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Neoplasias Renais/terapia , Neovascularização Patológica , Receptores de Superfície Celular/genética , Transcrição Gênica/genética , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Neoplasias Renais/irrigação sanguínea , Neoplasias Renais/genética , Neoplasias Renais/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Metástase Neoplásica , Transgenes/genética , Varfarina/farmacologiaRESUMO
Adenovirus serotype 5 (Ad5) vectors are well suited for gene therapy. However, tissue-selective transduction by systemically administered Ad5-based vectors is confounded by viral particle sequestration in the liver. Hexon-modified Ad5 expressing reporter gene under transcriptional control by the immediate/early cytomegalovirus (CMV) or the Roundabout 4 receptor (Robo4) enhancer/promoter was characterized by growth in cell culture, stability in vitro, gene transfer in the presence of human coagulation factor X, and biodistribution in mice. The obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Substitution of the hypervariable region 7 (HVR7) of the Ad5 hexon with HVR7 from Ad serotype 3 resulted in decreased liver tropism and dramatically altered biodistribution of gene expression. The results of these studies suggest that the combination of liver detargeting using a genetic modification of hexon with an endothelium-specific transcriptional control element produces an additive effect in the improvement of Ad5 biodistribution.