RESUMO
Inhibiting androgen signaling using androgen signaling inhibitors (ASI) remains the primary treatment for castrate-resistant prostate cancer. Acquired resistance to androgen receptor (AR)-targeted therapy represents a major impediment to durable clinical response. Understanding resistance mechanisms, including the role of AR expressed in other cell types within the tumor microenvironment, will extend the clinical benefit of AR-targeted therapy. Here, we show the ASI enzalutamide induces vascular catastrophe and promotes hypoxia and microenvironment adaptation. We characterize treatment-induced hypoxia, and subsequent induction of angiogenesis, as novel mechanisms of relapse to enzalutamide, highlighting the importance of two hypoxia-regulated cytokines in underpinning relapse. We confirmed AR expression in CD34+ vascular endothelium of biopsy tissue and human vascular endothelial cells (HVEC). Enzalutamide attenuated angiogenic tubule formation and induced cytotoxicity in HVECs in vitro, and rapidly induced sustained hypoxia in LNCaP xenografts. Subsequent reoxygenation, following prolonged enzalutamide treatment, was associated with increased tumor vessel density and accelerated tumor growth. Hypoxia increased AR expression and transcriptional activity in prostate cells in vitro. Coinhibition of IL8 and VEGF-A restored tumor response in the presence of enzalutamide, confirming the functional importance of their elevated expression in enzalutamide-resistant models. Moreover, coinhibition of IL8 and VEGF-A resulted in a durable, effective resolution of enzalutamide-sensitive prostate tumors. We conclude that concurrent inhibition of two hypoxia-induced factors, IL8 and VEGF-A, prolongs tumor sensitivity to enzalutamide in preclinical models and may delay the onset of enzalutamide resistance. IMPLICATIONS: Targeting hypoxia-induced signaling may extend the therapeutic benefit of enzalutamide, providing an improved treatment strategy for patients with resistant disease.
Assuntos
Antagonistas de Receptores de Andrógenos , Neoplasias de Próstata Resistentes à Castração , Antagonistas de Androgênios/farmacologia , Antagonistas de Receptores de Andrógenos/farmacologia , Androgênios/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Células Endoteliais/metabolismo , Humanos , Hipóxia/tratamento farmacológico , Interleucina-8/genética , Masculino , Recidiva Local de Neoplasia/tratamento farmacológico , Nitrilas/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Functional roles of neutrophil elastase (NE) have not been examined in distinct steps of the metastatic cascade. NE, delivered to primary tumors as a purified enzyme or within intact neutrophils or neutrophil granule content, enhanced human tumor cell intravasation and subsequent dissemination via NE-mediated formation of dilated intratumoral vasculature. These effects depended on picomole range of NE activity, sensitive to its natural inhibitor, α1PI. In Elane-negative mice, the lack of NE decreased lung retention of human tumor cells in experimental metastasis. Furthermore, NE was essential for spontaneous metastasis of murine carcinoma cells in a syngeneic orthotopic model of oral cancer. NE also induced tumor cell survival and migration via Src/PI3K-dependent activation of Akt signaling, vital for tumor cell dissemination in vivo. Together, our findings implicate NE, a potent host enzyme specific for first-responding innate immune cells, as directly involved in early metastatic events and a potential target for therapeutic intervention.
RESUMO
Background: CUB domain-containing protein 1 (CDCP1) is a cell surface receptor regulating key signalling pathways in malignant cells. CDCP1 has been proposed as a molecular target to abrogate oncogenic signalling pathways and specifically deliver anti-cancer agents to tumors. However, the development of CDCP1-targeting agents has been questioned by its frequent proteolytic processing which was thought to result in shedding of the CDCP1 extracellular domain limiting its targetability. In this study, we investigated the relevance of targeting CDCP1 in the context of pancreatic ductal adenocarcinoma (PDAC) and assess the impact of CDCP1 proteolysis on the effectiveness of CDCP1 targeting agents. Methods: The involvement of CDCP1 in PDAC progression was assessed by association analysis in several PDAC cohorts and the proteolytic processing of CDCP1 was evaluated in PDAC cell lines and patient-derived cells. The consequences of CDCP1 proteolysis on its targetability in PDAC cells was assessed using immunoprecipitation, immunostaining and biochemical assays. The involvement of CDCP1 in PDAC progression was examined by loss-of-function in vitro and in vivo experiments employing PDAC cells expressing intact or cleaved CDCP1. Finally, we generated antibody-based imaging and therapeutic agents targeting CDCP1 to demonstrate the feasibility of targeting this receptor for detection and treatment of PDAC tumors. Results: High CDCP1 expression in PDAC is significantly associated with poorer patient survival. In PDAC cells proteolysis of CDCP1 does not always result in the shedding of CDCP1-extracellular domain which can interact with membrane-bound CDCP1 allowing signal transduction between the different CDCP1-fragments. Targeting CDCP1 impairs PDAC cell functions and PDAC tumor growth independently of CDCP1 cleavage status. A CDCP1-targeting antibody is highly effective at delivering imaging radionuclides and cytotoxins to PDAC cells allowing specific detection of tumors by PET/CT imaging and superior anti-tumor effects compared to gemcitabine in in vivo models. Conclusion: Independent of its cleavage status, CDCP1 exerts oncogenic functions in PDAC and has significant potential to be targeted for improved radiological staging and treatment of this cancer. Its elevated expression by most PDAC tumors and lack of expression by normal pancreas and other major organs, suggest that targeting CDCP1 could benefit a significant proportion of PDAC patients. These data support the further development of CDCP1-targeting agents as personalizable tools for effective imaging and treatment of PDAC.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Moléculas de Adesão Celular/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/terapia , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias Pancreáticas/terapia , Medicina de Precisão , ProteóliseRESUMO
Latent transforming growth factor ß (TGFß)-binding proteins (LTBPs) are important for the secretion, activation, and function of mature TGFß, especially so in cancer cell physiology. However, specific roles of the LTBPs remain understudied in the context of the primary tumor microenvironment. Herein, we investigated the role of LTBP3 in the distinct processes involved in cancer metastasis. By using three human tumor cell lines of different tissue origin (epidermoid HEp-3 and prostate PC-3 carcinomas and HT-1080 fibrosarcoma) and several metastasis models conducted in both mammalian and avian settings, we show that LTBP3 is involved in the early dissemination of primary cancer cells, namely in the intravasation step of the metastatic cascade. Knockdown of LTBP3 in all tested cell lines led to significant inhibition of tumor cell intravasation, but did not affect primary tumor growth. LTBP3 was dispensable in the late steps of carcinoma cell metastasis that follow tumor cell intravasation, including vascular arrest, extravasation, and tissue colonization. However, LTBP3 depletion diminished the angiogenesis-inducing potential of HEp-3 cells in vivo, which was restorable by exogenous delivery of LTBP3 protein. A similar compensatory approach rescued the dampened intravasation of LTBP3-deficient HEp-3 cells, suggesting that LTBP3 regulates the induction of the intravasation-supporting angiogenic vasculature within developing primary tumors. Using our recently developed microtumor model, we confirmed that LTBP3 loss resulted in the development of intratumoral vessels with an abnormal microarchitecture incompatible with efficient intravasation of HEp-3 carcinoma cells. Collectively, these findings demonstrate that LTBP3 represents a novel oncotarget that has distinctive functions in the regulation of angiogenesis-dependent tumor cell intravasation, a critical process during early cancer dissemination. Our experimental data are also consistent with the survival prognostic value of LTBP3 expression in early-stage head and neck squamous cell carcinomas, further indicating a specific role for LTBP3 in cancer progression toward metastatic disease.
Assuntos
Proteínas de Ligação a TGF-beta Latente/fisiologia , Neoplasias/genética , Neoplasias/patologia , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Ligação a TGF-beta Latente/antagonistas & inibidores , Proteínas de Ligação a TGF-beta Latente/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/mortalidade , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , RNA Interferente Pequeno/farmacologia , Análise de SobrevidaRESUMO
Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvß3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvß3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvß3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvß3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease.
Assuntos
Plaquetas/patologia , Integrina alfaVbeta3/metabolismo , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes/patologia , Animais , Plaquetas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Humanos , Integrina alfaVbeta3/análise , Camundongos SCID , Células Neoplásicas Circulantes/metabolismoRESUMO
Many malignant characteristics of cancer cells are regulated through pathways induced by the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Herein, we show that besides directly affecting the biology of cancer cells per se, EGFR also regulates the primary tumor microenvironment. Specifically, our findings demonstrate that both the expression and signaling activity of EGFR are required for the induction of a distinct intratumoral vasculature capable of sustaining tumor cell intravasation, a critical rate-limiting step in the metastatic cascade. An intravasation-sustaining mode of intratumoral angiogenic vessels depends on high levels of tumor cell EGFR and the interplay between EGFR-regulated production of interleukin 8 by tumor cells, interleukin-8-induced influx of tumor-infiltrating neutrophils delivering their unique matrix metalloproteinase-9, and neutrophil matrix metalloproteinase-9-dependent release of the vascular permeability and endothelial growth factor, VEGF. Our data indicate that through VEGF-mediated disruption of endothelial layer integrity and increase of intratumoral vasculature permeability, EGFR activity significantly facilitates active intravasation of cancer cells. Therefore, this study unraveled an important but overlooked function of EGFR in cancer, namely, its ability to create an intravasation-sustaining microenvironment within the developing primary tumor by orchestrating several interrelated processes required for the initial steps of cancer metastasis through vascular routes. Our findings also suggest that EGFR-targeted therapies might be more effective when implemented in cancer patients with early-staged primary tumors containing a VEGF-dependent angiogenic vasculature. Accordingly, early EGFR inhibition combined with various anti-VEGF approaches could synergistically suppress tumor cell intravasation through inhibiting the highly permeable angiogenic vasculature induced by EGFR-overexpressing aggressive cancer cells.
Assuntos
Membrana Corioalantoide/irrigação sanguínea , Receptores ErbB/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/metabolismo , Membrana Corioalantoide/patologia , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Microscopia de Fluorescência , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neovascularização Patológica/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Microambiente Tumoral/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Metastasis is a distinct stage of cancer progression that requires the development of angiogenic blood vessels serving as conduits for tumor cell dissemination. An accumulated body of evidence indicates that metastasis-supporting neovasculature should possess certain structural characteristics allowing for the process of tumor cell intravasation, an active entry of cancer cells into the vessel interior. It appears that the development of tumor vessels with lumens of a distinctive size and support of these vessels by a discontinuous pericyte coverage constitute critical microarchitectural requirements to: (a) provide accessible points for vessel wall penetration by primary tumor cells; (b) provide enough lumen space for a tumor cell or cell aggregate upon intravasation; and (c) allow for sufficient rate of blood flow to carry away intravasated cells from the primary tumor to the next, proximal or distal site. This review will primarily focus on the functional roles of matrix metalloproteinases (MMPs), which catalytically trigger the development of an intravasation-sustaining neovasculature at the early stages of tumor growth and are also required for the maintenance of a metastasis-supporting state of blood vessels at later stages of cancer progression.
Assuntos
Metaloproteinases da Matriz/metabolismo , Neoplasias/patologia , Neovascularização Patológica/enzimologia , Animais , Humanos , Metástase Neoplásica , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Gelatinase B/matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many substrates and is produced by most cell types as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast with a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, AFM and TEM, we generated a 3D structure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers compared with monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1.
Assuntos
Precursores Enzimáticos/química , Metaloproteinase 9 da Matriz/química , Modelos Moleculares , Complexos Multiproteicos/química , Inibidor Tecidual de Metaloproteinase-1/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
According to established notion, one of the major angiogenesis-inducing factors, pro-matrix metalloproteinase-9 (proMMP-9), is supplied to the tumor microenvironment by tumor-associated macrophages (TAMs). Accumulated evidence, however, indicates that tumor-associated neutrophils (TANs) are also critically important for proMMP-9 delivery, especially at early stages of tumor development. To clarify how much angiogenic proMMP-9 is actually contributed by TAMs and TANs, we quantitatively evaluated TAMs and TANs from different tumor types, including human xenografts and syngeneic murine tumors grown in wild-type and Mmp9-knockout mice. Whereas host MMP-9 competence was required for full angiogenic potential of both normal and tumor-associated leukocytes, direct comparisons of neutrophils versus macrophages and TANs versus TAMs demonstrated that macrophages and TAMs secrete 40- to 50-fold less proMMP-9 than the same numbers of neutrophils or TANs. Correspondingly, the levels of MMP-9-mediated in vivo angiogenesis induced by neutrophils and TANs substantially exceeded those induced by macrophages and TAMs. MMP-9-delivering TANs were also required for development of metastasis-supporting intratumoral vasculature, characterized by ≥ 11-µm size lumens and partial coverage with stabilizing pericytes. Importantly, MMP-9-producing TAMs exhibit M2-skewed phenotype but do not express tissue inhibitor of metalloproteinases-1 (TIMP-1), a novel characteristic allowing them to secrete TIMP-1-free, neutrophil-like MMP-9 zymogen unencumbered by its natural inhibitor. Together, our findings support the notion whereby TANs, capable of immediate release of their pre-stored cargo, are the major contributors of highly angiogenic MMP-9, whereas tumor-influxing precursors of macrophages require time to differentiate, polarize into M2-skewed TAMs, shut down their TIMP-1 expression, and only then, initiate relatively low-level production of TIMP-free MMP-9 zymogen.
Assuntos
Indutores da Angiogênese/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neutrófilos/patologia , Microambiente Tumoral/fisiologia , Animais , Linhagem Celular Tumoral , Humanos , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Neutrófilos/metabolismo , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A proangiogenic function of tissue-infiltrating monocytes/macrophages has long been attributed to their matrix metalloproteinase-9 zymogen (proMMP-9). Herein, we evaluated the capacity of human monocytes, mature M0 macrophages, and M1- and M2-polarized macrophages to induce proMMP-9-mediated angiogenesis. Only M2 macrophages induced angiogenesis at levels comparable with highly angiogenic neutrophils previously shown to release their proMMP-9 in a unique form, free of tissue inhibitor of metalloproteinases-1 (TIMP-1). Macrophage differentiation was accompanied by induction of low-angiogenic, TIMP-1-encumbered proMMP-9. However, polarization toward the M2, but not the M1 phenotype, caused a substantial downregulation of TIMP-1 expression, resulting in production of angiogenic, TIMP-deficient proMMP-9. Correspondingly, the angiogenic potency of M2 proMMP-9 was lost after its complexing with TIMP-1, whereas TIMP-1 silencing in M0/M1 macrophages rendered them both angiogenic. Similar to human cells, murine bone marrow-derived M2 macrophages also shut down their TIMP-1 expression and produced proMMP-9 unencumbered by TIMP-1. Providing proof that angiogenic capacity of murine M2 macrophages depended on their TIMP-free proMMP-9, Mmp9-null M2 macrophages were nonangiogenic, although their TIMP-1 was severely downregulated. Our study provides a unifying molecular mechanism for high angiogenic capacity of TIMP-free proMMP-9 that would be uniquely produced in a pathophysiological microenvironment by influxing neutrophils and/or M2 polarized macrophages.
Assuntos
Diferenciação Celular/fisiologia , Precursores Enzimáticos/metabolismo , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neovascularização Fisiológica/fisiologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Animais , Embrião de Galinha , Regulação para Baixo/fisiologia , Precursores Enzimáticos/genética , Humanos , Macrófagos/citologia , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Mutantes , Neutrófilos/citologia , Neutrófilos/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genéticaRESUMO
Intravasation, the active entry of primary tumor cells into the vasculature, remains the least studied step in the metastatic cascade. Protease-mediated escape and stromal invasion of tumor cells represent widely accepted processes leading up to the intravasation step. However, molecular factors that contribute directly to tumor cell vascular penetration have not been identified. In this study, the in vivo role of the collagenolytic protease, MMP-1, in cancer cell intravasation and metastasis was analyzed by using a highly disseminating variant of human HEp3 epidermoid carcinoma, HEp3-hi/diss. Although naturally acquired or experimentally induced MMP-1 deficiency substantially suppressed HEp3-hi/diss intravasation, supplementation of recombinant MMP-1 to MMP-1-silenced primary tumors restored their impaired vascular dissemination. Surprisingly, abrogation of MMP-1 production and activity did not significantly affect HEp3-hi/diss migration or matrix invasion, suggesting noncollagenolytic mechanisms underlying MMP-1-dependent cell intravasation. In support of such noncollagenolytic mechanisms, MMP-1 silencing in HEp3-hi/diss cells modulated the microarchitecture and integrity of the angiogenic vasculature in a novel microtumor model. Concomitantly, MMP-1 deficiency led to decreased levels of intratumoral vascular permeability, tumor cell intravasation, and metastatic dissemination. Taking advantage of PAR1 deficiency of HEp3-hi/diss cells, we further show that endothelial PAR1 is a putative nontumor-cell/nonmatrix target, activation of which by carcinoma-produced MMP-1 regulates endothelial permeability and transendothelial migration. The inhibitory effects of specific PAR1 antagonists in live animals have also indicated that the mechanisms of MMP-1-dependent vascular permeability in tumors involve endothelial PAR1 activation. Together, our findings mechanistically underscore the contribution of a tumor MMP-1/endothelial PAR1 axis to actual intravasation events manifested by aggressive carcinoma cells.
Assuntos
Carcinoma de Células Escamosas/enzimologia , Metaloproteinase 1 da Matriz/metabolismo , Receptor PAR-1/metabolismo , Animais , Permeabilidade Capilar , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Embrião de Galinha , Endotélio/irrigação sanguínea , Endotélio/enzimologia , Endotélio/metabolismo , Endotélio/patologia , Humanos , Metaloproteinase 1 da Matriz/deficiência , Metaloproteinase 1 da Matriz/genética , Invasividade Neoplásica , Metástase Neoplásica , Neovascularização Patológica/enzimologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Receptor PAR-1/deficiência , Receptor PAR-1/genéticaRESUMO
The transcription factor Twist1 induces epithelial-mesenchymal transition and extracellular matrix degradation to promote tumor metastasis. Although Twist1 also plays a role in embryonic vascular development and tumor angiogenesis, the molecular mechanisms that underlie these processes are not as well understood. Here, we report a novel function for Twist1 in modifying the tumor microenvironment to promote progression. We found that expression of Twist1 in human mammary epithelial cells potently promoted angiogenesis. Surprisingly, Twist1 expression did not increase the secretion of the common proangiogenic factors VEGF and basic fibroblast growth factor but rather induced expression of the macrophage chemoattractant CCL2. Attenuation of endogenous Twist1 in vivo blocked macrophage recruitment and angiogenesis, whereas exogenous CCL2 rescued the ability of tumor cells lacking Twist1 to attract macrophages and promote angiogenesis. Macrophage recruitment also was essential for the ability of Twist1-expressing cells to elicit a strong angiogenic response. Together, our findings show that how Twist1 recruits stromal macrophages through CCL2 induction to promote angiogenesis and tumor progression. As Twist1 expression has been associated with poor survival in many human cancers, this finding suggests that anti-CCL2 therapy may offer a rational strategy to treat Twist1-positive metastatic cancers.
Assuntos
Neoplasias da Mama/irrigação sanguínea , Quimiocina CCL2/metabolismo , Macrófagos/fisiologia , Neovascularização Patológica , Proteínas Nucleares/fisiologia , Proteína 1 Relacionada a Twist/fisiologia , Animais , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Neoplasias Mamárias Experimentais/irrigação sanguínea , CamundongosRESUMO
Data accumulated over the latest two decades have established that the serine protease urokinase-type plasminogen activator (uPA) is a potential therapeutic target in cancer. When designing inhibitors of the proteolytic activity of serine proteases, obtaining sufficient specificity is problematic, because the topology of the proteases' active sites are highly similar. In an effort to generate highly specific uPA inhibitors with new inhibitory modalities, we isolated uPA-binding RNA aptamers by screening a library of 35 nucleotides long 2'-fluoro-pyrimidine RNA molecules using a version of human pro-uPA lacking the epidermal growth factor-like and kringle domains as bait. One pro-uPA-binding aptamer sequence, referred to as upanap-126, proved to be highly specific for human uPA. Upanap-126 delayed the proteolytic conversion of human pro-uPA to active uPA, but did not inhibit plasminogen activation catalyzed by two-chain uPA. The aptamer also inhibited the binding of pro-uPA to uPAR and the binding of vitronectin to the preformed pro-uPA/uPAR complex, both in cell-free systems and on cell surfaces. Furthermore, upanap-126 inhibited human tumor cell invasion in vitro in the Matrigel assay and in vivo in the chick embryo assay of cell escape from microtumors. Finally, upanap-126 significantly reduced the levels of tumor cell intravasation and dissemination in the chick embryo model of spontaneous metastasis. Together, our findings show that usage of upanap-126 represents a novel multifunctional mechanistic modality for inhibition of uPA-dependent processes involved in tumor cell spread.
Assuntos
Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Linhagem Celular Tumoral , Sistema Livre de Células , Embrião de Galinha , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Humanos , Masculino , Terapia de Alvo Molecular , Invasividade Neoplásica , Plasminogênio/genética , Plasminogênio/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Proteases/genética , Serina Proteases/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
After neoplastic cells leave the primary tumor and circulate, they may extravasate from the vasculature and colonize tissues to form metastases. ß1 integrins play diverse roles in tumorigenesis and tumor progression, including extravasation. In blood cells, activation of ß1 integrins can be regulated by "inside-out" signals leading to extravasation from the circulation into tissues. However, a role for inside-out ß1 activation in tumor cell metastasis is uncertain. Here we show that ß1 integrin activation promotes tumor metastasis and that activated ß1 integrin may serve as a biomarker of metastatic human melanoma. To determine whether ß1 integrin activation can influence tumor cell metastasis, the ß1 integrin subunit in melanoma and breast cancer cell lines was stably knocked down with shRNA and replaced with wild-type or constitutively-active ß1. When tumor cells expressing constitutively-active ß1 integrins were injected intravenously into chick embryos or mice, they demonstrated increased colonization of the liver when compared to cells expressing wild-type ß1 integrins. Rescue expression with mutant ß1 integrins revealed that tumor cell extravasation and hepatic colonization required extracellular ligand binding to ß1 as well as ß1 interaction with talin, an intracellular mediator of integrin activation by the Rap1 GTPase. Furthermore, shRNA-mediated knock down of talin reduced hepatic colonization by tumor cells expressing wild-type ß1, but not constitutively-active ß1. Overexpression in tumor cells of the tumor suppressor, Rap1GAP, inhibited Rap1 and ß1 integrin activation as well as hepatic colonization. Using an antibody that detects activated ß1 integrin, we found higher levels of activated ß1 integrins in human metastatic melanomas compared to primary melanomas, suggesting that activated ß1 integrin may serve as a biomarker of invasive tumor cells. Altogether, these studies establish that inside-out activation of ß1 integrins promotes tumor cell extravasation and colonization, suggesting diagnostic and therapeutic approaches for targeting of ß1 integrin signaling in neoplasia.
Assuntos
Integrina beta1/metabolismo , Metástase Neoplásica , Neoplasias/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias/metabolismoRESUMO
Plasmin, one of the most potent and reactive serine proteases, is involved in various physiological processes, including embryo development, thrombolysis, wound healing and cancer progression. The proteolytic activity of plasmin is tightly regulated through activation of its precursor, plasminogen, only at specific times and in defined locales as well as through inhibition of active plasmin by its abundant natural inhibitors. By exploiting the plasminogen activating system and overexpressing distinct components of the plasminogen activation cascade, such as pro-uPA, uPAR and plasminogen receptors, malignant cells can enhance the generation of plasmin which in turn, modifies the tumor microenvironment to sustain cancer progression. While plasmin-mediated degradation and modification of extracellular matrix proteins, release of growth factors and cytokines from the stroma as well as activation of several matrix metalloproteinase zymogens, all have been a focus of cancer research studies for decades, the ability of plasmin to cleave transmembrane molecules and thereby to generate functionally important cleaved products which induce outside-in signal transduction, has just begun to receive sufficient attention. Herein, we highlight this relatively understudied, but important function of the plasmin enzyme as it is generated de novo at the interface between cross-talking cancer and host cells.
Assuntos
Comunicação Celular/fisiologia , Membrana Celular/metabolismo , Fibrinolisina/metabolismo , Modelos Biológicos , Animais , HumanosAssuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Neutrófilos/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Humanos , Interleucina-8/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neutrófilos/efeitos dos fármacos , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismoRESUMO
Urokinase-type plasminogen activator (uPA) and plasmin have long been implicated in cancer progression. However, the precise contributions of the uPA/plasmin system to specific steps involved in cancer cell dissemination have not been fully established. Herein, we have used a highly disseminating variant of the human PC-3 prostate carcinoma cell line, PC-hi/diss, as a prototype of aggressive carcinomas to investigate the mechanisms whereby pro-uPA activation and uPA-generated plasmin functionally contribute to specific stages of metastasis. The PC-hi/diss cells secrete and activate significant amounts of pro-uPA, leading to efficient generation of plasmin in solution and at the cell surface. In a mouse orthotopic xenograft model, treatment with the specific pro-uPA activation-blocking antibody mAb-112 significantly inhibited local invasion and distant metastasis of the PC-hi/diss cells. To mechanistically examine the uPA/plasmin-mediated aspects of tumor cell dissemination, the anti-pro-uPA mAb-112 and the potent serine protease inhibitor, aprotinin, were used in parallel in a number of in vivo assays modeling various rate-limiting steps in early metastatic spread. Our findings demonstrate that, by generating plasmin, activated tumor-derived uPA facilitates early stages of PC-hi/diss dissemination, specifically the escape from the primary tumor and tumor cell intravasation. Moreover, through a series of in vitro and in vivo analyses, we suggest that PC-hi/diss-invasive escape and dissemination may be enhanced by cleavage of stromal fibronectin by uPA-generated plasmin. Together, our findings point to inhibition of pro-uPA activation at the apex of the uPA/plasmin cascade as a therapy-valid approach to control onset of tumor escape and ensuing metastatic spread.
Assuntos
Fibrinolisina/metabolismo , Metástase Neoplásica , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Aprotinina/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Embrião de Galinha , Ativação Enzimática , Fibronectinas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Inibidores de Serina Proteinase/farmacologia , Transplante Heterólogo , Evasão Tumoral , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
Tumor-associated neutrophils contribute to neovascularization by supplying matrix metalloproteinase-9 (MMP-9), a protease that has been genetically and biochemically linked to induction of angiogenesis. Specific roles of inflammatory neutrophils and their distinct proMMP-9 in the coordinate regulation of tumor angiogenesis and tumor cell dissemination, however, have not been addressed. We demonstrate that the primary tumors formed by highly disseminating variants of human fibrosarcoma and prostate carcinoma recruit elevated levels of infiltrating MMP-9-positive neutrophils and concomitantly exhibit enhanced levels of angiogenesis and intravasation. Specific inhibition of neutrophil influx by interleukin 8 (IL-8) neutralization resulted in the coordinated diminishment of tumor angiogenesis and intravasation, both of which were rescued by purified neutrophil proMMP-9. However, if neutrophil proMMP-9, naturally devoid of tissue inhibitor of metalloproteinases (TIMP), was delivered in complex with TIMP-1 or in a mixture with TIMP-2, the protease failed to rescue the inhibitory effects of anti-IL8 therapy, indicating that the TIMP-free status of proMMP-9 is critical for facilitating tumor angiogenesis and intravasation. Our findings directly link tumor-associated neutrophils and their TIMP-free proMMP-9 with the ability of aggressive tumor cells to induce the formation of new blood vessels that serve as conduits for tumor cell dissemination. Thus, treatment of cancers associated with neutrophil infiltration may benefit from specific targeting of neutrophil MMP-9 at early stages to prevent ensuing tumor angiogenesis and tumor metastasis.
Assuntos
Precursores Enzimáticos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neovascularização Patológica/enzimologia , Neutrófilos/metabolismo , Inibidores Teciduais de Metaloproteinases/fisiologia , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Fibrossarcoma/irrigação sanguínea , Fibrossarcoma/metabolismo , Fibrossarcoma/secundário , Humanos , Interleucina-8/farmacologia , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Invasividade Neoplásica , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/metabolismoRESUMO
Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Autólise/tratamento farmacológico , Precursores Enzimáticos/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Animais , Movimento Celular , Embrião de Galinha , Ativação Enzimática/efeitos dos fármacos , Precursores Enzimáticos/imunologia , Precursores Enzimáticos/metabolismo , Fibrinolíticos/farmacologia , Humanos , Hidrólise , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Plasminogênio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ressonância de Plasmônio de Superfície , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKCdelta to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein.