RESUMO
JMJD6 is known to localize in the nucleus, exerting histone arginine demethylase and lysyl hydroxylase activities. A novel localization of JMJD6 in the extracellular matrix, resulting from its secretion as a soluble protein, was unveiled by a new anti-JMJD6 mAb called P4E11, which was developed to identify new targets in the stroma. Recombinant JMJD6 binds with collagen type I (Coll-I), and distinct JMJD6 peptides interfere with collagen fibrillogenesis, collagen-fibronectin interaction, and adhesion of human tumor cells to the collagen substrate. P4E11 and collagen binding to JMJD6 are mutually exclusive because the amino acid sequences of JMJD6 necessary for the interaction with Coll-I are part of the conformational epitope recognized by P4E11. In mice injected with mouse 4T1 breast carcinoma cells, treatment with P4E11 reduced fibrosis at the primary tumor and prevented lung metastases. Reduction of fibrosis has also been documented in human breast and ovarian tumors (MDA-MB-231 and IGROV1, respectively) xenotransplanted into immunodeficient mice treated with P4E11. In summary, this study uncovers a new localization and function for JMJD6 that is most likely independent from its canonical enzymatic activities, and demonstrates that JMJD6 can functionally interact with Coll-I. P4E11 mAb, inhibiting JMJD6/Coll-I interaction, represents a new opportunity to target fibrotic and tumor diseases.-Miotti, S., Gulino, A., Ferri, R., Parenza, M., Chronowska, A., Lecis, D., Sangaletti, S., Tagliabue, E., Tripodo, C., Colombo, M. P. Antibody-mediated blockade of JMJD6 interaction with collagen I exerts antifibrotic and antimetastatic activities.
Assuntos
Anticorpos Monoclonais/metabolismo , Colágeno Tipo I/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Colágeno Tipo I/genética , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Osteonectina/genética , Osteonectina/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Receptores de Superfície Celular/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aim: Castration-resistant prostate cancer (CRPC) eventually becomes resistant to androgen receptor pathway inhibitors like enzalutamide. Immunotherapy also fails in CRPC. We propose a new approach to simultaneously revert enzalutamide resistance and rewire anti-tumor immunity. Methods: We investigated in vitro and in subcutaneous and spontaneous mouse models the effects of combining enzalutamide and GSK-126, a drug inhibiting the epigenetic modulator EZH2. Results: Enzalutamide and GSK-126 synergized to reduce CRPC growth, also restraining tumor neuroendocrine differentiation. The anti-tumor activity was lost in immunodeficient mice. Indeed, the combination treatment awoke cytotoxic activity and IFN-γ production of tumor-specific CD8+ T lymphocytes. Conclusion: These results promote the combination of enzalutamide and GSK-126 in CRPC, also offering new avenues for immunotherapy in prostate cancer.
Prostate cancer depends on hormones called androgens for its growth. Therefore, hormonal therapies are commonly used. However, the tumor often does not respond to these treatments and new therapeutic approaches are needed. Here, using cell and mouse models, we have tested a new combination between hormone therapy and a drug that restrains an enzyme regulating gene expression. Our results have shown that this combination therapy not only reduces the growth of the tumor but also stops it from becoming more aggressive. This is really important because aggressive prostate cancer is much harder to treat. We have also found that this approach helps the immune system recognizing and attacking cancer cells. More research is needed to identify the mechanism of action of this treatment. However, our findings suggest that this approach could pave the way for new therapeutic strategies, including using immunotherapy, typically unsuccessful in treating prostate cancer.
RESUMO
Neuroendocrine prostate cancer (NEPC) is an aggressive form of prostate cancer that emerges as tumors become resistant to hormone therapies or, rarely, arises de novo in treatment-naïve patients. The urgent need for effective therapies against NEPC is hampered by the limited knowledge of the biology governing this lethal disease. Based on our prior observations in the transgenic adenocarcinoma of the mouse prostate (TRAMP) spontaneous prostate cancer model, in which the genetic depletion of either mast cells (MC) or the matricellular protein osteopontin (OPN) increases NEPC frequency, we tested the hypothesis that MCs can restrain NEPC through OPN production, using in vitro co-cultures between murine or human tumor cell lines and MCs, and in vivo experiments. We unveiled a role for the intracellular isoform of OPN, so far neglected compared with the secreted isoform. Mechanistically, we unraveled that the intracellular isoform of OPN promotes TNFα production in MCs via the TLR2/TLR4-MyD88 axis, specifically triggered by the encounter with NEPC cells. We found that MC-derived TNFα, in turn, hampered the growth of NEPC. We then identified the protein syndecan-1 (SDC1) as the NEPC-specific TLR2/TLR4 ligand that triggered this pathway. Interrogating published single-cell RNA-sequencing data, we validated this mechanism in a different mouse model. Translational relevance of the results was provided by in silico analyses of available human NEPC datasets and by immunofluorescence on patient-derived adenocarcinoma and NEPC lesions. Overall, our results show that MCs actively inhibit NEPC, paving the way for innovative MC-based therapies for this fatal tumor. We also highlight SDC1 as a potential biomarker for incipient NEPC.
Assuntos
Mastócitos , Osteopontina , Neoplasias da Próstata , Fator de Necrose Tumoral alfa , Osteopontina/metabolismo , Osteopontina/genética , Masculino , Animais , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Humanos , Camundongos , Mastócitos/metabolismo , Mastócitos/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Linhagem Celular Tumoral , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/genética , Carcinoma Neuroendócrino/metabolismo , Carcinoma Neuroendócrino/patologia , Carcinoma Neuroendócrino/genética , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
Fatal neuroendocrine differentiation (NED) of castration-resistant prostate cancer is a recurrent mechanism of resistance to androgen deprivation therapies (ADT) and antiandrogen receptor pathway inhibitors (ARPI) in patients. The design of effective therapies for neuroendocrine prostate cancer (NEPC) is complicated by limited knowledge of the molecular mechanisms governing NED. The paucity of acquired genomic alterations and the deregulation of epigenetic and transcription factors suggest a potential contribution from the microenvironment. In this context, whether ADT/ARPI induces stromal cells to release NED-promoting molecules and the underlying molecular networks are unestablished. Here, we utilized transgenic and transplantable mouse models and coculture experiments to unveil a novel tumor-stroma cross-talk that is able to induce NED under the pressure of androgen deprivation. Castration induced upregulation of GRP78 in tumor cells, which triggers miR29-b-mediated downregulation of the matricellular protein SPARC in the nearby stroma. SPARC downregulation enabled stromal cells to release IL6, a known inducer of NED. A drug that targets GRP78 blocked NED in castrated mice. A public, human NEPC gene expression dataset showed that Hspa5 (encoding for GRP78) positively correlates with hallmarks of NED. Finally, prostate cancer specimens from patients developing local NED after ADT showed GRP78 upregulation in tumor cells and SPARC downregulation in the stroma. These results point to GRP78 as a potential therapeutic target and to SPARC downregulation in stromal cells as a potential early biomarker of tumors undergoing NED. SIGNIFICANCE: Tumor-stroma cross-talk promotes neuroendocrine differentiation in prostate cancer in response to hormone therapy via a GRP78/SPARC/IL6 axis, providing potential therapeutic targets and biomarkers for neuroendocrine prostate cancer.
Assuntos
Regulação para Baixo , Osteonectina/biossíntese , Neoplasias da Próstata/metabolismo , Células Estromais/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Chaperona BiP do Retículo Endoplasmático/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Neuroendócrinas/metabolismo , Transgenes , Microambiente TumoralRESUMO
We previously developed murine and chimeric antibodies against a specific epithelial ovarian carcinoma (EOC) marker, named folate receptor (FR), and promising results were obtained in phase II trials. More recently, we successfully generated a completely human Fab fragment, C4, by conversion of one of the murine anti-FR antibodies to human antibody using phage display and guided selection. However, subsequent efforts to obtain C4 in a dimer format, which seems especially desirable for EOC locoregional treatment, resulted in a highly heterogeneous product upon natural dimerization and in a very poor production yield upon chemical dimerization by a non-hydrolyzable linker to a di-Fab-maleimide (DFM). We therefore designed, constructed and characterized a large Fab dual combinatorial human antibody phage display library obtained from EOC patients and potentially biased toward an anti-tumor response in an effort to obtain new anti-FR human antibodies suitable for therapy. Using this library and guiding the selection on FR-expressing cells with murine/human antibody chains, we generated four new human anti-FR antibody (AFRA) Fab fragments, one of which was genetically and chemically manipulated to obtain a chemical dimer, designated AFRA-DFM5.3, with high yield production and the capability for purification scaled-up to clinical grade. Overall affinity of AFRA-DFM5.3 was in the 2-digit nanomolar range, and immunohistochemistry indicated that the reagent recognized the FR expressed on EOC samples. (131)I-AFRA-DFM5.3 showed high immunoreactivity, in vitro stability and integrity, and specifically accumulated only in FR-expressing tumors in subcutaneous preclinical in vivo models. Overall, our studies demonstrate the successful conversion of murine to completely human anti-FR antibodies through the combined use of antibody phage display libraries biased toward an anti-tumor response, guided selection and chain shuffling, and point to the suitability of AFRA5.3 for future clinical application in ovarian cancer.
Assuntos
Anticorpos/química , Especificidade de Anticorpos , Proteínas de Transporte/imunologia , Imunoterapia/métodos , Neoplasias Ovarianas/imunologia , Receptores de Superfície Celular/imunologia , Proteínas Recombinantes de Fusão/síntese química , Animais , Anticorpos/genética , Anticorpos/imunologia , Dimerização , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Receptores de Folato com Âncoras de GPI , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imuno-Histoquímica , Camundongos , Biblioteca de Peptídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
PURPOSE: The role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them. RESULTS: Compared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 µg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 µg/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells). CONCLUSIONS: These data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential.