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Immune checkpoint therapy has limited efficacy for patients with bone-metastatic castration-resistant prostate cancer (bmCRPC). To improve immunotherapy for bmCRPC, we aimed to identify the mechanism of bmCRPC-induced changes in the immune microenvironment. Among bmCRPC patients, higher levels of a 32-gene M2-like macrophage signature in bone metastasis samples correlated with shorter overall survival. Immunohistochemistry showed that CD206-positive (CD206+) macrophages were enriched in bmCRPC bone biopsy specimens compared with primary tumors or lymph node metastases. In preclinical osteogenic prostate cancer (Pca) xenograft models, CD206+ macrophages were recruited to areas with tumor-induced bone. RNA sequencing (RNAseq) analysis showed higher expression of an M2-like gene signature, with activated canonical and noncanonical Wnt pathways, in tumor-associated macrophages isolated from osteogenic tumors (bone-TAMs) than in TAMs isolated from nonosteogenic tumors (ctrl-TAMs). Mechanistic studies showed that endothelial cells (ECs) that had undergone EC-to-osteoblast (EC-to-OSB) transition, the precursors of tumor-induced OSBs, produced paracrine factors, including Wnts, CXCL14, and lysyl oxidase, which induced M2 polarization and recruited M2-like TAMs to the bone-tumor microenvironment (bone-TME). Bone-TAMs suppressed CD8+ T cells' proliferation and cytolytic activity, and these effects were partially reversed by treating bone-TAMs with Wnt inhibitors. Genetic or pharmacological inhibition of Pca-induced EC-to-OSB transition reduced the levels of M2-like macrophages in osteogenic tumors. Our study demonstrates that Pca-induced EC-to-OSB transition drives immunosuppression in the bone-TME, suggesting that therapies that reduce Pca-induced bone formation may improve immunotherapeutic outcomes for bmCRPC.
Assuntos
Neoplasias Ósseas , Células Endoteliais , Macrófagos , Osteoblastos , Microambiente Tumoral , Via de Sinalização Wnt , Masculino , Microambiente Tumoral/imunologia , Humanos , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/secundário , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Animais , Camundongos , Macrófagos/metabolismo , Macrófagos/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/imunologia , Osteoblastos/metabolismo , Osteoblastos/imunologia , Neoplasias de Próstata Resistentes à Castração/imunologia , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Linhagem Celular Tumoral , Neoplasias da Próstata/patologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/imunologiaRESUMO
OBJECTIVE: MicroRNA (miRNA) expression profile can be used as prognostic marker for human cancers. We aim to explore the significance of miRNAs in colorectal cancer (CRC) metastasis. DESIGN: We performed miRNA microarrays using primary CRC tissues from patients with and without metastasis, and validated selected candidates in 85 CRC samples by quantitative real-time PCR (qRT-PCR). We tested metastatic activity of selected miRNAs and identified miRNA targets by prediction algorithms, qRT-PCR, western blot and luciferase assays. Clinical outcomes were analysed in six sets of CRC cases (n=449), including The Cancer Genome Atlas (TCGA) consortium and correlated with miR-224 status. We used the Kaplan-Meier method and log-rank test to assess the difference in survival between patients with low or high levels of miR-224 expression. RESULTS: MiR-224 expression increases consistently with tumour burden and microsatellite stable status, and miR-224 enhances CRC metastasis in vitro and in vivo. We identified SMAD4 as a miR-224 target and observed negative correlation (Spearman Rs=-0.44, p<0.0001) between SMAD4 and miR-224 expression in clinical samples. Patients with high miR-224 levels display shorter overall survival in multiple CRC cohorts (p=0.0259, 0.0137, 0.0207, 0.0181, 0.0331 and 0.0037, respectively), and shorter metastasis-free survival (HR 6.51, 95% CI 1.97 to 21.51, p=0.0008). In the TCGA set, combined analysis of miR-224 with SMAD4 expression enhanced correlation with survival (HR 4.12, 95% CI 1.1 to 15.41, p=0.0175). CONCLUSIONS: MiR-224 promotes CRC metastasis, at least in part, through the regulation of SMAD4. MiR-224 expression in primary CRC, alone or combined with its targets, may have prognostic value for survival of patients with CRC.
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Adenocarcinoma/diagnóstico , Biomarcadores Tumorais/sangue , Neoplasias Colorretais/diagnóstico , MicroRNAs/sangue , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Áustria , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas In Vitro , Itália , Estimativa de Kaplan-Meier , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Valor Preditivo dos Testes , Romênia , Sensibilidade e Especificidade , Reino UnidoRESUMO
Radium 223 (Ra-223) is an α-emitting bone-homing radiopharmaceutical that targets tumor-induced osteoblasts and is used to reduce bone pain and prolong overall survival in men with bone-metastatic, castrate-resistant prostate cancer. However, increased fracture risk in skeletal sites with no bone metastasis has been observed in patients treated with Ra-223. Both luciferase- or green fluorescence protein (GFP)-labeled osteoblast reporter mice were used to monitor the effect of Ra-223 on resident osteoblasts and normal bone structure. Upon Ra-223 treatment, 70% of resident osteoblasts were reduced within 2 days, and the osteoblast reduction lasted for at least 18 weeks without detectable recovery, as measured by in vivo bioluminescent imaging. In GFP-labeled osteoblast reporter mice, Ra-223 mainly reduced osteoblasts localized in the trabecular bone areas; the osteoblasts in the growth plates were less affected. Micro-computed tomography analyses showed that Ra-223 significantly reduced bone mineral density and bone microstructure in the trabecular area of femurs but not in the cortical bone. Tumor-induced bone was generated by inoculating osteogenic TRAMP-BMP4 prostate cancer cells into the mouse femurs; Ra-223 treatment significantly reduced tumor-induced osteoblasts. Our study shows that Ra-223 affects bone structures that are not involved in bone metastasis. Strategies that improve bone health may reduce fracture risk in patients receiving Ra-223.
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Renal cell carcinoma (RCC) bone metastatis progression is driven by crosstalk between tumor cells and the bone microenvironment, which includes osteoblasts, osteoclasts, and osteocytes. RCC bone metastases (RCCBM) are predominantly osteolytic and resistant to antiresorptive therapy. The molecular mechanisms underlying pathologic osteolysis and disruption of bone homeostasis remain incompletely understood. We previously reported that BIGH3/TGFBI (transforming growth factor-beta-induced protein ig-h3, shortened to BIGH3 henceforth) secreted by colonizing RCC cells drives osteolysis by inhibiting osteoblast differentiation, impairing healing of osteolytic lesions, which is reversible with osteoanabolic agents. Here, we report that BIGH3 induces osteocyte apoptosis in both human RCCBM tissue specimens and in a preclinical mouse model. We also demonstrate that BIGH3 reduces Cx43 expression, blocking gap junction (GJ) function and osteocyte network communication. BIGH3-mediated GJ inhibition is blocked by the lysosomal inhibitor hydroxychloroquine (HCQ), but not osteoanabolic agents. Our results broaden the understanding of pathologic osteolysis in RCCBM and indicate that targeting the BIGH3 mechanism could be a combinational strategy for the treatment of RCCBM-induced bone disease that overcomes the limited efficacy of antiresorptives that target osteoclasts.
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Apoptose , Neoplasias Ósseas , Carcinoma de Células Renais , Proteínas da Matriz Extracelular , Junções Comunicantes , Neoplasias Renais , Osteócitos , Osteócitos/metabolismo , Osteócitos/patologia , Humanos , Animais , Neoplasias Ósseas/secundário , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/tratamento farmacológico , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/secundário , Apoptose/efeitos dos fármacos , Neoplasias Renais/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/tratamento farmacológico , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Progressão da Doença , Conexina 43/metabolismo , Linhagem Celular Tumoral , Fator de Crescimento Transformador beta/metabolismo , Osteólise/patologia , Osteólise/metabolismo , FemininoRESUMO
The receptor tyrosine kinase, MET, has been implicated in tumorigenesis and metastasis of many solid tumors, by multiple mechanisms, including cross talk with epidermal growth factor receptor. In this study, we examined the role of insulin-like growth factor receptor-1 (IGF-1R) signaling in MET activation, focusing on prostate cancer cells. Stimulation of the prostate cancer cell line PC3 with IGF-1 induces a delayed phosphorylation of MET at multiple sites (indicative of full activation), reaching a maximum 18 hr after IGF-1 addition. MET activation does not require the sole MET ligand hepatocyte growth factor (HGF), but does require transcription to occur. Furthermore, direct injection of IGF-1 is sufficient to induce MET activation in vivo, in a PC3 xenograft model. Pharmacologic or genetic inhibition of the tyrosine kinase, Src, abolishes MET phosphorylation, and expression of activated Src is sufficient to induce Met phosphorylation in the absence of IGF-1 stimulation. Activated MET is essential for IGF-1-mediated increased migration of PC3 cells, demonstrating an important biologic effect of IGF-1-mediated MET activation. Finally, we demonstrate that IGF-1-induced delayed MET activation occurs in multiple cell lines which express both the receptors, suggesting that IGF-1R-mediated MET activation may contribute to tumorigenic properties of multiple cancer types when both growth factor receptors are expressed. The results further suggest that MET may be activated by multiple receptor tyrosine kinase receptors, and dual targeting of these receptors may be important therapeutically.
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Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor IGF Tipo 1/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Ativação Enzimática , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Integrinas/metabolismo , Ligantes , Masculino , Camundongos , Transplante de Neoplasias , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Receptor IGF Tipo 1/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
BACKGROUND: Dasatinib, an inhibitor of Src-family kinases, combined with docetaxel in men with castrate-resistant prostate cancer (CRPC), affects bone turnover markers in a phase I/II clinical trial in metastatic CRPC. Only a subset of men benefit from this therapy, and predictive markers are lacking. We hypothesized a role for insulin-like growth factor-1 (IGF-1) as a predictive marker, since IGF-1 is important in both prostate cancer progression and bone development. Hence, we determined the association of IGF-1 expression to treatment response, and whether this expression resulted from tumor cells, the microenvironment, or their interactions. METHODS: We measured serum IGF-1 levels in men with CRPC treated with dasatinib plus docetaxel. To investigate the source of IGF-1, we utilized two different mouse models harboring human prostate cancer cells, and used species-specific IGF-1 ELISA kits (mouse vs. human). RESULTS: In men with CRPC, an increase in IGF-1 levels after one cycle of treatment with dasatinib and docetaxel is associated with a higher response rate and longer duration of treatment. Xenograft experiments with subcutaneous and intratibial injection of prostate cancer cells suggest that direct interaction of prostate cancer cells with bone microenvironment is necessary for IGF-1 induction, is entirely host-derived, and occurs only in mice that respond to dasatinib-based therapy. CONCLUSION: Our results support a role for serum IGF-1 as a potential biomarker for benefit from dasatinib-based combination treatments in CRPC.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/biossíntese , Fator de Crescimento Insulin-Like I/biossíntese , Neoplasias da Próstata/sangue , Neoplasias da Próstata/tratamento farmacológico , Células 3T3 , Animais , Biomarcadores Tumorais/sangue , Linhagem Celular Tumoral , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Dasatinibe , Docetaxel , Ensaio de Imunoadsorção Enzimática , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Camundongos Nus , Neoplasias Hormônio-Dependentes/sangue , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Osteoblastos/metabolismo , Pirimidinas/administração & dosagem , Taxoides/administração & dosagem , Tiazóis/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metastatic prostate cancer (PCa) in bone induces bone-forming lesions. We have previously shown that PCa-induced bone originates from endothelial cells (ECs) that have undergone EC-to-osteoblast (OSB) transition. Here, we investigated whether EC-to-OSB transition also occurs during normal bone formation. We developed an EC and OSB dual-color reporter mouse (DRM) model that marks EC-OSB hybrid cells with red and green fluorescent proteins. We observed EC-to-OSB transition (RFP and GFP co-expression) in both endochondral and intramembranous bone formation during embryonic development and in adults. Co-expression was confirmed in cells isolated from DRM. Bone marrow- and lung-derived ECs underwent transition to OSBs and mineralization in osteogenic medium. RNA-sequencing revealed GATA family transcription factors were upregulated in EC-OSB hybrid cells and knockdown of GATA3 inhibited BMP4-induced mineralization. Our findings support that EC-to-OSB transition occurs during normal bone development and suggest a new paradigm regarding the endothelial origin of OSBs.
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Disease progression following androgen ablation was shown to be associated with upregulation of the glucocorticoid receptor (GR). Longitudinal monitoring of GR expression in circulating extracellular vesicles (EV) may reflect changes in the tumor cell and facilitates detection of acquired resistance. We utilized LNCaP, LREX cells and a patient-derived xenograft, MDA PDX 322-2-6a, for in vitro and in vivo experiments. Plasma-derived EVs were isolated from patients with localized high-risk prostate cancer undergoing androgen ablation. The mRNA levels of GR in EVs and their responsive genes were detected by transcriptome analysis, qRT-PCR and the protein levels by Western blot analysis. We detected changes in GR expression at mRNA and protein levels in EVs derived from LNCaP and LREX cells in in vitro studies. In in vivo experiments, LNCaP and the PDX MDA 322-2-6a-bearing mice were treated with enzalutamide. GR levels in plasma-derived EVs were increased only in those tumors that did not respond to enzalutamide. Treatment of mice bearing enzalutamide-resistant tumors with a GR inhibitor in combination with enzalutamide led to a transient pause in tumor growth in a subset of tumors and decreased GR levels intracellular and in plasma-derived EVs. In a subgroup of patients with high-risk localized prostate cancer treated with androgen signaling inhibition, GR was found upregulated in matching tissue and plasma EVs. These analyses showed that GR levels in plasma-derived EVs may be used for monitoring the transition of GR expression allowing for early detection of resistance to androgen ablation treatment. SIGNIFICANCE: Longitudinal monitoring of GR expression in plasma-derived EVs from patients with prostate cancer treated with androgen signaling inhibitors facilitates early detection of acquisition of resistance to androgen receptor signaling inhibition in individual patients.
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Biomarcadores , Resistencia a Medicamentos Antineoplásicos , Vesículas Extracelulares , Neoplasias da Próstata , Receptores de Glucocorticoides , Receptores de Glucocorticoides/sangue , Receptores de Glucocorticoides/genética , Vesículas Extracelulares/metabolismo , Biomarcadores/sangue , Transdução de Sinais , Humanos , Animais , Camundongos , Masculino , Linhagem Celular Tumoral , Feniltioidantoína/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Mifepristona/farmacologiaRESUMO
BACKGROUND: Prostate cancer (PCa) typically spreads to the bone, and this distribution is attributed to the central role of the microenvironment in progression. However, metastasis to the adrenal glands, while not as common, does occur. The biology that accounts for adrenal metastases may be attributed to the unique local steroid metabolome and co-clinical characterization may elucidate the role steroid biosynthesis plays in PCa progression. METHODS: Three patients with metastatic PCa who had archived tumor tissue from an adrenalectomy were retrospectively identified, and one adrenal metastasis was developed into a xenograft (MDA-PCa-250). The adrenal metastases were characterized by performing somatic DNA whole exome sequencing (WES), RNA-Seq, immunohistochemistry (IHC), and steroid metabolite quantitation. The influence of steroid metabolites on adrenal metastasis cells and tumor growth was tested in vitro and in vivo. RESULTS: Clinically, adrenalectomy was performed during castration-resistant oligometastatic disease, and two men experienced resensitization to leuprolide. Somatic DNA WES revealed heterogeneous alterations in tumor suppressor and DNA damage repair pathway genes. Adrenal metastases had active androgen receptor (AR) signaling by IHC, and RNA-Seq supported a potential role for adrenal androgen precursor metabolism in activating the AR. Steroid quantitation suggested the adrenal androgen precursors were converted into testosterone in these metastases, and stable isotope tracing of an organoid from MDA-PCa-250 confirmed the capability of adrenal metastases to biosynthesize testosterone from adrenal precursors. In vitro testing of a cell line derived from MDA-PCa-250 showed that testosterone and cortisol stimulated tumor cell growth. In vivo experiments demonstrated that MDA-PCa-250 grew in intact mice with circulating testosterone, but not in castrated mice. CONCLUSIONS: PCa adrenal metastases depend upon AR signaling driven by androgen precursors, androstenedione and dehydroepiandrosterone, available in the microenvironment, despite the presence of heterogeneous somatic DNA alterations. Moreover, MDA-PCa-250 provides a preclinical model that can recapitulate the unique androgen-dependence of adrenal metastases. CLINICAL TRIAL REGISTRATION: This study does not report the clinical results of a clinical trial, but it does use samples from a completed clinical trial that is registered with clinicaltrials.gov (NCT01254864).
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Androgênios , Neoplasias da Próstata , Masculino , Humanos , Animais , Camundongos , Androgênios/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Estudos Retrospectivos , Esteroides/metabolismo , Testosterona/metabolismo , DNA , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
Immune checkpoint therapy has limited efficacy for patients with bone metastatic castrate-resistant prostate cancer (bmCRPC). In this study, we revealed a novel mechanism that may account for the relative resistance of bmCRPC to immune checkpoint therapy. We found that prostate cancer (PCa)-induced bone via endothelial-to-osteoblast (EC-to-OSB) transition causes an ingress of M2-like macrophages, leading to an immunosuppressive bone tumor microenvironment (bone-TME). Analysis of a bmCRPC RNA-seq dataset revealed shorter overall survival in patients with an M2-high versus M2-low signature. Immunohistochemical (IHC) analysis showed CD206 + M2-like macrophages were enriched in bmCRPC specimens compared with primary tumors or lymph node metastasis. In osteogenic PCa xenografts, CD206 + macrophages were enriched adjacent to tumor-induced bone. FACS analysis showed an increase in CD206 + cells in osteogenic tumors compared to non-osteogenic tumors. Genetic or pharmacological inhibition of the EC-to-OSB transition reduced aberrant bone and M2-like macrophages in osteogenic tumors. RNAseq analysis of tumor-associated macrophages from osteogenic (bone-TAMs) versus non-osteogenic (ctrl-TAMs) tumors showed high expression of an M2-like gene signature, canonical and non-canonical Wnt pathways, and a decrease in an M1-like gene signature. Isolated bone-TAMs suppressed T-cell proliferation while ctrl-TAMs did not. Mechanistically, EC-OSB hybrid cells produced paracrine factors, including Wnts, CXCL14 and LOX, which induced M2 polarization and recruited M2-like TAMs to bone-TME. Our study thus links the unique EC-to-OSB transition as an "upstream" event that drives "downstream" immunosuppression in the bone-TME. These studies suggest that therapeutic strategies that inhibit PCa-induced EC-to-OSB transition may reverse immunosuppression to promote immunotherapeutic outcomes in bmCRPC. Significance: The insight that prostate cancer-induced bone generates an immunosuppressive bone tumor microenvironment offers a strategy to improve responses to immunotherapy approaches in patients with bone metastatic castrate-resistant prostate cancer.
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Metastatic prostate cancer (PCa) in bone induces bone-forming lesions that enhance PCa progression. How tumor-induced bone formation enhances PCa progression is not known. We have previously shown that PCa-induced bone originates from endothelial cells (ECs) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition by tumor-secreted bone morphogenetic protein 4 (BMP4). Here, we show that EC-to-OSB transition leads to changes in the tumor microenvironment that increases the metastatic potential of PCa cells. We found that conditioned medium (CM) from EC-OSB hybrid cells increases the migration, invasion, and survival of PC3-mm2 and C4-2B4 PCa cells. Quantitative mass spectrometry (Isobaric Tags for Relative and Absolute Quantitation) identified Tenascin C (TNC) as one of the major proteins secreted from EC-OSB hybrid cells. TNC expression in tumor-induced OSBs was confirmed by immunohistochemistry of MDA PCa-118b xenograft and human bone metastasis specimens. Mechanistically, BMP4 increases TNC expression in EC-OSB cells through the Smad1-Notch/Hey1 pathway. How TNC promotes PCa metastasis was next interrogated by in vitro and in vivo studies. In vitro studies showed that a TNC-neutralizing antibody inhibits EC-OSB-CM-mediated PCa cell migration and survival. TNC knockdown decreased, while the addition of recombinant TNC or TNC overexpression increased migration and anchorage-independent growth of PC3 or C4-2b cells. When injected orthotopically, PC3-mm2-shTNC clones decreased metastasis to bone, while C4-2b-TNC-overexpressing cells increased metastasis to lymph nodes. TNC enhances PCa cell migration through α5ß1 integrin-mediated YAP/TAZ inhibition. These studies elucidate that tumor-induced stromal reprogramming generates TNC that enhances PCa metastasis and suggest that TNC may be a target for PCa therapy.
Assuntos
TenascinaRESUMO
Metastatic prostate cancer in the bone induces bone-forming lesions that contribute to progression and therapy resistance. Prostate cancer-induced bone formation originates from endothelial cells (EC) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition in response to tumor-secreted BMP4. Current strategies targeting prostate cancer-induced bone formation are lacking. Here, we show that activation of retinoic acid receptor (RAR) inhibits EC-to-OSB transition and reduces prostate cancer-induced bone formation. Treatment with palovarotene, an RARγ agonist being tested for heterotopic ossification in fibrodysplasia ossificans progressiva, inhibited EC-to-OSB transition and osteoblast mineralization in vitro and decreased tumor-induced bone formation and tumor growth in several osteogenic prostate cancer models, and similar effects were observed with the pan-RAR agonist all-trans-retinoic acid (ATRA). Knockdown of RARα, ß, or γ isoforms in ECs blocked BMP4-induced EC-to-OSB transition and osteoblast mineralization, indicating a role for all three isoforms in prostate cancer-induced bone formation. Furthermore, treatment with palovarotene or ATRA reduced plasma Tenascin C, a factor secreted from EC-OSB cells, which may be used to monitor treatment response. Mechanistically, BMP4-activated pSmad1 formed a complex with RAR in the nucleus of ECs to activate EC-to-OSB transition. RAR activation by palovarotene or ATRA caused pSmad1 degradation by recruiting the E3-ubiquitin ligase Smad ubiquitination regulatory factor1 (Smurf1) to the nuclear pSmad1/RARγ complex, thus blocking EC-to-OSB transition. Collectively, these findings suggest that palovarotene can be repurposed to target prostate cancer-induced bone formation to improve clinical outcomes for patients with bone metastasis. SIGNIFICANCE: This study provides mechanistic insights into how RAR agonists suppress prostate cancer-induced bone formation and offers a rationale for developing RAR agonists for prostate cancer bone metastasis therapy. See related commentary by Bhowmick and Bhowmick, p. 2975.
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Neoplasias Ósseas , Neoplasias da Próstata , Neoplasias Ósseas/metabolismo , Células Endoteliais/patologia , Humanos , Masculino , Osteoblastos/metabolismo , Neoplasias da Próstata/patologia , Receptores do Ácido Retinoico/metabolismo , Tretinoína/metabolismo , Tretinoína/farmacologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Clinical studies have shown that subsets of patients with cancer achieve a significant benefit from Aurora kinase inhibitors, suggesting an urgent need to identify biomarkers for predicting drug response. Chromodomain helicase DNA binding protein 1 (CHD1) is involved in chromatin remodeling, DNA repair, and transcriptional plasticity. Prior studies have demonstrated that CHD1 has distinct expression patterns in cancers with different molecular features, but its impact on drug responsiveness remains understudied. Here, we show that CHD1 promotes the susceptibility of prostate cancer cells to inhibitors targeting Aurora kinases, while depletion of CHD1 impairs their efficacy in vitro and in vivo. Pan-cancer drug sensitivity analyses revealed that high expression of CHD1 was associated with increased sensitivity to Aurora kinase A (AURKA) inhibitors. Mechanistically, KPNA2 served as a direct target of CHD1 and suppressed the interaction of AURKA with the coactivator TPX2, thereby rendering cancer cells more vulnerable to AURKA inhibitors. Consistent with previous research reporting that loss of PTEN elevates CHD1 levels, studies in a genetically engineered mouse model, patient-derived organoids, and patient samples showed that PTEN defects are associated with a better response to AURKA inhibition in advanced prostate cancer. These observations demonstrate that CHD1 plays an important role in modulating Aurora kinases and drug sensitivities, providing new insights into biomarker-driven therapies targeting Aurora kinases for future clinical studies. SIGNIFICANCE: CHD1 plays a critical role in controlling AURKA activation and promoting Aurora kinase inhibitor sensitivity, providing a potential clinical biomarker to guide cancer treatment.
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Aurora Quinase A , Proteínas de Ciclo Celular , DNA Helicases , Proteínas de Ligação a DNA , Proteínas Associadas aos Microtúbulos , Neoplasias da Próstata , Animais , Antineoplásicos , Aurora Quinase A/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Humanos , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Inibidores de Proteínas Quinases/farmacologiaRESUMO
A fraction of patients undergoing androgen deprivation therapy (ADT) for advanced prostate cancer (PCa) will develop recurrent castrate-resistant PCa (CRPC) in bone. Strategies to prevent CRPC relapse in bone are lacking. Here we show that the cholesterol-lowering drugs statins decrease castration-induced bone marrow adiposity in the tumor microenvironment and reduce PCa progression in bone. Using primary bone marrow stromal cells (BMSC) and M2-10B4 cells, we showed that ADT increases bone marrow adiposity by enhancing BMSC-to-adipocyte transition in vitro. Knockdown of androgen receptor abrogated BMSC-to-adipocyte transition, suggesting an androgen receptor-dependent event. RNAseq analysis showed that androgens reduce the secretion of adipocyte hormones/cytokines including leptin during BMSC-to-adipocyte transition. Treatment of PCa C4-2b, C4-2B4, and PC3 cells with leptin led to an increase in cell cycle progression and nuclear Stat3. RNAseq analysis also showed that androgens inhibit cholesterol biosynthesis pathway, raising the possibility that inhibiting cholesterol biosynthesis may decrease BMSC-to-adipocyte transition. Indeed, statins decreased BMSC-to-adipocyte transition in vitro and castration-induced bone marrow adiposity in vivo. Statin pre-treatment reduced 22RV1 PCa progression in bone after ADT. Our findings with statin may provide one of the mechanisms to the clinical correlations that statin use in patients undergoing ADT seems to delay progression to "lethal" PCa.
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Inibidores de Hidroximetilglutaril-CoA Redutases , Adiposidade , Humanos , Masculino , Neoplasias da PróstataRESUMO
Cell type transition occurs during normal development and under pathological conditions. In prostate cancer bone metastasis, prostate cancer-secreted BMP4 induces endothelial cell-to-osteoblast (EC-to-OSB) transition. Such tumor-induced stromal reprogramming supports prostate cancer progression. We delineate signaling pathways mediating EC-to-OSB transition using EC lines 2H11 and SVR. We found that BMP4-activated pSmad1-Notch-Hey1 pathway inhibits EC migration and tube formation. BMP4-activated GSK3ß-ßcatenin-Slug pathway stimulates Osx expression. In addition, pSmad1-regulated Dlx2 converges with the Smad1 and ß-catenin pathways to stimulate osteocalcin expression. By co-expressing Osx, Dlx2, Slug and Hey1, we were able to achieve EC-to-OSB transition, leading to bone matrix mineralization in the absence of BMP4. In human prostate cancer bone metastasis specimens and MDA-PCa-118b and C4-2b-BMP4 osteogenic xenografts, immunohistochemical analysis showed that ß-catenin and pSmad1 are detected in activated osteoblasts rimming the tumor-induced bone. Our results elucidated the pathways and key molecules coordinating prostate cancer-induced stromal programming and provide potential targets for therapeutic intervention.
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Yes-associated protein 1 (YAP1), a key player in the Hippo pathway, has been shown to play a critical role in tumor progression. However, the role of YAP1 in prostate cancer cell invasion, migration, and metastasis is not well defined. Through functional, transcriptomic, epigenomic, and proteomic analyses, we showed that prolyl hydroxylation of YAP1 plays a critical role in the suppression of cell migration, invasion, and metastasis in prostate cancer. Knockdown (KD) or knockout (KO) of YAP1 led to an increase in cell migration, invasion, and metastasis in prostate cancer cells. Microarray analysis showed that the EMT pathway was activated in Yap1-KD cells. ChIP-seq analysis showed that YAP1 target genes are enriched in pathways regulating cell migration. Mass spectrometry analysis identified P4H prolyl hydroxylase in the YAP1 complex and YAP1 was hydroxylated at multiple proline residues. Proline-to-alanine mutations of YAP1 isoform 3 identified proline 174 as a critical residue, and its hydroxylation suppressed cell migration, invasion, and metastasis. KO of P4ha2 led to an increase in cell migration and invasion, which was reversed upon Yap1 KD. Our study identified a novel regulatory mechanism of YAP1 by which P4HA2-dependent prolyl hydroxylation of YAP1 determines its transcriptional activities and its function in prostate cancer metastasis.
Assuntos
Prolil Hidroxilases/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas de Sinalização YAP/metabolismo , Animais , Movimento Celular/fisiologia , Humanos , Masculino , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias da Próstata/patologia , Proteínas de Sinalização YAP/antagonistas & inibidoresRESUMO
PURPOSE: Radium-223 prolongs survival in a fraction of men with bone metastatic prostate cancer (PCa). However, there are no markers for monitoring response and resistance to Radium-223 treatment. Exosomes are mediators of intercellular communication and may reflect response of the bone microenvironment to Radium-223 treatment. We performed molecular profiling of exosomes and compared the molecular profile in patients with favorable and unfavorable overall survival. EXPERIMENTAL DESIGN: We performed exosomal transcriptome analysis in plasma derived from our preclinical models (MDA-PCa 118b tumors, TRAMP-C2/BMP4 PCa) and from the plasma of 25 patients (paired baseline and end of treatment) treated with Radium-223. All samples were run in duplicate, and array data analyzed with fold changes +2 to -2 and P < 0.05. RESULTS: We utilized the preclinical models to establish that genes derived from the tumor and the tumor-associated bone microenvironment (bTME) are differentially enriched in plasma exosomes upon Radium-223 treatment. The mouse transcriptome analysis revealed changes in bone-related and DNA damage repair-related pathways. Similar findings were observed in plasma-derived exosomes from patients treated with Radium-223 detected changes. In addition, exosomal transcripts detected immune-suppressors (e.g., PD-L1) that were associated with shorter survival to Radium-223. Treatment of the Myc-CaP mouse model with a combination of Radium-223 and immune checkpoint therapy (ICT) resulted in greater efficacy than monotherapy. CONCLUSIONS: These clinical and coclinical analyses showed that RNA profiling of plasma exosomes may be used for monitoring the bTME in response to treatment and that ICT may be used to increase the efficacy of Radium-223.
Assuntos
Neoplasias Ósseas/secundário , Vesículas Extracelulares/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Proteínas de Checkpoint Imunológico/genética , Proteínas de Checkpoint Imunológico/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Compostos Radiofarmacêuticos/farmacologia , Compostos Radiofarmacêuticos/uso terapêutico , Rádio (Elemento)/farmacologia , Rádio (Elemento)/uso terapêutico , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Animais , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Exossomos/genética , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Neoplasias da Próstata/mortalidade , RNA/genética , Taxa de SobrevidaRESUMO
Renal cell carcinoma bone metastases (RCCBM) are typically osteolytic. We previously showed that BIGH3 (beta Ig-h3/TGFBI), secreted by 786-O renal cell carcinoma, plays a role in osteolytic bone lesion in RCCBM through inhibition of osteoblast (OSB) differentiation. To study this interaction, we employed three-dimensional (3D) hydrogels to coculture bone-derived 786-O (Bo-786) renal cell carcinoma cells with MC3T3-E1 pre-OSBs. Culturing pre-OSBs in the 3D hydrogels preserved their ability to differentiate into mature OSB; however, this process was decreased when pre-OSBs were cocultured with Bo-786 cells. Knockdown of BIGH3 in Bo-786 cells recovered OSB differentiation. Furthermore, treatment with bone morphogenetic protein 4, which stimulates OSB differentiation, or cabozantinib (CBZ), which inhibits VEGFR1 and MET tyrosine kinase activities, also increased OSB differentiation in the coculture. CBZ also inhibited pre-osteoclast RAW264.7 cell differentiation. Using RCCBM mouse models, we showed that CBZ inhibited Bo-786 tumor growth in bone. CBZ treatment also increased bone volume and OSB number, and decreased osteoclast number and blood vessel density. When tested in SN12PM6 renal cell carcinoma cells that have been transduced to overexpress BIGH3, CBZ also inhibited SN12PM6 tumor growth in bone. These observations suggest that enhancing OSB differentiation could be one of the therapeutic strategies for treating RCCBM that exhibit OSB inhibition characteristics, and that this 3D coculture system is an effective tool for screening osteoanabolic agents for further in vivo studies.
Assuntos
Anilidas/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Carcinoma de Células Renais/tratamento farmacológico , Diferenciação Celular , Neoplasias Renais/tratamento farmacológico , Osteoblastos/citologia , Osteólise/tratamento farmacológico , Piridinas/farmacologia , Animais , Apoptose , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Proliferação de Células , Técnicas de Cocultura , Humanos , Técnicas In Vitro , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos SCID , Osteoblastos/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genetic inactivation of PTEN is common in prostate cancer and correlates with poorer prognosis. We previously identified CHD1 as an essential gene in PTEN-deficient cancer cells. Here, we sought definitive in vivo genetic evidence for, and mechanistic understanding of, the essential role of CHD1 in PTEN-deficient prostate cancer. In Pten and Pten/Smad4 genetically engineered mouse models, prostate-specific deletion of Chd1 resulted in markedly delayed tumor progression and prolonged survival. Chd1 deletion was associated with profound tumor microenvironment (TME) remodeling characterized by reduced myeloid-derived suppressor cells (MDSC) and increased CD8+ T cells. Further analysis identified IL6 as a key transcriptional target of CHD1, which plays a major role in recruitment of immunosuppressive MDSCs. Given the prominent role of MDSCs in suppressing responsiveness to immune checkpoint inhibitors (ICI), our genetic and tumor biological findings support combined testing of anti-IL6 and ICI therapies, specifically in PTEN-deficient prostate cancer. SIGNIFICANCE: We demonstrate a critical role of CHD1 in MDSC recruitment and discover CHD1/IL6 as a major regulator of the immunosuppressive TME of PTEN-deficient prostate cancer. Pharmacologic inhibition of IL6 in combination with immune checkpoint blockade elicits robust antitumor responses in prostate cancer.This article is highlighted in the In This Issue feature, p. 1241.
Assuntos
Proteínas de Ligação a DNA/metabolismo , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Evasão Tumoral/genética , Microambiente Tumoral/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Masculino , Camundongos Transgênicos , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Proteína Smad4/genética , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Bone metastasis is common in renal cell carcinoma (RCC), and the lesions are mainly osteolytic. The mechanism of bone destruction in RCC bone metastasis is unknown. METHODS: We used a direct intrafemur injection of mice with bone-derived 786-O RCC cells (Bo-786) as an in vivo model to study if inhibition of osteoblast differentiation is involved in osteolytic bone lesions in RCC bone metastasis. RESULTS: We showed that bone-derived Bo-786 cells induced osteolytic bone lesions in the femur of mice. We examined the effect of conditioned medium of Bo-786 cells (Bo-786 CM) on both primary mouse osteoblasts and MC3T3-E1 preosteoblasts and found that Bo-786 CM inhibited osteoblast differentiation. Secretome analysis of Bo-786 CM revealed that BIGH3 (Beta ig h3 protein), also known as TGFBI (transforming growth factor beta-induced protein), is highly expressed. We generated recombinant BIGH3 and found that BIGH3 inhibited osteoblast differentiation in vitro. In addition, CM from Bo-786 BIGH3 knockdown cells (786-BIGH3 KD) reduced the inhibition of osteoblast differentiation compared to CM from vector control. Intrafemural injection of mice with 786-BIGH3 KD cells showed a reduction in osteolytic bone lesions compared to vector control. Immunohistochemical staining of 18 bone metastasis specimens from human RCC showed strong BIGH3 expression in 11/18 (61%) and moderate BIGH3 expression in 7/18 (39%) of the specimens. CONCLUSIONS: These results suggest that suppression of osteoblast differentiation by BIGH3 is one of the mechanisms that enhance osteolytic lesions in RCC bone metastasis, and raise the possibilty that treatments that increase bone formation may improve therapy outcomes.