RESUMO
BACKGROUND: Long-term prognosis remains poor for colorectal cancer (CRC) patients with advanced disease due to treatment resistance. The identification of novel targets is essential for the development of new therapeutic approaches. GPR56, an adhesion GPCR, is highly expressed in CRC tumours and correlates with poor survival. Here, we describe the generation and preclinical evaluation of a novel ADC consisting of an anti-GPR56 antibody (10C7) conjugated with the DNA-damaging payload duocarmycin. METHODS: RNA-seq dataset analysis was performed to determine GPR56 expression in CRC subtypes. The specificity of binding, epitope mapping, and internalisation of 10C7 was examined. 10C7 was conjugated to payload and ADC cytotoxicity was assessed against a panel of CRC cell lines and tumour organoids. Antitumour efficacy was evaluated in xenograft models of CRC cell lines and patient-derived tumours. RESULTS: High GPR56 was shown to be associated with the microsatellite stable (MSS) subtype that accounts for 80-85% of CRC. GPR56 ADC selectively induced cytotoxicity in CRC cells and tumour organoids at low nanomolar potency in a GPR56-dependent manner and showed significant antitumour efficacy against GPR56-expressing xenograft models. CONCLUSIONS: This study provides the rationale for the future development of a GPR56-targeted ADC approach to potentially treat a large fraction of MSS CRC patients.
Assuntos
Neoplasias Colorretais , Imunoconjugados , Humanos , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Prognóstico , Receptores Acoplados a Proteínas G/metabolismoRESUMO
OBJECTIVE: Enhancer aberrations are beginning to emerge as a key epigenetic feature of colorectal cancers (CRC), however, a comprehensive knowledge of chromatin state patterns in tumour progression, heterogeneity of these patterns and imparted therapeutic opportunities remain poorly described. DESIGN: We performed comprehensive epigenomic characterisation by mapping 222 chromatin profiles from 69 samples (33 colorectal adenocarcinomas, 4 adenomas, 21 matched normal tissues and 11 colon cancer cell lines) for six histone modification marks: H3K4me3 for Pol II-bound and CpG-rich promoters, H3K4me1 for poised enhancers, H3K27ac for enhancers and transcriptionally active promoters, H3K79me2 for transcribed regions, H3K27me3 for polycomb repressed regions and H3K9me3 for heterochromatin. RESULTS: We demonstrate that H3K27ac-marked active enhancer state could distinguish between different stages of CRC progression. By epigenomic editing, we present evidence that gains of tumour-specific enhancers for crucial oncogenes, such as ASCL2 and FZD10, was required for excessive proliferation. Consistently, combination of MEK plus bromodomain inhibition was found to have synergistic effects in CRC patient-derived xenograft models. Probing intertumour heterogeneity, we identified four distinct enhancer subtypes (EPIgenome-based Classification, EpiC), three of which correlate well with previously defined transcriptomic subtypes (consensus molecular subtypes, CMSs). Importantly, CMS2 can be divided into two EpiC subgroups with significant survival differences. Leveraging such correlation, we devised a combinatorial therapeutic strategy of enhancer-blocking bromodomain inhibitors with pathway-specific inhibitors (PARPi, EGFRi, TGFßi, mTORi and SRCi) for EpiC groups. CONCLUSION: Our data suggest that the dynamics of active enhancer underlies CRC progression and the patient-specific enhancer patterns can be leveraged for precision combination therapy.
Assuntos
Cromatina , Neoplasias Colorretais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Elementos Facilitadores Genéticos/genética , Humanos , Proteínas Nucleares , Fatores de Transcrição/genéticaRESUMO
GPR56 is a member of the adhesion G-protein-coupled receptor family shown to play important roles in cell adhesion, brain development, immune function, and tumorigenesis. GPR56 is highly upregulated in colorectal cancer and correlates with poor prognosis. Several studies have shown GPR56 couples to the Gα12/13 class of heterotrimeric G-proteins to promote RhoA activation. However, due to its structural complexity and lack of a high-affinity receptor-specific ligand, the complete GPR56 signaling mechanism remains largely unknown. To delineate the activation mechanism and intracellular signaling functions of GPR56, we generated a monoclonal antibody (mAb) that binds with high affinity and specificity to the extracellular domain (ECD). Using deletion mutants, we mapped the mAb binding site to the GAIN domain, which mediates membrane-proximal autoproteolytic cleavage of the ECD. We showed that GPR56 overexpression in 293T cells leads to increased phosphorylation of Src, Fak, and paxillin adhesion proteins and activation of the Gα12/13-RhoA-mediated serum response factor (SRF) pathway. Treatment with the mAb potentiated Src-Fak phosphorylation, RhoA-SRF signaling, and cell adhesion. Consistently, GPR56 knockdown in colorectal cancer cells decreased Src-Fak pathway phosphorylation and cell adhesion. Interestingly, GPR56-mediated activation of Src-Fak phosphorylation occurred independent of RhoA, yet mAb-induced potentiation of RhoA-SRF signaling was Src-dependent. Furthermore, we show that the C-terminal portion of the Serine-Threonine-Proline-rich (STP) region, adjacent to the GAIN domain, was required for Src-Fak activation. However, autoproteolytic cleavage of the ECD was dispensable. These data support a new ECD-dependent mechanism by which GPR56 functions to regulate adhesion through activation of Src-Fak signaling.
Assuntos
Neoplasias Colorretais/genética , Quinase 1 de Adesão Focal/genética , Receptores Acoplados a Proteínas G/genética , Fator de Resposta Sérica/genética , Proteína rhoA de Ligação ao GTP/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Carcinogênese/genética , Adesão Celular/imunologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Quinase 1 de Adesão Focal/imunologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Paxilina/genética , Paxilina/imunologia , Fosforilação/genética , Receptores Acoplados a Proteínas G/imunologia , Fator de Resposta Sérica/imunologia , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/imunologia , Quinases da Família src/genética , Quinases da Família src/imunologiaRESUMO
The four R-spondins (RSPO1-4) strongly potentiate Wnt signaling and play critical roles in normal development, adult stem cell survival, and cancer development and aggressiveness. All four RSPOs have been suggested to potentiate Wnt signaling by binding to three related receptors, i.e. leucine-rich repeat-containing, G protein-coupled receptors 4, 5, and 6 (LGR4/5/6), and then inducing the clearance of two E3 ubiquitin ligases (RNF43 and ZNRF3) that otherwise would ubiquitinate Wnt receptors for degradation. Here, we show that RSPO1-4 have differential dependence on LGRs in potentiating Wnt/ß-catenin signaling and that RSPO2 can enhance this pathway without any LGR. LGR4 knockout (LGR4KO) in HEK293 cells completely abrogated the Wnt/ß-catenin signaling response to RSPO1 and RSPO4 and strongly impaired the response to RSPO3. RSPO2, however, retained robust activity albeit with decreased potency. Complete rescue of RSPO1-4 activity in LGR4KO cells required the seven-transmembrane domain of LGR4. Furthermore, an RSPO2 mutant with normal binding affinity to ZNRF3 but no or little binding to LGR4 or LGR5 still potentiated Wnt/ß-catenin signaling in vitro, supported the growth of intestinal organoids ex vivo, and stimulated intestinal crypt growth in vivo Mechanistically, RSPO2 could increase Wnt receptor levels in the absence of any LGR without affecting ZNRF3 endocytosis and stability. These findings suggest that RSPO1-4 use distinct mechanisms in regulating Wnt and other signaling pathways, which have important implications for understanding the pleiotropic functions of RSPOs and LGRs in both normal and cancer development.
Assuntos
Transdução de Sinais , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Organoides/citologia , Organoides/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/genética , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers. LGR5 activation by R-spondin (RSPO) ligands potentiates Wnt/ß-catenin signaling in vitro; however, deletion of LGR5 in stem cells has little or no effect on Wnt/ß-catenin signaling or cell proliferation in vivo Remarkably, modulation of LGR5 expression has a major impact on the actin cytoskeletal structure and cell adhesion in the absence of RSPO stimulation, but the molecular mechanism is unclear. Here, we show that LGR5 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), an effector of Rac1/CDC42 GTPases, in the regulation of actin cytoskeleton dynamics and cell-cell adhesion. Specifically, LGR5 decreased levels of IQGAP1 phosphorylation at Ser-1441/1443, leading to increased binding of Rac1 to IQGAP1 and thus higher levels of cortical F-actin and enhanced cell-cell adhesion. LGR5 ablation in colon cancer cells and crypt stem cells resulted in loss of cortical F-actin, reduced cell-cell adhesion, and disrupted localization of adhesion-associated proteins. No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQGAP1-Rac1 pathway to strengthen cell-cell adhesion in normal adult crypt stem cells and colon cancer cells.
Assuntos
Adesão Celular , Neoplasias do Colo/patologia , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Células CHO , Células Cultivadas , Neoplasias do Colo/metabolismo , Cricetulus , Células HEK293 , Humanos , Células-Tronco/metabolismoRESUMO
Targeted therapies hold great promise for cancer treatment and may exhibit even greater efficacy when combined with patient selection tools. The clinical impact of identifying likely responders includes reducing the number of unnecessary and ineffective therapies as well as more accurately determining drug effects. Positron emission tomography (PET) imaging using zirconium-89 radiolabeled monoclonal antibodies (mAbs), also referred to as zirconium-89 (89Zr)-immuno-PET, provides a potential biomarker to measure target expression and verify optimal delivery of targeted agents to tumors. Antibody-drug conjugates (ADCs) combine the high affinity and specificity of mAbs with the potency of cytotoxic drugs to target tumor-expressing antigen and destroy cancer cells. Thus, 89Zr-immuno-PET of whole-body biodistribution, pharmacokinetics, and tumor targeting of antibodies and ADCs to predict toxicity and efficacy could help guide individualized treatment. Here, we review how 89Zr-immuno-PET is being used as a companion diagnostic with the development of ADCs. Furthermore, we discuss how 89Zr-immuno-PET may be utilized in future clinical trials as an adjunct tool with novel ADCs to select cancer patients who have the greatest potential to benefit from treatment and improve ADC dosing regimens.
Assuntos
Imunoconjugados/metabolismo , Tomografia por Emissão de Pósitrons , Animais , Antígenos/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Neoplasias/diagnóstico por imagem , Neoplasias/patologiaRESUMO
Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is highly expressed in colorectal tumors and marks colon cancer stem cells that drive tumor growth and metastasis. Recently, we showed that LGR5 is a promising target for antibody-drug conjugate (ADC) therapy. However, it is important to identify LGR5-positive tumors that would respond to ADC treatment. Prior to drug conjugation, we evaluated two different anti-LGR5 monoclonal antibodies (mAbs), 8F2 and 9G5, using 89Zr-immunoPET to select the optimal mAb for ADC development and tumor imaging. Binding, specificity, and internalization were compared, and mAbs were prescreened as ADC candidates against colon cancer cells using secondary ADCs. Both mAbs demonstrated strong, specific binding in 293T-LGR5 cells but not 293T-vector cells. In DLD-1 colorectal cancer cells, which express high levels of LGR5, the mAbs rapidly internalized into lysosomes and promoted ADC-induced cytotoxicity, with 8F2 exhibiting slightly higher potency. No binding was detected in DLD-1-shLGR5 (LGR5 knockdown) cells. 89Zr-DFO-LGR5 mAbs were generated and shown to retain high affinity and LGR5-dependent uptake in vitro. PET/CT imaging of DLD-1 tumors was performed 5 days postinjection of 89Zr-DFO-LGR5 mAbs, and findings were consistent with biodistribution data, which showed significantly higher tumor uptake (%ID/g) for 89Zr-DFO-8F2 (17.9 ± 2.2) compared to 89Zr-DFO-9G5 (5.5 ± 1.2) and 89Zr-DFO-IgG (3.8 ± 1.0). No significant uptake was observed in DLD-1-shLGR5 tumors. This study identifies 8F2 as the optimal candidate for ADC development and provides initial evidence that 89Zr-DFO-LGR5 mAbs may be utilized to stratify tumors which would respond best to LGR5-targeted ADC therapy.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Neoplasias Colorretais/diagnóstico por imagem , Imunoconjugados/administração & dosagem , Compostos Radiofarmacêuticos/administração & dosagem , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacocinética , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imunoconjugados/farmacocinética , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , RNA Interferente Pequeno/metabolismo , Radioisótopos , Compostos Radiofarmacêuticos/farmacocinética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto , ZircônioRESUMO
R-spondins (RSPOs) and their receptor leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) play pleiotropic roles in normal and cancer development as well as the survival of adult stem cells through potentiation of Wnt signaling. Current evidence indicates that RSPO-LGR4 functions to elevate levels of Wnt receptors through direct inhibition of two membrane-bound E3 ligases (RNF43 and ZNRF3), which otherwise ubiquitinate Wnt receptors for degradation. Whether RSPO-LGR4 is coupled to intracellular signaling proteins to regulate Wnt pathways remains unknown. We identified the intracellular scaffold protein IQ motif containing GTPase-activating protein 1 (IQGAP1) as an LGR4-interacting protein that mediates RSPO-LGR4's interaction with the Wnt signalosome. IQGAP1 binds to and modulates the activities of a plethora of signaling molecules, including MAP kinases, Rho GTPases, and components of the Wnt signaling pathways. Interaction of LGR4 with IQGAP1 brings RSPO-LGR4 to the Wnt signaling complex through enhanced IQGAP1-DVL interaction following RSPO stimulation. In this configuration, RSPO-LGR4-IQGAP1 potentiates ß-catenin-dependent signaling by promoting MEK1/2-medidated phosphorylation of LRP5/6 as well as ß-catenin-independent signaling through regulation of actin dynamics. Overall, these findings reveal that RSPO-LGR4 not only induces the clearance of RNF43/ZNRF3 to increase Wnt receptor levels but also recruits IQGAP1 into the Wnt signaling complex, leading to potent and robust potentiation of both the canonical and noncanonical pathways of Wnt signaling.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismo , Via de Sinalização Wnt , Proteínas Ativadoras de ras GTPase/metabolismo , Actinas/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Movimento Celular , Citoesqueleto/metabolismo , Adesões Focais/metabolismo , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Fosforilação , Ligação Proteica , Ubiquitina-Proteína Ligases/metabolismo , beta Catenina/metabolismoRESUMO
The Wnt/ß-catenin signaling system plays essential roles in embryonic development and in the self-renewal and maintenance of adult stem cells. R-spondins (RSPOs) are a group of secreted proteins that enhance Wnt/ß-catenin signaling and have pleiotropic functions in development and stem cell growth. LGR5, an orphan receptor of the G protein-coupled receptor (GPCR) superfamily, is specifically expressed in stem cells of the intestinal crypt and hair follicle. Knockout of LGR5 in the mouse results in neonatal lethality. LGR4, a receptor closely related to LGR5, also has essential roles in development, as its knockout leads to reduced viability and retarded growth. Overexpression of both receptors has been reported in several types of cancer. Here we demonstrate that LGR4 and LGR5 bind the R-spondins with high affinity and mediate the potentiation of Wnt/ß-catenin signaling by enhancing Wnt-induced LRP6 phosphorylation. Interestingly, neither receptor is coupled to heterotrimeric G proteins or to ß-arrestin when stimulated by the R-spondins, indicating a unique mechanism of action. The findings provide a basis for stem cell-specific effects of Wnt/ß-catenin signaling and for the broad range of functions LGR4, LGR5, and the R-spondins have in normal and malignant growth.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Ligantes , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Camundongos Knockout , Fosforilação , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Current therapies targeting the human epidermal growth factor receptor (HER) family, including monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs), are limited by drug resistance and systemic toxicities. Antibody-drug conjugates (ADCs) are one of the most rapidly expanding classes of anti-cancer therapeutics with 13 presently approved by the FDA. Importantly, ADCs represent a promising therapeutic option with the potential to overcome traditional HER-targeted therapy resistance by delivering highly potent cytotoxins specifically to HER-overexpressing cancer cells and exerting both mAb- and payload-mediated antitumor efficacy. The clinical utility of HER-targeted ADCs is exemplified by the immense success of HER2-targeted ADCs including trastuzumab emtansine and trastuzumab deruxtecan. Still, strategies to improve upon existing HER2-targeted ADCs as well as the development of ADCs against other HER family members, particularly EGFR and HER3, are of great interest. To date, no HER4-targeting ADCs have been reported. In this review, we extensively detail clinical-stage EGFR-, HER2-, and HER3-targeting monospecific ADCs as well as novel clinical and pre-clinical bispecific ADCs (bsADCs) directed against this receptor family. We close by discussing nascent trends in the development of HER-targeting ADCs, including novel ADC payloads and HER ligand-targeted ADCs.
RESUMO
Neuroblastoma (NB) is a cancer of the peripheral nervous system found in children under 15 years of age. It is the most frequently diagnosed cancer during infancy, accounting for ~12% of all cancer-related deaths in children. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a membrane receptor that is associated with the primary tumor formation and metastasis of cancers in the gastrointestinal system. Remarkably, high levels of LGR5 are found in NB tumor cells, and high LGR5 expression is strongly correlated with poor survival. Antibody-drug conjugates (ADCs) are monoclonal antibodies that are covalently linked to cell-killing cytotoxins to deliver the payloads into cancer cells. We generated an ADC with an anti-LGR5 antibody and pyrrolobenzodiazepine (PBD) dimer-based payload SG3199 using a chemoenzymatic conjugation method. The resulting anti-LGR5 ADC was able to inhibit the growth of NB cells expressing LGR5 with high potency and specificity. Importantly, the ADC was able to completely inhibit the growth of NB xenograft tumors in vivo at a clinically relevant dose for the PBD class of ADCs. The findings support the potential of targeting LGR5 using the PBD class of payload for the treatment of high-risk NBs.
RESUMO
In recent years, antibody-drug conjugates (ADCs) have emerged as promising anti-cancer therapeutic agents with several having already received market approval for the treatment of solid tumor and hematological malignancies. As ADC technology continues to improve and the range of indications treatable by ADCs increases, the repertoire of target antigens has expanded and will undoubtedly continue to grow. G protein-coupled receptors (GPCRs) are well-characterized therapeutic targets implicated in many human pathologies, including cancer, and represent a promising emerging target of ADCs. In this review, we will discuss the past and present therapeutic targeting of GPCRs and describe ADCs as therapeutic modalities. Moreover, we will summarize the status of existing preclinical and clinical GPCR-targeted ADCs and address the potential of GPCRs as novel targets for future ADC development.
Assuntos
Antineoplásicos , Neoplasias Hematológicas , Imunoconjugados , Neoplasias , Humanos , Imunoconjugados/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias Hematológicas/tratamento farmacológicoRESUMO
Leucine-rich repeat-containing, G protein-coupled receptor 5 (LGR5) is highly expressed in colorectal cancer and cancer stem cells (CSCs) that play important roles in tumor initiation, progression, and metastasis. Loss of LGR5 has been shown to enhance therapy resistance. However, the molecular mechanisms that mediate this resistance remain elusive. In this study, we demonstrate conversion of LGR5+ colorectal cancer cells to an LGR5- state in response to chemotherapy, LGR5- targeted antibody-drug conjugates (ADCs), or LGR5 gene ablation led to activation of STAT3. Further investigation revealed increased STAT3 activation occurred as a result of increased mesenchymal epithelial transition (MET) factor receptor activity. LGR5 overexpression decreased MET-STAT3 activity and sensitized colorectal cancer cells to therapy. STAT3 inhibition suppressed MET phosphorylation, while constitutively active STAT3 reduced LGR5 levels and increased MET activity, suggesting a potential feedback mechanism. Combination treatment of MET-STAT3 inhibitors with irinotecan or antibody-drug conjugates (ADCs) substantiated synergistic effects in colorectal cancer cells and tumor organoids. In colorectal cancer xenografts, STAT3 inhibition combined with irinotecan enhanced tumor growth suppression and prolonged survival. These findings suggest a mechanism by which drug-resistant LGR5- colorectal cancer cells acquire a survival advantage through activation of MET-STAT3 and provide rationale for new treatment strategies to target colorectal cancer.
Assuntos
Neoplasias Colorretais , Imunoconjugados , Humanos , Regulação para Baixo , Irinotecano/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Linhagem Celular Tumoral , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco Neoplásicas/metabolismo , Imunoconjugados/farmacologia , Fator de Transcrição STAT3/genéticaRESUMO
LGR4 and LGR5 are two homologous receptors that potentiate Wnt/ß-catenin signaling in response to R-spondin (RSPO) ligands. The RSPO and LGR4 complex binds to and inhibits activities of two related E3 ubiquitin ligases, RNF43 and ZNRF3, and thus protects Wnt receptors from the E3 ligase-mediated degradation. The RSPO and LGR5 complex, however, does not interact with the E3 ligases, and the structural basis of this difference remained unknown. Here we examined the affinities of monovalent and bivalent RSPO ligands in binding to LGR4, RNF43/ZNRF3, and LGR5 in whole cells and found unique features among the receptors and E3 ligases. Monovalent RSPO2 furin domain had much lower affinity in binding to LGR4 or RNF43/ZNRF3 than the bivalent form. In contrast, monovalent and bivalent forms had nearly identical affinity in binding to LGR5. Co-expression of ZNRF3 with LGR4 led to much higher binding affinity of the monovalent form whereas co-expression of ZNRF3 with LGR5 had no effect on the affinity. These results suggest that LGR4 and RNF43/ZNRF3 form a 2:2 dimer that accommodates bivalent binding of RSPO whereas LGR5 forms a homodimer that does not. Structural models are proposed to illustrate how RSPOs bind to LGR4, RNF43/ZNRF3, and LGR5 in whole cells.
Assuntos
Receptores Acoplados a Proteínas G , Via de Sinalização Wnt , Receptores Acoplados a Proteínas G/metabolismo , Ligantes , Ubiquitina-Proteína Ligases/metabolismo , Trombospondinas/metabolismoRESUMO
LGR4-6 (leucine-rich repeat-containing G-protein-coupled receptors 4, 5, and 6) are three related receptors with an upregulated expression in gastrointestinal cancers to various extents, and LGR5 is enriched in cancer stem cells. Antibody-drug conjugates (ADCs) targeting LGR5 showed a robust antitumor effect in vivo but could not eradicate tumors due to plasticity of LGR5-positive cancer cells. As LGR5-negative cancer cells often express LGR4 or LGR6 or both, we reasoned that simultaneous targeting of all three LGRs may provide a more effective approach. R-spondins (RSPOs) bind to LGR4-6 with high affinity and potentiate Wnt signaling. We identified an RSPO4 furin domain mutant (Q65R) that retains potent LGR binding but no longer potentiates Wnt signaling. Drug conjugates of a peptibody comprising the RSPO4 mutant and IgG1-Fc showed potent cytotoxic effects on cancer cell lines expressing any LGR in vitro and suppressed tumor growth in vivo without inducing intestinal enlargement or other adverse effects.
Assuntos
Neoplasias/tratamento farmacológico , Animais , Antineoplásicos , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Xenoenxertos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Receptores Acoplados a Proteínas G , Trombospondinas , Via de Sinalização WntRESUMO
Wnts are secreted lipid-modified glycoproteins that carry out various signaling functions during development and in adult tissue. Wnt signaling is mediated by frizzled receptors (Fzds) at the cell surface and can be modulated by the secreted frizzled-related proteins (SFRPs) and other molecular antagonists. Abnormal Wnt signaling has been implicated in several diseases. However, due to the complexity of the Wnt signal and the lack of knowledge pertaining to the binding properties of different Wnt ligands, no therapeutic agents that target this pathway exist. Using a novel enzyme-linked immunosorbent assay (ELISA)-based technique, we were able to determine the first measurements of binding affinity for specific Wnt interactions. This study shows that purified Wnt3a, Wnt7a, and Wnt5a have different binding specificities for Fzds and SFRPs.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Receptores Frizzled/metabolismo , Proteínas Wnt/metabolismo , Linhagem Celular Tumoral , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , MAP Quinase Quinase 4/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/metabolismoRESUMO
LGR4 and LGR5 encode two homologous receptors with critical, yet distinct, roles in organ development and adult stem cell survival. Both receptors are coexpressed in intestinal crypt stem cells, bind to R-spondins (RSPOs) with high affinity, and potentiate Wnt-ß-catenin signaling, presumably by the same mechanism: forming RSPO-bridged complexes with the E3 ligases RNF43 and ZNRF3 to inhibit ubiquitylation of Wnt receptors. However, direct evidence for RSPO-bound, full-length LGR5 interacting with these E3 ligases in whole cells has not been reported, and only LGR4 is essential for the self-renewal of intestinal stem cells. Here, we examined the mechanisms of action of LGR4 and LGR5 in parallel using coimmunoprecipitation, proximity ligation, competition binding, and time-resolved FRET assays in whole cells. Full-length LGR4 formed a tight complex with ZNRF3 and RNF43 even without RSPO, whereas LGR5 did not interact with either E3 ligase with or without RSPO. Domain-swapping experiments with LGR4 and LGR5 revealed that the seven-transmembrane domain of LGR4 conferred interaction with the E3 ligases. Native LGR4 and LGR5 existed as dimers on the cell surface, and LGR5 interacted with both FZD and LRP6 of the Wnt signalosome to enhance LRP6 phosphorylation and potentiate Wnt-ß-catenin signaling. These findings provide a molecular basis for the weaker activity of LGR5 in the potentiation of Wnt signaling that may underlie the distinct roles of LGR4 and LGR5 in organ development, as well as the self-renewal and fitness of adult stem cells.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/genética , Ubiquitina-Proteína Ligases/genética , beta Catenina/genéticaRESUMO
In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate beta-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH2-terminal kinase-responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4.
Assuntos
Neoplasias do Endométrio/metabolismo , Receptores Frizzled/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Endométrio/patologia , Feminino , Receptores Frizzled/análise , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Comunicação Parácrina , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/análise , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismoRESUMO
Drug resistance continues to be a major obstacle of effective therapy for colorectal cancer, leading to tumor relapse or treatment failure. Cancer stem cells (CSC) or tumor-initiating cells are a subpopulation of tumor cells which retain the capacity for self-renewal and are suggested to be implicated in drug resistance. LGR5 is highly expressed in colorectal cancer and marks CSCs that drive tumor growth and metastasis. LGR5(+) CSCs cells were shown to interconvert with more drug-resistant LGR5(-) cancer cells, and treatment with LGR5-targeted antibody-drug conjugates (ADC) eliminated LGR5(+) tumors, yet a fraction of LGR5(-) tumors eventually recurred. Therefore, it is important to identify mechanisms associated with CSC plasticity and drug resistance in order to develop curative therapies. Here, we show that loss of LGR5 in colon cancer cells enhanced resistance to irinotecan and 5-fluorouracil and increased expression of adhesion G-protein-coupled receptor, GPR56. GPR56 expression was significantly higher in primary colon tumors versus matched normal tissues and correlated with poor survival outcome. GPR56 enhanced drug resistance through upregulation of MDR1 levels via a RhoA-mediated signaling mechanism. Loss of GPR56 led to suppression of tumor growth and increased sensitivity of cancer cells to chemotherapy and monomethyl auristatin E-linked anti-LGR5 ADCs, by reducing MDR1 levels. These findings suggest that upregulation of GPR56 may be a mechanism associated with CSC plasticity by which LGR5(-) cancer cells acquire a more drug-resistant phenotype. IMPLICATIONS: Our findings suggest that targeting GPR56 may provide a new strategy for the treatment of colorectal cancer and combatting drug resistance.
Assuntos
Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Receptores Acoplados a Proteínas G/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Humanos , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Regulação para Cima , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Wnts are secreted glycoproteins that regulate important cellular processes including proliferation, differentiation, and cell fate. In the beta-catenin/canonical pathway, Wnt interacts with Fzd receptors to inhibit degradation of beta-catenin and promote its translocation into the nucleus where it regulates transcription of a number of genes. Dysregulation of this pathway has been attributed to a host of diseases including cancer. As a result, components of the beta-catenin/canonical pathway have been gaining recognition as promising targets for the discovery of novel therapeutic agents. Here, we show, using an ELISA-based protein-protein binding assay that purified Wnt7a binds to the extracellular cysteine-rich domain of Fzd5 in the nanomolar range. We have developed a novel split eGFP complementation assay to visually detect Wnt7a-Fzd5 interactions and subsequent pathway activation in cells. These biological tools could help lead to a better understanding of Wnt-Fzd interactions and the identification of new modulators of Wnt signaling.