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Colorectal cancer (CRC) is the third most common cancer globally. Regorafenib, a multi-target kinase inhibitor, has been approved for treating metastatic colorectal cancer patients who have undergone at least two prior standard anti-cancer therapies. However, regorafenib efficacy as a single agent remains suboptimal. A promising target at the crossroads of multiple signaling pathways is the Src homology 2 domain-containing protein tyrosine phosphatase (SHP2). However, a combination approach using SHP2 inhibitors (SHP099) and anti-angiogenic drugs (Regorafenib) has not been reported in current research. In this study, we conducted in vitro experiments combining SHP099 and regorafenib and established an MC-38 colon cancer allograft mouse model. Our results revealed that co-treatment with SHP099 and regorafenib significantly inhibited cell viability and altered the biological characteristics of tumor cells compared with treatment alone in vitro. Furthermore, the combination strategy demonstrated superior therapeutic efficacy compared to monotherapy with either drug. This was evidenced by reduced tumor size, decreased proliferation, increased apoptosis, normalized tumor microvasculature, and improved antitumor immune response in vivo. These findings suggest that the combination of an SHP2 inhibitor and regorafenib is a promising therapeutic approach for patients with colorectal cancer.
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Antineoplásicos , Neoplasias do Colo , Animais , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêuticoRESUMO
BACKGROUND AND OBJECTIVE: Colorectal cancer (CRC) is one of the most common malignancies in China. At present, there is a problem that the CRC treatment drugs SHP099, L-OHP and 5-FU are insensitive to tumor cells. Combination medication is an important means to solve the insensitivity of medication alone. The purpose of this project was to explore the effect and molecular mechanism of SHP099 combination on the malignant biological behavior of L-OHP/5-FU resistant strains of CRC. METHODS: HT29 and SW480 cells were cultured in media supplemented with L-OHP or 5-FU to establish drug-resistant strains. HT29 and SW480 drug-resistant cells were subcutaneously injected into the ventral nerves of nude mice at a dose of 5 × 106 to establish CRC drug-resistant animal models. CCK-8, Western blot, flow cytometry, Transwell and kit detection were used to detect the regulatory mechanism of energy metabolism reprogramming in drug-resistant CRC cells. RESULTS: Compared with nonresistant strains, L-OHP/5-FU-resistant strains exhibited greater metabolic reprogramming. Functionally, SHP099 can restrain the metabolic reprogramming of L-OHP/5-FU-resistant strains and subsequently restrain the proliferation, colony formation, migration and spheroid formation of L-OHP/5-FU-resistant strains. Downstream mechanistic studies have shown that SHP099 interferes with the metabolic reprogramming of L-OHP/5-FU drug-resistant strains by suppressing the PI3K/AKT pathway, thereby restraining the malignant biological behavior of L-OHP/5-FU drug-resistant strains and alleviating CRC. CONCLUSION: The combination of SHP099 can restrain the malignant biological behavior of L-OHP/5-FU-resistant CRC cells and alleviate the progression of CRC by interfering with the reprogramming of energy metabolism. This study explored the effect of SHP099 combination on dual-resistant CRC cells for the first time, and provided a new therapeutic idea for solving the problem of SHP099 insensitivity to CRC cells.
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Neoplasias Colorretais , Resistencia a Medicamentos Antineoplásicos , Fluoruracila , Reprogramação Metabólica , Animais , Humanos , Camundongos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fluoruracila/farmacologia , Células HT29 , Reprogramação Metabólica/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Src homology-2 domain-containing phosphatase 2 (SHP2) is a ubiquitously expressed phosphatase that is vital for skeletal development and maintenance of chondrocytes, osteoblasts, and osteoclasts. Study of SHP2 function in small animal models has led to insights in phenotypes observed in SHP2-mutant human disease, such as Noonan syndrome. In recent years, allosteric SHP2 inhibitors have been developed to specifically target the protein in neoplastic processes. These inhibitors are highly specific and have great potential for disease modulation in cancer and other pathologies, including bone disorders. In this review, we discuss the importance of SHP2 and related signaling pathways (e.g., Ras/MEK/ERK, JAK/STAT, PI3K/Akt) in skeletal development. We review rodent models of pathologic processes caused by germline mutations that activate SHP2 enzymatic activity, with a focus on the skeletal phenotype seen in these patients. Finally, we discuss SHP2 inhibitors in development and their potential for disease modulation in these genetic diseases, particularly as it relates to the skeleton.
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Neoplasias , Fosfatidilinositol 3-Quinases , Animais , Humanos , Transdução de Sinais , Esqueleto , Esterno/patologia , MutaçãoRESUMO
The E69K mutation is one of the most frequent protein tyrosine phosphatase-2 (SHP2) mutations in leukemia, and it can cause the increase in the protein activity. Recent studies have shown that the E69K mutation was fairly sensitive to the allosteric inhibitor of SHP2 (SHP099). However, the molecular mechanism of the allosteric drug SHP099 inhibiting SHP2E69K remains unclear. Thus, the molecular dynamic simulations and the post-dynamics analyses (RMSF, PCA, DCCM, RIN and the binding free energies) for SHP2WT, SHP2WT-SHP099, SHP2E69K and SHP2E69K-SHP099 were carried out, respectively. Owing to the strong binding affinity of SHP099 to residues Thr219 and Arg220, the flexibility of linker region (residues Val209-Arg231) was reduced. Moreover, the presence of SHP099 kept the autoinhibition state of the SHP2 protein through enhancing the interactions between the linker region and Q loop in PTP domain, such as Thr219/Val490, Thr219/Asn491, Arg220/Ile488 and Leu254/Asn491. In addition, it was found that the residues (Thr219, Arg220, Leu254 and Asn491) might be the key residues responsible for the conformational changes of protein. Overall, this study may provide an important basis for understanding how the SHP099 effectively inhibited the SHP2E69K activity at the molecular level.
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Regulação Alostérica , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Piperidinas/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Pirimidinas/química , Estabilidade de Medicamentos , Ligação de Hidrogênio , Estrutura Molecular , Piperidinas/farmacologia , Conformação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Protein tyrosine phosphatases (PTPs), which are ubiquitously expressed in hematopoietic and non-hematopoietic cells, are critical for regulating cell proliferation as well as differentiation in the physiology of multicellular organisms. PTPs regulate the intracellular signaling mechanism of immune cells via dephosphorylation of multiple targets and are associated with the onset of various autoimmune diseases through genomic alterations. PTPs also affect disease through their role in innate and/or acquired immunity. By modulating multiple substrates, PTPN12, a member of the proline-, glutamic acid-, serine- and threonine-rich (PEST) family of PTPs, is an important regulator of cell migration and adhesion. According to its newly identified roles and functions, PTPN12 is considered a promising therapeutic target against critical diseases, including cancer, diabetes, metabolic disease and autoimmune diseases. In this review, we provide an overview of PTPs and discuss the critical roles of PTPN12/PTP-PEST in tumor progression.
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Neoplasias/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 12/imunologia , Animais , Progressão da Doença , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Humanos , Imunidade/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 12/análise , Proteína Tirosina Fosfatase não Receptora Tipo 12/antagonistas & inibidoresRESUMO
Tumor-associated thrombus (TAT) accounts for a high proportion of venous thromboembolism. Traditional thrombolysis and anticoagulation methods are not effective due to various complications and contraindications, which can easily lead to patients dying from TAT rather than the tumor itself. These clinical issues demonstrate the need to research diverse pathways for adjuvant thrombolysis in antitumor therapy. Previously, the phenotypic and functional transformation of monocytes/macrophages is widely reported to be involved in intratribal collagen regulation. This study finds that myeloid deficiency of the oncogene SHP2 sensitizes Ly6Clow monocyte/macrophage differentiation and can alleviate thrombus organization by increasing thrombolytic Matrix metalloproteinase (MMP) 2/9 activities. Moreover, pharmacologic inhibition by SHP099, examined in mouse lung metastatic tumor models, reduces tumor and thrombi burden in tumor metastatic lung tissues. Furthermore, SHP099 increases intrathrombus Ly6Clow monocyte/macrophage infiltration and exhibits thrombolytic function at high concentrations. To improve the thrombolytic effect of SHP099, NanoSHP099 is constructed to achieve the specific delivery of SHP099. NanoSHP099 is identified to be simultaneously enriched in tumor and thrombus foci, exerting dual tumor-suppression and thrombolysis effects. NanoSHP099 presents a superior thrombus dissolution effect than that of the same dosage of SHP099 because of the higher Ly6Clow monocyte/macrophage proportion and MMP2/MMP9 collagenolytic activities in organized thrombi.
Assuntos
Monócitos , Trombose , Animais , Camundongos , Leucócitos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Terapia Trombolítica/métodos , Trombose/metabolismo , Piperidinas/farmacologia , Pirimidinas/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidoresRESUMO
Excessive pulmonary inflammation in acute lung injury (ALI) causes high patient mortality. Anti-inflammatory therapy, combined with infection resistance, can help to prevent ALI and save lives. The expression of Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2) was found to be significantly higher in macrophages and lung tissues with ALI, and SHP2-associated MAPK pathways were activated by lipopolysaccharide (LPS). The knockdown of the SHP2 gene suppressed the LPS-induced release of inflammatory factors and the phosphorylation of regulators in the NF-κB pathways in macrophages. Our findings showed crosstalk between the LPS-induced inflammatory pathway and the SHP2-associated MAPK pathways. SHP2 inhibition could be a valuable therapeutic approach for inhibiting excessive inflammation in ALI. We discovered that giving SHP099, a specific allosteric inhibitor of SHP2, to mice with ALI and sepsis relieves ALI and significantly increases animal survival. Our study highlights the important role of SHP2 in ALI development and demonstrates the potential application of SHP099 for treating ALI.
Assuntos
Lesão Pulmonar Aguda , Lipopolissacarídeos , Camundongos , Animais , Lipopolissacarídeos/farmacologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/prevenção & controle , Pulmão/metabolismo , NF-kappa B/metabolismoRESUMO
PURPOSE: To explore the possibility of a combination of dabrafenib and SHP2 inhibitor in the treatment of anaplastic thyroid carcinoma and to provide a new therapeutic strategy for the treatment of anaplastic thyroid cancer. PATIENTS AND METHODS: Firstly, a drug resistance model was established, and the expression levels of related RTK were detected by qPCR. Western blot was used to detect the protein expression levels of Akt and MAPK signaling pathways in the control group, single-drug group and two-drug combination group. The gene silencing of SHP2 was achieved by transfection of siRNA and verified by Western blot. CCK8 kit and clone formation assay were used to detect cell proliferation activity. In vivo model of mutant thyroid cancer cells was established by subcutaneous injection of mice and then divided into four groups. Tumor diameter was measured every two days. Immunohistochemistry was used to evaluate the expression of p-ERK, p-AKT and Ki67 in mouse tumors. RESULTS: In this study, dabrafenib-resistant ATC cells were first constructed, and the response of RTKs in drug-resistant cells was upregulated to activate Akt and MER/ERK pathways. The activation of Akt and MEK/ERK pathways in the combination group was significantly inhibited, and the proliferation ability of tumor cells was significantly reduced compared with Dabrafenib, SHP099 group and DMSO group. To verify that SHP099 was not off-target, we also silenced SHP2 expression by transfection with siRNA and obtained the same results. Finally, by building a mouse drug resistance model, we confirmed that dabrafenib and SHP099 can also play a powerful anti-cancer effect in vivo. CONCLUSION: The SHP2 inhibitor SHP099 can effectively reverse the drug resistance of dabrafenib through inhibiting the reactivated RAS signaling pathway in anaplastic thyroid cancer.The combination of dabrafenib with SHP2 inhibitor has shown significant tumor suppressive effects for dabrafenib-resistant cells and it may be a new therapeutic strategy with longer lasting therapeutic benefits.
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INTRODUCTION: The protein tyrosine phosphatase SHP2 (PTPN11) is a negative regulator of glycoprotein VI (GPVI)-induced platelet signal under certain conditions. Clinical trials with derivatives of the allosteric drug SHP099, inhibiting SHP2, are ongoing as potential therapy for solid cancers. Gain-of-function mutations of the PTPN11 gene are observed in part of the patients with the Noonan syndrome, associated with a mild bleeding disorder. Assessment of the effects of SHP2 inhibition in platelets from controls and Noonan syndrome patients. MATERIALS AND METHODS: Washed human platelets were incubated with SHP099 and stimulated with collagen-related peptide (CRP) for stirred aggregation and flow cytometric measurements. Whole-blood microfluidics assays using a dosed collagen and tissue factor coating were performed to assess shear-dependent thrombus and fibrin formation. Effects on clot formation were evaluated by thromboelastometry. RESULTS: Pharmacological inhibition of SHP2 did not alter GPVI-dependent platelet aggregation under stirring, but it enhanced integrin αIIbß3 activation in response to CRP. Using whole-blood microfluidics, SHP099 increased the thrombus buildup on collagen surfaces. In the presence of tissue factor and coagulation, SHP099 increased thrombus size and reduced time to fibrin formation. Blood from PTPN11-mutated Noonan syndrome patients, with low platelet responsiveness, after ex vivo treatment with SHP099 showed a normalized platelet function. In thromboelastometry, SHP2 inhibition tended to increase tissue factor-induced blood clotting profiles with tranexamic acid, preventing fibrinolysis. CONCLUSION: Pharmacological inhibition of SHP2 by the allosteric drug SHP099 enhances GPVI-induced platelet activation under shear conditions with a potential to improve platelet functions of Noonan syndrome patients.
Assuntos
Síndrome de Noonan , Trombose , Humanos , Plaquetas/metabolismo , Síndrome de Noonan/tratamento farmacológico , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Tromboplastina/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Colágeno/metabolismo , Fibrina/metabolismo , Glicoproteínas da Membrana de Plaquetas , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
Preclinical and clinical studies have evidenced that effective targeted therapy treatment against receptor tyrosine kinases (RTKs) in different solid tumor paradigms is predicated on simultaneous inhibition of both the PI3K and MEK intracellular signaling pathways. Indeed, re-activation of either pathway results in resistance to these therapies. Recently, oncogenic phosphatase SHP2 inhibitors have been developed with some now reaching clinical trials. To expand on possible indications for SHP099, we screened over 800 cancer cell lines covering over 25 subsets of cancer. We found HNSCC was the most sensitive adult subtype of cancer to SHP099. We found that, in addition to the MEK pathway, SHP2 inhibition blocks the PI3K pathway in sensitive HNSCC, resulting in downregulation of mTORC signaling and anti-tumor effects across several HNSCC mouse models, including an HPV+ patient-derived xenograft (PDX). Importantly, we found low levels of the RTK ligand epiregulin identified HNSCCs that were sensitive to SHP2 inhibitor, and, adding exogenous epiregulin mitigated SHP099 efficacy. Mechanistically, epiregulin maintained SHP2-GAB1 complexes in the presence of SHP2 inhibition, preventing downregulation of the MEK and PI3K pathways. We demonstrate HNSCCs were highly dependent on GAB1 for their survival and knockdown of GAB1 is sufficient to block the ability of epiregulin to rescue MEK and PI3K signaling. These data connect the sensitivity of HNSCC to SHP2 inhibitors and to a broad reliance on GAB1-SHP2, revealing an important and druggable signaling axis. Overall, SHP2 inhibitors are being heavily developed and may have activity in HNSCCs, and in particular those with low levels of epiregulin.
Assuntos
Neoplasias de Cabeça e Pescoço , Fosfatidilinositol 3-Quinases , Animais , Camundongos , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Epirregulina/metabolismo , Inibidores Enzimáticos/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Reoccurring/high-risk neuroblastoma (NB) tumors have the enrichment of non-RAS/RAF mutations along the mitogen-activated protein kinase (MAPK) signaling pathway, suggesting that activation of MEK/ERK is critical for their survival. However, based on preclinical data, MEK inhibitors are unlikely to be active in NB and have demonstrated dose-limiting toxicities that limit their use. Here, we explore an alternative way to target the MAPK pathway in high-risk NB. We find that NB models are among the most sensitive among over 900 tumor-derived cell lines to the allosteric SHP2 inhibitor SHP099. Sensitivity to SHP099 in NB is greater in models with loss or low expression of the RAS GTPase activation protein (GAP) neurofibromin 1 (NF1). Furthermore, NF1 is lower in advanced and relapsed NB and NF1 loss is enriched in high-risk NB tumors regardless of MYCN status. SHP2 inhibition consistently blocks tumor growth in high-risk NB mouse models, revealing a new drug target in relapsed NB.
Assuntos
Neuroblastoma , Neurofibromina 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Recidiva Local de Neoplasia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Osteoarthritis (OA), in which M1 macrophage polarization in the synovium exacerbates disease progression, is a major cause of cartilage degeneration and functional disabilities. Therapeutic strategies of OA designed to interfere with the polarization of macrophages have rarely been reported. Here, we report that SHP099, as an allosteric inhibitor of src-homology 2-containing protein tyrosine phosphatase 2 (SHP2), attenuated osteoarthritis progression by inhibiting M1 macrophage polarization. We demonstrated that M1 macrophage polarization was accompanied by the overexpression of SHP2 in the synovial tissues of OA patients and OA model mice. Compared to wild-type (WT) mice, myeloid lineage conditional Shp2 knockout (cKO) mice showed decreased M1 macrophage polarization and attenuated severity of synovitis, an elevated expression of cartilage phenotype protein collagen II (COL2), and a decreased expression of cartilage degradation markers collagen X (COL10) and matrix metalloproteinase 3 (MMP3) in OA cartilage. Further mechanistic analysis showed thatSHP099 inhibited lipopolysaccharide (LPS)-induced Toll-like receptor (TLR) signaling mediated by nuclear factor kappa B (NF-κB) and PI3K-AKT signaling. Moreover, intra-articular injection of SHP099 also significantly attenuated OA progression, including joint synovitis and cartilage damage. These results indicated that allosteric inhibition of SHP2 might be a promising therapeutic strategy for the treatment of OA.
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Colorectal cancer (CRC), a malignant tumor worldwide consists of microsatellite instability (MSI) and stable (MSS) phenotypes. Although SHP2 is a hopeful target for cancer therapy, its relationship with innate immunosuppression remains elusive. To address that, single-cell RNA sequencing was performed to explore the role of SHP2 in all cell types of tumor microenvironment (TME) from murine MC38 xenografts. Intratumoral cells were found to be functionally heterogeneous and responded significantly to SHP099, a SHP2 allosteric inhibitor. The malignant evolution of tumor cells was remarkably arrested by SHP099. Mechanistically, STING-TBK1-IRF3-mediated type I interferon signaling was highly activated by SHP099 in infiltrated myeloid cells. Notably, CRC patients with MSS phenotype exhibited greater macrophage infiltration and more potent SHP2 phosphorylation in CD68+ macrophages than MSI-high phenotypes, suggesting the potential role of macrophagic SHP2 in TME. Collectively, our data reveals a mechanism of innate immunosuppression mediated by SHP2, suggesting that SHP2 is a promising target for colon cancer immunotherapy.
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Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib. LC/MS-MS analysis identified multiple proteins, including PEAK1 and SHP2, which were validated as ALK interactors in NB cells. Further analysis of the ALK-SHP2 interaction confirmed that the ALK-SHP2 interaction as well as SHP2-Y542 phosphorylation was dependent on ALK activation. Use of the SHP2 inhibitors, SHP099 and RMC-4550, resulted in inhibition of cell growth in ALK-driven NB cells. In addition, we noted a strong synergistic effect of combined ALK and SHP2 inhibition that was specific to ALK-driven NB cells, suggesting a potential therapeutic option for ALK-driven NB.
Assuntos
Quinase do Linfoma Anaplásico/metabolismo , Neuroblastoma/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteômica/métodos , Aminopiridinas/farmacologia , Animais , Linhagem Celular Tumoral , Cromatografia Líquida , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Lactamas/farmacologia , Células PC12 , Fosforilação , Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Espectrometria de Massas em TandemRESUMO
Glioblastoma (GBM) is the most lethal primary brain tumor in adults with a median survival of around 15 months. A potential treatment strategy involves targeting glioma stem-like cells (GSCs) that are able to initiate, maintain, and repopulate the tumor mass. Here, we identify ACT001, a parthenolide derivative, targeting GSCs through regulation of adipocyte enhancer binding protein 1 (AEBP1) signaling. Methods: The effects of ACT001 on cell survival of normal human astrocytes (NHA) and patient-derived glioma stem-like cells (GSCs) were evaluated. RNA-Seq were performed to detect differentially expressed genes. ACT001 efficacy as a single agent or in combination with SHP-2 inhibitor SHP099 was assessed using a GSC orthotopic xenograft model. Results: GSCs exhibit high response to ACT001 in compared with normal human astrocytes. AEBP1 is a putative target of ACT001 by RNA-Seq analysis, which expression associates with prognosis of GBM patients. Knockdown of AEBP1 inhibits GSC proliferation and glioma sphere formation. Treatment with ACT001 or PI3K inhibitor or AEBP1 depletion would impair AKT phosphorylation and GSC proliferation, whereas constitutive AKT activation rescues ACT001 treatment or AEBP1 depletion-inhibited cell proliferation. Moreover, ACT001 blocks TGF-ß-activated AEBP1/AKT signaling in GSCs. ACT001 exhibits antitumor activity either as a single agent or in combination with SHP099, which provides significant survival benefits for GSC tumor xenograft-bearing animals. Conclusions: Our data demonstrate AEBP1 as a new druggable target in GBM and ACT001 as a potential therapeutic option for improving the clinical treatment of GBM in combination with SHP099.
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Antineoplásicos/farmacologia , Carboxipeptidases/antagonistas & inibidores , Furanos/farmacologia , Glioma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Sesquiterpenos/química , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inflammatory bowel disease (IBD) is a chronic inflammatory condition with complex pathogenesis that currently has no cure. α7 nicotinic acetylcholine receptor (α7nAChR) is known to regulate multiple aspects of immune function. The present study aimed to evaluate the protective effects of PNU282987 and SHP099, which are a selective agonist of α7nAChR and an SHP2 inhibitor, respectively, in dextran sulfate sodium (DSS)induced colitis in mice. Acute colitis was induced in mice using 3% DSS, and weight loss, colonic histology and cytokine production from colonic lamina propria were analyzed to evaluate disease severity. Bone marrowderived macrophages were treated with lipopolysaccharide (LPS) to induce an inflammatory response. Cytokine expression and reactive oxygen species (ROS) levels were quantified. The α7nAChR agonist, PNU282987, and the SHP2 inhibitor, SHP099, were administered alone or in combination to LPSinduced macrophages or to colitic model mice to evaluate the inflammatory response and protective efficacy in colitis. α7nAChR protein levels were found to be markedly increased in the colon of DSSinduced colitic mice, and were found to colocalize with macrophages. Consistently, α7nAChR mRNA and protein levels were upregulated with colitis progression in DSSinduced colitic mice. Colonic inflammation was attenuated by PNU282987 treatment in DSSinduced mice, as evidenced by reduced weight loss and alleviated colonic epithelial cell disruption. These effects of PNU282987 on colitis were enhanced when it was combined with SHP099. Cytokine production and ROS levels induced by LPS in macrophages were decreased by a combination treatment of PNU282987 and SHP099. These findings identified α7nAChR as an essential element in the role of intestinal macrophages in colonic repair and demonstrated a synergistic effect of PNU282987 and SHP099, suggesting a new potential therapy for IBD.
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Benzamidas/administração & dosagem , Compostos Bicíclicos com Pontes/administração & dosagem , Colite/tratamento farmacológico , Citocinas/genética , Sulfato de Dextrana/efeitos adversos , Piperidinas/administração & dosagem , Pirimidinas/administração & dosagem , Animais , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Colite/induzido quimicamente , Colite/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Piperidinas/farmacologia , Pirimidinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Resultado do Tratamento , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
The over-activation of Ras/mitogen-activated protein kinase (MAPK) signaling pathway associated with a variety of cancers is usually related with abnormal activation of Src-homology 2 domain-containing protein tyrosine phosphatase (SHP2). For this purpose, SHP2 has attracted extensive interest as a potential target for cancer treatment. RMC-4550, as a newly developed selective inhibitor of SHP2, possesses an overwhelming advantage over the previous generation inhibitor SHP099 in terms of in vitro activity. However, the binding mode of SHP2 with RMC-4550 and the reason for the high efficiency of RMC-4550 as SHP2 inhibitor at molecular level are still unclear. Therefore, in this study, the binding mode of RMC-4550 with SHP2 and the superiorities of RMC-4550 as inhibitor at binding affinity and dynamic interactive behavior with SHP2 were probed by molecular docking and molecular dynamics (MD) simulations. By comparing the results of molecular docking, it was found that SHP2 formed more tight interaction with RMC-4550 than that with SHP099. Subsequently, a series of post-dynamic analyses on three simulation trajectories (SHP2WT, SHP2SHP099 and SHP2RMC-4550) were performed and found that the SHP2 protein bound with RMC-4550 maintained a firmer interaction between N-Src-homology 2 (N-SH2) and PTP domain throughout the MD simulation, leading to a more stable protein conformation. The finding here provides new clues for the design of SHP2 inhibitor against the over-activation of Ras/MAPK pathway.Communicated by Ramaswamy H. Sarma.
Assuntos
Simulação de Dinâmica Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Simulação de Acoplamento Molecular , Conformação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de SinaisRESUMO
Some canine cases of histiocytic sarcoma (HS) carry an activating mutation in the src homology two domain-containing phosphatase 2 (SHP2) encoded by PTPN11. SHP099 is an allosteric inhibitor of SHP2 that stabilizes SHP2 in a folded, auto-inhibited conformation. Here, we examined the expression and mutation status of SHP2 in five canine HS cell lines and evaluated the growth inhibitory properties of SHP099 against these cell lines. All five of the canine HS cell lines expressed SHP2, with three of the lines each harbouring a distinct mutation in PTPN11/SHP2 (p.Glu76Gln, p.Glu76Ala and p.Gly503Val). In silico analysis suggested that p.Glu76Gln and p.Glu76Ala, but not p.Gly503Val, promote shifting of the SHP2 conformation from folded to open-active state. SHP099 potently suppressed the growth of two of the mutant cell lines (harbouring SHP2 p.Glu76Gln or p.Glu76Ala) but not that of the other three cell lines. In addition, SHP099 suppressed ERK activation in the cell line harbouring the SHP2 p.Glu76Ala mutation. The SHP2 p.Glu76Gln and p.Glu76Ala mutations are considered to be activating mutations, and the signal from SHP2 p.Glu76Ala is inferred to be transduced primarily via the ERK pathway. Moreover, SHP099-sensitive HS cells, including those with SHP2 p.Glu76Gln or p.Glu76Ala mutations, may depend on these mutations for growth. Therefore, targeting cells harbouring SHP2 p.Glu76Gln and p.Glu76Ala with SHP099 may be an approach for the treatment of canine HS.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sarcoma Histiocítico/tratamento farmacológico , Piperidinas/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Pirimidinas/farmacologia , Animais , Linhagem Celular Tumoral , Simulação por Computador , Cães , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
Tyrosine phosphatase SHP2 is a promising drug target in cancer immunotherapy due to its bidirectional role in both tumor growth promotion and T-cell inactivation. Its allosteric inhibitor SHP099 is known to inhibit cancer cell growth both in vitro and in vivo. However, whether SHP099-mediated SHP2 inhibition retards tumor growth in vivo via anti-tumor immunity remains elusive. To address this, a CT-26 colon cancer xenograft model was established in mice since this cell line is insensitive to SHP099. Consequently, SHP099 minimally affected CT-26 tumor growth in immuno-deficient nude mice, but significantly decreased the tumor burden in CT-26 tumor-bearing mice with intact immune system. SHP099 augmented anti-tumor immunity, as shown by the elevated proportion of CD8+IFN-γ + T cells and the upregulation of cytotoxic T-cell related genes including Granzyme B andPerforin, which decreased the tumor load. In addition, tumor growth in mice with SHP2-deficient T-cells was markedly slowed down because of enhanced anti-tumor responses. Finally, the combination of SHP099 and anti-PD-1 antibody showed a higher therapeutic efficacy than either monotherapy in controlling tumor growth in two colon cancer xenograft models, indicating that these agents complement each other. Our study suggests that SHP2 inhibitor SHP099 is a promising candidate drug for cancer immunotherapy.
RESUMO
BACKGROUND: Glioblastoma (GBM) is the most malignant primary brain tumor, with dismal median survival. Treatment of GBM is particularly challenging given the intrinsic resistance to chemotherapy and difficulty of drugs to reach the tumor beds due to the blood-brain barrier. Here, we examined the efficacy of SHP099, a potent, selective, and oral SHP-2 inhibitor for treating GBM with activated platelet derived growth factor receptor alpha (PDGFRα) signaling. METHODS: The effects of SHP099 on cell survival of neural progenitor cells (NPCs), GBM cell lines, and patient-derived glioma stem-like cells (GSCs) were evaluated. Brain and plasma pharmacokinetics of SHP099 and its ability to inhibit SHP-2 signaling were assessed. SHP099 efficacy as a single agent or in combination with temozolomide (TMZ) was assessed using transformed mouse astrocyte and GSC orthotopic xenograft models. RESULTS: Activated PDGFRα signaling in established GBM cells, GSCs, and transformed mouse astrocytes was significantly inhibited by SHP099 compared with NPCs in vitro and in vivo through targeting SHP-2-stimulated activation of extracellular signal-regulated protein kinases 1 and 2 in GBM. SHP099 treatment specifically inhibited expression of JUN, a downstream effector of PDGFR signaling, thereby attenuating cell cycle progression in GBM cells with activated PDGFRα. Moreover, SHP099 accumulated at efficacious concentrations in the brain and effectively inhibited orthotopic GBM tumor xenograft growth. SHP099 exhibited antitumor activity either as a single agent or in combination with TMZ and provided significant survival benefits for GBM tumor xenograft-bearing animals. CONCLUSIONS: Our data demonstrate the utility and feasibility of SHP099 as a potential therapeutic option for improving the clinical treatment of GBM in combination with TMZ.