RESUMO
Wnt signaling is a highly conserved metazoan pathway that plays a crucial role in cell fate determination and morphogenesis during development. Wnt ligands can induce disparate cellular responses. The exact mechanism behind these different outcomes is not fully understood but may be due to interactions with different receptors on the cell membrane. PTK7/Otk is a transmembrane receptor that is implicated in various developmental and physiological processes including cell polarity, cell migration, and invasion. Here, we examine two roles of Otk-1 and Otk-2 in patterning and neurogenesis. We find that Otk-1 is a positive regulator of signaling and Otk-2 functions as its inhibitor. We propose that PTK7/Otk functions in signaling, cell migration, and polarity contributing to the diversity of cellular responses seen in Wnt-mediated processes.
Assuntos
Padronização Corporal , Neurogênese , Receptores Proteína Tirosina Quinases , Via de Sinalização Wnt , Animais , Diferenciação Celular , Membrana Celular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Via de Sinalização Wnt/fisiologiaRESUMO
Protein tyrosine kinase 7 (PTK7), a catalytically defective receptor tyrosine kinase (RTK), is often upregulated in various cancers. This study aimed to validate PTK7 as a target for breast cancer (BC) and investigate its oncogenic signaling mechanism. BC tissue analysis showed significantly elevated PTK7 mRNA levels, especially in refractory triple-negative breast cancer (TNBC) tissues, compared with normal controls. Similarly, BC cell lines exhibited increased PTK7 expression. Knockdown of PTK7 inhibited the proliferation of T-47D and MCF-7 hormone-receptor-positive BC cell-lines and of HCC1187, MDA-MB-231, MDA-MB-436, and MDA-MB-453 TNBC cells. PTK7 knockdown also inhibited the adhesion, migration, and invasion of MDA-MB-231, MDA-MB-436, and MDA-MB-453 cells, and reduced the phosphorylation levels of crucial oncogenic regulators including extracellular signal-regulated kinase (ERK), Akt, and focal adhesion kinase (FAK). Furthermore, PTK7 interacts with fibroblast growth factor receptor 1 (FGFR1) and epidermal growth factor receptor (EGFR) expressed in MDA-MB-231 cells. Knockdown of PTK7 decreased the growth-factor-induced phosphorylation of FGFR1 and EGFR in MDA-MB-231 cells, indicating its association with RTK activation. In conclusion, PTK7 plays a significant role in oncogenic signal transduction by enhancing FGFR1 and EGFR activation, influencing BC tumorigenesis and metastasis. Hence, PTK7 represents a potential candidate for targeted BC therapy, including TNBC.
Assuntos
Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Transdução de Sinais , Fosforilação , Receptores ErbB/genética , Receptores ErbB/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Moléculas de Adesão Celular/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
While CAR-T cell therapy has shown success against hematological tumors, its effectiveness for solid tumors, including ovarian cancer, remains unsatisfactory. This study aimed to develop and evaluate the efficacy of novel chimeric antigen receptor T (CAR-T) cells targeting PTK7 through TREM1/DAP12 signaling against ovarian cancer. The expression of PTK7 in ovarian cancer tissues and cells was evaluated using immunohistochemical staining and flow cytometric analysis. The anti-tumor effects of PTK7 CAR-T cells were assessed in vitro using real-time cell analysis and enzyme-linked immunosorbent assay, and in vivo using a xenograft tumor model. PTK7 was significantly expressed in ovarian cancer tissues and cells. PTK7-targeting CAR-T cells based on TREM1/DAP12 signaling exhibited potent cytotoxicity against ovarian cancer cells expressing PTK7 in vitro, and effectively eradicated tumors in vivo. Our findings suggest that TREM1/DAP12-based PTK7 CAR-T cells have potential as a treatment strategy for ovarian cancer. Further studies are needed to evaluate the safety and efficacy of this approach in clinical trials.
Assuntos
Neoplasias Ovarianas , Receptores de Antígenos Quiméricos , Humanos , Feminino , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Linfócitos T , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Linhagem Celular Tumoral , Imunoterapia Adotiva , Ensaios Antitumorais Modelo de Xenoenxerto , Moléculas de Adesão Celular/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
The measurement of tumor biomarker levels is of great significance for early diagnosis of breast cancer. The combination diagnosis of multiple tumor biomarkers will significantly improve the accuracy of early diagnosis. Here, we successfully developed a dual-ratio fluorescent sensing platform for the detection of breast cancer biomarkers (PTK7, miRNA-21) using single excitation triple-signal detection. Introducing three types of fluorescence nanomaterials with narrow emission peaks and long Stokes shift as signal markers, the three peaks (430 nm, 530 nm and 640 nm) of which do not interfere with each other in fluorescence spectra under a single excitation (360 nm). The sensing platform linked aptamer (apt) modified green fluorescence quantum dots (gQDs-apt1) and aptamer modified red fluorescence quantum dots (rQDs-apt2) to Fe3O4-cDNA1 and Fe3O4-cDNA2, respectively, via base complementary pairing with aptamer molecules. When PTK7/miRNA-21 is present in the system, gQDs-apt1/rQDs-apt2 bound to the Fe3O4 MNPs surface will be released to recover fluorescence. Upon DNase I digestion of free apt1 and apt2, the target molecules will be released to bind to gQDs-apt1/rQDs-apt2 for signal amplification. After magnetic separation, PTK7 and miRNA-21 can be quantified using the fluorescence intensity ratio of gQDs with bCDs and rQDs with bCDs at a single excitation of 360 nm wavelength. This method has high sensitivity, good selectivity, and can quantify both PTK7 and miRNA-21 simultaneously with an LOD of 0.426 ng mL-1 and 0.072 nM, respectively. Additionally, the sensing platform was used for serum detection of health man and breast cancer patients with satisfactory results.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Neoplasias da Mama , MicroRNAs , Pontos Quânticos , Humanos , Feminino , Corantes Fluorescentes , Detecção Precoce de Câncer , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Biomarcadores Tumorais , Moléculas de Adesão Celular , Receptores Proteína Tirosina QuinasesRESUMO
The adhesion G-protein-coupled receptor GPR133 (ADGRD1) supports growth of the brain malignancy glioblastoma. How the extracellular interactome of GPR133 in glioblastoma modulates signaling remains unknown. Here, we use affinity proteomics to identify the transmembrane protein PTK7 as an extracellular binding partner of GPR133 in glioblastoma. PTK7 binds the autoproteolytically generated N-terminal fragment of GPR133 and its expression in trans increases GPR133 signaling. This effect requires the intramolecular cleavage of GPR133 and PTK7's anchoring in the plasma membrane. PTK7's allosteric action on GPR133 signaling is additive with but topographically distinct from orthosteric activation by soluble peptide mimicking the endogenous tethered Stachel agonist. GPR133 and PTK7 are expressed in adjacent cells in glioblastoma, where their knockdown phenocopies each other. We propose that this ligand-receptor interaction is relevant to the pathogenesis of glioblastoma and possibly other physiological processes in healthy tissues.
Assuntos
Glioblastoma , Humanos , Transdução de Sinais , Receptores Acoplados a Proteínas G/metabolismo , Membrana Celular/metabolismo , Regulação Alostérica , Ligantes , Sítio Alostérico , Moléculas de Adesão Celular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
GD2-targeting immunotherapies have improved survival in children with neuroblastoma, yet on-target, off-tumor toxicities can occur and a subset of patients cease to respond. The majority of neuroblastoma patients who receive immunotherapy have been previously treated with cytotoxic chemotherapy, making it paramount to identify neuroblastoma-specific antigens that remain stable throughout standard treatment. Cell surface glycoproteomics performed on human-derived neuroblastoma tumors in mice following chemotherapy treatment identified protein tyrosine kinase 7 (PTK7) to be abundantly expressed. Furthermore, PTK7 shows minimal expression on pediatric-specific normal tissues. We developed an anti-PTK7 chimeric antigen receptor (CAR) and find PTK7 CAR T cells specifically target and kill PTK7-expressing neuroblastoma in vitro. In vivo, human/murine binding PTK7 CAR T cells regress aggressive neuroblastoma metastatic mouse models and prolong survival with no toxicity. Together, these data demonstrate preclinical efficacy and tolerability for targeting PTK7 and support ongoing investigations to optimize PTK7-targeting CAR T cells for neuroblastoma.
Assuntos
Neuroblastoma , Receptores de Antígenos Quiméricos , Humanos , Criança , Animais , Camundongos , Neuroblastoma/terapia , Neuroblastoma/patologia , Imunoterapia , Receptores de Antígenos Quiméricos/genética , Proteínas Tirosina QuinasesRESUMO
Aptamers are emerging as a promising new class of functional nucleic acids because they can specifically bind to any target with high affinity and be easily modified chemically with different pharmacophoric subunits for therapy. The truncated aptamer, Sgc8-c, binds to tyrosine-protein kinase-like 7 receptor, a promising cancer therapeutic target, allowing the recognition of haemato-oncological malignancies, among others. We have previously developed aptamer-drug conjugates by chemical synthesis, hybridizing Sgc8-c and dasatinib, a drug proposed for lymphoma chemotherapy. One of the best-characterised Sgc8-c-dasatinib hybrids, namely Sgc8-c-carb-da, was capable of releasing dasatinib at an endosomal-pH. Herein, we probed the therapeutic potential of this aptamer-drug conjugate. Sgc8-c-carb-da specifically inhibited murine A20 B lymphocyte growth and produced cell death, mainly by late apoptosis and necrosis. In addition, Sgc8-c-carb-da generated an arrest in cell proliferation, with a cell cycle arrest in the Sub-G1-peak. The mitochondrial potential was altered accordingly to these pathways. Moreover, using an in vitro cell-targeting assay that mimics in vivo conditions, we showed that Sgc8-c-carb-da displayed higher (2.5-fold) cytotoxic effects than dasatinib. These findings provide proof-of-concept of the therapeutic value of Sgc8-c-carb-da for lymphoma, creating new opportunities for the chemical synthesis of targeted biotherapeutics.
RESUMO
BACKGROUND & AIMS: Metastasis is found in most advanced hepatocellular carcinoma (HCC) patients, and it drives tumor recurrence and systemic failure. There is no effective treatment owing to its complex biological features. Many of the molecular drivers of metastasis are crucial players in normal physiology but behave unconventionally during cancer progression. Targeting these molecular drivers for therapy and differentiating them from a physiological background require a detailed examination of the novel mechanisms involved in their activation during metastasis. METHODS: Publicly available transcriptomic data such as that of TCGA-LIHC and Gene Expression Omnibus were utilized to identify novel targets upregulated in advanced and metastatic HCC. Validation of candidates was assisted by immunohistochemistry performed on tissue microarrays derived from more than 100 HCC patients. Expression of protein tyrosine kinase 7 (PTK7) was studied under the treatment of transforming growth factor-ß1 and knockdown of SRY-Box Transcription Factor 9 (SOX9) to delineate upstream regulation, while CRISPR-mediated knockout and lentiviral overexpression of PTK7 in HCC cells were performed to study their functional and signaling consequences. Manipulated HCC cells were injected into mice models either by orthotopic or tail-vein injection to observe for any in vivo pro-metastatic effects. RESULTS: PTK7 was discovered to be the kinase most significantly upregulated in advanced and metastatic HCC, at both transcriptomic and proteomic level. Bioinformatic analyses and functional assays performed in HCC cell lines revealed transforming growth factor-ß signaling and SOX9 to be important activators of PTK7 expression. Functionally, enrichment of PTK7 expression could positively regulate metastatic potential of HCC cells in vitro and in lung metastasis models performed in immunodeficient mice. The up-regulation of PTK7 recruited the epithelial-mesenchymal transition components, zinc finger protein SNAI2 (SLUG) and zinc finger E-box-binding homeobox 1 (ZEB1). CONCLUSIONS: Our study proposes PTK7 as a novel molecular driver in metastatic HCC, particularly in a transforming growth factor-ß-activated microenvironment. The preferential expression of PTK7 resulted in a previously unobserved regulatory effect on the recruitment of epithelial-mesenchymal transition components, which established PTK7 as a potential determinant of specific epithelial-mesenchymal transition status. Therefore, our data support the continual development of PTK7-targeted agents as antimetastatic therapies.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Receptores Proteína Tirosina Quinases , Fatores de Transcrição SOX9 , Animais , Camundongos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Neoplasias Hepáticas/patologia , Proteômica , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de Transcrição SOX9/metabolismoRESUMO
Tyrosine protein kinase 7 (PTK7) is overexpressed in breast cancer, which is considered as a cancer marker for breast cancer diagnosis. Therefore, a simple fluorescent probe for PTK7 detection and cell imaging was developed. In the developed probe, Fe3O4 magnetic nanoparticles were used as the fluorescent separator, and the fluorescence of carbon dots were used as the detection signal. The probe was worked by control the configurations of the aptamer of PTK7, the aptamer would be open chains by recognition of PTK7, which bond with carbon dots and show fluorescent signal. Based on the remarkably high affinity and selectivity of aptamer for PTK7, the excellent fluorescence property of carbon dots and the outstanding magnetism of Fe3O4 magnetic nanoparticles, the developed probe showed satisfied results for PTK7 detection in serum and MCF-7 cell imaging. The probe detected PTK7 in the range of 0.2-200 ng mL-1 with a detection limit of 0.0347 ng mL-1, and successfully imaged the cancer cell expressed PTK7. The results indicate that the nano-fluorescent probe has great potential for clinical applications.
Assuntos
Neoplasias da Mama , Corantes Fluorescentes , Humanos , Feminino , Corantes Fluorescentes/química , Células MCF-7 , Proteínas Tirosina Quinases , Carbono , Moléculas de Adesão Celular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
BACKGROUND: Gemcitabine (GEM) is one of the first-line chemotherapies for bladder cancer (BC), but the GEMs cannot recognize cancer cells and have a low long-term response rate and high recurrence rate with side effects during the treatment of BC. Targeted transport of GEMs to mediate cytotoxicity to tumor and avoid the systemic side effects remains a challenge in the treatment of BC. METHODS: Based on a firstly confirmed biomarker in BC-protein tyrosine kinase 7 (PTK7), which is overexpressed on the cell membrane surface in BC cells, a novel targeting system protein tyrosine kinase 7 aptamer-Gemcitabine conjugate (PTK7-GEMs) was designed and synthesized using a specific PTK7 aptamer and GEM through auto-synthesis method to deliver GEM against BC. In addition, the antitumor effects and safety evaluation of PTK7-GEMs was assessed with a series of in vitro and in vivo assays. RESULTS: PTK7-GEMs can specifically bind and enter to BC cells dependent on the expression levels of PTK7 and via the macropinocytosis pathway, which induced cytotoxicity after GEM cleavage from PTK7-GEMs respond to the intracellular phosphatase. Moreover, PTK7-GEMs showed stronger anti-tumor efficacy and excellent biosafety in three types of tumor xenograft mice models. CONCLUSION: These results demonstrated that PTK7-GEMs is a successful targeted aptamer-drug conjugates strategy (APDCs) to treat BC, which will provide new directions for the precision treatment of BC in the field of biomarker-oriented tumor targeted therapy.
RESUMO
PTK7 is a catalytically defective receptor protein tyrosine kinase upregulated in various cancers, including esophageal squamous cell carcinoma (ESCC). In previous studies, we observed a positive correlation between PTK7 expression levels and tumorigenicity in various ESCC cell lines and xenograft mice with ESCC KYSE-30 cells. In this study, we analyzed the effects of anti-PTK7 monoclonal antibodies (mAbs) on the tumorigenic activity in KYSE-30 cells and in mouse xenograft models. PTK7 mAb-32 and mAb-43 bind with a high affinity to the extracellular domain of PTK7. PTK7 mAbs significantly reduced three-dimensional cell proliferation, adhesion, wound healing, and migration. PTK7 mAbs also reduce chemotactic invasiveness by decreasing MMP-9 secretion. PTK7 mAbs decreased actin cytoskeleton levels in the cortical region of KYSE-30 cells. PTK7 mAbs reduced the phosphorylation of ERK, SRC, and FAK. In a mouse xenograft model of ESCC using KYSE-30 cells, PTK7 mAbs reduced tumor growth in terms of volume, weight, and the number of Ki-67-positive cells. These results demonstrated that PTK7 mAbs can inhibit the tumorigenicity of ESCC at the cellular level and in vivo by blocking the function of PTK7. Considering the anticancer activities of PTK7 mAbs, we propose that PTK7 mAbs can be used in an effective treatment strategy for PTK7-positive malignancies, such as ESCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Camundongos , Animais , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/patologia , Neoplasias Esofágicas/patologia , Metaloproteinase 9 da Matriz , Carcinoma de Células Escamosas/patologia , Xenoenxertos , Anticorpos Monoclonais/farmacologia , Antígeno Ki-67 , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Receptores Proteína Tirosina Quinases/metabolismo , Proliferação de CélulasRESUMO
PTK7, a catalytically defective receptor protein tyrosine kinase, promotes angiogenesis by activating KDR through direct interaction and induction of KDR oligomerization. This study developed anti-PTK7 monoclonal antibodies (mAbs) to regulate angiogenesis by inhibiting PTK7 function. The effect of anti-PTK7 mAbs on vascular endothelial growth factor (VEGF)-induced angiogenic phenotypes in human umbilical vascular endothelial cells (HUVECs) was examined. Analysis of mAb binding with PTK7 deletion mutants revealed that mAb-43 and mAb-52 recognize immunoglobulin (Ig) domain 2 of PTK7, whereas mAb-32 and mAb-50 recognize Ig domains 6-7. Anti-PTK7 mAbs inhibited VEGF-induced adhesion and wound healing in HUVECs. mAb-32, mAb-43, and mAb-52 dose-dependently mitigated VEGF-induced migration and invasion in HUVECs without exerting cytotoxic effects. Additionally, mAb-32, mAb-43, and mAb-52 inhibited capillary-like tube formation in HUVECs, and mAb-32 and mAb-43 suppressed angiogenesis ex vivo (aortic ring assay) and in vivo (Matrigel plug assay). Furthermore, mAb-32 and mAb-43 downregulated VEGF-induced KDR activation and downstream signaling and inhibited PTK7-KDR interaction in PTK7-overexpressing and KDR-overexpressing HEK293 cells. Thus, anti-PTK7 mAbs inhibit angiogenic phenotypes by blocking PTK7-KDR interaction. These findings indicate that anti-PTK7 mAbs that neutralize PTK7 function can alleviate impaired angiogenesis-associated pathological conditions, such as cancer metastasis.
RESUMO
ROR1, ROR2, and PTK7 are Wnt ligand-binding members of the receptor tyrosine kinase family. Despite their lack of catalytic activity, these receptors regulate skeletal, cardiorespiratory, and neurological development during embryonic and fetal stages. However, their overexpression in adult tissue is strongly connected to tumor development and metastasis, suggesting a strong pharmacological potential for these molecules. Wnt5a ligand can activate these receptors, but lead to divergent signaling and functional outcomes through mechanisms that remain largely unknown. Here, we developed a cellular model by stably expressing ROR1, ROR2, and PTK7 in BaF3 cells that allowed us to readily investigate side-by-side their signaling capability and functional outcome. We applied proteomic profiling to BaF3 clones and identified distinctive roles for ROR1, ROR2, and PTK7 pseudokinases in modulating the expression of proteins involved in cytoskeleton dynamics, apoptotic, and metabolic signaling. Functionally, we show that ROR1 expression enhances cell survival and Wnt-mediated cell proliferation, while ROR2 and PTK7 expression is linked to cell migration. We also demonstrate that the distal C-terminal regions of ROR1 and ROR2 are required for receptors stability and downstream signaling. To probe the pharmacological modulation of ROR1 oncogenic signaling, we used affinity purification coupled to mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID) to map its interactome before and after binding of GZD824, a small molecule inhibitor previously shown to bind to the ROR1 pseudokinase domain. Our findings bring new insight into the molecular mechanisms of ROR1, ROR2, and PTK7, and highlight the therapeutic potential of targeting ROR1 with small molecule inhibitors binding to its vestigial ATP-binding site.
Assuntos
Proteômica , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Proliferação de Células , Ligantes , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Transdução de SinaisRESUMO
Exosomes (30-100 nm in diameter) are a group of cell-derived membrane vesicles, packaged as valuable cargo with lipid, proteins, and genetic materials from their parent cells. With the increasing interest in exosomes for diagnostic and therapeutic applications, the rapid isolation of pure exosome populations has become a hot topic. In this paper, we propose modified microchannels with aptamer in a microfluidics system for rapid and efficient isolation of exosomes by targeting exosome-carrying CD63 and PTK 7. The capture efficiency in surface-modified channels reaches around 107-108 particles/mL in 20 min, and purified exosomes with reliable size can be achieved.
Assuntos
Exossomos , Microfluídica , Oligonucleotídeos , Proteínas/metabolismoRESUMO
Recent biotechnological applications in the field of clinical oncology led to the identification of new biomarkers as molecular targets of cancer, and to broad developments in the field of personalized medicine. Aptamers are oligonucleotides (ssDNA or RNA) that are selected to specifically recognize a molecular target with high affinity and specificity. Based on this, new horizons for their use as molecular imaging probes are being explored. The objective of this work was to evaluate the Sgc8-c aptamer conjugated with Alexa Fluor 647 fluorophore as an imaging probe in a colon tumor xenograft mouse model, with potential application in molecular imaging. In this study, the LS174T cell line was used to induce colorectal adenocarcinoma in nude mice. After confirmation of PTK7 overexpression by immunohistochemistry, in vivo studies were performed. Pharmacokinetic, in vivo and ex vivo biodistribution imaging, and a competition assay were evaluated by fluorescence imaging. In vivo visualization of the probe in the tumors was assessed two hours after aptamer probe administration, exhibiting excellent tumor-to-background ratios in biodistribution studies and high specificity in the competition test. Our results demonstrated the functionality of Scg8-c as an imaging probe for colon cancer, with potential clinical applications.
Assuntos
Aptâmeros de Nucleotídeos , Neoplasias do Colo , Animais , Aptâmeros de Nucleotídeos/química , Moléculas de Adesão Celular , Linhagem Celular Tumoral , Neoplasias do Colo/diagnóstico por imagem , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Imagem Molecular , Sondas Moleculares , Receptores Proteína Tirosina Quinases , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
It has been acknowledged that circulating tumor cells (CTCs) are promising biomarkers in liquid biopsy for cancer diagnosis and prognosis. However, the relationship between the CTC number and gastric cancer has scarcely been quantitatively investigated. Moreover, the single criterion of epithelial cell adhesion molecule (EpCAM) antibody/aptamer to specifically recognize epithelial CTCs cannot be universally applied for clinical applications, as it fails to recognize EpCAM-negative CTCs. Herein, we propose simple, low-cost, dual-aptamer (EpCAM and PTK7)-modified immunomagnetic Fe3O4 particles (IMNs) for efficient capture of heterogeneous CTCs and downstream analysis in gastric cancer patients. High PTK7 expression and a significant negative correlation between PTK7 and EpCAM expression were observed in primary gastric cancer tissues. Taking MGC-803 and BGC-823 cells as CTC models, the obtained dual-targeting IMNs could distinguishably recognize these cells with both high or low EpCAM and PTK7 expressions, which enhanced the accuracy of CTC recognition in gastric cancer. More than 95% of these two kinds of cells could be captured within 20 min of incubation, which was significantly more efficient than that of single EpCAM- or PTK7-modified IMNs. With this strategy, as low as five CTCs could be captured from phosphate-buffered saline (PBS), a cell mixture containing THP-1 cells, and lysed blood mediums. Moreover, the obtained CTCs can be used for subsequent gene analysis. Finally, the fabricated IMNs were successfully applied for CTC capture in 1.0 mL of peripheral blood samples from patients with gastric cancer. The detected CTC numbers in 72 participants were found to have close relationships with chemotherapy sensitivity, diagnosis, stage, and distant metastasis of patients. This work provides important references for further investigations on CTC-related diagnosis and individualized treatment.
Assuntos
Nanopartículas , Células Neoplásicas Circulantes , Neoplasias Gástricas , Biomarcadores Tumorais/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/metabolismo , Humanos , Células Neoplásicas Circulantes/metabolismo , Receptores Proteína Tirosina Quinases , Neoplasias Gástricas/diagnósticoRESUMO
Colorectal cancer (CRC) is the third most frequently diagnosed malignancy worldwide. Only 5% of all CRC cases are due to germline mutations in known predisposition genes, and the remaining genetic burden still has to be discovered. In this study, we performed whole-exome sequencing on six members of a Polish family diagnosed with CRC and identified a novel germline variant in the protein tyrosine kinase 7 (inactive) gene (PTK7, ENST00000230419, V354M). Targeted screening of the variant in 1705 familial CRC cases and 1674 healthy elderly individuals identified the variant in an additional familial CRC case. Introduction of this variant in HT-29 cells resulted in increased cell proliferation, migration, and invasion; it also caused down-regulation of CREB, p21 and p53 mRNA and protein levels, and increased AKT phosphorylation. These changes indicated inhibition of apoptosis pathways and activation of AKT signaling. Our study confirmed the oncogenic function of PTK7 and supported its role in genetic predisposition of familial CRC.
Assuntos
Moléculas de Adesão Celular/genética , Neoplasias Colorretais/genética , Receptores Proteína Tirosina Quinases/genética , Idoso , Moléculas de Adesão Celular/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Família , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Oncogenes , Linhagem , Proteínas Proto-Oncogênicas c-akt/genética , Receptores Proteína Tirosina Quinases/metabolismo , Proteína Supressora de Tumor p53/genética , Sequenciamento do Exoma/métodosRESUMO
Extracellular vesicles (EV) are released by virtually all cells and they transport biologically important molecules from the release site to target cells. Colorectal cancer (CRC) is a leading cause of cancer-related death cases, thus, it represents a major health issue. Although the EV cargo may reflect the molecular composition of the releasing cells and thus, EVs may hold a great promise for tumor diagnostics, the impact of intratumoral heterogeneity on the intensity of EV release is still largely unknown. By using CRC patient-derived organoids that maintain the cellular and molecular heterogeneity of the original epithelial tumor tissue, we proved that CD44high cells produce more organoids with a higher proliferation intensity, as compared to CD44low cells. Interestingly, we detected an increased EV release by CD44high CRC cells. In addition, we found that the miRNA cargos of CD44high and CD44low cell derived EVs largely overlapped and only four miRNAs were specific for one of the above subpopulations. We observed that EVs released by CD44high cells induced the proliferation and activation of colon fibroblasts more strongly than CD44low cells. However, this effect was due to the higher EV number rather than to the miRNA cargo of EVs. Collectively, we identified CRC subpopulations with different EV releasing capabilities and we proved that CRC cell-released EVs have a miRNA-independent effect on fibroblast proliferation and activation.
Assuntos
Neoplasias Colorretais , Vesículas Extracelulares , MicroRNAs , Comunicação Celular , Neoplasias Colorretais/patologia , Vesículas Extracelulares/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Organoides/metabolismoRESUMO
Protein tyrosine kinase 7 (PTK7), a catalytically defective receptor protein tyrosine kinase, is upregulated in tumor tissues and cell lines of esophageal squamous cell carcinoma (ESCC). We showed that PTK7 plays an oncogenic role in various ESCC cell lines. However, its role as an oncogene has not been demonstrated in vivo. Here, we examined the influence of PTK7 on the tumorigenic potential of ESCC KYSE-30 cells, which are known to establish xenograft tumors. Overexpression of PTK7 enhanced the proliferation, adhesion, wound healing, and migration of KYSE-30 cells, and these effects were reversed by the knockdown of PTK7. PTK7 overexpression and knockdown, respectively, increased and decreased the tyrosine phosphorylation of cellular proteins and the phosphorylation of ERK, AKT, and FAK, which are important for cell proliferation, survival, adhesion, and migration. Additionally, PTK7 overexpression and silencing, respectively, increased and decreased the weight, volume, and number of Ki-67-positive proliferating cells in xenograft tumors of KYSE-30 cells. Therefore, we propose that PTK7 plays an important role in the tumorigenesis of ESCC cells in vivo and is a potential therapeutic target for ESCC.
Assuntos
Carcinogênese/genética , Moléculas de Adesão Celular/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Xenoenxertos/metabolismo , Oncogenes/genética , Receptores Proteína Tirosina Quinases/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Fenótipo , Fosforilação/genética , Transdução de Sinais/genéticaRESUMO
Depletion of ptk7 is associated with both congenital scoliosis (CS) and adolescent idiopathic scoliosis (AIS) in zebrafish models. However, only one human variant of PTK7 has been reported previously in a patient with AIS. In this study, we systemically investigated the variant landscape of PTK7 in 583 patients with CS and 302 patients with AIS from the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO) study. We identified a total of four rare variants in CS and four variants in AIS, including one protein truncating variant (c.464_465delAC) in a patient with CS. We then explored the effects of these variants on protein expression and sub-cellular location. We confirmed that the c.464_465delAC variant causes loss-of-function (LoF) of PTK7. In addition, the c.353C>T and c.2290G>A variants identified in two patients with AIS led to reduced protein expression of PTK7 as compared to that of the wild type. In conclusion, LoF and hypomorphic variants are associated with CS and AIS, respectively.