Quantitative Membrane Proteomics for Discovery of Actionable Drug Targets at the Surface of RAS-Driven Human Cancer Cells.

With the advent of promising lung cancer immunotherapies targeting proteins at the cell surface of RAS-driven human cancers, the mass spectrometry (MS)-based surfaceomics remains a feasible strategy for therapeutic target discovery. This chapter describes a protocol for discovery of druggable protein targets at the surface of RAS-driven human cancer cells. This method relies on bottom-up MS-based quantitative surfaceomics that employs in parallel, targeted hydrazide-based cell-surface glycoproteomics and global shotgun membrane proteomics to enable unbiased quantitative profiling of thousands of cell surface membrane proteins. A large-scale molecular map of the KRASG12V surface was attained, resulting in confident detection and quantitation of more than 500 cell surface membrane proteins that were found to be unique or upregulated at the surface of cells harboring the KRASG12V mutant. A multistep bioinformatic progression revealed a subset of unique and/or significantly upregulated proteins as priority drug targets selected for orthogonal cross-validation using immunofluorescence, structured illumination microscopy, and western blotting. Among cross-validated targets, CUB domain containing protein 1 (CDCP1) and basigin (BSG-CD147) were selected as leading targets due to their involvement in cell adhesion and migration, consistent with the KRASG12V malignant phenotype as revealed by scanning electron microscopy and phenotypic cancer cell assays. Follow-up studies confirmed CDCP1 as an actionable therapeutic target, resulting in development of recombinant antibodies capable of killing KRAS-transformed cancer cells in preclinical setting. The present MS-based surfaceomics workflow represents a powerful drug target discovery platform that enables development of innovative immunotherapeutics (e.g., antibody drug conjugate against CDCP1) for attacking oncogenic RAS-driven cancers at the cell surface.

CD6 and Its Interacting Partners: Newcomers to the Block of Cancer Immunotherapies.

Cancer management still requires more potent and safer treatments, of which immunomodulatory receptors on the lymphocyte surface have started to show promise in new cancer immunotherapies (e.g., CTLA-4 and PD-1). CD6 is a signal-transducing transmembrane receptor, mainly expressed by all T cells and some B and NK cell subsets, whose endogenous ligands (CD166/ALCAM, CD318/CDCP-1, Galectins 1 and 3) are overexpressed by malignant cells of different lineages. This places CD6 as a potential target for novel therapies against haematological and non-haematological malignancies. Recent experimental evidence for the role of CD6 in cancer immunotherapies is summarised in this review, dealing with diverse and innovative strategies from the classical use of monoclonal antibodies to soluble recombinant decoys or the adoptive transfer of immune cells engineered with chimeric antigen receptors.

Ascites-derived CDCP1+ extracellular vesicles subcluster as a novel biomarker and therapeutic target for ovarian cancer.

Introduction: Ovarian cancer (OVCA) is one of the most prevalent malignant tumors of the female reproductive system, and its diagnosis is typically accompanied by the production of ascites. Although liquid biopsy has been widely implemented recently, the diagnosis or prognosis of OVCA based on liquid biopsy remains the primary emphasis. Methods: In this study, using proximity barcoding assay, a technique for analyzing the surface proteins on single extracellular vesicles (EVs). For validation, serum and ascites samples from patients with epithelial ovarian cancer (EOC) were collected, and their levels of CDCP1 was determined by enzyme-linked immunosorbent assay. Tissue chips were prepared to analyze the relationship between different expression levels of CDCP1 and the prognosis of ovarian cancer patients. Results: We discovered that the CUB domain-containing protein 1+ (CDCP1+) EVs subcluster was higher in the ascites of OVCA patients compared to benign ascites. At the same time, the level of CDCP1 was considerably elevated in the ascites of OVCA patients. The overall survival and disease-free survival of the group with high CDCP1 expression in EOC were significantly lower than those of the group with low expression. In addition, the receiver operating characteristic curve demonstrates that EVs-derived CDCP1 was a biomarker of early response in OVCA ascites. Discussion: Our findings identified a CDCP1+ EVs subcluster in the ascites of OVCA patients as a possible biomarker for EOC prevention.

Metastatic prostate cancer-derived extracellular vesicles facilitate osteoclastogenesis by transferring the CDCP1 protein.

Bone metastases are still incurable and result in the development of clinical complications and decreased survival for prostate cancer patients. Recently, a number of studies have shown that extracellular vesicles (EVs) play important roles in tumour progression. Here, we show that EVs from metastatic prostate cancer cells promote osteoclast formation in the presence of receptor activator of NF-κB ligand (RANKL). EV characterization followed by functional siRNA screening identified CUB-domain containing protein 1 (CDCP1), a transmembrane protein, as an inducer of osteoclastogenesis. Additionally, CDCP1 expression on plasma-derived EVs was upregulated in bone metastatic prostate cancer patients. Our findings elucidate the effect of EVs from metastatic prostate cancer cells on osteoclast formation, which is promoted by CDCP1 located on EVs. Furthermore, our data suggested that CDCP1 expression on EVs might be useful to detect bone metastasis of prostate cancer.

Detection and Isolation of Circulating Tumor Cells from Breast Cancer Patients Using CUB Domain-Containing Protein 1.

In cancer metastasis, single circulating tumor cells (CTCs) in the blood and disseminated tumor cells (DTCs) in the bone marrow mediate cancer metastasis. Because suitable biomarker proteins are lacking, CTCs and DTCs with mesenchymal attributes are difficult to isolate from the bulk of normal blood cells. To establish a procedure allowing the isolation of such cells, we analyzed the cell line BC-M1 established from DTCs in the bone marrow of a breast cancer patient by stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry. We found high levels of the transmembrane protein CUB domain-containing protein 1 (CDCP1) in breast cancer cell lines with mesenchymal attributes. Peripheral blood mononuclear cells were virtually negative for CDCP1. Confirmation in vivo by CellSearch revealed CDCP1-positive CTCs in 8 of 30 analyzed breast cancer patients. Only EpCam-positive CTCs were enriched by CellSearch. Using the extracellular domain of CDCP1, we established a magnetic-activated cell sorting (MACS) approach enabling also the enrichment of EpCam-negative CTCs. Thus, our approach is particularly suited for the isolation of mesenchymal CTCs with downregulated epithelial cancer that occur, for example, in triple-negative breast cancer patients who are prone to therapy failure.

The roles of proteases in prostate cancer.

Since the proposition of the pro-invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well-characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein-related peptidases, including KLK3 (prostate-specific antigen, PSA), the most well-known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration-resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease-activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens.

CUB Domain-Containing Protein 1 (CDCP1) is a rational target for the development of imaging tracers and antibody-drug conjugates for cancer detection and therapy.

Rationale: An antibody-drug conjugate (ADC) is a targeted therapy consisting of a cytotoxic payload that is linked to an antibody which targets a protein enriched on malignant cells. Multiple ADCs are currently used clinically as anti-cancer agents significantly improving patient survival. Herein, we evaluated the rationale of targeting the cell surface oncoreceptor CUB domain-containing protein 1 (CDCP1) using ADCs and assessed the efficacy of CDCP1-directed ADCs against a range of malignant tumors. Methods: CDCP1 mRNA expression was evaluated using large transcriptomic datasets of normal/tumor samples for 23 types of cancer and 15 other normal organs, and CDCP1 protein expression was examined in 34 normal tissues, >300 samples from six types of cancer, and in 49 cancer cell lines. A recombinant human/mouse chimeric anti-CDCP1 antibody (ch10D7) was labelled with 89Zirconium or monomethyl auristatin E (MMAE) and tested in multiple pre-clinical cancer models including 36 cancer cell lines and three mouse xenograft models. Results: Analysis of CDCP1 expression indicates elevated CDCP1 expression in the majority of the cancers and restricted expression in normal human tissues. Antibody ch10D7 demonstrates a high affinity and specificity for CDCP1 inducing cell signalling via Src accompanied by rapid internalization of ch10D7/CDCP1 complexes in cancer cells. 89Zirconium-labelled ch10D7 accumulates in CDCP1 expressing cells enabling detection of pancreatic cancer xenografts in mice by PET imaging. Cytotoxicity of MMAE-labelled ch10D7 against kidney, colorectal, lung, ovarian, pancreatic and prostate cancer cells in vitro, correlates with the level of CDCP1 on the plasma membrane. ch10D7-MMAE displays robust anti-tumor effects against mouse xenograft models of pancreatic, colorectal and ovarian cancer. Conclusion: CDCP1 directed imaging agents will be useful for selecting cancer patients for personalized treatment with cytotoxin-loaded CDCP1 targeting agents including antibody-drug conjugates.

Src activation in lipid rafts confers epithelial cells with invasive potential to escape from apical extrusion during cell competition.

Abnormal/cancerous cells within healthy epithelial tissues undergo apical extrusion to protect against carcinogenesis, although they acquire invasive capacity once carcinogenesis progresses. However, the molecular mechanisms by which cancer cells escape from apical extrusion and invade surrounding tissues remain elusive. In this study, we demonstrate a molecular mechanism for cell fate switching during epithelial cell competition. We found that during competition within epithelial cell layers, Src transformation promotes maturation of focal adhesions and degradation of extracellular matrix. Src-transformed cells underwent basal delamination by Src activation within sphingolipid/cholesterol-enriched membrane microdomains/lipid rafts, whereas they were apically extruded when Src was outside of lipid rafts. A comparative analysis of contrasting phenotypes revealed that activation of the Src-STAT3-MMP axis through lipid rafts was required for basal delamination. CUB-domain-containing protein 1 (CDCP1) was identified as an Src-activating scaffold and as a Met regulator in lipid rafts, and its overexpression induced basal delamination. In renal cancer models, CDCP1 promoted epithelial-mesenchymal transition-mediated invasive behavior by activating the Src-STAT3-MMP axis through Met activation. Overall, these results suggest that spatial activation of Src signaling in lipid rafts confers resistance to apical extrusion and invasive potential on epithelial cells to promote carcinogenesis.

Molecular mechanism by which CDCP1 promotes proneural-mesenchymal transformation in primary glioblastoma.

BACKGROUND: Compared with the proneural (PN) subtype of glioblastoma (GBM), the mesenchymal (MES) subtype is more invasive and immune evasive and is closely related to poor prognosis. Here, we used transcriptome data and experimental evidence to indicate that CUB domain-containing protein 1 (CDCP1) is a novel regulator that facilitates the transformation of PN-GBM to MES-GBM. METHODS: The mRNA expression data of CDCP1 in glioma were collected from the TCGA, CGGA and GEO databases, and in vitro experiments verified CDCP1 expression in glioma tissue samples. Independent prognostic analysis revealed the correlation of the CDCP1 expression level and patient survival. Bioinformatics analysis and experiments verified the biological function of CDCP1. Multivariate proportional hazards models and a PPI network were used to select key genes. A prognostic risk model for predicting the survival of glioma patients was constructed based on the selected genes. RESULTS: The results showed that the expression of CDCP1 increased with increasing tumor grade and that the overexpression of CDCP1 correlated with a poor prognosis. CDCP1 was highly expressed in MES-GBM but weakly expressed in PN-GBM. The risk model (considering CDCP1 combined with CD44 and ITGAM expression) could represent a tool for predicting survival and prognosis in glioma patients. CONCLUSIONS: Our study indicates that CDCP1 plays an important role in facilitating the transformation of PN-GBM to MES-GBM.

Elevated levels of serum CDCP1 in individuals recovering from severe COVID-19 disease.

BACKGROUND: COVID-19 survivors report residual lung abnormalities after discharge from the hospital. The aim of this study was to identify biomarkers in serum and induced sputum samples from patients after hospitalization for COVID-19. METHODS: Patients admitted to hospitals in Spain with laboratory-confirmed COVID-19 were recruited for this study. SARS-CoV-2-infected patients were divided into groups with mild/moderate and severe disease according to the severity of their symptoms during hospitalization. Levels of 92 biomarkers were measured in serum and induced sputum samples. RESULTS: A total of 108 patients (46.2% severe cases) were included in this study. The median number of days after the onset of symptoms was 104. A significant difference was observed in diffusing capacity for carbon monoxide (DLCO), an indicator of lung function, whereby DLCO <80% was significantly lower in severe cases (p <0.001). Differences in inflammatory biomarkers were observed between patients with mild/moderate and severe disease. For some biomarkers, correlations in serum and induced sputum levels were detected. Independent predictors of severe disease were DLCO <80% and the serum CDCP1 value. CONCLUSIONS: Higher levels of CDCP1 remain after hospital discharge and are associated with the severity of COVID-19. The possible prognostic implications warrant further investigation.

CDCP1 Expression Is a Potential Biomarker of Poor Prognosis in Resected Stage I Non-Small-Cell Lung Cancer.

BACKGROUND: Although early-stage lung cancer has increased owing to the introduction of screening programs, high recurrence rate remains a critical concern. We aimed to explore biomarkers related to the prognosis of surgically resected non-small-cell lung cancer (NSCLC). METHODS: In this retrospective study, we collected medical records of patients with NSCLC and matched tissue microarray blocks from surgical specimens. Semiquantitative immunohistochemistry was performed for measuring the expression level of fibroblast activation protein-alpha (FAP-α), Jagged-1 (JAG1), and CUB-domain-containing protein 1 (CDCP1). RESULTS: A total of 453 patients who underwent complete resection between January 2011 and February 2012 were enrolled; 55.2% patients had stage I NSCLC, and 31.1% presented squamous cell carcinoma. Disease stage was a significant risk factor for recurrence and death, and age ≥ 65 years and male sex were associated with poor overall survival. FAP-a and JaG1 were not related to survivals, while CDCP1-expressing patients exhibited poor disease-free and overall survival. Moreover, CDCP1 expression in stage I NSCLC was significantly associated with recurrence. CONCLUSIONS: Old age, male sex, and high pathological stage were poor prognostic factors in patients with NSCLC who underwent surgical resection. Furthermore, CDCP1 expression could serve as a biomarker for poor prognosis in stage I NSCLC.

hsa_circ_0005358 suppresses cervical cancer metastasis by interacting with PTBP1 protein to destabilize CDCP1 mRNA.

Metastasis is the main cause of cervical cancer lethality, but to date, no effective treatment has been developed to block metastasis. Circular RNAs (circRNAs) were recently found to be involved in cancer metastasis. In this study, we identified a downregulated circRNA derived from the host gene Gli1 (hsa_circ_0005358) in cervical cancer tissues, which was expressed at lower levels in tissues with extracervical metastasis than in those without extracervical metastasis. Upregulation of hsa_circ_0005358 significantly suppressed the migration and invasion of cervical cancer cells in vitro, and downregulation of hsa_circ_0005358 had the opposite effect. A mouse model revealed that cervical cancer cells overexpressing hsa_circ_0005358 possessed weaker metastatic potential in vivo. RNA-pull-down assay, mass spectrometry, and RNA immunoprecipitation validated the findings that hsa_circ_0005358 functions via its 215-224 sequence, which interacts with polypyrimidine tract-binding protein 1 (PTBP1). RNA-sequencing profiling revealed that CUB-domain-containing protein 1 (CDCP1) is a common target for hsa_circ_0005358 and PTBP1. We further confirmed that hsa_circ_0005358 sequestered PTBP1, preventing it from stabilizing CDCP1 mRNA, reducing CDCP1 protein translation and ultimately suppressing cancer metastasis. Our findings reveal the function of hsa_circ_0005358 in tumor metastasis, which may be applied to a potential therapeutic approach for patients with metastatic cervical cancer.

SRC kinase activator CDCP1 promotes hepatocyte growth factor-induced cell migration/invasion of a subset of breast cancer cells.

Cancer invasion and metastasis are the major causes of cancer patient mortality. Various growth factors, including hepatocyte growth factor (HGF), are known to promote cancer invasion and metastasis, but the regulatory mechanisms involved are not fully understood. Here, we show that HGF-promoted migration and invasion of breast cancer cells are regulated by CUB domain-containing protein 1 (CDCP1), a transmembrane activator of SRC kinase. In metastatic human breast cancer cell line MDA-MB-231, which highly expresses the HGF receptor MET and CDCP1, we show that CDCP1 knockdown attenuated HGF-induced MET activation, followed by suppression of lamellipodia formation and cell migration/invasion. In contrast, in the low invasive/nonmetastatic breast cancer cell line T47D, which had no detectable MET and CDCP1 expression, ectopic MET expression stimulated the HGF-dependent activation of invasive activity, and concomitant CDCP1 expression activated SRC and further promoted invasive activity. In these cells, CDCP1 expression dramatically activated HGF-induced membrane remodeling, which was accompanied by activation of the small GTPase Rac1. Analysis of guanine nucleotide exchange factors revealed that ARHGEF7 was specifically required for CDCP1-dependent induction of HGF-induced invasive ability. Furthermore, immunofluorescence staining demonstrated that CDCP1 coaccumulated with ARHGEF7. Finally, we confirmed that the CDCP1-SRC axis was also crucial for HGF and ARHGEF7-RAC1 signaling in MDA-MB-231 cells. Altogether, these results demonstrate that the CDCP1-SRC-ARHGEF7-RAC1 pathway plays an important role in the HGF-induced invasion of a subset of breast cancer cells.

Evaluation of CDCP1 (CD318) and endoglin (CD105) expression as prognostic markers in acute myeloid leukemia.

BACKGROUND: The most commonly used prognostic factors in acute myeloid leukemia (AML) are cytogenetic, molecular, and morphological markers. However, AML prognosis is still unfavorable particularly in adults. So, further reliable markers are urgently needed to improve the risk stratification and treatment decisions. CUB domain-containing protein 1 (CDCP1; CD318) and endoglin (CD105) are new markers correlated with poor prognosis in different solid tumors, but their role in AML prognosis is not fully evaluated. OBJECTIVES: This work aimed to evaluate the prognostic role of CD318 and CD105 in AML and their impact on the outcomes. METHODS: Sixty-five newly diagnosed AML patients were included in this study. CD318 and CD105 expression was assessed by quantitative real-time polymerase chain reaction. Patients were followed up for ∼ 2 years to evaluate the prognostic impact of gene expression on the outcomes. RESULTS: Patients with high CD318 and CD105 showed higher white blood cell (WBC) count, M2 subtype, poor cytogenetic risk, reduced complete remission, and a greater number of deaths compared to low CD318 and CD105. CD318 was correlated with CD105, and both were correlated with WBC count, bone marrow blasts, and peripheral blood blasts. After a follow-up period of up to 24 months, relapse-free survival for high CD318 and CD105 was significantly different (42.1% and 52.6% vs. 64.5% and 58.1% for low CD318 and CD105, respectively). Survival was worse in patients with high CD318 and CD105, as the mean survival time was 13.9 and 13.3 months compared to 24 and 22.7 months in low CD318 and CD105, respectively. CONCLUSIONS: CD318 and CD105 are upregulated in AML patients. Their overexpression was associated with poor response to treatment and poor outcomes. Therefore, CD318 and CD105 can be useful prognostic markers in AML.

Inhibition of cotranslational translocation by apratoxin S4: Effects on oncogenic receptor tyrosine kinases and the fate of transmembrane proteins produced in the cytoplasm.

Receptor tyrosine kinases (RTKs) have become major targets for anticancer therapy. However, resistance and signaling pathway redundancy has been problematic. The marine-derived apratoxins act complementary to direct kinase inhibitors by downregulating the levels of multiple of these receptors and additionally prevent the secretion of growth factors that act on these receptors by targeting Sec61α, therefore interfering with cotranslational translocation. We have profiled the synthetic, natural product-inspired apratoxin S4 against panels of cancer cells characterized by differential sensitivity to RTK inhibitors due to receptor mutations, oncogenic KRAS mutations, or activation of compensatory pathways. Apratoxin S4 was active at low-nanomolar to sub-nanomolar concentrations against panels of lung, head and neck, bladder, and pancreatic cancer cells, concomitant with the downregulation of levels of several RTKs, including EGFR, MET and others. However, the requisite concentration to inhibit certain receptors varied, suggesting some differential substrate selectivity in cellular settings. This selectivity was most pronounced in breast cancer cells, where apratoxin S4 selectively targeted HER3 over HER2 and showed greater activity against ER+ and triple negative breast cancer cells than HER2+ cancer cells. Depending on the breast cancer subtype, apratoxin S4 differentially downregulated transmembrane protein CDCP1, which is linked to metastasis and invasion in breast cancer and modulates EGFR activity. We followed the fate of CDCP1 through proteomics and found that nonglycosylated CDCP1 associates with chaperone HSP70 and HUWE1 that functions as an E3 ubiquitin ligase and presumably targets CDCP1, as well as potentially other substrates inhibited by apratoxins, for proteasomal degradation. By preventing cotranslational translocation of VEGF and other proangiogenic factors as well as VEGFR2 and other receptors, apratoxins also possess antiangiogenic activity, which was validated in endothelial cells where downregulation of VEGFR2 was observed, extending the therapeutic scope to angiogenic diseases.

Loss of CDCP1 triggers FAK activation in detached prostate cancer cells.

A major metastasis suppressing mechanism is the rapid apoptotic death of cancer cells upon detachment from extracellular matrix, a process called anoikis. Focal adhesion kinase (PTK2/FAK) is a key enzyme involved in evasion of anoikis. We show that loss of the Cub-domain containing protein-1 (CDCP1), paradoxically stimulates FAK activation in the detached state of prostate cancer cells. In CDCP1low DU145 and PC3 prostate cancer cells, detachment-activation of FAK occurs through local production of PI(4,5)P2. PI(4,5)P2 is generated by the PIP5K1c-201 splicing isoform of PIP5K1c, which contains a unique SRC phosphorylation site. In the detached state, reduced expression of CDCP1 and an alternative CDCP1-independent SRC activation mechanism triggers PIP5K1c-pY644 phosphorylation by SRC. This causes a switch of Talin binding from ß1-integrin to PIP5K1c-pY644 and leads to activation of PIP5K1c-FAK. Reduced CDCP1 expression also inactivates CDK5, a negative regulator of PIP5K1c. Furthermore, immersion of prostate cancer cells in 10% human plasma or fetal bovine serum is required for activation of PIP5K1c-FAK. The PIP5K1c induced detachment-activation of FAK in preclinical models sensitizes CDCP1low prostate cancer cells to FAK inhibitors. In patients, CDCP1High versus CDCP1low circulating tumor cells differ in expression of AR-v7, ONECUT2 and HOXB13 oncogenes and TMPRSS2 and display intra-patient heterogeneity of FAK-pY397 expression. Taken together, CDCP1low and CDCP1high detached prostate cancer cells activate distinct cytoplasmic kinase complexes and targetable transcription factors, which has important therapeutic implications.

Preclinical Evaluation of a Fluorescent Probe Targeting Receptor CDCP1 for Identification of Ovarian Cancer.

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.

Methylation of microRNA-338-5p by EED promotes METTL3-mediated translation of oncogene CDCP1 in gastric cancer.

Unmasking the complex regulatory pathways that mediate the malignant phenotypes of cancer cells can provide novel targets for therapies that could limit the recurrence and metastasis of gastric cancer (GC). Herein, we intended to clarify the role of embryonic ectoderm development protein (EED), microRNA-228-5p (miR-338-5p), methyltransferase like 3 (METTL3) and CUB domain containing protein 1 (CDCP1) in GC. Differentially expressed miRNAs and their target genes were extracted by in silico analysis. The studies revealed high expression of EED in GC tissues and cell lines and it high expression in GC patients was shown to be associated with poor prognosis. The chromatin immunoprecipitation assay identified that EED methylated miR-338-5p to inhibit its expression. EED knockdown could restrain the proliferative and invasive abilities of GC cells by inducing miR-338-5p. Furthermore, miR-338-5p targeted m6A methylase METTL3, while METTL3 amplified the translation of CDCP1 via m6A activity which led to accelerated proliferation and invasion of GC cells. Moreover, in vivo experiments validated that EED promoted the progression of GC through mediating the miR-338-5p/METTL3/CDCP1 axis. Collectively, EED downregulated miR-338-5p through histone methylation, which in turn impaired miR-338-5p-dependent METTL3 inhibition and enhanced CDCP1 translation, therefore contributing to the development of GC.

Repurposing Tranexamic Acid as an Anticancer Agent.

Tranexamic Acid (TA) is a clinically used antifibrinolytic agent that acts as a Lys mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic Lys and Arg, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. Further, TA inhibited uptake of Lys and Arg by cancer cells. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

The emerging role of the MiR-1272-ADAM9-CDCP1 signaling pathway in the progression of glioma.

Glioma is a primary, malignant, and aggressive brain tumor in adults. To develop new therapeutic strategies for glioma, we must determine its underlying mechanisms. In the present study, we aimed to investigate the potential role of miR-1272-ADAM9-CDCP1 signaling in the progression of glioma. We found that ectopic expression of miR-1272 produced significant inhibitory effects on cell proliferation and migration and was associated with cell cycle G0/G1 arrest in A172 and SHG44 glioma cells. Using the luciferase reporter assay, we identified ADAM9 as a target of miR-1272. The expression of ADAM9 was markedly decreased or increased after overexpression or inhibition, respectively, of miR-1272 in glioma cells. Moreover, overexpression of ADAM9 reversed the inhibitory effects of miR-1272 on glioma cell progression. Furthermore, CDCP1 served as a potential downstream molecule of miR-1272/ADAM9 signaling in glioma and promoted the proliferation and migration of glioma. Results derived from clinical samples and online databases confirmed correlations between the expression of ADAM9 and CDCP1 and both the severity and prognosis of glioma. In conclusion, these results suggest that miR-1272 and CDCP1 may act as novel regulators in glioma. The miR-1272/ADAM9/CDCP1 pathway may serve as a potential candidate pathway for the prevention of glioma.