ABSTRACT
BACKGROUNDS: KLKs have been proved to be key regulators of the tumor microenvironment. In this study, we explored the potential of Kallikrein-related peptidases (KLKs) as clinical diagnostic and prognostic markers in patients with kidney renal clear cell carcinoma (KIRC) as well as their relationship with common immuno-inhibitor and immune cell infiltration in the tumor microenvironment to provide new targets and novel ideas for KIRC therapy. METHODS: Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), UCSC Xena, Genotype-Tissue Expression (GTEx), Kaplan-Meier plotter, cBioPortal, STRING, GeneMANIA, and TISIDB were used to analyze the differential expression, prognostic value, gene changes, molecular interaction, and immune infiltration of KLKs in patients with KIRC. RESULTS: From the gene expression level, it can be determined that KLK1, KLK6, and KLK7 are differentially expressed in KIRC and normal tissues. From the perspective of clinical prognosis, KLK1, KLK13, and KLK14 are highly correlated with the clinical prognosis of KIRC. The expression of KLKs is regulated by various immunosuppressive agents, with KDR, PVRL2, and VTCN1 being the most significant. The expression of KLKs is significantly correlated with the infiltration of various immune cells, of which Eosinophils and Neutrophils are the most significant. CONCLUSIONS: KLK1, KLK6, KLK7, KLK13, and KLK14 have potential as diagnostic and prognostic biomarkers, among which KLK1 is the most significant. This study may provide detailed immune information and promising targets for KIRC immunotherapy to assist in designing new immunotherapies.
Subject(s)
Carcinoma, Renal Cell , Computational Biology , Kallikreins , Kidney Neoplasms , Humans , Kallikreins/genetics , Kallikreins/metabolism , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Kidney Neoplasms/immunology , Computational Biology/methods , Gene Expression Regulation, Neoplastic , Prognosis , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Gene Expression ProfilingABSTRACT
Porcine placental extract (PPE) is commonly used in various health foods and cosmetics. PPE use in cosmetics predominantly consist of the water-soluble fraction derived from the entire placenta. In this report, we examined the effect of the hydrophobic constituents of the PPE, specifically the sphingolipid-enriched fraction designated as the sphingolipid-enriched porcine placental extract (SLPPE), on the expression of genes associated with skin function in cultured normal human epidermal keratinocytes. Using quantitative RT-PCR (qRT-PCR) analysis, we found that SLPPE concentrations ranging from 25 to 100 µg/mL upregulated the gene expression of key components associated with the cornified envelope structure (filaggrin (FLG), involucrin (IVL) and loricrin (LOR)), cornification enzymes (transglutaminase 1 (TGM1) and TGM5) and the desquamation enzymes (kallikrein 5 (KLK5) and KLK7). Additionally, KLK5p and FLG protein (FLGp) were detected in the culture supernatants of keratinocytes treated with SLPPE at these concentrations. These findings suggest that SLPPE is possible to promote the cornification and desquamation in epidermal keratinocytes, and it may offer potential benefits in cosmetics.
Subject(s)
Filaggrin Proteins , Kallikreins , Keratinocytes , Sphingolipids , Transglutaminases , Keratinocytes/drug effects , Keratinocytes/metabolism , Humans , Animals , Transglutaminases/metabolism , Transglutaminases/genetics , Swine , Sphingolipids/metabolism , Kallikreins/metabolism , Kallikreins/genetics , Placental Extracts/pharmacology , Cells, Cultured , Female , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Precursors/genetics , Protein Precursors/metabolism , PregnancyABSTRACT
Liver cancer is one of the most pivotal global health problems, leading hepatocellular carcinoma (HCC) with a significant increase in cases worldwide. The role of non-coding-RNA in cancer proliferation and carcinogenesis has attracted much attention in the last decade; however, microRNAs (miRNAs), as non-coding RNA, are considered master mediators in various cancer progressions. Yet the role of miR-141 as a modulator for specific cellular processes in liver cancer cell proliferation is still unclear. This study identified the role of miR-141 and its potential functions in liver carcinogenesis. The level of miR-141 in HepG2 and HuH7 cells was assessed using quantitative real-time PCR (qRT-PCR) and compared with its expression in normal hepatocytes. A new miR-141 construct has been performed in a CMV promoter vector tagged with GFP. Using microarray analysis, we identified the potentially regulated genes by miR-141 in transfected HepG2 cells. The protein profile of the kallikrein-related peptidase 10 (KLK10) and tumor necrosis factor TNFSF-15 was investigated in HepG2 cells transfected with either an inhibitor, antagonist miR-141, or miR-141 overexpression vector using immunoblotting and flow cytometry assay. Finally, ELISA assay has been used to monitor the produced inflammatory cytokines from transfected HepG2 cells. Our findings showed that the expression of miR-141 significantly increased in HepG2 and HuH7 cells compared to the normal hepatocytes. Transfection of HepG2 cells with an inhibitor, antagonist miR-141, showed a significant reduction of HepG2 cell viability, unlike the transfection of miR-141 overexpression vector. The microarray data of HepG2 cells overexpressed miR-141 provided a hundred downregulated genes, including KLK10 and TNFSF-15. Furthermore, the expression profile of KLK10 and TNFSF-15 markedly depleted in HepG2 cells transfected with miR-141 overexpression accompanied by a decreasing level of interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α), indicating the role of miR-141 in HepG2 cell proliferation and programmed cell death. Interestingly, the experimental rats with liver cancer induced by Diethylnitrosamine injection further confirmed the upregulation of miR-141 level, IL-10, and TNF-α and the disturbance in KLK10 and TNFSF-15 gene expression compared with their expression in normal rats. The in-silico online tools, IntaRNA and miRWalk were used to confirm the direct interaction and potential binding sites between miR-141 and identified genes. Thus, the seeding regions of potential targeted sequences was cloned upstream of luciferase reporter gene in pGL3 control vector. Interestingly, the luciferase activities of constructed vectors were significantly decreased in HepG2 cells pre-transfected with miR-141 overexpression vector, while increasing in cells pre-transfected with miR-141 specific inhibitor. In summary, these data suggest the crucial role of miR-141 in liver cancer development via targeting KLK10 and TNFSF-15 and provide miR-141 as an attractive candidate in liver cancer treatment and protection.
Subject(s)
Gene Expression Regulation, Neoplastic , Hepatoblastoma , Liver Neoplasms , MicroRNAs , Humans , Carcinogenesis/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cell Proliferation , Hep G2 Cells , Hepatoblastoma/genetics , Hepatoblastoma/metabolism , Hepatoblastoma/pathology , Kallikreins/genetics , Kallikreins/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , MicroRNAs/genetics , MicroRNAs/metabolismABSTRACT
Advancing healthcare for elderly men requires a deeper understanding of testicular aging processes. In this study, we conducted transcriptomic profiling of 43,323 testicular single cells from young and old mice, shedding light on 1032 telocytes-an underexplored testicular cell type in previous research. Our study unveiled 916 age-related differentially expressed genes (age-DEGs), with telocytes emerging as the cell type harboring the highest count of age-DEGs. Of particular interest, four genes (Klk1b21, Klk1b22, Klk1b24, Klk1b27) from the Kallikrein family, specifically expressed in Leydig cells, displayed down-regulation in aged testes. Moreover, cell-type-level splicing analyses unveiled 1838 age-related alternative splicing (AS) events. While we confirmed the presence of more age-DEGs in somatic cells compared to germ cells, unexpectedly, more age-related AS events were identified in germ cells. Further experimental validation highlighted 4930555F03Rik, a non-coding RNA gene exhibiting significant age-related AS changes. Our study represents the first age-related single-cell transcriptomic investigation of testicular telocytes and Kallikrein genes in Leydig cells, as well as the first delineation of cell-type-level AS dynamics during testicular aging in mice.
Subject(s)
Aging , Alternative Splicing , Gene Expression Profiling , Kallikreins , Single-Cell Analysis , Testis , Animals , Male , Mice , Kallikreins/genetics , Kallikreins/metabolism , Testis/metabolism , Aging/genetics , Transcriptome , Leydig Cells/metabolismABSTRACT
Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.
Subject(s)
Biomarkers , Carboxypeptidases , Cellular Senescence , Endothelial Cells , Humans , Endothelial Cells/metabolism , Biomarkers/metabolism , Carboxypeptidases/metabolism , Carboxypeptidases/genetics , Prekallikrein/metabolism , Prekallikrein/genetics , Bradykinin/pharmacology , Bradykinin/metabolism , Pulmonary Artery/metabolism , Pulmonary Artery/cytology , Cells, Cultured , Kininogen, High-Molecular-Weight/metabolism , Signal Transduction , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide Synthase Type III/genetics , Kallikreins/metabolism , Kallikreins/geneticsABSTRACT
PURPOSE: Prostate-specific membrane antigen (PSMA)-based images, which visually quantify PSMA expression, are used to determine prostate cancer micrometastases. This study evaluated whether a circulating tumor cell (CTC)-based transcript platform, including PSMA mRNA, could help identify potential prognostic markers in prostate cancer. EXPERIMENTAL DESIGN: We prospectively enrolled 21 healthy individuals and 247 patients with prostate cancer [localized prostate cancer (LPCa), n = 94; metastatic hormone-sensitive prostate cancer (mHSPC), n = 44; and metastatic castration-resistant prostate cancer (mCRPC), n = 109]. The mRNA expression of six transcripts [PSMA, prostate-specific antigen (PSA), AR, AR-V7, EpCAM, and KRT 19] from CTCs was measured, and their relationship with biochemical recurrence (BCR) in LPCa and mCRPC progression-free survival (PFS) rate in mHSPC was assessed. PSA-PFS and radiological-PFS were also calculated to identify potential biomarkers for predicting androgen receptor signaling inhibitor (ARSI) and taxane-based chemotherapy resistance in mCRPC. RESULTS: CTC detection rates were 75.5%, 95.3%, and 98.0% for LPCa, mHSPC, and mCRPC, respectively. In LPCa, PSMA [hazard ratio (HR), 3.35; P = 0.028) and PSA mRNA (HR, 1.42; P = 0.047] expressions were associated with BCR. Patients with mHSPC with high PSMA (HR, 4.26; P = 0.020) and PSA mRNA (HR, 3.52; P = 0.042) expressions showed significantly worse mCRPC-PFS rates than those with low expression. Increased PSA and PSMA mRNA expressions were significantly associated with shorter PSA-PFS and radiological PFS in mCPRC, indicating an association with drug resistance. CONCLUSIONS: PSMA and PSA mRNA expressions are associated with BCR in LPCa. In advanced prostate cancer, PSMA and PSA mRNA can also predict rapid progression from mHSPC to mCRPC and ARSI or taxane-based chemotherapy resistance.
Subject(s)
Antigens, Surface , Biomarkers, Tumor , Glutamate Carboxypeptidase II , Neoplasm Staging , Neoplastic Cells, Circulating , Prostate-Specific Antigen , Humans , Male , Neoplastic Cells, Circulating/metabolism , Neoplastic Cells, Circulating/pathology , Prostate-Specific Antigen/blood , Aged , Glutamate Carboxypeptidase II/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/blood , Antigens, Surface/genetics , Antigens, Surface/metabolism , Middle Aged , Prognosis , RNA, Messenger/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/blood , Prostatic Neoplasms/mortality , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/pathology , Prostatic Neoplasms, Castration-Resistant/blood , Prostatic Neoplasms, Castration-Resistant/drug therapy , Aged, 80 and over , Prospective Studies , Kallikreins/blood , Kallikreins/genetics , Gene Expression Regulation, NeoplasticABSTRACT
Alterations of the androgen receptor (AR) are associated with resistance to AR-directed therapy in prostate cancer. Thus, it is crucial to develop robust detection methods for AR alterations as predictive biomarkers to enable applicability in clinical practice. We designed and validated five multiplex droplet digital PCR assays for reliable detection of 12 AR targets including AR amplification, AR splice variant 7, and 10 AR hotspot mutations, as well as AR and KLK3 gene expression from plasma-derived cell-free DNA and cell-free RNA. The assays demonstrated excellent analytical sensitivity and specificity ranging from 95% to 100% (95% CI, 75% to 100%). Intrarun and interrun variation analyses revealed a high level of repeatability and reproducibility. The developed assays were applied further in peripheral blood samples from 77 patients with advanced prostate cancer to assess their feasibility in a real-world scenario. Optimizing the reverse transcription of RNA increased the yield of plasma-derived cell-free RNA by 30-fold. Among 23 patients with castration-resistant prostate cancer, 6 patients (26.1%) had one or a combination of several AR alterations, whereas only 2 of 54 patients (3.7%) in the hormone-sensitive stage showed AR alterations. These findings were consistent with other studies and suggest that implementation of comprehensive AR status detection in clinical practice is feasible and can support the treatment decision-making process.
Subject(s)
Receptors, Androgen , Humans , Receptors, Androgen/genetics , Male , Biomarkers, Tumor/genetics , Biomarkers, Tumor/blood , Aged , Prostatic Neoplasms/genetics , Prostatic Neoplasms/diagnosis , Reproducibility of Results , Mutation , Sensitivity and Specificity , Middle Aged , Cell-Free Nucleic Acids/genetics , Cell-Free Nucleic Acids/blood , Kallikreins/blood , Kallikreins/genetics , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/diagnosis , Prostatic Neoplasms, Castration-Resistant/blood , Multiplex Polymerase Chain Reaction/methodsABSTRACT
Tumor endothelial cells (TECs), which are thought to be structurally and functionally different from normal endothelial cells (NECs), are increasingly attracting attention as a therapeutic target in hypervascular malignancies. Although colorectal liver metastasis (CRLM) tumors are hypovascular, inhibitors of angiogenesis are a key drug in multidisciplinary therapy, and TECs might be involved in the development and progression of cancer. Here, we analyzed the function of TEC in the CRLM tumor microenvironment. We used a murine colon cancer cell line (CT26) and isolated TECs from CRLM tumors. TECs showed higher proliferation and migration than NECs. Coinjection of CT26 and TECs yielded rapid tumor formation in vivo. Immunofluorescence analysis showed that coinjection of CT26 and TECs increased vessel formation and Ki-67+ cells. Transcriptome analysis identified kallikrein-related peptide 10 (KLK10) as a candidate target. Coinjection of CT26 and TECs after KLK10 downregulation with siRNA suppressed tumor formation in vivo. TEC secretion of KLK10 decreased after KLK10 downregulation, and conditioned medium after KLK10 knockdown in TECs suppressed CT26 proliferative activity. Double immunofluorescence staining of KLK10 and CD31 in CRLM tissues revealed a significant correlation between poor prognosis and positive KLK10 expression in TECs and tumor cells. On multivariate analysis, KLK10 expression was an independent prognostic factor in disease-free survival. In conclusion, KLK10 derived from TECs accelerates colon cancer cell proliferation and hematogenous liver metastasis formation. KLK10 in TECs might offer a promising therapeutic target in CRLM.
Subject(s)
Cell Proliferation , Colonic Neoplasms , Endothelial Cells , Kallikreins , Liver Neoplasms , Animals , Female , Humans , Male , Mice , Cell Line, Tumor , Cell Movement , Colonic Neoplasms/pathology , Colonic Neoplasms/metabolism , Colonic Neoplasms/genetics , Endothelial Cells/metabolism , Endothelial Cells/pathology , Kallikreins/metabolism , Kallikreins/genetics , Liver Neoplasms/secondary , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/genetics , Mice, Inbred BALB C , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Tumor MicroenvironmentABSTRACT
The identification of surfaceome proteins is a main goal in cancer research to design antibody-based therapeutic strategies. T cell engagers based on KLK2, a kallikrein specifically expressed in prostate cancer (PRAD), are currently in early clinical development. Using genomic information from different sources, we evaluated the immune microenvironment and genomic profile of prostate tumors with high expression of KLK2. KLK2 was specifically expressed in PRAD but it was not significant associated with Gleason score. Additionally, KLK2 expression did not associate with the presence of any immune cell population and T cell activating markers. A mild correlation between the high expression of KLK2 and the deletion of TMPRSS2 was identified. KLK2 expression associated with high levels of surface proteins linked with a detrimental response to immune checkpoint inhibitors (ICIs) including CHRNA2, FAM174B, OR51E2, TSPAN1, PTPRN2, and the non-surface protein TRPM4. However, no association of these genes with an outcome in PRAD was observed. Finally, the expression of these genes in PRAD did not associate with an outcome in PRAD and any immune populations. We describe the immunologic microenvironment on PRAD tumors with a high expression of KLK2, including a gene signature linked with an inert immune microenvironment, that predicts the response to ICIs in other tumor types. Strategies targeting KLK2 with T cell engagers or antibody-drug conjugates will define whether T cell mobilization or antigen release and stimulation of immune cell death are sufficient effects to induce clinical activity.
Subject(s)
Kallikreins , Prostatic Neoplasms , Receptors, Odorant , Humans , Male , Genomics , Kallikreins/genetics , Kallikreins/immunology , Kallikreins/metabolism , Neoplasm Proteins , Prostatic Neoplasms/genetics , Prostatic Neoplasms/immunology , Prostatic Neoplasms/metabolism , Tetraspanins , Tumor Microenvironment/geneticsABSTRACT
Lung squamous cell carcinoma (LUSC) is the second most common lung cancer worldwide, leading to millions of deaths annually. Although immunotherapy has expanded the therapeutic choices for LUSC and achieved considerable efficacy in a subset of patients, many patients could not benefit, and resistance was pervasive. Therefore, it is significant to investigate the mechanisms leading to patients' poor response to immunotherapies and explore novel therapeutic targets. Using multiple public LUSC datasets, we found that Kallikrein-8 (KLK8) expression was higher in tumor samples and was correlated with inferior survival. Using a LUSC cohort (n = 190) from our center, we validated the bioinformatic findings about KLK8 and identified high KLK8 expression as an independent risk factor for LUSC. Function enrichment showed that several immune signaling pathways were upregulated in the KLK8 low-expression group and downregulated in the KLK8 high-expression group. For patients with low KLK8 expression, they were with a more active TME, which was both observed in the TCGA database and immune marker immunohistochemistry, and they had extensive positive relations with immune cells with tumor-eliminating functions. This study identified KLK8 as a risk factor in LUSC and illustrated the associations between KLK8 and cancer immunity, suggesting the potentiality of KLK8 as a novel immune target in LUSC.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Carcinoma, Squamous Cell , Lung Neoplasms , Humans , Tumor Microenvironment , Carcinoma, Squamous Cell/genetics , Lung Neoplasms/genetics , Lung , Prognosis , Kallikreins/geneticsABSTRACT
PURPOSE: Prostate cancer (PCa) is the leading cause of death among men in 48 countries. Genetic alterations play a significant role in PCa carcinogenesis. For the hypothesis of this research, five unique polymorphisms (SNP) were investigated in different genes that showed to be associated in different ways with PCa: rs4430796, rs2735839, rs4792311, rs12329760, and rs28931588, respectively for the genes HNF1B, KLK3, ELAC2, TMPRSS2-ERG, and CTNNB1. PATIENTS AND METHODS: Blood samples from 426 subjects were evaluated: 290 controls (161 females and 129 males) and 136 PCa patients. SNP were determined by real-time polymerase chain reaction. TaqMan SNP genotyping assay. In the control samples, the SNPs were defined in association with the self-reported ethnicity, and in 218 control samples with markers with ancestry indicators. The genes were in Hardy-Weinberg equilibrium. One hundred and seventy control samples were matched by ethnicity for comparison with the PCa samples. RESULTS: The G allele at rs28931588 was monomorphic in both patients and controls studied. Significant differences were observed in allelic and genotypic frequencies between the control and Pca samples in rs2735839 (KLK3; p = 0.002 and χ2 = 8.73 and p = 0.01, respectively), by the global frequency and in the dominant model rs2735839_GG (odds ratio [OR] = 0.51, p = 0.02). AA and GA genotypes at rs4792311 (ELAC2) were more frequent in patients with Gleason 7(4 + 3), 8, and 9 (n = 37%-59.7%) compared to patients with Gleason 6 and 7(3 + 4) (n = 26%-40.0%) conferring a protective effect on the GG genotype (OR = 0.45, p = 0.02). The same genotype showed an OR = 2.71 (p = 0.01) for patients with low severity. The HNF1B-KLK3-ELAC2-TMPRSS2-ERG haplotypes: GAAT, AAAT, GAGT, and AAGT were more frequent in patients with Pca with OR ranging from 4.65 to 2.48. CONCLUSIONS: Higher frequencies of risk alleles were confirmed in the SNPs, KLK3 rs2735839_A, ELAC2 rs4792311_A, and TMPRSS2 rs12329760_T in patients with Pca. Rs2735839_A was associated with risk of Pca and rs4792311_A with severity and Gleason score of 7(4 + 3) or greater. There is a need for careful observation of rs2735839 and rs4792311 in association with the prostatic biopsy due to the increased risk of Pca.
Subject(s)
Prostate-Specific Antigen , Prostatic Neoplasms , Male , Humans , Kallikreins/genetics , Genetic Predisposition to Disease , Prostatic Neoplasms/pathology , Genotype , Polymorphism, Single Nucleotide , Transcriptional Regulator ERG/genetics , Hepatocyte Nuclear Factor 1-beta/genetics , Neoplasm Proteins , beta Catenin/geneticsABSTRACT
Endometriosis is a common benign gynecologic condition. Endometriosis lesions are associated with endometrial cell proliferation, migration, invasion, and neovascularization, while the specific molecular mechanisms are still elusive. Transcriptome sequencing has been used for the identification of diagnostic markers in endometriosis. Here, transcriptome profiling revealed that kallikrein-related peptidase 4 (KLK4) expression was up-regulated in ectopic endometrium (EC) tissues of patients with endometriosis. KLK4 mediates the degradation of extracellular matrix proteins, and its proteolytic activity activates many tumorigenic and metastatic pathways via tumor invasion and migration. Nevertheless, whether KLK4 serves as an important regulatory factor in endometriosis remains unclear. This study confirmed that KLK4 was highly expressed in ectopic endometrial stromal cells (EC-ESCs). KLK4 overexpression promoted proliferation and suppressed apoptosis of EC-ESCs, induced cell migration and invasion, and enhanced angiogenesis in vivo. Mechanistically, KLK4 overexpression mediated the protein cleavage of pro-brain-derived neurotrophic factor in EC-ESCs. Finally, brain-derived neurotrophic factor was a vital downstream substrate of KLK4 maintained the proliferation, metastasis, and pro-angiogenesis abilities and inhibited apoptosis of ESCs through a rescue study. Together, these findings demonstrate the promotive role of KLK4 in endometriosis development. In addition, the study provides a new insight that KLK4 might be a potential therapeutic target and prognostic marker for patients with endometriosis.
Subject(s)
Endometriosis , Female , Humans , Angiogenesis , Brain-Derived Neurotrophic Factor , Cell Movement , Cell Proliferation , Endometriosis/pathology , Endometrium/pathology , Kallikreins/genetics , Kallikreins/metabolism , Stromal Cells/metabolismABSTRACT
ABSTRACT: Hereditary angioedema (HAE) is associated with episodic kinin-induced swelling of the skin and mucosal membranes. Most patients with HAE have low plasma C1-inhibitor activity, leading to increased generation of the protease plasma kallikrein (PKa) and excessive release of the nanopeptide bradykinin from high-molecular-weight kininogen (HK). However, disease-causing mutations in at least 10% of patients with HAE appear to involve genes for proteins other than C1-inhibitor. A point mutation in the Kng1 gene encoding HK and low-molecular weight kininogen (LK) was identified recently in a family with HAE. The mutation changes a methionine (Met379) to lysine (Lys379) in both proteins. Met379 is adjacent to the Lys380-Arg381 cleavage site at the N-terminus of the bradykinin peptide. Recombinant wild-type (Met379) and variant (Lys379) versions of HK and LK were expressed in HEK293 cells. PKa-catalyzed kinin release from HK and LK was not affected by the Lys379 substitutions. However, kinin release from HK-Lys379 and LK-Lys379 catalyzed by the fibrinolytic protease plasmin was substantially greater than from wild-type HK-Met379 and LK-Met379. Increased kinin release was evident when fibrinolysis was induced in plasma containing HK-Lys379 or LK-Lys379 compared with plasma containing wild-type HK or LK. Mass spectrometry revealed that the kinin released from wild-type and variant kininogens by PKa is bradykinin. Plasmin also released bradykinin from wild-type kininogens but cleaved HK-Lys379 and LK-Lys379 after Lys379 rather than Lys380, releasing the decapeptide Lys-bradykinin (kallidin). The Met379Lys substitutions make HK and LK better plasmin substrates, reinforcing the relationship between fibrinolysis and kinin generation.
Subject(s)
Angioedemas, Hereditary , Bradykinin , Humans , Lysine , Angioedemas, Hereditary/genetics , Fibrinolysin , Methionine , HEK293 Cells , Kininogens , Kallikreins/genetics , RacemethionineABSTRACT
Kirsten rat sarcoma virus (KRAS) gene mutation is common in colorectal cancer (CRC) and is often predictive of treatment failure and poor prognosis. To understand the mechanism, we compared the transcriptome of CRC patients with wild-type and mutant KRAS and found that KRAS mutation is associated with the overexpression of a secreted serine protease, kallikrein-related peptidase 10 (KLK10). Moreover, using in vitro and in vivo models, we found that KLK10 overexpression favors the rapid growth and liver metastasis of KRAS mutant CRC and can also impair the efficacy of KRAS inhibitors, leading to drug resistance and poor survival. Further functional assays revealed that the oncogenic role of KLK10 is mediated by protease-activated receptor 1 (PAR1). KLK10 cleaves and activates PAR1, which further activates 3-phosphoinositide-dependent kinase 1 (PDK1)-AKT oncogenic pathway. Notably, suppressing PAR1-PDK1-AKT cascade via KLK10 knockdown can effectively inhibit CRC progression and improve the sensitivity to KRAS inhibitor, providing a promising therapeutic strategy. Taken together, our study showed that KLK10 promotes the progression of KRAS mutant CRC via activating PAR1-PDK1-AKT signaling pathway. These findings expanded our knowledge of CRC development, especially in the setting of KRAS mutation, and also provided novel targets for clinical intervention.
Subject(s)
Colorectal Neoplasms , Receptor, PAR-1 , Humans , Cell Line, Tumor , Colorectal Neoplasms/metabolism , Kallikreins/genetics , Kallikreins/metabolism , Mutation/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Receptor, PAR-1/genetics , Receptor, PAR-1/metabolism , Signal Transduction , 3-Phosphoinositide-Dependent Protein Kinases/metabolismABSTRACT
The incidence and mortality rates of colorectal cancer (CRC) have significantly increased in recent years. It has been shown that early diagnosis of CRC improves the five-year survival of patients compared to late diagnosis, as patients with stage I disease have a five-year survival rate as high as 90 %. Through bioinformatics analysis, we identified Kallikrein 10 (KLK10), a member of the Kallikrein family, as a reliable predictor of CRC progression, particularly in patients with early-stage CRC. Furthermore, single-cell analysis revealed that KLK10 was highly expressed in tumor and partial immune cells. Analysis of the biological functions of KLK10 using the Kyoto encyclopedia of genes and genomes and gene ontology indicated that KLK10 plays a role in the proliferation and differentiation of cancer cells, along with the maintenance of tumor function and immune regulation, explicitly by T cells and macrophages. EdU cell proliferation staining, plate clone formation assay, and cell scratch assay demonstrated that KLK10 inhibition by siRNA affected the proliferation and migration of CRC cells. Cell cycle detection by flow cytometry demonstrated that KLK10 inhibition led to cell cycle arrest in the G1 phase. In addition, the proportion of M1 and M2 macrophages in 45 tumor specimens was analyzed by immunohistochemistry, the proportion of CD4+ T cells and CD8+ T cells in plasma was identified by flow cytometry, and their correlation with KLK10 was analyzed. The effects of KLK10 on T cells and macrophages were verified in independent cell experiments. The results revealed that KLK10 also activates CD4+ T cells, mediating M2-type macrophage polarization.
Subject(s)
CD8-Positive T-Lymphocytes , Colorectal Neoplasms , Humans , CD8-Positive T-Lymphocytes/metabolism , Colorectal Neoplasms/pathology , Kallikreins/genetics , Kallikreins/metabolismABSTRACT
Investigation on a competitive endogenous RNA (ceRNA) network attracted lots of attention due its function in cancer regulation. Here, we probed into the possible molecular mechanism of circSSPO/microRNA-6820-5p (miR-6820-5p)/kallikrein-related peptidase 8 (KLK8)/PKD1 network in the esophageal squamous cell carcinoma (ESCC). Following whole-transcriptome sequencing and differential analysis in collected ESCC tissue samples, circRNA-miRNA-mRNA regulatory network affecting ESCC was investigated. After interaction measurement among circSSPO/miR-6820-5p/KLK8/PKD1, their regulatory roles in ESCC cell functions in vitro and xenograft tumor growth and lung metastasis in vivo were analyzed. The bioinformatics prediction and sequencing results screened that circSSPO, miR-6820-5p, KLK8, and PKD1 were associated with ESCC development. In ESCC, miR-6820-5p was expressed at very low levels, while circSSPO, KLK8, and PKD1 were highly expressed. In vitro cell experiments further proved that circSSPO competitively inhibited miR-6820-5p to induce ESCC cell malignant properties. Moreover, knockdown of KLK8 or PKD1 inhibited ESCC cell malignant properties. circSSPO also promoted the tumorigenic and metastasis of ESCC through the upregulation of KLK8 and PKD1 expression in vivo. We found that circSSPO was an oncogenic circRNA that was significantly abundant in ESCC tissues and circSSPO exhibited an oncogenic activity in ESCC by elevating expression of KLK8 and PKD1 through suppressing miR-6820-5p expression.
Subject(s)
Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , MicroRNAs , RNA, Circular , Humans , Cell Line, Tumor , Cell Movement , Cell Proliferation/genetics , Esophageal Neoplasms/genetics , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/metabolism , Esophageal Squamous Cell Carcinoma/pathology , Gene Expression Regulation, Neoplastic , Kallikreins/genetics , Kallikreins/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , Up-Regulation/geneticsABSTRACT
BACKGROUND: Multiple treatment options are available for the management of psoriasis, but clinical response varies among individual patients and no biomarkers are available to facilitate treatment selection for improved patient outcomes. OBJECTIVES: To utilize retrospective data to conduct a pharmacogenetic study to explore the potential genetic pathways associated with drug response in the treatment of psoriasis. METHODS: We conducted a retrospective pharmacogenetic study using self-evaluated treatment response from 1942 genotyped patients with psoriasis. We examined 6 502 658 genetic markers to model their associations with response to six treatment options using linear regression, adjusting for cohort variables and demographic features. We further utilized an integrative approach incorporating epigenomics, transcriptomics and a longitudinal clinical cohort to provide biological implications for the topmost signals associated with drug response. RESULTS: Two novel markers were revealed to be associated with treatment response: rs1991820 (P = 1.30 × 10-6) for anti-tumour necrosis factor (TNF) biologics; and rs62264137 (P = 2.94 × 10-6) for methotrexate, which was also associated with cutaneous mRNA expression levels of two known psoriasis-related genes KLK7 (P = 1.0 × 10-12) and CD200 (P = 5.4 × 10-6). We demonstrated that KLK7 expression was increased in the psoriatic epidermis, as shown by immunohistochemistry, as well as single-cell RNA sequencing, and its responsiveness to anti-TNF treatment was highlighted. By inhibiting the expression of KLK7, we further illustrated that keratinocytes have decreased proinflammatory responses to TNF. CONCLUSIONS: Our study implicates the genetic regulation of cytokine responses in predicting clinical drug response and supports the association between pharmacogenetic loci and anti-TNF response, as shown here for KLK7.
Subject(s)
Psoriasis , Humans , Kallikreins/genetics , Kallikreins/therapeutic use , Pharmacogenetics , Pharmacogenomic Testing , Psoriasis/drug therapy , Psoriasis/genetics , Psoriasis/pathology , Retrospective Studies , Tumor Necrosis Factor Inhibitors/therapeutic use , Tumor Necrosis Factor-alpha/geneticsABSTRACT
INTRODUCTION: Endothelial injury is a major characteristic of sepsis and contributes to sepsis-induced multiple-organ dysfunction. In this study, we investigated the role of miR-107-3p in sepsis-induced endothelial injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to 20 µg/mL of lipopolysaccharide (LPS) for 6-48 h. The levels of miR-107-3p and kallikrein-related peptidase 5 (KLK5) were examined. HUVECs were treated with LPS for 12 h and subsequently transfected with miR-107-3p inhibitor, KLK5 siRNA, or cotransfected with KLK5 siRNA and miR-107-3p inhibitor/negative control inhibitor. Cell survival, apoptosis, invasion, cell permeability, inflammatory response, and the Toll-like receptor 4/nuclear factor κB signaling were evaluated. In addition, the relationship between miR-107-3p and KLK5 expression was predicted and verified. RESULTS: LPS significantly elevated miR-107-3p levels, which peaked at 12 h. Conversely, the KLK5 level was lower in the LPS group than in the control group and was lowest at 12 h. MiR-107-3p knockdown significantly attenuated reductions in cell survival and invasion, apoptosis promotion, hyperpermeability and inflammation induction, and activation of the NF-κB signaling caused by LPS. KLK5 knockdown had the opposite effect. Additionally, KLK5 was demonstrated as a target of miR-107-3p. MiR-107-3p knockdown partially reversed the effects of KLK5 depletion in LPS-activated HUVECs. CONCLUSIONS: Our findings indicate that miR-107-3p knockdown may protect against sepsis-induced endothelial cell injury by targeting KLK5. This study identified a novel therapeutic target for sepsis-induced endothelial injury.
Subject(s)
MicroRNAs , Sepsis , Humans , Apoptosis/genetics , Human Umbilical Vein Endothelial Cells , Kallikreins/genetics , Kallikreins/metabolism , Kallikreins/pharmacology , Lipopolysaccharides/pharmacology , MicroRNAs/metabolism , NF-kappa B/metabolism , RNA, Small Interfering/metabolism , Sepsis/complications , Sepsis/genetics , Sepsis/metabolismABSTRACT
Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti-metastatic activity. Consequently, there is an urgent need for new agents targeting the metastatic process. Emerging evidence correlates in vitro and in vivo cancer invasion and metastasis with increased activity of the proteases mesotrypsin (prostate and breast cancer) and kallikrein 6 (KLK6; ovarian cancer). Thus, mesotrypsin and KLK6 are attractive putative targets for therapeutic intervention. As potential therapeutics for advanced metastatic prostate, breast, and ovarian cancers, we report novel mesotrypsin- and KLK6-based therapies, based on our previously developed mutants of the human amyloid ß-protein precursor Kunitz protease inhibitor domain (APPI). These mutants, designated APPI-3M (prostate and breast cancer) and APPI-4M (ovarian cancer), demonstrated significant accumulation in tumors and therapeutic efficacy in orthotopic preclinical models, with the advantages of long retention times in vivo, high affinity and favorable pharmacokinetic properties. The applicability of the APPIs, as a novel therapy and for imaging purposes, is supported by their good safety profile and their controlled and scalable manufacturability in bioreactors.