Hepatocyte transformation is induced by laminin γ2 monomer.

Serum laminin-γ2 monomer (Lm-γ2m) is a potent predictive biomarker for hepatocellular carcinoma (HCC) onset in patients with hepatitis C infection who achieve a sustained virologic response with liver cirrhosis (LC) and for the onset of extrahepatic metastases in early-stage HCC. Although Lm-γ2m involvement in late-stage cancer progression has been well investigated, its precise roles in HCC onset remain to be systematically investigated. Therefore, we analyzed an HCC model, human hepatocytes and cholangiocytes, and surgically resected liver tissues from patients with HCC to understand the roles of Lm-γ2m in HCC onset. Ck-19- and EpCAM-positive hepatic progenitor cells (HPCs) in the liver of pdgf-c transgenic HCC mouse model with ductular reaction showed ectopic expression of Lm-γ2m. Forced expression of Lm-γ2m in hepatocytes adjacent to HPCs resulted in enhanced tumorigenicity, cell proliferation, and migration in immortalized hepatocytes, but not in cholangiocytes in vitro. Further, pharmacological inhibition of epidermal growth factor receptor (EGFR) and c-Jun activator JNK suppressed Lm-γ2m-induced hepatocyte transformation, suggesting the involvement of EGFR/c-Jun signaling in the transformation, leading to HCC development. Finally, immunohistochemical staining of HCC tissues revealed a high level of Lm-γ2 expression in the HPCs of the liver with ductular reaction in normal liver adjacent to HCC tissues. Overall, HPC-derived Lm-γ2m in normal liver with ductular reaction acts as a paracrine growth factor on surrounding hepatocytes and promotes their cellular transformation through the EGFR/c-Jun signaling pathway. Furthermore, this is the first report on Lm-γ2m expression detected in the normal liver with ductular reaction, a human precancerous lesion of HCC.

EphA2 Proteolytic Fragment as a Sensitive Diagnostic Biomarker for Very Early-stage Pancreatic Ductal Carcinoma.

Cleavage of erythropoietin-producing hepatocellular ephrin receptor A2 (EphA2) triggers malignant progression and yields an N-terminal fragment (EphA2-NF) detectable in sera from patients with pancreatic ductal carcinoma. We established a quantitative automated chemiluminescence immunoassay for EphA2-NF and evaluated serum EphA2-NF levels as a biomarker to diagnose pancreatic ductal carcinoma in the test and validation cohorts. The EphA2-NF value was elevated (above the cutoff: mean ± SD) in more than half of the patients with stage I/II pancreatic ductal carcinoma. Among patients receiving standard chemotherapy for pancreatic ductal carcinoma [gemcitabine plus nab-paclitaxel (GnP)], the median survival time of patients with elevated serum EphA2-NF was half that of patients with values below the cutoff. Patients with intraductal papillary mucinous neoplasm (IPMN), a precancerous pancreatic ductal carcinoma lesion, also show high serum EphA2 levels, which are associated with an increase in pancreatic duct size and the development of pancreatic ductal carcinoma in some cases. IHC showed loss of EphA2-NF staining in IPMN with pancreatic ductal carcinoma, but not in the normal epithelium or IPMN without pancreatic ductal carcinoma, regardless of the histologic grade. These results suggest that EphA2 cleavage is an essential event that occurs very early in pancreatic ductal carcinoma development, and that the consequent release of EphA2-NF can be detected in the serum. Thus, serum EphA2-NF could be a diagnostic biomarker for very early-stage pancreatic ductal carcinoma and pancreatic ductal carcinoma development from high-risk IPMN and as a prognostic biomarker after chemotherapy with GnP. SIGNIFICANCE: EphA2 N-terminus deletion is involved in pancreatic ductal carcinoma development from high-risk IPMN and EphA2-NF produced by cleavage can be used as a serum biomarker to diagnose pancreatic ductal carcinoma and predict pancreatic ductal carcinoma development from high-risk IPMN.

Proteolytic cleavage of membrane proteins by membrane type-1 MMP regulates cancer malignant progression.

Strategies to develop cancer therapies using inhibitors that target matrix metalloproteinases (MMPs), particularly membrane type-1 MMP (MT1-MMP), have failed. This is predominantly attributed to the specificity of MMP inhibitors and numerous functions of MMPs; therefore, targeting substrates with such broad specificity can lead to off-target effects. Thus, new drug development for cancer therapeutics should focus on the ability of MT1-MMP to break down substrates, such as functional cell membrane proteins, to regulate the functions of these proteins that promote tumor malignancy. In this review, we discuss the mechanism by which proteolysis of cell surface proteins by MT1-MMP promotes progression of malignant tumor cells. In addition, we discuss the two protein fragments generated by limited cleavage of erythropoietin-producing hepatoma receptor tyrosine kinase A2 (EphA2-NF, -CF), which represent a promising basis for developing new cancer therapies and diagnostic techniques.

Clinical evaluation of urine laminin-γ2 monomer as a potent biomarker for non-muscle invasive bladder cancer.

BACKGROUND: To evaluate whether urine laminin-γ2 monomer (Ln-γ2m) offers a useful biomarker for patients with non-muscle-invasive bladder cancer (NMIBC). METHODS: Participants comprised 297 patients, including 111 patients with NMIBC, 136 patients with benign genitourinary disease (BD) and 50 healthy donors (HD). Urine Ln-γ2m was prospectively measured and accuracy was analyzed. Receiver operating characteristic (ROC) curves were determined and area under the ROC curve (AUC) was calculated for urine Ln-γ2m, and compared to those of traditional urine tumor markers such as nuclear matrix protein 22 (NMP22), bladder tumor antigen (BTA) and cytology. The net benefits of combining urine markers were analyzed by decision curve analysis. RESULTS: Mean urine Ln-γ2m was significantly higher in NMIBC than in BD or HD. The AUC for urine Ln-γ2m was significantly higher than those for urine NMP22, BTA or cytology when comparing NMIBC with HD. In patients with low-grade NMIBC, the AUC for urine Ln-γ2m was higher than the AUCs for NMP22, BTA or cytology. A net benefit of combined examination using urine Ln-γ2m/uCRN with NMP22 was demonstrated. CONCLUSION: These results suggest urine Ln-γ2m as a potentially useful biomarker for NMIBC, particularly in cases of low-grade cancer.

Chronological Change in EPHA2 Protein Expression Is Associated With Recurrence of Bladder Cancer.

BACKGROUND/AIM: Bladder cancer is the most common urinary tract cancer. Patients diagnosed with advanced T-stage/muscle-invasive bladder cancer through transurethral resection of bladder tumors (TURBT) are treated with total radical cystectomy; however, there is a high chance of recurrence. Nevertheless, markers for predicting this recurrence are not currently available. Here, we evaluated the chronological change of ephrin type-A receptor 2 (EPHA2) expression, a molecule known for its role in cell adhesion, to predict bladder cancer recurrence after cystectomy, using TURBT and cystectomy specimens. MATERIALS AND METHODS: An immunostaining evaluation method that combines whole-slide images and image analysis software was developed to quantify and evaluate stainability objectively. We assessed the correlation between EPHA2 expression and bladder cancer recurrence using this novel immunostaining method and chronological changes in target protein expression in TURBT and radical cystectomy samples. RESULTS: In TURBT specimens, the number of cases with a high N-terminal/C-terminal EPHA2 ratio in the group with recurrence was significantly higher than in the non-recurrent group (p=0.019). The number of cases with a high level of C-terminal EPHA2 positivity in the radical cystectomy specimen when compared to the TURBT specimen obtained from the same patient was significantly higher in the recurrent group than in the non-recurrent group (p=0.0034). CONCLUSION: EPHA2 appears to be a promising marker for bladder tumor recurrence after cystectomy and its evaluation may enable the selection of appropriate cases for adjuvant therapy among patients undergoing radical cystectomy. Further studies, including mass-scale analysis, are required to confirm these results.

Metalloproteinase-Dependent and TMPRSS2-Independent Cell Surface Entry Pathway of SARS-CoV-2 Requires the Furin Cleavage Site and the S2 Domain of Spike Protein.

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently, virus variants that can evade the immunity in a host achieved through vaccination have emerged. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread, are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner and is TMPRSS2 independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific small interfering RNAS (siRNAs) revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytium formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosomal pathways could be an effective strategy by which to cure COVID-19 in the future. IMPORTANCE To develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2. SARS-CoV-2 binds to the cell surface receptor ACE2 via the spike protein, and then the spike protein is cleaved by host proteases to enable entry. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection in addition to the TMPRSS2-mediated pathway and the endosomal pathway. The metalloproteinase-mediated pathway requires both the prior cleavage of spike into two domains and a specific sequence in the second domain, S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. Besides the contribution of metalloproteinases to SARS-CoV-2 infection, inhibition of metalloproteinases was important in preventing cell death, which may cause organ damage. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.

Novel LAMC2 fusion protein has tumor-promoting properties in ovarian carcinoma.

Laminins are heterotrimeric ECM proteins composed of α, ß, and γ chains. The γ2 chain (Lm-γ2) is a frequently expressed monomer and its expression is closely associated with cancer progression. Laminin-γ2 contains an epidermal growth factor (EGF)-like domain in its domain III (DIII or LEb). Matrix metalloproteinases can cleave off the DIII region of Lm-γ2 that retains the ligand activity for EGF receptor (EGFR). Herein, we show that a novel short form of Lm-γ2 (Lm-γ2F) containing DIII is generated without requiring MMPs and chromosomal translocation between LAMC2 on chromosome 1 and NR6A1 gene locus on chromosome 9 in human ovarian cancer SKOV3 cells. Laminin-γ2F is expressed as a truncated form lacking domains I and II, which are essential for its association with Lm-α3 and -ß3 chains of Lm-332. Secreted Lm-γ2F can act as an EGFR ligand activating the EGFR/AKT pathways more effectively than does the Lm-γ2 chain, which in turn promotes proliferation, survival, and motility of ovarian cancer cells. LAMC2-NR6A1 translocation was detected using in situ hybridization, and fusion transcripts were expressed in ovarian cancer cell tissues. Overexpression and suppression of fusion transcripts significantly increased and decreased the tumorigenic growth of cells in mouse models, respectively. To the best of our knowledge, this is the first report regarding a fusion gene of ECM showing that translocation of LAMC2 plays a crucial role in the malignant growth and progression of ovarian cancer cells and that the consequent product is a promising therapeutic target against ovarian cancers.

Expression of Cancer Stem Cell Markers EpCAM and CD90 Is Correlated with Anti- and Pro-Oncogenic EphA2 Signaling in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Additionally, the efficacy of targeted molecular therapies with multiple tyrosine kinase inhibitors is limited. In this study, we focused on the cellular signaling pathways common to diverse HCC cells and used quantitative reverse phase protein array (RPPA) and statistical analyses to elucidate the molecular mechanisms determining its malignancy. We examined the heterogeneity of 17 liver cancer cell lines by performing cluster analysis of their expression of CD90 and EpCAM cancer stem cell markers. Gaussian mixture model clustering identified three dominant clusters: CD90-positive and EpCAM-negative (CD90+), EpCAM-positive and CD90-negative (EpCAM+) and EpCAM-negative and CD90-negative (Neutral). A multivariate analysis by partial least squares revealed that the former two cell populations showed distinct patterns of protein expression and phosphorylation in the EGFR and EphA2 signaling pathways. The CD90+ cells exhibited higher abundance of AKT, EphA2 and its phosphorylated form at Ser897, whereas the EpCAM+ cells exhibited higher abundance of ERK, RSK and its phosphorylated form. This demonstrates that pro-oncogenic, ligand-independent EphA2 signaling plays a dominant role in CD90+ cells with higher motility and metastatic activity than EpCAM+ cells. We also showed that an AKT inhibitor reduced the proliferation and survival of CD90+ cells but did not affect those of EpCAM+ cells. Taken together, our results suggest that AKT activation may be a key pro-oncogenic regulator in HCC.

Serum Laminin γ2 Monomer as a Diagnostic and Predictive Biomarker for Hepatocellular Carcinoma.

BACKGROUNDS AND AIMS: Structural dynamics of basement membrane components are still to be elucidated in the process of hepatocarcinogenesis. We evaluated the characteristics of HCC expressing laminin γ2 monomer (LG2m), a basement membrane component not detected in normal tissues, for HCC diagnosis. We further determined whether elevated serum LG2m is a risk factor for HCC development in patients with chronic hepatitis C (CHC). APPROACH AND RESULTS: In HCC cell lines, LG2m was expressed in alpha-fetoprotein (AFP)-negative, CD90-positive cells characterized by highly metastatic natures. Using 14 cell lines and 258 HCC microarray data, we identified that LG2m gene signature was associated with Hoshida's S1/Boyault's G3 molecular subclasses with poor prognosis, which could not be recognized by AFP. Serum LG2m was assessed in 24 healthy donors, 133 chronic liver disease patients, and 142 HCC patients, and sensitivity and specificity of LG2m testing for HCC diagnosis were 62.9% and 70.5%, respectively (cutoff, 30 pg/mL). We evaluated the consequence of LG2m elevation in two independent HCC cohorts (n = 47 and n = 81), and LG2m-high HCC showed poor prognosis with later development of distant organ metastasis (cutoff, 60 pg/mL). LG2m was slightly elevated in a subset of CHC patients, and Kaplan-Meier analysis indicated a high incidence of HCC (n = 70). For validation, we enrolled 399 CHC patients with sustained virological response (SVR) as a multicenter, prospective study, and serum LG2m elevation correlated with a high incidence of HCC in the CHC patients with SVR (P < 0.0001). CONCLUSIONS: LG2m is a predictive biomarker for the development of metastatic HCC. Elevated serum LG2m is an HCC risk in CHC patients who have achieved SVR.

Evaluation of the RAS signaling network in response to MEK inhibition using organoids derived from a familial adenomatous polyposis patient.

RAS signaling is a promising target for colorectal cancer (CRC) therapy, and a variety of selective inhibitors have been developed. However, their use has often failed to demonstrate a significant benefit in CRC patients. Here, we used patient-derived organoids (PDOs) derived from a familial adenomatous polyposis (FAP) patient to analyze the response to chemotherapeutic agents targeting EGFR, BRAF and MEK. We found that PDOs carrying KRAS mutations were resistant to MEK inhibition, while those harboring the BRAF class 3 mutation were hypersensitive. We used a systematic approach to examine the phosphorylation of RAS effectors using reverse-phase protein array (RPPA) and found increased phosphorylation of MEK induced by binimetinib. A high basal level of ERK phosphorylation and its rebound activation after MEK inhibition were detected in KRAS-mutant PDOs. Notably, the phosphorylation of EGFR and AKT was more closely correlated with that of MEK than that of ERK. Transcriptome analysis identified MYC-mediated transcription and IFN signaling as significantly correlated gene sets in MEK inhibition. Our experiments demonstrated that RPPA analysis of PDOs, in combination with the genome and transcriptome, is a useful preclinical research platform to understand RAS signaling and provides clues for the development of chemotherapeutic strategies.

NH2 -terminal fragment of ZF21 protein suppresses tumor invasion via inhibiting the interaction of ZF21 with FAK.

Cellular migration, coupled with the degradation of the extracellular matrix (ECM), is a key step in tumor invasion and represents a promising therapeutic target in malignant tumors. Focal adhesions (FAs) and invadopodia, which are distinct actin-based cellular structures, play key roles in cellular migration and ECM degradation, respectively. The molecular machinery coordinating the dynamics between FAs and invadopodia is not fully understood, although several lines of evidence suggest that the disassembly of FAs is an important step in triggering the formation of invadopodia. In a previous study, we identified the ZF21 protein as a regulator of both FA turnover and invadopodia-dependent ECM degradation. ZF21 interacts with multiple factors for FA turnover, including focal adhesion kinase (FAK), microtubules, m-Calpain, and Src homology region 2-containing protein tyrosine phosphatase 2 (SHP-2). In particular, the dephosphorylation of FAK by ZF21 is a key event in tumor invasion. However, the precise role of ZF21 binding to FAK remains unclear. We established a method to disrupt the interaction between ZF21 and FAK using the FAK-binding NH2 -terminal region of ZF21. Tumor cells expressing the ZF21-derived polypeptide had significantly decreased FA turnover, migration, invadopodia-dependent ECM degradation, and Matrigel invasion. Furthermore, the expression of the polypeptide inhibited an early step of experimental lung metastasis in mice. These findings indicate that the interaction of ZF21 with FAK is necessary for FA turnover as well as ECM degradation at the invadopodia. Thus, ZF21 is a potential regulator that coordinates the equilibrium between FA turnover and invadopodia activity by interacting with FAK.

Membrane type 1 matrix metalloproteinase regulates anaplastic thyroid carcinoma cell growth and invasion into the collagen matrix.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive cancer types; however, the molecular mechanism contributing to the aggressive characteristics remain unclear. Membrane type 1 matrix metalloproteinase (MT1-MMP) plays an important role in cancer invasion and has been associated with a poor prognosis in various malignant neoplasms. In this study, we investigated the relationship between MT1-MMP expression and the proliferation and invasion of ATC cells, along with the association with clinicopathologic factors in patients with ATC. Suppression of MT1-MMP reduced the proliferation and invasion of ATC cells, and suppressed ERK activity, indicating a role in cancer cell proliferation in collagen matrix culture conditions. The expression of MT1-MMP was detected in 29 of 34 (85.3%) surgical specimens from ATC patients. In addition, the expression of MT1-MMP in the tumor lesion was higher than that of normal and stromal tissues. Collectively, these results suggest that elevated MT1-MMP expression plays a role in the pathogenesis of ATC, which may promote its aggressive characteristics such as proliferation and invasion, highlighting a potential new therapeutic target.

Vasoactive Intestinal Peptide Derived From Liver Mesenchymal Cells Mediates Tight Junction Assembly in Mouse Intrahepatic Bile Ducts.

Formation of intrahepatic bile ducts (IHBDs) proceeds in accordance with their microenvironment. Particularly, mesenchymal cells around portal veins regulate the differentiation and ductular morphogenesis of cholangiocytes in the developing liver; however, further studies are needed to fully understand the arrangement of IHBDs into a continuous hierarchical network. This study aims to clarify the interaction between biliary and liver mesenchymal cells during IHBD formation. To identify candidate factors contributing to this cell-cell interaction, mesenchymal cells were isolated from embryonic day 16.5 matrix metalloproteinase 14 (MMP14)-deficient (knockout [KO]) mice livers, in which IHBD formation is retarded, and compared with those of the wild type (WT). WT mesenchymal cells significantly facilitated the formation of luminal structures comprised of hepatoblast-derived cholangiocytes (cholangiocytic cysts), whereas MMP14-KO mesenchymal cells failed to promote cyst formation. Comprehensive analysis revealed that expression of vasoactive intestinal peptide (VIP) was significantly suppressed in MMP14-KO mesenchymal cells. VIP and VIP receptor 1 (VIPR1) were mainly expressed in periportal mesenchymal cells and cholangiocytic progenitors during IHBD development, respectively, in vivo. VIP/VIPR1 signaling significantly encouraged cholangiocytic cyst formation and up-regulated tight junction protein 1, cystic fibrosis transmembrane conductance regulator, and aquaporin 1, in vitro. VIP antagonist significantly suppressed the tight junction assembly and the up-regulation of ion/water transporters during IHBD development in vivo. In a cholestatic injury model of adult mice, exogenous VIP administration promoted the restoration of damaged tight junctions in bile ducts and improved hyperbilirubinemia. Conclusion: VIP is produced by periportal mesenchymal cells during the perinatal stage. It supports bile duct development by establishing tight junctions and up-regulating ion/water transporters in cholangiocytes. VIP contributes to prompt recovery from cholestatic damage through the establishment of tight junctions in the bile ducts.

Utility of a Reverse Phase Protein Array to Evaluate Multiple Biomarkers in Diffuse Large B-Cell Lymphoma.

PURPOSE: Diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin lymphoma, is a heterogeneous lymphoma with different clinical manifestations and molecular alterations, and several markers are currently being measured routinely for its diagnosis, subtyping, or prognostication by immunohistochemistry (IHC). Here, the utility of a reverse-phase-protein-array (RPPA) as a novel supportive tool to measure multiple biomarkers for DLBCL diagnosis is validated. EXPERIMENTAL DESIGN: The expression of seven markers (CD5, CD10, BCL2, BCL6, MUM1, Ki-67, and C-MYC) is analyzed by RPPA and IHC using 37 DLBCL tissues, and the correlation between the two methods is determined. To normalize tumor content ratio in the tissues, the raw RPPA values of each marker are adjusted by that of CD20 or PAX-5. RESULTS: The CD20-adjusted data for CD5, MUM1, BCL2, Ki-67, and C-MYC has better correlation with IHC results than PAX-5-adjusted data. Receiver operating characteristic (ROC) analysis reveals that CD5, MUM1, BCL2, and C-MYC exhibit a better sensitivity and specificity >0.750. Furthermore, the CD20-adjusted C-MYC value strongly correlates with that of IHC, and has a particularly high specificity (0.882). CONCLUSIONS AND CLINICAL RELEVANCE: Although further investigation using a large number of DLBCL specimens needs to be conducted, these results suggest that RPPA could be applicable as a supportive tool for determining lymphoma prognosis.

Unique Biological Activity and Potential Role of Monomeric Laminin-γ2 as a Novel Biomarker for Hepatocellular Carcinoma: A Review.

Laminin (Ln)-332 consists of α3, ß3, and γ2 chains, which mediate epithelial cell adhesion to the basement membrane. Ln-γ2, a component of Ln-332, is frequently expressed as a monomer in the invasion front of several types of malignant tissues without simultaneous expression of Ln-α3 and/or Ln-ß3 chains. Moreover, monomeric Ln-γ2 induces tumor cell proliferation and migration in vitro. These unique biological activities indicate that monomeric Ln-γ2 could be a candidate biomarker for early cancer surveillance. However, the present immune method for monomeric Ln-γ2 detection can only predict its expression, since no antibody that specifically reacts with monomeric γ2, but not with heterotrimeric γ2 chain, is commercially available. We have, therefore, developed monoclonal antibodies to specifically detect monomeric Ln-γ2, and devised a highly sensitive method to measure serum monomeric Ln-γ2 levels using a fully automated chemiluminescent immunoassay (CLIA). We evaluated its diagnostic value in sera from patients with several digestive cancers, including hepatocellular carcinoma (HCC), and found serum monomeric Ln-γ2 to be a clinically available biomarker for HCC surveillance. The combination of monomeric Ln-γ2 and prothrombin induced by Vitamin K Absence II (PIVKA-II) may be more sensitive for clinical diagnosis of HCC than any currently used combination.

Activated EphA2 Processing by MT1-MMP Is Involved in Malignant Transformation of Ovarian Tumours In Vivo.

BACKGROUND/AIM: Erythropoietin-producing hepatocellular receptor-2 (EphA2) is overexpressed in ovarian cancer. The N-terminals of EphA2 are processed by membrane-type 1 matrix metalloproteinase (MT1-MMP) and can subsequently induce ligand-independent signal activation to promote motility, invasion, and metastasis. The aim of this study was to investigate whether EphA2 processing occurs in benign, borderline, and malignant ovarian tumours. MATERIALS AND METHODS: Overall 107 ovarian epithelial carcinomas (OECs; 47 serous, 24 endometrioid, 16 mucinous, and 20 clear cell), 54 ovarian borderline tumours (OBTs; 12 serous, 42 mucinous), and 45 adenomas (15 serous, 17 mucinous, and 13 endometriotic cysts) were evaluated. Expression and processing of EphA2 were semi-quantitatively analyzed. EphA2 processing was also investigated by immunoblotting. RESULTS: EphA2 and MT1-MMP co-expression were detected. N-terminal EphA2 levels were significantly lower than those of C-terminal EphA2 in OECs and OBTs, but not in adenomas. Immunoblotting revealed processed fragments in OEC and OBTs. CONCLUSION: EphA2 processing by MT1-MMP is associated with malignant transformation in ovarian tumours.

Identification of Proteolytic Cleavage Sites of EphA2 by Membrane Type 1 Matrix Metalloproteinase on the Surface of Cancer Cells.

Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification . The purified fragment was digested with trypsin to generate proteolytic peptides , and the amino acid sequences of these peptides were determined by nano-LC-mass spectrometry to identify the MT1-MMP-mediated cleavage site(s) of EphA2.

Study on the tumor-induced angiogenesis using mathematical models.

We studied angiogenesis using mathematical models describing the dynamics of tip cells. We reviewed the basic ideas of angiogenesis models and its numerical simulation technique to produce realistic computer graphics images of sprouting angiogenesis. We examined the classical model of Anderson-Chaplain using fundamental concepts of mass transport and chemical reaction with ECM degradation included. We then constructed two types of numerical schemes, model-faithful and model-driven ones, where new techniques of numerical simulation are introduced, such as transient probability, particle velocity, and Boolean variables.

Specific detection of soluble EphA2 fragments in blood as a new biomarker for pancreatic cancer.

Because membrane type 1-matrix metalloproteinase 1 (MT1-MMP) and erythropoietin-producing hepatocellular receptor 2 (EphA2) expression are upregulated by the Ras/mitogen-activated protein kinase pathway, they are frequently coexpressed in malignant tumors. MT1-MMP cleaves the N-terminal ligand-binding domain of EphA2 and inactivates its ligand-dependent tumor-suppressing activity. Therefore, specific detection of the cleaved N-terminal EphA2 fragment in blood might be an effective biomarker to diagnose malignant tumors. To evaluate this possibility, we developed three monoclonal antibodies against the soluble EphA2 fragment. One of them recognized this fragment specifically, with negligible cross-reactivity to the intact form. We used the cleaved form-specific antibody to develop a quantitative enzyme-linked immunosorbent assay and confirmed the linear reactivity to the recombinant fragment. We applied this assay on commercially available serum specimens obtained from patients with several types of cancer including gastric, pancreatic, esophageal, gastroesophageal, and head-and-neck cancers, and healthy donors. Soluble EphA2 fragment levels in cancer-patient sera were higher than those in healthy donors (n=50). In particular, levels of eight out of nine (89%) pancreatic cancer patients and ten out of seventeen (59%) gastric cancer patients significantly exceeded cutoff values obtained from the healthy donors, whereas those of esophageal and head-and-neck cancer-patient sera were low. The preliminary receiver operating characteristic curve analysis for pancreatic cancer demonstrated that the sensitivity and specificity were 89.0% and 90.0%, respectively, whereas those of the conventional digestive tumor marker CA19-9 were 88.9% and 72.0%, respectively. These results indicated that specific detection of soluble EphA2 fragment levels in serum could be potentially useful as a biomarker to diagnose pancreatic cancer.

Development of a fully automated chemiluminescence immunoassay for urine monomeric laminin-γ2 as a promising diagnostic tool of non-muscle invasive bladder cancer.

BACKGROUND: Monomeric laminin-γ2 in urine is a potential biomarker for bladder cancer. However, the current detection system uses an antibody that cannot discriminate between monomeric laminin-γ2 and the heterotrimeric γ2 chain of laminin-332, which may cause false-positive reactions. The present study aimed to develop a fully automated chemiluminescence immunoassay system using a specific monoclonal antibody against monomeric laminin-γ2. METHODS: In total, 237 urine specimens (84 from patients with bladder cancer, 48 from patients with benign urological disease, and 105 from healthy donors) were collected, and monomeric laminin-γ2 values in the urine were measured using a fully automated chemiluminescence immunoassay. RESULTS: The results revealed that laminin-γ2 values in patients with benign urological disease were comparable to those of healthy donors and that the chemiluminescence immunoassay's lower limit of detection was 10 pg/mL (approximately 20-fold better than the sandwich enzyme-linked immunosorbent assay's limit of 200 pg/mL). Moreover, the chemiluminescence immunoassay demonstrated that patients with bladder cancer, including non-muscle invasive bladder cancer (≤pT1), had higher laminin-γ2 values than patients with benign urological disease or healthy donors. CONCLUSIONS: These results suggest that urine monomeric laminin-γ2 may be a promising biomarker to diagnose cases of non-muscle invasive bladder cancer using a fully automated chemiluminescence immunoassay system.