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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.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.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).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.

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.

To evaluate whether urine laminin-γ2 monomer (Ln-γ2m) offers a useful biomarker for patients with non-muscle-invasive bladder cancer (NMIBC).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.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.These results suggest urine Ln-γ2m as a potentially useful biomarker for NMIBC, particularly in cases of low-grade cancer.

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

Supplementary Methods, Table 1, Figures 1-8 from Membrane Type 1-Matrix Metalloproteinase Cleaves Off the NH2-Terminal Portion of Heparin-Binding Epidermal Growth Factor and Converts It into a Heparin-Independent Growth Factor

Supplementary Figure S3. TMOD1 positively regulates MMP13 expression and activity.

Supplementary Table S1. Primers for the cDNA amplification and semiquantitative and real time RT-PCR.

Supplementary Figure S3. Cleavage of EphA2 in vitro.

Supplementary Methods, Figure Legends and References

Epidermal growth factor (EGF) receptors (ErbB) and EGF family members represent promising targets for cancer therapy. Heparin-binding EGF (HB-EGF) is a member of the EGF family and is an important target for therapy in some types of human cancers. Processing of HB-EGF by proprotein convertases, and successively, by ADAM family proteases, generates a soluble growth factor that requires heparin as a cofactor. Although heparin potentiates HB-EGF activity in vitro, it is not clear how the heparin-binding activity of HB-EGF is regulated. Here, we show that membrane type 1-matrix metalloproteinase (MT1-MMP; MMP14), a potent invasion-promoting protease, markedly enhances HB-EGF–dependent tumor formation in mice. MT1-MMP additionally cleaves HB-EGF and removes the NH2-terminal 20 amino acids that are important for binding heparin. Consequently, the processing of HB-EGF by MT1-MMP converts HB-EGF into a heparin-independent growth factor with enhanced mitogenic activity, and thereby, expression of both proteins costimulates tumor cell growth in vitro and in vivo. The ErbB family of receptors expressed in human gastric carcinoma cells play a role in mediating enhanced HB-EGF activity by MT1-MMP during invasive cell growth in collagen. Thus, we shed light on a new mechanism whereby HB-EGF activity is regulated that should be considered when designing HB-EGF–targeted cancer therapy. Cancer Res; 70(14); 6093–103. ©2010 AACR.

Laminins are heterotrimeric ECM proteins composed of α, β, 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 ‐β3 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 LAMC2‐NR6A1 fusion transcripts in human ovarian carcinomas.

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.

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.

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 have emerged that can evade the immunity in a host achieved through vaccination. 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 is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific 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 syncytia 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 endosome pathways could be an effective strategy by which to cure COVID-19 in the future.Author SummaryTo develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2, including recently emerging variants. 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. Selection of target cells by expression of these tissue-specific proteases contributes to pathogenesis. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection, variants included. This 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. The contribution of several proteases, including metalloproteinases, to SARS-CoV-2 infection was cell type dependent, especially in cells derived from kidney, ovary, and endometrium, in which SARS-CoV-2 infection was metalloproteinase-dependent. In these cells, inhibition of metalloproteinases by treatment with marimastat or prinomastat, whose safety was previously confirmed in clinical trials, was important in preventing cell death. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.

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.

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., 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 also contributes to prompt recovery from cholestatic liver damage through the establishment of tight junctions in the bile ducts.

Backgrounds and aims Structural dynamics of basement membrane components remained to be elucidated in the process of hepatocarcinogenesis. We evaluated the characteristics of hepatocellular carcinoma (HCC) expressing laminin γ2 monomer (LG2m), a previously unrecognized basement membrane component not detected in normal tissues, for HCC diagnosis. We further determined if 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-negative cluster of differentiation 90-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 alpha-fetoprotein. Serum LG2m was assessed in 24 healthy donors, 133 chronic liver disease and 142 HCC patients, and sensitivity and specificity of LG2m testing for HCC diagnosis were 62.9% and 70.5%, respectively (cutoff: 30pg/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: 60pg/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 Conclusions LG2m is a novel predictive biomarker for the development of metastatic HCC. Elevated serum LG2m is a HCC risk in CHC patients who have achieved SVR.

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.

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., ZF21 is a possible regulator coordinating the equilibrium between FA turnover and invadopodia activity by interacting with FAK.

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.

The diagnosis of hepatocellular carcinoma (HCC) in the early stages is important for successful clinical management. Laminin (Ln)-γ2 expression has been reported in various types of malignant carcinomas. We recently developed a highly sensitive method to measure serum monomeric Ln-γ2 levels using a fully automated chemiluminescent immunoassay (CLIA). Using our CLIA, we evaluated its diagnostic value in sera from patients with chronic liver disease (CLD) and patients with hepatocellular carcinoma (HCC). Serum alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) were also examined in these subjects. Median levels of Ln-γ2 were significantly higher in patients with HCC (173.2 pg/mL; range: 39.5-986 pg/mL) compared with patients with CLD (76.7 pg/mL; range: 38.7-215.9 pg/mL) and with healthy volunteers (41.1 pg/mL; range: 10.9-79.0 pg/mL). The optimal cutoff value for Ln-γ2 that allowed us to distinguish between HCC and nonmalignant CLD was 116.6 pg/mL. Elevated Ln-γ2 levels were observed in 0% of healthy volunteers, 17% of patients with CLD, and 63% of patients with HCC. The positivity rate in patients with HCC for the combination of Ln-γ2 and DCP was 89.5%, which was better than that for either of the two markers alone (63% and 68%, respectively). Among patients with early-stage HCC (T1 or T2), the positivity rates for monomeric Ln-γ2, AFP and DCP were 61%, 39% and 57%, respectively. Serum Ln-γ2 may be a potential biomarker for HCC surveillance. The combination of Ln-γ2 and DCP may be more sensitive for laboratory diagnosis of HCC than the combination of AFP and DCP.

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.

BackgroundInteraction between cancer cells and fibroblasts mediated by extracellular matrix metalloproteinase inducer (emmprin, CD147) is important in the invasion and proliferation of cancer cells. However, the exact mechanism of emmprin mediated stimulation of matrix metalloprotease-2 (MMP-2) production from fibroblasts has not been elucidated. Our previous studies using an inhibitory peptide against emmprin suggested the presence of a molecule on the cell membrane which forms a complex with emmprin. Here we show that CD73 expressed on fibroblasts interacts with emmprin and is a required factor for MMP-2 production in co-cultures of sarcoma cells with fibroblasts.MethodsCD73 along with CD99 was identified by mass spectrometry analysis as an emmprin interacting molecule from a co-culture of cancer cells (epithelioid sarcoma cell line FU-EPS-1) and fibroblasts (immortalized fibroblasts cell line ST353i). MMP-2 production was measured by immunoblot and ELISA. The formation of complexes of CD73 with emmprin was confirmed by immunoprecipitation, and their co-localization in tumor cells and fibroblasts was shown by fluorescent immunostaining and proximity ligation assays.ResultsStimulated MMP-2 production in co-culture of cancer cells and fibroblasts was completely suppressed by siRNA knockdown of CD73, but not by CD99 knockdown. MMP-2 production was not suppressed by CD73-specific enzyme inhibitor (APCP). However, MMP-2 production was decreased by CD73 neutralizing antibodies, suggesting that CD73-mediated suppression of MMP-2 production is non-enzymatic. In human epithelioid sarcoma tissues, emmprin was immunohistochemically detected to be mainly expressed in tumor cells, and CD73 was expressed in fibroblasts and tumor cells: emmprin and CD73 were co-localized predominantly on tumor cells.ConclusionThis study provides a novel insight into the role of CD73 in emmprin-mediated regulation of MMP-2 production.

Laminin (Ln)-332 consists of α3, β3, 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-β3 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.

Erythropoietin-producing hepatocellular receptor-2 (EphA2) overexpression is prevalent in many types of human cancers, and it has been reported that high EphA2 expression is correlated with malignancy. Recent studies revealed that processing of EphA2 by cleaving off the N-terminal portion by membrane-type 1 matrix metalloproteinase (MT1-MMP) promotes invasion via stimulation of Ras in cancer cells in vitro. The objectives of this study were to investigate the presence and role of EphA2 processing in cutaneous squamous cell carcinoma (SCC) tissues. EphA2 (C-terminal and N-terminal) and MT1-MMP expression patterns and levels were analyzed immunohistochemically in SCC (n = 70) and Bowen disease (BD; n = 20). Levels of MT1-MMP and EphA2 expression were evaluated using digital image analysis. Proximity between MT1-MMP and EphA2 in cancer cells and its effect on EphA2 processing were investigated using a combination of in situ proximity ligation assay (PLA) and Western blotting. Immunohistochemical analyses showed that levels of EphA2 N-terminal expression were significantly lower than those of EphA2 C-terminal expression in SCC, whereas levels of EphA2 C- and N-terminal expression were similar in BD. Western blotting showed processed EphA2 fragments in human SCC tissues. Expression levels of MT1-MMP, EphA2, and processed EphA2 fragments were higher in SCC than BD. Proximity between MT1-MMP and EphA2 in SCC was demonstrated by in situ PLA. Our results suggest possible involvement of MT1-MMP processing of EphA2 in invasiveness of cutaneous SCC.

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.

Expression of the protein deacetylase SIRT1 is associated with either poor or favorable prognosis in cancer patients, depending on the cancer type. In head and neck squamous cell carcinoma (HNSCC), SIRT1 expression is associated with favorable prognosis. However, the molecular mechanism underlying the tumor-suppressive function of SIRT1 in HNSCC is unknown. SIRT1 promotes differentiation in epithelial cells; therefore, we investigated whether SIRT1 promotes differentiation in HNSCC cells by studying the correlations between the expression of SIRT1 and several genes implicated in stemness or differentiation in HNSCC-derived cell lines. Our results suggest that SIRT1 does not contribute to differentiation in HNSCC cells. RNA interference-mediated reduction of SIRT1 revealed that SIRT1 supports the expression of TAp63, which has been implicated in tumor suppression, in addition to epithelial differentiation. A positive correlation was observed between SIRT1 and TAp63 expression in HNSCC tissues, as determined by quantitative reverse transcription-polymerase chain reaction analysis of RNA extracted from formalin-fixed paraffin-embedded biopsy samples. Together, these results suggest that although SIRT1 does not regulate differentiation of HNSCC cells, it functions as a tumor suppressor in HNSCC by supporting the transcription of tumor-suppressive TAp63. This finding supports the notion that SIRT1-activating drugs could be useful for the treatment of HNSCC.

MT6-MMP/MMP-25 is the latest member of the membrane-type matrix metalloproteinase (MT-MMP) subgroup in the MMP family and is expressed in neutrophils and some brain tumors. The proteolytic activity of MT6-MMP has been studied using recombinant catalytic fragments and shown to degrade several components of the extracellular matrix. However, the activity is possibly modulated further by the C-terminal hemopexin-like domain, because some MMPs are known to interact with other proteins through this domain. To explore the possible function of this domain, we purified a recombinant MT6-MMP with the hemopexin-like domain as a soluble form using a Madin-Darby canine kidney cell line as a producer. Mature and soluble MT6-MMP processed at the furin motif was purified as a 45-kDa protein together with a 46-kDa protein having a single cleavage in the hemopexin-like domain. Interestingly, 73- and 70-kDa proteins were co-purified with the soluble MT6-MMP by forming stable complexes. They were identified as clusterin, a major component of serum, by N-terminal amino acid sequencing. MT1-MMP that also has a hemopexin-like domain did not form a complex with clusterin. MT6-MMP forming a complex with clusterin was detected in human neutrophils as well. The enzyme activity of the soluble MT6-MMP was inactive in the clusterin complex. Purified clusterin was inhibitory against the activity of soluble MT6-MMP. On the other hand, it had no effect on the activities of MMP-2 and soluble MT1-MMP. Because clusterin is an abundant protein in the body fluid in tissues, it may act as a negative regulator of MT6-MMP in vivo.

In invasive cancer cells, specialized sub-cellular membrane structures which carry out a pivotal process in cancer invasion, termed invadopodia, are observed. Invadopodia appear irregularly within the cytoplasm and their general shape is small punctuated finger-like protrusions with dimension up to several μm long. They may exist and persist on a timescale between several tens of minutes to one hour. The formation of invadopodia requires the integration of several processes that include actin reorganization, extracellular matrix (ECM) degradation, signaling processes through receptors such as the epidermal growth factor receptor (EGFR) and matrix metalloproteinase (MMP) synthesis and delivery to the location of the invading front. In this paper, we consider a mathematical model investigating the coupling of these fundamental processes, and we investigate how invadopodia appear in this model. We investigate the spatio-temporal dynamics of the model in two spatial dimensions by using numerical computational simulations. We show that in a special parameter region of the model, random fluctuations of ECM degradation and a positive feedback loop regarding the up-regulation of MMPs allow us to reproduce finger-like protrusions which have similar size and lifetime as invadopodia. This study provides a new insight into how invadopodia appear in cancer cells and why space and time scales exist for invadopodia.

Cells are usually surrounded by the extracellular matrix (ECM), and adhesion of the cells to the ECM is a key step in their migration through tissues. Integrins are important receptors for the ECM and form structures called focal adhesions (FAs). Formation and disassembly of FAs are regulated dynamically during cell migration. Adhesion to the ECM has been studied mainly using cells cultured on an ECM-coated substratum, where the rate of cell migration is determined by the turnover of FAs. However, the molecular events underlying the disassembly of FAs are less well understood. We have recently identified both a new regulator of this disassembly process and its interaction partners. Here, we summarize our understanding of FA disassembly by focusing on the proteins implicated in this process.

Directional migration of adherent cells on an extracellular matrix requires repeated formation and disassembly of focal adhesions (FAs). Directional migration of adherent cells We have identified ZF21 as a regulator of disassembly of FAs and cell migration, and increased expression of the gene has been linked to metastatic colon cancer. ZF21 is a member of a protein family characterized by the presence of the FYVE domain, which is conserved among Fab1p, YOPB, Vps27p, and EEA1 proteins, and has been shown to mediate the binding of such proteins to phosphoinositides in the lipid layers of cell membranes. ZF21 binds multiple factors that promote disassembly of FAs such as FAK, β-tubulin, m-calpain, and SHP-2. ZF21 does not contain any other known protein motifs other than the FYVE domain, but a region of the protein C-terminal to the FYVE domain is sufficient to mediate binding to β-tubulin. In this study, we demonstrate that the C-terminal region is important for the ability of ZF21 to induce disassembly of FAs and cell migration, and to promote an early step of experimental metastasis to the lung in mice. In light of the importance of the C-terminal region, we analyzed its ternary structure using NMR spectroscopy. We demonstrate that this region exhibits a structure similar to that of a canonical pleckstrin homology domain, but that it lacks a positively charged interface to bind phosphatidylinositol phosphate. Thus, ZF21 contains a novel noncanonical PH-like domain that is a possible target to develop a therapeutic strategy to treat metastatic cancer.

Background: Laminin-5 (Ln5), a heterotrimer composed of three chains (α3, β3, and γ2), is a major component of the basement membrane in most adult tissues. One of the chains, Ln5-γ2, is a marker of invasive tumours because it is frequently expressed as a monomer in malignant tumours. Recent studies from our laboratories detected higher levels of Ln5-γ2 expression in basal cell carcinoma (BCC) than in trichoblastoma. Furthermore, Ln5-γ2 overexpression tended to correlate with aggressiveness in BCC. Methods: In this study, we compared the expression of Ln5-γ2 in invasive squamous cell carcinoma (SCC, n=62) of the skin to that in preinvasive Bowen's disease (BD, n=51), followed by analysis of the role of Ln5-γ2 in cancer invasion in vitro. Results: Immunohistochemically, the proportion of SCC cases (86%) strongly positive for Ln5-γ2 expression was higher than that of BD (16%). Real-time RT–PCR showed Ln5-γ2 overexpression in SCC cell line, A431, compared with normal keratinocyte cell line, HaCaT. Ln5-γ2 monomer and proteolytically cleaved, biologically active fragments of Ln5-γ2 were identified in SCC tumour extracts. In in vitro raft cultures, which simulate in vivo conditions, Ln5-γ2 siRNA significantly suppressed epidermal growth factor (EGF)-stimulated A431 cell invasion. Conclusion: Our results indicate that Ln5-γ2 has a role in cutaneous SCC invasion.

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.

Rho family proteins regulate multiple cellular functions including motility and invasion through regulation of the actin cytoskeleton and gene expression. Activation of Rho proteins is controlled precisely by multiple regulators in a spatiotemporal manner. RhoA and/or RhoC are key players that regulate the metastatic activity of malignant tumor cells, and it is therefore of particular interest to understand how activation of these Rho proteins is controlled. We recently identified an upstream regulator of RhoA activation, p27RF-Rho (p27(kip1) releasing factor from RhoA) that acts by freeing RhoA from inhibition by p27(kip1). p27(kip1) is a cell cycle regulator when it is localized to the nucleus, but it binds RhoA and inhibits activation of the latter when it is localized to the cytoplasm. Here, we show that a metastatic variant of mouse melanoma B16 cells (F10) exhibits greater expression of p27RF-Rho, RhoA, and RhoC than the nonmetastatic parental cells (F0). Injection of F10 cells into mouse tail vein resulted in the formation of metastatic lung colonies, whereas prior knockdown of expression of either one of the three proteins using specific shRNA sequences decreased metastasis markedly. p27RF-Rho regulated the activation of RhoA and RhoC and thereby modulated cellular adhesion and motility, in addition to pericellular proteolysis. The Rho activities enhanced by p27RF-Rho had a marked effect upon efficiency of lodging of F10 cells in the lung, which represents an early step of metastasis. p27RF-Rho also regulated metastasis of human melanoma and fibrosarcoma cells. Thus, p27RF-Rho is a key upstream regulator of RhoA and RhoC that controls spreading of tumor cells.

Increased expression of heparin-binding EGF-like growth factor (HB-EGF) and membrane-type matrix metalloproteinase-1 (MT1-MMP) is frequently associated with various types of malignant tumor. HB EGF-like growth factor has been reported to promote the malignant progression of ovarian carcinoma. Based on this finding, inhibition of HB-EGF activity with CRM197 is now under phase I clinical evaluation. On the other hand, MT1-MMP expressed in ovarian carcinoma cells is thought to promote invasion and growth of tumor cells by degrading the extracellular matrix. However, we recently demonstrated that co-expression of MT1-MMP and HB-EGF in gastric carcinoma cells leads to cleavage of HB-EGF within its N-terminal heparin-binding region, converting it into a potent heparin-independent growth factor. In this study, we evaluated the importance of regulation of HB-EGF by MT1-MMP in clinical samples of ovarian carcinoma. We detected co-expression of HB-EGF and MT1-MMP in clear cell ovarian carcinoma tissues, particularly at the invasion front and in tumor cells that had disseminated into the ascites, whereas HB-EGF alone was expressed in non-invasive borderline ovarian tumor tissue. Furthermore, a soluble HB-EGF fragment that corresponds to that processed by MT1-MMP was detected in malignant ascites obtained from patients with metastatic ovarian carcinoma. Ovarian carcinoma cells that express MT1-MMP and HB-EGF exhibited enhanced cell growth in a 3D-collagen matrix and anchorage-independent growth in suspension. These results indicate that MT1-MMP co-expressed with HB-EGF in ovarian carcinoma cells potentiates the activity of HB-EGF to promote invasive tumor growth and spreading in vivo.

Membrane-type 1 matrix metalloproteinase 1 (MT1-MMP) is a potent modulator of the pericellular microenvironment and regulates cellular functions in physiological and pathological settings in mammals. MT1-MMP mediates its biological effects through cleavage of specific substrate proteins. However, our knowledge of MT1-MMP substrates remains limited. To identify new substrates of MT1-MMP, we purified proteins associating with MT1-MMP in human epidermoid carcinoma A431 cells and analyzed them by mass spectrometry. We identified 163 proteins, including membrane proteins, cytoplasmic proteins, and functionally unknown proteins. Sixty-four membrane proteins were identified, and they included known MT1-MMP substrates. Of these, eighteen membrane proteins were selected, and we confirmed their association with MT1-MMP using an immunoprecipitation assay. Co-expression of each protein together with MT1-MMP revealed that nine proteins were cleaved by MT1-MMP. Lutheran blood group glycoprotein (Lu) is one of the proteins cleaved by MT1-MMP, and we confirmed the cleavage of the endogenous Lu protein by endogenous MT1-MMP in A431 cells. Mutation of the cleavage site of Lu abrogated processing by MT1-MMP. Lu protein expressed in A431 cells bound to laminin-511, and knockdown of MT1-MMP in these cells increased both their binding to laminin-511 and the amount of Lu protein on the cell surface. Thus, the identified membrane proteins associated with MT1-MMP are an enriched source of physiological MT1-MMP substrates.