Development of UTX-143, a selective sodium-hydrogen exchange subtype 5 inhibitor, using amiloride as a lead compound.

NHE5, an isoform of the Na+/H+ exchanger (NHE) protein, is an ion-transporting membrane protein that regulates intracellular pH and is highly expressed in colorectal adenocarcinoma. Therefore, we hypothesized that NHE5 inhibitors can be used as anticancer drugs. However, because NHE1 is ubiquitously expressed in all cells, it is extremely important to demonstrate its selective inhibitory activity against NHE5. We used amiloride, an NHE non-selective inhibitor, as a lead compound and created UTX-143, which has NHE5-selective inhibitory activity, using a structure-activity relationship approach. UTX-143 showed selective cytotoxic effects on cancer cells and reduced the migratory and invasive abilities of cancer cells. These results suggest a new concept wherein drugs exhibit cancer-specific cytotoxic effects through selective inhibition of NHE5 and the possibility of UTX-143 as a lead NHE5-selective inhibitor.

m6A RNA methylation regulates the transcription factors JUN and JUNB in TGF-ß-induced epithelial-mesenchymal transition of lung cancer cells.

N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during transforming growth factor-beta-induced epithelial-mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and nonredundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by methyltransferase-like 3 (METTL3) m6A methyltransferase. METTL3-mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3'UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during transforming growth factor-beta-induced EMT of lung cancer cells.

ASH2L, a COMPASS core subunit, is involved in the cell invasion and migration of triple-negative breast cancer cells through the epigenetic control of histone H3 lysine 4 methylation.

ASH2L (Absent-Small-Homeotic-2-Like protein) is a core subunit of the COMPASS (COMplex of Proteins ASsociated with Set1) complex, the most notable writer of the methylation of histone H3 lysine 4 (H3K4). The COMPASS complex regulates active promoters or enhancers for gene expression, and its dysfunction is associated with aberrant development and disease. Here, we demonstrated that ASH2L mediated the cell invasion and migration activity of triple-negative breast cancer cells through the interaction with the COMPASS components and the target genomic regions. Transcriptome analysis indicated a potential correlation between ASH2L and the genes involved in inflammatory/immune responses. Among them, we found that the intrinsic expression of IL1B (interleukin 1 beta), an essential proinflammatory gene, was directly regulated by ASH2L. These results revealed a novel role of ASH2L on the maintenance of breast cancer malignancy possibly through H3K4 methylation of the target inflammatory/immune responsive genes.

KDM2B is involved in the epigenetic regulation of TGF-ß-induced epithelial-mesenchymal transition in lung and pancreatic cancer cell lines.

Polycomb repressive complex-1 (PRC1) induces transcriptional repression by regulating monoubiquitination of lysine 119 of histone H2A (H2AK119) and as such is involved in a number of biological and pathological processes including cancer development. Previously we demonstrated that PRC2, which catalyzes the methylation of histone H3K27, has an essential function in TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung and pancreatic cancer cell lines. Since the cooperative activities of PRC1 and PRC2 are thought to be important for transcriptional repression in EMT program, we investigated the role of KDM2B, a member of PRC1 complex, on TGF-ß-induced EMT in this study. Knockdown of KDM2B inhibited TGF-ß-induced morphological conversion of the cells and enhanced cell migration and invasion potentials as well as the expression changes of EMT-related marker genes. Overexpression of KDM2B influenced the expression of several epithelial marker genes such as CDH1, miR200a, and CGN and enhanced the effects of TGF-ß. Mechanistic investigations revealed that KDM2B specifically recognized the regulatory regions of CDH1, miR200a, and CGN genes and induced histone H2AK119 monoubiquitination as a component of PRC1 complex, thereby mediating the subsequent EZH2 recruitment and histone H3K27 methylation process required for gene repression. Studies using KDM2B mutants confirmed that its DNA recognition property but not its histone H3 demethylase activity was indispensable for its function during EMT. This study demonstrated the significance of the regulation of histone H2A ubiquitination in EMT process and provided the possibility to develop novel therapeutic strategies for the treatment of cancer metastasis.

Structure-Activity Relationships of UTX-121 Derivatives for the Development of Novel Matrix Metalloproteinase-2/9 Inhibitors.

Celecoxib, a nonsteroidal anti-inflammatory drug, has been reported to have antitumor and antimetastatic activities, and it has potential for application in cancer treatments. The expression of matrix metalloproteinase (MMP)-2/9 is strongly correlated with cancer malignancy, and inhibition of these MMPs is believed to be effective in improving the antitumor and antimetastatic effects of drugs. We have previously revealed that UTX-121, which converted the sulfonamide of celecoxib to methyl ester, has more potent MMP-2/9 inhibitory activity than celecoxib. Based on these findings, we identified compounds with improved MMP inhibitory activity through a structure-activity relationship (SAR) study, using UTX-121 as a lead compound. Among them, compounds 9c and 10c, in which the methyl group of the p-tolyl group was substituted for Cl or F, showed significantly higher antitumor activity than UTX-121, and suppressed the expression of MMP-2/9 and activation of pro MMP-2. Our findings suggest that compounds 9c and 10c may be potent lead compounds for the development of more effective antitumor drugs targeting MMP.

A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by modulating membrane-type 1 matrix metalloproteinase.

We designed and synthesized a celecoxib derivative UTX-121 to enhance its anti-tumor activity. Similar to celecoxib, this compound could also inhibit matrix metalloproteinase (MMP)-9 activity. In addition, UTX-121 suppressed membrane-type 1 MMP (MT1-MMP)-mediated pro-MMP-2 activation by disturbing the cell surface expression of MT1-MMP. UTX-121 also impeded the glycosylation of cell surface proteins, resulting in the suppression of cell attachment to fibronectin. This inhibition by UTX-121 caused the reduction of fibronectin-stimulated focal adhesion kinase activation, Akt activation, and cell migration. Consequently, UTX-121 treatment significantly inhibited fibronectin-induced HT1080 cell invasion into the Matrigel. UTX-121 may be a potent lead compound that can be used to develop a novel anti-tumor drug.

The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB.

N6-Methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the role of m6A methyltransferase METTL3 in TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung cancer cell lines. The expression of METTL3 and m6A RNA modification were increased during TGF-ß-induced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 inhibited TGF-ß-induced morphological conversion of the cells, enhanced cell migration potential and the expression changes of EMT-related marker genes such as CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin. Mechanistic investigations revealed that METTL3 knockdown decreased the m6A modification, total mRNA level and mRNA stability of JUNB, one of the important transcriptional regulators of EMT. Over-expression of JUNB partially rescued the inhibitory effects of METTL3 knockdown in the EMT phenotypes. This study demonstrates that m6A methyltransferase METTL3 is indispensable for TGF-ß-induced EMT of lung cancer cells through the regulation of JUNB.

Activation of MMP-9 by membrane type-1 MMP/MMP-2 axis stimulates tumor metastasis.

An artificial receptor for proMMP-9 was created by fusing tissue inhibitor of MMP-1 (TIMP-1) with type II transmembrane mosaic serine protease (MSP-T1). Expression of MSP-T1 in 293T cells induced binding of proMMP-9, which was processed by MMP-2 activated by membrane type 1 MMP (MT1-MMP). HT1080 cells transfected with the MSP-T1 gene produced activated MMP-9 in collagen gel, and addition of proMMP-2 to the culture augmented it, which resulted in intensive collagen digestion. These cells metastasized into chick embryonic liver more than control cells. Treatment of HT1080 cells with concanavalin A in the presence of exogenous proMMP-2 induced activation of not only proMMP-2 but also proMMP-9. Knockdown of MT1-MMP or TIMP-2 expression with siRNA suppressed activation of both proMMP-2 and proMMP-9. Transfection of TIMP-1 siRNA suppressed cell binding and activation of proMMP-9, but not proMMP-2 activation. Knockdown of a disintegrin and metalloproteinase 10 (ADAM10) expression reduced cell binding and processing of proMMP-9. These results suggest that proMMP-9, which binds to a receptor complex containing TIMP-1 and ADAM10, is activated by the MT1-MMP/MMP-2 axis, and MMP-9 thus activated stimulates cellular proteolysis and metastasis.

Vinculin negatively regulates transcription of MT1-MMP through MEK/ERK pathway.

Vinculin regulates a variety of cellular functions partly through stabilization of tumor suppressor PTEN. In order to study the role of vinculin in tumor progression other than PTEN stabilization, vinculin was knocked down in PTEN-deficient squamous cell carcinoma HSC-4 cells. Knockdown of vinculin induced phenotypical change by reducing cell-cell and cell-extracellular matrix adhesions, and enhanced MT1-MMP expression at transcription level and subsequent cell migration. Up-regulation of MT1-MMP transcription by vinculin knockdown was abrogated by ERK inhibition. These results suggest that vinculin negatively regulates malignant phenotype of tumor cells including MT1-MMP transcription through MEK/ERK pathway.

Membrane-type 1 matrix metalloproteinase regulates fibronectin assembly and N-cadherin adhesion.

Fibronectin matrix formation requires the increased cytoskeletal tension generated by cadherin adhesions, and is suppressed by membrane-type 1 matrix metalloproteinase (MT1-MMP). In a co-culture of Rat1 fibroblasts and MT1-MMP-silenced HT1080 cells, fibronectin fibrils extended from Rat1 to cell-matrix adhesions in HT1080 cells, and N-cadherin adhesions were formed between Rat1 and HT1080 cells. In control HT1080 cells contacting with Rat1 fibroblasts, cell-matrix adhesions were formed in the side away from Rat1 fibroblasts, and fibronectin assembly and N-cadherin adhesions were not formed. The role of N-cadherin adhesions in fibronectin matrix formation was studied using MT1-MMP-silenced HT1080 cells. MT1-MMP knockdown promoted fibronectin matrix assembly and N-cadherin adhesions in HT1080 cells, which was abrogated by double knockdown with either integrin ß1 or fibronectin. Conversely, inhibition of N-cadherin adhesions by its knockdown or treatment with its neutralizing antibody suppressed fibronectin matrix formation in MT1-MMP-silenced cells. These results demonstrate that fibronectin assembly initiated by MT1-MMP knockdown results in increase of N-cadherin adhesions, which are prerequisite for further fibronectin matrix formation.

MT1-MMP prevents growth inhibition by three dimensional fibronectin matrix.

The extracellular microenvironment plays a key role in regulation of cellular functions and growth control. We show here that membrane-type 1 matrix metalloproteinase (MT1-MMP) acts as a growth promoter in confluent culture. When MT1-MMP was silenced in HT1080 fibrosarcoma cells, cells created three dimensional (3D) fibronectin matrix in a confluent culture, and growth of cells embedded within it was retarded. Formation of 3D fibronectin matrix initiated by MT1-MMP silencing was impeded by knockdown of either FN or integrin ß1, which resulted in restoration of cell growth. When cells in 3D fibronectin matrix were treated with integrin ß1 inhibitory antibody, cells underwent S phase entry. These results suggest that MT1-MMP prevents growth suppression by 3D fibronectin matrix, which is mediated through integrin ß1.

Cleavage of hepatocyte growth factor activator inhibitor-1 by membrane-type MMP-1 activates matriptase.

Co-expression of membrane-type 1 (MT1)-MMP with hepatocyte growth factor activator inhibitor-1 (HAI-1) in HEK293T cells resulted in cleavage of HAI-1 to produce three fragments. Recombinant MT1-MMP was shown to cleave HAI-1 protein in vitro. Hepatocyte growth factor activator inhibitor-1 was initially identified as the cognate inhibitor of matriptase, a transmembrane serine protease that processes urokinase-type plasminogen activator (uPA). Co-expression of HAI-1 with matriptase suppressed matriptase protease activity, and co-expression of MT1-MMP with them resulted in recovery of matriptase activity by stimulating shedding of HAI-1 fragments. Matriptase protein was detected in squamous carcinoma-derived HSC-4 cells, however, matriptase protease activity was undetectable. Transfection of siRNA for HAI-1 enhanced serine protease activity, which was suppressed by cotransfection of matriptase siRNA. Collagen-gel culture or treatment with concanavalin A (ConA) of HSC-4 cells enhanced MT1-MMP activity, which induced shedding of HAI-1 fragments and conversely stimulated uPA activation by these cells. Serine protease activity, including uPA activation of cells treated with ConA, was abrogated by downregulation of either matriptase or MT1-MMP through the transfection of each siRNA. These results suggest that MT1-MMP induced by collagen-gel culture or ConA treatment causes cleavage and shedding of HAI-1 protein, which allows activation of matriptase in HSC-4 cells. HSC-4 cells showed a characteristic invasive growth by forming vacuole-like structures in collagen gel, which was suppressed by transfection of siRNA for either MT1-MMP or matriptase, suggesting that activation of matriptase through the cleavage of HAI-1 is one of the MT1-MMP multifunctions essential for invasive growth of HSC-4 cells.

Erratum: TGF-ß1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells (BBREP_101072).

[This corrects the article DOI: 10.1016/j.bbrep.2021.101072.].

TGF-ß1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells.

Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-ß1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-ß1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-ß1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-ß1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-ß1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-ß1-induced cell invasion into collagen gel. Thus, TGF-ß1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.

Coordinate action of membrane-type matrix metalloproteinase-1 (MT1-MMP) and MMP-2 enhances pericellular proteolysis and invasion.

Membrane-type matrix metalloproteinase-1 (MT1-MMP) mediates cleavage of not only MMP-2/gelatinase A for activation, but also a variety of substrates including type I collagen (reviewed in Cancer Sci 2005; 96: 212-7). MMP-2 activation involves tissue inhibitor of MMP (TIMP)-2 as a bridging molecule between MT1-MMP and pro-MMP-2. Thus, net activity of MT1-MMP and MMP-2 is regulated in a complex manner depending on TIMP-2 concentration. During invasive growth of tumor cells in type I collagen matrix, MT1-MMP initiates denaturation of collagen into gelatin, which is subsequently digested further by MMP-2 adjacent to MT1-MMP. Coordinate action of MT1-MMP and MMP-2 may facilitate pericellular proteolysis, and enhance not only tumor invasion/migration but also cell growth. Tetraspanins as binding proteins of MT1-MMP regulate MT1-MMP subcellular localization and compartmentalization, leading to efficient MMP-2 activation and proteolysis coupled with cellular function.

GI24 enhances tumor invasiveness by regulating cell surface membrane-type 1 matrix metalloproteinase.

GI24, an immunoglobulin superfamily member, has been cloned from a placenta cDNA library as a gene product that promoted activation of matrix metalloproteinase (MMP)-2 mediated by membrane type (MT) 1-MMP. Co-expression of GI24 with MT1-MMP in HEK293T cells increased the cell-surface level of MT1-MMP concomitant with the cleavage of the GI24 at the juxtamembrane site to shed the extracellular domain. HT1080 fibrosarcoma cells stably transfected with the GI24 gene expressed a higher level of MT1-MMP and showed more invasive ability in collagen gel than the control cells. GI24 was cleaved in HT1080 cells, which was blocked by the administration of MMP inhibitor BB94 or transfection of small interfering RNA (siRNA) targeting MT1-MMP. GI24 expression is relatively high in some squamous carcinoma and hepatocarcinoma cell lines. Transfection of siRNA for GI24 into oral squamous carcinoma-derived HSC-4 cells, which express GI24 and MT1-MMP genes reduced the expression of not only GI24 but also MT1-MMP, and attenuated invasive growth in the collagen matrix. These results suggest that GI24 contributes to tumor-invasive growth in the collagen matrix by augmenting cell surface MT1-MMP.

MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21(WAF1) and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin alpha(v)beta(3) were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.

Isolation of Highly Migratory and Invasive Cells in Three-Dimensional Gels.

We developed a modified invasion assay in three-dimensional (3D) gels that permits isolation of invading cells as living cells, termed an invading cell-trapping (iCT) assay. A small cell strainer consisting of nylon mesh with pores of 40-µm square is used in this assay. A layer of gel composed of extracellular-matrix components is formed on each side of the nylon mesh, which permits cell migration or invasion from one gel layer to the other. At the end of the assay, the two gel layers are removed from the apparatus and easily separated from each other. Invading cells from the primary gel are trapped in the secondary gel, which maintains the morphology and other properties of the invasive cells in a 3D matrix. The cells that have invaded are observed and counted with a standard light microscope without cell staining. There is no need for a specialized microscope, imaging analysis software, or advanced cell-biological technical knowledge in this assay. This assay can also reduce measurement of nonspecific cell invasion by monitoring the upward invasion of cells. The viability of both invading and non-invading cells trapped in the gels can be assessed by typical colorimetric assays, if desired. This assessment characterizes the total number of cells (invading and non-invading cells) and the ratio of invading cells to total cells. By repeating the iCT assay, further enrichment of invasive and noninvasive cells can be attained. Thus, this assay improves comparative analyses between invasive and noninvasive cells. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Measuring upward cell invasion into collagen gel Basic Protocol 2: Measuring cell invasion from Matrigel into collagen gel Basic Protocol 3: Isolation and enrichment of highly invasive cells.

Cathepsin G, a neutrophil protease, induces compact cell-cell adhesion in MCF-7 human breast cancer cells.

Cathepsin G is a serine protease secreted by activated neutrophils that play a role in the inflammatory response. Because neutrophils are known to be invading leukocytes in various tumors, their products may influence the characteristics of tumor cells such as the growth state, motility, and the adhesiveness between cells or the extracellular matrix. Here, we demonstrate that cathepsin G induces cell-cell adhesion of MCF-7 human breast cancer cells resulting from the contact inhibition of cell movement on fibronectin but not on type IV collagen. Cathepsin G subsequently induced cell condensation, a very compact cell colony, resulting due to the increased strength of E-cadherin-mediated cell-cell adhesion. Cathepsin G action is protease activity-dependent and was inhibited by the presence of serine protease inhibitors. Cathepsin G promotes E-cadherin/catenin complex formation and Rap1 activation in MCF-7 cells, which reportedly regulates E-cadherin-based cell-cell junctions. Cathepsin G also promotes E-cadherin/protein kinase D1 (PKD1) complex formation, and Go6976, the selective PKD1 inhibitor, suppressed the cathepsin G-induced cell condensation. Our findings provide the first evidence that cathepsin G regulates E-cadherin function, suggesting that cathepsin G has a novel modulatory role against tumor cell-cell adhesion.

The scaffold protein c-Jun NH2-terminal kinase-associated leucine zipper protein regulates cell migration through interaction with the G protein G(alpha 13).

Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH(2)-terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of full-length shRNA-resistant mouse JLP rescued the impaired cell migration of the JLP-depleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein G(alpha13), showed little or no effect on the cell migration defect. Furthermore, although a constitutively active G(alpha13) enhanced the migration of control HeLa cells, the G(alpha13)-induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with G(alpha13).