MMP-9 Promoter Activity Research Articles

Temozolomide promotes matrix metalloproteinase 9 expression through p38 MAPK and JNK pathways in glioblastoma cells

Glioblastoma (GBM) is a highly aggressive and deadly brain cancer. Temozolomide (TMZ) is the standard chemotherapeutic agent for GBM, but the majority of patients experience recurrence and invasion of tumor cells. We investigated whether TMZ treatment of GBM cells regulates matrix metalloproteinases (MMPs), which have the main function to promote tumor cell invasion. TMZ effectively killed GL261, U343, and U87MG cells at a concentration of 500 µM, and surviving cells upregulated MMP9 expression and its activity but not those of MMP2. TMZ also elevated levels of MMP9 mRNA and MMP9 promoter activity. Subcutaneous graft tumors survived from TMZ treatment also exhibited increased expression of MMP9 and enhanced gelatinolytic activity. TMZ-mediated MMP9 upregulation was specifically mediated through the phosphorylation of p38 and JNK. This then stimulates AP-1 activity through the upregulation of c-Fos and c-Jun. Inhibition of the p38, JNK, or both pathways counteracted the TMZ-induced upregulation of MMP9 and AP-1. This study proposes a potential adverse effect of TMZ treatment for GBM: upregulation of MMP9 expression potentially associated with increased invasion and poor prognosis. This study also provides valuable insights into the molecular mechanisms by which TMZ treatment leads to increased MMP9 expression in GBM cells.

Abstract 5407: Temozolomide promotes matrix metalloproteinase 9 expression through p38 MAPK and JNK pathways in glioblastoma cells

Abstract Glioblastoma multiforme (GBM) is a highly aggressive and deadly brain cancer. Temozolomide (TMZ) is the standard chemotherapeutic agent for GBM, but many patients experience recurrence and invasion of tumor cells. We investigated whether TMZ treatment of GBM cells regulates matrix metalloproteinases (MMPs), which have main function to promote tumor cell invasion. TMZ effectively killed GL261, U343, and U87MG cells at a concentration of 500 μM, and surviving cells upregulated MMP9 expression and its activity but not those of MMP2. TMZ also elevated levels of MMP9 mRNA and MMP9 promoter activity. Subcutaneous graft tumors survived from TMZ treatment also exhibited increased expression of MMP9 and enhanced gelatinolytic activity. TMZ- mediated-MMP9 upregulation was specifically mediated through the phosphorylation of p38 and JNK. This then stimulates AP-1 activity through the upregulation of c-Fos and c-Jun. Inhibition of the p38, JNK, or both pathways counteracted the TMZ-induced upregulation of MMP9 and AP-1. This study proposes a potential adverse effect of TMZ treatment for GBM: upregulation of MMP9 expression associated with increased invasion and poor prognosis. This study also provides valuable insights into the molecular mechanisms by which TMZ treatment leads to increased MMP9 expression in GBM cells. Citation Format: Chaeyong Jung, Hien Duong Thanh, Sueun Lee, Kyung-Sup Moon. Temozolomide promotes matrix metalloproteinase 9 expression through p38 MAPK and JNK pathways in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5407.

Steroid receptor coactivator 1 promotes human hepatocellular carcinoma invasiveness through enhancing MMP-9.

SRC-1 functions as a transcriptional coactivator for steroid receptors and various transcriptional factors. Notably, SRC-1 has been implicated in oncogenic roles in multiple cancers, including breast cancer and prostate cancer. Previous investigations from our laboratory have established the high expression of SRC-1 in human HCC specimens, where it accelerates HCC progression by enhancing Wnt/beta-catenin signalling. In this study, we uncover a previously unknown role of SRC-1 in HCC metastasis. Our findings reveal that SRC-1 promotes HCC metastasis through the augmentation of MMP-9 expression. The knockdown of SRC-1 effectively mitigated HCC cell metastasis both invitro and invivo by suppressing MMP-9 expression. Furthermore, we observed a positive correlation between SRC-1 mRNA levels and MMP-9 mRNA levels in limited and larger cohorts of HCC specimens from GEO database. Mechanistically, SRC-1 operates as a coactivator for NF-κB and AP-1, enhancing MMP-9 promoter activity in HCC cells. Higher levels of SRC-1 and MMP-9 expression are associated with worse overall survival in HCC patients. Treatment with Bufalin, known to inhibit SRC-1 expression, significantly decreased MMP-9 expression and inhibited HCC metastasis in both invitro and invivo settings. Our results demonstrated the pivotal role of SRC-1 as a critical modulator in HCC metastasis, presenting a potential therapeutic target for HCC intervention.

Rhamnetin Prevents Bradykinin-Induced Expression of Matrix Metalloproteinase-9 in Rat Brain Astrocytes by Suppressing Protein Kinase-Dependent AP-1 Activation.

Bradykinin (BK) has been recognized as a stimulant for matrix metalloproteinase (MMP)-9 expression, contributing to neuroinflammation. Modulating the BK/MMP-9 pathway offers potential in the treatment of neuroinflammatory disorders. Rhamnetin (RNT), a flavonoid compound known for its antioxidant and anti-inflammatory effects, has shown promise. However, the specific mechanisms through which RNT inhibits BK-induced MMP-9 expression remain unclear. Therefore, this study aims to delve into the intricate mechanisms underlying this process. Here, we initially demonstrated that RNT effectively attenuated BK-induced MMP-9 expression and its associated cell migration in rat brain astrocyte-1 (RBA-1) cells. Further investigation revealed that BK-driven MMP-9 protein, mRNA, and promoter activity linked to cell migration relied on c-Src, Pyk2, EGFR, PDGFR, PI3K/Akt, JNK1/2, and c-Jun. This was validated by the inhibition of these effects through specific inhibitors, a finding substantiated by the introduction of siRNAs targeting these signaling molecules. Notably, the phosphorylated levels of these signaling components induced by BK were significantly reduced by their respective inhibitors and RNT, underscoring the inhibitory role of RNT in this process. These findings indicate that, in RBA-1 cells, RNT diminishes the heightened induction of MMP-9 triggered by BK through the inhibition of c-Src/Pyk2/PDGFR and EGFR/PI3K/Akt/JNK1/2-dependent AP-1 activation. This suggests that RNT holds promise as a potential therapeutic approach for addressing neuroinflammation in the brain.

Opposing MMP-9 Expression in Mesenchymal Stromal Cells and Head and Neck Tumor Cells after Direct 2D and 3D Co-Culture

Bone marrow-derived mesenchymal stromal cells (BMSCs) respond to a variety of tumor cell-derived signals, such as inflammatory cytokines and growth factors. As a result, the inflammatory tumor microenvironment may lead to the recruitment of BMSCs. Whether BMSCs in the tumor environment are more likely to promote tumor growth or tumor suppression is still controversial. In our experiments, direct 3D co-culture of BMSCs with tumor cells from the head and neck region (HNSCC) results in strong expression and secretion of MMP-9. The observed MMP-9 secretion mainly originates from BMSCs, leading to increased invasiveness. In addition to our in vitro data, we show in vivo data based on the chorioallantoic membrane (CAM) model. Our results demonstrate that MMP-9 induces hemorrhage and increased perfusion in BMSC/HNSCC co-culture. While we had previously outlined that MMP-9 expression and secretion originate from BMSCs, our data showed a strong downregulation of MMP-9 promoter activity in HNSCC cells upon direct contact with BMSCs using the luciferase activity assay. Interestingly, the 2D and 3D models of direct co-culture suggest different drivers for the downregulation of MMP-9 promoter activity. Whereas the 3D model depicts a BMSC-dependent downregulation, the 2D model shows cell density-dependent downregulation. In summary, our data suggest that the direct interaction of HNSCC cells and BMSCs promotes tumor progression by significantly facilitating angiogenesis via MMP-9 expression. On the other hand, data from 3D and 2D co-culture models indicate opposing regulation of the MMP-9 promoter in tumor cells once stromal cells are involved.

FOSL1 knockdown ameliorates DSS-induced inflammation and barrier damage in ulcerative colitis via MMP13 downregulation.

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon. Fos-related antigen 1 is highly expressed in mild UC and affects the maintenance and delayed recurrence of inflammatory bowel disease. Therefore the present study aimed to investigate the role of Fos-like antigen-1 (FOSL1) in UC. Use of the JASPAR database predicted the possible interaction of FOSL1 and the MMP13 promoter. FOSL1 and MMP13 mRNA and protein expression levels in dextran sodium sulfate (DSS)-induced HT29 cells and colon tissues of a UC mice model were determined using reverse transcription-quantitative PCR and western blotting, respectively. MMP13 promoter activity was determined using the dual-luciferase reporter assay, the relationship between FOSL1 and MMP13 promoter was determined using chromatin immunoprecipitation, inflammatory factor levels were quantified using ELISA, cell monolayer permeability analysis was performed using transepithelial electrical resistance measurements and tight junction protein expression levels were determined by western blotting. After constructing a UC mice model, mice colonic injury was determined with the quantification of colon length, H&E staining and disease activity index. The results demonstrated that FOSL1 and MMP13 were upregulated in DSS-induced HT29 cells and tissues. FOSL1 was also determined to be bound to the MMP13 promoter region and was demonstrated to upregulate MMP13 expression levels. Furthermore, FOSL1 knockdown inhibited DSS-induced inflammation and barrier damage in HT29 cells via MMP13 downregulation. FOSL1 knockdown also ameliorated colonic injury, inflammation and barrier damage in the tissues of the UC mice model via MMP13 downregulation. Overall, FOSL1 knockdown was demonstrated to potentially ameliorate DSS-induced inflammatory injury and protect the intestinal barrier integrity in the UC mice model via MMP13 downregulation.

Identification and characterization of TGF-β1-responsive Runx2 acetylation sites for matrix Metalloproteinase-13 expression in osteoblastic cells

In skeletal tissues, transforming growth factor-beta 1 (TGF-β1) serves a number of activities. For example, in osteoblastic cells, TGF-β1 stimulates the expression of matrix metalloproteinase-13 (MMP-13, a bone remodeling gene), which requires the bone transcription factor Runx2. Although TGF-β1 is known to stimulate Runx2 acetylation, the sites involved in MMP-13 gene activation remain unknown. Mass spectrometry analysis revealed that Runx2 was acetylated at one site (K134) and three sites (K24, K134, and K169) following control and TGF-β1-treatment, respectively, in osteoblastic cells. In addition, we mutated the lysine residues in the Runx2 construct into arginine and transfected the construct into mouse mesenchymal stem cells (C3H10T1/2). Wild-type Runx2 expression and acetylation were significantly increased by TGF-β1-treatment, whereas this effect was decreased in the presence of the Runx2 double mutant construct (K24 + K169) in C3H10T1/2 cells. TGF-β1 enhanced MMP-13 promoter activity in cells transfected with the wild-type Runx2 construct, but this effect was considerably reduced in cells transfected with the Runx2 double mutant construct (K24 + K169), according to a luciferase reporter test. Hence, the stability of Runx2 may be mediated by TGF-β1-induced acetylation at K24 and K169 and is required for MMP-13 expression in osteoblastic cells. These findings add to our knowledge of TGF-β1, Runx2, and MMP-13's physiological roles in bone metabolism.

Interleukin-1β activated c-FOS transcription factor binds preferentially to a specific allele of the matrix metalloproteinase-13 promoter and increases susceptibility to endometriosis.

Endometriosis is a benign gynecological condition characterized by increased growth, inflammation, invasion, and angiogenesis, partly regulated by a class of enzymes called matrix metalloproteinases (MMPs). The importance of a few MMPs, e.g., MMP-9, -3, and -7 has been studied in endometriosis progression. Although MMP-13 plays an essential role in bone regeneration and cancer, no report has been found on the part of MMP-13 and endometriosis progression. We found the upregulation of MMP-13 expression and activity in patients having endometriosis in the eastern Indian population. In addition, the -77A/G polymorphism of the MMP13 promoter (rs: 2252070) is associated with regulating transcription and subsequent susceptibility to disease. In eastern Indian case-control groups, the effect of the -77A/G single-nucleotide polymorphism on MMP13 promoter activity and its relationship with endometriosis susceptibility was studied. The AG genotype was shown to be more predisposed to endometriosis risk than the GG genotype (p:0.02; odds ratio [OR]: 1.65, 95% confidence interval [CI]: 1.10-2.49), also AG genotype was more frequent in late-stage patients compared to early-stage (p: 0.03, OR: 2.0, 95% CI: 1.09-3.66). Furthermore, the MMP13 gene levels were greater in AA compared to GG individuals. Additionally, MMP13 promoter-reporter experiments in cultured endometrial epithelial cells and in silico analyses both demonstrated increased transcriptional activity near the G to A transition under basal/IL-1β -induced/c-FOS overexpressed condition. Overall, c-FOS tighter binding to the A allele-carrying promoter enhances MMP13 transcription, which is further amplified by IL-1β due to increased c-FOS phosphorylation, promoting MMP-13 production and endometriosis risk.

Study on the Mechanism of Capillary Leakage Caused by Hypoxia-Inducible Factor-1α through Inducing High Expression of Matrix Metalloproteinase-9.

Purposes This study mainly explored the mechanism of capillary leakage caused by hypoxia-inducible factor-1α through inducing high expression of matrix metalloproteinase-9. Method. We established a monolayer endothelial cell model by culturing human umbilical vein endothelial cells (HUVEC) in vitro, used tumor necrosis factor (TNFα) and HIF-1α inhibitor 2-methoxyestradiol (2ME2) to act on HUVEC, and at the same time constructed siRNA-transfected HUVEC to interfere with the expression of HIF-1α. The permeability of monolayer endothelial cells was measured by transwell chamber method, the concentration of MMP-9 in the supernatant was measured by ELISA method, the expression of key molecules related to permeability (HIF- 1α, MMP-9, claudin-5, and ZO-1) was measured by RT-PCR and Western blot method, and the localization and expression of claudin-5 and ZO-1 were measured by immunofluorescence method. We searched for 7 HIF-1α hypoxia response elements within 4000 bp before the transcription start site in the MMP-9 promoter region, constructed the MMP-9 promoter-luciferase reporter gene recombinant plasmid, transfected and stimulated HUVEC with TNFα, and detected the effect of 7 hypoxia response element plasmids on the transcription activity of MMP-9 promoter. Results Under the action of TNFα, the permeability of monolayer endothelial cells increased, and the concentration of MMP-9 in the cell supernatant increased. 2ME2 and HIF-1α-siRNA transfection can improve the above situation (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can inhibit the high expression of HIF-1α and MMP-9 caused by TNFα, thereby increasing the expression of claudin-5 and ZO-1 (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can reduce the inhibition of TNFα on the expression of cell membrane protein claudin-5 and tight junction protein ZO-1. Element 1, element 5, and element 7 are the sites where HIF-1α interacts with MMP-9 at the transcription level. Conclusion This study shows that HIF-1α can increase the permeability of monolayer epithelial cells by inducing the high expression of MMP-9, leading to capillary leakage. Its target is at the −3798 bp, −1878 bp, and −1489 bp points of the transcription initiation site in the MMP-9 promoter region.

Retraction Note to: Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF

Prostate-derived Ets factor (PDEF) is expressed in tissues of high epithelial content including prostate, although its precise function has not been fully established. Conventional therapies produce a high rate of cure for patients with localized prostate cancer, but there is, at present, no effective treatment for intervention in metastatic prostate cancer. These facts underline the need to develop new approaches for early diagnosis of aggressive prostate cancer patients, and mechanism based anti-metastasis therapies that will improve the outlook for hormone-refractory prostate cancer. In this study we evaluated role of prostate-derived Ets factor (PDEF) in prostate cancer. We observed decreased PDEF expression in prostate cancer cell lines correlated with increased aggressive phenotype, and complete loss of PDEF protein in metastatic prostate cancer cell lines. Loss of PDEF expression was confirmed in high Gleason Grade prostate cancer samples by immuno-histochemical methods. Reintroduction of PDEF profoundly affected cell behavior leading to less invasive phenotypes in three dimensional cultures. In addition, PDEF expressing cells had altered cell morphology, decreased FAK phosphorylation and decreased colony formation, cell migration, and cellular invasiveness. In contrast PDEF knockdown resulted in increased migration and invasion as well as clonogenic activity. Our results also demonstrated that PDEF downregulated MMP9 promoter activity, suppressed MMP9 mRNA expression, and resulted in loss of MMP9 activity in prostate cancer cells. These results suggested that loss of PDEF might be associated with increased MMP9 expression and activity in aggressive prostate cancer. To confirm results we investigated MMP9 expression in clinical samples of prostate cancer. Results of these studies show increased MMP9 expression correlated with advanced Gleason grade. Taken together our results demonstrate decreased PDEF expression and increased MMP9 expression during the transition to aggressive prostate cancer. These studies demonstrate for the first time negative regulation of MMP9 expression by PDEF, and that PDEF expression was lost in aggressive prostate cancer and was inversely associated with MMP9 expression in clinical samples of prostate cancer. Based on these exciting results, we propose that loss of PDEF along with increased MMP9 expression should serve as novel markers for early detection of aggressive prostate cancer.

Gel-Free 3D Tumoroids with Stem Cell Properties Modeling Drug Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer

Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have been reported using 2D culture systems, whereas 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aimed to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyrosine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter the tumoroid growth of metastatic colorectal cancer (mCRC). Gene expression signatures of highly metastatic aggregative CRC (LuM1 cells) vs. low-metastatic, non-aggregative CRC (Colon26 and NM11 cells) were analyzed using microarray. To establish a 3D culture-based multiplexing reporter assay system, LuM1 was stably transfected with the Mmp9 promoter-driven ZsGreen fluorescence reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate®, a gel-free 3D culture device. LuM1 cells highly expressed mRNA encoding ABCG2 (a drug resistance pump, i.e., CSC/CIC marker), other CSC/CIC markers (DLL1, EpCAM, podoplanin, STAT3/5), pluripotent stem cell markers (Sox4/7, N-myc, GATA3, Nanog), and metastatic markers (MMPs, Integrins, EGFR), compared to the other two cell types. Hoechst efflux stem cell-like side population was increased in LuM1 (7.8%) compared with Colon26 (2.9%), both of which were markedly reduced by verapamil treatment, an ABCG2 inhibitor. Smaller cell aggregates of LuM1 were more sensitive to Cisplatin (at 10 μM), whereas larger tumoroids with increased ABCG2 expression were insensitive. Notably, Cisplatin (2 μM) and Imatinib (10 μM) at low concentrations significantly promoted tumoroid formation (cell aggregation) and increased Mmp9 promoter activity in mCRC LuM1/m9, while not cytotoxic to them. On the other hand, 5-FU significantly inhibited tumoroid growth, although not completely. Thus, drug resistance in cancer with increased stem cell properties was modeled using the gel-free 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance.

Galangin Inhibits LPS-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent AP-1 and FoxO1 Activation in Rat Brain Astrocytes.

PurposeNeuroinflammation, characterized by the increased expression of inflammatory proteins such as matrix metalloproteinases (MMPs), plays a critical role in neurodegenerative disorders. Lipopolysaccharide (LPS) has been shown to upregulate MMP-9 expression through the activation of various transcription factors, including activator protein 1 (AP-1) and forkhead box protein O1 (FoxO1). The flavonoid 3,5,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one (galangin) has been demonstrated to possess antioxidant and anti-inflammatory properties in various types of cells. Here, we investigated the mechanisms underlying the inhibitory effect of galangin on LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells).MethodsPharmacological inhibitors and siRNAs were employed to explore the effects of galangin on LPS-challenged RBA-1 cells. Gelatin zymography, Western blotting, real-time PCR, and a luciferase reporter assay were used to detect MMP-9 activity, protein expression, mRNA levels, and promoter activity, respectively. The protein kinases involved in the LPS-induced MMP-9 expression were determined by Western blot. A chromatin immunoprecipitation (ChIP) assay was employed to evaluate the activity of c-Jun at the MMP-9 promoter.ResultsGalangin treatment attenuated the LPS-mediated induction of MMP-9 protein and mRNA expression, as well as the activity at the MMP-9 promoter. In addition, galangin exerted its inhibitory effects on MMP-9 expression through suppressing the LPS-stimulated activation of proline-rich tyrosine kinase (Pyk2), platelet-derived growth factor receptor beta (PDGFRβ), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPKs). Pretreatment with galangin attenuated the LPS-induced phosphorylation of c-Jun and FoxO1. LPS-induced cell migration was also suppressed by galangin pretreatment.ConclusionGalangin attenuates the LPS-induced inflammatory responses, including the induction of MMP-9 expression and cell migration, via inhibiting Pyk2/PDGFRβ/PI3K/Akt/mTOR/JNK1/JNK2 and p44/p42 MAPK cascade-dependent AP-1 and FoxO1 activities. These results provide new insights into the mechanisms through which galangin mitigates LPS-induced inflammatory responses, and suggest novel strategies for the management of LPS-related brain diseases.

Transcription factor EGR-1 transactivates the MMP1 gene promoter in response to TNFα in HaCaT keratinocytes

Matrix metalloproteinase 1 (MMP-1), a calcium-dependent zinc- containing collagenase, is involved in the initial degradation of native fibrillar collagen. Tissue necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that is rapidly produced by dermal fibroblasts, monocytes/macrophages, and keratinocytes and regulates inflammation and damaged-tissue remodeling. MMP-1 is induced by TNFα and plays a critical role in tissue remodeling and skin aging processes. However, the regulation of the MMP1 gene by TNFα is not fully understood. We aimed to find additional cis-acting elements involved in the regulation of TNFα-induced MMP1 gene transcription in addition to the nuclear factor-kappa B (NF-kB) and activator protein 1 (AP1) sites. Assessments of the 5’-regulatory region of the MMP1 gene, using a series of deletion constructs, revealed the requirement of the early growth response protein 1 (EGR-1)-binding sequence (EBS) in the proximal region for proper transcription by TNFα. Ectopic expression of EGR-1, a zinc-finger transcription factor that binds to G-C rich sequences, stimulated MMP1 promoter activity. The silencing of EGR-1 by RNA interference reduced TNFα-induced MMP-1 expression. EGR-1 directly binds to the proximal region and transactivates the MMP1 gene promoter. Mutation of the EBS within the MMP1 promoter abolished EGR-1-mediated MMP-1 promoter activation. These data suggest that EGR-1 is required for TNFα-induced MMP1 transcriptional activation. In addition, we found that all three MAPKs, ERK1/2, JNK, and p38 kinase, mediate TNFα-induced MMP-1 expression via EGR-1 upregulation. These results suggest that EGR-1 may represent a good target for the development of pharmaceutical agents to reduce inflammation-induced MMP-1 expression.

Lipopolysaccharide-Induced Matrix Metalloproteinase-9 Expression Associated with Cell Migration in Rat Brain Astrocytes.

Neuroinflammation is a landmark of neuroinflammatory and neurodegenerative diseases. Matrix metalloproteinase (MMP)-9, one member of MMPs, has been shown to contribute to the pathology of these brain diseases. Several experimental models have demonstrated that lipopolysaccharide (LPS) exerts a pathological role through Toll-like receptors (TLRs) in neuroinflammation and neurodegeneration. However, the mechanisms underlying LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1) are not completely understood. Here, we applied pharmacological inhibitors and siRNA transfection to assess the levels of MMP-9 protein, mRNA, and promoter activity, as well as protein kinase phosphorylation in RBA-1 cells triggered by LPS. We found that LPS-induced expression of pro-form MMP-9 and cell migration were mediated through TLR4, proto-oncogene tyrosine-protein kinase (c-Src), proline-rich tyrosine kinase 2 (Pyk2), platelet-derived growth factor receptor (PDGFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), p38 mitogen-activated protein kinase (MAPK), and Jun amino-terminal kinase (JNK)1/2 signaling molecules in RBA-1 cells. In addition, LPS-stimulated binding of c-Jun to the MMP-9 promoter was confirmed by chromatin immunoprecipitation (ChIP) assay, which was blocked by pretreatment with c-Src inhibitor II, PF431396, AG1296, LY294002, Akt inhibitor VIII, p38 MAP kinase inhibitor VIII, SP600125, and tanshinone IIA. These results suggest that in RBA-1 cells, LPS activates a TLR4/c-Src/Pyk2/PDGFR/PI3K/Akt/p38 MAPK and JNK1/2 pathway, which in turn triggers activator protein 1 (AP-1) activation and ultimately induces MMP-9 expression and cell migration.

A Reporter System Evaluates Tumorigenesis, Metastasis, β-catenin/MMP Regulation, and Druggability.

Cancer invasion, metastasis, and therapy resistance are the crucial phenomena in cancer malignancy. The high expression of matrix metalloproteinase 9 (MMP9) is a biomarker as well as a causal factor of cancer invasiveness and metastatic activity. However, a regulatory mechanism underlying MMP9 expression in cancer is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming an important clue. In the present study, we aimed (i) to develop a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional tumoroid model and Mmp9 promoter and (ii) to examine pharmacological actions of anticancer medications using this reporter system. High expression and genetic amplification of MMP9 were found in colon cancer cases. We found that proximal promoter sequences of MMP9 in murine and human contained conserved binding sites for transcription factors β-catenin/TCF/LEF, glucocorticoid receptor (GR), and nuclear factor kappa-B (NF-κB). The murine Mmp9 promoter (-569 to +19) was markedly activated in metastatic colon cancer cells and additionally activated by tumoroid formation and by β-catenin signaling stimulator lithium chloride. The Mmp9 promoter-driven fluorescent reporter cells enabled the monitoring of activities of MMP9/gelatinase, tumorigenesis, invasion, and metastasis in syngeneic transplantation experiments. We also demonstrated pharmacological actions as follows: dexamethasone and hydrocortisone, steroidal medications binding to GR, inhibited the Mmp9 promoter but did not inhibit tumorigenesis. On the contrary, antimetabolite 5-fluorouracil, a gold standard for colon cancer chemotherapy, inhibited tumoroid formation but did not inhibit Mmp9 promoter activity. Notably, antimalaria medication artesunate inhibited both tumorigenesis and the Mmp9 promoter in vitro, potentially through inhibition of β-catenin/TCF/LEF signaling. Thus, this novel reporter system enabled monitoring tumorigenesis, invasiveness, metastasis, key regulatory signalings such as β-catenin/MMP9 axis, and druggability. Impact Statement Cancer invasion and metastasis have been shown to be driven by matrix metalloproteinase 9 (MMP9), whose expression mechanism is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming important. We established a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional (3D) tumoroid model and Mmp9 promoter. Using this reporter system, we demonstrated pharmacological actions of anticancer medications such as antimetabolite 5-fluorouracil (5-FU) and antimalaria medication artesunate (ART), which inhibited both tumorigenesis and β-catenin/MMP regulatory signaling. Our study impacts the translational fields of oncology, drug discovery, and organoid model.

Anti-inflammatory property of quercetin through downregulation of ICAM-1 and MMP-9 in TNF-α-activated retinal pigment epithelial cells

Quercetin is a flavonoid polyphenolic compound present in fruits and vegetables that has proven anti-inflammatory activity. The goal of the present investigation was to investigate the effects of quercetin on tumor necrosis factor-α (TNF-α)-induced inflammatory responses via the expression of ICAM-1 and MMP-9 in human retinal pigment epithelial cells (ARPE-19 cells). Real-time PCR, gelatin zymography, and Western blot analysis showed that TNF-α induced the expression of ICAM-1 and MMP-9 protein and mRNA in a time-dependent manner. These effects were attenuated by pretreatment of ARPE-19 cells with quercetin. Quercetin inhibited the TNF-α-induced phosphorylation of PKCδ, JNK1/2, ERK1/2. Quercetin, rottlerin, SP600125 and U0126 attenuated TNF-α-stimulated c-Jun phosphorylation and AP-1–Luc activity. Pretreatment with quercetin, rottlerin, SP600125, or Bay 11-7082 attenuated TNF-α-induced NF-κB (p65) phosphorylation, translocation and RelA/p65–Luc activity. TNF-α significantly increased MMP-9 promoter activity and THP-1 cell adherence, and these effects were attenuated by pretreatment with quercetin, rottlerin, SP600125, U0126, tanshinone IIA or Bay 11-7082. These results suggest that quercetin attenuates TNF-α-induced ICAM-1 and MMP-9 expression in ARPE-19 cells via the MEK1/2–ERK1/2 and PKCδ–JNK1/2–c-Jun or NF-κB pathways.

Alterations of the Immunologic Co-Stimulator B7 and TNFR Families Correlate with Hepatocellular Carcinoma Prognosis and Metastasis by Inactivating STAT3.

Blockade of the immunosuppressive checkpoint receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA4) or programmed death 1 (PD-1) and its cognate ligand, programmed death 1 ligand (PD-L1), has altered the landscape of anti-tumor immunotherapy. B7 family and tumor necrosis factor receptor (TNFR) superfamily play a crucial role in T cell activation, tolerance, and anergy through co-stimulatory and inhibitory signal transduction. Investigating the immune molecular landscapes of the B7 and TNFR families is critical in defining the promising responsive candidates. Herein, we performed comprehensive alteration analysis of the B7 and TNFR family genes across six hepatocellular carcinoma (HCC) datasets with over 1000 patients using cBioPortal TCGA data. About 16% of patients had both B7 and TNFR gene alterations. TNFR gene amplifications were relatively more common (1.73–8.82%) than B7 gene amplifications (1.61–2.94%). Analysis of 371 sequenced samples revealed that all genes were upregulated: B7 and TNFR mRNA were upregulated in 23% of cases (86/371) and 28% of cases (105/371), respectively. Promoter methylation analysis indicated an epigenetic basis for B7 and TNFR gene regulation. The mRNA levels of B7 and TNFR genes were inversely correlated with promoter methylation status. B7-H6 expression was significantly associated with worse overall survival, and B7-H6 mRNA was increased gradually in cases with gene copy number alterations. B7-H6 overexpression was associated with aggressive clinicopathologic features and poor prognosis in HCC. Downregulation of B7-H6 in HCC cells significantly inhibited cell adhesion, proliferation, migration, and invasion. Knockdown of B7-H6 in HCC cells inhibited tumor growth and metastasis in vivo. B7-H6 promoted HCC metastasis via induction of MMP-9 expression and STAT3 activation. B7-H6 and STAT3 performed functional overlapping roles on enhancing the MMP-9 promoter activity in HCC cells. These results suggest that alterations of the immunologic co-stimulator B7 and TNFR families correlate with HCC metastasis and prognosis, and especially B7-H6 plays a critical role in promoting metastasis of HCC.

Preventive effect of Curcuma zedoaria extract on UVB-induced skin inflammation and photoaging

We sought to investigate whether Curcuma zedoaria extract (CZE) has preventive effects against UVB-induced skin inflammation and photoaging. CZE was identified as a promising anti-inflammatory and photoaging candidate based on a matrix metalloproteinase (MMP)-1 promoter assay and the suppressive effects of CZE on UVB-induced cyclooxygenases (COX)-2 and MMP-13 expression were confirmed in immortalized skin human keratinocytes. CZE suppressed UVB-induced phosphorylation of c-Jun N-terminal kinases, mitogen-activated protein kinase kinases 3/6/p38, B-Raf/ERK kinase (MEK)1/2/extracellular signal-regulated kinases (ERK)1/2, and Akt as well as phosphorylation of epidermal growth factor receptor (EGFR) and Src. Using a chronic skin inflammation and photoaging model, we observed that CZE significantly suppressed repetitive UVB-induced wrinkle formation and COX-2 and MMP-13 expression in vivo. Among the compounds investigated, curcumin was found to exhibit the strongest inhibitory effect on UVB-induced MMP-1 promoter activity. These results demonstrate that CZE has a potent preventive activity for UVB-induced skin inflammation and photoaging, which occurs via the suppression of EGFR, Src, MAPKKs/MAPKs, and Akt phosphorylation. Practical applications This study is a study to develop materials that can prevent skin inflammation and photoaging by UVB exposure. Therefore, it can be applied to functional cosmetics or functional foods after standardization and clinical studies on this material.

Abstract 2872: Mechanism of anti-proliferative and anti-invasive actions of ω3-polyunsaturated fatty acids in human hypopharyngeal carcinoma cells

Abstract Hypopharyngeal carcinoma is one of head and neck cancers. According to the latest statistics from the United States National Cancer Institute, it is estimated that about 17,000 Americans will be diagnosed with pharyngeal cancer in 2017. Although ω3-polyunsaturated fatty acids (ω3-PUFAs) have anti-tumorigenic properties in the several cancers, the anti-cancer effect of ω3-PUFAs on hypopharyngeal carcinoma has been not known yet. In this study, we report inhibitory mechanisms of ω3-PUFAs on cell growth and invasion in hypopharyngeal carcinoma. DHA and EPA inhibited the cell growth of FaDu and SNU1041 in a dose- and time-dependent manner; in contrast, arachidonic acid, a ω6-PUFA, had no significant effect. In flow cytometry analysis, subG1 population increased in DHA-treated FaDu and SNU1041. Moreover, apoptotic cell death was confirmed by TUNEL assay, and the induction of cleaved PARP and caspase-3. Furthermore, treatment of hypopharyngeal carcinoma cells with DHA resulted in a significant increase in autophagic activity, as revealed by increased LC3-II levels, GFP-LC3 puncta, and autophagic flux activation. EGF-induced phosphorylation of EGFR, which is frequently overexpressed in hypopharyngeal carcinoma, was also suppressed after DHA pretreatment, and levels of p-Akt (p- Aktthr308 and AktSer473), which is a downstream signal, was also inhibited in the FaDu and SNU1041 cells. The invasiveness of cells was significantly inhibited by DHA treatment in vitro transwell assay as well. The MMP-2 and MMP-9 promoter activities were inhibited after DHA treatment. Cox-2 and VEGF promoter activities were also suppressed by DHA. Furthermore, DHA decreased the levels of reporter activity of NF-κB, which is transcription factor that regulates MMPs, Cox-2 and VEGF expression. Fat1-stably expressing FaDu cells (fFaDu-sc) was established (fFaDu-sc express a Caenorhabditis elegans ω3-desaturase converting ω6- to ω3-PUFAs endogenously), and the proliferation of fFaDu-sc was more attenuated than that of cells expressing control vectors (fFaDu-cc). Moreover, fFaDu-sc showed reduction of cell invasion compared with fFaDu-cc. in transwell chamber. The MMPs promoter activities were also suppressed in fFaDu-sc. Taken together, these findings provide evidence that ω3-PUFAs may inhibit invasion as well as cell growth through suppression of p-EGFR and MMPs expression in hypopharyngeal carcinoma cells, indicating that the utilization of ω3-PUFAs may represent a potential effective therapy for the chemoprevention and treatment of human hypopharyngeal carcinoma. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)]. Citation Format: Seung-Hyeon Han, Soyeon Shin, Young-Joo Jeon, Jun-Young Heo, Gi-Ryang Kweon, Seung-Kiel Park, Jong-Il Park, Kyu Lim. Mechanism of anti-proliferative and anti-invasive actions of ω3-polyunsaturated fatty acids in human hypopharyngeal carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2872.

Transcription Factor SOX5 Promotes the Migration and Invasion of Fibroblast-Like Synoviocytes in Part by Regulating MMP-9 Expression in Collagen-Induced Arthritis.

Fibroblast-like synoviocytes (FLS) exhibit a unique aggressive phenotype in rheumatoid arthritis (RA). Increased FLS migration and subsequent invasion of the extracellular matrix are essential to joint destruction in RA. Our previous research reported that transcription factor SOX5 was highly expressed in RA-FLS. Here, the effects of SOX5 in RA-FLS migration and invasion will be investigated. The migration and invasion of RA-FLS were evaluated using a transwell chamber assay. The expression of several potential SOX5-targeted genes, including matrix metalloproteinases (MMP-1, 2, 3 and 9), chemokines (CCL4, CCL2, CCR5 and CCR2), and pro-inflammatory cytokines (TNF-α and IL-6), were examined in RA-FLS using SOX5 gain- and loss-of-function study. The molecular mechanisms of SOX5-mediated MMP-9 expressions were assayed by luciferase reporter gene and chromatin immunoprecipitation (ChIP) studies. The in vivo effect of SOX5 on FLS migration and invasion was examined using collagen-induced arthritis (CIA) in DBA/1J mice. Knockdown SOX5 decreased lamellipodium formation, migration, and invasion of RA-FLS. The expression of MMP-9 was the only gene tested to be concomitantly affected by silencing or overexpressing SOX5. ChIP assay revealed that SOX5 was bound to the MMP-9 promoter in RA-FLS. The overexpression of SOX5 markedly enhanced the MMP-9 promoter activity, and specific deletion of a putative SOX5-binding site in MMP-9 promoter diminished this promoter-driven transcription in FLS. Locally knocked down SOX5 inhibited MMP-9 expression in the joint tissue and reduced pannus migration and invasion into the cartilage in CIA mice. SOX5 plays a novel role in mediating migration and invasion of FLS in part by regulating MMP-9 expression in RA.