In this encyclopedia, the followings are reviewed: 1. Structures of MMPs; 2. Extracellular roles of MMPs; 3. "Intracellular roles" of MMPs; 4. "Extracellular vesicle"-associated MMPs; 5. Roles of MMPs in cancers.

1. Structures of MMPs ^[1]

   The matrix metalloproteinase family consists of

The matrix metalloproteinase family consists of

about 30 members that share similarities in their structure, regulation, and function [4]. Earlier studies showed MMPs constitute a large family of zinc/calcium-dependent endopeptidases. All MMPs have principal domains, including (1) A

that share similarities in their structure, regulation, and function ^[2]. Earlier studies showed MMPs constitute a large family of zinc/calcium-dependent endopeptidases. All MMPs have principal domains, including (1) A

signal peptide (SP) sequenc

e found at the very N-terminus of all MMPs, (2) a

pro-domain

that functions as an intramolecular inhibitor to maintain the enzyme in an inactive state, (3) a

metalloproteinase catalytic domain

that can exert the proteolytic activity, (4) A

linker sequence

connecting the catalytic domains with a following domain, and (5) a

hemopexin-like repeat (PEX or HPX) domain

, which interacts with other molecules and determines the substrate specificity. The PEX domain is present in all MMPs except for MMP-7, -23, and -26.

   Proline residues in the middle of the SP can structurally weaken the secretory activities of MMPs [2,5] and thus generate intracellular MMPs. Besides, human MMP-3 contains six

Proline residues in the middle of the SP can structurally weaken the secretory activities of MMPs ^[3][4] and thus generate intracellular MMPs. Besides, human MMP-3 contains six

nuclear localization signals (NLS)

composed of basic amino acid clusters [5]. MMP3 thus has both extracellular and nuclear functions.

Additionally, gelatinases (MMP-2, -9) contain the

fibronectin type II inserts

in the middle of the catalytic domain. Membrane type (MT) -MMPs contain

type I transmembrane (TM) domains

followed by cytoplasmic tails at the C-terminus. MT-MMPs are composed of MMP-14, -15, -16, and -24. Only MMP-23 contains a cysteine array region and an IgG-like domain.

2. Extracellular roles of MMPs

   MMPs cleave substrate proteins in the extracellular space. MMP-dependent proteolysis of

MMPs cleave substrate proteins in the extracellular space. MMP-dependent proteolysis of

extracellular matrix (ECM)

and

intercellular adhesion molecules

enable cells to migrate and invade tissue

microenvironment

. Proteolysis of ECM also triggers the

release of cytokines, chemokines, and growth factors

that activate their

receptors and intracellular signaling pathways. In addition, MMPs also directly cleave and alter activities of growth factors, cytokines, chemokines, and their receptors. For example, MMPs can alter the activity of connective tissue growth factor (CTGF, recently known as cellular communication network factor 2 (CCN2)) by direct cleavage [6]. MMP cleavage of CCNs alters the angiogenic activities of CCNs and VEGF. A disintegrin and metalloproteinases (ADAMs) family members as well as MMPs cleave membrane-bound heparin-binding EGF-like growth factor (HB-EGF) and then release soluble HB-EGF, which stimulates EGFR/ERBB signaling [7].

and intracellular signaling pathways. In addition, MMPs also directly cleave and alter activities of growth factors, cytokines, chemokines, and their receptors. For example, MMPs can alter the activity of connective tissue growth factor (CTGF, recently known as cellular communication network factor 2 (CCN2)) by direct cleavage ^[5]. MMP cleavage of CCNs alters the angiogenic activities of CCNs and VEGF. A disintegrin and metalloproteinases (ADAMs) family members as well as MMPs cleave membrane-bound heparin-binding EGF-like growth factor (HB-EGF) and then release soluble HB-EGF, which stimulates EGFR/ERBB signaling ^[6].

   Extracellular MMPs are known to be components of the

Extracellular MMPs are known to be components of the

senescence-associated secretory phenotype (SASP) that includes interleukins (IL-1β, IL-1α, IL-6, IL-8) and chemokines (CCL2) as well [8].

that includes interleukins (IL-1β, IL-1α, IL-6, IL-8) and chemokines (CCL2) as well ^[7].

3. Intracellular roles of MMPs

     Intracellular and intranuclear roles for MMPs

have also been discovered. MMP-3 possesses several

NLSs

and can translocate into cellular nuclei, at which site MMP-3 can

bind to DNA and chromatin proteins

leading to

transcriptional regulation

of

CTGF/CCN2 gene [2,3]. Promoter analysis of the

gene ^[3][8]. Promoter analysis of the

CCN2/CTGF

gene revealed a

cis-element, designated transcriptional enhancer dominant in chondrocytes (TRENDIC) [2,9]. One of the TRENDIC-binding proteins was identified to be MMP-3 [2]. MMP-3 overexpression enhanced

-element, designated transcriptional enhancer dominant in chondrocytes (TRENDIC) ^[3][9]. One of the TRENDIC-binding proteins was identified to be MMP-3 ^[3]. MMP-3 overexpression enhanced

CCN2/CTGF promoter activity in human chondrosarcoma-derived chondrocytic cell line HCS-2/8 and non-basal type, triple-negative breast cancer cell line MDA-MB-231 [2].

promoter activity in human chondrosarcoma-derived chondrocytic cell line HCS-2/8 and non-basal type, triple-negative breast cancer cell line MDA-MB-231 ^[3].

Intranuclear translocation of recombinant MMP-3, as well as endogenous MMP-3, were observed under confocal laser scanning microscopy (CLSM) [2].

of recombinant MMP-3, as well as endogenous MMP-3, were observed under confocal laser scanning microscopy (CLSM) ^[3].

DNA-binding

of MMP-3 was demonstrated by gel shift and chromatin immunoprecipitation (ChIP) assays. An MMP-3 specific inhibitor inhibited the activity of the

CCN2/CTGF promoter [2], suggesting that MMP-3 proteolytic activity was partly involved in the transcriptional role for this enzyme, although the general MMP inhibitor GM6001 or an MMP2/9 inhibitor were each ineffective in this regard [2]. MMP-3 was strongly immunostained in cell nuclei in

promoter ^[3], suggesting that MMP-3 proteolytic activity was partly involved in the transcriptional role for this enzyme, although the general MMP inhibitor GM6001 or an MMP2/9 inhibitor were each ineffective in this regard ^[3]. MMP-3 was strongly immunostained in cell nuclei in

cartilage tissues in the normal and arthritic mouse model [2]. These findings demonstrated the role of MMP-3 in

^[3]. These findings demonstrated the role of MMP-3 in

gene regulation

.

     MMPs also undergo endogenous

MMPs also undergo endogenous

auto-cleavage

MMPs themselves to generate fragments containing the PEX domain. The

PEX domain

transcriptionally activates some members of

heat shock protein (HSP)

genes ^[10], a process that can contribute to anti-apoptosis and drug resistance. MMP-3 directly interacts with

heterochromatin proteins (HP1)

, members of the

chromobox protein (CBX) family that involve transcriptional and chromosomal control [2,5,10].

family that involve transcriptional and chromosomal control ^[3][4][10].

     MMPs also impact

MMPs also impact

oxidative stress, DNA damage

, and

chromosome instability

in cell nuclei. Intranuclear activities of MMP-2 and MMP-9 were shown to cleave PARP-1 and XRCC1, nuclear matrix proteins, promoting

oxidative DNA damage

, and apoptosis in an ischemic injury model ^[11].

4. Extracellular vesicle-associated MMPs

   Members of the MMP family are often associated with

Members of the MMP family are often associated with

extracellular vesicles (EVs)

^[12]. MMP3-rich EVs enhance cancer cell migration and invasion, molecular transmission, and gene activation while

CRISPR/Cas9-based knockout of MMP3 reduced these pro-tumorigenic roles of EVs [3,13].

-based knockout of MMP3 reduced these pro-tumorigenic roles of EVs ^[8][13].

EV-associated MMP3

was transmissive into recipient cell nuclei, trans-activated the

CCN2/CTGF promoter, and induced CCN2/CTGF production in vitro [3].

promoter, and induced CCN2/CTGF production in vitro ^[8].

   The CRISPR/Cas9-mediated knockout of

The CRISPR/Cas9-mediated knockout of

Mmp3

gene significantly

reduced 3D-tumoroid formation

in vitro, reduced the levels of tetraspanins (CD9 and CD63) in EVs, and resulted in

destabilizing EV structural integrity

^[13]. Indeed, the

Mmp3

gene loss was associated with abnormal, disorganized shapes of EVs such as crescent moon-like and broken EVs ^[13]. MMP3-enriched EVs were highly

penetrative

and transferred deeply into the recipient MMP3-null tumoroids ^[13]. The addition of MMP3-rich EVs fostered the tumorigenicity and increased the proliferation of MMP3-null cells as judged by the highly significant increase in Ki-67 expression index. Thus, MMP3-rich EVs were highly

transmissive and pro-tumorigenic

in vitro ^[13].

5. Roles of MMPs in cancers

     MMPs represent the most prominent family of proteinases involved in

MMPs represent the most prominent family of proteinases involved in

tumor progression

and are regulators of the

tumor microenvironment [14,15]. MMPs have also been reported to be potent biomarkers of tumor progression as well constituting some of the causal factors that promote multiple processes of tumorigenesis, including oxidative stress-dependent DNA damage and chromosomal instability,

^[14][15]. MMPs have also been reported to be potent biomarkers of tumor progression as well constituting some of the causal factors that promote multiple processes of tumorigenesis, including oxidative stress-dependent DNA damage and chromosomal instability,

epithelial-to-mesenchymal transition (EMT)

^[16], migration and invasion of cancer cells ^[17],

angiogenesis

, and

metastasis [14,15,18]. MMP3-induced EMT and genomic instability are mediated by the

^[14][15][18]. MMP3-induced EMT and genomic instability are mediated by the

small GTPase Rac1b

and a

reactive oxygen species (ROS) in breast adenocarcinoma and pancreatic cancer [16,19], indicating potent roles for MMPs in proteotoxic and genotoxic stress.

in breast adenocarcinoma and pancreatic cancer ^[16][19], indicating potent roles for MMPs in proteotoxic and genotoxic stress.

     MMPs appear to be appropriate target molecules in treatments of aggressive types of cancers. Although more than 50 types of MMP inhibitors have been investigated in clinical trials for various cancers, all of those trials have so far failed [18]. The involvement of

MMPs appear to be appropriate target molecules in treatments of aggressive types of cancers. Although more than 50 types of MMP inhibitors have been investigated in clinical trials for various cancers, all of those trials have so far failed ^[18]. The involvement of

intracellular and non-proteolytic roles for MMPs

in cancer has, however, not been well-investigated yet.

      High expression of

High expression of

MMP3

mRNA was

prognostically unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers [3]. The

in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers ^[8]. The

RNA interference (RNAi)

-mediated knockdown of MMP-3 and MMP-9

significantly inhibited tumor growth and metastasis

in the tumor allograft mouse model ^[20].

CRISPR/Cas9-based knockout of MMP3 revealed that MMP3 is essential for the integrities of tumors and their EVs [13].

6. References

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7. Prenzel, N.; Zwick, E.; Daub, H.; Leserer, M.; Abraham, R.; Wallasch, C.; Ullrich, A. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 1999, 402, 884-888, doi:10.1038/47260.
8. Eguchi, T.; Taha, E.A. Extracellular Vesicle-associated Moonlighting Proteins: Heat Shock Proteins and Metalloproteinases. In Heat Shock Proteins, Asea, A.A.A., Kaur, P., Eds. Springer, Dordrecht: 2020; 10.1007/7515_2020_25pp. 1-18.
9. Eguchi, T.; Kubota, S.; Kawata, K.; Mukudai, Y.; Ohgawara, T.; Miyazono, K.; Nakao, K.; Kondo, S.; Takigawa, M. Different transcriptional strategies for ccn2/ctgf gene induction between human chondrocytic and breast cancer cell lines. Biochimie 2007, 89, 278-288, doi:10.1016/j.biochi.2006.12.006.
10. Eguchi, T.; Calderwood, S.K.; Takigawa, M.; Kubota, S.; Kozaki, K. Intracellular MMP3 Promotes HSP Gene Expression in Collaboration With Chromobox Proteins. J Cell Biochem 2017, 118, 43-51, doi:10.1002/jcb.25607.
11. Yang, Y.; Candelario-Jalil, E.; Thompson, J.F.; Cuadrado, E.; Estrada, E.Y.; Rosell, A.; Montaner, J.; Rosenberg, G.A. Increased intranuclear matrix metalloproteinase activity in neurons interferes with oxidative DNA repair in focal cerebral ischemia. Journal of Neurochemistry 2010, 112, 134-149, doi:10.1111/j.1471-4159.2009.06433.x.
12. Shimoda, M.; Khokha, R. Metalloproteinases in extracellular vesicles. Biochimica et biophysica acta. Molecular cell research 2017, 1864, 1989-2000, doi:10.1016/j.bbamcr.2017.05.027.
13. Taha, E.A.; Sogawa, C.; Okusha, Y.; Kawai, H.; Oo, M.W.; Elseoudi, A.; Lu, Y.; Nagatsuka, H.; Kubota, S.; Satoh, A., et al. Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles. Cancers (Basel) 2020, 12, doi:10.3390/cancers12051260.
14. Kessenbrock, K.; Dijkgraaf, G.J.; Lawson, D.A.; Littlepage, L.E.; Shahi, P.; Pieper, U.; Werb, Z. A role for matrix metalloproteinases in regulating mammary stem cell function via the Wnt signaling pathway. Cell Stem Cell 2013, 13, 300-313, doi:10.1016/j.stem.2013.06.005.
15. Kessenbrock, K.; Plaks, V.; Werb, Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 2010, 141, 52-67, doi:10.1016/j.cell.2010.03.015.
16. Radisky, D.C.; Levy, D.D.; Littlepage, L.E.; Liu, H.; Nelson, C.M.; Fata, J.E.; Leake, D.; Godden, E.L.; Albertson, D.G.; Nieto, M.A., et al. Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature 2005, 436, 123-127, doi:10.1038/nature03688.
17. Sato H; Takino T; Okada Y; Cao J; Shinagawa A; Yamamoto E; Seiki, M. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 1994, 370, 61-65, doi:10.1038/370061a0.
18. Vandenbroucke, R.E.; Libert, C. Is there new hope for therapeutic matrix metalloproteinase inhibition? Nat Rev Drug Discov 2014, 13, 904-927, doi:10.1038/nrd4390.
19. Mehner, C.; Miller, E.; Khauv, D.; Nassar, A.; Oberg, A.L.; Bamlet, W.R.; Zhang, L.; Waldmann, J.; Radisky, E.S.; Crawford, H.C., et al. Tumor cell-derived MMP3 orchestrates Rac1b and tissue alterations that promote pancreatic adenocarcinoma. Mol Cancer Res 2014, 12, 1430-1439, doi:10.1158/1541-7786.MCR-13-0557-T.
20. Okusha, Y.; Eguchi, T.; Sogawa, C.; Okui, T.; Nakano, K.; Okamoto, K.; Kozaki, K. The intranuclear PEX domain of MMP involves proliferation, migration, and metastasis of aggressive adenocarcinoma cells. J Cell Biochem 2018, 119, 7363-7376, doi:10.1002/jcb.27040.

-based knockout of MMP3 revealed that MMP3 is essential for the integrities of tumors and their EVs ^[13].