Matrix Metalloproteinases (MMPs)

Matrix metalloproteinases (MMPs) are a family of Zinc-dependent endopeptidase that degrade various proteins of the extracellular matrix (ECM). As members of the metzincin group of proteases, they share the conserved zinc-binding motif in their catalytic active site. Initially, MMPs were thought to function mainly in degrading ECM, but recent studies has shown that they also function significantly as regulators of extracellular tissue signaling networks.

MMPs are defined by the presence of two conserved zinc-binding motifs. One motif is a cysteine-containing pro-domain, whose function is partly to restrain catalytic; whereas the other motif is a histidine-rich catalytic domain, responsible for the endopeptidase activity.

MMPs Are Involved in Various Physiological and Pathological Processes.

MMPs are involved in numerous physiological and pathological processes. In physiological process, MMPs are involved in embryonic development, wound repair, ovulation, bone remodeling, macrophage function and in neutrophil function. They are also involved in pathological processes such as inflammation, tumor metastasis, rheumatoid arthritis, gastric ulcer, among others.

 Matrix Metalloproteinases and EMT in Tumor Metastasis

Epithelia-mesenchymal transition (EMT) plays its central role in normal embryonic development. But recent studies have shown its roles in pathological processes, such as cancer progression, fibrosis, and chronic inflammation.

MMPs associate with EMT in cancer progression through three mechanisms:

  • Elevated levels of MMPs in the tumor microenvironment induce EMT in epithelia cells.
  • EMT in cancer then produces more MMPs, thereby facilitating cell invasion and metastasis.
  • EMT can generate activated stromal-like cells that drive cancer progression through further MMPs production.

Role of MMPs in Tumor Metastasis

The role of MMPs in tumor metastasis was initially believed to be limited to the degradation of ECM and basement membrane collagen. But now, we know that MMPs paly critical roles at every step of tumor progression. MMPs influence several biological functions, such as modification of signaling pathways, regulation of cytokines involved in immune responses, and tumor growth by stimulating angiogenesis, which leads to the spread of cancer.

MMP-11 Plays Dual Roles in Tumors

Unlike many members of MMPs, MMP-8 and MMP-12 were reported to exert antitumor effects, thereby suppressing tumor growth. MMP-11 on the other hand plays a dual role in cancer progression. In one hand, MMP-11 promotes cancer development by inhibiting apoptosis and enhancing the invasion of cancer cells; on the other hand, however, MMP-11 plays a negative role against cancer development through the suppression of metastasis.

MMPs play Major Roles In Rheumatoid Arthritis

Rheumatoid arthritis (RA) is characterized by progressive joint destruction with loss of bone and cartilage as well as the aggressive activation of synovian fibroblasts (SFs) bearing a tumor-like appearance.

During joint destruction, RASFs secrete various proteases, including MMPs that degrade ECM
components, mainly proteoglycans and collagens, of articular cartilage in the affected joints.

However, among the various MMPs involved in articular degradation, MMP-1 and MMP-13 cleave collagens, whereas MMP-3 and MMP-9 target proteoglycans which are comprised of aggrecan.

This way, the degradation of proteoglycans at the surface and the subsequent degradation of collagen fibrils in the deep zone together result in the destruction of articular cartilage.

MMPs Play Major Roles In Infectious Diseases

In a normal immune responses, when the host immune system is challenged by an invading
organism, it must first recruit leucocytes to the site of infection, eradicate the pathogen and then dampen the response to allow the resolution of inflammation.

Matrix metalloproteinases play an important role in this process both by degrading components of the extracellular matrix and by modulating cytokine and chemokine activity.

 

REFERENCES

Hook D, Keller R. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech Dev. 2003;120(11):1351–83.

Cheng S, Lovett DH. Gelatinase A (MMP-2) is necessary and sufficient for renal tubular cell epithelial-mesenchymal transformation. Am J Pathol. 2003;162(6):1937–49.

Lopez-Otin C., Matrisian L.M. Emerging roles of proteases in tumour suppression. Nat. Rev. Cancer. 2007;7:800–808.

Zhang X., Huang S., Guo J., Zhou L., You L., Zhang T., Zhao Y. Insights into the distinct roles of MMP-11 in tumor biology and future therapeutics (Review) Int. J. Oncol. 2016;48:1783–1793.

Araki, Y., & Mimura, T. (2017). Matrix Metalloproteinase Gene Activation Resulting from Disordred Epigenetic Mechanisms in Rheumatoid Arthritis. International journal of molecular sciences, 18(5), 905. https://doi.org/10.3390/ijms18050905

Elkington, P. T., O’Kane, C. M., & Friedland, J. S. (2005). The paradox of matrix metalloproteinases in infectious disease. Clinical and experimental immunology142(1), 12–20. https://doi.org/10.1111/j.1365-2249.2005.02840.x

What is Angiogenesis?

Angiogenesis is the formation of new blood vessels. It is the process by which the body forms new blood vessels from existing ones. This process occurs throughout the life of an individual, starting in the uterus and continuing to old age. Angiogenesis occurs both in healthy tissues as well as in diseased ones, such as in cancer cell growths.

Why Does Angiogenesis Occur in the Body?

Angiogenesis takes place when a particular body part or tissue requires the supply of nutrients and to it. In hypoxia tissues, the need for oxygen supply to the parenchymal cells is detected by the oxygen sensing mechanisms, which then demands the formation of new blood vessels to meet this need.

Types of Angiogenesis

There are two types of angiogenesis, they are:

  1. Sprouting angiogenesis
  2. Intrussusceptive angiogenesis

Both sprouting and intrussusceptive angiogenesis, occur in the uterus and in adults.

Sprouting Angiogenesis (SA)

Sprouting angiogenesis is the process of growing new blood capillary vessels from pre-existing ones. When this occurs, the new blood vessels shall provide oxygen to expanding tissues and organs.

Sprouting angiogenesis plays important roles in many diseases, such as diabetes, rheumatoid arthritis, cardiovascular ischemic complications and cancer. In cancer, SA is involved not only in primary tumor but also in metastasis formation and further outgrowth of metastasis.

Steps in Sprouting Angiogenesis

Sprouting angiogenesis involves several steps, which include:

  1. Initiation of growth factors responsible for angiogenesis by low oxygen tension, low pH and high lactate levels.
  2. Expression of a transcription factor, hypoxia-inducible factor (HIF), by endothelia cells, which regulates expression of vascular endothelia growth factor (VEGF) and stimulates angiogenesis.
  3. Binding of growth factors to their receptors on endothelia cells and activating them. This is followed by detachment of pericytes.
  4. Binding of VEGF to its receptors and inducing a signaling cascades which enables one endothelia cell to form a tip cell while adjacent cells form stalk cells.
  5. Tip cells express VEGFR, delta-like ligand-4 (DLL-4), and matrix metalloproteinases (MMPs). They form filopodia, which are slender protrusions of the plasma membrane containing parallel bundles of actin filaments.
  6. Rhoa, Racl and Cdc42, members of Rho small GTPases, regulate the formation of filopodia.
  7. Activation of VEGFR leads to the extension of filopodia and migration of the tip cells forward.
  8. Activated endothelia cells secrete proteases, which are essential for the degradation of basement membrane. They allow tip cells to escape from the parent vessels and allow formation of sprouts and guidance of sprouts through the extracellular matrix (ECM).

Intrussusceptive Angiogenesis (IA)

In intrussusceptive angiogenesis, also called splitting angiogenesis, the blood vessel wall extends into the lumen, thereby causing a single vessel to split into two. It is fast and efficient compared to SA. This is because IA only requires reorganization of the existing endothelia cells; it does not rely on endothelia proliferation and migration. Like SA, however, IA also occur throughout the life of an individual, but it plays prominent roles in vascular development in embryos, where growth is fast with limited resources.

Promoters of Angiogenesis

Angiogenesis is regulated by a balance between pro-angiogenic and anti-angiogenic factors. The promoters include, HIF-1, VEGF, FGF, PDGE, TGF-β and angiopoietin and proteases.

Hypoxia-induced Factor-1 (HIF-1)

This is the most potent inducer of the expression of genes such as those encoding for glycolytic enzymes, VEGF and erythropoietin. HIF-1 is upregulated in hypoxia tumor cells; it activates transcription of target genes by binding to Cis-actin enhancers, hypoxia response element (HRE) close to the promoters of those genes.

Vascular Endothelia Growth factor (VEGF)

VEGF functions in angiogenesis by inducing the expression of DLL-4 in tip cells. It promotes the migration of endothelia cells by inducing expression of intergrins. It also stimulates production of MMPs, plasminogen activator and proteolytic enzymes by endothelia cells, which in turn promote the degradation of ECM.

Fibroblast Growth Factor (FGF)

FGF promotes proteases production and upregulates VEGF expression by endothelia cells. It also stimulates endothelia cells proliferation and migration.

Inhibition of Angiogenesis by Thymoquinone

Thymoquinone exerts its inhibitory effects on VEGF, FGF, PIGF, PDGF, which are pro-angiogenic factors, by suppressing the Akt/ERK signaling pathway.

 

REFERENCES

Carmeliet, P., & Jain, R. K. (2000). Angiogenesis in cancer and other diseases. Nature, 407, 249–257.

Martin, A., Komada, M. R., & Sane, D. C. (2003). Abnormal angiogenesis in diabetes mellitus. Medicinal Research Reviews, 23, 117–145.

Koch, A. E. (2003). Angiogenesis as a target in rheumatoid arthritis. Annals of the Rheumatic Diseases, 62 Suppl 2, ii60–67.

Cao, Y., Hong, A., Schulten, H., & Post, M. J. (2005). Update on therapeutic neovascularization. Cardiovascular Research, 65, 639–648.

Carmeliet, P. (2005). Angiogenesis in life, disease and medicine. Nature, 438, 932–936.

Hanahan, D., & Weinberg, R. A. (2000). The hallmarks of cancer. Cell, 100, 57–70.

Kerbel, R. S. (2000). Tumor angiogenesis: Past, present and the near future. Carcinogenesis, 21, 505–515.

Yi T, Cho SG, Yi Z, Pang X, Rodriguez M, Wang Y, Sethi G, Aggarwal BB, Liu M. Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and extracellular signal-regulated kinase signaling pathways. Mol Cancer Ther. 2008 Jul;7(7):1789-96.

Nuclear Factor-Kappa B (NF-kB)

Nuclear factor-kappa B (NF-kB) is a transcription factor that plays critical functional roles in inflammation, immunity, cell proliferation, differentiation, and survival. It exists in an inactive state in the cytosol and can be stimulated by molecules such as TNFα, and other cell stressors.

NF-kB is found in almost all cellular cell types and is identified as a regulator of kB light chain expression in mature B and plasma cells.

Because NF-kB has the ability to influence the expression of numerous genes, its activity is tightly regulated at multiple levels. The primary mechanism for regulating NF-kB is through inhibitory IkB proteins and the IKK complex, which phosphorylates IkBs.

Nuclear Factor-kappa B (NF-kB) Activation

NF-kB activation is initiated by TNFα. When TNFα binds to TNF receptors and activate them, IkB kinase (IKK) is ultimately triggered, which leads to the phosphorylation of IkB. The phosphorylation of IkB results in ubiquitination and degradation of IkB. When this happens, the remaining NF-kB dimmer (e.g., p65/p50 or p50/p50 subunits) translocates to the nucleus, where it then binds to a DNA consensus sequence of target genes.

 

Nuclear Factor-kappa B (NF-kB) Structure-Function relationship

NF-kB is a multiple-gene family of proteins that can form stable homo- and heterodimeric complexes, which vary in their DNA binding specificity and transcriptional activation potential.

There are five proteins of the NF-kB family in mammalian cells; they are RelA (P65), C-Rel, RelB, and NF-kB1 (p50 and its precursor p105), and NF-kB2 (p52 and its precursor p100). NF-kB and Rel proteins share a highly conserved 300 amino acid N-terminal Rel homology domain (RHD), which is responsible for DNA binding, dimerization, and association with IkB inhibitory proteins.

The p50/p65 complex shows strong transcriptional activation, whereas p50/p50 and p52/p52 homodimers suppress transcription of NF-kB target genes.

 

Nuclear Factor-kappa B (NF-kB) Is Inhibited By IkB Family Proteins

NF-kB is inhibited by IkB family proteins through NF-kB/IkB complex formation. There are seven inhibitory protein members of IkB family. They are IkBα, IkBβ, IkBε, IkBϒ, Bcl3, NF-kB1 precursor and NFkB2 precursor.

The IkB family members have a common ankyrin repeat domain. They regulate the subcellular localization, and hence, the DNA binding and transcriptional activity of NF-kB proteins.

 

IkB Regulates NF-kB Translocation to the Nucleus.

NF-kB is localized in the cytoplasm in an NF-kB/IkB complex, which is inactive. The inactive NF-kB/IkB co2mplex is a result of masking of the nuclear localization signals (NLS) on the NF-kB subunits by the IkB proteins. Hence, the degradation of IkB would lead to unmasking of the NLS, allowing NF-kB to undergo translocation to the nucleus.

The IkB proteins show a preference for specific NF-kB/Rel complexes, which provides a means to regulate the activation of distinct Rel/NF-kB complexes.

After the binding and transcriptional activity of NF-kB on DNA, it induces the expression of IkBα, which enters the nucleus and remove NF-kB  from DNA by forming NF-kB/IkB complex with the released NF-kB. The complex is then expelled from the nucleus back to the cytoplasm as a result of potent nuclear export signals on IkB and p65.

 

IkB Degradation Is Mediated By Ubiquiti/Proteasome System

The activation of NF-kB is achieved through the signal induced proteolytic degradation of IkB. This degradation is initiated by the stimuli dependent phosphorylation of IkB at specific N-terminal residues (S32/S36 for IkBα, S19/S23 for IkBβ), and is mediated by the ubiquitin/proteasome system.

Phosphorylation of IkB however, is not enough to initiate degradation of IkB. Ubiquitination and subsequent degradation depends on the recognition of phosphorylated IkB by the β-TrCp, an F-box/WD containing component of the Skp1-cullin-F-box (SCF) class of E3 ubiquitin ligases.

 

IKKs Mediates The Phosphorylation Of IkB On Serine 32 and 36

Phosphorylation of IkBa on the Serine 32 and 36 is mediated by IkB kinases (IKKs), whose activity is induced by activators of the NF-kB pathway. IKK contains two subunits, IKK1 (IKKα) and IKK2 (IKKβ). It also contains a regulatory subunit, NEMO ( IKKy, IKKAPI, FIP3).

IKK1 and IKK2 are homologous kinases, and both contain an N-terminal kinase domain and a C-terminal region with two protein interaction motifs, a leucine zipper (LZ), and ahelix-loop-helix (HLH) motif.

The LZ domain is responsible for demerization of IKK1 and IKK2. It is also essential for IKK complex activity. The IKK1/2 complex associates with NEMO through a short interaction motif located at the C-terminus of either catalytic subunit. Short peptides derived from the interaction motif can be used to disrupt the IKK complex and prevent its activation. NEMO connects the IKK complex to upstream activators through its C-terminus, which contains a zinc finger motif. NEMO also undergoes stimulus dependent interaction with components of TNF receptor complex.

 

 

REFERENCES

 

Hacker H, Karin M. 2006. Regulation and function of IKK and IKK-related kinases. Sci STKE 2006: re13.

Harhaj EW, Maggirwar SB, Sun SC. 1996. Inhibition of p105 processing by NF-kB proteins in transiently transfected cells. Oncogene 12: 2385–2392.

Hatakeyama S, Kitagawa M, Nakayama K, Shirane M, Matsumoto M, Hattori K, Higashi H, Nakano H, Okumura K, Onoe K, et al. 1999. Ubiquitin-dependent degradation of IkB is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1. Proc Natl Acad Sci 96: 3859–3863.

Chen G , Cao P , Goeddel DV . TNF-induced recruitment and activation of the IKK complex require Cdc37 and Hsp90 . Mol Cell 2002 ; 9 : 401 – 10.

Hu Y , Baud V , Delhase M , et al . Abnormal morphogenesis but intact IKK activation in mice lacking the IKKalpha subunit of IkappaB kinase . Science 1999 ; 284 : 316 – 20 .

Li Q , Lu Q , Hwang JY , et al . IKK1-deficient mice exhibit abnormal development of skin and skeleton . Genes Dev 1999 ; 13 : 1322 – 8.

Li Q , Van Antwerp D , Mercurio F , et al . Severe liver degeneration in mice lacking the IkappaB kinase 2 gene . Science 1999 ; 284 : 321 – 5.

Takeda K , Takeuchi O , Tsujimura T , et al . Limb and skin abnormalities in mice lacking IKKalpha . Science 1999 ; 284 : 313 – 16.

Ghosh S , Karin M . Missing pieces in the NFkappaB puzzle . Cell 2002 ; 109 ( Suppl. ) : S81 – 96.

Yang J , Lin Y , Guo Z , et al . The essential role of MEKK3 in TNFinduced NFkappaB activation . Nat Immunol 2001 ; 2 : 620 – 4.

Li ZW , Chu W , Hu Y , et al . The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis . J Exp Med 1999 ; 189 : 1839 – 45.

 

 

 

 

 

CLC-3

 

CLC-3 is an intracellular chloride transport protein known to reside on the endosomes and synaptic vesicles, which plays a role in cancer cell proliferation. It is a member of the CLC gee family that encodes Cl channels and Cl/H+ exchangers, and is predominantly expressed in membranes of the endosomal system. It may also be present in synaptic vesicles and synaptic-like microvesicles (SLMVs).

CLC-3 assists in acidification of endosomes and other compartments by shunting the electrical current of the vessicular V-type H+-ATPase. Also, CLC-3 either changes the vesicular voltage or lead to luminal chloride accumulation, as directly shown for the lysosomal 2Cl/H+-exchanger, CLC-7.

 

CLC protein structure

CLC has three highly conserved Cl binding sites, which feature a partial positive charge formed by amino acid residues located in the N-terminal portion of specific alpha-helices. In the crystal structure, Cl could be found at three specific sites made up by these amino acids: (1) an internal site (Sint) in contact with the intracellular environment, (2) a central site (Scen) buried in the membrane bilayer, and (3) an external site (Sext) in contact with the extracellular solution.

Both Sint and Scen are occupied by Cl, whereas Sext is occupied by the negatively charged side-chain of a conserved glutamate (E148) named Gluext. In Scen, Cl ions are coordinated mainly by residues S107 and Y445 also called Sercen and Tyrcen, respectively. Following a mutation or protonation of E148, a Cl occurs in Sext, which renders CLC gating proton-dependency.

Mutation of this glutamate residue abolishes voltage and Chloride-dependent gating in CLC channels and uncouples Cl/H+ exchange, turning the proteins into passive chloride conductors.

 

CLC-3 plays a role in Glioblastoma multiforme

Glioblastoma multiforme, the major malignant glioma cell, expresses three members of the CLC family, CLC-2, CLC-3 and CLC-5, however, CLC-3 in particular is the a critical regulator of cell volume changes associated with cycle. There are evidences that CLC-3 is involved in neuronal excitability, proliferation and migration. It has been shown to have an important role in the invasiveness of human glioma cells as CLC-3 is abundantly expressed in the cytoplasmic membrane and in intracellular vesicles of glioma cells.

A knockdown of CLC-3 reduces resting outwardly-rectifying chloride current in glioma cells, suggesting that CLC-3 mediates resting chloride current in the plasma membrane and thus, may be involved in cell shrinkage during invasion.

 

Activation and inhibition of CLC-3 in glioma cells

CLC-3 activity is regulated through phosphorylation via CaMKII. Intracellular infusion of autoactivated CaMKII via patch pipette enhanced chloride currents3-fold. This regulation of CaMKII was however inhibited by autocamtide-2 related inhibitory peptide, a CaMKII-specific inhibitor.

 

GLIOBLASTOMA MULTIFORME (GBM)

 

Glioblastoma multiforme (GBM) is one of the most aggressive types of cancer. It exhibits rapid cellular growth and highly invasive behavior. GBM is the most common primary intracranial tumor with a very poor prognosis representing 57% of all gliomas.

GBM can be divided into Isocitrate dehydrogenase (IDH) wild type, which is clinically defined as primary or de novo glioblastoma and also corresponds to approximately 90% of GBM cases, and IDH mutant, corresponding to secondary glioblastoma that progressively develops from low-grade astrocytoma and frequently manifests in patients aged 40-50 years old. GBM remains and incurable disease with a median survival rate o 15 months.

 

Classification of Glioblastoma multiforme

Glioblastoma multiforme can be classified into four known subtypes with specific alteration in NF1 and PDGFRA/IDH1. The four types of GBM are Proneural, Neural, Classical and Mesenchymal subtypes.

 

Ethiology of Glioblastoma multiforme

Many genetic and environmental factors have been studied in glioblastoma multiforme, however, no risk factor that accounts for a large proportion of GBM has been identified. An exposure to high ionizing radiation is the only confirmed risk factor of GBM. It has also been reported that relatively low doses of radiation that are used to treat tinea Capitis and skin hemangioma in children have also been associated with relative risks for gliomas.

Furthermore, patients who received treatment for Acute lymphoid leukemia (ALL) are more prone to develop GBM, which could be a result of complications arising from the leukemia or the chemotherapeutic agents used to treat ALL. There is no concluding association between GBM and environmental factors such as smoking, dietary risks factors, cell phones or electromagnetic field. Few studies have however shown the possible role of ovarian steroid hormones in development of GBM.

 

Epidemiology of Glioblastoma Multiforme

Although GBM is a rare tumor with global incidence of less than 10 per 100,000 persons, its poor prognosis with survival rate of 14-15 months after diagnosis makes it a crucial public health issue. GBM accounts for 50% of all gliomas in all age groups. It is primarily diagnosed in adults with a median age but is rare among children.

The ratio of GBM incidence is higher in men as compared to women. It is also more prevalent in the western world than in developing nations. And blacks are less prone to GBM. GBM incidence is higher in other ethnic groups, including, Asians, Latinaos, and Whites.

 

Pathogenesis of Glioblastoma Multiforme

The most frequent location for GBM is the cerebral hemisphere, with 95% of these tumors arising in supratentorial region, while only a few percentage of tumors occur in cerebellum, brain stem, and spinal cord.

 

Genetic and molecular pathogenesis of Glioblastoma Multiforme

Based on clinical characteristic, GBM can be subdivided into primary and secondary GBMs. Primary GBM arise de novo without clinical and histological evidences of precursor lesion. In contrast, secondary GBMs progress slowly from preexisting lower-grade astrocytoma.

Primary GBM is marked by epidermal growth factor receptor (EGFR) gene mutation and amplification, over expression of mouse double minute 2 (MDM2), deletion of p16 and loss of heterozygosity (LOH) of chromosome 10q holding phosphatase and tensin homolog (PTEN) and TERT promoter mutation.

Genetic and molecular pathogenesis of secondary Glioblastoma Multiforme

The molecular pathogenesis of secondary GBM include over expression of platelet-derived growth factor A and platelet-derived growth factor receptor alpha (PDGFA/PDGFRα), retinoblastoma (RB), LOH of 19q and mutation of IDH1/2, TP53 and ATRX.

An assimilated analysis of the numerous genetic abrasions has shown that these genetic lesion are grouped into three main signaling pathways; they are (I) receptor tyrosine kinase (RAS/PI3K), (II) P53 pathway and (III) RB signaling pathway.

 

FIBROBLAST ACTIVATION PROTEIN-α (FAP)

Fibroblast activation protein-α (FAP) is an inducible cell surface glycoprotein originally identified in 1986 in cultured fibroblasts using the monoclonal antibody (mAb) F19. It is a type ii integral serine protease that is specifically expressed by activated fibroblast.

Cancer-associated fibroblasts (CAFs) in the tumor stroma have an abundant and stable expression of FAP, which plays an important role in promoting tumor growth, invasion, metastasis, and immunosupression. For example, in females with a high incidence of breast cancer, CAFs account for 50-70% of the cells in the tumor’s microenvironment.

CAF over expression of FAP promotes tumor development and metastasis by extracellular matrix remodeling, intracellular signaling, angiogenesis, epithelia-to-mesenchymal transition, and immunosuppression.

 

The structure of Fibroblast activation protein

Fibroblast activation protein-α (FAP) is a type ii transmembrane glycoprotein consisting of 760 amino acids. It belongs to the family of post-proline dipeptidyl aminopeptidase, known as dipeptidyl peptidase-4 activity (DPP4). At the genetic level, human FAP and dipeptidyl peptidase-4 (DPP4) genes share substantial homology. Human FAP gene is located on chromosome 2q23 and contain 26 exons (total length: 73kb) while DPP4 is located on chromosome 2q24.3 and contains 26 exons (total length: 70kb)

The FAP protein is a 170-kda homodimer with two N-terminal glycosylated subunits. The 97-kda type ii transmembrane serine protease is a member of the polylpeptidase family, which includes DPP-4 (most similar to FAP) DPP7, DPP8, DPP9 and prolyl carboxypeptidase. The FAP and DPP-4 proteins have a 70% identity at the amino acid sequence and share a catalytic triad of serine, aspartic acid, and histidine residues.

Serine plays a nucleophilic role, which allows DPP-4 to cleave N-terminal Pro-“X” peptide bond (where “X” is any amino acid except proline or hydroxylproline). Unlike FAP, DPP-4 is expressed in a variety of human tissues under normal conditions and is related to many physiological processes, including glucose homeostasis and T-cell activation.

 

Enzymatic activity of Fibroblast activation protein-α

The FAP protein has dipeptidyl peptidase and endopeptidase activities, which are sometimes described as gelatinase activity. Although both FAP and DPP-4 have dipeptidase activity, the unique endopeptidase activity of FAP makes it preferentially cleave to the Gly-pro”X” sequence, with the most effective cleavage when “X” is Phe or Met and the least effective one when “X” is His or Glu.

The cleavage activity of FAP can be impaired when P4 and P2 residues are heavily charged amino acids. Therefore, endopeptidase activity can be used to specifically detect FAP and are the basis of nanomaterial treatments that aim to specifically inhibit FAP.

Studies have shown that DPP-4 can cleave neuropeptide Y, peptide YY, Sp (substance P), and brain natriuretic peptide 32, which can also be cleaved by FAP. The known active substrates of endopeptidase include collagens I, ii, and v, as well as α-2-antiplasmin and fibroblast growth factor 2. The ability of FAP o cleave collagen depends on previous matrix metalloproteinase activity o thermal degradation.

Soluble FAP is known as α-2-antiplasmin cleaving enzymes (APCE), which has pro-coagulation poperties. After FAP cleaves α-2-antiplasmin, it is converted into a more effective plasmin inhibitor, which slows the dissolution of the fibrin clot and reduces bleeding during tissue repair.

 

Expression of Fibroblast activation-α in Tumors

Fibroblast activation protein-α level is generally undetectable in normal tissues. However, FAP is over expressed in many tumor tissues, including breast, pancreatic, lung, colorectal, brain, intrahepatic bile duct, and ovarian cancers. High level of FAP can also be detected in some tumors that are derived from non-epithelial tissues, such mas melanoma and myeloma. In these tumors, FAP over expression is typically observed in the intestitium, which has led to FAP being considered a universal marker for CAFs.

 

Roles of fibroblast activation protein-α in tumors

Fibroblast activation protein-α expression influences tumor growth by impacting tumor cell proliferation and invasion, angiogenesis, epithelia-to-mesenchymal transition, immunosuppression and drug resistance.

Fibroblast activation protein-α promotion of Tumor cell proliferation and invasion

Fibroblast activation protein promotes tumor cell proliferation, migration and invasion, which ultimately leads to tumor growth. There are two hypothesis regarding the underlying mechanisms. The first hypothesis involves an indirect mechanism, whereby FAP regulates extracellular matrix remodeling that leads to enhanced tumor growth and invasion. It remains unclear whether FAP regulates this extracellular matrix remodeling through its enzymatic or non-enzymatic activity.

The second hypothesis involves a direct mechanism, in which FAP expression influences signaling pathways that control the cell cycle and proliferation, which ultimately promote tumor growth.

In the indirect hypothesis, transfection of small interfering RNA targeting FAP inhibits the proliferation of ovarian CAFs, leading to cell cycle arrest. In squamous lung cancer cell, FAP overexpresion promotes proliferation, migration and invasion, accompanied by upregulaton of the PI3k/protein kinase B and sonic hedgehog/glioma-associated oncogene signaling pathways. Other studies on oral squamous cell carcinoma have indicated that FAP is an upstream regulation of phosphatase and tensin homolog, PI3k/protein kinase B and Ras-ERK signaling pathways.

Pro-tumorigenic activity of fibroblast activation protein

Fibroblast activation protein as a tumor suppressor

Studies suggest that FAP has tumor suppressive activity and show that this activity is independent of its enzymatic activities. Elevated expression of Fibroblast activation protein in cancer causes dramatic promotion or suppression of tumor growth, depending on the model system investigated. There is however obvious discrepancy between FAP function in tumor promotion and tumor suppression. Some researchers propose that the factor that determines this must reside in the signaling molecules that are available for interaction with FAP on the cell.