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BK21PLUS R-FIND team, College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea
Metastasis is one of hallmarks of cancer and a major cause of cancer death. Combatting metastasis is highly challenging. To overcome these difficulties, researchers have focused on physical properties of metastatic cancer cells. Metastatic cancer cells from patients are softer than benign cancer or normal cells. Changes of viscoelasticity of cancer cells are related to the keratin network. Unexpectedly, keratin network is dynamic and regulation of keratin network is important to the metastasis of cancer. Keratin is composed of heteropolymer of type I and II. Keratin connects from the plasma membrane to nucleus. Several proteins including kinases, and protein phosphatases bind to keratin intermediate filaments. Several endogenous compounds or toxic compounds induce phosphorylation and reorganization of keratin network in cancer cells, leading to increased migration. Continuous phosphorylation of keratin results in loss of keratin, which is one of the features of epithelial mesenchymal transition (EMT). Therefore, several proteins involved in phosphorylation and reorganization of keratin also have a role in EMT. It is likely that compounds controlling phosphorylation and reorganization of keratin are potential candidates for combating EMT and metastasis.
Metastasis is critical hallmark of cancer and contributes to the 90% of cancer death (Hanahan and Weinberg, 2011). Diverse approaches have been attempted to combat the metastasis of cancer. The spot light has been on matrix metalloproteinase inhibitors but the clinical outcome of matrix metalloproteinase inhibitors in most cancer metastasis is poor (Coussens
Recently, several researchers investigated physical properties of cancer cells and found that metastatic cancer cells are significantly softer than other benign or normal cells (Cross
Because the physical properties and mechanotransduction of cancer cells are crucial in various steps of the metastatic process, control of physical properties of cancer cell may be an effective therapeutic approach for patients suffering cancer (Stroka and Konstantopoulos, 2014).
However, measuring changes of physical properties of cancer cells is not easy to most researchers in pharmacology fields. We are interested in the biological phenomena reflecting the changes of physical properties such as keratin reorganization via phosphorylation, which is changed by sphingosylphosphorylcholine (SPC) and related to viscoelasticity of metastatic cancer cells (Beil
Epithelial cell keratins are composed of heteropolymer of one type I keratin and one type II keratin proteins (Table 1) (Coulombe and Omary, 2002). Keratin contains a common α-helical rod domain of ∼310 amino acid, sided by non-helical head and tail domains of diverse length and sequence having several phosphorylation sites (Ku
Simple epithelia of liver, intestine, and pancreas, are discovered as pairs of K7, K8, K18, K19, and K20, but the ratio of type I and type II keratins is 1:1 in all cells (Moll
Several situations including diverse stress requires the changes of keratins (Leube
In the epithelia tissues, a network of proteins links the nucleus to membrane of cell through keratin filaments, in which transmembrane proteins gives the ground for cell to cell and cell to extracellular matrix adhesion (Fig. 4) (Omary
Keratin is connected to desmosome in the cell to cell adhesion site through desmoplakin (Green and Simpson, 2007). The cadherin family, the desmogleins and desmocollins, join the adhesion point (Getsios
Hemidesmosomes are junction complexes contributing to the adherence of epithelial cells to the basal layer (Borradori and Sonnenberg, 1999). The molecular structure of hemidesmosome is composed of 3 kinds of proteins: the cytoplasmic linker proteins for intermediate filaments at the cytoplasmic leaflet of the plasma membrane, the transmemebrane proteins acting as receptors linking the inside of cell to the proteins of the basal layers (Borradori and Sonnenberg, 1999). Keratin is linked to plectin and BPAG1e at hemidesmosome cell-matrix adhesions (Guo
Lamins underlie the inner face of nuclear membrane and also make stable structures within the nucleus interior which contains emerin, lamin B-receptor, and SUN (Sad1 and UNC84 domain containing) 1/2 (Friedl
Nesprins are a core member of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex that cross over both nuclear membranes to link the cytoplasm and the inside of nucleus (Neumann and Noegel, 2014).
Nesprins interact with SUN proteins through perinuclear space via their KASH (Klarsicht, ANC-1, Syne Homology) domain and directly link to actin filaments (nesprin-1 and -2) and keratins via plectin (nesprin-3) (Padmakumar
Keratin particles emerge from the vicinity of the plasma membrane, maneuver continuously toward the central part of cell, and consolidate into the peripheral keratin network (Kolsch
Keratin particles also moves fast via microtubules (Liovic
Spectroplakins are big cytoskeletal linking proteins that bind to all 3 members of the cytoskeleton such as actin filaments, microtubules, and intermediate filaments (Suozzi
A wide range of post-translational modifications have been reported on keratins such as phosphorylation, ubiquitylation, acetylation, glycosylation, and, sumoylation, which seem to control the solubility of keratins in several situations (Omary
Phosphorylation is a key reaction of keratins, and K1, K8, K18, and K19 are the fully studied among keratin family (Steinert, 1988; Zhou
Numerous kinases are involved in phosphorylation of keratins (Snider and Omary, 2014). Phosphorylation of serine residue of keratin leads to disintegration of the stable structure and increased solubility of keratin in the cytoplasm (Omary
ERK is one of the kinases involved in SPC or leukotriene B4 (LTB4)- evoked K8 phosphorylation and reorganization (Fig. 5, Table 2) (Busch
Serine-73 (Ser-73) of K8 is a residue of phosphorylation by c-Jun N-terminal kinase (JNK) (Fig. 5, Table 2). Furthermore, we found that JNK phosphorylates serine-431 (Ser-431) in SPC-induced phosphorylation and reorganization of K8 (He
p38 mitogen activated protein kinase (MAPK) is also involved in phosphorylation of Ser-73 induced by treatment with okadaic acid or orthovanadate (Ku
cAMP-dependent protein kinase (PKA) and Ca2+- dependent protein kinase C (PKC) almost exclusively phosphorylates serine of K8 (Fig. 5, Table 2) (Yano
Calmodulin-dependent protein kinase II (CAMK II) phosphorylates K8 at serine and threonine amino acids (Yano
Predicted phosphorylation sites for Akt exist in several keratins and Akt binds K8 but not K18 (Fig. 5, Table 2) (Paramio
K17, a type I keratin, is heavily induced in epidermis after injury, and in psoriasis and cancer (Pan
Casein kinase Iα (CK-Iα) plays an essential role in the phosphorylation and degradation of β-catenin (Knippschild
Ser-35 of K19, which is a type I keratin, is a well-known residue of phosphorylation (Zhou
The AMP-activated protein kinase (AMPK) is important in the biological response induced by metabolic changes and is turned on by AMP (Velasco
US3 is a specific serine/threonine protein kinase found in herpes simplex virus (Murata
Several kinases are reportedly implicated in the SPC-induced phosphorylation of K8. For example, ERK and JNK are involved in SPC-induced K8 phosphorylation (Park
Phosphatase of regenerating liver-3 (PRL-3) belongs to the PRL protein tyrosine phosphatase family and highly PRL-3 expressed cancer cells demonstrate reduction of K8 phosphorylation, especially at the front of invasion and metastasis to liver (Fig. 5) (Mizuuchi
Pharmacological inhibition of the protein-tyrosine phosphatase PTP1B increases phosphorylation of Tyr-267 of K8, decreases solubility, and increases K8 filament bundling, whereas PTP1B overexpression has the opposite effects (Fig. 5) (Snider
It seems that effects on K8 structure and stability by phosphorylation of serine differ from those of tyrosine phosphorylation. Further study is needed to elucidate the role of different phosphorylated keratins on structure and reorganization.
High-risk human papillomaviruses (HPV) such as HPV16, are the major cause of cervical cancer and one of HPV16 proteins, E1–E4 binds to keratins leading to keratin network disorganization (Fig. 5) (Wang
Death effector domain with DNA binding protein (DEDD), is present mostly as mono- or diubiquitinated form, and diubiquitinated DEDD bind to the K8 and K18 (Fig. 5) (Lee
Association of small heat shock proteins (HSP) with intermediate filament including keratins, may regulate filament interactions in cellular networks. For example, the chaperone HSP27 affects assembly dynamics and organization of K8 and K18 cytoskeleton through direct keratin interactions (Fig. 5) (Perng
14-3-3 protein binds to several kinases of signal transduction (Liao and Omary, 1996). 14-3-3 proteins also interact with phosphorylated form of keratin in simple epithelia during the course of cell cycle and plays a role of cofactor for solubilization of keratins (Liao and Omary, 1996). Ser-33 phosphorylation of K18 influences binding of K18 to 14-3-3 proteins in the course of mitosis and interaction of K18 with 14-3-3 proteins regulates keratin filaments and mitotic progression of hepatic cells (Fig. 5) (Ku
Epidermal growth factor (EGF) leads to phosphorylation of keratin in rat hepatocyte before keratin reorganization (Fig. 5) (Baribault
Interleukin-6 (IL-6) significantly up-regulates K8 and K18 in intestinal epithelial cells such as Caco2-BBE (brush border expressing) cell line and IL-6 evoked K8 phosphorylation at serine residue (Fig. 5) (Wang
K17, the myoepithelial keratin, is expressed in psoriasis but is not present in healthy skin (Komine
Exposure of the hepatocytes to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (150 nM), a typical activator of protein kinase C, leads to phosphorylation of K8 but not K18 (Cadrin
Treatment of several human breast cancer cells including MCF7, T47D, SKBR3 with vitamin K3 (50–100 μM) leads to K8 phosphorylation at Ser-73 via MEK (MAPK/ERK kinase) 1/2 signaling (Fig. 6) (Scott
Acrolein is a primary mediator of pulmonary edema and induces phosphorylation of K8 at Ser-73 in bronchiolar lung epithelia (Fig. 6) (Burcham
Griseofulvin induces Mallory-Denk bodies in hepatocytes of mice (Fortier
Pervanadate, tyrosine phosphatase inhibitor, induces phosphorylation of tyrosine residue in K8, and K19, but not K18 via p38 MAP kinase (Feng
Withaferin A (WFA) binds to the vimentin and modifies perinuclear aggregates of intermediates filaments including keratin (Grin
Compressive loads induce K8 phosphorylation in human disc generation by activating protein kinase C (Sun
Several reports support an active role of keratins as versatile regulators in carcinogenesis (Karantza, 2011). However, roles of phosphorylation of keratin in carcinogenesis and metastasis are controversial. For example, loss of K8 Ser-73 and Ser-431 phosphorylation is also observed in human oral squamous cell carcinoma (OSCC) tissues evaluated by immunohistochemistry, in which dephosphorylation greatly associated with size, and progression of the tumor (Alam
On the other hand, loss of K8 and K18 leads to increased collective emigration and invasiveness of breast cancer cells (Fortier
The probable differences in results might be by use of different kinds of cells and methods (Windoffer
Epithelial-mesencymal transition (EMT) is an important event that permit a polarized epithelial cell, to experience numerous biochemical conversions to deduce a mesenchymal phenotype of cell including increased migration, invasiveness, and significantly elevated resistance to apoptosis (Kalluri and Neilson, 2003).
Loss of keratin by phosphorylation is one of hallmarks in EMT (Kalluri and Weinberg, 2009). Therefore it is plausible that players implicated in perinuclear reorganization of keratin by phosphorylation are also involved in EMT. Accordingly, Tgase-2 involved in SPC or TPA-induced K8 phosphorylation and reorganization, is also implicated in TGF-β1-induced EMT (Park
Several phosphatases involved in dephosphorylation of keratins are also implicated in process of EMT. PRL-3 or PTP1B involved in keratin dephosphorylation also induced EMT (Wang
Modulation of keratin phosphorylation and reorganization is potential new way for controlling EMT and metastasis of cancer (Beil
We also developed SPC blocker based on structure of SPC. Several compounds derived from SPC, suppressed SPC-induced K8 phosphorylation, reorganization and migration (Lee
However, additional inducers released from tumor microenvironment that affect keratin phosphorylation and reorganization have not been identified. If several factors are released from tumor microenvironment and induced keratin phosphorylation and reorganization, blocking the common pathway would be an optimal strategy. Hence PP2A activator or inducers also might be good candidate for controlling keratin reorganization by dephosphorylating the phosphor serine residue of keratins or phosphorylated kinases (active forms) involved in phosphorylation of keratins.
Metastatic cancer cell is much softer than non-metastatic cancer cells (Cross
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