Figure 1. Rho signaling pathway
The Rho family of proteins is the first cloned protein in the Ras superfamily. They are a group of guanosine triphosphate (GTP) binding proteins with a relative molecular mass of about 2025 KD, which have GTPase activity and are called the "GTP enzymes". The Rho GTPase plays an important role in the regulation of cytoskeletal reorganization. In recent years, studies have found that Rho GTPase is highly expressed in a variety of malignant tumors and is closely related to tumorigenesis, invasion and metastasis.
So far, the Rho GTPase superfamily has found about 20 members (Figure 2), which are roughly divided into five subfamilies based on their structure and function, including: (1) Rho subfamily, including RhoA, RhoB and RhoC, which are highly sequenced homologous and highly expressed in a variety of cells, and are mainly involved in the formation of tensile fibers and focal adhesion complexes (FACs); (2) Rac subfamily, including Rac1, Rac2, Rac3 and RhoG, the promoting layer Pseudopodia and membrane fold formation; (3) Cdc42 subfamily, including Cdc42, TC10, TCL, Wrch1 and chp/Wrch2, in which Cdc42 promotes filopodia formation; (4) Rnd subfamily, including Rnd1, Rnd3 /RhoE and Rnd2, which are constitutively activated in cells and have different tissue distributions that antagonize the Rho signaling pathway; (5) Rho BTB subfamily, including Rho BTB1 and Rho BTB2, and the specific function is unclear. Among all Rho GTPase superfamily members, Cdc42, Rac1 and RhoA are the most studied Rho GTPases. Each member of the Rho family has 50% to 55% homology in the amino acid sequence, and a functional region capable of binding to GTP near the catalytic site is closely related to the catalytic GTP hydrolysis. Like the other members of the Ras superfamily, the carboxy terminus of Rho GTPase usually has a common domain—a terminus consisting of a cysteine residue, an aliphatic residue and other amino acid residues, which is the site of post-translational modification. The post-translational modification of Rho GTPase is related to its plasma membrane localization. Only the post-translationally modified Rho GTPase is active and binds to appropriate lipid molecules on the cell membrane. Under the action of prenyltransferase, the sulfhydryl group and the isoprene group of the cysteine covalently form a thioether bond, and under the action of the endonuclease, the remaining 3 residues at the end are hydrolyzed, and finally the prenylated cysteine residue is methylated by the action of a methyltransferase to complete post-translational modification. The Rho family of proteins, like all members of the Ras superfamily, circulate between the active/GTP-restricted and inactive/guanosine diphosphate (GDP) restricted conformations.
Figure 2. Phylogenetic tree of the Rho-family GTPases and representatives of other Ras-superfamily GTPases.
Rho signaling pathway
We introduce the Rho signaling pathway cascade by the signal pathway used in the cancer mediation. The movement of tumor cells in the matrix consists of four cycles of reciprocating steps, namely the formation and extension of the pseudopods of the head, the establishment of new adhesion sites, the contraction of the cell bodies and the retraction of the tails, the four repeated processes. The molecular mechanisms that precisely regulate this process are complex and involve multiple intracellular signaling pathways. Among the multiple signal cascade pathways, Rho GTPase, especially RhoA, Rac1 and Cdc42, are key regulators involved in cell morphology, cell-matrix adhesion and cytoskeletal reorganization, regulating tumor cell invasion. Wiskott Aldrich syndrome protein (WASP) is a key molecule regulating cell migration, including neural tissue source WASP, WASP family rich in proline homologous protein 1, WASP family verprolin homologous protein1 (WAVE1), WASP family verprolin homologous protein 2 (WAVE2) and other members. It is also an important effector downstream of Rac and Cdc42, and is highly expressed in tumor cells. Rac and Cdc42 pseudopod formation and matrix degradation are induced by activation of WASP family members. For the first time, Lorenz et al. used a fluorescence resonance energy sensor to distinguish between active and inactive NWASP conformations and simulated endogenous NWASP function discovery. NWASP plays an important role in the formation of stratified pseudopods in the migrating tumor cells. The highly dynamic pseudopod structure of the cell migration head is dependent on the polymerization of actin monomers and the elongation of actin fibers. Different members of the WASP family are activated by binding of different domains to Rac and Cdc42, whereas actin-associated 2 directly regulate the polymerization of actin monomers by binding to the common domain of the carboxy terminus of the activated WASP family, respectively. The Arp2/3 complex is the core of actin assembly, which can synthesize actin monomers from the head into actin filaments, which promotes the formation of filopodia and lamellar pseudopods. The pseudopod forms a process of initiating cell migration, but the continuous migration of the cell depends on the stable adhesion of the cell pseudopod to the extracellular matrix (ECM), providing a traction fulcrum for the cell to migrate forward. The adhesion of the migrated cell heads to the ECM and the constant de-adhesion of the tail to the ECM causes the cells to migrate forward, and the Rho GTPase exerts precise regulation of this process. The integrin receptor on the cell surface binds to a specific ligand in the ECM, and aggregates into a cluster to form FACs, while the intracellular region of the integrin receptor is associated with paxillin, vinculin and prion protein. A variety of actin-binding proteins, such as talin, interact to form a molecular bridge and are linked to the cytoskeleton to provide anchor sites for cell migration. Activated Rac can induce the polymerization of actin and the formation of lamellar pseudopods and induce the formation of new FACs, which in turn can activate Rac. This loss of positive feedback can increase the invasive ability of tumor cells. Jung found that activated Rac1 and Cdc42 can activate the focal adhesion kinase (FAK) downstream of PAK1 phosphorylation and activated FAK as a molecular scaffold to recruit cytoplasmic paxillin, globulin and prion protein to FACs and promote the formation of FACs. The p65-activated kinase also indirectly regulates the activity of cofilin via LIMK. The circulation of cofilin between active and inactive forms regulates the dissociation and polymerization of actin subunits from the ends of actin filaments and promotes actin fibers. The occurrence of treadmilling is very necessary during assembly. Tumor cell invasion and metastasis are closely related to the degradation of ECM. Rho GTPase can directly or indirectly regulate downstream effectors to promote ECM degradation. The study of human breast cancer cell line MDAMB435 revealed that Rac1 and Cdc42 can up-regulate LIMK1 to up-regulate the serine protease urokinase type plasminogen activator (uPA) system, increase uPA promoter activity, induce uPA and uPA receptor mRNA and protein expression and uPA secretion, degradation of ECM collagen and other components, and contribute to cell invasion and metastasis. The sustained movement of invading and metastatic tumor cells relies on the contraction of tonic fibers and the elongation of actin filaments, which regulate cell cytoskeletal reorganization and provide power for cell migration. Tensile fiber is a stable, parallel-arranged microfilament structure in eukaryotic cells. It is composed of actin, myosin, tropomyosin, etc. The contractile force generated by the relative motion of myosin is cell migration power. Rho and its downstream Rho-associated forming protein kinase (ROCK) increase the phosphorylation level of myosin light chain (MLC) and increase the contraction of myosin-myosin. ROCK is an important effector molecule downstream of Rho, including Rho kinase and p160 ROCK 2 members. Activated ROCK enhances the phosphorylation level of MLC through two pathways. On the one hand, ROCK phosphorylates the myosin binding subunit (MBS) of its substrate myosin light chain phosphatase (MLCP), inhibits phosphatase activity of MLCP and reduces hydrolysis of MLC phosphate groups; on the other hand, ROCK directly phosphorylates MLC, and increases the phosphorylation level of MLC, thereby increasing the contractile force produced by cross-linking with actin filaments. The inhibition of Rho/Rock pathway inhibits contraction and cell invasion of tumor cell tension fibers, and plasmid-transfected human ovarian cancer cells with dominant active p160 ROCK have stronger invasion and migration ability. Rho can also act on another important downstream effector molecule, mDia (Mammalian Diaphanous Related Protein). The activated mDia protein can bind actin monomer to the end of actin filament and prevent the binding of cap protein, and induce muscle movement. Prolongation of the protein filaments contributes to cell migration.
Nowadays, the regulation of Rho signaling pathway is widely used in the development of anticancer drugs, so the development of anticancer targeted drugs against Rho protein family members and their regulatory proteins can be started from the following aspects. (1) The use of specific Rho small G protein and its regulatory protein and downstream effector protein inhibitor; (2) small molecule RNA interference technology; (3) Rho small G protein translation of prenylation modification inhibitor. Since RhoA, Cdc42, and Rac are involved in many cellular functions, direct inhibition of their function leads to significant cytotoxicity. Therefore, inhibition of the regulatory protein GEF and downstream effector proteins by small molecule inhibitors has practical application value. Most of the Rho protein downstream kinase inhibitors have been used to date, and such inhibitors can compete with kinase proteins for ATP to block kinase activity. The most commonly used chemical inhibitors are small molecules that inhibit ROCK and PAK downstream of the Rho signaling pathway, such as Y27632, Y32885, HA1077, and H1152P, which inhibit ROCK activity; PAK4 inhibitor PF-3758309 inhibits cells anchor growth and proliferation, and induces apoptosis and cytoskeletal rearrangement; chemical inhibitor SP600125 can indirectly attenuate the effect of PAK1 on colorectal cancer proliferation; allosteric inhibitor IPA-3 can block the PAK1 autoinhibitory domain. The inactivation and autophosphorylation of activated monomers inhibits PAK1 activity, and this method of inhibiting the self-regulating domain of kinases can provide higher specificity than methods that compete with kinase proteins for ATP binding, thus reducing the drug. There are also some inhibitors that prevent the activation of the latter by inhibiting the binding of GTP or GEF to the Rho protein. For example, ITX3 can inhibit the activity of the N-terminal domain of the Rho GEF protein Trio, thereby inhibiting its activation of Rac1 and RhoG. MLS000532223 is a compound that can inhibit the binding of GTP to Rho protein by high-throughput drug screening. It can inhibit the binding of GTP to Rho protein in a dose-dependent manner. It is a wide spectral range of Rho protein inhibitor. Most proteins of the Rho protein family contain a CAAX motif at the C-terminus (C: cysteine; A: any aliphatic amino acid; X: any amino acid), and the CAAX motif undergoes prenylation after translation. On the endoplasmic reticulum, the AAX moiety is cleaved by RAS-converting enzyme-1 (RCE1), and the carbon group of cysteine is methylated. After translation, the modified Rho protein localizes to the cell membrane and undergoes upstream stimulation to activate normal physiological functions. Therefore, the farnesyl transferase and geranylgeranyl transferase involved in post-translational modification are potential anti-cancer therapeutic targets. Inhibitors of farnesyl transferase and bovine transferase competitively bind to CAAX substrates, or isoprenoid pyrophosphate donors, some of which have entered clinical trials, such as Zarnestra® (Tipifarnib/R115777; Phase III), Sarasar® (Lonafarnib/SCH66336; Phase II), L-778 123 (Phase II) and BMS-214662 (Phase I). Prenylation of the Rho protein can also be inhibited by mevalonate inhibitors, such as statins or diphosphates.
As mentioned above, the Rho signaling pathway plays an important role in the occurrence, expansion and metastasis of cancer, and it is also expected to treat cancer by developing inhibitors of the Rho signaling pathway.
Coronary heart disease
In recent years, research on the mechanism of Rho/Rho kinase pathway in AS has become a hot topic. In-depth research is helpful for the prevention and treatment of coronary heart disease; and it may be of great significance to prevent restenosis after percutaneous coronary intervention. In addition, Rho kinase inhibitors are expected to be the preferred anti-ischemic drugs in patients who are intolerant to nitrates, but the safety and efficacy of long-term use of such drugs still require large clinical trials.