Targeting Rho GTPase Signaling Networks in Cancer

Abstract

As key regulators of cytoskeletal dynamics, Rho GTPases coordinate a wide range of cellular processes, including cell polarity, cell migration, and cell cycle progression. The adoption of a pro-migratory phenotype enables cancer cells to invade the stroma surrounding the primary tumor and move toward and enter blood or lymphatic vessels. Targeting these early events could reduce the progression to metastatic disease, the leading cause of cancer-related deaths. Rho GTPases play a key role in the formation of dynamic actin-rich membrane protrusions and the turnover of cell-cell and cell-extracellular matrix adhesions required for efficient cancer cell invasion. Here, we discuss the roles of Rho GTPases in cancer, their validation as therapeutic targets and the challenges of developing clinically viable Rho GTPase inhibitors. We review other therapeutic targets in the wider Rho GTPase signaling network and focus on the four best characterized effector families: p21-activated kinases (PAKs), Rho-associated protein kinases (ROCKs), atypical protein kinase Cs (aPKCs), and myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs).

Keywords: GTPase; Rho; cancer; invasion; metastasis.

FIGURE 1

(A) Stages of cancer metastasis. A subset of cancer cells in the primary tumor acquire an invasive phenotype and spread into the surrounding stroma, either collectively or as single cells. Some invading cells migrate toward the tumor neovasculature and enter the blood stream by migrating through vascular endothelial cell junctions in a process known as intravasation. These cancer cells can be transported by the circulation to distal tissues, where they enter narrower vessels that permit their attachment to vascular endothelial cells. Following attachment, cancer cells commonly extravasate as single cells by migrating through endothelial cell junctions and then invade into the stroma of the secondary organ. These cells may form a metastatic niche if supported by survival and growth signals in the new micro-environment. Further cell proliferation will give rise to micro- and macro-metastases and a secondary tumor is established by the formation of a new blood supply through neo-angiogenesis. (B) Modes of cell migration. Elongated cell migration involves the extension of actin-rich protrusions at the front of the cell and the localized release of matrix metalloproteinases (MMPs), which degrade extracellular matrix (ECM) proteins and create space into which the cell can move. The formation of new adhesions at the front of the cell and contraction of the cell body pull the cell in the direction of movement, whilst loss of ECM adhesions at the rear allows the cell to migrate forward. During collective cell migration, neighboring cells within a tissue remain physically linked by adherens junctions. Cells at the invasive front extend actin-rich protrusions facing the direction of movement, which form new adhesions with the ECM and enable the generation of traction forces that pull neighboring cells forward. During rounded cell migration, high actomyosin contractility produces hydrostatic pressure, which leads to the formation of membrane blebs devoid of filamentous actin at the front of the migrating cell. Highly dynamic membrane blebs fill pre-existing spaces in the matrix and form only weak attachments to the ECM.

FIGURE 2

Schematic representation of the classical Rho GTPase regulatory cycle. GDP-bound Rho GTPases are prenylated at a C-terminal CAAX sequence by farnesyltransferase (FTase) and/or geranylgeranyltransferase type I (GGTase-I), which mediates their association with biological membranes. Rho guanine nucleotide exchange factors (RhoGEFs) promote the dissociation of GDP and uptake of GTP, which permits the interaction of Rho GTPases with effector proteins. GTPase-effector interactions are terminated by the hydrolysis of bound GTP to GDP, which is accelerated by Rho GTPase-activating proteins (RhoGAPs). Both GDP- and GTP-bound Rho GTPases can be negatively regulated by Rho guanine nucleotide dissociation inhibitors (RhoGDIs), which bind to and sequester the prenyl group, resulting in relocalization of the Rho GTPase to the cytoplasm. RhoGDI-Rho GTPase binding can be regulated by phosphorylation of RhoGDIs by kinases Src, PAK, and PKC.

FIGURE 3

Domain organization of PAKs, ROCKs, MRCKs, and aPKCs. C, Cdc42- and Rac-Interactive Binding (CRIB) domain; AI, autoinhibitory domain; KD, kinase domain; AIL, autoinhibitory-like domain; RBD, Rho-binding domain; CC, coiled-coil region; PH, pleckstrin homology domain; CRD, cysteine-rich domain; C1, C1 domain; CH, citron homology domain; PB1, Phox Bem1 domain; PS, pseudosubstrate motif. Proline-rich regions are indicated by pink bars.

FIGURE 4

Cancer-associated PAK signaling pathways activated downstream of Rho GTPases. PAK1 phosphorylates LIM Kinase-1 (LIMK-1), which stabilizes actin filaments through inhibition of cofilin. PAK1 also binds to and stimulates the GEF α-PIX, leading to an increase in Cdc42 activation. PAK1 activates PLK1, which leads to phosphorylation and degradation of claspin. In the absence of claspin, ATR is uncoupled from its effectors, resulting in an impaired DNA damage response. PAK1 also phosphorylates the pro-apoptotic protein Bad, which prevents its association with Bcl-2 and inhibits apoptosis. PAK4 binding protects RhoU from proteasomal degradation and promotes focal adhesion turnover by providing a scaffold that promotes the phosphorylation of paxillin. PAK4 also inhibits PDZ-RhoGEF, which suppresses the activation of RhoA and promotes invadopodia maturation.

FIGURE 5

Role of Cdc42-MRCK signaling and RhoA-ROCK signaling in regulation of the actin cytoskeleton. RhoA directly binds and activates the formin mDia, which nucleates the formation of unbranched actin filaments. ROCK activation downstream of RhoA leads to phosphorylation of LIMK-1 and LIMK-2, which phosphorylate and inactivate cofilin, leading to a reduction in actin depolymerization. ROCK activation leads to an increase in actomyosin contractility via phosphorylation of myosin light chain (MLC) and inhibition of myosin light chain phosphatase (MLCP). ROCK also phosphorylates ERM proteins (ezrin, radixin, and moesin) and NHE1 (Na+/H+ -Exchanger 1) to enhance coupling of the actin cytoskeleton to integral membrane proteins. Like ROCK, MRCK activation leads to decreased actin depolymerization via phosphorylation LIMK-1 and LIMK-2 and increased actomyosin contractility via MLC phosphorylation. Phosphorylation of moesin by MRCKα may enhance coupling of the actin cytoskeleton to integral membrane proteins.

FIGURE 6

Roles of ROCK, MRCK, and aPKC in the maintenance of epithelial cell polarity. Apical MRCK activation by Cdc42 leading to a local increase in actomyosin contractility, via phosphorylation of myosin light chain (MLC) and inhibition of myosin light chain phosphatase (MLCP). Cdc42 stimulates a Par-aPKC complex, which inhibits junctional Rho-ROCK signaling and establishes an intracellular actomyosin contractility gradient, leading to the segregation of Par proteins into distinct cellular domains. Apical GTP-bound Cdc42 binds to Par6, which in turn binds aPKC and Par3. Activation of aPKC leads to the phosphorylation of key polarity proteins that maintain apical-basal cell polarity.