Integrin-mediated function of Rab GTPases in cancer progression

Rab25, a 28-kDa protein also referred to as Rab11c, is involved in cell proliferation and protection from apoptosis. It has high levels of expression in ovarian and breast cancers, in which it has been shown to have a key function in both in vitro and in vivo cancer progression [13‐15]. Increased expression of Rab25 has also been noted in prostate cancer [16] and transitional cell carcinoma of the bladder [17], indicating that it has a pathological role in tumor progression in several epithelial lineages. Reports indicate that Rab25 is involved in tumor progression by regulating the localization of integrin-recycling vesicles to enhance tumor invasion (Figure 2).

Rab25 has been shown to decrease apoptosis, as well as to increase both the proliferation and aggressiveness of ovarian and breast cancer [18]. Regulation of cell survival is mediated by antiapoptotic molecules such as Bcl-2, BAX, BAK, and phosphoinositide-3-kinase [19, 20]. Forced expression of Rab25 in ovarian cancer cells decreases levels of BAX and BAK [21] and increases AKT phosphorylation, which in turn activates the phosphoinositide-3-kinase pathway. Down-regulation of Rab25 reverses apoptotic signaling through BAK and BAX protein signaling, as well as AKT activity. The Bcl-2 and phosphoinositide-3-kinase pathways are enhanced by Rab25-mediated signaling for cell survival (Figure 2)[18].

Goldenring et al. [22] demonstrated that Rab 25 expression is increased in colon cancer cells. Mills and colleagues [14] showed that 1q22 amplicon containing Rab25 is amplified in 50% of ovarian cancers. Further in vivo studies confirmed that expression of Rab25 increases ovarian tumor growth. In addition, chromosome 1q is gained in 50% of breast cancer patients. Cheng and colleagues[23] showed high expression of Rab25 in 92% of ER-positive samples and loss of expression in 83% ER-negative and PR-negative samples of basal and triple-negative tumors. These findings suggest that Rab25 may have different functions in different subtypes of breast cancer. In addition, Rab 25 is overexpressed in Wilms tumor, early-stage transitional cell carcinoma of the bladder, and hepatocellular carcinoma.

In contrast, Rab25 has also been shown to function as a tumor suppressor; its expression is lost in hormonally insensitive breast tumors [24]. Another recent study indicated that Rab25 may function as a tumor suppressor in intestinal cells. This study demonstrates that loss of Rab25 leads to increased tumorigenesis through alteration in the regulation of protein trafficking to the cell surface. These findings suggest that Rab 25 functions as a tumor suppressor in the intestinal mucosa and leading to human colorectal adenocarcinoma [25]. Rab25 knockdown by shRNA shows less proliferation, more apoptosis, and decreased ovarian tumor growth both in vitro and in vivo[13]. Studies on human breast cancer tissues have shown that loss of Rab25 expression occurs through a mutation in locus 1q22-23. Thus, Rab25 could be used as a biological marker of breast cancer [23].

Rab5 regulates the fusogenic properties of early endosomes through GTP-dependent recruitment and activation of effector proteins. Defective Rab5 causes the formation of enlarged multivesicular endosomes with many intraluminal vesicles; endosomes with early and late endocytic markers are frequently observed in the presence of defective Rab5. Inactive Rab5 is defective in sorting EGF receptor and transferrin receptor [26]. Cdc42 interacting protein-4 (CIP-4) localizes to vesicles with EGFR and the small GTPase Rab5. In giant endosomes, expression of constitutively active Rab5 leads to the accumulation of CIP-4 and related adaptor Toca-1. Downregulation of CIP-4 expression in A431 epidermoid carcinoma cells causes elevation of EGFR but, surprisingly, does not affect surface expression of EGFR [27]. Thus, understanding the function of Rab5 in EGFR signaling will be of great interest.

Rab5 guanine nucleotide exchange factor Rin1, which is activated by Ras, is important in growth-factor receptor trafficking in fibroblasts and endocytosis. Rin1 is highly expressed in the lung adenocarcinoma cell lines Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Cell proliferation is reduced by depletion of Rin1 in A549 cells, and is correlated with a decrease in EGFR signaling. Thus, Tomshine et al proposed not only that EGFR-mediated signaling essentially requires proper internalization and endocytic trafficking, but that upregulation of Rab5 guanine nucleotide exchange factor Rin1 may be involved in the proliferative pathway in A549 cells [28].

Rab11 co-localizes with insulin-containing granules in the mouse insulin-secreting cell line MIN6. Overexpression of Rab11B inhibits insulin secretion, indicating its involvement in regulating insulin secretion. Rip11, an effector of Rab11, is a substrate for PKA, which regulates exocytosis in pancreatic β-cells [29]. Rip11, which participates in insulin granule exocytosis, also has been reported to regulate the recycling of internalized cell-surface proteins from the early endosome through a trans-Golgi network. Expression of Rip11 is higher in ductal carcinoma in situ (DCIS) than it is in normal breast epithelium. Rab11a influences EGFR recycling, enhances proliferation, and prevents motility of an immortal breast cell line (MCF 10A), which is consistent with the phenotype of DCIS [30]. Rab11a and Rab11-family interacting protein 2 (FIP2) are involved in the regulation of plasma membrane recycling in epithelial cells. The phosphorylation of Rab11-FIP2 on serine 227 by MARK2 is required for epithelial cell polarity [31]. EGFR, which initiates many biochemical pathways to potentiate physiological responses, also has an important part in cancer progression. The recycling of EGFR and its signaling pathway is regulated by the endocytic pathway [32]. ADP-ribosylation factor 6 (Arf6), a regulator of cytoskeletal dynamics during cell movement, binds with protein FIP3, which interacts with Arf6 and Rab11. Both Arf6 and Rab11 appear to be involved in regulating cell motility. MDA-MB-231 breast cancer cells require FIP3 for motility. FIP3 also is involved in regulating the localization of Arf6 in MDA-MB-231 breast cancer cells and polarization of Rac1 [33].

The Rab-coupling protein RCP (RAB11FIP1), located on chromosome 8p11-12, encodes for a protein with Ras-activating function. RCP is frequently amplified in breast cancer. An in-vitro study with normal human mammary epithelial cells (MCF10A) has shown that overexpression of RCP not only increases loss of contact inhibition, growth-factor independence, and anchorage-independent growth, but also promotes Ras activation and ERK phosphorylation. Consistent with this, knockdown of RCP inhibits tumor formation and metastasis in a xenograft model [34]. Rab11 and the Eps15 homology domain 1 (EAD1) regulate the exit of internalized molecules from endocytic recycling to the plasma membrane. Thus, coordination between Rab11 and EAD proteins occurs in the mediation of endocytic recycling [35].

Rab27A is involved in exocytosis of endocrine cells and is associated with the invasive and metastatic potential of breast cancer, promoting the secretion of insulin-like growth factor-II (IGF-II). The rate of secretion controls the expression of vascular endothelial growth factor, matrix metalloproteinase-9 (MMP-9), cathepsin D, cyclin D1, p16, and urokinase-type plasminogen activator [36]. Mutations in Rab27A lead to an autosomal-recessive immunodeficiency called Griscelli syndrome type 2. Clinically, this syndrome is characterized by partial albinism and hemophagocytic lymphohistocytosis. Understanding the mechanisms of Rab27A mutations may be helpful in early diagnosis and treatment [37]. An elegant study recently demonstrated that Rab 27B controls vesicle exocytosis and releases important growth regulators into the tumor microenvironment, leading to regulation of invasive growth and metastasis in ER-positive human breast tumors [38]. This study also suggested that increased expression of Rab27 is associated with poor prognosis in humans [38] suggesting Rab 27b may function like an oncogene.

Microarray analysis data indicate that Rab35 is highly expressed in ovarian cancer. It also has been shown that androgens may affect the expression of oncogenic GTPases in ovarian cancer [29]. Rab35 is involved in T-cell receptor and oocyte yolk protein recycling and cytokinesis. Chua et al. (2010) have shown that Rab35 causes actin to reconcile protrusion through either Rho GTPases or effector fascin of actin bundling protein [39].

Rab4 and Rab14 are also involved in vesicle trafficking and associate with the C-terminal domain of P-glycoprotein (P-gp), which induces signaling for a multi-drug-resistance (MDR) mechanism in tumors. Overexpression of Rab4, but not Rab14, decreases P-gp on the cell surface of K562ADR cells, reducing the MDR phenotype by elevating the intracellular addition of daunomycin [40]. Accordingly, modulation of the spatial and temporal distribution of P-gp may be a good therapeutic strategy for suppressing the MDR phenotype.

Integrins are members of a glycoprotein family that form heterodimeric receptors involved in interaction between the cell and the surrounding extracellular matrix (ECM). Nineteen alpha subunits and eight beta subunits are paired to form 25 different integrins. Integrins control several biological processes, including cell adhesion, cell migration, and the progression of cancer and some other diseases [43, 44]. Integrins are also involved in transducing signals from outside the cells to inside and from inside to outside the cells. The ligand binding abilities of integrins are determined by the α/β subunit heterodimerization. The ligands for integrins are several extracellular matrix proteins including collagen, laminin, vitronectin and fibronectin. Some integrins such as α5β1 interact with a single ECM protein, fibronectin, however, usually integrins recognize several distinct matrix proteins [45].

The cytoplasmic domains of integrins provide a key connection between the ECM and intracellular proteins and signaling mechanisms [43]. Although both alpha and beta subunits make important contributions to various aspects of integrin functions including signal transduction, cell motility, cell adhesion and integrin activation, the beta cytoplasmic domains are critical for recruitment of integrins to focal contacts (the specialized structures at cell-matrix junctions). Integrins can also respond to signals generated inside the cells that may impact on the cytoplasmic domain, which is often referred to as inside-out signaling. For example, truncation of some integrin cytoplasmic domains prevents integrin activation [46].